TWI418629B - Transgenic animal with monocyte chemotactic protein 1 promoter - Google Patents
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本發明提供一種轉殖基因動物，其在單核細胞趨化蛋白1(MCP-1)啟動子之控制下表現一或多種報導蛋白，並提供其應用。The present invention provides a transgenic animal that exhibits one or more reporter proteins under the control of the monocyte chemoattractant protein 1 (MCP-1) promoter and provides its use.
發炎是一種對抗細胞或組織之損傷或感染所產生的局部或全身性之保護反應。然而，發炎會因發炎性細胞激素之失衡或是作用細胞間之交互作用而導致諸多疾病。主要的發炎性疾病包括：鼻炎及鼻竇炎，諸如，感染性鼻炎、過敏性鼻炎、慢性鼻炎、急性鼻竇炎、及慢性鼻竇炎；肺炎，諸如，細菌性肺炎、支氣管肺炎、大葉性肺炎、退伍軍人症肺炎、及病毒性肺炎；中耳炎，諸如，急性化膿性中耳炎及慢性化膿性中耳炎；急性或慢性胃炎；腸炎，諸如，感染性腸炎、克隆氏病、特發性潰瘍性大腸炎、及偽膜性大腸炎；關節炎，諸如，敗血性關節炎、結核性關節炎、退化性關節炎、及類風濕性關節炎；以及糖尿病性眼疾。Inflammation is a local or systemic protective response to damage or infection from cells or tissues. However, inflammation can lead to many diseases due to the imbalance of inflammatory cytokines or the interaction between cells. The main inflammatory diseases include: rhinitis and sinusitis, such as infectious rhinitis, allergic rhinitis, chronic rhinitis, acute sinusitis, and chronic sinusitis; pneumonia, such as bacterial pneumonia, bronchial pneumonia, lobar pneumonia, retiring Military pneumonia, and viral pneumonia; otitis media, such as acute suppurative otitis media and chronic suppurative otitis media; acute or chronic gastritis; enteritis, such as infectious enteritis, Crohn's disease, idiopathic ulcerative colitis, and pseudomembrane Crohn's disease; arthritis, such as septic arthritis, tuberculous arthritis, degenerative arthritis, and rheumatoid arthritis; and diabetic eye disease.
MCP-1，亦稱為趨化激素(C-C基序)配位體2(CCL2)，是一種屬於CC趨化激素家族之小型細胞激素。MCP-1可聚集單核細胞以及淋巴細胞亞群(Mol Med Today (1996) 2:198-204;J Leukoc Biol (1999) 65:482-491)。在先前的研究中，曾在肺類肉瘤症病患之支氣管肺泡沖洗液(BALF)及血清中偵測到高含量之MCP-1(Internal Med (1997) 36:856-860;Clin Exp Immunol (1998) 111:604-610;Eur Res J (2002) 20:1206-1212;Res Med (2004) 98:945-951)，且提出MCP-1是主導肉芽腫進程之重要中介因子。MCP-1亦已知在諸多疾病之致病機制中扮演重要角色，諸如，氣喘、間質性肺炎、及腫瘤。然而，大部分的研究皆是以試管內之實驗進行。MCP-1, also known as chemokine (CC motif) ligand 2 (CCL2), is a small cytokine belonging to the CC chemokine family. MCP-1 aggregates monocytes as well as lymphocyte subsets ( Mol Med Today (1996) 2: 198-204; J Leukoc Biol (1999) 65: 482-491). In previous studies, high levels of MCP-1 were detected in bronchoalveolar lavage fluid (BALF) and serum in patients with pulmonary sarcoma ( Internal Med (1997) 36:856-860; Clin Exp Immunol ( 1998) 111:604-610; Eur Res J (2002) 20:1206-1212; Res Med (2004) 98:945-951), and suggested that MCP-1 is an important mediator of the dominant granuloma process. MCP-1 is also known to play an important role in the pathogenesis of many diseases, such as asthma, interstitial pneumonia, and tumors. However, most of the research was conducted in vitro.
因此，需要研發出一種動物模式，以研究MCP-1之體內表現，並進一步篩選可調控MCP-1表現之作用劑或可治療發炎病症之作用，特別是治療其中MCP-1扮演關鍵角色之發炎病症。Therefore, there is a need to develop an animal model to study the in vivo performance of MCP-1 and to further screen for agents that modulate the performance of MCP-1 or to treat inflammatory conditions, particularly in the treatment of inflammation in which MCP-1 plays a key role. Illness.
在一方面，本發明提供一種轉殖基因動物，其在基因組中包含重組核酸分子，該重組核酸分子包含編碼一或多種報導蛋白之聚核苷酸，以及單核細胞趨化蛋白-1(MCP-1)啟動子，其中該一或多種報導蛋白是在該MCP-1啟動子之控制下表現。特定言之，該一或多種報導蛋白是選自以下所組成之群：可由螢光顯影偵測之報導蛋白、可由生物發光顯影偵測之報導蛋白、可由核顯影偵測之報導蛋白、及其任何組合。在本發明之實例中，該報導蛋白是EGFP、冷光酶(luciferase)、單純皰疹病毒第一型胸苷激酶(HSV1-TK)之融合蛋白。In one aspect, the invention provides a transgenic animal comprising a recombinant nucleic acid molecule comprising a polynucleotide encoding one or more reporter proteins, and monocyte chemoattractant protein-1 (MCP) in the genome -1) A promoter, wherein the one or more reporter proteins are expressed under the control of the MCP-1 promoter. Specifically, the one or more reporter proteins are selected from the group consisting of: a reporter protein detectable by fluorescence development, a reporter protein detectable by bioluminescence, a reporter protein detectable by nuclear development, and Any combination. In an embodiment of the invention, the reporter protein is a fusion protein of EGFP, luciferase, herpes simplex virus type 1 thymidine kinase (HSV1-TK).
在另一方面，本發明提供一種用於在體內監測MCP-1之內源性表現的方法，其包含：In another aspect, the invention provides a method for monitoring endogenous performance of MCP-1 in vivo, comprising:
(a)提供一種轉殖基因動物，其在基因組中包含重組核酸分子，該重組核酸分子包含編碼一或多種報導蛋白之聚核苷酸，以及單核細胞趨化蛋白-1(MCP-1)啟動子，其中該一或多種報導蛋白是在該MCP-1啟動子之控制下表現，以及(a) Providing a transgenic animal comprising a recombinant nucleic acid molecule comprising a polynucleotide encoding one or more reporter proteins, and monocyte chemoattractant protein-1 (MCP-1) in the genome a promoter, wherein the one or more reporter proteins are expressed under the control of the MCP-1 promoter, and
(b)在該轉殖基因動物中偵測該一或多種報導蛋白之存在或含量，做為內源性MCP-1表現之指標。(b) detecting the presence or amount of the one or more reporter proteins in the transgenic animal as an indicator of endogenous MCP-1 performance.
在本發明之一具體實例中，該方法可用於辨識候選作用劑是否可在哺乳動物體內調控MCP-1表現；其中如該一或多種報導蛋白之含量在投予該候選作用劑後降低，則表示該候選作用劑是MCP-1表現之抑制劑；如該一或多種報導蛋白之含量在投予該候選作用劑後增加，則表示該候選作用劑是MCP-1表現之誘發劑。In one embodiment of the invention, the method can be used to identify whether a candidate agent can modulate MCP-1 expression in a mammal; wherein, if the amount of the one or more reporter proteins is decreased following administration of the candidate agent, Indicates that the candidate agent is an inhibitor of MCP-1 expression; if the content of the one or more reporter proteins increases after administration of the candidate agent, it indicates that the candidate agent is an inducer of MCP-1 expression.
在本發明之另一具體實例中，該方法可用於決定候選作用劑是否為抗發炎劑；其中如該一或多種報導蛋白之含量在投予該候選作用劑後降低，則表示該候選作用劑為抗發炎劑。In another embodiment of the present invention, the method can be used to determine whether a candidate agent is an anti-inflammatory agent; wherein, if the content of the one or more reporter proteins is decreased after administration of the candidate agent, the candidate agent is indicated It is an anti-inflammatory agent.
以下詳述本發明之各種具體實例。本發明之其他特徵將可由下文有關此等具體實例之詳細說明及圖式以及申請專利範圍而清楚地呈現。Various specific examples of the invention are detailed below. Other features of the present invention will be apparent from the following detailed description of the embodiments and the appended claims.
在不須進一步說明之情形下，相信一般熟習本發明所屬技藝者根據本文之敘述可將本發明應用至其最廣範圍。因此，下文之說明應僅被視為具有例示目的，而非以任何方式限制本發明之範圍。Without further elaboration, it is believed that the present invention may be applied to the broadest scope of the invention. Accordingly, the descriptions below are to be considered as illustrative only and are not intended to limit the scope of the invention.
本發明提供一種新穎的轉殖基因動物，其特徵為重組核酸分子，該核酸分子帶有編碼一或多種報導蛋白之聚核苷酸，以及MCP-1啟動子，其中該一或多種報導蛋白是在該MCP-1啟動子之控制下表現。該轉殖基因動物可作為動物模式，俾以基於由該(等)報導蛋白所產生之信號而監測MCP-1之體內表現，且特定言之，俾以篩選可調控MCP-1表現且因而可有效作為抗發炎劑之試劑，特別是針對與MCP-1相關之發炎。The present invention provides a novel transgenic animal characterized by a recombinant nucleic acid molecule having a polynucleotide encoding one or more reporter proteins, and a MCP-1 promoter, wherein the one or more reporter proteins are Performed under the control of the MCP-1 promoter. The transgenic animal can be used as an animal model to monitor the in vivo performance of MCP-1 based on signals generated by the (etc.) reporter protein, and in particular, to screen for modulate MCP-1 expression and thus Effective as an anti-inflammatory agent, especially for inflammation associated with MCP-1.
除非另外定義，本文中所用之所有技術及科學辭彙具有熟習本文所屬技藝者所通常明瞭之相同意義。在本文中，除非另有明示，下列之術語具有其所述意義。All technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art, unless otherwise defined. In this document, the following terms have their meanings unless otherwise indicated.
在本文中，冠詞「一」係指一個或一個以上(亦即，至少一個)該冠詞語法上之受詞。舉例而言，「一元件」意謂一個元件或一個以上之元件。In the present context, the article "a" refers to one or more (ie, at least one) of the words of the lexical grammar. For example, "a component" means one element or more than one element.
本文所用之術語「聚核苷酸」、「核酸」、「核酸分子」係指由核苷酸單元組成之聚合物，包括天然存在之核酸，諸如，去氧核糖核酸(DNA)及核糖核酸(RNA)，以及核酸類似物，包括具有非天然存在之核苷酸者。聚核苷酸可經合成，例如，使用自動化DNA合成儀而製備。術語「核酸」或「核酸分子」一般而言係指大型之聚核苷酸。可明瞭的是，當核酸片段以DNA序列表示時(亦即，A、T、G、C)，其亦包括其中以「U」取代「T」之RNA序列(亦即，A、U、G、C)。The terms "polynucleotide", "nucleic acid", and "nucleic acid molecule" as used herein, refer to a polymer composed of nucleotide units, including naturally occurring nucleic acids, such as deoxyribonucleic acid (DNA) and ribonucleic acid ( RNA), as well as nucleic acid analogs, including those having non-naturally occurring nucleotides. Polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" or "nucleic acid molecule" generally refers to a large polynucleotide. It will be understood that when the nucleic acid fragment is represented by a DNA sequence (ie, A, T, G, C), it also includes an RNA sequence in which "U" is substituted for "T" (ie, A, U, G). , C).
本文所用之術語「重組」係指具有非天然連結之序列的聚核苷酸、核酸、或核酸分子。重組核酸分子可以構築體之形式表示。本文所用之術語「構築體」可包括一或多種指定的核苷酸序列，以及用以表現該(等)指定核苷酸序列之調控序列，諸如啟動子序列。構築體可用以表現該指定的核苷酸序列或是維持該指定的核苷酸序列以對其進行複製、操縱、或是使其在不同之位置間傳遞(如，在不同之生物體間)。構築體可被引入適當之宿主細胞中以完成上述目的。The term "recombinant" as used herein, refers to a polynucleotide, nucleic acid, or nucleic acid molecule having a sequence that is not naturally linked. The recombinant nucleic acid molecule can be expressed in the form of a construct. The term "construct" as used herein may include one or more specified nucleotide sequences, as well as regulatory sequences, such as promoter sequences, to represent the (or) designated nucleotide sequence. The construct can be used to represent the specified nucleotide sequence or to maintain the specified nucleotide sequence for replication, manipulation, or transfer between different locations (eg, between different organisms) . The construct can be introduced into a suitable host cell to accomplish the above objectives.
構築體之實例包括，但不限於，質體、黏粒、噬菌體、YAC或PAC。一般而言，在構築體中，指定的核苷酸序列係以可操作之方式連接至調控序列，使得當構築體被引入宿主細胞中時，指定的核苷酸序列可在調控序列之控制下而在宿主細胞中表現。調控序列可包含，例如且不限於，啟動子序列、起始密碼子、複製起點、增強子、操作子序列、分泌信號序列、及其他控制序列(如，Shine-Dalgano序列及終止序列)。Examples of constructs include, but are not limited to, plastids, cosmids, phage, YAC or PAC. Generally, in a construct, a specified nucleotide sequence is operably linked to a regulatory sequence such that when the construct is introduced into a host cell, the designated nucleotide sequence is under the control of the regulatory sequence It is expressed in host cells. Regulatory sequences can include, for example and without limitation, promoter sequences, initiation codons, origins of replication, enhancers, operator sequences, secretion signal sequences, and other control sequences (e.g., Shine-Dalgano sequences and termination sequences).
本文所用之術語「編碼」係指聚核苷酸(如，基因、cDNA、或mRNA)中特定的核苷酸序列之固有特性，使其可作為模板以在生物過程中合成出其他具有指定核苷酸序列(亦即，rRNA、tRNA、及mRNA)或是指定胺基酸序列及由其所產生的生物特性的聚合物及巨分子。因此，如由一基因所產生之mRNA的轉錄及轉譯作用在細胞或其他生物系統中產生了蛋白，表示該基因編碼該蛋白。熟習技藝者可明瞭，不同的聚核苷酸及核酸可因遺傳密碼之簡併性而編碼相同之多肽。亦明瞭的是，熟習技藝者可使用常規技術進行核苷酸取代，而不影響該聚核苷酸所編碼之多肽序列，以反映出要表現該多肽的任何特定宿主生物的密碼子使用偏好。因此，除非另有明示，「編碼一胺基酸序列之核苷酸序列」或其類似用語包括所有互為彼此之簡併版本且編碼相同胺基酸序列的核苷酸序列。編碼蛋白及RNA之核苷酸序列亦可包括內含子。The term "coding" as used herein, refers to the intrinsic property of a particular nucleotide sequence in a polynucleotide (eg, gene, cDNA, or mRNA) that allows it to serve as a template for the synthesis of other designated cores in a biological process. The nucleotide sequence (i.e., rRNA, tRNA, and mRNA) is either a polymer or a macromolecule that specifies the amino acid sequence and the biological properties produced therefrom. Thus, transcription and translation of mRNA, as produced by a gene, produces a protein in a cell or other biological system, indicating that the gene encodes the protein. It will be apparent to those skilled in the art that different polynucleotides and nucleic acids can encode the same polypeptide due to the degeneracy of the genetic code. It will also be apparent to those skilled in the art that nucleotide substitutions can be made using conventional techniques without affecting the polypeptide sequence encoded by the polynucleotide to reflect the codon usage preferences of any particular host organism in which the polypeptide is to be expressed. Thus, unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" or the like includes all nucleotide sequences which are degenerate versions of each other and which encode the same amino acid sequence. The nucleotide sequence encoding the protein and RNA may also include an intron.
本文所用之術語「啟動子」意指可調控與啟動子以可操作之方式連結之特定基因的表現之DNA序列，其可在細胞中達成該特定基因之轉錄作用。啟動子含有特定之DNA序列(反應元件)，其可為RNA聚合酶及轉錄因子提供結合位點以進行轉錄作用。一般而言，啟動子是位於接近其所調控基因之相同股及上游，即朝向有義股(sense strand)之5'區域。因此，在本文中，「MCP-1啟動子」意指基因MCP-1之啟動子。MCP-1啟動子之序列係為此技藝中所熟知，參見，例如，Biochem. Biophys. Res. Commun .169 (1990),pp. 346-351。The term "promoter" as used herein, refers to a DNA sequence that modulates the expression of a particular gene that is operably linked to a promoter, which can effect transcription of that particular gene in a cell. The promoter contains a specific DNA sequence (reaction element) which provides a binding site for RNA polymerase and transcription factors for transcription. In general, the promoter is located in the same strand and upstream of the gene it is regulating, i.e., towards the 5' region of the sense strand. Thus, as used herein, "MCP-1 promoter" means the promoter of the gene MCP-1. The sequence of the MCP-1 promoter is well known in the art, see, for example, Biochem. Biophys. Res. Commun . 169 (1990), pp. 346-351.
在本文中，報導蛋白是可在其表現時經由，例如，顏色或酶活性而可受到特異性偵測之任何蛋白。報導蛋白可用於評估負責該報導蛋白表現之調控序列的活性，諸如，啟動子序列。舉例而言，將編碼報導蛋白之核苷酸序列以可操作之方式連結天然存在之啟動子序列，可針對該啟動子序列對於不同刺激之活化反應進行活體內研究。在本發明中，該報導蛋白是作為標記，以顯示啟動子序列之活化。根據本發明，諸多此技藝中已知之報導蛋白皆可使用，其包括但不限於β-半乳糖苷酶、冷光酶、及鹼性磷酸酶，其可產生特定之可偵測性產物。螢光報導蛋白包括，例如，綠螢光蛋白(GFP)、藍螢光蛋白(CFP)、紅螢光蛋白(RFP)、及黃螢光蛋白(YFP)，以及其經修飾形式，如，強化型GFP(EGFP)、強化型CFP(ECFP)、強化型RFP(ERFP)、及強化型YEP(EYEP)。在本發明之實施例中，使用GFP作為報導蛋白，其螢光可在不添加受質之情形下，於接觸紫外光時進行觀察。此外，核顯影是一種在投予放射性追蹤物質後，藉著偵測身體不同部分之輻射而產生影像之方法；此等影像記錄在電腦底片上，使用諸如γ-相機、SPECT、或正電子發射斷層攝影(PET)。部分其他可以非侵襲性核顯影而偵測之報導蛋白亦可用於本發明，諸如，單純皰疹病毒第一型胸苷激酶(HSV1-TK)、正腎上腺素轉運蛋白(NET)、鈉碘同向轉運蛋白(NIS)。在本發明之實例中，針對HSV1-TK可使用放射性追蹤物質，諸如經放射性標示的非阿尿苷(fialuridine,FIAU)、2'-氟-5-乙基***呋喃基尿嘧啶(FEAU)、3-羥基甲基丁基鳥嘌呤(FHBG)、及噴昔洛弗(penciclovir,PCV)，針對NIS可使用經標示的碘及高鎝酸鹽(pertechnetate)，以及針對NET可使用經標示的間碘苄胍(meta-iodobenzyl-guanidine,MIBG)。As used herein, a reporter protein is any protein that can be specifically detected by its expression, for example, color or enzymatic activity. The reporter protein can be used to assess the activity of a regulatory sequence responsible for the expression of the reporter protein, such as a promoter sequence. For example, a nucleotide sequence encoding a reporter protein is operably linked to a naturally occurring promoter sequence for in vivo studies of activation responses to different stimuli for the promoter sequence. In the present invention, the reporter protein is used as a marker to show activation of the promoter sequence. In accordance with the present invention, a variety of reporter proteins known in the art can be used including, but not limited to, beta-galactosidase, luminescent enzymes, and alkaline phosphatase, which produce specific detectable products. Fluorescent reporter proteins include, for example, green fluorescent protein (GFP), blue fluorescent protein (CFP), red fluorescent protein (RFP), and yellow fluorescent protein (YFP), as well as modified forms thereof, such as enhanced GFP. (EGFP), enhanced CFP (ECFP), enhanced RFP (ERFP), and enhanced YEP (EYEP). In an embodiment of the invention, GFP is used as a reporter protein, and its fluorescence can be observed when exposed to ultraviolet light without adding a substrate. In addition, nuclear imaging is a method of generating images by detecting radiation from different parts of the body after administration of radioactive trace substances; such images are recorded on computer negatives using gamma-camera, SPECT, or positron emission. Tomography (PET). Some other reporter proteins detectable by non-invasive nuclear development may also be used in the present invention, such as herpes simplex virus type 1 thymidine kinase (HSV1-TK), norepinephrine transporter (NET), sodium iodine Transporter (NIS). In an example of the invention, radioactive tracing substances such as radiolabeled nonaluridine (FIAU), 2'-fluoro-5-ethyl arabinofuryl uracil (FEAU), can be used for HSV1-TK, 3-hydroxymethylbutylguanine (FHBG), and penciclovir (PCV), labeled iodine and pertechnetate for NIS, and labeled rooms for NET Meta-iodobenzyl-guanidine (MIBG).
本文所用之術語「轉殖基因動物」是指帶有經設計***其基因組中之外源基因物質(亦稱為轉殖基因)的動物。該轉殖基因可使用重組DNA方法而構築。轉殖基因動物之典型實例為鼠類，如，小鼠或大鼠。轉殖基因動物之產生可依諸多習知之方法進行，諸如描述於以下文獻之方法：Hogan等人，小鼠胚胎之操縱(Manipulating the Mouse Embryo)，Cold Spring Harbor Laboratory Press,1986；以及Kraemer等人，早期哺乳動物胚胎之基因操縱(Genetic Manipulation of the Early Mammalian Embryo)，Cold Spring harbor Laboratory Press,1985。The term "transgenic animal" as used herein refers to an animal having a genetic material (also referred to as a transgenic gene) that is designed to be inserted into its genome. The transgenic gene can be constructed using recombinant DNA methods. A typical example of a transgenic animal is a mouse, such as a mouse or a rat. The production of transgenic animals can be carried out according to a number of conventional methods, such as those described in Hogan et al., Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, 1986; and Kraemer et al. , Genetic Manipulation of the Early Mammalian Embryo, Cold Spring Harbor Laboratory Press, 1985.
在一方面，本發明提供一種轉殖基因動物，其在基因組中包含重組核酸分子，該重組核酸分子包含編碼一或多種報導蛋白之聚核苷酸，以及單核細胞趨化蛋白-1(MCP-1)啟動子，其中該一或多種報導蛋白是在該MCP-1啟動子之控制下表現。特定言之，該聚核苷酸是以可操作之方式連結至該MCP-1啟動子。根據本發明，該動物是非人類哺乳動物，其包括但不限於靈長類、有蹄類、犬、貓。在本發明之實例中，該動物是小鼠。In one aspect, the invention provides a transgenic animal comprising a recombinant nucleic acid molecule comprising a polynucleotide encoding one or more reporter proteins, and monocyte chemoattractant protein-1 (MCP) in the genome -1) A promoter, wherein the one or more reporter proteins are expressed under the control of the MCP-1 promoter. In particular, the polynucleotide is operably linked to the MCP-1 promoter. According to the invention, the animal is a non-human mammal including, but not limited to, primates, ungulates, dogs, cats. In an embodiment of the invention, the animal is a mouse.
根據本發明，MCP-1啟動子可衍生自人類或非人類哺乳動物。非人類哺乳動物包括，但不限於，靈長類、有蹄類、犬、貓、及鼠。在本發明之具體實例中，MCP啟動子具有SEQ ID NO:1之核苷酸序列。According to the invention, the MCP-1 promoter can be derived from a human or non-human mammal. Non-human mammals include, but are not limited to, primates, ungulates, dogs, cats, and rats. In a particular embodiment of the invention, the MCP promoter has the nucleotide sequence of SEQ ID NO: 1.
根據本發明，該聚核苷酸可編碼一或多種報導蛋白，其係選自該等技藝中已知者，或是其任何組合。特定言之，該根據本發明之一或多種報導蛋白是選自以下所組成之群：可由螢光顯影偵測之報導蛋白、可由生物發光顯影偵測之報導蛋白、可由核顯影偵測之報導蛋白、及其任何組合。一般而言，可由螢光顯影偵測之報導蛋白可為GFP、CFP、RFP、或YFP；可由生物發光顯影偵測之報導蛋白可為冷光酶(螢火蟲冷光酶或水母冷光酶)；以及可由核顯影偵測之報導蛋白可為HSV1-TK、NIS、或NET。According to the present invention, the polynucleotide may encode one or more reporter proteins selected from those known in the art, or any combination thereof. In particular, the reporter protein according to one or more of the present invention is selected from the group consisting of: a reporter protein detectable by fluorescence development, a reporter protein detectable by bioluminescence, and a report detectable by nuclear development. Protein, and any combination thereof. In general, the reporter protein detectable by fluorescence development may be GFP, CFP, RFP, or YFP; the reporter protein detectable by bioluminescence development may be a luminescent enzyme (firefly luciferase or jellyfish luminescent enzyme); The reporter protein for development detection can be HSV1-TK, NIS, or NET.
在本發明之具體實例中，該聚核苷酸編碼單一報導蛋白，其係可由螢光顯影偵測之報導蛋白、可由生物發光顯影偵測之報導蛋白、或可由核顯影偵測之報導蛋白。在本發明之實施例中，該報導蛋白是EGFP，其係在MCP-1啟動子之控制下表現。在特定實施例中，該重組核酸分子包含SEQ ID NO:2之核苷酸序列，其構成為EGFP編碼序列，以可操作之方式連結至SEQ ID NO:1之核苷酸序列(亦即，MCP-1啟動子)並接續多聚A序列。In a particular embodiment of the invention, the polynucleotide encodes a single reporter protein, which is a reporter protein detectable by fluorescence imaging, a reporter protein detectable by bioluminescence, or a reporter protein detectable by nuclear development. In an embodiment of the invention, the reporter protein is EGFP, which is expressed under the control of the MCP-1 promoter. In a particular embodiment, the recombinant nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 2, which is constructed as an EGFP coding sequence operably linked to the nucleotide sequence of SEQ ID NO: 1 (ie, The MCP-1 promoter) is followed by a poly A sequence.
在本發明之另一具體實例中，該聚核苷酸是編碼二或多種報導蛋白之融合蛋白，該等報導蛋白是選自可由螢光顯影偵測之報導蛋白、可由生物發光顯影偵測之報導蛋白、及可由核顯影偵測之報導蛋白。在本發明之一實施例中，為了提供可用不同方式偵測之轉殖基因動物，使用了編碼可由螢光顯影偵測之報導蛋白、可由生物發光顯影偵測之報導蛋白、及可由核顯影偵測之報導蛋白的融合蛋白之聚核苷酸。特定言之，該聚核苷酸編碼EGFP、冷光酶、及HSV1-TK之融合蛋白。具體言之，該重組核酸分子包含前述三種報導蛋白的符合譯讀框(in-frame)編碼序列，其以可操作之方式連結SEQ ID NO:1之核苷酸序列。In another embodiment of the present invention, the polynucleotide is a fusion protein encoding two or more reporter proteins, which are selected from a reporter protein detectable by fluorescence development and detectable by bioluminescence development. Report proteins, and reporter proteins that can be detected by nuclear development. In an embodiment of the present invention, in order to provide a transgenic animal that can be detected in different ways, a reporter protein encoding a fluorescently detectable protein, a reporter protein detectable by bioluminescence, and a nuclear imaging assay can be used. A polynucleotide encoding a fusion protein of a reporter protein. In particular, the polynucleotide encodes a fusion protein of EGFP, luminescent enzyme, and HSV1-TK. In particular, the recombinant nucleic acid molecule comprises an in-frame coding sequence of the aforementioned three reporter proteins operably linked to the nucleotide sequence of SEQ ID NO: 1.
可明瞭的是，可根據常用或已知之技術及知識，取得一或多種報導蛋白以及MCP-1啟動子之核苷酸序列，以製備本發明之重組核酸分子。可使用任何基因工程技術以製備本發明之重組核酸分子，諸如，聚核苷酸合成、聚合酶連鎖反應(PCR)、選殖、核酸純化、載體構築、以及載體與核酸片段之酶處理及定序，參見，例如，Sambrook等人，分子選殖：實驗室手冊(Molecular Cloning:A Laboratory Manual)，第二版，Cold Spring Harbor Laboratory Press(1989)，以及Frederick M.A.等人，分子生物學之現代操作流程(Current Protocols in Molecular Biology)，John Wiley ＆ Sons,Inc.(2001)。有關製備本發明重組聚核苷酸之細節述於下文之實施例中。It will be appreciated that one or more reporter proteins and the nucleotide sequence of the MCP-1 promoter can be obtained according to commonly used or known techniques and knowledge to prepare recombinant nucleic acid molecules of the invention. Any of the genetic engineering techniques can be used to prepare recombinant nucleic acid molecules of the invention, such as, for example, polynucleotide synthesis, polymerase chain reaction (PCR), colonization, nucleic acid purification, vector construction, and enzymatic treatment of vectors and nucleic acid fragments. For example, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), and Frederick MA et al., Modern Molecular Biology Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (2001). Details regarding the preparation of the recombinant polynucleotides of the present invention are described in the Examples below.
亦可明瞭的是，任何熟習技藝者皆可根據本文所提供之教示，使用已知的標準技術，諸如上述者，製備本發明之轉殖基因動物。舉例而言，可將本發明之重組聚核苷酸直接注射進入受精卵之雄原核中，並接著將注射後之受精卵重新移置至假懷孕受體母體之子宮中，而所得之子代將會具有一或多份複製數之嵌入基因組的轉殖基因。接著繁殖此等「種源(founder)」動物以建立轉殖基因品系，再進行回交而產生具有所選擇的基因背景之動物。轉殖基因動物可藉由以下方式予以辨識，例如，以特定引子進行PCR而偵測該轉殖基因之存在，或是以特定探針進行南方點漬分析，較佳為使用尾DNA之南方點漬分析。It will also be apparent that any skilled artisan can prepare a transgenic animal of the present invention using known standard techniques, such as those described above, in accordance with the teachings provided herein. For example, the recombinant polynucleotide of the present invention can be directly injected into the male pronucleus of the fertilized egg, and then the fertilized egg after injection can be relocated to the uterus of the pseudopregnant recipient parent, and the resulting progeny will be A transgenic gene with one or more copies of the embedded genome. These "founder" animals are then propagated to establish a transgenic gene line, which is then backcrossed to produce an animal with the selected genetic background. The transgenic animal can be identified by, for example, PCR for specific primers to detect the presence of the transgene, or Southern blotting with a specific probe, preferably using the southern point of the tail DNA. Stain analysis.
根據本發明，由MCP-1啟動子所驅動之一或多種報導基因的表現可模擬體內之內源性MCP-1表現。如下列實例所示，由人類MCP-1啟動子所驅動之EGFP表現與本發明轉殖基因動物之內源性MCP-1表現有相關性。因此，本發明之轉殖基因動物可用於監測體內之內源性MCP-1表現，其有助於對MCP-1在各種不同疾病中所扮演之角色獲得更好的理解。同時，本發明之轉殖基因動物可用於辨識能夠調控MCP-1表現之作用劑或是可治療發炎病症之作用劑，特別是治療其中MCP-1扮演關鍵角色之發炎病症。According to the present invention, the expression of one or more reporter genes driven by the MCP-1 promoter mimics endogenous MCP-1 expression in vivo. As shown in the examples below, the EGFP expression driven by the human MCP-1 promoter correlates with the endogenous MCP-1 expression of the transgenic animals of the present invention. Thus, the transgenic animal of the present invention can be used to monitor endogenous MCP-1 expression in vivo, which contributes to a better understanding of the role of MCP-1 in various diseases. Meanwhile, the transgenic animal of the present invention can be used to identify an agent capable of regulating the expression of MCP-1 or an agent capable of treating an inflammatory condition, particularly an inflammatory condition in which MCP-1 plays a key role.
因此，本發明尚提供一種用於在體內監測MCP-1之內源性表現的方法，其包含：Accordingly, the present invention still provides a method for monitoring the endogenous performance of MCP-1 in vivo, comprising:
(a)提供一種轉殖基因動物，該動物在基因組中包含重組核酸分子，該重組核酸分子包含編碼一或多種報導蛋白之聚核苷酸，以及單核細胞趨化蛋白-1(MCP-1)啟動子，其中該一或多種報導蛋白是在該MCP-1啟動子之控制下表現，以及(a) Providing a transgenic animal comprising a recombinant nucleic acid molecule comprising a polynucleotide encoding one or more reporter proteins, and monocyte chemoattractant protein-1 (MCP-1) in the genome a promoter, wherein the one or more reporter proteins are expressed under the control of the MCP-1 promoter, and
(b)在該轉殖基因動物中偵測該一或多種報導蛋白之存在或含量，做為內源性MCP-1表現之指標。(b) detecting the presence or amount of the one or more reporter proteins in the transgenic animal as an indicator of endogenous MCP-1 performance.
可明瞭的是，報導蛋白之偵測可經由技藝中已知之各種方法達成，包括，但不限於，比色性、螢光測定性、發光測定性之分析、或是核顯影，可根據所用報導基因之性質而決定。It will be appreciated that detection of reporter proteins can be accomplished by a variety of methods known in the art, including, but not limited to, colorimetric, fluorometric, luminescence assays, or nuclear development, depending on the report used. The nature of the gene is determined.
在本發明之實施例中，該聚核苷酸編碼單一報導蛋白，諸如EGFP；且在步驟(b)中是使用螢光顯影以偵測EGFP。具體言之，在步驟(b)中，取得來自轉殖基因動物之新鮮切除組織，並在具有GFP濾鏡之螢光實體顯微鏡下觀察，以偵測該報導蛋白之表現，其係轉殖基因動物之內源性MCP-1表現之指標。In an embodiment of the invention, the polynucleotide encodes a single reporter protein, such as EGFP; and in step (b), fluorescence development is used to detect EGFP. Specifically, in step (b), fresh excised tissue from the transgenic animal is obtained and observed under a fluorescent solid microscope with a GFP filter to detect the expression of the reporter protein, which is a transgenic gene. An indicator of endogenous MCP-1 performance in animals.
在本發明之另一實施例中，該聚核苷酸編碼三種報導蛋白之融合蛋白，諸如，EGFP、冷光酶、及HSV1-TK之融合蛋白；且在步驟(b)中是使用螢光顯影以偵測EGFP；使用生物發光顯影以偵測冷光酶、及/或使用核顯影以偵測HSV1-TK。在本發明之實施例中，在步驟(b)中，為進行核顯影，投予放射性追蹤物質，並使用例如γ-相機、SPECT、或正電子發射斷層攝影(PET)，非侵襲性之方式，偵測所產生之影像。可明瞭的是，此等多模態顯影系統可對活體動物之細胞或組織中的基因表現進行有效及有彈性的分析。In another embodiment of the invention, the polynucleotide encodes a fusion protein of three reporter proteins, such as EGFP, a luminescent enzyme, and a fusion protein of HSV1-TK; and in step (b), a fluorescent development is used. To detect EGFP; use bioluminescence to detect luminescent enzymes, and/or use nuclear development to detect HSV1-TK. In an embodiment of the invention, in step (b), a radioactive tracking substance is administered for nuclear development, and using, for example, gamma-camera, SPECT, or positron emission tomography (PET), non-invasive means , detecting the image produced. It will be appreciated that such multimodal development systems provide an efficient and flexible analysis of gene expression in cells or tissues of living animals.
在本發明之具體實例中，所述方法可用於決定候選作用劑是否可在哺乳動物體內調控MCP-1表現；其中如報導蛋白之含量在投予該候選作用劑後降低，則表示該候選作用劑是MCP-1表現之抑制劑；而如報導蛋白之含量在投予該候選作用劑後增加，則表示該候選作用劑是MCP-1表現之誘發劑。In a specific embodiment of the invention, the method can be used to determine whether a candidate agent can modulate MCP-1 expression in a mammal; wherein, if the content of the reporter protein is decreased after administration of the candidate agent, the candidate effect is indicated The agent is an inhibitor of MCP-1 expression; and if the content of the reporter protein is increased after administration of the candidate agent, it indicates that the candidate agent is an inducer of MCP-1 expression.
在本發明之另一具體實例中，所述方法可用於決定候選作用劑是否為抗發炎劑；其中如該報導蛋白之含量在投予該候選作用劑後降低，則表示該候選作用劑為抗發炎劑。In another embodiment of the present invention, the method can be used to determine whether a candidate agent is an anti-inflammatory agent; wherein if the content of the reporter protein is decreased after administration of the candidate agent, it indicates that the candidate agent is resistant Inflammatory agent.
本文所用之術語「抗發炎劑」是指具有可抑制、降低、消除、治療、或改善由發炎病症所造成之發炎反應的特徵之作用劑。特定言之，該發炎病症與MCP-1相關，包括但不限於，關節炎、氣喘、動脈粥狀硬化、神經退化、肥胖誘發性脂肪組織發炎、胰島素抗性、腎炎、狼瘡、自體免疫淋巴增生性、腫瘤溶瘤性作用、肺肉芽腫、以及自體免疫疾病。在本發明之實施例中，該發炎病症是肺部之發炎病症，諸如，肺肉芽腫發炎。The term "anti-inflammatory agent" as used herein refers to an agent having the characteristics of inhibiting, reducing, eliminating, treating, or ameliorating the inflammatory response caused by an inflammatory condition. In particular, the inflammatory condition is associated with MCP-1, including but not limited to, arthritis, asthma, atherosclerosis, neurodegeneration, obesity-induced adipose tissue inflammation, insulin resistance, nephritis, lupus, autoimmune lymph Proliferative, tumor oncolytic effects, pulmonary granuloma, and autoimmune diseases. In an embodiment of the invention, the inflammatory condition is an inflammatory condition of the lungs, such as inflammation of the pulmonary granuloma.
本發明現將參照下列之具體實例而進行更具體之敘述，該等具體實例係為說明而非限制之目的而提供。The invention will now be described more particularly with reference to the particular embodiments illustrated herein
如Biochem. Biophys. Res. Commun. 169 (1990),pp. 346-351所述，對MCP1-540-Luc載體進行Xho1及BamH1酶切消化，取得534bp之人類立即早期啟動子(SEQ ID NO:1)。接著在pEGFP-C2載體(Clonetech,USA)中，將該產物接合至EGFP編碼序列及SV 40多聚腺苷酸化序列之前，以產生編碼在MCP-1啟動子控制下之EGFP的轉殖基因載體，稱為MCP-1-EGFP轉殖基因載體。圖1顯示所得MCP-1-EGFP轉殖基因載體的結構圖譜。圖2顯示MCP-1立即早期啟動子在MCP-1-EGFP轉殖基因載體中之位置。The MCP1-540-Luc vector was digested with Xho1 and BamH1 as described in Biochem. Biophys. Res. Commun. 169 (1990), pp. 346-351, and a 534 bp human immediate early promoter (SEQ ID NO: 1). This product was then ligated into the EGFP coding sequence and the SV 40 polyadenylation sequence in the pEGFP-C2 vector (Clonetech, USA) to generate a transgenic gene vector encoding EGFP under the control of the MCP-1 promoter. It is called MCP-1-EGFP transgenic gene vector. Figure 1 shows the structural map of the resulting MCP-1-EGFP transgenic gene vector. Figure 2 shows the location of the MCP-1 immediate early promoter in the MCP-1-EGFP transgene vector.
此外，進行DNA定序分析，顯示MCP-1-EGFP轉殖基因載體包含SEQ ID NO:2之核苷酸序列，其構成為MCP-1啟動子、EGFP編碼序列、以及多聚A序列。Furthermore, DNA sequencing analysis was performed, showing that the MCP-1-EGFP transgene vector contains the nucleotide sequence of SEQ ID NO: 2, which is composed of the MCP-1 promoter, the EGFP coding sequence, and the poly A sequence.
使用取自實施例1之MCP-1-EGFP轉殖基因載體以產生MCP-1-EGFP轉殖基因小鼠。簡言之，提供FVB/NarL小鼠(MHC型2q )以製備轉殖基因小鼠。以ApaL1及Ssp1進行酶切消化，將純化之MCP1-EGFP-PolyA片段由該載體中切出，並注射至FVB小鼠胚胎中，根據描述於以下文獻之流程：Nagy等人(2003)小鼠胚胎操縱：實驗室手冊(Manipulating the mouse embryo:A laboratory manual)，第三版，Cold Spring Harbor Laboratory,Cold Spring Harbor,NY.，於進階基因轉殖中心(Level Transgenic Center)(台北，臺灣)進行。產生轉殖基因小鼠後，在標準之無特定病原設施條件下飼養。所有涉及使用小鼠之研究皆係根據國家衛生研究院(Institute of National Health Research Institutes)(臺灣)之IACUC所訂定之規定進行。The MCP-1-EGFP transgenic gene vector from Example 1 was used to generate MCP-1-EGFP transgenic mice. Briefly, there is provided FVB / NarL mice (MHC type 2 q) to prepare a gene transfected mouse colonization. The purified MCP1-EGFP-PolyA fragment was excised from the vector by ApaL1 and Ssp1, and injected into the FVB mouse embryo according to the procedure described in the following literature: Nagy et al. (2003) Manipulating the mouse embryo: A laboratory manual, third edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY., at the Advanced Transgenic Center (Taipei, Taiwan) get on. After the transgenic mouse is produced, it is raised under standard conditions without specific pathogenic facilities. All studies involving the use of mice were conducted in accordance with the regulations established by the IACUC of the Institute of National Health Research Institutes (Taiwan).
以南方點漬分析篩選所得轉殖基因小鼠，以確認導入了MCP-1-EGFP轉殖基因。使用Ncol對鼠尾DNA樣本進行酶切消化，接著以0.8%之瓊脂凝膠進行電泳。用於進行南方點漬分析之探針為EGFP編碼序列之752bp片段(SEQ ID NO:3)。The resulting transgenic mouse was screened by Southern blot analysis to confirm the introduction of the MCP-1-EGFP transgene. The rat tail DNA sample was digested with Ncol, followed by electrophoresis on a 0.8% agar gel. The probe used for Southern blot analysis was a 752 bp fragment of the EGFP coding sequence (SEQ ID NO: 3).
在所試驗之小鼠中，種源品系第4號(2008年9月3日出生)經確認帶有單一或低複製份數之轉殖基因，將來自於種源品系第4號之小鼠用於後續之研究中。於此，使用基因型鑑定PCR方法篩選轉殖基因陽性之仔畜。在代表性之實施例中，用於該基因型鑑定PCR法之引子如下：ACCGAGCTCGGATCCACTAGTAACG(作為位於人類MCP1啟動子中之有義引子；SEQ ID NO:4)及CTCACCATGGTGGCGACCGG(作為位於EGFP中之反義引子；SEQ ID NO:5)，且如圖5所示，仔畜m2、m3、m6、及f1帶有來自於種源品系第4號之轉殖基因。Among the mice tested, the provenance line No. 4 (born on September 3, 2008) was confirmed to have a single or low copy number of the transgenic gene, and the mouse from the provenance line No. 4 was used. In the follow-up study. Here, the genotype identification PCR method was used to screen the transgenic gene-positive litter. In a representative example, the primers used for the genotyping PCR method are as follows: ACCGAGCTCGGATCCACTAGTAACG (as a sense primer located in the human MCP1 promoter; SEQ ID NO: 4) and CTCACCATGGTGGCGACCGG (as antisense located in EGFP) Primer; SEQ ID NO: 5), and as shown in Figure 5, the animals m2, m3, m6, and f1 carry the transgenic gene from the provenance line No. 4.
在8至10週齡之MCP-1-EGFP轉殖基因小鼠體內，使用經修飾之流程(Am J Pathol (1995) 147:1001-1015;J Immunol (2002) 168:3004-3016)，以Sephadex(SDX) G-50超細微珠(Amersham Biosciences)之氣管內(i.t.)滴注，誘發異物肺肉芽腫發炎。簡言之，以磷酸緩衝鹽水(PBS,pH 7.4)清洗SDX微珠數次，進行高溫高壓滅菌，再以2.0×105 微珠/ml之濃度重新懸浮於PBS中。接著將50μl之SDX-PBS溶液以氣管內滴注至麻醉小鼠之肺中。假手術控制組小鼠則僅滴注50μl之PBS。在SDX或PBS滴注後之第1天、第3天、及第5天結束時犧牲經處理之小鼠，並犧牲未處理之小鼠作為陰性控制組。In a 10 to 10 week old MCP-1-EGFP transgenic mouse, a modified procedure ( Am J Pathol (1995) 147: 1001-1015; J Immunol (2002) 168: 3004-3016) was used to Intratracheal (it) instillation of Sephadex (SDX) G-50 ultrafine beads (Amersham Biosciences) induces inflammation of foreign body pulmonary granuloma. Briefly, phosphate-buffered saline (PBS, pH 7.4) washing several times the beads SDX, autoclaved, and then to 2.0 × 10 5 beads / ml concentration of resuspended in PBS. Next, 50 μl of the SDX-PBS solution was intratracheally instilled into the lungs of anesthetized mice. In the sham operation control group, only 50 μl of PBS was instilled. Treated mice were sacrificed on the 1st, 3rd, and 5th day after SDX or PBS instillation, and untreated mice were sacrificed as a negative control group.
以SPSS/7.5軟體對所有之分組數據進行統計評估。假設檢定法包括單因子變異數分析(ANOVA)接著進行最小顯著差異(LSD)檢定。將P <0.05視為統計顯著。所有之結果皆以各組4至5隻動物之平均值±SD.表示。Statistical evaluation of all packet data was performed using SPSS/7.5 software. The hypothesis assay included a one-way variance analysis (ANOVA) followed by a least significant difference (LSD) assay. P < 0.05 was considered statistically significant. All results are expressed as the mean ± SD of 4 to 5 animals in each group.
如先前所述方法(Clin Immunol Immunopathol (1994) 73:312-329)，經由氣管導管，以1ml之分裝液，將6ml之無菌PBS溫和滴注至肺中，接著將其抽出，以收集BALF，並在各個沖洗流程結束時，大致取回80%之滴注液。在除去紅血球後，使用血球計數器，計算沖洗液中之細胞數目。以細胞離心機製備細胞學玻片，並以經修飾之Wright-Giemsa染劑(Sigma)染色，以決定個別細胞類型之總數。As described previously ( Clin Immunol Immunopathol (1994) 73: 312-329), 6 ml of sterile PBS was gently instilled into the lungs via a tracheal tube in 1 ml, followed by extraction to collect BALF. And at the end of each flushing process, approximately 80% of the instillation is retrieved. After removing the red blood cells, the number of cells in the rinse solution was calculated using a hemocytometer. Cytology slides were prepared in a cell centrifuge and stained with modified Wright-Giemsa stain (Sigma) to determine the total number of individual cell types.
結果顯示，相較於未處理或注射PBS之控制組，SDX之滴注會造成在1及3天後回收之BALF中的總白血球計數大量增加。在第5天結束時，在PBS或SDX處理組中並未觀察到總細胞計數之顯著差異(圖6A)。可注意到，未經處理小鼠之BALF僅含有單核細胞但不含有粒細胞(圖6B及6C)。PBS之滴注僅在第1天結束時誘發嗜中性細胞數目之顯著增加，並在其後回復正常。在進行SDX滴注時，嗜中性細胞之數目在第1天時有最大量之增加，在第3天時仍維持高量，而在第5天後幾乎不存在(圖6B)。相對地，在第1天時觀察到單核細胞/巨噬細胞數目之增加，在第3天時到達最大量，而在其後漸減(圖6C)。The results showed that the SDX instillation resulted in a substantial increase in total white blood cell counts in BALF recovered after 1 and 3 days compared to the untreated or injected PBS control group. At the end of day 5, no significant difference in total cell count was observed in the PBS or SDX treated groups (Fig. 6A). It can be noted that the BALF of untreated mice contained only monocytes but no granulocytes (Figs. 6B and 6C). The instillation of PBS induced a significant increase in the number of neutrophils only at the end of day 1 and returned to normal thereafter. At the time of SDX instillation, the number of neutrophils increased the most in the first day, remained high on the third day, and hardly disappeared after the fifth day (Fig. 6B). In contrast, an increase in the number of monocytes/macrophages was observed on day 1, reaching the maximum amount on day 3 and decreasing gradually thereafter (Fig. 6C).
將肺固定在10%福馬林中，接著以石蠟包埋。以蘇木精及伊紅(H＆E)染色薄切片(4μm)，並予以檢驗以進行組織學評估。針對免疫組織化學分析，以檸檬酸鈉法修復抗原，藉由預置於3% H2 O2 溶液中而使內源性過氧化物酶活性淬滅。The lungs were fixed in 10% fumarin and then embedded in paraffin. Thin sections (4 μm) were stained with hematoxylin and eosin (H&E) and examined for histological evaluation. For immunohistochemical analysis, the antigen was repaired by the sodium citrate method, and the endogenous peroxidase activity was quenched by presetting in a 3% H 2 O 2 solution.
根據結果顯示，經SDX處理小鼠之肺的組織學檢驗有顯著之發炎性細胞浸潤，但未經處理或經PBS處理小鼠之肺中則無(數據未示)。在第1天時，明顯出現含嗜中性細胞浸潤的肉芽腫，到第3天時尺寸逐漸增加，接著逐漸減小。在第3天結束時，在微珠之周圍出現濃密之嗜中性細胞但僅有極少之巨噬細胞，在第5天時，在肉芽腫之周邊，SDX微珠則受到濃密之單核細胞浸潤但是極少之嗜中性細胞圍繞。According to the results, histological examination of the lungs of SDX-treated mice showed significant inflammatory cell infiltration, but not in the lungs of untreated or PBS-treated mice (data not shown). On day 1, granulomas containing neutrophil infiltration were apparently present, and gradually increased in size on day 3, and then gradually decreased. At the end of the third day, dense neutrophils appeared around the beads but only very few macrophages. On day 5, around the granuloma, the SDX beads were densely mononuclear cells. Infiltrated but very few neutrophils surround.
在經處理及未經處理之小鼠取得新鮮切除之肺組織，並在具有GFP濾鏡(激發480nm，發光510nm)之螢光實體顯微鏡(Olympus SZX10)下觀察。此外，在各個實驗結束時，輕微麻醉小鼠，切開橫隔膜及胸廓，並暴露心臟及肺。經由心內抽吸除去鼠血，分離氣管，再經由一個小切口置入窄鈍緣導管(20G)並綁住固定。使用結核菌素注射器，以1ml之分裝液，在室溫下，將6ml之無菌PBS輸液至肺中，並在各次輸液後溫和抽吸以吸出該液體，接著記錄其體積。在各個沖洗流程結束時大致取回80%之滴注液。離心(400g×10分鐘)以清洗所收集到之細胞；在含有FCS 10%、見大黴素(gentamicin,50μg/ml)、及麩醯胺酸(2mM)之RPMI 1640中清洗二或三次；並溫和震盪而重新懸浮於培養基中。以Tris緩衝性氯化銨裂解污染之紅血球，並使用血球計數器決定細胞數目。風乾BALF細胞(0.5至1.0×105 個細胞/玻片)之細胞離心製備物(1200rpm×2分鐘)，並以經修飾之Wright-Giemsa染劑(Sigma)染色，以決定個別細胞類型之總數。為偵測螢光，快速進行細胞收集，並使用螢光顯微鏡(Olympus BX51)觀察。Freshly excised lung tissue was obtained from treated and untreated mice and observed under a fluorescent solid microscope (Olympus SZX10) with a GFP filter (excitation 480 nm, luminescence 510 nm). In addition, at the end of each experiment, mice were lightly anesthetized, the diaphragm and thorax were dissected, and the heart and lungs were exposed. The rat blood was removed by intracardiac aspiration, the trachea was separated, and a narrow blunt edge catheter (20G) was placed through a small incision and tied for fixation. Using a tuberculin syringe, a 1 ml portion of the solution was filled, and 6 ml of sterile PBS was infused into the lungs at room temperature, and gently aspirated after each infusion to aspirate the liquid, and then the volume was recorded. At the end of each rinsing process, approximately 80% of the instillation was retrieved. Centrifuge (400 g × 10 minutes) to wash the collected cells; wash two or three times in RPMI 1640 containing FCS 10%, gentamicin (50 μg / ml), and glutamic acid (2 mM); It was resuspended in the medium with gentle shaking. The contaminated red blood cells are cleaved with Tris buffered ammonium chloride and the number of cells is determined using a hemocytometer. Air-dried cytospin preparations BALF cells (0.5 to 1.0 × 10 5 cells / slide) of (1200rpm × 2 min), and to the modified Wright-Giemsa stain (Sigma) staining to determine the total number of individual cell types . To detect fluorescence, cells were collected quickly and observed using a fluorescence microscope (Olympus BX51).
根據該等結果(數據未顯示)，相較於經PBS處理之小鼠，在經SDX處理MCP-1 EGFP小鼠之肺中發現螢光之增加，並在經SDX處理小鼠之BALF中發現綠螢光細胞數目及綠螢光強度之增加。Based on these results (data not shown), an increase in fluorescence was found in the lungs of SDX-treated MCP-1 EGFP mice compared to PBS-treated mice and was found in BALF of SDX-treated mice. The number of green fluorescent cells and the increase in green fluorescence intensity.
使用RNA分離套組(Roche)，在未經處理小鼠之肺部以及在PBS或SDX滴注後之不同時點，分離總細胞RNA。以DNase 1處理RNA樣本以除去基因組DNA污染。使用逆轉錄套組(Qiagen)，對5μg之總RNA進行第一股cDNA合成，並在使用SYBR green master mix(Applied Biosystems)及基因特異性引子之定量PCR(qPCR)中，使用該產物作為模板進行擴增。用於進行qPCR之引子組為MCP-1之引子(GCCCCACTCACCTGCTGCTA(SEQ ID NO:6);TTTACGGGTCAACTTCACATTCAA(SEQ ID NO:7))，EGFP之引子(CTGCTGCCCGACAACCA(SEQ ID NO:8);GAACTCCAGCAGGACCATGTG(SEQ ID NO:9))，以及作為內部控制組的GAPDH之引子(AGAATGGGAAGCTTGTCATC(SEQ ID NO:10);GTAGACTCCACGACATACTC(SEQ ID NO:11))。循環條件為95℃下10分鐘，接著為45循環的95℃下之15秒變性，60℃下之黏連，以及95℃下之15秒延伸。mRNA之含量由下式定量：循環閾值(ΔCt)=Ct(MCP-1)-Ct(GAPDH)。Total cellular RNA was isolated using the RNA isolation kit (Roche) at the lungs of untreated mice and at different points after PBS or SDX instillation. RNA samples were treated with DNase 1 to remove genomic DNA contamination. The first strand cDNA synthesis was performed on 5 μg of total RNA using a reverse transcription kit (Qiagen), and the product was used as a template in quantitative PCR (qPCR) using SYBR green master mix (Applied Biosystems) and gene-specific primers. Amplification was performed. The primer set for performing qPCR is the primer for MCP-1 (GCCCCACTCACCTGCTGCTA (SEQ ID NO: 6); TTTACGGGTCAACTTCACATTCAA (SEQ ID NO: 7)), the primer for EGFP (CTGCTGCCCGACAACCA (SEQ ID NO: 8); GAACTCCAGCAGGACCATGTG (SEQ ID) NO: 9)), and an introduction of GAPDH as an internal control group (AGAATGGGAAGCTTGTCATC (SEQ ID NO: 10); GTAGACTCCACGACATACTC (SEQ ID NO: 11)). The cycling conditions were 10 minutes at 95 ° C followed by a 15 second denaturation at 95 ° C for 45 cycles, a bond at 60 ° C, and a 15 second extension at 95 ° C. The content of mRNA was quantified by the following formula: cycle threshold (ΔCt) = Ct(MCP-1)-Ct(GAPDH).
同時，亦進行RT-PCR，並以1.0%瓊脂凝膠分析擴增之PCR片段。用於進行RT-PCR之引子如下：MCP-1之引子(CTCACCTGCTGCTACTCATTC(SEQ ID NO:12);GCTTGAGGTGGTTGTGGAAAA(SEQ ID NO:13))；EGFP之引子(CCTACGGCGTGCAGTGCTTCAGC(SEQ ID NO:14);TGCTCAGGTAGTGGTTGT(SEQ ID NO:15))；以及GAPDH之引子(GTGGCAAAGT GGAGATTGTTGCC(SEQ ID NO:16);GATGATGAC CCGTTTGGCTCC(SEQ ID NO:17))。At the same time, RT-PCR was also performed, and the amplified PCR fragment was analyzed on a 1.0% agar gel. The primers used for RT-PCR were as follows: MCP-1 primer (CTCACCTGCTGCTACTCATTC (SEQ ID NO: 12); GCTTGAGGTGGTTGTGGAAAA (SEQ ID NO: 13)); EGFP primer (CCTACGGCGTGCAGTGCTTCAGC (SEQ ID NO: 14); TGCTCAGGTAGTGGTTGT ( SEQ ID NO: 15)); and the primer for GAPDH (GTGGCAAAGT GGAGATTGTTGCC (SEQ ID NO: 16); GATGATGAC CCGTTTGGCTCC (SEQ ID NO: 17)).
圖7顯示RT-PCR之結果。在未經處理MCP-1-EGFP小鼠之肺中含有可偵測表現量之MCP-1及EGFP轉錄物。SDX之滴注造成第1天、第3天結束時有顯著的MCP-1及EGFP誘發，並在第5天後朝向基線含量減少。SDX之最大MCP-1誘發在第3天後為約18倍，並在其後迅速減少，而EGFP之誘發在第1天至第5天為約3倍。PBS之滴注亦在第1天後誘發MCP-1及EGFP，但在第3天後朝向基線含量回復。應注意的是，由於PBS係誘發瞬間的MCP-1表現，因此在PBS處理組中並未形成肉芽腫。Figure 7 shows the results of RT-PCR. The detectable amount of MCP-1 and EGFP transcripts was contained in the lungs of untreated MCP-1-EGFP mice. SDX instillation resulted in significant MCP-1 and EGFP induction at the end of Day 1 and Day 3, and decreased towards baseline after Day 5. The maximum MCP-1 induction of SDX was about 18-fold after day 3, and rapidly decreased thereafter, and the induction of EGFP was about 3-fold from day 1 to day 5. Instillation of PBS also induced MCP-1 and EGFP after day 1, but recovered to baseline content after day 3. It should be noted that granuloma was not formed in the PBS-treated group because the PBS system induced transient MCP-1 expression.
以含有蛋白酶抑制劑之RIPA裂解緩衝液(Upstate,CA)萃取組織均質物中之蛋白，並以Bradford分析測定(Bio-Rad,CA)其濃度。根據製造商之指示，以MCP-1 ELISA套組(R＆D Systems,Minneapolis,MN)測定肺組織均質物中之MCP-1濃度。對蛋白表現物進行15% SDS-PAGE分析，接著使用抗-EGFP(Abcam)及抗-β-肌動蛋白(Chemicon)進行西方點漬分析，接著再以適當之HRP綴合性次級抗體及ECL偵測系統進行偵測。Proteins in tissue homogenates were extracted with RIPA lysis buffer (Upstate, CA) containing protease inhibitors and assayed by Bradford assay (Bio-Rad, CA). The MCP-1 concentration in the lung tissue homogenate was determined using the MCP-1 ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. 15% SDS-PAGE analysis of protein expressions followed by Western blot analysis using anti-EGFP (Abeam) and anti-beta-actin (Chemicon) followed by appropriate HRP-conjugated secondary antibodies and The ECL detection system performs detection.
如圖8A所示，SDS處理小鼠中之MCP-1蛋白表現高於PBS處理小鼠，且如圖6B所示，SDS處理小鼠中之EGFP蛋白表現高於PBS處理小鼠。注意到的是，在未經處理MCP-1-EGFP小鼠之肺的西方點漬中，EGFP表現之基線含量(圖8B)係以兩條帶之形式出現。該雙重信號之較上方條帶可能代表新合成之EGFP的中間物形式。該兩條帶皆因PBS之滴注有些微增加，且因SDX而強烈增加。As shown in Fig. 8A, the MCP-1 protein expression in the SDS-treated mice was higher than that in the PBS-treated mice, and as shown in Fig. 6B, the EGFP protein expression in the SDS-treated mice was higher than that in the PBS-treated mice. It is noted that in Western blotting of the lungs of untreated MCP-1-EGFP mice, the baseline content of EGFP expression (Fig. 8B) appeared in the form of two bands. The upper band of the double signal may represent an intermediate form of the newly synthesized EGFP. Both of these bands are slightly increased by the instillation of PBS and are strongly increased by SDX.
如實施例3.3所述取得並將肺切片予以染色。為偵測MCP-1蛋白，在5%正常驢血清中對該等肺切片進行阻斷，接著與山羊抗-小鼠CCL2/JE/MCP-1(R＆D Systems)、HRP-標記性驢抗-山羊次級抗體進行培育，接著再以DAB套組(Vector Laboratories)進行呈色，以蘇木精對比染色，最後以光學顯微鏡檢驗。為偵測EGFP蛋白，在5%山羊血清中對該等肺切片進行阻斷，接著與對抗GFP之兔多株抗體進行培育，接著再與HRP-標記性山羊抗-兔次級抗體進行培育，以AEC套組(Vector Laboratories)進行呈色，並以蘇木精對比染色。Lung sections were obtained as described in Example 3.3 and stained. To detect MCP-1 protein, these lung sections were blocked in 5% normal sputum serum, followed by goat anti-mouse CCL2/JE/MCP-1 (R&D Systems), HRP-labeled 驴 anti- Goat secondary antibodies were incubated, then stained with DAB kits (Vector Laboratories), stained with hematoxylin and stained with light microscopy. To detect EGFP protein, these lung sections were blocked in 5% goat serum, followed by incubation with multiple antibodies against GFP rabbits, followed by incubation with HRP-labeled goat anti-rabbit secondary antibody. Coloring was performed with an AEC kit (Vector Laboratories) and stained with hematoxylin.
根據該等結果(數據未顯示)，MCP-1及DGFP兩者之陽性染色在未經處理及注射PBS之控制組中反應極低。然而，在SDX處理之肺中，發現MCP-1及EGFP之表現不僅存在於肺泡巨噬細胞、SDX微珠周圍之巨噬細胞的細胞質中，亦存在於細支氣管上皮細胞中。EGFP陽性染色之分布模式與MCP-1染色相符，表示MCP-1及EGFP之表現分布在共同位置上。Based on these results (data not shown), positive staining for both MCP-1 and DGFP was extremely low in the untreated and PBS-injected control group. However, in the SDX-treated lung, it was found that the expression of MCP-1 and EGFP was not only present in the cytoplasm of alveolar macrophages, macrophages surrounding the SDX microbeads, but also in the bronchiole epithelial cells. The distribution pattern of EGFP positive staining was consistent with MCP-1 staining, indicating that the distribution of MCP-1 and EGFP was in a common position.
為產生LPS誘發性發炎之動物模式，以LPS(6mg/kg;i.p.)對如實例2所取得之MCP-1-EGFP轉殖基因小鼠進行處理24小時。提供野生型小鼠及無任何處理之MCP-1-EGFP轉殖基因小鼠作為控制組進行以下研究。To generate an LPS-induced inflammatory animal model, MCP-1-EGFP transgenic mice obtained as in Example 2 were treated with LPS (6 mg/kg; i.p.) for 24 hours. Wild type mice and MCP-1-EGFP transgenic mice without any treatment were provided as control groups for the following studies.
進行Gr-1(粒細胞之標記)及Mac-1(單核細胞巨噬細胞之標記)之表面染色，以分辨分離的骨髓細胞中之細胞類型。簡言之，以PBS沖洗脛骨及股骨而取得細胞懸浮液並清洗該分離細胞懸浮液，接著以下列抗體標記該混合物：綴合藻紅蛋白(PE)之Mac-1(Biolegend)、生物素化之Gr-1抗體、以及綴合PE-C7次級抗體之鏈黴親和素(eBioscience)，以進行細胞表面染色。Surface staining of Gr-1 (marker of granulocytes) and Mac-1 (marker of monocyte macrophages) was performed to distinguish the cell types in the isolated bone marrow cells. Briefly, the tibia and femur were washed with PBS to obtain a cell suspension and the isolated cell suspension was washed, followed by labeling the mixture with the following antibodies: phycoerythrin (PE) Mac-1 (Biolegend), biotinylated The Gr-1 antibody, and streptavidin (eBioscience) conjugated to the PE-C7 secondary antibody, were used for cell surface staining.
根據所得結果(數據未顯示)，在野生型及未經處理之MCP-1控制組中具有可比較的骨髓Mac-1(+)及Gr-1(+)亞種群分布。在LPS處理後，造成Gr-1(+)之減少並同時導致具有高Mac-1表現之Gr-1(+)及Mac-1(+)細胞的獨特亞種群之增加。以FACS分析偵測到此一亞種群具有高度之綠螢光。此一結果表示，具有因LPS而產生之較高Mac-1表現的骨髓細胞亦具有較高之MCP-1啟動子活性，因而造成高度之綠螢光。Based on the results obtained (data not shown), comparable bone marrow Mac-1 (+) and Gr-1 (+) subpopulations were distributed in the wild-type and untreated MCP-1 control groups. After LPS treatment, a decrease in Gr-1(+) was caused and at the same time an increase in the unique subpopulation of Gr-1(+) and Mac-1(+) cells with high Mac-1 expression was caused. This sub-population was detected by FACS analysis with a high degree of green fluorescence. This result indicates that bone marrow cells with higher Mac-1 expression due to LPS also have higher MCP-1 promoter activity, resulting in a high degree of green fluorescence.
綜上所述，藉由構築螢光MCP-1報導蛋白轉殖基因小鼠，我們得以建立EGFP報導蛋白表現與肺肉芽腫發炎後之MCP-1誘發之間的相關性。此種MCP-1-EGFP小鼠模式將可提供一種有價值的工具，以監測單核細胞巨噬細胞之活化並有助於研究MCP-1基因在各種發炎性疾病中之角色。In summary, by constructing a fluorescent MCP-1 reporter protein transgenic mouse, we were able to establish a correlation between EGFP-reported protein expression and MCP-1 induction after pulmonary granulomatous inflammation. This MCP-1-EGFP mouse model will provide a valuable tool to monitor the activation of monocyte macrophages and to help investigate the role of the MCP-1 gene in various inflammatory diseases.
就說明本發明之目的，在圖式中顯示目前為較佳之具體實例。然而，應明瞭的是，本發明並不限於所示之較佳具體實例。For the purposes of the present invention, the presently preferred embodiments are shown in the drawings. However, it should be understood that the invention is not limited to the preferred embodiments shown.
在圖式中：In the schema:
圖1顯示實例1所取得MCP-1-EGFP轉殖基因載體之結構圖譜。Figure 1 shows the structure map of the MCP-1-EGFP transgenic gene vector obtained in Example 1.
圖2顯示MCP-1立即早期啟動子及EGFP編碼序列在該MCP-1-EGFP轉殖基因載體中之位置；其中箭號指出用以進行實例2之基因型鑑定PCR之引子的結合位置；而標記底線之區域則表示用以進行實例2之南方點漬分析之探針的位置。Figure 2 shows the position of the MCP-1 immediate early promoter and the EGFP coding sequence in the MCP-1-EGFP transgene vector; wherein the arrow indicates the binding position of the primer used to carry out the genotypic PCR of Example 2; The area marked with the bottom line indicates the position of the probe used to perform the Southern spot analysis of Example 2.
圖3顯示MCP-1-EGFP轉殖基因載體中之MCP-1啟動子的序列。Figure 3 shows the sequence of the MCP-1 promoter in the MCP-1-EGFP transgenic gene vector.
圖4顯示包含在MCP-1-EGFP轉殖基因載體中之由MCP-1啟動子、EGFP編碼序列、以及多聚A序列所構成之核苷酸片段的序列。Figure 4 shows the sequence of the nucleotide fragment consisting of the MCP-1 promoter, the EGFP coding sequence, and the poly A sequence contained in the MCP-1-EGFP transgene vector.
圖5顯示由尾DNA樣本擴增轉殖基因片段(如圖2所示由箭號標記)之代表性基因型鑑定PCR的結果。Figure 5 shows the results of PCR analysis of representative genotypes of amplified transgenic gene fragments (marked by arrows as shown in Figure 2) from tail DNA samples.
圖6顯示存在於不同實驗組之BALF中的不同細胞類型(A：總體白細胞；B：嗜中性細胞；及C：巨噬細胞)；其中該等結果係以4至5隻動物之平均值±SD表示。*P<0.05，相較於未經處理組；，相較於PBS處理組。Figure 6 shows the different cell types (A: total white blood cells; B: neutrophils; and C: macrophages) present in BALF of different experimental groups; wherein the results are averaged from 4 to 5 animals. ±SD indicates. *P<0.05 compared to the untreated group; Compared to the PBS treatment group.
圖7顯示揭露MCP-1(A)及EGFP(B)之mRNA含量在SDX投予後第1、3、及5天時之增加的RT-PCR結果；其中MCP-1或EGFP之表現係相對於GAPDH正常化，而變化倍數係由兩次實驗取得。Figure 7 shows the results of RT-PCR showing the increase in mRNA levels of MCP-1 (A) and EGFP (B) on days 1, 3, and 5 after administration of SDX; the expression of MCP-1 or EGFP is relative to GAPDH was normalized, and the fold change was obtained from two experiments.
圖8A顯示在肺均質物中於SDX投予後第1、3、及5天時由SDX所誘發之MCP-1蛋白表現的ELISA結果；其中此等結果係以4至5隻動物之平均值±SD表示；符號“*”意謂相較於未經處理組P<0.05；而符號“”意謂相較於PBS處理組P<0.05。Figure 8A shows ELISA results of SDX-induced MCP-1 protein expression on day 1, 3, and 5 after SDX administration in lung homogenates; these results are averaged from 4 to 5 animals ± SD indicates; the symbol "*" means P<0.05 compared to the untreated group; and the symbol " "meaning" P < 0.05 compared to the PBS treated group.
圖8B顯示在SDX投予後第1、3、及5天時之增加EGFP蛋白之表現量的西方點漬分析結果；其中發現約28kDa之兩條位置密切接近的條帶是EGFP蛋白(箭號)；亦顯示26kDa之分子量標記(箭頭)；使用β-肌動蛋白作為內部標準物。Figure 8B shows the results of Western blotting analysis of increasing the expression of EGFP protein on days 1, 3, and 5 after SDX administration; it was found that the two closely spaced bands of about 28 kDa were EGFP proteins (arrows). ; molecular weight marker (arrow) of 26 kDa; also using β-actin as an internal standard.
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