TWI491616B - Use of pedf-derived polypeptides for promoting muscle or tendon regeneration or arteriogenesis - Google Patents

Use of pedf-derived polypeptides for promoting muscle or tendon regeneration or arteriogenesis Download PDF

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TWI491616B
TWI491616B TW101128705A TW101128705A TWI491616B TW I491616 B TWI491616 B TW I491616B TW 101128705 A TW101128705 A TW 101128705A TW 101128705 A TW101128705 A TW 101128705A TW I491616 B TWI491616 B TW I491616B
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muscle
tendon
pedf
synthetic peptide
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TW201406777A (en
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Yeou Ping Tsao
Tsung Chuan Ho
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Mackay Memorial Hospital
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色素上皮衍生因子衍生之多胜肽於促進肌肉或肌腱再生或動脈血管生成之用途Use of pigmented epithelial-derived factor-derived multi-peptide to promote muscle or tendon regeneration or arterial angiogenesis

本發明是關於組織損傷的治療，且特別是關於利用來自色素上皮衍生因子(pigment epithelium-derived factor，簡稱PEDF)的合成胜肽來促進肌肉或肌腱組織再生或促進動脈血管生成，以治療組織損傷。The present invention relates to the treatment of tissue damage, and in particular to the use of a synthetic peptide derived from a pigment epithelium-derived factor (PEDF) to promote muscle or tendon tissue regeneration or to promote arterial angiogenesis for the treatment of tissue damage. .

肌肉組織可分為骨骼肌、心肌或平滑肌。肌肉能夠修復其所受到的損傷。以骨骼肌為例，在受傷後，骨骼肌會藉由自發的過程來移除受損的肌纖維，並合成新的肌纖維。然而，在某些組織損傷的情形下，可能不會發生這種自發性的組織修復機制，或其修復程度不足以完全修復組織所受的損傷。舉例來說，某些病理因素或遺傳缺陷可能導致組織無法完全癒合；常見的病理因素如重傷、年邁、肌肉廢用(muscle disuse)、癌症與組織缺血(tissue ischemia)；上述遺傳缺陷如肌肉萎縮症(muscular dystrophy)。組織無法修復可能會導致肌肉質量(muscle mass)的永久損失、疾病進展與組織功能缺失。Muscle tissue can be divided into skeletal muscle, myocardial or smooth muscle. The muscles are able to repair the damage they are exposed to. In the case of skeletal muscle, after injury, skeletal muscles remove the damaged muscle fibers and synthesize new muscle fibers through a spontaneous process. However, in the case of certain tissue damage, this spontaneous tissue repair mechanism may not occur, or its degree of repair is insufficient to completely repair the damage to the tissue. For example, certain pathological factors or genetic defects may cause tissue to fail to heal completely; common pathological factors such as severe injury, old age, muscle disuse, cancer and tissue ischemia; the above genetic defects such as muscle Atrophic disease (muscular dystrophy). Tissue failure to repair can result in permanent loss of muscle mass, disease progression, and loss of tissue function.

肌腱是有韌性且成束狀的纖維性結締組織，通常用於將肌肉連接至骨骼。肌腱損傷通常會導致發炎以及肌腱退化或弱化，最終可能導致肌腱斷裂。肌腱修復是漫長且複雜的過程，通常要花上數個月甚至超過一年，在這段時間中，肌腱組織會由纖維狀慢慢變成疤狀。這種疤狀組織可 能會使得肌腱的彈性與可動性降低，且使得再次受傷的可能性偏高。肌腱幹細胞(tendon-derived stem cell，簡稱TSC)與骨髓間葉幹細胞(bone marrow-derived mesenchymal stem cell，簡稱BM-MSC)對於肌腱炎病變所提供的自體療癒效果有限。Tendons are flexible, bundled fibrous connective tissue that is commonly used to connect muscle to bone. Tendon damage usually leads to inflammation and degeneration or weakening of the tendon, which may eventually lead to tendon rupture. Tendon repair is a long and complicated process that usually takes months or even more than a year, during which time the tendon tissue slowly turns into a braid from fibrous. This sickle tissue can It can reduce the elasticity and mobility of the tendon and make the possibility of re-injury high. Tendon-derived stem cell (TSC) and bone marrow-derived mesenchymal stem cell (BM-MSC) have limited autologous healing effects on tendonitis lesions.

缺血現象也是造成明顯組織損傷的常見原因。缺血現象引起的組織損傷會造成心肌梗塞、中風與其他疾病。細胞通常能夠修復因短暫缺血現象所造成的輕微損傷；然而長時間缺血通常會造成不可回復的細胞損傷，進而導致細胞死亡。在長時間缺血的情形下，就算之後重新建立血液循環，仍無法完全回復受損細胞的功能。此外，功能喪失通常會早於細胞死亡而發生。Ischemia is also a common cause of significant tissue damage. Tissue damage caused by ischemia can cause myocardial infarction, stroke and other diseases. Cells are usually able to repair minor damage caused by transient ischae; however, prolonged ischemia usually causes irreversible cell damage, which in turn leads to cell death. In the case of prolonged ischemia, even if the blood circulation is re-established, the function of the damaged cells cannot be fully recovered. In addition, loss of function usually occurs earlier than cell death.

到目前為止，仍未發展出能有效治療受損且失去功能的組織或協助此類組織再生的方法。因此，相關領域亟需提出一種促進組織再生的方法。具體來說，需提供能夠促進動脈血管生成的藥學組合物與方法，以便促進損傷組織區域附近或其中的血流量，進而使得組織功能回復到近乎正常的程度。To date, no methods have been developed to effectively treat damaged and functionally disabled tissues or to assist in the regeneration of such tissues. Therefore, there is a need in the related art to propose a method for promoting tissue regeneration. In particular, it is desirable to provide pharmaceutical compositions and methods that promote arterial angiogenesis in order to promote blood flow in or near the damaged tissue region, thereby allowing tissue function to return to near normal levels.

發明內容旨在提供本揭示內容的簡化摘要，以使閱讀者對本揭示內容具備基本的理解。此發明內容並非本揭示內容的完整概述，且其用意並非在指出本發明實施例的重要/關鍵元件或界定本發明的範圍。發明內容旨在提供本揭 示內容的簡化摘要，以使閱讀者對本揭示內容具備基本的理解。此發明內容並非本揭示內容的完整概述，且其用意並非在指出本發明實施例的重要/關鍵元件或界定本發明的範圍。SUMMARY OF THE INVENTION The Summary of the Disclosure is intended to provide a basic understanding of the present disclosure. This Summary is not an extensive overview of the disclosure, and is not intended to be an SUMMARY OF THE INVENTION A simplified summary of the content is presented to give the reader a basic understanding of the disclosure. This Summary is not an extensive overview of the disclosure, and is not intended to be an

本發明至少部分是基於發現PEDF衍生合成胜肽能夠促進一個體體內的肌肉再生、肌腱再生或動脈血管生成。因此，此處所述的PEDF衍生合成胜肽可作為治療組織損傷(特別是由缺血現象引發的組織損傷)的製劑或藥劑。The present invention is based, at least in part, on the discovery that PEDF-derived synthetic peptides can promote muscle regeneration, tendon regeneration, or arterial angiogenesis in a body. Thus, the PEDF-derived synthetic peptides described herein are useful as a formulation or agent for the treatment of tissue damage, particularly tissue damage caused by ischemia.

有鑑於上述，本發明的一態樣是關於一種用以促進一個體體內肌肉或肌腱再生的合成胜肽。In view of the above, an aspect of the present invention relates to a synthetic peptide for promoting regeneration of muscles or tendons in a body.

根據本說明書多個實施例，所述的合成胜肽長度為20-39個胺基酸殘基，且其序列至少80%與序列編號：1相同。此外，上述胺基酸序列包含至少20個連續胺基酸殘基，其序列至少90%與序列編號：1的胺基酸殘基11-30相同，而使得此合成胜肽可用於促進一個體體內肌肉或肌腱再生。According to various embodiments of the present specification, the synthetic peptide is 20-39 amino acid residues in length, and the sequence thereof is at least 80% identical to the sequence number: 1. Furthermore, the above amino acid sequence comprises at least 20 contiguous amino acid residues, the sequence of which is at least 90% identical to the amino acid residues 11-30 of SEQ ID NO: 1, such that the synthetic peptide can be used to promote a body Regeneration of muscles or tendons in the body.

在可任選的實施例中，上述合成胜肽中有至少四個連續的胺基酸，其序列與序列編號：1的胺基酸殘基11-14相同。此種合成胜肽的非限制性例示包括胺基酸序列如序列編號：1(39-mer)、序列編號：2(34-mer)、序列編號：3(29-mer)、序列編號：5(24-mer)、序列編號：6(20-mer)、序列編號：8(MO 29-mer)與序列編號：9(MO 20-mer)所示者。根據本發明某些實施例，上述合成胜肽的胺基酸序列為序列編號：3(29-mer)、序列編號：5(24-mer)或 序列編號：6(20-mer)任一者所示。In an optional embodiment, the above synthetic peptide has at least four consecutive amino acids having the same sequence as the amino acid residues 11-14 of SEQ ID NO: 1. Non-limiting examples of such synthetic peptides include amino acid sequences such as SEQ ID NO: 1 (39-mer), SEQ ID NO: 2 (34-mer), SEQ ID NO: 3 (29-mer), SEQ ID NO: 5 (24-mer), SEQ ID NO: 6 (20-mer), SEQ ID NO: 8 (MO 29-mer) and SEQ ID NO: 9 (MO 20-mer). According to some embodiments of the invention, the amino acid sequence of the above synthetic peptide is SEQ ID NO: 3 (29-mer), SEQ ID NO: 5 (24-mer) or Serial number: 6 (20-mer) is shown as either.

在本發明另一態樣中，提出一種用以促進一個體體內肌肉或肌腱再生的藥學組合物。所述的個體/患者可以是任何哺乳類動物，包括人類。In another aspect of the invention, a pharmaceutical composition for promoting regeneration of muscle or tendon in a body is provided. The individual/patient can be any mammal, including a human.

根據本發明一實施方式，上述藥學組合物包含任一根據本發明上述態樣/實施例的合成胜肽，且此合成胜肽的含量足以促進該個體體內的肌肉或肌腱再生。此藥學組合物亦包含可用以攜帶該合成胜肽的一藥學上可接受載體。According to an embodiment of the present invention, the pharmaceutical composition comprises any of the synthetic peptides according to the above aspect/embodiment of the present invention, and the synthetic peptide is present in an amount sufficient to promote muscle or tendon regeneration in the individual. The pharmaceutical composition also includes a pharmaceutically acceptable carrier that can be used to carry the synthetic peptide.

在本發明的可任選實施例中，上述藥學上可接受載體可為一高分子材料，譬如海藻酸鹽(alginate)、明膠(gelatin)、膠原蛋白(collagen)或聚乳酸/甘醇酸共聚物(poly(lactide-co-glycolide)，簡稱PLGA)。In an optional embodiment of the present invention, the pharmaceutically acceptable carrier may be a polymer material such as alginate, gelatin, collagen or polylactic acid/glycolic acid copolymerization. (poly(lactide-co-glycolide), referred to as PLGA).

根據本發明的可任選實施例，此藥學組合物中所述合成胜肽的含量為約1-100 μM；較佳為約10 μM。According to an optional embodiment of the invention, the synthetic peptide is present in the pharmaceutical composition in an amount of from about 1 to 100 μM; preferably about 10 μM.

在本發明的又另一個態樣中，提出了一種方法，其可用以促進個體體內於鄰近一損傷區域或該損傷區域中的肌肉或肌腱再生。所述的個體/患者可以是任何哺乳類動物，包括人類。In still another aspect of the invention, a method is presented that can be used to promote regeneration of muscles or tendons in an individual adjacent to a damaged area or the damaged area. The individual/patient can be any mammal, including a human.

於一實施例中，上述方法包含對該個體體內的一治療區域投予一治療有效量的根據本發明上述態樣/實施例之合成胜肽，其中上述治療區域係鄰近該損傷區域，藉以促進該個體體內於鄰近該損傷區域或其中的肌肉或肌腱再生。In one embodiment, the method comprises administering to a therapeutic region of the subject a therapeutically effective amount of a synthetic peptide according to the above aspect/embodiment of the invention, wherein the therapeutic region is adjacent to the damaged region, thereby promoting The individual is regenerated in the body adjacent to the damaged area or muscle or tendon therein.

於可任選的實施例中，可將上述合成胜肽調製成根據 本發明上述態樣/實施例所述的藥學組合物。在實際操作時，可透過肌肉內注射(intradermal injection)的方式來投予該組合物。In an optional embodiment, the above synthetic peptide can be prepared according to A pharmaceutical composition according to the above aspect/embodiment of the invention. In actual practice, the composition can be administered by intradermal injection.

根據某些實施例，該個體可能因為肌肉損傷、肌肉廢用、肌肉萎縮症、肌萎縮性側索硬化(amyotrophic lateral sclerosis)、肌腱損傷、組織缺血、腦缺血(cerebral ischemia)、周邊動脈疾病(peripheral arterial diseases)或心肌梗塞等原因，而使損傷區域中的肌肉或肌腱受損。According to certain embodiments, the individual may be due to muscle damage, muscle wasting, muscular dystrophy, amyotrophic lateral sclerosis, tendon injury, tissue ischemia, cerebral ischemia, peripheral arteries Causes of damage to muscles or tendons in the injured area, such as peripheral arterial diseases or myocardial infarction.

此外，本發明於另一態樣中提出了一種用以促進一個體體內動脈血管生成的合成胜肽。Furthermore, the present invention provides, in another aspect, a synthetic peptide for promoting arterial angiogenesis in a body.

根據本說明書多個實施例，所述的合成胜肽長度為20-39個胺基酸殘基，且其序列至少80%與序列編號：1相同。此外，上述胺基酸序列包含至少20個連續胺基酸殘基，其序列至少90%與序列編號：1的胺基酸殘基11-30相同，而使得此合成胜肽可用於促進一個體體內動脈血管生成。According to various embodiments of the present specification, the synthetic peptide is 20-39 amino acid residues in length, and the sequence thereof is at least 80% identical to the sequence number: 1. Furthermore, the above amino acid sequence comprises at least 20 contiguous amino acid residues, the sequence of which is at least 90% identical to the amino acid residues 11-30 of SEQ ID NO: 1, such that the synthetic peptide can be used to promote a body Intra-arterial angiogenesis.

在可任選的實施例中，上述合成胜肽中有至少四個連續的胺基酸，其序列與序列編號：1的胺基酸殘基11-14相同。此種合成胜肽的非限制性例示包括胺基酸序列如序列編號：1(39-mer)、序列編號：2(34-mer)、序列編號：3(29-mer)、序列編號：5(24-mer)、序列編號：6(20-mer)、序列編號：8(MO 29-mer)與序列編號：9(MO 20-mer)所示者。根據本發明某些實施例，上述合成胜肽的胺基酸序列為序列編號：3(29-mer)、序列編號：5(24-mer)或 序列編號：6(20-mer)任一者所示。In an optional embodiment, the above synthetic peptide has at least four consecutive amino acids having the same sequence as the amino acid residues 11-14 of SEQ ID NO: 1. Non-limiting examples of such synthetic peptides include amino acid sequences such as SEQ ID NO: 1 (39-mer), SEQ ID NO: 2 (34-mer), SEQ ID NO: 3 (29-mer), SEQ ID NO: 5 (24-mer), SEQ ID NO: 6 (20-mer), SEQ ID NO: 8 (MO 29-mer) and SEQ ID NO: 9 (MO 20-mer). According to some embodiments of the invention, the amino acid sequence of the above synthetic peptide is SEQ ID NO: 3 (29-mer), SEQ ID NO: 5 (24-mer) or Serial number: 6 (20-mer) is shown as either.

在本發明另一態樣中，提出一種用以促進一個體體內動脈血管生成的藥學組合物。所述的個體/患者可以是任何哺乳類動物，包括人類。In another aspect of the invention, a pharmaceutical composition for promoting arterial angiogenesis in a body is provided. The individual/patient can be any mammal, including a human.

根據本發明一實施方式，上述藥學組合物包含任一根據本發明上述態樣/實施例的合成胜肽，且此合成胜肽的含量足以促進該個體體內的動脈血管生成。此藥學組合物亦包含可用以攜帶該合成胜肽的一藥學上可接受載體。According to an embodiment of the present invention, the pharmaceutical composition comprises any of the synthetic peptides according to the above aspect/embodiment of the present invention, and the synthetic peptide is present in an amount sufficient to promote arterial angiogenesis in the individual. The pharmaceutical composition also includes a pharmaceutically acceptable carrier that can be used to carry the synthetic peptide.

在本發明的可任選實施例中，上述藥學上可接受載體可為一高分子材料，譬如海藻酸鹽(alginate)、明膠(gelatin)、膠原蛋白(collagen)或聚乳酸/甘醇酸共聚物(poly(lactide-co-glycolide)，簡稱PLGA)。In an optional embodiment of the present invention, the pharmaceutically acceptable carrier may be a polymer material such as alginate, gelatin, collagen or polylactic acid/glycolic acid copolymerization. (poly(lactide-co-glycolide), referred to as PLGA).

根據本發明的可任選實施例，此藥學組合物中所述合成胜肽的含量為約1-100 μM；較佳為約10 μM。According to an optional embodiment of the invention, the synthetic peptide is present in the pharmaceutical composition in an amount of from about 1 to 100 μM; preferably about 10 μM.

在本發明的又另一個態樣中，提出了一種方法，其可用以促進個體體內於鄰近一缺血區域或該缺血區域中的動脈血管生成。所述的個體/患者可以是任何哺乳類動物，包括人類。In still another aspect of the invention, a method is presented that can be used to promote arterial angiogenesis in an individual adjacent to an ischemic region or the ischemic region. The individual/patient can be any mammal, including a human.

於一實施例中，上述方法包含對該個體體內的一治療區域投予一治療有效量的根據本發明上述態樣/實施例之合成胜肽，其中上述治療區域係鄰近該缺血區域，藉以促進該個體體內於鄰近該缺血區域或其中的動脈血管生成。In one embodiment, the method comprises administering to a therapeutic region of the subject a therapeutically effective amount of a synthetic peptide according to the above aspect/embodiment of the invention, wherein the therapeutic region is adjacent to the ischemic region, whereby Promoting angiogenesis in the body adjacent to the ischemic region or therein.

於可任選的實施例中，可將上述合成胜肽調製成根據本發明上述態樣/實施例所述的藥學組合物。在實際操作 時，可透過肌肉內注射(intradermal injection)的方式來投予該組合物。In an optional embodiment, the above synthetic peptide can be formulated into a pharmaceutical composition according to the above aspects/embodiments of the invention. In actual operation The composition can be administered by intradermal injection.

根據某些實施例，該個體可能因為肌肉損傷、肌肉廢用、肌肉萎縮症、肌萎縮性側索硬化(amyotrophic lateral sclerosis)、肌腱損傷、組織缺血、腦缺血(cerebral ischemia)、周邊動脈疾病(peripheral arterial diseases)或心肌梗塞等原因，而使缺血區域中的血流受阻。According to certain embodiments, the individual may be due to muscle damage, muscle wasting, muscular dystrophy, amyotrophic lateral sclerosis, tendon injury, tissue ischemia, cerebral ischemia, peripheral arteries Causes of blood flow in the ischemic area, such as peripheral arterial diseases or myocardial infarction.

在參閱下文實施方式後，本發明所屬技術領域中具有通常知識者當可輕易瞭解本發明之基本精神及其他發明目的，以及本發明所採用之技術手段與實施態樣。The basic spirit and other objects of the present invention, as well as the technical means and implementations of the present invention, will be readily apparent to those skilled in the art of the invention.

為了使本揭示內容的敘述更加詳盡與完備，下文針對了本發明的實施態樣與具體實施例提出了說明性的描述；但這並非實施或運用本發明具體實施例的唯一形式。實施方式中涵蓋了多個具體實施例的特徵以及用以建構與操作這些具體實施例的方法步驟與其順序。然而，亦可利用其他具體實施例來達成相同或均等的功能與步驟順序。The description of the embodiments of the present invention is intended to be illustrative and not restrictive. The features of various specific embodiments, as well as the method steps and sequences thereof, are constructed and manipulated in the embodiments. However, other specific embodiments may be utilized to achieve the same or equivalent function and sequence of steps.

除非本說明書另有定義，此處所用的科學與技術詞彙之含義與本發明所屬技術領域中具有通常知識者所理解與慣用的意義相同。此外，在不和上下文衝突的情形下，本說明書所用的單數名詞涵蓋該名詞的複數型；而所用的複數名詞時亦涵蓋該名詞的單數型。The scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains, unless otherwise defined herein. In addition, the singular noun used in this specification covers the plural of the noun in the case of no conflict with the context; the plural noun of the noun is also included in the plural noun used.

雖然用以界定本發明較廣範圍的數值範圍與參數界是 約略的數值，已盡可能精確地呈現具體實施例的相關數值。然而，任何數值本質上不可避免地含有因個別測試方法所致的標準偏差。在此處，「約」通常係指實際數值在一特定數值或範圍的正負10%、5%、1%或0.5%之內。或者是，「約」一詞代表實際數值落在平均值的可接受標準誤差之內，視本發明所屬技術領域中具有通常知識者的考量而定。除了實驗例之外，或除非另有明確的說明，當可理解此處所用的所有範圍、數量、數值與百分比(例如用以描述材料用量、時間長短、溫度、操作條件、數量比例及其他相似者)均經過「約」的修飾。因此，除非另有相反的說明，本說明書與附隨申請專利範圍所揭示的數值參數皆為約略的數值，且可視需求而更動。至少應將這些數值參數理解為所指出的有效位數與套用一般進位法所得到的數值。Although the numerical ranges and parameter boundaries used to define the broader scope of the invention are The approximate values have been presented to the relevant values of the specific embodiments as precisely as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the term "about" means that the actual value falls within the acceptable standard error of the average, depending on the considerations of those of ordinary skill in the art to which the invention pertains. Except for the experimental examples, or unless otherwise explicitly stated, all ranges, quantities, values, and percentages used herein are understood (eg, to describe the amount of material used, the length of time, the temperature, the operating conditions, the quantity ratio, and the like. Are all modified by "about". Therefore, unless otherwise indicated to the contrary, the numerical parameters disclosed in the specification and the appended claims are intended to be At a minimum, these numerical parameters should be understood as the number of significant digits indicated and the values obtained by applying the general carry method.

在此處「胜肽」(peptide)一詞係指胺基酸殘基所組成的高分子。「合成胜肽」(synthetic peptide)一詞則代表此胜肽並未包含存在於自然界的完整蛋白質分子。此種胜肽之所以是「合成的」，表示其乃是由人類利用技術手段所得，譬如化學合成、重組遺傳技術或將整個抗原切段。於本說明書中，任何胺基酸殘基於一胜肽中的位置係由該胜肽的N端起算。The term "peptide" as used herein refers to a polymer composed of amino acid residues. The word "synthetic peptide" means that the peptide does not contain intact protein molecules found in nature. The reason why such a peptide is "synthetic" is that it is obtained by humans using technical means such as chemical synthesis, recombinant genetic techniques or segmentation of the entire antigen. In the present specification, any amino acid residue based on the position in a peptide is counted from the N-terminus of the peptide.

「幹細胞」(stem cell)一詞在此係指此細胞在某些條件下保有能夠增殖而不實質分化的能力，且亦能夠在某些條件下分化成更專一或分化程度較高的細胞。The term "stem cell" as used herein means that the cell retains the ability to proliferate without substantial differentiation under certain conditions and is capable of differentiating into more specific or highly differentiated cells under certain conditions.

在此處「增殖」的各種詞性(如proliferate或proliferation)係指族群中的細胞數目透過細胞***而增加之情形。The various parts of the word "proliferation" or "proliferation" herein refer to the situation in which the number of cells in a population increases through cell division.

「肌肉細胞」(muscle cells)一詞在此係指任何對肌肉組織有貢獻的細胞，且包含肌母細胞(myoblasts)、衛星細胞(satellite cells)、肌小管細胞(myotubes)及肌原纖維組織(myofibril tissues)。「肌肉再生」(muscle regeneration)一詞在此是指由肌肉先驅細胞(muscle progenitor cells)形成新的肌纖維之任何過程。可透過多種指標來評估肌肉在損傷區域中或鄰近區域的再生情形，這些指標如在損傷區域中或附近的肌纖維數目、直徑(大小)、濕重和/或蛋白質含量等。此外，亦可透過損傷區域中或附近之肌肉細胞和/或衛星細胞的增殖活性，來觀察肌肉再生之情形。The term "muscle cells" as used herein refers to any cell that contributes to muscle tissue and includes myoblasts, satellite cells, myotubes, and myofibrils. (myofibril tissues). The term "muscle regeneration" as used herein refers to any process in which new muscle fibers are formed by muscle progenitor cells. Multiple indicators can be used to assess the regeneration of muscles in or adjacent to the injured area, such as the number of muscle fibers, diameter (size), wet weight and/or protein content in or near the damaged area. In addition, muscle regeneration can be observed through the proliferative activity of muscle cells and/or satellite cells in or near the injured area.

在本說明書中，「肌腱」(tendon)一詞係指由平行排列且緊密相接的膠原蛋白纖維所形成的纖維狀組織，其可將肌肉連接至骨骼。肌腱損傷的復原過程非常緩慢的過程，且通常會形成疤痕，因而使得肌腱產生缺陷，而無法回復正常或原本的肌腱功能。在此處「肌腱再生」(tendon regeneration)一詞係指在肌腱復原的過程中，產生第一型膠原蛋白(type I ollagen)，且新形成的膠原蛋白纖維平行於張力方向，因而將復原過程中的疤痕形成降至最低。在損傷區域之中或附近呈有組織排列的膠原蛋白原纖維(fibrils)之數目，可用以評估肌腱再生之程度。此外，亦可透過損傷區域中或附近之肌腱幹細胞的增殖活性，來觀 察肌腱再生之情形。In the present specification, the term "tendon" refers to a fibrous tissue formed by collagen fibers arranged in parallel and closely connected, which can connect muscles to bones. The recovery process of tendon injury is a very slow process, and usually a scar is formed, which causes the tendon to become defective and unable to return to normal or original tendon function. Here, the term "tendon regeneration" refers to the production of type I ollagen during the recovery of tendon, and the newly formed collagen fibers are parallel to the direction of tension, thus restoring the process Scar formation is minimized. The number of organized fibrils in or near the lesion area can be used to assess the extent of tendon regeneration. In addition, it can also be observed through the proliferative activity of tendon stem cells in or near the injured area. Check the condition of tendon regeneration.

於本說明書中，「動脈血管生成」(arteriogenesis)一詞與「血管新生」(angiogenesis)並不相同。血管新生是指由既有血管分枝形成微血管的過程；這些新生成的微血管並沒有脈管平滑肌細胞(vascular smooth muscle cells，簡稱vSMCs)，因此較為脆弱且容易破裂。此種微血管無法進行血管重塑(vasculature remodeling)過程，且因而不能對損傷區域或鄰近區域供應和/或回復適當的血流循環。與血管新生相較之下，動脈血管生成係指由既有動脈管路的內皮細胞與平滑肌細胞增殖，而在原地補充與擴展動脈或側枝動脈(collateral arteries)。這些新形成的動脈或側枝動脈可發展成動脈(或側枝動脈)網路，而能夠形成天然的繞道已供應足夠的血液至受損或缺血組織或發炎部位。In this specification, the term "arteriogenesis" is not the same as "angiogenesis". Angiogenesis refers to the process of forming microvessels from existing blood vessel branches; these newly formed microvessels are not vascular smooth muscle cells (vSMCs) and are therefore relatively fragile and easily broken. Such microvasculature is unable to undergo a vasculature remodeling process and thus cannot supply and/or restore an appropriate blood flow cycle to the damaged area or adjacent areas. In contrast to angiogenesis, arterial angiogenesis refers to the proliferation of endothelial cells and smooth muscle cells from existing arterial lines, and the in situ recruitment and expansion of collateral arteries. These newly formed arteries or collateral arteries can develop into a network of arteries (or collaterals) that can form a natural bypass that has supplied sufficient blood to damaged or ischemic tissue or inflammatory sites.

「缺血」(ischemia)一詞係指在任何組織和/或器官中，因為缺乏氧氣供應和/或因為代謝物不正常累積，而導致的狀況。此種情形通常是由於不正常的灌流(perfusion)，而使得氧氣供需不平衡所引起。已知有多種情況可能會引發上述不正常的灌流，如動脈粥狀硬化(atherosclerosis)、血管狹窄(restenotic lesions)、貧血(anemia)、中風(stroke)或動脈阻塞(clogged arteries)等，這些情況會導致組織(如，肌肉心臟或腦部)的氧氣量不足。然而，缺血狀況不限於上述器官或組織，而可能發生在任何器官/組織。The term "ischemia" refers to a condition in any tissue and/or organ that results from a lack of oxygen supply and/or because of abnormal accumulation of metabolites. This is usually caused by abnormal perfusion, which causes an imbalance in oxygen supply and demand. A variety of conditions are known to cause such abnormal perfusion, such as atherosclerosis, restenotic lesions, anemia, stroke or clogged arteries. It can cause insufficient oxygen in tissues (eg, muscle heart or brain). However, the ischemic condition is not limited to the above-mentioned organs or tissues, but may occur in any organ/tissue.

「促進」及其各種時態(如promote或promoting)在此係指一種正面的改變；特別是指在統計上具有顯著意義的正面改變。在一實施例中，正面的改變係指相較於一參考標準，增加了至少10%。"Promotion" and its various tenses (such as promotion or promotion) refer to a positive change here; in particular, a positive change that is statistically significant. In one embodiment, the change in front refers to an increase of at least 10% compared to a reference standard.

此處針對合成胜肽序列所述的「胺基酸序列相似度百分比」(Percent% amino acid sequence identity)係指該候選合成胜肽之胺基酸殘基與一參考多胜肽之胺基酸殘基完全相同的百分比。於進行上述比對時，可將該候選合成胜肽與該參考多胜肽並排，並於必要時引入間隙，以使二序列形成最高的序列相似度，且在計算相似度時，並未將保守性置換之胺基酸殘基納入考量。相關領域已有多種方法可供進行上述並排，譬如可公開取得的軟體如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)等。本發明所屬技術領域中具有通常知識者在進行並排時，可選擇適當的參數與計算方式，以得到最佳的排列方式。在本說明書中，二胺基酸序列間的序列比較是採用美國國家生物科技資訊中心(Nation Center for Biotechnology Information，簡稱NCBI)所提供的蛋白質-蛋白質BLAST分析軟體Blastp來進行。一候選胺基酸序列A相較於一參考胺基酸序列B的胺基酸序列相似度(在本說明書中亦稱之為序列A與序列B具有特定百分比(%)的胺基酸序列相似度)的計算方式如下： 其中X是利用Blastp軟體對序列A、B進行排列後所得到 的相同胺基酸殘基數目(identical matches)，而Y是A、B二序列中較短者的胺基酸殘基總數。The "Percent% amino acid sequence identity" as used herein for the synthetic peptide sequence refers to the amino acid residue of the candidate synthetic peptide and the amino acid of a reference polypeptide. The percentage of residues that are identical. When performing the above alignment, the candidate synthetic peptide can be side by side with the reference polypeptide, and a gap is introduced as necessary to form the highest sequence similarity of the two sequences, and when calculating the similarity, the Amino acid residues of conservative substitutions are taken into account. A variety of methods are available for the above-described side-by-side, such as publicly available software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR). Those skilled in the art to which the present invention pertains may select appropriate parameters and calculation methods when performing side-by-side to obtain an optimal arrangement. In the present specification, sequence comparison between diamine acid sequences is carried out using the protein-protein BLAST analysis software Blastp provided by the National Center for Biotechnology Information (NCBI). Amino acid sequence similarity of a candidate amino acid sequence A compared to a reference amino acid sequence B (also referred to herein as sequence A and sequence B have a specific percentage (%) of amino acid sequence similarity Degree) is calculated as follows: Where X is the identity of the same amino acid residue obtained by aligning the sequences A and B with Blastp software, and Y is the total number of amino acid residues of the shorter of the A and B sequences.

在此處「藥學上可接受載體」(pharmaceutically acceptable carrier)係指一種藥學上可接受的材料、組合物或媒劑(vehicle)，諸如液體或固體填充物(liquid or solid filler)、稀釋劑(diluent)、賦型劑(excipient)、溶劑(solvent)或包覆材料(encapsulating material)，其可用以攜帶或運送標的成分由身體的一器官或部分到達身體的另一器官或部份。「可接受」的載體係指其可含組合物中的其他成分相容。載體可為固態、半固態、液態、乳霜或膠囊等形式。By pharmaceutically acceptable carrier herein is a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, a diluent ( A diluent, an excipient, a solvent, or an encapsulating material that can be used to carry or transport a target component from one organ or part of the body to another organ or part of the body. By "acceptable" carrier is meant that it may be compatible with the other ingredients in the composition. The carrier can be in the form of a solid, semi-solid, liquid, cream or capsule.

於本說明書中，「治療」一詞的各種詞性與時態(如treat、treating或treatment)係指預防性(如，預防用藥)、療癒性或緩和性的處置。「治療」一詞可用以指稱將此處提出的組合物投予或施用於一個體，此個體可能有一醫療疾患、症狀、疾病或與疾患相關的異常或易於罹患一疾患，以期能部分地或完全地緩和、改善、減輕一特定異常和/或病症的一或多種症狀或特徵，或延緩其發生、阻礙其進展、減輕其嚴重性和/或減低發生率。亦可對並未表出現疾病、異常和/或病症之徵兆的個體和/或呈現早期徵兆的個體進行治療，以期降低發展出與該疾病、異常和/或病症相關之病理變化的風險。在本說明書中，當一或更多種症狀或臨床指標減少時，通常認為該治療是「有效」的。或者是，有效的治療意味著一疾病的進展減少或暫停。亦即，「治療」不僅包含改善症狀或降低疾病指標，還涵蓋了使得原本在 不進行治療的情形下，極有可能發生的疾病進展或症狀惡化等情形停止或減緩。有益的或的臨床結果包括，但不限於：一或多種症狀的緩解、疾病程度的縮減、疾病狀態的穩定(如，並未惡化)、疾病進程的延遲或延緩、疾病狀態的部分或完全改善或緩和，以上所述包含可偵測或不可偵測的症狀、程度、狀態或進程。In this specification, the various parts of speech and tense (eg, treat, treating, or treatment) refer to prophylactic (eg, prophylactic), healing, or palliative treatment. The term "therapeutic" may be used to refer to the administration or administration of a composition presented herein to a subject which may have a medical condition, symptom, disease or disorder associated with the condition or is susceptible to a condition, with a view to being able to partially or Completely alleviating, ameliorating, alleviating, or delaying the onset, hindering the progression, reducing the severity, and/or reducing the incidence of a particular abnormality and/or condition. Individuals who are not indicative of signs of disease, abnormality, and/or condition and/or individuals presenting early signs may also be treated with a view to reducing the risk of developing pathological changes associated with the disease, disorder, and/or condition. In the present specification, when one or more symptoms or clinical indicators are reduced, the treatment is generally considered to be "effective." Or, effective treatment means a reduction or pause in the progression of a disease. That is, "treatment" includes not only improving symptoms or reducing disease indicators, but also covering the original In the case of no treatment, it is highly probable that the progression of the disease or the deterioration of the symptoms will stop or slow down. Beneficial or clinical outcomes include, but are not limited to, relief of one or more symptoms, reduction in disease severity, stabilization of the disease state (eg, no deterioration), delay or delay in disease progression, partial or complete improvement in disease status Or alleviation, the above includes symptoms, degrees, status or progression that are detectable or undetectable.

在此處，「有效量」一詞係指一成分的用量足以招致所欲的反應或效果。「治療有效量」一詞則是指藥學組合物中的治療藥劑之含量足以產生上文定義的所欲「有效治療」。具體的治療有效量取決於多種因素，諸如欲治療的特定狀況、個體的生理條件(如，個體體重、年齡或性別)、接受治療的哺乳動物或動物的類型、治療持續時間、目前療法(如果有的話)的本質以及所用的具體配方和化合物或其衍生物的結構。治療有效量亦指於此用量下，該化合物或組合物的毒性或負面效果不及於其所帶來的正面療效。As used herein, the term "effective amount" means that the amount of a component is sufficient to induce a desired response or effect. The term "therapeutically effective amount" means that the therapeutic agent is present in an amount sufficient to produce the desired "effective treatment" as defined above. The particular therapeutically effective amount depends on a variety of factors, such as the particular condition being treated, the physiological condition of the individual (eg, the individual's weight, age, or sex), the type of mammal or animal being treated, the duration of treatment, current therapy (if The nature of any) and the specific formulation and structure of the compound or derivative thereof used. A therapeutically effective amount also means that the toxic or negative effect of the compound or composition is less than the positive effect of the compound or composition.

「患者」或「個體」等詞在此係指可接受本發明之合成胜肽、藥學組合物和/或方法來進行治療的哺乳類動物(包括人類)。除非另有指明，「個體」一般包含雄性與雌性。The terms "patient" or "individual" are used herein to mean a mammal (including a human) that can receive the synthetic peptide, pharmaceutical composition and/or method of the present invention for treatment. Unless otherwise indicated, "individual" generally includes males and females.

色素上皮衍生因子(Pigment epithelium-derived factor，簡稱PEDF)是一種多功能的分泌性蛋白質，其具有抗血管新生(anti-angiogenic)、抗腫瘤生成(anti-tumorigenic)與神經滋養(neurotrophic)等功能。人類PEDF蛋白質(序列編號：14)是一種大小約50 kDa 的分泌性蛋白質，具有418個胺基酸。已知PEDF的34-mer片段(相當於PEDF之第44-77號殘基)與44-mer片段(相當於PEDF之第78-121號殘基)分別具有抗血管新生與神經滋養性質。Pigment epithelium-derived factor (PEDF) is a multifunctional secreted protein with anti-angiogenic, anti-tumorigenic and neurotrophic functions. . The human PEDF protein (SEQ ID NO: 14) is a size of approximately 50 kDa a secreted protein with 418 amino acids. It is known that the 34-mer fragment of PEDF (corresponding to residues 44-77 of PEDF) and the 44-mer fragment (corresponding to residues 78-121 of PEDF) have anti-angiogenic and neurotrophic properties, respectively.

本說明書至少部分是基於發現來自PEDF的合成胜肽能夠促進一個體體內的肌肉或肌腱再生與動脈血管生成。特別是，本發明率先發現局部施用此PEDF衍生合成胜肽與受損或缺血的肌肉或肌腱組織的復原以及在缺血區域中或附近的動脈(或側枝動脈)生成之間的關聯。本發明的另一創新特徵在於此處所提出的合成胜肽最長只有39個胺基酸殘基，比起先前技術所述的全長PEDF短了許多；因此，本發明克服了傳統蛋白質在臨床使用上經常面臨的困境，諸如製造成本高昂、生體可用率(bioavailability)低落與藥物動力學(pharmacokinetics)表現不佳等。因此，這些合成胜肽可用以治療肌肉或肌腱損傷以及缺血的組織或器官。This specification is based, at least in part, on the discovery that synthetic peptides derived from PEDF can promote muscle or tendon regeneration and arterial angiogenesis in a body. In particular, the present invention is the first to discover the association between the local administration of this PEDF-derived synthetic peptide and the recovery of damaged or ischemic muscle or tendon tissue and the formation of arteries (or collaterals) in or near the ischemic area. Another innovative feature of the present invention is that the synthetic peptide proposed herein has a maximum of only 39 amino acid residues, which is much shorter than the full length PEDF described in the prior art; therefore, the present invention overcomes the clinical use of conventional proteins. Frequently faced dilemmas such as high manufacturing costs, low bioavailability, and poor pharmacokinetics. Thus, these synthetic peptides can be used to treat muscle or tendon injuries as well as ischemic tissues or organs.

因此，本發明的一態樣是關於用以促進一個體體內肌肉或肌腱再生的合成胜肽。本發明的另一態樣則是關於用以促進一個體體內動脈血管生成的合成胜肽。下文敘述了適用於上述兩種態樣或其中之一的實施例。Accordingly, one aspect of the present invention relates to a synthetic peptide for promoting regeneration of muscle or tendon in a body. Another aspect of the invention relates to a synthetic peptide for promoting arterial angiogenesis in a body. Embodiments suitable for either or both of the above aspects are described below.

根據本說明書多個實施例，此合成胜肽有20-39個胺基酸殘基，且其胺基酸序列與序列編號：1(LSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGT)有至少80%的胺基酸序列相似度。舉例來說，此合成胜肽 與序列編號：1的胺基酸序列相似度可為約80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99或100%。另外，此合成胜肽包含至少20個連續的胺基酸殘基，其與序列編號：1第11-30個殘基有至少90%的胺基酸序列相似度。具體來說，這20個連續胺基酸殘基與序列編號：1第11-30個殘基的胺基酸序列相似度可為約90、91、92、93、94、95、96、97、98、99或100%。According to various embodiments of the present specification, the synthetic peptide has 20 to 39 amino acid residues, and the amino acid sequence thereof has at least 80% amino acid sequence similarity to SEQ ID NO: 1 (LSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGT). For example, this synthetic peptide The similarity to the amino acid sequence of SEQ ID NO: 1 may be about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97. , 98, 99 or 100%. In addition, the synthetic peptide comprises at least 20 contiguous amino acid residues having at least 90% amino acid sequence similarity to SEQ ID NO: 1 from 11 to 30 residues. Specifically, the 20 consecutive amino acid residues may have a similarity to the amino acid sequence of the 11th to 30th residues of SEQ ID NO: 1 of about 90, 91, 92, 93, 94, 95, 96, 97. , 98, 99 or 100%.

在本發明一實施例中，所述的合成胜肽有39個胺基酸殘基，其序列即如序列編號：1所示。在下文實驗例中，亦將此合成胜肽稱為39-mer。此種39-mer胜肽是來自人類PEDF的已知44-mer片段(PEDF第78-121號殘基)的一種較短的變形。In one embodiment of the invention, the synthetic peptide has 39 amino acid residues, the sequence of which is as shown in SEQ ID NO: 1. In the experimental examples below, this synthetic peptide was also referred to as a 39-mer. Such a 39-mer peptide is a shorter variant of the known 44-mer fragment from human PEDF (PEDF residues 78-121).

本案發明人先前所做的實驗(如，審查中的美國專利申請號13/428996，在此將該先申請案的內容一併納入為本說明書的一部分)與本說明書所提供的實驗例顯示還有其他數種來自此39-mer的短PEDF合成胜肽能夠有效促進一個體體內的肌肉或肌腱再生和/或動脈血管生成。The experiments previously performed by the inventors of the present invention (e.g., U.S. Patent Application Serial No. 13/428,996, the entire contents of which is incorporated herein by reference in its entirety) There are several other short PEDF synthetic peptides from this 39-mer that are effective in promoting muscle or tendon regeneration and/or arterial angiogenesis in a body.

舉例來說，由下文與先申請案的實驗例可知，如序列編號：2(ALSALSLGAEQRTESIIHRALYYDLISSPDIHGT)所示的34-mer合成胜肽能夠有效促進個體體內的肌肉或肌腱再生和/或動脈血管生成。34-mer合成胜肽相當於人類PEDF的第88-121號胺基酸殘基；根據上文所述的胺基酸序列相似度的計算方法，此34-mer與39-mer的胺基酸序 列相似度為100%，且34-mer的第6-25號胺基酸殘基與39-mer的第11-30號胺基酸殘基的胺基酸序列相似度也是100%。For example, it can be seen from the experimental examples of the following application that the 34-mer synthetic peptide as shown in SEQ ID NO: 2 (ALSALSLGAEQRTESIIHRALYYDLISSPDIHGT) is effective for promoting muscle or tendon regeneration and/or arterial angiogenesis in an individual. The 34-mer synthetic peptide corresponds to amino acid residues 88-121 of human PEDF; the 34-mer and 39-mer amino acids are calculated according to the amino acid sequence similarity calculation method described above. sequence The column similarity is 100%, and the amino acid sequence residues of amino acids 6-25 of the 34-mer and the amino acid residues 11-30 of the 39-mer are also 100% similar.

此外，下文多個實驗例證明如序列編號：3(SLGAEQRTESIIHRALYYDLISSPDIHGT)所示的29-mer合成胜肽能夠有效促進個體體內的肌肉或肌腱再生，還能促進動脈血管生成。此29-mer合成胜肽相當於人類PEDF的第93-121號胺基酸殘基；其與39-mer的胺基酸序列相似度為100%，且29-mer的第1-20號胺基酸殘基與39-mer的第11-30號胺基酸殘基的胺基酸序列相似度亦為100%。In addition, the following experimental examples demonstrate that the 29-mer synthetic peptide represented by SEQ ID NO: 3 (SLGAEQRTESIIHRALYYDLISSPDIHGT) can effectively promote muscle or tendon regeneration in an individual and promote arterial angiogenesis. This 29-mer synthetic peptide corresponds to amino acid residues 93-121 of human PEDF; its similarity to the amino acid sequence of 39-mer is 100%, and the amine of No. 1-20 of 29-mer The amino acid sequence has a similarity to the amino acid sequence of the amino acid residues 11-30 of the 39-mer.

某些實驗例證實，一24-mer合成胜肽亦可有效促進個體體內的肌肉或肌腱再生以及動脈血管生成；此24-mer的序列如序列編號：5(SLGAEQRTESIIHRALYYDLISSP)所示，且相當於人類PEDF第93-116號胺基酸殘基。此外，24-mer序列與39-mer的第11-30號胺基酸殘基完全相同(胺基酸序列相似度：100%)且其相較於39-mer的胺基酸序列相似度同樣也是100%。In some experiments, a 24-mer synthetic peptide can also effectively promote muscle or tendon regeneration and arterial angiogenesis in an individual; the 24-mer sequence is shown in SEQ ID NO: 5 (SLGAEQRTESIIHRALYYDLISSP) and is equivalent to human PEDF No. 93-116 amino acid residue. In addition, the 24-mer sequence is identical to the amino acid residues 11-30 of the 39-mer (amino acid sequence similarity: 100%) and is similar to the amino acid sequence similarity of the 39-mer. It is also 100%.

某些實驗例證實，如序列編號：6(SLGAEQRTESIIHRALYYDL)所示的20-mer亦可有效促進個體體內的肌肉或肌腱再生以及動脈血管生成。此20-mer的序列相當於人類PEDF第93-112號胺基酸殘基，其與39-mer的第11-30號胺基酸殘基完全相同(胺基酸序列相似度：100%)且其相較於39-mer的胺基酸序列相似度同樣也是100%。In some experiments, the 20-mer shown in SEQ ID NO: 6 (SLGAEQRTESIIHRALYYDL) is also effective in promoting muscle or tendon regeneration and arterial angiogenesis in an individual. The sequence of this 20-mer corresponds to the amino acid residue of human PEDF No. 93-112, which is identical to the amino acid residues 11-30 of the 39-mer (amino acid sequence similarity: 100%) And its similarity to the amino acid sequence of the 39-mer is also 100%.

本案與先申請案所揭載的實驗例亦顯示，另外有兩種衍生自小鼠PEDF的短、合成胜肽同樣也能夠有效地促進個體體內的肌肉或肌腱再生和/或動脈血管生成。第一種衍生自小鼠的合成胜肽在本說明書中稱為Mo 29-mer，其序列如序列編號：8(SLGAEHRTESVIHRALYYDLITNPDIHST)所示，此序列與39-mer的胺基酸序列相似度為83%，且其前20個胺基酸殘基與39-mer第11-30個殘基的胺基酸序列相似度為90%。另一種衍生自小鼠PEDF的合成胜肽稱為Mo 20-mer，其序列如序列編號：9(SLGAEHRTESVIHRALYYDL)所示。Mo 20-mer與39-mer或39-mer的第11-30號胺基酸殘基的胺基酸序列相似度皆為90%。The experimental examples disclosed in the present application and the prior application also show that two other short, synthetic peptides derived from mouse PEDF are also effective in promoting muscle or tendon regeneration and/or arterial angiogenesis in an individual. The first synthetic peptide derived from mouse is referred to in this specification as Mo 29-mer, the sequence of which is shown in SEQ ID NO: 8 (SLGAEHRTESVIHRALYYDLITNPDIHST), which has a similarity to the 39-mer amino acid sequence of 83. %, and its first 20 amino acid residues have a similarity to the amino acid sequence of the 11- to 30-residue of the 39-mer. Another synthetic peptide derived from mouse PEDF is called Mo 20-mer and its sequence is shown as SEQ ID NO: 9 (SLGAEHRTESVIHRALYYDL). The amino acid sequence residues of the Mo 20-mer and the 39-mer or 39-mer amino acid residues are all 90% similar.

在可任選的實施例中，上述合成胜肽中有至少四個連續的胺基酸，其序列與序列編號：1的胺基酸殘基11-14相同。本案發明人所進行的實驗結果顯示，序列編號：1所示序列的第11-14號胺基酸殘基(即，SLGA)對於維持短PEDF合成胜肽之生理功能扮演的重要的角色。舉例來說，下文提出的多個實驗例顯示，不具有此SLGA片段的18-mer合成胜肽(EQRTESIIHRALYYDLIS；序列編號：7)無法促進個體體內的動脈血管生成。此外，由本案與先申請案所載的實驗例可以發現，25-mer合成胜肽(EQRTESIIHRALYYDLISSPDIHGT；序列編號：4)無法有效促進個體體內的肌肉或肌腱再生和/或動脈血管生成。In an optional embodiment, the above synthetic peptide has at least four consecutive amino acids having the same sequence as the amino acid residues 11-14 of SEQ ID NO: 1. The experimental results conducted by the inventors of the present invention show that the amino acid residues No. 11-14 of the sequence of SEQ ID NO: 1 (i.e., SLGA) play an important role in maintaining the physiological function of the short PEDF synthetic peptide. For example, the various experimental examples presented below show that an 18-mer synthetic peptide (EQRTESIIHRALYYDLIS; SEQ ID NO: 7) without this SLGA fragment fails to promote arterial angiogenesis in an individual. Furthermore, it can be found from the experimental examples contained in the present application and the prior application that the 25-mer synthetic peptide (EQRTESIIHRALYYDLISSPDIHGT; SEQ ID NO: 4) is not effective in promoting muscle or tendon regeneration and/or arterial angiogenesis in an individual.

可利用任何習用的技術來合成此處所述的合成胜肽， 譬如t-BOC或FMOC來保護α-氨基(alpha-amino groups)。這兩種方法都採用了逐步合成法，其係由該胜肽的C端開始，每次加上一個胺基酸。亦可利用其他已知的固態胜肽合成(solid phase peptide synthesis，簡稱SPPS)法來合成此處所述的合成胜肽。Any of the conventional techniques can be utilized to synthesize the synthetic peptides described herein, For example, t-BOC or FMOC protects alpha-amino groups. Both methods employ a stepwise synthesis starting with the C-terminus of the peptide, each time adding an amino acid. Other known solid phase peptide synthesis (SPPS) methods can also be utilized to synthesize the synthetic peptides described herein.

本發明之範圍亦涵蓋了其他相較於39-mer具有保守性置換的合成胜肽。在此處，「保守性置換」一詞係指利用另一種在生物學上相似的殘基來取代某一殘基。保守性置換的例示包括親水性殘基(如異白胺酸、纈胺酸、白胺酸與甲硫胺酸)彼此間的置換，或相近極性殘基(如精胺酸與離胺酸；或麩胺酸與天冬胺酸)彼此間的置換，以及其他類似的置換模式。「保守性置換」在此亦指利用一具有取代基的胺基酸來取代一不具有取代基的原始胺基酸，只要可和此具有取代基之胺基酸反應的抗體亦可和原始胺基酸進行免疫反應即可。Also included within the scope of the invention are other synthetic peptides that have conservative substitutions compared to the 39-mer. Here, the term "conservative substitution" refers to the replacement of a residue with another biologically similar residue. Illustrative of conservative substitutions include substitutions of hydrophilic residues (such as isoleucine, valine, leucine, and methionine) with each other, or similar polar residues (such as arginine and lysine; Or glutamic acid and aspartate) replacement with each other, and other similar modes of substitution. "Conservative substitution" as used herein also refers to the use of a substituted amino acid to replace an unsubstituted amino acid, as long as the antibody reactive with the substituted amino acid can also react with the original amine. The base acid can be used for an immune reaction.

亦可將上述實施方式所述的各種合成胜肽調製成用以促進個體體內的肌肉或肌腱再生和/或動脈血管生成之藥學組合物，此藥學組合物即屬於本發明另一態樣之範圍。The various synthetic peptides described in the above embodiments may also be formulated into pharmaceutical compositions for promoting muscle or tendon regeneration and/or arterial angiogenesis in an individual, the pharmaceutical composition being within the scope of another aspect of the present invention. .

根據本發明一實施例，上述藥學組合物包含根據本發明上述態樣/實施方式所述的任一種合成胜肽，且此合成胜肽的含量足以促進個體體內的肌肉或肌腱再生和/或動脈血管生成。此藥學組合物亦包適用於該合成胜肽的藥學上可接受載體。According to an embodiment of the present invention, the pharmaceutical composition comprises any one of the synthetic peptides according to the above aspect/embodiment of the present invention, and the synthetic peptide is present in an amount sufficient to promote muscle or tendon regeneration and/or arteries in the individual. Angiogenesis. This pharmaceutical composition also includes a pharmaceutically acceptable carrier suitable for use in the synthetic peptide.

在選擇適用於投遞合成胜肽之藥學上可接受載體時， 主要需考量該藥學組合物的給藥途徑。根據本發明的可任選實施例，此藥學組合物可透過肌肉內注射的方式來給藥。在此種情形中，可利用無菌水溶液作為藥學上可接受載體，此水溶液較佳為與接受者血液等張的溶液。舉例來說，可將活性成分溶解或懸浮於含有生理可相容物質的水中，所述的生理可相容物質如氯化鈉、甘胺酸及與其相似者，且其pH值經緩衝可與生理條件相容，以得到一水溶液，而後再進行滅菌。In selecting a pharmaceutically acceptable carrier suitable for delivery of a synthetic peptide, It is primarily necessary to consider the route of administration of the pharmaceutical composition. According to an optional embodiment of the invention, the pharmaceutical composition can be administered by intramuscular injection. In this case, a sterile aqueous solution may be utilized as a pharmaceutically acceptable carrier, and the aqueous solution is preferably an isotonic solution with the recipient's blood. For example, the active ingredient may be dissolved or suspended in water containing a physiologically compatible substance such as sodium chloride, glycine, and the like, and the pH thereof may be buffered. The physiological conditions are compatible to obtain an aqueous solution which is then sterilized.

於可任選的實施例中，亦可將所述合成胜肽調製於一緩釋劑型中，以確保該藥學組合物可提供較長效的治療效果。已知有多種高分子材料可延長藥物的釋放，其實施例包括但不限於：海藻酸鹽、明膠、膠原蛋白及聚乳酸/甘醇酸共聚物。In an optional embodiment, the synthetic peptide can also be formulated in a sustained release dosage form to ensure that the pharmaceutical composition provides a longer-lasting therapeutic effect. A variety of polymeric materials are known to extend drug release, examples of which include, but are not limited to, alginate, gelatin, collagen, and polylactic acid/glycolic acid copolymers.

根據本發明某些實驗例，所述的合成胜肽係包埋於由經交聯的海藻膠所組成的基質中，此時該合成胜肽的最終濃度約為1-100 μM；且較佳為約10 μM。舉例來說，上述合成胜肽的濃度可為約1、2、3、4、5、6、7、8、9、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95或100 μM。According to some experimental examples of the present invention, the synthetic peptide is embedded in a matrix composed of cross-linked seaweed, and the final concentration of the synthetic peptide is about 1-100 μM; It is about 10 μM. For example, the concentration of the above synthetic peptide may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55. , 60, 65, 70, 75, 80, 85, 90, 95 or 100 μM.

本發明之組合物亦可包含各種本領域習知的添加物。舉例來說，可使用溶劑(包括少量乙醇)來穩定某些藥物。其他可任選的藥學上可接受添加物包括：乳白劑、抗氧化劑、香料、色素、膠化劑、增稠劑、安定劑、界面活性劑及與其相似者。亦可添加其他物質以防止組合物因儲存而 變質，譬如可添加抗菌劑來抑制微生物(如酵母菌與霉菌)的生長。本發明的組合物中亦可包括滲透促進劑和/或降低刺激性的添加物。The compositions of the present invention may also contain various additives known in the art. For example, solvents (including small amounts of ethanol) can be used to stabilize certain drugs. Other optional pharmaceutically acceptable additives include: opacifiers, antioxidants, perfumes, colors, gelling agents, thickeners, stabilizers, surfactants, and the like. Other substances may also be added to prevent the composition from being stored Deterioration, such as the addition of antibacterial agents to inhibit the growth of microorganisms such as yeasts and molds. Permeation enhancers and/or irritating additives may also be included in the compositions of the present invention.

在又一態樣中，本發明提出了一種用以於一個體的損傷區域中或鄰近處促進肌肉或肌腱再生的方法；且本發明的另一態樣是關於用以於一個體的缺血區域中或鄰近處促進動脈血管生成的方法。在以上任一種實施態樣中，所述個體/患者可以是任何哺乳類動物，包括人類。下文敘述了適用於上述兩種態樣或其中之一的實施例。In still another aspect, the invention provides a method for promoting muscle or tendon regeneration in or adjacent to a lesion of a body; and another aspect of the invention relates to ischemia for a body A method of promoting arterial angiogenesis in or adjacent to a region. In any of the above embodiments, the individual/patient can be any mammal, including a human. Embodiments suitable for either or both of the above aspects are described below.

於一實施例中，上述用以促進肌肉或肌腱再生的方法包含對該個體體內的一治療區域投予一治療有效量的根據本發明上述態樣/實施例之合成胜肽，其中上述治療區域係鄰近該損傷區域，藉以促進該個體體內於鄰近該損傷區域或其中的肌肉或肌腱再生。In one embodiment, the method for promoting regeneration of muscle or tendon comprises administering to a therapeutic region of the subject a therapeutically effective amount of a synthetic peptide according to the above aspect/embodiment of the present invention, wherein the therapeutic region is Adjacent to the damaged area, thereby promoting regeneration of the muscle or tendon adjacent to the damaged area or the body thereof.

於另一實施例中，上述促進動脈血管生成的方法包含對該個體體內的一治療區域投予一治療有效量的根據本發明上述態樣/實施例之合成胜肽，其中上述治療區域係鄰近該缺血區域，藉以促進該個體體內於鄰近該缺血區域或其中的動脈血管生成。In another embodiment, the method of promoting arterial angiogenesis comprises administering to a therapeutic region of the subject a therapeutically effective amount of a synthetic peptide according to the above aspect/embodiment of the invention, wherein the therapeutic region is adjacent The ischemic region is thereby promoted angiogenesis in the body adjacent to the ischemic region or therein.

於可任選的實施例中，可將所述合成胜肽調製為上述本發明態樣/實施例所述的藥學組合物。在實際操作時，可透過肌肉內注射(intradermal injection)的方式來投予該組合物。In an optional embodiment, the synthetic peptide can be formulated into the pharmaceutical compositions described above in the aspects/embodiments of the invention. In actual practice, the composition can be administered by intradermal injection.

根據某些實施例，該個體可能因為肌肉損傷、肌肉廢 用、肌肉萎縮症、肌萎縮性側索硬化、肌腱損傷、組織缺血、腦缺血、周邊動脈疾病或心肌梗塞等原因，而使缺血區域中的血流受阻。According to some embodiments, the individual may be due to muscle damage, muscle waste Use, muscular dystrophy, amyotrophic lateral sclerosis, tendon injury, tissue ischemia, cerebral ischemia, peripheral arterial disease, or myocardial infarction, etc., cause blood flow in the ischemic area to be blocked.

下文提出多個實驗例來說明本發明的某些態樣，以利本發明所屬技術領域中具有通常知識者實作本發明。不應將這些實施例視為對本發明範圍的限制。據信習知技藝者在閱讀了此處提出的說明後，可在不需過度解讀的情形下，完整利用並實踐本發明。此處所引用的所有公開文獻，其全文皆視為本說明書的一部分。A number of experimental examples are set forth below to illustrate certain aspects of the present invention, and the present invention may be practiced by those of ordinary skill in the art. These examples should not be construed as limiting the scope of the invention. It is believed that the skilled artisan, after reading the description set forth herein, may fully utilize and practice the invention without undue interpretation. All publications cited herein are hereby incorporated by reference in their entirety.

實驗例Experimental example 材料與方法Materials and Methods

<材料><material>

經HEPES緩衝的DMEM培養基(Dulbecco’s modified Eagle’s medium)、胎牛血清(fetal bovine serum，FBS)、0.25%胰蛋白酶，抗-BrdU抗體、MCDB131培養基、TRIzol與Dynabeads皆購自Invitrogen(Carlsbad,CA)。超純海藻酸鹽(6000 Da)、二甲亞碸(dimethyl sulfoxide，DMSO)、牛血清白蛋白(bovine serum albumin，BSA)、5-溴-2'-去氧尿苷(5-bromo-2'-deoxyuridine，BrdU)、Hoechst 33258染料與曼森氏三色染劑(Masson's Trichrome)皆購自Sigma-Aldrich(St.Louis,MO)。第一型膠原蛋白酶(collagenase type I)與第二型中性蛋白酶(dispase II)係購自Roche(Indianapolis,IN)。各種螢光染料-結合二級 抗體皆購自BioLegend(San Diego,CA)。蘇木色素與伊紅(Hematoxylin and eosin，簡稱H&E)染料購自Merck(Rayway,NJ,USA)。抗-膠原蛋白1A1抗體係購自Santa Cruz Biotechnology(Santa Cruz,CA)。基質膠(Matrigel)係購自BD Biosciences(Bedford,MA)。抗-α-平滑肌肌動蛋白(anti-α-smooth muscle actin，簡稱anti-α-SMA)抗體(ab5694)與抗-核幹細胞因子(anti-nucleostemin)抗體係購自Abcam(Cambridge,MA)。抗-Pax7抗體(GTX62311)購自GeneTex(Taipei,Taiwan)。凝集素B4(Isolectin B4，簡稱IB4)-Alexa Fluor 568螢光耦合物係購自Molecular Probes(Eugene,OR)。HEPES buffered DMEM medium (Dulbecco's modified Eagle's medium), fetal bovine serum (FBS), 0.25% trypsin, anti-BrdU antibody, MCDB131 medium, TRIzol and Dynabeads were purchased from Invitrogen (Carlsbad, CA). Ultrapure alginate (6000 Da), dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), 5-bromo-2'-deoxyuridine (5-bromo-2) '-deoxyuridine, BrdU), Hoechst 33258 dye and Masson's Trichrome were purchased from Sigma-Aldrich (St. Louis, MO). Collagenase type I and type 2 neutral protease (dispase II) were purchased from Roche (Indianapolis, IN). Various fluorescent dyes - combined with secondary Antibodies were purchased from BioLegend (San Diego, CA). Hematoxylin and eosin (H&E) dyes were purchased from Merck (Rayway, NJ, USA). The anti-collagen 1A1 anti-system was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Matrigel was purchased from BD Biosciences (Bedford, MA). Anti-α-smooth muscle actin (anti-α-SMA) antibody (ab5694) and anti-nucleostemin anti-system were purchased from Abcam (Cambridge, MA). Anti-Pax7 antibody (GTX62311) was purchased from GeneTex (Taipei, Taiwan). Lectin B4 (IB4)-Alexa Fluor 568 fluorescent coupler was purchased from Molecular Probes (Eugene, OR).

短合成胜肽(包括29-mer(序列編號：3)、25-mer(序列編號：4)、24-mer(序列編號：5)、20-mer(序列編號：6)、18-mer(序列編號：7)、Mo 29-mer(序列編號：8)與Mo 20-mer(序列編號：9))係購自GenScript(Piscataway,NJ)，於合成後，將其N端醯化(acetylated)並將C端醯胺化(amidated)以提升安定性，並以質譜儀定性(純度>95%)。Short synthetic peptides (including 29-mer (SEQ ID NO: 3), 25-mer (SEQ ID NO: 4), 24-mer (SEQ ID NO: 5), 20-mer (SEQ ID NO: 6), 18-mer ( SEQ ID NO: 7), Mo 29-mer (SEQ ID NO: 8) and Mo 20-mer (SEQ ID NO: 9)) were purchased from GenScript (Piscataway, NJ), and after synthesis, the N-terminal was cyanated (acetylated). The C-terminus is amidated to enhance stability and is characterized by mass spectrometry (purity >95%).

本說明書所載實施例所用的所有動物皆飼育於有溫度與濕度控制的飼養籠中，飼養溫度約24℃至25℃，光暗循環為12小時。試驗過程中提供飲水與標準齧齒類飼料供任食。實驗計畫皆通過馬偕紀念醫院(新北市，台灣)倫理委員會核准，並遵循國家動物保護相關規範。All animals used in the examples contained in this specification were housed in a cage with temperature and humidity control at a temperature of about 24 ° C to 25 ° C and a light dark cycle of 12 hours. Drinking water and standard rodent feed are provided for the meal during the test. The experimental plans were approved by the Ethics Committee of the Ma Rong Memorial Hospital (New Taipei City, Taiwan) and followed the national animal protection regulations.

<PEDF合成胜肽/海藻膠配方及丸劑配方><PEDF Synthetic peptide/seaweed formula and pill formulation>

以DMSO分別將每一合成胜肽(29-mer、25-mer、24-mer、20-mer、18-mer、MO 29-mer或MO 20-mer)復水備用(5 mM)。之後，將備用液與超純海藻酸鹽混合，以得到濃度2%(重量/體積)的海藻酸鹽溶液，其中PEDF合成胜肽的最終濃度約10 μM。接著以薄膜過濾器(孔洞大小：0.22 μm)過濾海藻酸鹽溶液，並和經過濾的硫酸鈣漿液(每毫升蒸餾水含0.21克硫酸鈣)以25：1的比例混合(每毫升經過濾的海藻酸鹽溶液和40 μL的硫酸鈣漿液混合)。將混合物靜置於室溫下1小時，以使海藻酸鹽進行交聯反應。所得到的緩釋配方之後可用以治療肌肉或肌腱損傷與缺血。Each synthetic peptide (29-mer, 25-mer, 24-mer, 20-mer, 18-mer, MO 29-mer or MO 20-mer) was rehydrated (5 mM) in DMSO. Thereafter, the stock solution was mixed with ultrapure alginate to obtain a 2% (w/v) alginate solution in which the final concentration of the PEDF synthase was about 10 μM. The alginate solution was then filtered through a membrane filter (pore size: 0.22 μm) and mixed with filtered calcium sulphate slurry (0.21 g of calcium sulphate per ml of distilled water) in a ratio of 25:1 (filtered algae per ml) The acid salt solution is mixed with 40 μL of calcium sulfate slurry). The mixture was allowed to stand at room temperature for 1 hour to allow the alginate to undergo a crosslinking reaction. The resulting sustained release formulation can then be used to treat muscle or tendon injuries and ischemia.

欲進行一次大量給藥(bolus delivery)時，將5 mM備用液連續稀釋，以得到PEDF胜肽最終濃度為10 μM的丸劑配方(bolus formulation)。For a bolus delivery, a 5 mM stock solution was serially diluted to obtain a bolus formulation with a final concentration of 10 μM of PEDF peptide.

<組織學、免疫螢光與定量分析><Histology, Immunofluorescence and Quantitative Analysis>

股薄肌(gracilis)、內收長肌(adductor magnus)、比目魚肌(soleus)與脛骨肌(tibialis)分別以4%三聚甲醛(paraformaldehyde)固定，之後以梯度乙醇脫水，接著以石蠟包埋。經固定的樣本以二甲苯(xylene)脫臘，並以梯度乙醇復水。將組織切成約5-μm的切片。利用H&E染料進行一般組織學分析。The gracilis, adductor magnus, soleus and tibialis were fixed with 4% paraformaldehyde, then dehydrated with gradient ethanol, then embedded in paraffin. . The fixed sample was dewaxed with xylene and rehydrated with a gradient of ethanol. The tissue was cut into sections of about 5-μm. General histological analysis was performed using H&E dyes.

脫臘的組織切片以10%山羊血清處理約1小時以進行 阻斷。在4℃下利用BrdU初級抗體(GTX42641；稀釋比例1：50)或抗第一型膠原蛋白1A1初級抗體稀釋比例1：50)染色一夜，之後在室溫下先和適當的過氧化物酶-標記驢免疫球蛋白(peroxidase-labeled donkey immunoglobulin)反應約30分鐘，而後和顯色原基質(chromogen substrate)3,3-二氨基聯苯胺(3，3’-diaminobenzidine，簡稱DAB)反應2分鐘，接著利用蘇木色素進行對比染色。利用Nikon Eclipse 80i光學顯微鏡擷取高品質影像(解析度1208 X 960畫素)，以供進行定量分析。Dewaxed tissue sections were treated with 10% goat serum for about 1 hour. Blocked. Staining at 4 ° C with BrdU primary antibody (GTX42641; dilution ratio 1:50) or anti-type 1 collagen 1A1 primary antibody dilution ratio 1:50), then at room temperature with appropriate peroxidase - The reaction was carried out by peroxidase-labeled trophic immunoglobulin for about 30 minutes, and then reacted with 3,3'-diaminobenzidine (DAB) on a chromogen substrate for 2 minutes. Contrast staining was then carried out using hematoxylin. High-quality images (resolution 1208 X 960 pixels) were taken using a Nikon Eclipse 80i optical microscope for quantitative analysis.

利用經H&E染色之肌肉切片來決定肌纖維直徑，並以與顆粒邊緣(particle borders)相切的一對平行線之間的最小距離進行定量，此即「最小費里特直徑」(minimal Feret’s diameter)。利用Nikon Eclipse 80i光學顯微鏡擷取影像後，以Image-Pro Plus 4.5.1軟體(Media Cybernetics)來計算最小費里特直徑。以每一張照片中肌纖維總數為基準，將各纖維費里特群組(以每5 μm為單位)中的纖維數目標準化。The muscle fiber diameter is determined by H&E-stained muscle sections and quantified by the minimum distance between a pair of parallel lines tangent to the particle borders, which is the "minimal Feret's diameter". . After capturing images using a Nikon Eclipse 80i optical microscope, the minimum Feret diameter was calculated using Image-Pro Plus 4.5.1 software (Media Cybernetics). The number of fibers in each fiber Ferrite group (in units of 5 μm) was normalized based on the total number of muscle fibers in each photograph.

為了確認細胞核中心移位(centrally nucleated)肌纖維的數目，切片樣本以H&E染色後，以上述裝置擷取影像。於每一照片中隨機選擇至少100條經染色之纖維；若該纖維中有一或多細胞核並未處於靠近纖維周圍的位置，則將該肌纖維判定為細胞核中心移位纖維。將數據表示為所計算之總纖維數的百分比。每一實驗組別10隻老鼠，每一肌肉部位中選擇6個切片進行計算。In order to confirm the number of centurally nucleated muscle fibers, the slice samples were stained with H&E and images were taken with the above apparatus. At least 100 dyed fibers are randomly selected in each photograph; if one or more nuclei in the fiber are not in a position close to the periphery of the fiber, the muscle fiber is determined to be a nuclear center shifting fiber. The data is expressed as a percentage of the total number of fibers counted. For each experimental group, 10 mice were selected and 6 slices were selected for each muscle site for calculation.

根據製造商的指示，以曼森氏三色染劑將脫臘的肌腱組織染色。對膠原蛋白面積進行半定量(semi-quantitative)分析時，在光學顯微鏡下，由每一玻片樣本中隨機選擇10個視野(field)，並以Image-Pro Plus 4.5.1軟體來計算樣本中修復區域面積佔未受損肌腱面積的比例(mm2 /mm2 )。The dewaxed tendon tissue was stained with Manson's trichrome dye according to the manufacturer's instructions. For semi-quantitative analysis of collagen area, 10 fields were randomly selected from each slide sample under an optical microscope, and the sample was calculated using Image-Pro Plus 4.5.1 software. The area of the repaired area as a percentage of the area of the intact tendon (mm 2 /mm 2 ).

<肌腱幹細胞的分離與培養><Isolation and culture of tendon stem cells>

本實驗採用紐西蘭白兔(6-8個月大，3.0-4.0 kg)。順著兔子的肌腱韌帶骨附著處(bony attachment)切下，以取得阿基里斯肌腱。剝除腱翹後，將肌腱芯部切成小片。於DMEM-高葡萄糖溶液中加入3 mg/ml的第一型膠原蛋白酶與4 mg/ml的中性蛋白酶，將每100 mg的肌腱芯部片段加入1 ml上述溶液中，於37℃下進行2小時的水解反應。之後將所得細胞懸浮液在1,000 rpm的轉速下離心15分鐘，取出細胞沈澱物後重新懸浮於DMEM生長培養基中，此培養基添加了10%經熱不活化胎牛血清(FBS)、100 μM的2-巰乙醇(2-mercaptoethanol)與100 U/ml的青黴素(Penicillin)及100 μg/ml的鏈黴素(streptomycin)。欲進行繼代時，利用0.25%胰蛋白來收集幾近長滿的細胞，且之後將密度1 X 105 的繼代培養細胞培養於培養基中。New Zealand white rabbits (6-8 months old, 3.0-4.0 kg) were used in this experiment. Cut along the rabbit's tendon attachment to obtain the Achilles tendon. After peeling off the tendon, cut the core of the tendon into small pieces. Add 3 mg/ml of type I collagenase and 4 mg/ml of neutral protease to DMEM-high glucose solution, add 100 ml of the tendon core fragment to 1 ml of the above solution, and perform at 37 °C. Hour hydrolysis reaction. The resulting cell suspension was then centrifuged at 1,000 rpm for 15 minutes, and the cell pellet was taken out and resuspended in DMEM growth medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 μM 2 - 2-mercaptoethanol with 100 U/ml penicillin and 100 μg/ml streptomycin. For subculture, 0.25% trypsin was used to collect nearly overgrown cells, and then subcultured cells at a density of 1×10 5 were cultured in the medium.

<肌腱幹細胞增殖分析><muscle stem cell proliferation analysis>

將繼代4的肌腱幹細胞以每孔2 X 105 個細胞的密度植入經明膠塗覆玻片上置於六孔盤中，並加入生長培養基 (DMEM+10% FBS)培養24小時，而後將培養基置換成僅含5% FBS的基礎生長培養基(對照組)或含有5% FBS加上50 nM的PEDF合成胜肽(即，29-mer、24-mer、20-mer、18-mer、Mo 29-mer或Mo 20-mer)並培養24小時。欲進行BrdU標記分析時，將BrdU(最終濃度10 μM)加入培養系統中約4小時。在以4%三聚甲醛固定後，將細胞置於冷的甲醇中約2分鐘，而後在室溫下以1N氯化氫處理約1小時，而後再進行免疫螢光分析。透過核幹細胞因子(nucleostemin)與第一型膠原蛋白的免疫化學分析，來決定繼代4的肌腱幹細胞的表現型。幾乎所有經增殖的肌腱幹細胞都是核幹細胞因子與第一型膠原蛋白-雙陽性細胞。The 4th tendon stem cells were implanted into a gelatin coated slide at a density of 2×10 5 cells per well, placed in a six-well plate, and added to the growth medium (DMEM + 10% FBS) for 24 hours, and then The medium was replaced with a basal growth medium containing only 5% FBS (control) or a PEDF synthetic peptide containing 5% FBS plus 50 nM (ie, 29-mer, 24-mer, 20-mer, 18-mer, Mo 29-mer or Mo 20-mer) and cultured for 24 hours. For BrdU labeling analysis, BrdU (final concentration 10 μM) was added to the culture system for approximately 4 hours. After fixation with 4% paraformaldehyde, the cells were placed in cold methanol for about 2 minutes and then treated with 1 N hydrogen chloride for about 1 hour at room temperature before immunofluorescence analysis. The phenotype of the tendon stem cells of the secondary 4 was determined by immunochemical analysis of the nucleostemin and the first type of collagen. Almost all of the proliferating tendon stem cells are nuclear stem cell factors and type I collagen-double positive cells.

<DNA合成的活體偵測><In vivo detection of DNA synthesis>

欲偵測細胞增殖時，將BrdU重新溶於DMSO中備用(80 mM)。將10 μl的BrdU備用液和90 μl的PBS混合後經腹膜內注射至小鼠體內，並於16小時後將小鼠進行安樂死。大白鼠則是經腹膜內注射150 μl的BrdU備用液與350 μl的PBS，並同樣於16小時後安樂死。利用抗-BrdU抗體來標記BrdU，評估以DNA合成。To detect cell proliferation, BrdU was redissolved in DMSO for use (80 mM). 10 μl of BrdU stock solution and 90 μl of PBS were mixed and intraperitoneally injected into mice, and the mice were euthanized after 16 hours. The rats were injected intraperitoneally with 150 μl of BrdU stock solution and 350 μl of PBS, and were also euthanized after 16 hours. BrdU was labeled with an anti-BrdU antibody and assessed for DNA synthesis.

<免疫螢光分析><Immunofluorescence analysis>

脫臘組織切片或經4%三聚甲醛固定的兔子肌腱幹細胞(TSC)以10%山羊血清與5% BSA處理約1小時以進行阻斷。利用抗α-SMA(稀釋比例1：100)、IB4(5 μg/ml)、 Pax7(稀釋比例1：100)、核幹細胞因子(稀釋比例1：100)或第一型膠原蛋白1A1(稀釋比例1：50)的初級抗體於37℃下染色2小時，之後在室溫下和適當的玫紅-或FITC-結合驢免疫球蛋白反應約1小時。利用Hoechst 33258對細胞核進行對比染色(約7分鐘)。利用帶有CCD相機的蔡司(Zeiss)螢光顯微鏡來擷取影像，並利用Axiovert軟體照相。The demineralized tissue sections or rabbit tendon stem cells (TSC) fixed with 4% paraformaldehyde were treated with 10% goat serum and 5% BSA for about 1 hour to block. Use anti-α-SMA (dilution ratio 1:100), IB4 (5 μg/ml), Primary antibodies with Pax7 (diluted 1:100), nuclear stem cell factor (diluted 1:100) or type 1 collagen 1A1 (diluted 1:50) were stained at 37 °C for 2 hours, then at room temperature The appropriate rose- or FITC-conjugated immunoglobulin is reacted for about 1 hour. The nuclei were contrast-stained using Hoechst 33258 (about 7 minutes). Images were taken using a Zeiss fluorescence microscope with a CCD camera and photographed using Axiovert software.

每一內收長肌樣本中，隨機選取10個區域來擷取影像(放大倍率：200×)，並據以計算小動脈(圍繞於血管整個周邊的α-SMA陽性細胞)之密度。以事先不知處理組別的實驗人員來計算各樣本中的陽性細胞，並重複三次；之後將來自五個樣本的數值平均，並表示成每平方公釐的小動脈(arteriole)密度。In each of the adducted long muscle samples, 10 regions were randomly selected to capture images (magnification: 200×), and the density of the small arteries (α-SMA positive cells surrounding the entire circumference of the blood vessel) was calculated. Positive cells in each sample were counted by an experimenter who did not know the treatment group beforehand and repeated three times; the values from the five samples were then averaged and expressed as arteriole density per square millimeter.

<骨髓間葉幹細胞的分離、培養與處理><Separation, culture and treatment of mesenchymal stem cells>

由雄性Sprague-Dawley大鼠(300-450 g)的股骨(femur)分離出初級大鼠骨髓間葉幹細胞(BM-MSC)。在無菌環境下移出股骨，並切除附著(adhering)組織，之後將DMEM培養基注入骨髓腔內沖洗。將收集到的骨髓細胞培養於100×15-mm的Petri碟中，使用DMEM培養基(添加10% FBS、100 U/ml青黴素與100 μg/ml鏈黴素)在5%二氧化碳與37℃的環境下培養2週，並每2-3天更換新鮮培養基。欲進行繼代時，利用0.25%胰蛋白來收集幾近長滿的細胞，且之後將2 X 105 的繼代培養細胞培植於6孔盤 的每一孔中，並培養於10% FBS-DMEM培養基中。在進行處理前，改用添加1% FBS的DMEM培養基使細胞飢餓12小時，而後在新鮮的1% FBS-DMEM中加入50 nM的PEDF合成胜肽(29-mer或20-mer)，並培養24或48小時。Primary rat bone marrow mesenchymal stem cells (BM-MSCs) were isolated from femurs of male Sprague-Dawley rats (300-450 g). The femur was removed in a sterile environment and the adhering tissue was excised, after which the DMEM medium was injected into the medullary cavity for rinsing. The collected bone marrow cells were cultured in a 100×15-mm Petri dish using DMEM medium (10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin) in a 5% carbon dioxide and 37 ° C environment. The culture was carried out for 2 weeks and fresh medium was replaced every 2-3 days. For subculture, 0.25% trypsin was used to collect nearly overgrown cells, and then 2×10 5 subcultured cells were plated in each well of a 6-well plate and cultured in 10% FBS- In DMEM medium. Before treatment, cells were starved for 12 hours in DMEM medium supplemented with 1% FBS, and then 50 nM PEDF synthetic peptide (29-mer or 20-mer) was added to fresh 1% FBS-DMEM and cultured. 24 or 48 hours.

<RNA萃取與反轉錄聚合酶連鎖反應><RNA extraction and reverse transcription polymerase chain reaction>

利用TRIzol自細胞內萃取出全RNA，並以不含核糖核酸水解酶(Rnase-free)的第一型去氧核糖核酸水解酶(DNase I)(Qiagen,Santa Clarita,CA)處理以移除基因組DNA，之後以RNA純化套組Dynabeads進行純化。在20 μl的反應緩衝液(含0.25 μg的隨機引子與0.8 mM去氧核糖核苷三磷酸鹽(dNTPs))中加入1 μg取自BM-MSC的全RNA與200單位的反轉錄酶(Roche,Mannheim,Germany)，於42℃下反應1小時，以將RNA反轉錄為cDNA。在後續的PCR反應中，取2 μl的cDNA作為反應模板。Total RNA was extracted from the cells by TRIzol and treated with DNase I (Qiagen, Santa Clarita, CA) without ribohydrolysis (Rnase-free) to remove the genome. DNA was then purified using RNA purification kit Dynabeads. Add 1 μg of total RNA from BM-MSC to 200 units of reverse transcriptase in 20 μl of reaction buffer (containing 0.25 μg of random primer and 0.8 mM deoxyribonucleoside triphosphate (dNTPs)) (Roche , Mannheim, Germany), reacted at 42 ° C for 1 hour to reverse transcribe RNA into cDNA. In the subsequent PCR reaction, 2 μl of cDNA was taken as a reaction template.

PCR反應的反應體積為30 μl，其中含有15 μl的EconoTaq® PLUS GREEN 2× Master Mix(Lucigen® Corp.)、1 μM的各種引子以2 μl的模板DNA。合成cDNA的增殖反應約18-22個循環(變性：20秒、94℃；黏合：30秒、57℃：以及聚合：40秒、72℃)。每種引子組所用的循環數落在擴充的線性範圍內。用以增殖大鼠腱調蛋白(Tenomodulin，簡稱TNMD)基因(註冊號：NM_022290)的引子組包含正向引子(AGAATGAGCAATGGGTGGTC； 序列編號：10)與反向引子(CTCGACCTCCTTGGTAGCAG；序列編號：11)，其PCR產物大小約240 bp。另外以大鼠甘油醛3-磷酸去氫酶(glyceraldehyde 3-phosphate dehydrogenase，簡稱GAPDH)基因(註冊號：X02231.1)作為管家基因(housekeeping gene)，以將基因表現量標準化。用以增殖GAPDH基因的引子組包括正向引子(AGACAGCCGCATCTTCTTGT；序列編號：12)與反向引子(CTTGCCGTGGGTAGAGTCAT；序列編號：13)，而PCR產物的大小約207 bp。The PCR reaction has a reaction volume of 30 μl containing 15 μl of EconoTaq® PLUS GREEN 2× Master Mix (Lucigen® Corp.) and 1 μM of various primers with 2 μl of template DNA. The proliferative reaction of the synthetic cDNA was about 18-22 cycles (denaturation: 20 seconds, 94 ° C; adhesion: 30 seconds, 57 ° C: and polymerization: 40 seconds, 72 ° C). The number of cycles used for each primer set falls within the linear range of the extension. The primer set used to propagate the rat Tenomodulin (TNMD) gene (Registration No.: NM_022290) contains a forward primer (AGAATGAGCAATGGGTGGTC; SEQ ID NO: 10) and reverse primer (CTCGACCTCCTTGGTAGCAG; SEQ ID NO: 11), the PCR product size is about 240 bp. In addition, a rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene (Registration No.: X02231.1) was used as a housekeeping gene to standardize gene expression. The primer set for proliferating the GAPDH gene includes a forward primer (AGACAGCCGCATCTTCTTGT; SEQ ID NO: 12) and a reverse primer (CTTGCCGTGGGTAGAGTCAT; SEQ ID NO: 13), and the PCR product has a size of about 207 bp.

利用含有溴化乙錠(ethidium bromide)的2%洋菜膠對PCR產物進行電泳分析，並以UV光照使其顯影。利用FUJI LAS-3000系統與Multi Gauge Ver.1.01軟體(Fujifilm,Tokyo,Japan)對PCR產物進行光密度法定量分析。The PCR product was electrophoresed using 2% acacia gum containing ethidium bromide and developed by UV light. The PCR products were quantitatively analyzed by optical density method using a FUJI LAS-3000 system and Multi Gauge Ver. 1.01 software (Fujifilm, Tokyo, Japan).

<統計分析><Statistical Analysis>

將結果表示為平均值±平均值的標準差(standard error of the mean，SEM)。利用單向(one-way)ANOVA分析來進行統計比較。除非另有說明，P <0.05時具有統計上的顯著差異。The results are expressed as mean ± standard error of the mean (SEM). Statistical comparisons were made using one-way ANOVA analysis. Statistically significant differences were obtained at P < 0.05 unless otherwise stated.

實驗例1Experimental example 1

PEDF合成胜肽可自海藻膠基質中持續釋放PEDF synthetic peptide can be continuously released from seaweed matrix

為了決定29-mer與20mer的釋放動力學，將100 μg的FITC-結合PEDF合成胜肽與100μL的海藻酸鹽溶液混 合，之後根據「材料與方法」中所述的步驟製備水凝膠。其後，在微量離心管中加入100 mg的水凝膠與1.5 ml的PBS(pH 7.4)，並於迴轉式振盪培養箱(orbital shaking incubator)中在37℃下培養6天。分別在預定的時間點將微量離心管離心後取出200 μL的上清液並儲存於-80℃下以供後續分析，同時將200 μL的新鮮PBS加入離心管中，以補足所抽取的上清液。利用96孔的螢光計(fluorimeter)來測定所收取之上清液中FITC-結合PEDF合成胜肽的含量。利用已知濃度且未經包埋之FITC-胜肽來產生一標準曲線。本分析採用三次重複實驗所得數據，並整理於第1圖。To determine the release kinetics of 29-mer and 20mer, 100 μg of FITC-bound PEDF synthetic peptide was mixed with 100 μL of alginate solution. Then, the hydrogel is prepared according to the procedure described in "Materials and Methods". Thereafter, 100 mg of a hydrogel and 1.5 ml of PBS (pH 7.4) were added to a microcentrifuge tube, and cultured at 37 ° C for 6 days in a rotary shake incubator (orbital shaking incubator). Centrifuge the microcentrifuge tube at the predetermined time point and take out 200 μL of the supernatant and store at -80 °C for subsequent analysis. Add 200 μL of fresh PBS to the centrifuge tube to make up the extracted supernatant. . A 96-well fluorimeter was used to determine the amount of FITC-bound PEDF synthetic peptide in the supernatant obtained. A standard curve was generated using a known concentration of unembedded FITC-peptide. The analysis used the data from three replicate experiments and compiled them in Figure 1.

如第1圖所示，在6天的試驗過程中，經包埋的PEDF合成胜肽會自海藻膠基質持續地釋出。更明確地說，在試驗開始後24小時，海藻膠基質中仍有約48%的29-mer與35%的20-mer合成胜肽。大多數(約90%)的29-mer合成胜肽會在試驗前四天釋出，其後釋放速率會明顯減緩，因而在累積釋放曲線中可以觀察到一個平線區(plateau)。20-mer合成胜肽的釋放速率比起29-mer稍微快了一些，在試驗前三天就釋出了約90%的20-mer。As shown in Figure 1, the embedded PEDF synthetic peptide was continuously released from the alginic matrix during the 6-day test. More specifically, about 48% of the 29-mer and 35% of the 20-mer synthetic peptide remained in the alginate matrix 24 hours after the start of the test. Most (about 90%) of the 29-mer synthetic peptides were released four days before the test, and the rate of release was significantly slowed down, so a plateau was observed in the cumulative release profile. The release rate of the 20-mer synthetic peptide was slightly faster than that of the 29-mer, and about 90% of the 20-mer was released three days before the test.

實驗例2Experimental example 2

緩釋PEDF合成胜肽可降低缺血損傷Slow release of PEDF synthetic peptide can reduce ischemic injury

缺血性肌肉損傷通常會導致局部壞死(necrosis)以及組織與功能的喪失。因此，以下實驗例中採用缺血性動物 模型，以探究局部投予PEDF合成胜肽/海藻膠配方(下稱「緩釋配方」)是否能促進遭受缺血性損傷之組織或器官的組織或器官功能的回復。在下文的實驗例中，分別分析了與缺血性損傷相關的多種情形，例如，肢體灌流(limb perfusion)、組織壞死、動脈血管生成與新血管分枝(neovessel sprouting)等。Ischemic muscle damage usually leads to local necrosis and loss of tissue and function. Therefore, ischemic animals are used in the following experimental examples. Models to explore whether topical administration of PEDF synthetic peptide/alginic gel formula (hereinafter referred to as "sustained release formulation") can promote the recovery of tissue or organ function of tissues or organs suffering from ischemic injury. In the following experimental examples, various conditions associated with ischemic injury were analyzed, for example, limb perfusion (limb perfusion), tissue necrosis, arterial angiogenesis, and neovascular sprouting.

本實驗採用六週大的雌性C57BL/6野生型小鼠，動物經腹腔內注射舒泰(zoletil，用量為每公斤體重6毫克)與若朋(xylazine，用量為每公斤體重3毫克)混合液，以進行麻醉。以脫毛霜移除後腿與臀部的毛髮。將單邊外腸股動脈(external iliac artery)和股動脈(femoral artery)及周邊血管紮起並切除，以得到後肢缺血小鼠。手術後，將小鼠隨機分成數個實驗群組(每一群組樣本數為6)並分別給予如下處置。在空白對照組中，小鼠給予50 μl的空白海藻膠；而在丸劑對照組中，小鼠接受含有29-mer的丸劑配方。在PEDF合成胜肽/海藻膠處理組中，小鼠接受50 μl的緩釋配方，其分別含有29-mer、24-mer或20-mer。此外，於PEDF 18-mer對照組中，小鼠接受含PEDF 18-mer合成胜肽的緩釋配方。在手術後，立刻以單一劑肌肉內注射的方式將藥物投遞至股薄肌。傷口以無菌生理食鹽水沖洗後縫合。In this experiment, six-week-old female C57BL/6 wild-type mice were used. The animals were intraperitoneally injected with a mixture of zoletil (6 mg/kg body weight) and xylazine (3 mg/kg body weight). To perform anesthesia. Remove the hair from the hind legs and buttocks with a depilatory cream. The unilateral external iliac artery and the femoral artery and peripheral blood vessels were ligated and excised to obtain hind limb ischemic mice. After the surgery, the mice were randomly divided into several experimental groups (the number of samples per group was 6) and the following treatments were respectively administered. In the blank control group, the mice were given 50 μl of blank seaweed gum; while in the pill control group, the mice received a pill formulation containing 29-mer. In the PEDF synthetic peptide/alginate treated group, the mice received a 50 μl sustained release formulation containing 29-mer, 24-mer or 20-mer, respectively. In addition, in the PEDF 18-mer control group, mice received a sustained release formulation containing a PEDF 18-mer synthetic peptide. Immediately after the operation, the drug is delivered to the gracilis muscle by a single intramuscular injection. The wound was washed with sterile physiological saline and sutured.

實驗例2.1Experimental Example 2.1

緩釋PEDF合成胜肽可增加肢體灌流Slow release of PEDF synthetic peptide can increase limb perfusion

利用雷射都卜勒灌流影像(Laser Doppler Perfusion imaging，簡稱LDPI)分析儀(Moor Instruments,USA)來定量手術前(術前；pre OP)、手術後即時(術後；post OP)以及手術後一段時間的血液灌流情形。為了最小化因麻醉熱損失(anesthetic heat loss)所造成的血管收縮，在測量前將動物置於37℃的加熱板上5分鐘。第2圖為代表性的LDPI照片，這些照片顯示了在手術後四週間缺血性後肢的血液灌流情形，其中深藍色代表血流量較低。另外，以LDPI指數來表示血液灌流情形，此一數值為同一動物之經手術(缺血性)後肢相對於未經手術(非缺血性)後肢的血流量比例，其結果摘要整理於第3圖與表1(n6)。以雷射頻率(不同顏色的像素)的改變來表示血流量。Pre-operative (pre-operative; pre-OP), immediate post-operative (postoperative; post OP), and post-surgery using a Laser Doppler Perfusion Imaging (LDPI) analyzer (Moor Instruments, USA) Blood perfusion for a period of time. To minimize vasoconstriction due to anesthetic heat loss, animals were placed on a 37 ° C hot plate for 5 minutes prior to measurement. Figure 2 is a representative LDPI photograph showing the blood perfusion of the ischemic hind limbs during the four weeks after surgery, with dark blue representing a lower blood flow. In addition, the blood perfusion condition is represented by the LDPI index, which is the ratio of the blood flow of the surgically operated (ischemic) hind limb to the unoperated (non-ischemic) hind limb of the same animal, and the results are summarized in the third. Figure and Table 1 (n 6). Blood flow is represented by a change in the laser frequency (pixels of different colors).

如第3圖所示，一如預期地，在所有處理組別中，局部血流在手術後(術後)都立刻下降為同一動物非缺血性後肢的約8%。在空白對照組(僅以海藻膠處理)中，可以 觀察到重新灌流隨著時間緩慢成長的現象。另外，可注意到丸劑給藥的效果與空白對照組相似，意味著PEDF合成胜肽的持續釋出可能是這些PEDF合成胜肽產生保護效果所必須的。再者，相較於空白對照組或丸劑對照組而言，以含有18-mer合成胜肽之緩釋配方處理的小鼠並未觀察到血液灌流提升之現象；代表18-mer合成胜肽無法有效治療缺血損傷。相較之下，以本發明實施例提出的PEDF合成胜肽處理的小鼠，其血液灌流皆有顯著的提升(相較於空白對照組、丸劑對照組與PEDF 18-mer對照組。具體來說，經含有29-mer、24-mer或20-mer的緩釋配方處理的動物，在手術後約第二週開始，皆可觀察到血流量有明顯的提升(至少為正常肢體的約60%)。在術後第4週，經含有29-mer、24-mer或20-mer的緩釋配方處理的動物，其灌流狀況的復原情形良好，分別達到正常肢體的約105%、92%與93%；相較之下，空白對照組的灌流比例只有約50%，而丸劑對照組的灌流比例也只有約55%。As shown in Figure 3, as expected, local blood flow decreased immediately after surgery (postoperative) to approximately 8% of the non-ischemic hind limbs of the same animal in all treatment groups. In the blank control group (treated only with seaweed gum), A phenomenon of reperfusion that slowly grows over time is observed. In addition, it can be noted that the effect of bolus administration is similar to that of the blank control group, meaning that sustained release of the PEDF synthetic peptide may be necessary for the protective effect of these PEDF synthetic peptides. Furthermore, compared with the blank control group or the pill control group, the mice treated with the sustained release formulation containing the 18-mer synthetic peptide did not observe the increase of blood perfusion; the representative peptide of 18-mer could not be synthesized. Effective treatment of ischemic injury. In contrast, the blood perfusion of the PEDF-treated peptide treated by the present invention was significantly improved (compared with the blank control group, the pill control group and the PEDF 18-mer control group). It is said that animals treated with a slow release formulation containing 29-mer, 24-mer or 20-mer have a significant increase in blood flow (at least 60% of normal limbs) at about the second week after surgery. %). At the 4th week after surgery, the animals treated with the slow-release formula containing 29-mer, 24-mer or 20-mer had a good recovery of perfusion status, reaching about 105% and 92% of the normal limbs, respectively. Compared with 93%; the perfusion ratio of the blank control group was only about 50%, while the perfusion ratio of the pill control group was only about 55%.

實驗例2.2Experimental Example 2.2

緩釋PEDF合成胜肽可預防缺血誘發的組織壞死Slow release of PEDF synthetic peptide can prevent tissue necrosis induced by ischemia

在大多數的後肢缺血模型中，位於膝蓋下方的肌肉通常會發生組織壞死的情形。舉例來說，在股動脈切除後，位於投予治療之股薄肌遠端的脛骨前肌(tibialis anterior muscle)通常會出現大量肌肉壞死與再生的情形。樣本經曼森氏三色染劑後，藍色染料的強度取決於樣本組織中膠 原蛋白纖維的含量，而壞死則會導致這種纖維化的情形。因此，在手術與治療後第二週與第七週分別自脛骨前肌取得組織樣本，並以曼森氏三色染劑染色，以評估纖維化(與壞死)程度。第4A與4B圖提供了代表性樣本的照片。In most hindlimb ischemia models, tissue located under the knee usually undergoes tissue necrosis. For example, after femoral resection, the tibialis anterior muscle located at the distal end of the femoral muscle of the treatment usually has a large amount of muscle necrosis and regeneration. After the sample is dyed by Manson's three-color dye, the intensity of the blue dye depends on the glue in the sample tissue. The content of the original protein fiber, while necrosis can lead to this fibrosis. Therefore, tissue samples were taken from the tibialis anterior muscles at the second and seventh weeks after surgery and treatment, respectively, and stained with Manson's trichrome stain to assess the degree of fibrosis (and necrosis). Figures 4A and 4B provide photographs of representative samples.

如第4A圖所示，在術後兩週，空白對照組動物的肌肉組織出現大量纖維化的情形(藍色染色區域)，而經本發明提出之緩釋配方處理的動物，其肌肉組織中纖維化區域相對較小。此外，在第4A圖中還可發現，於術後七週，此處提出之緩釋配方可有效降低壞死與纖維化區域，且因而可使肌肉組織完全復原。As shown in Fig. 4A, in the two weeks after the operation, the muscle tissue of the blank control group showed a large amount of fibrosis (blue stained area), and the animal treated by the sustained release formulation proposed by the present invention had fibers in the muscle tissue. The area is relatively small. In addition, it can be found in Fig. 4A that, at seven weeks after surgery, the sustained release formulation proposed herein can effectively reduce the area of necrosis and fibrosis, and thus the muscle tissue can be completely restored.

在缺血性損傷之後，可透過衛星細胞增殖來進行肌纖維再生。新生成的肌纖維的特點之一為中心位移的細胞核。此外，壞死區域中出現壞死肌原纖維，其呈現淺色嗜伊紅細胞質且具有水腫與欠缺周邊細胞核等現象。如第4B圖所示，於手術後兩週，經本緩釋配方處理的小鼠中，具有中心位移之細胞核的肌原纖維之再生情形比起空白對照組小鼠更為明顯。參照第4B圖上方照片，空白對照組之樣本中的較大淺紅色區域(相較於經20-mer治療之樣本)代表此處提出的緩釋配方能夠有效地預防組織壞死。在手術後7週，空白對照組之動物的脛骨前肌中，還有15%的肌肉區域可見到小型的肌纖維束，且有脂肪滴交錯分布於其間(第4B圖；左下方)。After ischemic injury, muscle fiber regeneration can be performed by satellite cell proliferation. One of the characteristics of newly generated muscle fibers is the centrally displaced nuclei. In addition, necrotic myofibrils appear in the necrotic area, which exhibits light eosinophilic cytoplasm and edema and lack of peripheral nuclei. As shown in Fig. 4B, in the mice treated with the sustained release formulation, the regeneration of myofibrils with centrally displaced nuclei was more pronounced than in the blank control mice two weeks after surgery. Referring to the photograph above in Figure 4B, the larger light red region of the blank control sample (compared to the 20-mer treated sample) represents the sustained release formulation proposed herein to be effective in preventing tissue necrosis. At 7 weeks after surgery, small muscle fiber bundles were observed in 15% of the tibialis anterior muscle of the blank control group, and fat droplets were staggered between them (Fig. 4B; lower left).

針對術後兩週的損傷區域(壞死區域與纖維化區域)以及細胞核中心移位之纖維數目進行統計分析，結果摘要 整理於表2。將損傷區域面積表示成總染色區域面積的百分比(%)，而將細胞核中心移位之纖維表示成所計算之肌纖維總數的百分比(%)。Statistical analysis of the number of lesions (necrotic and fibrotic areas) and the number of fibers displaced by the center of the nucleus for two weeks after surgery, summary of results Organized in Table 2. The area of the damaged area is expressed as a percentage (%) of the area of the total stained area, and the fiber displaced by the center of the core is expressed as a percentage (%) of the total number of calculated muscle fibers.

由表2所載的數據可以看出，注射此處提出的緩釋配方可實質降低組織損傷(相較於空白或丸劑對照組)。具體來說，經PEDF處理之群組損傷區域可降低至空白或丸劑對照組的約45-48%。此外，這些資料顯示以29-mer、24-mer或20-mer配方治療會使得脛骨前肌中細胞核中心移位之纖維數目增加約3-3.7倍(相較於空白或丸劑對照組)。As can be seen from the data contained in Table 2, injection of the sustained release formulation set forth herein can substantially reduce tissue damage (compared to the blank or bolus control group). Specifically, the PEDF-treated group lesion area can be reduced to about 45-48% of the blank or pill control group. In addition, these data show that treatment with a 29-mer, 24-mer or 20-mer formulation increases the number of fibers displaced in the nucleus of the tibialis anterior muscle by about 3-3.7 fold (compared to the blank or bolus control group).

綜上所述，實驗例2.2所述的結果顯示以含29-mer、24-mer或20-mer之緩釋配方進行處置，可預防缺血所引發的組織壞死與纖維化，且因而可促進肌肉組織的復原。此外，以此處提出之緩釋配方治療的小鼠，其脛骨肌復原情形較佳，可進一步佐證此緩釋配方於促進缺血性肢體中血液灌流的效果(參見上文實驗例2.1)。In summary, the results described in Experimental Example 2.2 show that treatment with a slow release formulation containing 29-mer, 24-mer or 20-mer can prevent tissue necrosis and fibrosis induced by ischemia, and thus promote Restoration of muscle tissue. In addition, the mice treated with the sustained release formulation proposed herein have better iliac muscle recovery, and can further demonstrate the effect of the sustained release formulation on promoting blood perfusion in ischemic limbs (see Experimental Example 2.1 above).

實驗例2.3Experimental example 2.3

緩釋PEDF合成胜肽可刺激動脈血管生成而藉由側枝循環供應血流至缺血性組織Sustained-release PEDF synthetic peptide can stimulate arterial angiogenesis and supply blood flow to ischemic tissue through collateral circulation

在主要動脈(如冠狀動脈或股動脈)急性阻塞的情形中，可以運用既有的動脈連接來繞過阻塞部位。此一過程稱為「動脈血管生成」，且和「血管新生」過程有諸多不同之處。從解剖學的角度來看，這些既有的側枝動脈是由一內皮細胞層(endothelial lining)、一內彈性層(internal elastic lamina)以及一或兩層平滑肌細胞所組成的具有薄管壁的微型血管管路；此一結構與血管新生過程中生成的微血管不同。在一般的情形下，不會使用這些原生的既有小動脈來提供血液灌流。然而，在主要動脈阻塞後，這些血管會經由生長作用而大幅提升其管腔容量，並繞過阻塞區域，而能提供較高的血液灌流至受損的缺血區域。在動物體內許多器官或組織(如後肢、心臟、腦、腎等)中，當主要動脈發生慢性或急性阻塞時，這些側枝動脈可緩減隨後可能發生的各種不利狀況。當注意到，動脈血管生成血管新生並非使既有側枝動脈被動擴張的簡單過程；反之，動脈血管生成涉及將既有動脈連結經主動增殖與重塑而形成真正的側枝動脈。已知血管半徑對血流量具有關鍵性的影響；因此，側枝動脈在經過適應性的生長後，能夠在單位時間內輸送相對大量的血液。因而，刺激動脈血管生成比起管新生能夠更有效率地保護缺血的後肢或其他器 官(如心臟、腦等)。相較之下，血管新生指由既有血管形成內皮細胞結構的微血管過程，其對於提供較高的血液灌流至受損的缺血區域沒有幫助。因此，新形成的微血管，不論數量有多少，其供應血流至可能缺血組織的能力，都比不上二或三條側枝動脈所增加的血流量。In the case of acute obstruction of the main artery (such as the coronary or femoral artery), the existing arterial connection can be used to bypass the obstruction site. This process is called "arterial angiogenesis" and has many differences from the "angiogenesis" process. From an anatomical point of view, these existing collateral arteries are microscopically thin, composed of an endothelial lining, an internal elastic lamina, and one or two smooth muscle cells. Vascular line; this structure is different from the microvessels generated during angiogenesis. Under normal circumstances, these native small arteries are not used to provide blood perfusion. However, after major arterial occlusion, these blood vessels greatly increase their lumen volume via growth and bypass the occlusion area, providing higher blood perfusion to the damaged ischemic area. In many organs or tissues in animals (such as hind limbs, heart, brain, kidney, etc.), when the main artery is chronically or acutely blocked, these collateral arteries can alleviate various adverse conditions that may occur subsequently. It is noted that arterial angiogenesis is not a simple process for passive expansion of existing collateral vessels; conversely, arterial angiogenesis involves the coupling of existing arteries through active proliferation and remodeling to form true collateral arteries. It is known that the vascular radius has a critical effect on blood flow; therefore, after adaptive growth, the collateral artery can deliver a relatively large amount of blood per unit time. Thus, stimulating arterial angiogenesis can protect the ischemic hind limbs or other organs more efficiently than tube births. Official (such as heart, brain, etc.). In contrast, angiogenesis refers to a microvascular process in which endothelial cells are formed by existing blood vessels, which does not help to provide higher blood perfusion to the damaged ischemic area. Thus, newly formed microvessels, regardless of their number, are less able to supply blood to potentially ischemic tissue than the increased blood flow of two or three collateral arteries.

為了探究此處提出的緩釋配方對動脈血管生成的效果，於術後兩週取得每一處理組別之動物的內收長肌(其深度與股動脈切除位置相同，且預期該處會出現用以建立側枝循環之動脈血管生成)。對脈管平滑肌細胞(α-SMA；褐色)染色，以標記肌肉剖面中的小動脈，並以蘇木色素來標記細胞核；代表性照片見第5圖。此外，亦進行定量分析，其結果表示為損傷區域周邊(peri-injury region)內每平方公釐(mm2 )的α-SMA-陽性小動脈之數目，如表3所示。In order to explore the effect of the sustained-release formulation proposed here on arterial angiogenesis, the adductor longus muscle of each treatment group was obtained two weeks after surgery (the depth was the same as the femoral artery resection position, and it is expected to appear there. Used to establish arterial angiogenesis in the collateral circulation). Vascular smooth muscle cells (α-SMA; brown) were stained to mark small arteries in the muscle section, and the nuclei were labeled with hematoxylin; see Figure 5 for representative photos. In addition, quantitative analysis was also performed, and the results were expressed as the number of α-SMA-positive arterioles per square millimeter (mm 2 ) in the peri-injury region, as shown in Table 3.

這些資料顯示，相較於空白與丸劑對照組，給予此處 提供的緩釋配方能夠增加鄰近股動脈移除處之內收長肌內的小動脈密度。因而，在急性阻斷血液供應後，PEDF合成胜肽的持續釋出能夠提供動脈血管生成活性，以建立側枝循環。透過生長作用而大幅提升這些血管的管腔容量，能夠促進受損之缺血區域的灌流；而此種發展良好的側枝循環使得組織或器官能夠自缺血情況中復原。These data show that compared to the blank and the pill control group, given here The sustained release formulation is provided to increase the density of the small arteries in the elongate muscles adjacent to the femoral artery removal. Thus, after acute blockade of blood supply, sustained release of PEDF synthetic peptides can provide arterial angiogenic activity to establish collateral circulation. Significantly increasing the lumen volume of these vessels through growth can promote perfusion of the damaged ischemic area; and such well-developed collateral circulation allows tissue or organs to recover from ischemic conditions.

實驗例2.4Experimental Example 2.4

PEDF合成胜肽可刺激完整組織培養(ex vivo )之新血管分枝PEDF synthetic peptide can stimulate new blood vessel branches in complete tissue culture ( ex vivo )

為了進一步確認由PEDF合成胜肽促進之新血管發展，進行大鼠主動脈環分枝(aortic ring sprouting)分析。自安樂死的大鼠取出胸主動脈(thoracic aortas)，並輕輕地剝除大動脈周邊的纖維脂肪(fibroadipose)組織。將大動脈切片成長度約2 mm的環，之後將其嵌入於低生長因子基質膠(growth factor-reduced Matrigel)中。將含有主動脈環的膠體置於12孔的孔盤內，在37℃下培育30分鐘使膠體聚合。將1 ml的MCDB131培養基加入含有基質膠的培植體中，上述培養基內添加了100 U/ml的青黴素、100 ng/ml的鏈黴素、1% FBS以及不同的添加劑(50 ng/ml的VEGF-A、20 ng/ml的FGF-2；或50 ng/ml的29-mer、24-mer、20-mer、Mo 29-mer、Mo 20-mer、25-mer或18-mer)。將培植體置於有濕度調節的保溫箱中在37℃下培養4天，並每2天更換新鮮培養基。在培養後第4天， 利用具有明視野鏡頭(bright-field optics)的倒立顯微鏡(inverted microscope)平台(Leica)來觀察新血管分枝的情形；代表性照片見第6圖。此外，利用Image-Pro Plus 6.0軟體(Dendrites program)對新血管分枝進行定量分析，結果表示成相對於未經處理之主動脈環的倍數，如表4所示。本實驗重複三次。To further confirm the development of new blood vessels promoted by the PEDF synthetic peptide, a rat aortic ring sprouting analysis was performed. The thoracic aortas (thoracic aortas) were removed from the euthanized rats and the fibroadipose tissue surrounding the aorta was gently stripped. The aorta was sectioned into a loop of approximately 2 mm in length, which was then embedded in a growth factor-reduced Matrigel. The colloid containing the aortic ring was placed in a 12-well well plate and incubated at 37 ° C for 30 minutes to polymerize the colloid. Add 1 ml of MCDB131 medium to Matrigel-containing culture medium containing 100 U/ml penicillin, 100 ng/ml streptomycin, 1% FBS, and different additives (50 ng/ml VEGF). -A, 20 ng/ml of FGF-2; or 50 ng/ml of 29-mer, 24-mer, 20-mer, Mo 29-mer, Mo 20-mer, 25-mer or 18-mer). The culture bodies were placed in a humidity-conditioned incubator for 4 days at 37 ° C, and fresh medium was replaced every 2 days. On the 4th day after the training, The situation of new blood vessel branches was observed using an inverted microscope platform (Leica) with bright-field optics; see Figure 6 for representative photographs. In addition, new blood vessel branches were quantified using the Image-Pro Plus 6.0 software (Dendrites program) and the results were expressed as multiples relative to the untreated aortic rings, as shown in Table 4. This experiment was repeated three times.

在未經處理的對照組(UT)中，培養基並未添加任何生長因子，在第4天僅可觀察到少量的新血管分枝情形。可注意到，相較於未經處理的對照組，添加對照組PEDF合成胜肽(即，25-mer及18-mer)並未實質促進新血管分枝。In the untreated control (UT), the medium was not added with any growth factors, and only a small amount of new blood vessel branching was observed on the fourth day. It can be noted that the addition of the control PEDF synthetic peptide (i.e., 25-mer and 18-mer) did not substantially promote new blood vessel branching compared to the untreated control group.

一如預期地，已知的血管新生因子包括VEGF與FGF2皆可實質提升新血管分枝。經VEGF或FGF-2處理之樣本 的新血管分枝分別是未經處理之對照組的約3.4倍與約3.5倍。As expected, known angiogenic factors, including both VEGF and FGF2, substantially enhance neovascular branching. Samples treated with VEGF or FGF-2 The new blood vessel branches were about 3.4 times and about 3.5 times, respectively, of the untreated control group.

表4的資料亦顯示，此處提出的PEDF合成胜肽(包括29-mer、24-mer、20-mer、Mo 29-mer與Mo 20-mer)於刺激新血管分枝的效果更勝於VEGF或FGF2。此外，對這些新血管進行雙重染色免疫分析，分別標記α-平滑肌肌動蛋白(小動脈壁平滑肌肉細胞(SMC)的標記)與凝集素B4(IB4，內皮細胞的標記)，代表性照片見第7圖。由第7圖可以看出，以此處提出之PEDF合成胜肽(29-mer或20-mer)處理的樣本顯現出小動脈的表現型而具有SMC披覆。相反地，在以對照組PEDF 18-mer處理的樣本中，幾乎很難觀察到初級管狀構造(endothelial tube)的形成與及SMC增殖的情形。此一結果顯示根據本發明實施例的PEDF合成胜肽能夠刺激新血管生成，而不僅止於只含內皮細胞之微血管的血管新生。此外，此實驗結果亦可支持此處提出的PEDF合成胜肽可於活體內刺激動脈血管生成之論述。The data in Table 4 also shows that the PEDF synthetic peptides proposed here (including 29-mer, 24-mer, 20-mer, Mo 29-mer and Mo 20-mer) are more effective at stimulating new blood vessel branches than VEGF or FGF2. In addition, a double staining immunoassay was performed on these new blood vessels, and α-smooth muscle actin (a marker of small arterial wall smooth muscle cells (SMC)) and lectin B4 (IB4, a marker of endothelial cells) were labeled, respectively. Figure 7. As can be seen from Figure 7, samples treated with the PEDF synthetic peptide (29-mer or 20-mer) presented herein exhibited a phenotype of the arteriole with SMC coverage. In contrast, in the samples treated with the control PEDF 18-mer, it was almost difficult to observe the formation of the primary tubular tube and the proliferation of SMC. This result shows that the PEDF synthetic peptide according to the embodiment of the present invention is capable of stimulating neovascularization, and not only angiogenesis of microvessels containing only endothelial cells. In addition, the results of this experiment can also support the discussion of the PEDF synthetic peptide proposed here to stimulate arterial angiogenesis in vivo.

總結來說，實驗例2(包含實驗例2.1至2.4)所記載的結果顯示此處提出的PEDF合成胜肽可有效促進肢體灌流、減少組織壞死與纖維化以及促進動脈血管生成與新血管分枝；且因而投予上述PEDF合成胜肽(特別是含有任一種所述PEDF合成胜肽的緩釋配方)能夠降低缺血性損傷並有助於組織或器官之結構與功能的復原。當注意到，先前研究顯示人類PEDF有一34-mer片段(PEDF第44-77 號殘基)具有抗血管新生(anti-angiogenic)性質，且人類PEDF另有一44-mer片段(PEDF第78-121號殘基)具有神經滋養(neurotrophic)性質。然而，本發明首度證實，短的PEDF片段(至少包含上述29-mer、24-mer、20-mer、Mo 29-mer與Mo 20-mer)具有促進動脈血管生成(arteriogenic)的活性。In summary, the results of Experimental Example 2 (including Experimental Examples 2.1 to 2.4) show that the PEDF synthetic peptide proposed here can effectively promote limb perfusion, reduce tissue necrosis and fibrosis, and promote arterial angiogenesis and new blood vessel branching. And thus administration of the above PEDF synthetic peptide (especially a sustained release formulation containing any of the PEDF synthetic peptides) can reduce ischemic injury and contribute to the recovery of the structure and function of the tissue or organ. When noted, previous studies have shown that human PEDF has a 34-mer fragment (PEDF 44-77) Residues have anti-angiogenic properties, and human PEDF has a 44-mer fragment (PEDF residues 78-121) with neurotrophic properties. However, the present invention demonstrates for the first time that a short PEDF fragment (containing at least the above 29-mer, 24-mer, 20-mer, Mo 29-mer and Mo 20-mer) has an arteriogenic-promoting activity.

實驗例3Experimental example 3

緩釋PEDF合成胜肽可促進肌肉再生Slow release of PEDF synthetic peptide can promote muscle regeneration

將單一劑丁胍卡因(bupivacaine)注射至大鼠之比目魚肌，致使大鼠肌肉壞死(myonecrosis)，並用此模型探究此處提出的PEDF合成胜肽對於肌肉再生的影響。本實驗採用十週大的雄性Sprague-Dawley大鼠(試驗開始的平均體重為312±11 g)，動物經腹腔內注射舒泰(zoletil，用量為每公斤體重10毫克)以進行麻醉。以脫毛霜移除後腿與臀部的毛髮。之後，以拋棄式注射器與26號(26-gauge)針頭將0.5 ml的丁胍卡因(AstraZeneca)單邊注射到動物的比目魚肌中。簡言之，將針頭***至比目魚肌的遠端，且之後縱向收回針頭至進端，同時均勻地注射丁胍卡因溶液。在緩慢收回針頭的過程中，使溶液注射於肌肉的全長範圍中。A single dose of bupivacaine was injected into the soleus muscle of the rat, causing myonecrosis in the rat, and this model was used to explore the effect of the PEDF synthetic peptide proposed herein on muscle regeneration. In this experiment, ten-week-old male Sprague-Dawley rats (average body weight of 312±11 g at the start of the experiment) were used, and the animals were intraperitoneally injected with zoletil (10 mg/kg body weight) for anesthesia. Remove the hair from the hind legs and buttocks with a depilatory cream. Thereafter, 0.5 ml of AstraZeneca was unilaterally injected into the soleus muscle of the animal using a disposable syringe and a 26-gauge needle. Briefly, the needle is inserted into the distal end of the soleus muscle, and then the needle is longitudinally retracted to the leading end while uniformly injecting the cardiocaine solution. During the slow withdrawal of the needle, the solution is injected over the full length of the muscle.

在注射丁胍卡因之後，將大鼠平均分成四個實驗群組(每群樣本數為10隻)，並分別給予以下處理。在空白對照組中，給予小鼠50 μl的空白海藻膠。在治療組中，給予 小鼠50 μl的緩釋配方(含29-mer或20-mer)。丸劑對照組之小鼠則接受丸劑配方(含29-mer)。在丁胍卡因灌流後，立刻以單一劑肌肉內注射的方式，將不同的處理施用至動物的比目魚肌。After the injection of dysproin, the rats were equally divided into four experimental groups (the number of samples per group was 10), and the following treatments were separately administered. In the blank control group, 50 μl of blank seaweed gum was administered to the mice. In the treatment group, given A 50 μl sustained release formulation of mice (containing 29-mer or 20-mer). The mice in the pill control group received a pill formulation (containing 29-mer). Immediately after the perfusion of the dysprosium, different treatments were applied to the soleus muscle of the animal by a single intramuscular injection.

在注射丁胍卡因後第4天，取比目魚肌的切片進行組織學分析，結果顯示不同組別皆出現一般肌肉壞死的情形，比目魚肌樣本中可見到肌纖維崩解與大量浸潤發炎細胞(照片未示出)。只有在周邊區域還有少數肌纖維保有相對正常的結構；在空白對照組與經PEDF胜肽處理的組別中，肌纖維壞死的程度都很相近。以上結果顯示丁胍卡因對不同組別的原有肌纖維引起相同程度的肌纖維崩解。On the 4th day after the injection of dysproin, histological analysis of the soleus muscle section showed that general muscle necrosis occurred in different groups. Muscle fiber disintegration and massive infiltration of inflammatory cells were observed in the soleus muscle sample (photograph) Not shown). Only a few muscle fibers in the surrounding area maintained a relatively normal structure; in the blank control group and the PEDF peptide-treated group, the degree of muscle fiber necrosis was similar. The above results show that dipyridamine causes the same degree of muscle fiber disintegration in different groups of original muscle fibers.

實驗例3.1Experimental example 3.1

緩釋PEDF合成胜肽可促進細胞增殖Slow release of PEDF synthetic peptide can promote cell proliferation

肌肉再生過程涉及了肌纖維或肌肉細胞的增殖；於本實驗例中，將BrdU引入增殖的細胞核中，以觀察肌纖維增殖之情形。衛星細胞的增殖是肌肉再生的關鍵；因此，亦對比目魚肌進行染色，藉由偵測衛星細胞標記Pax7來探究肌肉再生活性。詳細的檢測方法如「材料與方法」一節所述。以標記指數(labeling index)來表示BrdU-陽性細胞的數量，此一數值是將經標記的細胞數目除以細胞總數，並以百分比(%)表示。Pax7-陽性細胞的標記指數(%)則是將經標記的細胞數目除以具有細胞核的細胞總數。自每一實驗群組的10隻小鼠分別取得肌肉切片，且每一切片 取六個部分進行定量分析，結果摘要整理於表5。The process of muscle regeneration involves the proliferation of muscle fibers or muscle cells; in this experimental example, BrdU was introduced into the proliferating cell nucleus to observe the proliferation of muscle fibers. The proliferation of satellite cells is the key to muscle regeneration; therefore, staining is also performed on the target fish muscle, and muscle regeneration activity is explored by detecting the satellite cell marker Pax7. Detailed testing methods are described in the section "Materials and Methods". The number of BrdU-positive cells is indicated by a labeling index, which is the number of labeled cells divided by the total number of cells and expressed as a percentage (%). The labeling index (%) of Pax7-positive cells is the number of labeled cells divided by the total number of cells with nuclei. Muscle sections were taken from 10 mice in each experimental group, and each slice was taken Six parts were taken for quantitative analysis, and the results are summarized in Table 5.

由表5可以看出，經含有29-mer或20-mer之緩釋配方治療的小鼠傷口中，有明顯較多的BrdU-陽性細胞(相較於經空白或丸劑對照組處理的小鼠)。關於衛星細胞的增殖活性，表5的數據顯示，相較於空白與丸劑對照組，此處提出的緩釋配方可導致較高的Pax7-陽性細胞比例。總結來看，這些資料顯示給予此處提出的緩釋配方能夠提升肌纖維和/或衛星細胞的增殖活性，而此一特性又能夠促進肌肉再生。As can be seen from Table 5, there were significantly more BrdU-positive cells in the wounds treated with the slow release formulation containing 29-mer or 20-mer (compared to mice treated with blank or pellet control). ). Regarding the proliferative activity of satellite cells, the data in Table 5 shows that the sustained release formulation proposed herein can result in a higher proportion of Pax7-positive cells compared to the blank and the pill control group. In summary, these data show that the sustained-release formulation proposed here can increase the proliferative activity of muscle fibers and/or satellite cells, which in turn promotes muscle regeneration.

實驗例3.2Experimental Example 3.2

緩釋PEDF合成胜肽可促進肌纖維再生Slow release of PEDF synthetic peptide can promote muscle fiber regeneration

在肌肉再生的過程中，新生成的肌纖維的細胞核通常位於中心。因此，這種細胞核中心移位之肌纖維的百分比亦可作為肌肉之再生活性的指標。在注射丁胍卡因後第7天，對細胞核中心移位之纖維數目進行統計分析，結果摘要整理於表6。During the process of muscle regeneration, the nuclei of newly formed muscle fibers are usually located at the center. Therefore, the percentage of muscle fibers displaced by the center of the nucleus can also be used as an indicator of the regenerative activity of the muscle. On the 7th day after the injection of dyscaine, the number of fibers displaced at the center of the nucleus was statistically analyzed, and the results are summarized in Table 6.

由表6可以看出，相較於空白或丸劑對照組，經含有29-mer或20-mer之緩釋配方處理的動物中，含有中心位移之細胞核的肌纖維比例較高。這些結果顯示給予此處提出的緩釋配方能夠有效促進肌肉再生。As can be seen from Table 6, the proportion of muscle fibers containing centrally displaced nuclei was higher in animals treated with a slow release formulation containing 29-mer or 20-mer compared to the blank or bolus control group. These results show that the sustained release formulation proposed herein can effectively promote muscle regeneration.

此外，在注射丁胍卡因後第14天，在所有實驗群組中，比目魚肌內都形成了新的肌肉細胞來取代已壞死的肌纖維。然而，在以空白或丸劑對照組處理的動物中，其再生肌肉仍可見到許多細胞核中心移位之纖維(第8圖)，這意味著這些群組中的肌肉再生尚未完成。相反地，在經過含有29-mer或20-mer之緩釋配方處理的動物中，其肌肉切片內的細胞核中心移位之纖維明顯少了許多。總結來看，這些資料顯示PEDF合成胜肽的持續釋放有利於肌纖維的再生。In addition, on day 14 after the injection of dyscaine, new muscle cells were formed in the soleus muscle to replace the necrotic muscle fibers in all experimental groups. However, in animals treated with a blank or pellet control group, many of the core-displaced fibers were still visible in the regenerated muscle (Fig. 8), which means that muscle regeneration in these groups has not been completed. Conversely, in animals treated with a slow release formulation containing 29-mer or 20-mer, the core of the nucleus displaced within the muscle section was significantly less. In summary, these data show that the sustained release of PEDF synthetic peptide is beneficial to the regeneration of muscle fibers.

實驗例3.3Experimental Example 3.3

緩釋PEDF合成胜肽可促進再生之肌纖維的成熟Slow release of PEDF synthetic peptide can promote the maturation of regenerated muscle fibers

在肌肉再生過程的後期，新生成的肌纖維會開始變 大。於受傷後第14天取得肌肉樣本，並根據「材料與方法」一節所述的步驟，分別測量纖維直徑，結果摘要整理於第9圖。In the later stages of the muscle regeneration process, the newly formed muscle fibers will begin to change. Big. Muscle samples were taken on the 14th day after the injury and the fiber diameters were measured according to the procedure described in the section "Materials and Methods". The results are summarized in Figure 9.

平均來看，以含有29-mer或20-mer之緩釋配方處理的動物之肌肉纖維直徑大於空白或丸劑對照組中之動物。此外，經20-mer治療的動物肌肉之尺寸分布非常接近未受傷的完整肌肉之尺寸分布。具體來說，經20-mer治療的動物約有56.6%的肌纖維其最小費里特直徑為15-25 μm，而未受傷的動物則有約53.2%的肌纖維落於此一範圍中；相較之下，空白及丸劑對照組則分別有約59.6%與約56.2%的再生纖維的丸劑對照組最小費里特直徑介於約10-20 μm之間。這些資料顯示與此處提出的緩釋配方能夠有效增加再生之肌肉的質量。On average, animals treated with a slow release formulation containing 29-mer or 20-mer had larger muscle fiber diameters than animals in the blank or pill control group. In addition, the size distribution of the muscles of the 20-mer treated animals is very close to the size distribution of the intact muscles. Specifically, about 56.6% of the muscle fibers treated with 20-mer had a minimum Feret diameter of 15-25 μm, while uninjured animals had about 53.2% of the muscle fibers falling within this range; Below, the blank and pill control groups had a minimum Feret diameter of about 10-20 μm in the pill control group of about 59.6% and about 56.2% regenerated fiber, respectively. These data show that the sustained release formulation proposed here can effectively increase the quality of regenerated muscle.

總結而論，實驗例3(包括實驗例3.1至3.3)所提出的資料證實此處提出的PEDF合成胜肽能夠有效促進肌纖維與衛星細胞的增殖、肌纖維的再生以及再生之肌纖維的成熟；且因而投予所述PEDF合成胜肽(特別是含有任一種所述PEDF合成胜肽之緩釋配方)能夠有效促進肌肉再生過程並有助於肌肉組織結構與功能的復原。本發明率先發現短的PEDF片段(至少包含29-mer與20-mer)能夠促進肌肉再生。In summary, the data presented in Experimental Example 3 (including Experimental Examples 3.1 to 3.3) confirm that the PEDF synthetic peptide proposed here can effectively promote the proliferation of muscle fibers and satellite cells, the regeneration of muscle fibers, and the maturation of regenerated muscle fibers; Administration of the PEDF synthetic peptide (especially a sustained release formulation containing any of the PEDF synthetic peptides) can effectively promote the muscle regeneration process and contribute to the recovery of muscle tissue structure and function. The present invention pioneered the discovery that short PEDF fragments (containing at least 29-mer and 20-mer) are capable of promoting muscle regeneration.

實驗例4Experimental example 4

緩釋PEDF合成胜肽可促進肌腱再生Slow release of PEDF synthetic peptide can promote tendon regeneration

本實驗例採用大鼠肌腱損傷模型來探究此處提出的PEDF合成胜肽對肌腱再生的效果。採用十週大的成年雄性Sprague-Dawley大鼠(總樣本數50隻，試驗開始的平均體重為312±11 g)，動物經腹腔內注射若朋(用量為每公斤體重10毫克)以進行麻醉。接著，將18號(18-gauge)針頭***穿過大鼠左腿阿基里斯腱的完整厚度，並在跟骨(calcaneum)上方約1公分處將阿基里斯腱橫切切斷，以得到左阿基里斯腱缺損的動物模型。此一方式所得到的水平傷口兩側有完整的肌腱組織，可防止兩斷裂端的收縮。In this experimental example, a rat tendon injury model was used to investigate the effect of the PEDF synthetic peptide proposed herein on tendon regeneration. Ten-week-old adult male Sprague-Dawley rats (50 in total, starting at 312 ± 11 g) were injected intraperitoneally with an injection of 10 mg per kilogram of body weight for anesthesia. . Next, insert the 18-gauge needle through the full thickness of the Achilles's left leg, and cross-cut the Achilles tendon about 1 cm above the calcaneum to get the left. Animal model of the Kiris 腱 defect. In this way, the horizontal wound obtained on both sides has intact tendon tissue, which can prevent the contraction of the two fracture ends.

將大鼠隨機分派於五個實驗群組(每群樣本數為10隻)，並分別接受以下處置。空白對照組的小鼠接受150 μl的空白海藻膠。丸劑對照組的小鼠給予150 μl的丸劑配方(29-mer)。在PEDF處理組中，小鼠分別接受150 μl的緩釋配方(含29-mer、24-mer或20-mer)。傷後立刻將各處理以皮下注射的方式注射至鄰近肌腱損傷處，傷口以無菌生理食鹽水沖洗後縫合。Rats were randomly assigned to five experimental groups (10 per group) and received the following treatments, respectively. The mice in the blank control group received 150 μl of blank seaweed gum. Pills in the control group were given 150 μl of the pellet formulation (29-mer). In the PEDF treatment group, mice received 150 μl of a sustained release formulation (containing 29-mer, 24-mer or 20-mer), respectively. Immediately after the injury, each treatment was injected subcutaneously into the adjacent tendon lesion, and the wound was washed with sterile physiological saline and sutured.

實驗例4.1Experimental example 4.1

緩釋PEDF合成胜肽可促進肌腱癒合Slow release of PEDF synthetic peptide can promote tendon healing

在肌腱損傷後第3週，進行組織學分析以觀察肌腱復原的情形，代表性照片如第10圖所示。第10圖上方為空白對照組之動物的復原情形，由圖中可看到在兩斷裂端之間，形成了由不規則的纖維所組成的疤痕組織，且在此疤痕組織中經染色的膠原蛋白束(粉紅色)非常少。相較之 下，經含有29-mer之緩釋配方處理後，肌腱二斷裂端之間形成的癒合組織中的疤痕組織較少(相較於空白對照組樣本)，且有成熟膠原蛋白束(粉紅色)延伸至肌腱損傷處；此外，在某些區域中，來自二斷裂端的肌腱纖維似乎有接合的情形(第10圖下方照片)。再者，經29-mer處理處理的群組中，疤痕中的纖維組織似乎較為規則，且平行排列。At the 3rd week after the tendon injury, histological analysis was performed to observe the tendon recovery, and a representative photograph is shown in Fig. 10. Figure 10 above shows the recovery of the animals in the blank control group. It can be seen that between the two fracture ends, scar tissue composed of irregular fibers is formed, and the stained collagen in the scar tissue is formed. The protein bundle (pink) is very small. Compared Next, after treatment with a slow-release formulation containing 29-mer, there was less scar tissue in the healing tissue formed between the two fracture ends of the tendon (compared to the blank control sample), and there was a mature collagen bundle (pink) It extends to the tendon lesion; in addition, in some areas, the tendon fibers from the two fracture ends appear to be joined (photograph below in Figure 10). Furthermore, in the 29-mer treated group, the fibrous tissue in the scar appeared to be relatively regular and arranged in parallel.

第11圖為較高倍率的組織學分析照片。由照片中可以看出，正常肌腱在膠原蛋白纖維之間所含的細胞數相對較少，且細胞核幾乎都呈狹長形。在空白與丸劑對照組中，在經過三週的復原後，可在肌腱中觀察到較多的纖維母細胞(fibroblast，其特徵在於有圓形或紡錘形的似纖維母細胞細胞核)，且新形成的膠原蛋白纖維結構較不規則(照片中未損傷組織以星號(*)標記)。這些型態學上的改變顯示空白及丸劑對照組中的肌腱傷口癒合較差。Figure 11 is a photograph of histological analysis of higher magnification. As can be seen from the photograph, the normal tendon contains relatively few cells between the collagen fibers, and the nuclei are almost elongated. In the blank and the pill control group, after three weeks of recovery, more fibroblasts (fibroblasts, characterized by round or spindle-shaped fibroblast-like nuclei) were observed in the tendon, and new formation was observed. The collagen fiber structure is more irregular (the undestroyed tissue in the photo is marked with an asterisk (*)). These changes in morphology showed poor healing of tendon wounds in the blank and pill control groups.

相較之下，第11圖顯示在以此處提出之緩釋配方處理的肌腱中，癒合區域可見到較薄、細長形的細胞核，其型態近似成熟肌腱細胞(tenocyte)的細胞核，而其中的膠原蛋白纖維排列規則且和原生肌腱(深粉紅色deep pink)相平行；以上型態代表著肌腱傷口的癒合情形較佳。這些結果顯示此處提出的緩釋配方有利於肌腱傷口之癒合。In contrast, Figure 11 shows that in the tendon treated with the sustained release formulation proposed herein, a thin, elongated nuclei can be seen in the healing region, which is similar in morphology to the nuclei of mature tenocytes. The collagen fibers are regularly arranged in parallel with the native tendon (deep pink); the above pattern represents a better healing of the tendon wound. These results show that the sustained release formulation proposed here is beneficial for the healing of tendon wounds.

此外，樣本經曼森氏三色染劑染色，以評估膠原蛋白纖維的結構與排置，代表性樣本照片如第12圖所示。在未受傷的肌腱中，膠原蛋白纖維實質上彼此平行(第12圖左方)。相反地，在空白對照組中，受傷肌腱之復原區域內的 膠原蛋白纖維成不規則排列(第12圖中；傷口邊緣以星號(*)標記)。然而，以此處提出的緩釋配方處理的受傷肌腱中，可觀察到排列規則的膠原蛋白纖維，且其方向與受傷邊緣之外的未受傷肌腱組織大致相同(第12圖右方；傷口邊緣以星號(*)標記)。這些高度規則排列的膠原蛋白纖維意味著經此處提出的緩釋配方處理的動物，其肌腱傷口復原情形較佳。In addition, samples were stained with Manson's trichrome dye to evaluate the structure and arrangement of collagen fibers. Representative sample photographs are shown in Figure 12. In the uninjured tendon, the collagen fibers are substantially parallel to each other (Fig. 12 left). Conversely, in the blank control group, within the recovery area of the injured tendon Collagen fibers are arranged in an irregular pattern (in Figure 12; the edges of the wound are marked with an asterisk (*)). However, in the injured tendon treated with the sustained release formulation proposed herein, regular arrangement of collagen fibers was observed, and the direction was approximately the same as that of the uninjured tendon tissue outside the injured edge (Fig. 12 right; wound edge) Marked with an asterisk (*)). These highly regularly arranged collagen fibers mean that the animals treated with the sustained release formulation set forth herein have better tendon wound healing.

此外，以定量分析來評估於再生區域中的膠原蛋白百分比(%)，結果摘要整於表7。In addition, the percentage (%) of collagen in the regeneration area was evaluated by quantitative analysis, and the results are summarized in Table 7.

由表7可以看出，PEDF(29-mer、24-mer或20-mer)處理組中的動物其再生區域內的膠原蛋白含量較高(相較於空白或丸劑對照組)。這些數據顯示，施用此處提出的緩釋配方能夠促進創傷區域內的膠原蛋白生合成。As can be seen from Table 7, the animals in the PEDF (29-mer, 24-mer or 20-mer) treated group had higher collagen content in the regeneration area (compared to the blank or the pill control group). These data show that administration of the sustained release formulation set forth herein promotes collagen biosynthesis in the wound area.

此外，對樣本中的第一型膠原蛋白進行免疫染色，並以蘇木色素來染色細胞核，代表性的樣本照片如第13圖所示，其中下方圖式為上方圖式虛線部分的放大圖。當可想見，未受傷肌腱中的膠原蛋白纖維彼此有良好的交鏈，且 因而抗-膠原蛋白1A1抗體可能不易辨識這些膠原蛋白纖維。因此，在第13圖左方照片中，僅可觀察到非常少量的第一型膠原蛋白(褐色)。藉由比較空白對照組的照片(第13圖中)與PEDF處理組的照片(第13圖右方)，可以發現經PEDF處理的動物中，第一型膠原蛋白(褐色)的含量高於空白對照組之動物。In addition, the first type of collagen in the sample was immunostained, and the nuclei were stained with hematoxylin. The representative sample photograph is shown in Fig. 13, wherein the lower graph is an enlarged view of the dotted line portion of the upper graph. It is conceivable that the collagen fibers in the uninjured tendon have a good cross-linking with each other, and Therefore, anti-collagen 1A1 antibodies may not easily recognize these collagen fibers. Therefore, in the photograph on the left of Fig. 13, only a very small amount of collagen type I (brown) was observed. By comparing the photos of the blank control group (Fig. 13) with the photos of the PEDF treatment group (Fig. 13 right), it can be found that the content of type I collagen (brown) in PEDF-treated animals is higher than that in the blank. Animals in the control group.

總結來看，這些結果顯示投予含有所述PEDF合成胜肽之緩釋配方能夠刺激受傷肌腱組織內的細胞之第一型膠原蛋白的生合成，有助於癒合損傷組織膠原蛋白的沈積，且可促使膠原蛋白纖維排列更為規則，因而有利於肌腱再生。In conclusion, these results show that administration of a sustained-release formulation containing the PEDF synthetic peptide can stimulate the biosynthesis of type I collagen in cells in injured tendon tissue, and contribute to healing the deposition of collagen in damaged tissues, and It can promote the arrangement of collagen fibers more regularly, thus facilitating tendon regeneration.

實驗例4.2Experimental example 4.2

PEDF合成胜肽可誘導活體外(in vitro )肌腱幹細胞增殖PEDF synthetic peptide can induce proliferation of tendon stem cells in vitro

已知在肌腱癒合的過程中，肌腱幹細胞(tendon stem cell，簡稱TSC)會擴增並分化為肌腱細胞。為了探討此處提出的PEDF合成胜肽是否可誘使肌腱幹細胞於活體外增殖，利用「材料與方法」一節所述的步驟來分離並培養肌腱幹細胞。並利用肌腱幹細胞標記(核幹細胞因子)以及肌腱幹細胞可表現第一型膠原蛋白的特性來確認肌腱幹細胞的純度(近百分之百；資料未示出)。再利用BrdU脈衝標記2小時，來確認細胞增殖。採用上文所述的方法對BrdU-陽性細胞進行定量分析，結果摘要整理於表8。It is known that in the process of tendon healing, tendon stem cells (TSCs) are expanded and differentiated into tendon cells. To investigate whether the PEDF synthetic peptide proposed herein can induce tendon stem cells to proliferate in vitro, the steps described in the "Materials and Methods" section were used to isolate and culture tendon stem cells. The purity of the tendon stem cells (nearly 100%; data not shown) was confirmed by the use of tendon stem cell markers (nuclear stem cell factor) and tendon stem cells to express the characteristics of type 1 collagen. Cell proliferation was confirmed by pulse labeling with BrdU for 2 hours. The BrdU-positive cells were quantitatively analyzed by the method described above, and the results are summarized in Table 8.

這些結果顯示，相較於培養於對照組培養基中的肌腱幹細胞，培養於含此處提出之PEDF合成胜肽(29-mer、24-mer、20-mer、Mo 29-mer或Mo 20-mer)之培養基中的肌腱幹細胞的增殖活性較佳。此外，亦可注意到，此處所用的Mo 29-mer與Mo 20-mer是來自老鼠PEDF胜肽，且這兩個序列和39-mer的11-30個胺基酸殘基的序列相似度並非100%；然而，這兩個序列促進有絲***的效果與來自人類PEDF的短PEDF合成胜肽(如，29-mer、24-mer與20-mer)不相上下。These results show that the PEDF synthetic peptide (29-mer, 24-mer, 20-mer, Mo 29-mer or Mo 20-mer) is cultured in comparison with tendon stem cells cultured in the control medium. The proliferation activity of the tendon stem cells in the medium is preferred. In addition, it can also be noted that the Mo 29-mer and Mo 20-mer used herein are derived from the mouse PEDF peptide, and the sequence similarity between the two sequences and the 11-30 amino acid residues of the 39-mer. Not 100%; however, the effect of these two sequences on promoting mitosis is comparable to that of short PEDF synthetic peptides from human PEDF (eg, 29-mer, 24-mer, and 20-mer).

實驗例4.3Experimental example 4.3

緩釋PEDF合成胜肽於肌腱損傷後可促進活體內(in vivo )肌腱幹細胞增殖Sustained-release PEDF synthetic peptide promotes in vivo viability of tendon stem cells after injury to tendon

由實驗例4.1中的各組動物身上取得樣本，並對核幹細胞因子進行染色(綠色)，以探究此處提出的緩釋配方在肌腱傷口復原的過程中，是否能夠促進活體內的肌腱幹細 胞增殖。進行定量分析時，由每一實驗群組的樣本中隨機選取10個顯微視野並擷取影像，接著計算在所有細胞中(以Hoechst 33258進行對比染色；藍色)，核幹細胞因子-陽性細胞的百分，結果摘要整理於表9。Samples were taken from each group of animals in Experimental Example 4.1, and the nuclear stem cell factor was stained (green) to investigate whether the sustained release formulation proposed herein can promote the tendon dryness in the process of tendon wound healing. Cell proliferation. For quantitative analysis, 10 microscopic fields were randomly selected from the samples of each experimental group and images were taken, and then calculated in all cells (contrast staining with Hoechst 33258; blue), nuclear stem cell factor-positive cells The percentages are summarized in Table 9.

這些資料顯示，在經過29-mer、24-mer或20-mer處理的動物中，呈核幹細胞因子-陽性之肌腱幹細胞較多(相較於空白與丸劑對照組中之動物)。將實驗例4.1與4.3的實驗結果綜合分析，可以發現藉由給予此處提出的緩釋配方而促進活體內肌腱幹細胞擴增的現象，和肌腱復原情形較佳(相較於自然的復原過程)的現象可互相呼應。These data show that there are more nuclear stem cell factor-positive tendon stem cells in animals treated with 29-mer, 24-mer or 20-mer (compared to animals in the blank and pill control groups). By comprehensively analyzing the experimental results of Experimental Examples 4.1 and 4.3, it was found that the phenomenon of promoting tendon stem cell expansion in vivo was promoted by administering the sustained-release formulation proposed herein, and the tendon recovery was better (compared to the natural recovery process). The phenomena can echo each other.

實驗例4.4Experimental example 4.4

PEDF合成胜肽可誘導由骨髓間葉幹細胞產生似肌腱細胞PEDF synthetic peptide can induce the production of tendon-like cells from mesenchymal stem cells

近來研究顯示，可利用成體間葉幹(mesenchymal stem cell，簡稱MSC)來再生具有功能的肌腱。於本實驗例中，將骨髓間葉幹細胞培養於對照組培養基或含有PEDF 29-mer或20-mer的培養基中，以探究此處提出的PEDF合成胜肽於促進骨髓間葉幹細胞分化為肌腱細胞的能力。腱調蛋白(TNMD)基因大量表現於肌腱中，且被認為是肌腱細胞系(lineage)最可靠的表現型標記之一。因此，本實驗藉由TNMD的表現量，來評估肌腱細胞的分化。RT-PCR分析的代表性照片見第14圖。Recent studies have shown that mesenchymal stem cells (MSCs) can be used to regenerate functional tendons. In this experimental example, bone marrow mesenchymal stem cells were cultured in control medium or contained PEDF. The medium of 29-mer or 20-mer was used to explore the ability of the PEDF synthetic peptide proposed herein to promote differentiation of bone marrow mesenchymal stem cells into tendon cells. The TNMD gene is abundantly expressed in tendons and is considered to be one of the most reliable phenotypic markers of the tendon cell lineage. Therefore, in this experiment, the differentiation of tendon cells was evaluated by the amount of TNMD expression. Representative photographs of RT-PCR analysis are shown in Figure 14.

分析結果顯示，在體外培養的骨髓間葉幹細胞內，此處提出的PEDF合成胜肽(如29-mer或20-mer)可有效地誘導似肌腱細胞的細胞分化。由於骨髓間葉幹細胞的移動(mobilization)與分化是活體內肌腱修復的可能機制之一，上述結果意味著此處提出的PEDF合成胜肽似乎能夠藉由促進骨髓間葉幹細胞分化為肌腱細胞，而修復肌腱損傷。這些結果亦顯示出，此處提出的PEDF合成胜肽有潛力用於由骨髓間葉幹細胞組成的骨架基質培養系統(scaffold matrix culture)中，以促進人工肌腱的合成。The analysis results show that the PEDF synthetic peptide (such as 29-mer or 20-mer) proposed herein can effectively induce cell differentiation of tendon-like cells in the mesenchymal stem cells cultured in vitro. Since the mobilization and differentiation of mesenchymal stem cells are one of the possible mechanisms of tendon repair in vivo, the above results suggest that the PEDF synthetic peptide proposed here can promote the differentiation of mesenchymal stem cells into tendon cells. Repair tendon damage. These results also show that the PEDF synthetic peptide proposed here has potential for use in a scaffold matrix culture composed of mesenchymal stem cells to promote the synthesis of artificial tendons.

總結而論，實驗例4(包括實驗例4.1至4.4)所示的資料顯示此處提出的PEDF合成胜肽能夠有效促進規則排列之膠原蛋白(特別是第一型膠原蛋白)纖維的生合成以及肌腱幹細胞的增殖；且因而投予此處提出的PEDF合成胜肽(特別是，含有任一種所述PEDF合成胜肽之緩釋配方)能夠促進肌腱再生過程，且有利肌腱組織在結構與功能方面的復原。本發明率先證實短的PEDF胜肽片段(至少29-mer與20-mer)能夠促進肌腱再生，亦能促使骨髓間葉幹細胞分化為肌腱細胞。In summary, the data shown in Experimental Example 4 (including Experimental Examples 4.1 to 4.4) show that the PEDF synthetic peptide proposed here can effectively promote the biosynthesis of regularly arranged collagen (especially type I collagen) fibers and Proliferation of tendon stem cells; and thus administration of the PEDF synthetic peptide proposed herein (in particular, a sustained release formulation containing any of the PEDF synthetic peptides) can promote tendon regeneration and facilitate structural and functional aspects of tendon tissue Recovery. The present invention is the first to demonstrate that short PEDF peptide fragments (at least 29-mer and 20-mer) can promote tendon regeneration and also promote differentiation of mesenchymal stem cells into tendon cells.

綜上所述，上開實施例所載的結果證實此處提出的PEDF合成胜肽(例如29-mer、24-mer、20-mer、Mo 29-mer、與Mo 20-mer)能夠促進缺血區域中或其鄰近區域內的動脈血管生成，亦能促進損傷區域中或其鄰近區域內的肌肉與肌腱再生。因而，此處提出的PEDF合成胜肽可作為一種用以促進肌肉與肌腱傷口復原以及降低缺血性損傷的治療藥劑。In summary, the results of the above examples demonstrate that the PEDF synthetic peptides proposed herein (eg, 29-mer, 24-mer, 20-mer, Mo 29-mer, and Mo 20-mer) can promote the deficiency. Arterial angiogenesis in or adjacent to the blood region can also promote muscle and tendon regeneration in or adjacent to the injured region. Thus, the PEDF synthetic peptide proposed herein can be used as a therapeutic agent for promoting muscle and tendon wound healing and reducing ischemic injury.

雖然上文實施方式中揭露了本發明的具體實施例，然其並非用以限定本發明，本發明所屬技術領域中具有通常知識者，在不悖離本發明之原理與精神的情形下，當可對其進行各種更動與修飾，因此本發明之保護範圍當以附隨申請專利範圍所界定者為準。Although the embodiments of the present invention are disclosed in the above embodiments, the present invention is not intended to limit the invention, and the present invention may be practiced without departing from the spirit and scope of the invention. Various changes and modifications may be made thereto, and the scope of the invention is defined by the scope of the appended claims.

為讓本發明的上述與其他目的、特徵、優點與實施例能更明顯易懂，所附圖式之說明如下。The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood.

第1圖以折線圖繪示在37℃下，海藻膠將PEDF合成胜肽於活體外釋放至PBS中的累積釋放量。結果以三次獨立試驗結果的平均值±標準差來表示。Figure 1 is a line graph showing the cumulative release of PEDF synthetic peptide from PBS in vitro at 37 °C. Results are expressed as mean ± standard deviation of three independent test results.

第2圖為雷射都卜勒灌流影像(LDPI)，繪示缺血後肢在手術後4週內的血流灌注情形。Figure 2 shows the laser Doppler perfusion image (LDPI), showing the blood perfusion of the ischemic hind limbs within 4 weeks after surgery.

第3圖以折線圖繪示小鼠經單獨海藻膠(空白對照)、含29-mer、24-mer、20-mer或18-mer之緩釋配方以及含29-mer的丸劑配方處理後，其後肢血流灌注分析的結果。 果以三次獨立試驗結果的平均值±標準差來表示；n6。*P <0.05；相較於空白對照組。Figure 3 is a line drawing showing the mice treated with a separate algae gel (blank control), a 29-mer, 24-mer, 20-mer or 18-mer sustained release formulation and a 29-mer pellet formulation. The results of blood flow perfusion analysis of the hind limbs. Fruit expressed as the mean ± standard deviation of three independent test results; n 6. * P <0.05; compared to the blank control group.

第4A圖為經曼森氏三色染劑染色後之脛骨肌樣本的代表性照片(原始倍率：×40)；而第4B圖為相同樣本在較高倍率下的照片，以更清楚地呈現以手術誘發後肢缺血後2週與7週的肌肉壞死程度(原始倍率：×200)。Figure 4A is a representative photograph of the tibia muscle sample stained with Manson's trichrome dye (original magnification: ×40); and Fig. 4B is a photograph of the same sample at a higher magnification for more clear presentation The degree of muscle necrosis at 2 weeks and 7 weeks after surgery was induced by surgery (original magnification: ×200).

第5圖為缺血後兩週於內收長肌中之小動脈的代表性免疫顯微照片。小動脈以抗-α-SMA(褐色)標記，而細胞核以蘇木色素標記。Figure 5 is a representative immunomicrograph of the arterioles in the adductor longus two weeks after ischemia. The small arteries are labeled with anti-α-SMA (brown) and the nuclei are labeled with hematoxylin.

第6圖為經過不同培養基培養四天後之主動脈環培植體的代表性照片，所用的培養基分別為：基礎MCDB131培養基(未處理對照組，UT)以及添加了已知血管新生因子(FGF2或VEGF)、對照組PEDF合成胜肽(25-mer或18-mer)或根據本發明實施例之PEDF合成胜肽(29-mer、24-mer、20-mer、Mo 29-mer或Mo 20-mer)的培養基。Figure 6 is a representative photograph of aortic ring cultures after four days of culture in different media. The media used were: basic MCDB131 medium (untreated control, UT) and the addition of known angiogenic factors (FGF2 or VEGF), control PEDF synthetic peptide (25-mer or 18-mer) or PEDF synthetic peptide according to embodiments of the invention (29-mer, 24-mer, 20-mer, Mo 29-mer or Mo 20- Mer) medium.

第7圖為經過不同培養基培養四天後之主動脈環培植體的代表性雙重免疫染色照片，以觀察在添加了不同PEDF合成胜肽(29-mer、20-mer或18-mer)的培養基中，由主動脈環向外長出脈管平滑肌細胞(vSMCs)的情形。照片中內皮細胞係以Alexa Fluor 594-標記之凝集素B4(IB4；紅色，左方)標記，而vSMCs則以抗-α-SMA(綠色；中間)標記。右方為合併圖式(黃色)。以Hoechst 33258來染色細胞核。原始倍率：×400。所示照片為四次獨立實驗的代表性照片。Figure 7 is a representative double immunostained photograph of aortic ring cultures after four days of culture in different media to observe media supplemented with different PEDF synthetic peptides (29-mer, 20-mer or 18-mer). In the case where vascular smooth muscle cells (vSMCs) grow outward from the aortic annulus. In the photograph, the endothelial cell line was labeled with Alexa Fluor 594-labeled lectin B4 (IB4; red, left), while vSMCs were labeled with anti-α-SMA (green; middle). The right side is the merged pattern (yellow). The nuclei were stained with Hoechst 33258. Original magnification: ×400. The photographs shown are representative photographs of four independent experiments.

第8圖為注射丁胍卡因後第14天之比目魚肌的代表性H&E染色照片。Figure 8 is a representative H&E staining of the soleus muscle on day 14 after the injection of dariccaine.

第9圖為折線圖，繪示了在不同實驗狀況下的肌肉之肌纖維直徑分布。Figure 9 is a line graph showing the distribution of muscle fiber diameters of muscles under different experimental conditions.

第10圖為受傷後3週之肌腱內部之再生組織(↑)的代表性照片。原始倍率：×100。Fig. 10 is a representative photograph of the regenerated tissue (↑) inside the tendon 3 weeks after the injury. Original magnification: ×100.

第11圖為受傷後3週阿基里斯(Achillis)肌腱的代表性H&E染色照片。原始倍率：×400；比例尺：50 μM。 所示照片為三次獨立實驗的代表性照片。Figure 11 is a representative H&E staining of the Achillis tendon 3 weeks after injury. Original magnification: ×400; scale bar: 50 μM. The photographs shown are representative photographs of three independent experiments.

第12圖為受傷後3週經曼森氏三色染劑染色以呈現膠原蛋白纖維之組織切片的代表性照片。星號(*)代表肌腱中未受傷的區域。原始倍率：×400；比例尺：50 μM。所示照片為三次獨立實驗的代表性照片。Figure 12 is a representative photograph of a tissue section of collagen fibers stained with Manson's trichrome dye 3 weeks after injury. The asterisk (*) represents the uninjured area of the tendon. Original magnification: ×400; scale bar: 50 μM. The photographs shown are representative photographs of three independent experiments.

第13圖為受傷後3週的第一型膠原蛋白(褐色)的代表性免疫染色照片。細胞核以蘇木色素標記。下方以較高倍率顯示方塊區域。比例尺：50 μM。所示照片為三次獨立實驗的代表性照片。Figure 13 is a representative immunostained photograph of type I collagen (brown) 3 weeks after injury. The nucleus is labeled with hematoxylin. The area of the square is displayed at a higher magnification below. Scale bar: 50 μM. The photographs shown are representative photographs of three independent experiments.

第14圖為根據本發明一實驗例的代表性電泳照片，其顯示此處提出的PEDF合成胜肽(29-mer與20-mer)可促進腱調蛋白(TNMD)基因的表現。腱調蛋白(TNMD)基因的表現顯示BM-MSC分化為肌腱細胞。所示照片為三次獨立實驗的代表性照片。Figure 14 is a representative electrophoresis photograph showing an example of an experimental example of the present invention showing that the PEDF synthetic peptide (29-mer and 20-mer) proposed herein can promote the expression of the hedgehog protein (TNMD) gene. The expression of the taumumodulin (TNMD) gene showed that BM-MSCs differentiated into tendon cells. The photographs shown are representative photographs of three independent experiments.

<110> 財團法人台灣基督長老教會馬偕紀念社會事業基金會馬偕紀念醫院<120> 色素上皮衍生因子衍生之多胜肽於促進肌肉或肌腱再生或動脈血管生成之用途<130> P2717-US <160> 14 <210> 1 <211> 39 <212> PRT <213> 人工序列<220> <223> 合成胜肽<400> 1<210> 2 <211> 34 <212> PRT <213> 人工序列<220> <223> 合成胜肽<400> 2<210> 3 <211> 29 <212> PRT <213> 人工序列<220> <223> 合成胜肽<400> 3<210> 4 <211> 25 <212> PRT <213> 人工序列<220> <223> 合成胜肽<400> 4<210> 5 <211> 24 <212> PRT <213> 人工序列<220> <223> 合成胜肽<400> 5<210> 6 <211> 20 <212> PRT <213> 人工序列<220> <223> 合成胜肽<400> 6<210> 7 <211> 18 <212> PRT <213> 人工序列<220> <223> 合成胜肽<400> 7<210> 8 <211> 29 <212> PRT <213> 人工序列<220> <223> 合成胜肽<400> 8<210> 9 <211> 20 <212> PRT <213> 人工序列<220> <223> 合成胜肽<400> 9<210> 10 <211> 20 <212> DNA <213> 人工序列<220> <223> Primer <400> 10<210> 11 <211> 20 <212> DNA <213> 人工序列<220> <223> Primer <400> 11<210> 12 <211> 20 <212> DNA <213> 人工序列<220> <223> Primer <400> 12<210> 13 <211> 20 <212> DNA <213> 人工序列<220> <223> Primer <400> 13<210> 14 <211> 418 <212> PRT <213> Homo sapiens <400> 14 <110> Taiwan Christ Presbyterian Church Stables Social Welfare Foundation Stables Hospital <120> Use of pigment epithelium-derived factor-derived peptides to promote muscle or tendon regeneration or arterial angiogenesis <130> P2717-US <160> 14 <210> 1 <211> 39 <212> PRT <213> Artificial sequence <220><223> Synthetic peptide <400> 1 <210> 2 <211> 34 <212> PRT <213> Artificial sequence <220><223> Synthetic peptide <400> 2 <210> 3 <211> 29 <212> PRT <213> Artificial sequence <220><223> Synthetic peptide <400> 3 <210> 4 <211> 25 <212> PRT <213> Artificial sequence <220><223> Synthetic peptide <400> 4 <210> 5 <211> 24 <212> PRT <213> Artificial sequence <220><223> Synthetic peptide <400> 5 <210> 6 <211> 20 <212> PRT <213> Artificial sequence <220><223> Synthetic peptide <400> 6 <210> 7 <211> 18 <212> PRT <213> Artificial sequence <220><223> Synthetic peptide <400> 7 <210> 8 <211> 29 <212> PRT <213> Artificial sequence <220><223> Synthetic peptide <400> 8 <210> 9 <211> 20 <212> PRT <213> Artificial sequence <220><223> Synthetic peptide <400> 9 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220><223> Primer <400> 10 <210> 11 <211> 20 <212> DNA <213> Artificial sequence <220><223> Primer <400> 11 <210> 12 <211> 20 <212> DNA <213> Artificial sequence <220><223> Primer <400> 12 <210> 13 <211> 20 <212> DNA <213> Artificial sequence <220><223> Primer <400> 13 <210> 14 <211> 418 <212> PRT <213> Homo sapiens <400> 14

Claims (2)

一種合成胜肽之用途，其係用以製備可促進一個體體內肌肉再生、肌腱再生或血管動脈生成之藥學組合物，該合成胜肽係由一長度為20-39個胺基酸殘基的胺基酸序列所組成，其中該胺基酸序列包含至少20個連續胺基酸殘基，其與序列編號：1的第11-30個胺基酸殘基具有至少95%的胺基酸序列相似度，且至少四個連續胺基酸殘基與序列編號：1的第11-14個胺基酸殘基相同。 A use of a synthetic peptide for the preparation of a pharmaceutical composition for promoting muscle regeneration, tendon regeneration or vascular arteriogenesis in a body, the synthetic peptide being composed of a length of 20-39 amino acid residues Amino acid sequence consisting of the amino acid sequence comprising at least 20 contiguous amino acid residues having at least 95% amino acid sequence with the 11-30 amino acid residues of SEQ ID NO: 1. Similarity, and at least four consecutive amino acid residues are identical to the 11-14 amino acid residues of SEQ ID NO: 1. 如請求項1所述的合成胜肽之用途，其中該合成胜肽的該胺基酸序列為序列編號：1(39-mer)、序列編號：2(34-mer)、序列編號：3(29-mer)、序列編號：5(24-mer)或序列編號：6(20-mer)所示的胺基酸序列。The use of the synthetic peptide according to claim 1, wherein the amino acid sequence of the synthetic peptide is SEQ ID NO: 1 (39-mer), SEQ ID NO: 2 (34-mer), SEQ ID NO: 3 ( 29-mer), SEQ ID NO: 5 (24-mer) or SEQ ID NO: 6 (20-mer) amino acid sequence.
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US20090069241A1 (en) * 2006-02-15 2009-03-12 Yale University Compositions and Methods for Use of Pigment Epithelial Derived Factor (PEDF) Peptide Fragments
US20100047212A1 (en) * 2006-02-17 2010-02-25 Universitat De Valencia Estudi General Use of the pedf factor to induce cell regeneration
TW201309724A (en) * 2011-03-23 2013-03-01 Mackay Memorial Hospital Use of PEDF-derived polypeptides for promoting stem cells proliferation and wound healing

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US20090069241A1 (en) * 2006-02-15 2009-03-12 Yale University Compositions and Methods for Use of Pigment Epithelial Derived Factor (PEDF) Peptide Fragments
US20100047212A1 (en) * 2006-02-17 2010-02-25 Universitat De Valencia Estudi General Use of the pedf factor to induce cell regeneration
TW201309724A (en) * 2011-03-23 2013-03-01 Mackay Memorial Hospital Use of PEDF-derived polypeptides for promoting stem cells proliferation and wound healing

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