TW201012928A - Composition made with in vitro culture of non-hematopoietic human somatic stem cells and use of same - Google Patents

Abstract

This invention utilizes a process culturing and proliferating non-hematopoietic human somatic stem cells in vitro to yield quantity ready for further separation and application. During culturing and proliferation, non-hematopoietic human somatic stem cells produce proliferation-promoting releasing factors, which are secreted into a culture medium, and mixed with the culture medium to form a composition. Both the cultured and proliferated non-hematopoietic human somatic stem cells and releasing factors complex as being capable of facilitating the activation and proliferation of neighboring cells could be applied on skin wound repair, activation of hair follicle cells and melanocytes, relevant dermatology medication and cosmeceuticals as an active or auxiliary component.

Description

201012928 九、發明說明： 【發明所屬之技術領域】 本發明係關於根據-種藉由體外培養人類非造血201012928 IX. Description of the invention: [Technical field to which the invention pertains] The present invention relates to the cultivation of human non-hematopoietic cells by in vitro culture

體幹細胞的程序所製造具有Μ周圍細胞活化與增生功能 之組成物、其製造方法及其應用。 B 【先前技術】 人類成體幹細胞（somatic stem cell)係源自人類胚幹細 參 胞（embry〇nic stem cell)，在生長、增殖與分化過程中經歷 全能（topipotent)、萬能（plurip〇tent)期，再成為多能 (multipotent)幹細胞。醫學上最早、亦最為廣泛應用人類 成體幹細胞的方式，是以源自於同種異體的骨髓之未分化 造血（hematopoietic)幹細胞，移植到造血功能異常的病患 身上。造金幹細胞屬於成體幹細胞的一種，是所有血球細 胞的前身（progenitor·) ’其分化的功能僅限於進一步成熟為 各式血球細胞。 φ 人類成體幹細胞包括有人類非造血型成體幹細胞。許 多人類組織中可分離出人類非造血型成體幹細胞（例如骨 趙、臍帶血、乳牙、脂肪組織、内皮組織等），可進一步分 化為其他種類的細胞（例如骨細胞、肌細胞等）前身，具有 醫學上更多元的應用價值。然而以往人類非造血型成體幹 細胞的取得來源均牵涉到人體組織，在應用上必需考慮其 可獲得性（availability)與可利用性（practicality)。 不論是以上提及或其他可能的人類非造血裂成體幹細 201012928 胞來源，現有的體外培養技術均不足快速有 以#晝接麻用·*κ本 ^ 量;增殖 胞體醫療程序中直接使用活體細 胞，即使疋侷限於本發明所議及之 M , a ^ w ^ , J種自體或同種異體途 :二Γ 度不一的法規限制。可知就既有 人類非“型成體幹細胞在應用技術方面， :到難以大量增殖的技術問題及法規限制的影響，不能夠 有效地實施應用，是亟待改善的缺點。 【發明内容】 有鐾於前述人類非造血型成體幹細胞體外培養技術在 :施應用方面的缺點，本發明之目的在於提供一種組成 ’其包括有取自人類非造血型成體幹細胞培養過程中的 =放因子，以提供有效的實施或應S。藉由穩定有效的體 二養方法，可增殖足量的成體幹細胞並自培養過程中獲 =前述釋放因子。本發明並根據上述體外培養方法，延伸 -不涉及活體細胞的物質，提#一種方法以製造前述包括 :釋放因子的組成物’以提供促進周圍細胞活化與增生功 此之效果。本發明的另__方面，則關於前述組成物的應用。 為達到上述目的，本發明分別以源自於諸如人類骨 趙，、人類脂肪、人類臍帶、與人類乳牙等人類組織的人類 非k血型成體幹細胞，進行體外培養，並自其中獲取前述 人類非造血型成體幹細胞在培養時所釋出的釋放因子，進 而藉由該釋放因子製造一組成物。 本發明利用人類成體幹細胞體外培養，在較受偏好的 201012928 情況下，為提供具有促進周目細胞活化與增生功能之板成 物的製造方法包括有：以不含酵素的t合劑將前述貼附於 培養皿的細胞與培養皿分離後，㈣懸浮於-生理緩衝 液’成為—人類非造▲型成體幹細胞；同時將前述體外培 養所置換之細胞懸浮液收集，經由冷咖得到一脫水： =，將刚达脫水粉末以生理緩衝液回溶，得到一人類成體 幹細胞體外培養的釋放因子組成物等步驟。 ❹ ❿ 本發明分別以分離自人類骨髓、人類脂肪、人類臍帶 間質、與人類乳牙的四種非造▲型成體幹細胞，同時以前 述步驟進行體外培養驗證；前述體外培養的方法主要包括 有：以前述各人類組織分離出屬於人類非造血型成體幹細 =之单核間質幹細胞；將前述各單核間質幹細胞分別置於 含有一培養基之培養皿；#前述各培養皿置於一提供二氧 化碳並維m與濕度的培養器，使前述培養基轉變為一 細胞懸浮液；以前述培養基更換前述細胞懸浮液；將維持 貼:於各培養皿之細胞，以前述培養基培養使細胞繼續增 殖-段時間’同時亦將前述培養基轉變為—細胞懸浮液等 步驟°在前述體外培養的過程，前單核間f幹細胞將釋出 釋放因子至前述培養基亦即細胞懸浮液之中。 在已元成的實驗中，四種來源之非造血型成體幹細胞 在培養過程中均維持良好的貼附性，因此應維持有正常的 細胞間交互作用、並有利於方便快速的更新培養基；培養 :並且未觀察到任何潛在腫瘤細胞生成。四組體外培養驗 中源自人類濟帶間質之人類非造血型成體幹細胞 201012928 平均處於GO至CM成長期時間約為74%、處於s + g2+m穩 定期約為12%、倍增時間約為28至32小時、可維持二十 .代以上增殖（Proliferation)而未發生表面特殊抗原的分佈變 •化；以上性質略優於同時比對進行體外培養的其他人類非 造血型成體幹細胞。 本發明利用前述具有促進活化與增生功能之增殖為足 量的成體幹細胞、與製造出的釋放因子組成物，所引申的 應用範圍包括有：促進皮膚傷口修復，例如加速傷口的癒 參 合、降低傷口感染、抑制傷口發炎、減少疤痕等；活化毛 囊細胞，例如促進毛囊細胞生長、增生毛髮、治療禿髮； 活化黑色素細胞，例如促進黑色素細胞生長、深色化淡色 毛髮、治療白斑等。因此製備處理前述狀況之藥物的應用， 亦屬於本發明之範疇。 【實施方式】 參 非造血型成體幹細胞（non-hematopoietic somatic stem cell)」於本文定義為胚幹細胞在成熟過程中進入多能 期’成為同時具有***增殖為同性質的細胞與分化成專一 性細胞的成體幹細胞’其中一類分化成各式血球細胞，屬 於造血型成體幹細胞；而其他的成體幹細胞則分化成例如 肌肉、血管、神經、骨骼等各種組織細胞，屬於非造血型 成體幹細胞。 「同種自體（autol〇g〇Us )」於本文定義為生物學分 201012928 類中同一物種且同一生物個體之意。此術語尤指將器官、 組織、細胞或其衍生物植入或移殖至其所由來之生物個體 - 之狀況。在此狀況下’由取得及接受前述器官、組織、細 • 胞或其衍生物之生物個體係同一生物個體，因此即使前述 生物個體具有可發揮相當免疫能力之免疫系統，通常亦不 會對於前述器官、組織、細胞或其衍生物發動排斥反應。 「同種異體（allogenic )」於本文定義為生物學分類 中同一物種不同生物個體之意。此術語尤指將來自一生物 參個體之器官、組織、細胞或其衍生物植入或移殖至另一同 種之生物個體之狀況。在此狀況下，接受前述器官、組織、 細胞或其衍生物之生物個體若具有可發揮相當免疫能力之 免疫系統’將可能針對前述器官、組織、細胞或其衍生物 發動排斥（reject)反應。 「細胞懸浮液（supernatant)」於本文定義為以液態培 養基進行人類非造血型成體幹細胞體外培養時，包括細胞 所釋放之分泌物與前述液態培養基，且可進一步包括有懸 〇 浮分散於其中之細胞。 「釋放因子（secreted factors )」於本文定義為體外 培養之人類非造血型成體幹細胞時所釋放到細胞外之物 質’較佳的是’以液態培養基體外培養源自人類臍帶間質 幹細胞時’由該細胞釋放到液態培養基之物質。 「傷口（wound)」於本文定義為生物個體之體表受到破 壞而呈現割裂、穿刺、撕裂、或受燒燙之形態。較佳的是， 皮膚之表皮層（epidermis)、真皮層（dermis)受到破壞，形成 201012928 開口而露出皮下組織（hypodermis)，或皮下組織亦遭受破 壞之形態。 - 「癒合dealing or repairing)」於本文定義為形成於皮 • 膚之傷口藉由皮膚組織新生（regeneration)而重建的過程， 尤指表皮與真皮組織新生使受到破壞而形成之開口恢復為 封閉狀態的過程。 「感染（infection)」於本文定義為諸如細菌或病毒等 作為病原（pathogen)的其他物種，進入宿主（h〇st)之生物個 瞻體内或生物個體之細胞内，進行有害的複製、繁殖之過程。 就形成開口之開放性傷口而言，該感染通常導因於前述之 其他物種經該傷口進入前述生物個體内而發生，因此隨著 前述開放性傷口曝露於前述其他物種的時間增長，前述生 物個體受到感染的機會亦隨之增加。 「發炎（inflammation)」於本文定義為生物個體對於諸 如細胞受損或外來病原等有害刺激所產生的防禦反應，其 係位於形成傷口處或傷口周圍的組織釋出包括組織胺在内 Φ 的化學物質，而引發免疫細胞及巨噬細胞（macrophage)的 侵潤，並刺激附近的血管，導致局部血管擴張及微血管通 透性增大。 「疤痕（scar)」於本文定義為在傷口癒合的修復過程中 所造成之永久痕跡。前述痕跡係由於傷口癒合時產生纖維 化組織替代原來皮膚組織而形成。 「毛囊細胞（follicle)」於本文定義為存在於包圍在毛 髮根部周圍的囊狀組織中的細胞，具有控制毛髮生長的功 201012928 能。當毛生長到一定長度，毛根部由圓柱狀變成棒狀時， 毛囊萎縮，毛髮脫落，經過一段休息期後，毛囊細胞再， 活躍’重建毛球，再生新的毛髮。 度 「禿髮（Alopecia)」於本文定義為指頭髮不正常脫 落，可以是由荷爾蒙失調引起。營養不良、遺傳、疾病、 心理壓力、情緒、内分泌失調、接受癌症治療均有可能 致禿髮。 b导 「黑色素細胞（melanocyte )」於本文定義為—種動 參 物細胞，帶有黑色素或是其他類似的色素。通常位於皮膚 的表皮與眼睛的葡萄膜（虹膜後面的色素層）中。恆溫動 物的黑色素細胞，除了黑色素以外還能夠製造一些紅色或 黃色的色素。變溫動物的黑色素細胞則只能製造黑色的色 素。黑色素細胞的代謝若是受到破壞或抑制，會產生一些 疾病。此外皮膚、毛髮和眼睛的顏色，以及黑痣、雀斑等 皮膚上的斑點，也都與黑色素細胞有關。 「白斑（Vitiligo)」於本文定義為一種常見的色素退化 ❹ 消失的疾病，全身到處都可能產生。一般身體兩側都會出 現，呈現乳白色斑塊。常見侵犯部位包括臉部、嘴唇、手 部、手臂、腿部、生殖器。白斑的患部呈現乳白色，但個 別部位脫色的程度不一，有時白斑病灶周圍會有一圈較為 深色。 發明詳述 (一）一種組成物。 201012928 本發明之-個方面關於一種組成物。前述組成物主要 係以源自-人體組織之人類非造血型成體幹細胞，使用液 態培養基進行體外培養增殖所製得者。 前述人類非造血型成體幹細胞係可取自但不限於骨 趙、脂肪、臍帶或乳牙，其他可以取得符合本發明之發明 概念的人體組織亦可作為r、+、，& ^ P匈取得則述人類非造血型成體幹細 胞的來源。 在一受偏好的實施態樣令，前述用以液態培養基包括 ❹有：alpha-最小必需培養基（mem)、1%抗生素、10%胎牛 血清、lmML-麩醯胺、lmM非必需胺基酸及imM丙酮酸鈉。 藉由包3以上成为之液態培養基，可對於前述人類非造灰 型成體幹細胞進行培養，並令之在培養的過程中增殖以達 到適用之數量。 由於前述組成物主要係取得經過培養之培養基，該培 養基經過前述人類非造血型成體幹細胞在培養過程中消耗 並代謝’並在細胞增殖達到適用之數量前經過數次以新鮮 碜=。養基更換，因此前述組成物可能進一步包括有最小必 2培養基、h生素、胎牛血清、胺基酸、丙酮酸納，但在The body stem cell process produces a composition having a cell activation and proliferation function around the sputum, a method for producing the same, and an application thereof. B [Prior Art] Human somatic stem cells are derived from human embryonic stem cells, undergoing topopotent and plurip〇tent during growth, proliferation and differentiation. In the period, it becomes a multipotent stem cell. The earliest and most widely used method of human adult stem cells is the transplantation of undifferentiated hematopoietic stem cells derived from allogeneic bone marrow into patients with abnormal hematopoietic function. Gold-forming stem cells are a type of adult stem cells and are the precursors of all blood cells (progenitor·). The function of differentiation is limited to further maturation into various types of blood cells. φ Human adult stem cells include human non-hematopoietic adult stem cells. Human non-hematopoietic adult stem cells (such as bone marrow, cord blood, deciduous teeth, adipose tissue, endothelial tissue, etc.) can be isolated from many human tissues, and can be further differentiated into precursors of other kinds of cells (such as bone cells, muscle cells, etc.). It has more application value in medicine. However, in the past, the sources of human non-hematopoietic adult stem cells have been involved in human tissues, and their availability and practicality must be considered in application. Regardless of the above mentioned or other possible human non-hematopoietic stem fines of the 201012928 cell source, the existing in vitro culture techniques are insufficient to quickly use the #昼接麻用·*κ本量; directly in the proliferating cell body medical procedure The use of living cells, even if limited to M, a ^ w ^ , J, autologous or allogeneic, as discussed in the present invention: regulatory limitations of varying degrees of dichotomy. It can be seen that there are disadvantages that human non-"type adult stem cells in the application technology, the technical problems and regulatory restrictions that are difficult to proliferate, and the inability to implement the application effectively are urgently needed to be improved. The above-mentioned human non-hematopoietic adult stem cell in vitro culture technique has disadvantages in application, and the object of the present invention is to provide a composition which includes a factor of a factor obtained from a human non-hematopoietic adult stem cell culture process to provide Effective implementation or should be S. By a stable and effective body-boosting method, a sufficient amount of adult stem cells can be proliferated and obtained from the culture process = the aforementioned release factor. The present invention is extended according to the above-mentioned in vitro culture method - does not involve a living body A substance of a cell, a method for producing the aforementioned composition comprising: a release factor to provide an effect of promoting activation and proliferation of surrounding cells. Another aspect of the invention relates to the use of the aforementioned composition. For the above purposes, the present invention is derived from, for example, human bones, human fat, human umbilical cord, human deciduous teeth, and the like. Organized human non-k blood group adult stem cells are cultured in vitro, and a release factor released by the aforementioned human non-hematopoietic adult stem cells during culture is obtained therefrom, and a composition is produced by the release factor. In vitro culture using human adult stem cells, in the case of the preferred 201012928, a method for producing a plate having a function of promoting activation and proliferation of the cells of the eye includes: attaching the aforementioned to the enzyme-free t-mixer After the cells of the culture dish are separated from the culture dish, (4) suspended in the physiological buffer to become human non-made type ▲ adult stem cells; at the same time, the cell suspension replaced by the above-mentioned in vitro culture is collected, and a dehydration is obtained via cold coffee: The Ganda dehydrated powder is reconstituted in a physiological buffer to obtain a release factor composition for in vitro culture of human adult stem cells. ❹ ❿ The present invention is isolated from human bone marrow, human fat, human umbilical cord interstitial, and human, respectively. Four non-made ▲ type adult stem cells of deciduous teeth, and verified in vitro by the above steps; the above-mentioned in vitro culture The method mainly comprises: separating mononuclear mesenchymal stem cells belonging to a human non-hematopoietic adult fine tissue = each of the aforementioned human tissues; and placing each of the aforementioned mononuclear mesenchymal stem cells in a culture dish containing a medium; The culture dish is placed in an incubator that supplies carbon dioxide and maintains m and humidity, and the medium is converted into a cell suspension; the cell suspension is replaced with the aforementioned medium; and the cells are maintained in the culture medium, and cultured in the aforementioned medium. The cells continue to proliferate for a period of time - while also converting the aforementioned medium into a cell suspension or the like. In the above-mentioned in vitro culture process, the pre-mononuclear f stem cells will release the release factor to the aforementioned medium, that is, the cell suspension. In the experiments of Yuancheng, the non-hematopoietic adult stem cells from four sources maintained good adhesion during the culture process, so normal cell-cell interaction should be maintained and the medium should be easily and quickly updated. ; Culture: and no potential tumor cell formation was observed. The four groups of human non-hematopoietic adult stem cells derived from the human mesenchyme in the in vitro culture test were in the range of about 74% in the GO to CM growth period, about 12% in the s + g2+m stabilization period, and the doubling time. It is about 28 to 32 hours, and can maintain the proliferation of more than twenty generations without the distribution of surface specific antigens. The above properties are slightly better than other human non-hematopoietic adult stem cells cultured in vitro. . The present invention utilizes the aforementioned adult stem cells having a proliferation promoting proliferative and proliferative function to a sufficient amount, and the released release factor composition, and the scope of application includes: promoting skin wound repair, for example, accelerating wound healing and reducing Wound infection, inhibition of wound inflammation, reduction of scars, etc.; activation of hair follicle cells, such as promoting hair follicle cell growth, proliferating hair, treating alopecia; activation of melanocytes, such as promoting melanocyte growth, darkening pale hair, treating white spots, and the like. Therefore, the application of a medicament for treating the aforementioned conditions is also within the scope of the present invention. [Embodiment] Non-hematopoietic somatic stem cells are defined herein as embryonic stem cells entering a pluripotent phase during maturation, and become cells with differentiation and proliferation to the same nature and differentiation into specificity. One of the adult stem cells of the cell differentiates into various hematopoietic cells and belongs to hematopoietic adult stem cells; while other adult stem cells differentiate into various tissue cells such as muscle, blood vessels, nerves, bones, etc., belonging to non-hematopoietic adult bodies. stem cell. "Autologous autologous (autol〇g〇Us)" is defined herein as the same species in the class of biology 201012928 and the same organism. The term particularly refers to the condition in which an organ, tissue, cell or derivative thereof is implanted or transplanted to the individual from which it is derived. In this case, 'the biological entity of the organ, tissue, cell or derivative thereof is obtained and accepted by the same biological entity. Therefore, even if the aforementioned biological individual has an immune system capable of exerting considerable immunity, it is usually not Organs, tissues, cells or derivatives thereof initiate rejection. "Allogenic" is defined herein as the meaning of a different organism in the same species in a biological classification. The term particularly refers to the condition of implanting or transplanting an organ, tissue, cell or derivative thereof from a biological subject to another biological individual of the same species. In this case, an organism having an organ, a tissue, a cell or a derivative thereof, if it has an immune system capable of exerting a relatively immune ability, will likely initiate a rejection reaction against the aforementioned organ, tissue, cell or derivative thereof. "Supernatant" is defined herein as a liquid medium for in vitro culture of human non-hematopoietic adult stem cells, including secretions released by the cells and the aforementioned liquid medium, and may further comprise a suspension floating in the liquid medium. The cells. "Secreted factors" are defined herein as substances that are released outside the cell when human non-hematopoietic adult stem cells are cultured in vitro. 'Better when in vitro cultured from human umbilical cord mesenchymal stem cells in liquid medium' A substance released from the cell into a liquid medium. "Wound" is defined herein as the form in which the body surface of a biological individual is damaged, which is split, pierced, torn, or burnt. Preferably, the epidermis and dermis of the skin are destroyed, forming a 201012928 opening to expose the hypodermis, or the subcutaneous tissue is also damaged. - "healing or repairing" is defined herein as the process of reconstitution of a skin wound formed by the regeneration of skin tissue, especially the opening of the epidermis and dermal tissue that is destroyed and restored to a closed state. the process of. "Infection" is defined herein as other species of pathogens, such as bacteria or viruses, that enter the host (h〇st) organisms or in the cells of biological individuals for harmful replication and reproduction. The process. In the case of an open wound forming an opening, the infection usually occurs as a result of the aforementioned other species entering the aforementioned biological individual through the wound, and thus the aforementioned biological individual grows as the aforementioned open wound is exposed to the aforementioned other species. The chances of getting infected also increase. "Inflammation" is defined herein as the defensive response of a biological individual to a noxious stimulus, such as a damaged cell or a foreign pathogen, which is located at the site where the wound is formed or around the wound, releasing the chemistry of Φ including histamine. The substance, which invades immune cells and macrophage, stimulates nearby blood vessels, causing local vasodilation and increased microvascular permeability. "Scar" is defined herein as a permanent trace of healing during wound healing. The aforementioned traces are formed by the formation of fibrotic tissue instead of the original skin tissue when the wound heals. "Follic follicles" are defined herein as cells present in the saclike tissue surrounding the hair roots and have the ability to control hair growth 201012928. When the hair grows to a certain length and the hair root changes from a cylindrical shape to a rod shape, the hair follicles shrink and the hair falls off. After a rest period, the hair follicle cells are again active to rebuild the hair bulb and regenerate new hair. Degree "Alopecia" is defined herein as a condition in which the hair is abnormally detached and may be caused by a hormonal imbalance. Malnutrition, genetics, disease, psychological stress, mood, endocrine disorders, and cancer treatment may all cause alopecia. b. "Melanocyte" is defined herein as a kind of kinetic cell with melanin or other similar pigments. Usually located in the epidermis of the skin and the uvea of the eye (the pigmented layer behind the iris). Thermophilic melanocytes, in addition to melanin, can produce some red or yellow pigments. Melanocytes from warming animals can only produce black pigments. If the metabolism of melanocytes is destroyed or inhibited, some diseases will occur. In addition, the color of the skin, hair and eyes, as well as the spots on the skin such as black sputum and freckles, are also related to melanocytes. "Vitiligo" is defined herein as a common disease in which pigmentation is degraded and disappears throughout the body. It usually appears on both sides of the body, showing milky white patches. Common areas of invasion include the face, lips, hands, arms, legs, and genitals. The affected part of the white spot appears milky white, but the degree of discoloration varies from place to place, and sometimes there is a dark circle around the white spot lesion. DETAILED DESCRIPTION OF THE INVENTION (a) A composition. 201012928 An aspect of the invention relates to a composition. The above-mentioned composition is mainly produced by human non-hematopoietic adult stem cells derived from human tissues, and cultured in vitro using a liquid medium. The aforementioned human non-hematopoietic adult stem cell line can be obtained from, but not limited to, bone radish, fat, umbilical cord or deciduous teeth, and other human tissues which can obtain the inventive concept according to the present invention can also be obtained as r, +, & The source of human non-hematopoietic adult stem cells. In a preferred embodiment, the aforementioned liquid medium includes: alpha-minimum essential medium (mem), 1% antibiotic, 10% fetal bovine serum, lmML-glutamine, lmM non-essential amino acid And imM sodium pyruvate. The human non-ash-forming adult stem cells can be cultured by the liquid medium of the above-mentioned package 3 or higher, and allowed to proliferate in the course of culture to obtain a suitable amount. Since the above composition is mainly obtained by culturing a culture medium, the culture medium is consumed and metabolized by the aforementioned human non-hematopoietic adult stem cells during the culture and is subjected to fresh 碜 = several times before the cell proliferation reaches an appropriate amount. Nutrient replacement, so the aforementioned composition may further include a minimum of 2 medium, h vitamins, fetal bovine serum, amino acid, sodium pyruvate, but in

别述組成物中的冬暮》β L 置及配比不必然同於在新鮮培養基中的 含量及配比。 由於在培養過程中，部分人類非造血型成體幹細胞在 培養基中漂浮，且前述人類非造也型成體幹細胞將释出釋 ;σ養基中，因此前述組成物將包括有經過體外培 曰殖所獲取的人類非造^型成體幹細胞、前述人類非造 12 201012928 血型成體幹細胞進行體外培養增殖所獲取的一釋放因子。 在前述受偏好的實施態樣中，前述組成物將進一步包括有 最小必需培養基、抗生素、胎牛血清、胺基酸、丙酮酸鈉。 在取得前述組成物時，可進一步分離前述成體幹細 胞。而分離之手段包括但不限於過濾、沉降、離心、層析、 萃取、或其它物理性分離純化之技術程序，進而使得前述 人類非造血型成體幹細胞處於前述分離程序處理過的狀 態。 ❿ 在本發明中’前述釋放因子係生長因子、胞核嘧咬、 訊息核苷酸、調節性蛋白質或前述物質之組合，且前述釋 放因子具有促進周圍細胞活化與增生的功能。 為便於應用本發明所提供之組成物，可將之進一步處 理而令其狀態處於液體狀態、冷凍乾燥狀態、或回溶於生 理緩衝液狀態。另外，就本發明所屬技術領域中具有通常 知識者可得而知的其他處理與應用方法，在不脫離本發明 概念的原則下，仍被涵蓋於本發明之範轉。 ® (一）一種包括有人類非造血型成體幹細胞釋放因子之 組成物的製造方法 本發明之一個方面係關於一種自人體組織分離人類非 造血型成體幹細胞後進行冑外培養，料增殖之人類非造 血型成體幹細胞與釋放因子組成物的方法。 在本發明的實施態樣中，該方法主要包括有： k供一人體組織； 以胰蛋白分解酵素及膠原蛋白分解酵素水解前述人體 13 201012928 組織； 以離心方, t 八自水解之前述人體組織分離並獲取單核成 體幹細胞； 將則述單核成體幹細胞植入含培養基之培養皿； 經過24小時後，以一新鮮培養基更換前述培養基並 除未貼附於培_ 養皿之單核成體幹細胞； 將貼附於培養皿之單核成體幹細胞留置於該培養皿， 使之於新鮮培養基中增殖至一定數量；In the composition, the composition and ratio of β L in the winter scorpion are not necessarily the same as those in the fresh medium. Since some human non-hematopoietic adult stem cells float in the culture medium during the culture process, and the aforementioned human non-synthetic adult stem cells will be released and released; in the sputum, the aforementioned composition will include in vitro culture. A release factor obtained by in vitro culture and proliferation of human non-typed adult stem cells obtained from the colony, and the aforementioned human non-made 12 201012928 blood group adult stem cells. In the foregoing preferred embodiment, the aforementioned composition will further comprise minimal essential medium, antibiotics, fetal bovine serum, amino acids, sodium pyruvate. When the above composition is obtained, the aforementioned adult stem cells can be further separated. The means of separation include, but is not limited to, filtration, sedimentation, centrifugation, chromatography, extraction, or other physical separation and purification techniques, thereby allowing the aforementioned human non-hematopoietic adult stem cells to be subjected to the aforementioned separation procedure. ❿ In the present invention, the aforementioned release factor is a growth factor, a nuclear nucleoside, a message nucleotide, a regulatory protein or a combination of the foregoing, and the aforementioned release factor has a function of promoting activation and proliferation of surrounding cells. In order to facilitate the application of the composition provided by the present invention, it may be further treated to bring its state into a liquid state, freeze-dried state, or to be dissolved in a physiological buffer state. In addition, other processing and application methods that are known to those of ordinary skill in the art to which the present invention pertains, are still encompassed by the present invention without departing from the principles of the invention. ® (I) A method for producing a composition comprising a human non-hematopoietic adult stem cell releasing factor. One aspect of the present invention relates to a method for isolating human non-hematopoietic adult stem cells from human tissues and then performing extrauterine culture. A method of human non-hematopoietic adult stem cells and a release factor composition. In an embodiment of the present invention, the method mainly comprises: k for a human tissue; hydrolyzing the human body 13 with a trypsin and collagen decomposing enzyme; 201012928 tissue; Separating and obtaining mononuclear adult stem cells; implanting the mononuclear adult stem cells into a culture dish containing the medium; after 24 hours, replacing the medium with a fresh medium and removing the single core which is not attached to the culture vessel Adult stem cells; mononuclear adult stem cells attached to the culture dish are left in the culture dish, and propagated to a certain amount in fresh medium;

將增瘦的幹細胞自前述培養皿脫附； 以流式細胞儀確認幹細胞之表面抗原標記特徵； 獲取人類非造血型成體幹細胞。 /尤：發明所屬㈣中具有通常知冑者可得而知之技術 手奴’則述之方法進行適度之調整與變化。因&，在本發 明的另-實施態樣中，該方法主要包括有： 自水解之人體組織分離單核成體幹細胞； .b.將前述單核成體幹細胞置於一含有一培養基之培養 ，，、——％ iU吸业难符 度的培養器，使前述培養基轉變為一細胞懸浮液； d.以前述的培養基更換前述細胞懸浮液； 舍U前述培養基培養使貼附於培養孤的細胞增殖至 畜數S ’同時亦將前述培養基轉變為_細胞懸浮液； 、f.以—細胞脫附試劑將貼附於培養皿的細胞與培養 分離，以-生理缓衝液將細胞重新懸浮後保存’成為\ 201012928 非造血型成體幹細胞；以及 g·將前述細胞懸浮液收集後以冷凍乾燥法製得一脫水 的粉末，以適當量的生理緩衝液將該粉末回溶，即得到一 釋放因子組成物；等步驟。 ❹The thinned stem cells were desorbed from the culture dish; the surface antigen labeling characteristics of the stem cells were confirmed by flow cytometry; human non-hematopoietic adult stem cells were obtained. / You: In the invention (4), there is a technique that can be obtained by the general knowledge, and the method described is moderately adjusted and changed. In another embodiment of the present invention, the method mainly comprises: separating mononuclear adult stem cells from the hydrolyzed human tissue; and b. placing the mononuclear adult stem cells in a medium containing a medium. Cultivate,,, -% iU incubator, the above medium is converted into a cell suspension; d. replace the aforementioned cell suspension with the aforementioned medium; The cells proliferate to the number of animals S' and also convert the aforementioned medium into a cell suspension; f. separate the cells attached to the culture dish with the cell desorption reagent, and resuspend the cells with physiological buffer After saving 'become \201012928 non-hematopoietic adult stem cells; and g· collecting the aforementioned cell suspension, lyophilized to obtain a dehydrated powder, and re-dissolving the powder with an appropriate amount of physiological buffer to obtain a release Factor composition; steps. ❹

為有效獲取足量的原始成體幹細胞，需對於前述屬於 間質之人體組織以蛋白分解酵素在適當的環境條件下進行 分解等處理。該方法之a步驟可採用胰蛋白酵素㈣㈣ 或膠原蛋白酵素（c〇llagenase)，或採用兩者之組合等作對 前述人體組織進行水解處理。進行處理時’可令^於攝氏 3 7度下處理前述組織1小時。 該方法之^步驟可採用離心之方式分離前述成體幹細 胞。其中一可行之離心環境條件係於14〇G下離心、分鐘 進行。配合適當的離心機，可採用每分鐘8〇〇〇轉之轉速。 在該方法之c步驟中，可令前述恆溫恒濕培養器提供 外之二氧化碳並維持攝& 37度之溫度。其二氧化碳之濃 度與溫度可配合需要加以適當之調整。 在該方法之e步驟中，可採用的培養基組成至少 包括有：alpha-最小必需培養基⑽M)、l%抗生素、1〇%胎 牛血清、ImM L-麩醯胺、lmM非必需胺基酸及imM丙酮 酸納。 的在該方法t f步驟中，培養條件可使用怪溫怔濕培養 窃提供5/〇之一氧化碳並維持攝氏度之溫度，可以進 行至y 84 Λΐ、_ ’使細胞增造至相f數量時，貝占附於培養皿 的細胞密度可為大於107個/平方公分；在前述培養的84 15 201012928 小時之中，可以俞、+. 魂培養基至少更換一次前述細胞懸 液。亦即，藉著每缌m ^ 前述細胞懸浮液之方彳 臀土更換 〈万式，可周而復始的循環實施該方法 d步驟與g步驟，你〆万法的 死而持續不斷地培養增殖至相當之數量。 、☆由2 =該方法之c步驟中前述培養基轉變為一細胞懸 '子液，故可於d舟聰、， 驟獲取刖述細胞懸浮液，並可重覆e步 驟與d步驟’進而重覆收集前述細胞懸浮液；該方法之' 步驟中的適當時間可為24至48小時。In order to effectively obtain a sufficient amount of the original adult stem cells, it is necessary to decompose the proteolytic enzymes in the above-mentioned interstitial human tissues under appropriate environmental conditions. In the step a of the method, trypsin (4) (4) or collagenase (c〇llagenase) may be used, or a combination of the two may be used to hydrolyze the aforementioned human tissue. When the treatment is carried out, the aforementioned tissue can be treated at 37 ° C for 1 hour. The method of the method can separate the aforementioned adult stem cells by centrifugation. One of the possible centrifugal environmental conditions is carried out by centrifugation at minute 〇G. With a suitable centrifuge, it can be used at 8 rpm. In the step c of the method, the aforementioned constant temperature and humidity incubator is allowed to supply carbon dioxide outside and maintain a temperature of 37 °C. The concentration of carbon dioxide and the temperature can be adjusted as appropriate. In the e step of the method, the medium composition that can be used includes at least: alpha-minimum essential medium (10) M), 1% antibiotic, 1% fetal bovine serum, 1 mM L-glutamate, lmM non-essential amino acid and imM pyruvate sodium. In the tf step of the method, the culture condition can be used to provide 5/〇 of carbon monoxide and maintain the temperature of Celsius using a strange temperature and humidity culture, and can be carried out until y 84 Λΐ, _ ' to increase the number of cells to the phase f, The cell density attached to the culture dish may be greater than 107 cells per square centimeter; during the above-mentioned culture of 84 15 201012928 hours, the cell suspension may be replaced at least once by the soul medium. That is to say, by replacing the square cell of the cell suspension with 缌m ^, the method can be carried out in a cycle of repeated steps, and the steps d and g can be carried out in a cycle that is repeated and continuously cultivated to a considerable extent. The number. ☆ By 2 = the above-mentioned medium in the c step of the method is converted into a cell suspension 'sub-liquid, so the cell suspension can be obtained in d Zhou Cong, and the step e can be repeated and the step d can be repeated. The aforementioned cell suspension is collected; the appropriate time in the 'step of the method may be 24 to 48 hours.

在該方法的f步驟中，為了使細胞自培養皿脫附，可 使用不含任何蛋白酵素與乙婦二胺四乙酸的脫附試劑，令 脫附反應在攝氏37度的溫度下進行2至5分鐘，完成時可 使用不含鈣、鎂離子的標準磷酸鹽緩衝液將細胞重新懸浮 後保存。 在該方法的g步驟中，可採用前揭的標準磷酸鹽緩衝 液，選擇一重量百分率介於〇.1%至1〇%的濃度計算，將 前述細胞懸浮液的脫水粉末回溶，成為釋放因子組成物。 又，前述人類非造血型成體幹細胞得進一步經過諸過 濾、"L降、離心、層析、萃取、或其它物理性分離純化程 序處理。 (三）組成物的應用 本發明之另一個方面係關於前述組成物的應用。如前 述，本發明基於前揭源自人類臍帶間質幹細胞體外培養的 步驟，取得前述組成物。在本發明之實施例中，該組成物 201012928 包括有人類成體幹細胞與釋放因子。前述人類成體幹細胞 與釋放因子皆具有促進周圍細胞活化盥婵峰 a王功能，可以作 為與皮膚醫療保健相關的醫藥或含藥化粧品之活性或輔助 成分。 本發明以一實驗動物皮膚上的人造傷口為例，使用前 述組成物塗抹該傷口，可觀察到兩者皆能發揮促進傷口癒 合’降低傷口感染、抑制發炎及減少疤痕之作用，故可引 申前述兩種物質能夠應用於促進皮膚傷口修復，進而可將 ® 前述兩種物質應用於製造具有加速傷口癒合，降低傷口感 染、抑制傷口發炎及減少疤痕等作用之醫藥組成物。 本發明另以一實驗動物皮膚上一定面積之區塊予以剃 毛’使用前述組成物塗抹該區塊，可觀察到兩者皆能促進 該區塊的毛髮生長’故可引申前述兩種物質具有活化毛囊 細胞的功能，可應用於製造具有促進毛囊細胞生長、增生 毛髮及治療禿髮等作用之醫藥組成物。 本發明另以一實驗動物皮膚表面因衰老而毛髮變白的 ^ 區塊，使用前述組成物塗抹，可觀察到兩者皆能減少白毛、 增加黑色或溧色的毛髮生長。實驗動物因衰老而發生皮膚 黑色素細胞活性降低或死亡，但其毛囊細胞仍持續作用而 長出白毛，因此，本試驗觀察結果可引申前述兩種物質可 活化皮膚黑色素細胞，可應用於製造具有促進黑色素細胞 生長、深色化淡色毛髮及治療白斑等作用之醫藥組成物。 易言之’本發明所屬技術領域中具有通常知識者應能 理解’本發明所提供之組成物可以應用於，促進皮膚傷口 17 201012928 修復，例如加速傷口的癒合、降低傷口感染、抑制傷口發 炎、減少疤痕等；活化毛囊細胞，例如促進毛囊細胞生長、 增生毛髮、治療禿髮；活化黑色素細胞，例如促進黑色素 細胞生長、深色化淡色毛髮、治療白斑等方面。從而，前 述組成物於製備處理前述各種狀況之藥物的應用，亦為本 發明之範疇所涵蓋。 實施例 囑^ 本發明之實際實施態樣及實施方法如下所示，但本發 明所屬領域中常用、眾所周知或可得而知的各種已確立之 技術手段’則酌減冗贅重覆之說明。又，各實施例係用以 說明本發明而非限定本發明於該等實施例上。 實施例1 组成物的製造舆共培養可行性實驗 (一）獲取組成物 本發明係關於一種包括有人類成體幹細胞與釋放因子 之組成物。為取得前述組成物，在本實施例中所採取的具 體技術手段係自一來源人類個體獲取一人類臍帶；除去血 管後切碎臍帶組織，接著分離在分離出花頓氏膠後，進一 步利用胰蛋白分解酵素與膠原蛋白分解酵素進行分解處 理。在本實施例中所採取的具體技術手段係以0.05%之胰 蛋白分解酵素與0.3 wuensch單位之膠原蛋白分解酵素， 於攝氏37度下反應1小時，然後以140G下離心1〇分鐘 18 201012928 的方式分離出一群單核間質幹細胞，植入盛有培養基之培 養皿。前述培養基的成分包括alpha-最小必需培養基、丨❶/。抗 生素、10%胎牛血清、1 mM L-麵醯胺、1 mM非必需胺基 酸及ImM丙酮酸鈉（此培養基係一周知的先前技術，參見 Stem Cells 21:105-110,2003; Stem Cells 22:649-658,2004) » 並於攝氏37度、含5%二氧化碳的恆濕培養箱令培養。在 本實施例中所採取的具體技術手段係經過24小時後，以新 鮮的前述培養基更換前更換含有不貼附細胞之懸浮液，呈 ❹ 貼附形態之細胞則留置於原培養皿繼續以前述條件培養。 將貼附於培養皿的細胞，以前述培養基培養一段適當 時間使細胞增殖至相當數量，在本實施例中所採取的具艘 技術手段係經過至少84小時’使貼附於培養皿的細胞密度 大於107個/平方公分；在前述培養的84小時之中，可以 前述培養基至少更換一次前述細胞懸浮液。 為取得前述組成物’在本實施例中所採取的具體技術 手段係將前述體外培養增殖的人類臍帶間質幹細胞，以不 參 含任何種類蛋白酵素與乙烯二胺四乙酸的脫附試劑，令脫 附反應在攝氏37度的溫度下進行2至5分鐘，將貼附的細 胞與培養孤分離；完成時以懸浮後每毫升有1〇5至1〇6個 細胞數計算，使用不含鈣、鎂離子的標準磷酸鹽缓衝液將 細胞重新懸浮’製備成為人類成體幹細胞；亦可進一步溶 於10%的試藥級二甲基亞職中，保存於可調控升降溫速率 的冷藏庫中。 將前述細胞懸浮液收集後以冷凍乾燥法製得一脫水的 19 .201012928 粉末，在本實施例中所採取的具體技術手段係以冷凍乾燥 機’在攝氏零下4度將前述細胞懸浮液抽至i〇_5Bar並維 持24小時，製得脫水粉末後選擇一重量百分率介於〇丨％至 1 的濃度計算’使用適當量之不含鈣、鎂離子的標準碟 酸鹽緩衝液將該脫水粉末回溶，得到釋放因子組成物。 (二）人類臍帶間質幹細胞與脂肪幹細胞體外共培養 在本實施例中所採用的臍帶間質幹細胞來源與上節之 © 人類非造血型成體幹細胞相同。又，在本實施例中所採用 的脂肪幹細胞係源自於人類脂肪組織。前述脂肪組織係於 抽脂手術中經低壓抽取技術收集之人類腹部體脂肪混合 物’由財團法人長庚紀念醫院美容醫學中心提供，捐贈人 為一成年女性’體脂指數（body mass index，BMI)為26.1。 在本實施例中所採取的具體技術手段係將體脂肪混合 物分層為脂質層與液態層’將脂質層以〇.〇5%之肤蛋白分 解酵素與0.3 wuensch單位之膠原蛋白分解酵素於攝氏37 _ 度下反應1小時後’在8〇〇g下離心1 〇分鐘；同時將液態 層以400g離心10分鐘，收集兩組離心所得之懸浮液與懸 浮顆粒’以一紅丘球水解試劑溶液（使用eBi〇seience lx rbC LysisBuffer’此試劑組成與使用方法係一周知的先前技術） 在常溫下反應5分鐘後重新懸浮，再通過一平均孔徑1 〇〇 微米的濾、膜。 脂肪幹細胞同時存在於脂質層與液態層，而且在原有 組織中的含量比例’抽脂手術前與體外分離純化之後可能 20 201012928 因機械力影響而有很大的差異（參見journal 〇f Cellular Physiology 208:64-76, 2006)，因此在本實施例中所採取的 具體技術手段係將兩相^分離純化產物混合使用。In the f step of the method, in order to desorb the cells from the culture dish, a desorption reagent containing no proteoglycan and ethylenediaminetetraacetic acid may be used, and the desorption reaction is carried out at a temperature of 37 degrees Celsius 2 to After 5 minutes, the cells can be resuspended and stored in standard phosphate buffer without calcium or magnesium ions. In the g step of the method, the previously used standard phosphate buffer solution can be used to select a concentration percentage of 〇.1% to 1% by weight, and the dehydrated powder of the aforementioned cell suspension is dissolved back to be released. Factor composition. Further, the aforementioned human non-hematopoietic adult stem cells are further subjected to filtration, <L drop, centrifugation, chromatography, extraction, or other physical separation and purification procedures. (III) Use of Composition Another aspect of the present invention relates to the use of the aforementioned composition. As described above, the present invention is based on the step of in vitro culture of human umbilical cord mesenchymal stem cells, and obtains the aforementioned composition. In an embodiment of the invention, the composition 201012928 comprises human adult stem cells and a release factor. Both the aforementioned human adult stem cells and the release factor have the function of promoting the activation of peripheral cells, and can be used as an active or auxiliary component of a medical or medicated cosmetic related to skin health care. The invention uses an artificial wound on the skin of an experimental animal as an example, and the wound is applied by using the above composition, and it can be observed that both can promote wound healing, reduce wound infection, inhibit inflammation and reduce scar, so the above-mentioned one can be extended. Both substances can be used to promote skin wound repair, which in turn can be applied to the manufacture of pharmaceutical compositions that have the effects of accelerating wound healing, reducing wound infection, inhibiting wound inflammation and reducing scarring. The invention further shaves a certain area of the skin of an experimental animal. 'The above composition is used to smear the block, and both can be observed to promote the hair growth of the block. The function of activating hair follicle cells can be applied to the manufacture of a pharmaceutical composition having the effects of promoting growth of hair follicle cells, proliferating hair, and treating baldness. In the present invention, the surface of the skin of the experimental animal whose skin is whitened due to aging is coated with the above composition, and it can be observed that both of them can reduce white hair, increase black or ochre hair growth. The animal's melanocyte activity decreases or dies due to aging, but the hair follicle cells continue to act and grow white hair. Therefore, the results of this experiment can be extended to activate the skin melanocytes, which can be applied to manufacturing. A pharmaceutical composition that promotes the growth of melanocytes, darkening and lightening hair, and the treatment of leukoplakia. It is to be understood by those of ordinary skill in the art to which the present invention pertains that the composition provided by the present invention can be applied to promote repair of skin wounds 17 201012928, such as accelerating wound healing, reducing wound infection, inhibiting wound inflammation, Reduce scars, etc.; activate hair follicle cells, such as promoting hair follicle cell growth, proliferating hair, treating alopecia; activating melanocytes, such as promoting melanocyte growth, darkening pale hair, treating white spots, and the like. Thus, the use of the foregoing compositions for the preparation of a medicament for treating the various conditions described above is also encompassed within the scope of the invention. EXAMPLES The actual embodiments and implementation methods of the present invention are as follows, but various established technical means that are commonly used, well-known or available in the art to which the present invention pertains are described as redundant and redundant. Further, the embodiments are intended to illustrate the invention and not to limit the invention to the embodiments. Example 1 Manufacture of composition 舆 Co-culture feasibility test (I) Obtaining composition The present invention relates to a composition comprising human adult stem cells and a release factor. In order to obtain the foregoing composition, the specific technical means adopted in the present embodiment is to obtain a human umbilical cord from a source human body; after removing the blood vessel, the umbilical cord tissue is chopped, and then separating and separating the Watson's gum, further utilizing the pancreas Proteolytic enzymes and collagen decomposing enzymes are decomposed. The specific technical means adopted in this example is to react with 0.05% trypsin and 0.3 wuensch unit of collagen decomposing enzyme at 37 ° C for 1 hour, then centrifuge at 140 G for 1 〇 18 201012928 A group of mononuclear mesenchymal stem cells were isolated and implanted in a culture dish containing medium. The components of the aforementioned medium include alpha-minimum essential medium, 丨❶/. Antibiotics, 10% fetal bovine serum, 1 mM L-face decylamine, 1 mM non-essential amino acid, and 1 mM sodium pyruvate (this medium is a well-known prior art, see Stem Cells 21: 105-110, 2003; Stem Cells 22: 649-658, 2004) » Cultured in a constant humidity chamber containing 5% CO2 at 37 °C. The specific technical means adopted in this embodiment is that after 24 hours, the suspension containing the unattached cells is replaced before the fresh medium is replaced, and the cells in the sputum attachment form are left in the original culture dish to continue. Conditional culture. The cells attached to the culture dish are cultured in the aforementioned medium for a suitable period of time to proliferate the cells to a considerable amount. In the present embodiment, the technical means adopted is to make the cell density attached to the culture dish after at least 84 hours. More than 107 / cm ^ 2; during the 84 hours of the aforementioned culture, the aforementioned medium suspension may be replaced at least once with the aforementioned medium. In order to obtain the aforementioned composition, the specific technical means adopted in the present embodiment is to use the above-mentioned in vitro cultured human umbilical cord mesenchymal stem cells to be desorbed without any kind of proteinase and ethylenediaminetetraacetic acid. The desorption reaction is carried out at a temperature of 37 ° C for 2 to 5 minutes, and the attached cells are separated from the culture; at the time of completion, the number of cells per ml is 1 to 5 to 1 6 cells after suspension, and calcium-free is used. The standard phosphate buffer of magnesium ion resuspend the cells to prepare human adult stem cells; it can be further dissolved in 10% of the reagent-grade dimethyl sub-sector, and stored in a refrigerator capable of regulating the temperature rise and fall. . After the above cell suspension was collected, a dehydrated 19.201012928 powder was obtained by freeze-drying, and the specific technical means adopted in this example was to freeze the aforementioned cell suspension at a temperature of minus 4 degrees Celsius in a freeze dryer. 〇_5Bar and maintain for 24 hours, after the dehydrated powder is prepared, select a concentration percentage of 〇丨% to 1 to calculate 'Use the appropriate amount of standard discate buffer containing no calcium or magnesium ions to return the dehydrated powder. Dissolved to obtain a release factor composition. (II) Co-culture of human umbilical cord mesenchymal stem cells and adipose stem cells in vitro The source of umbilical cord mesenchymal stem cells used in this example is the same as that of the human non-hematopoietic adult stem cells in the previous section. Further, the adipose stem cell line used in the present example is derived from human adipose tissue. The aforementioned adipose tissue is a human abdominal body fat mixture collected by a low pressure extraction technique in liposuction surgery. The donor's body mass index (BMI) is 26.1. . The specific technical means adopted in this embodiment is to layer the body fat mixture into a lipid layer and a liquid layer. The lipid layer is 〇. 5% of the skin protein decomposing enzyme and 0.3 wuensch unit of collagen decomposing enzyme in Celsius. After 1 hour of reaction at 37 °C, 'centrifuge at 8 〇〇g for 1 〇 minute; while centrifuging the liquid layer at 400 g for 10 minutes, collect two suspensions and suspension particles obtained by centrifugation to a red sorbate hydrolysis reagent solution (Use eBi〇seience lx rbC LysisBuffer' This reagent is a well-known prior art method of use.) Re-suspension after 5 minutes of reaction at room temperature, and then pass through a filter and membrane with an average pore size of 1 μm. Adipose-derived stem cells are present in both the lipid layer and the liquid layer, and the ratio of the content in the original tissue is different from that before the liposuction and after in vitro purification. 20 201012928 is greatly different due to mechanical influence (see journal 〇f Cellular Physiology 208). : 64-76, 2006), therefore, the specific technical means adopted in this embodiment is to use a mixture of two phases separated and purified products.

在本實施例中所採取之體外共培養增殖的具體技術手 段’係將自人體組織分離出的臍帶與脂肪幹細胞分別以前 述培養基在攝氏37度、含5%二氧化碳的恆濕培養箱中培 養24小時後’以細胞計數器計算貼附細胞的數量，再以前 述之脫附試劑，令脫附反應在攝氏3 7度的溫度下進行2至 5分鐘，將兩種貼附的細胞與培養皿分離，以生理緩衝液 重新懸浮後，將相同數量的兩種細胞置入含新鮮培養基之 培養皿重新貼附增殖。 在本實施例中所採取之實驗方法係將此體外共培養的 成體幹細胞，與前述單一來源成體幹細胞體外培養同步進 行，以同樣的每48小時間隔收集一次細胞懸浮液並更換新 鮮培養基，並記錄細胞增殖數；記錄結果顯示混合培養成 體幹細胞生長速度明顯低於臍帶間質幹細胞單獨培養（細胞 數超過1〇7個/平方公分的時間各為⑼與84小時)，然而 兩組體外培養所收集的細胞懸浮液平均蛋白質濃度非常接 近，混合培養組的平均標準相因前兩組濃度㈣偏低而 放大（平均值與標準差分別為2 + & π 匈 2,295±540ppm 與 2，173士 1 72ppm)。 由本實施例可合理推測 胞依照等比例混合培養的方 胞體外培養增殖與細胞懸浮 以其他來源非造血型成體幹細 式’亦可得到類似的成體幹細 液收集結果；以人類臍帶與脂 21 201012928 肪間質幹細胞為例，脂肪幹細胞表面抗原特徵在培養前一 般已知為 CD31-，CD34 +，CD45-,CD90 +，CD105-，CD146+ ,但 在體外培養增殖過程中產生以下特徵變化：CD34-，CD45-， CD29 +，CD105 +，與臍帶間質幹細胞性質接近，尤其以CD34 與CD 105這兩項已知與間質幹細胞表現之線性關係最明顯 的抗原特徵顯示，不同來源之非造血型成體幹細胞的體外 共培養及衍生物，在體外培養增殖過程中其性質類似於單 一來源非造血型成體幹細胞體外培養之前例，由此亦可推 論至其他例如骨髓、臍帶血、上皮組織、乳牙等來源的人 類非造血型成體幹細胞》易言之，前述人類非造血型成體 幹細胞係得與一種或一種以上的另種人類非造血型成體幹 細胞共培養’並得進而藉由前述技術手段獲取前述組成 物。前述另種人類非造血型成體幹細胞可為源於骨趙、臍 帶血、上皮組織、乳牙或脂肪組織者。 實施例2 ❹ 以免疫不全小鼠測試人類成體幹細胞组成物與 釋放因子組成物之傷口癒合促進效果 (一）測試人類成體幹細胞促進傷口癒合的效果 在本實施例中所採用的人類成體幹細胞，均製備自繼 代十次以内的前述人類臍帶間質幹細胞體外培養。 在本實施例中所採用的實驗動物均為來自財團法人 國家動物中心、實驗鼠齡為8週大的免疫不全小鼠（Nude mice: BLAB/cAnN-Cg-Foxninu/CrlNarl)，飼養過程均處在 22 201012928 無菌環境中。 在本實施例中所採用的實驗方法是先將前述小鼠分為 相同隻數（例如每組3隻）的人類成體幹細胞治療組與控制 組。將所有小鼠背部以穿刺刀製造 且1約5毫米之深及皮 下組織的傷口。傷口建立完成後， #又人類成體幹細胞治 療組的小氟傷口各滴入5〇微升的人 J入類成體幹細胞（含有約 H)·個細胞）’每隻控制組的小鼠傷口各滴人Μ微升新鮮The specific technical means for in vitro co-culture proliferation adopted in the present embodiment is to culture the umbilical cord and the adipose-derived stem cells isolated from the human tissue in the above-mentioned culture medium at 37 degrees Celsius and a humidified incubator containing 5% carbon dioxide. After the hour, calculate the number of attached cells by the cell counter, and then remove the desorption reaction with the aforementioned desorption reagent for 2 to 5 minutes at a temperature of 37 ° C to separate the two attached cells from the culture dish. After resuspending in physiological buffer, the same number of the two cells were placed in a petri dish containing fresh medium and re-adhered to proliferation. The experimental method adopted in this example is to carry out the in vitro co-culture of adult stem cells in synchronization with the aforementioned single-source adult stem cells in vitro, and collect the cell suspension and replace the fresh medium at the same interval every 48 hours. The number of cell proliferation was recorded. The results showed that the growth rate of mixed cultured stem cells was significantly lower than that of umbilical cord mesenchymal stem cells alone (the number of cells exceeded 1〇7/cm 2 (9) and 84 hours, respectively). The average protein concentration of the collected cell suspensions was very close, and the average standard phase of the mixed culture group was amplified due to the low concentration of the first two groups (four) (average and standard deviation were 2 + & π Hungarian 2,295 ± 540 ppm and 2, respectively). 173 士 1 72ppm). According to the present embodiment, it can be reasonably estimated that the cells are cultured in an equal proportion and cultured in vitro, and the cells are suspended in a non-hematopoietic form of other sources, and a similar dry liquid collection result can be obtained; Lipid 21 201012928 For example, adipose-derived stem cells are generally known as CD31-, CD34+, CD45-, CD90+, CD105-, CD146+ before culture, but produce the following characteristic changes during in vitro culture and proliferation. : CD34-, CD45-, CD29 +, CD105 +, close to the nature of umbilical cord mesenchymal stem cells, especially the antigenic features of CD34 and CD 105, which are known to have the most linear relationship with mesenchymal stem cell expression, showing different sources. The in vitro co-culture and derivatives of non-hematopoietic adult stem cells are similar in nature to the single-source non-hematopoietic adult stem cells in vitro, and can be inferred to other such as bone marrow, cord blood, Human non-hematopoietic adult stem cells derived from epithelial tissues, deciduous teeth, etc. It is easy to say that the aforementioned human non-hematopoietic adult stem cell line has one One or more other human non-hematopoietic adult stem cells are co-cultured and the aforementioned composition is obtained by the aforementioned technical means. The aforementioned other human non-hematopoietic adult stem cells may be derived from bone, cord blood, epithelial tissue, deciduous teeth or adipose tissue. Example 2 伤口 Test of wound healing promoting effect of human adult stem cell composition and release factor composition in immunocompromised mice (I) Effect of testing human adult stem cells for promoting wound healing Human adult body used in this example The stem cells were prepared by in vitro culture of the aforementioned human umbilical cord mesenchymal stem cells within ten times of subculture. The experimental animals used in this example were all from the National Animal Center of the consortium, and the experimental mice were 8 weeks old (Nude mice: BLAB/cAnN-Cg-Foxninu/CrlNarl). In the sterile environment of 22 201012928. The experimental method employed in this example was to first divide the aforementioned mice into the same number (e.g., 3 per group) of human adult stem cell treatment and control groups. All mice were made with a puncture knife and a wound of about 5 mm deep and subcutaneous tissue. After the wound was established, the small fluoride wounds of the human adult stem cell treatment group were each instilled into 5 μL of human J into adult adult stem cells (containing about H)·cells' wounds in each control group. Each drop of people is slightly new

的前述培養基;所有小鼠的傷口皆以透明薄膜狀的貼布(〇.p site)貼附。 天；實驗觀察結果發 口在第14天已經完全 皮層受傷後所產生的 在本實施例中本實驗共進行14 現，人類成體幹細胞治療組小鼠的傷 癒合’表皮層已完全覆蓋傷口，因真 傷口在第14天並未完全癒 仍有痂皮及明顯之真皮缺 凹陷疤痕也較淺；控制組小鼠的 合’表皮層仍未完全覆蓋傷口， 陷之凹陷症痕。 此實驗證明臨床上可利用前述之人類成體幹細胞來應 用於加速皮膚傷口癒合；又根據前述實驗觀察之傷口癒合 結果，所屬領域中具有通常知識者可合理推知包括傷口癒 合不良、燒烫傷、青春痘、電射美容術後傷口，及一切未 提及所有可能之皮膚傷口，均可能藉由前述之人類成體幹 細胞組成物達到促進傷口癒合之效果。 由本實施例可合理推測以前述之人類成體幹細胞作為 有效成分的促進傷口癒合藥劑，可提供使過敏與交叉感染 降至最低的效果。 23 201012928 (二）測試錢釋放因子組成物促進傷口癒合的效果 在本實施例中所採用的釋放因子組成物，均製備自繼 代十次以内的前述人類擠帶間質幹細胞體外培養。 在本實施例中所採用的實驗動物均來自財團法人國家 動物中心、實驗鼠齡8週大的免疫不全小鼠（Nude mice: BLAB/CAnN-Cg-F〇xninu/CrlNarl)，飼養過程均處在無菌環 境中。 〇 在本實施例中所採用的實驗方法是先將前述小鼠分為 相同隻數的釋放因子組成物治療組與控制組。將所有小鼠 背部以穿刺刀製造直徑約5毫米之深及皮下組織的傷口。 傷口建立完成後，每隻釋放因子組成物治療組的小鼠傷口 母天各滴入20微升的釋放因子組成物，每隻控制組的小鼠 傷口每天各滴入20微升新鮮的前述培養基；所有小鼠的傷 口皆以透明薄膜狀的貼布（O.p. site)貼附。 在本實施例中本實驗共進行14天；實驗觀察結果發 ® 現’釋放因子組成物治療組小鼠的傷口在第14天已部份癒 合為直徑約1.5到2.0毫米，表皮層已部份覆蓋傷口；控 制組小鼠的傷口在第14天直徑均大於3.0毫米，表皮層仍 未覆蓋傷〇。 此實驗證明臨床上可利用前述之釋放因子組成物來應 用於加速皮膚傷口癒合；又根據前述實驗觀察之傷口癒合 結果’所屬領域中具有通常知識者可合理推知包括傷口癒 合不良、燒燙傷、青春痘、電射美容術後傷口，及一切未 24 201012928 提及所有可能之皮膚傷口，均 成物達到促進傷口癒合之效果 由本實施例可合理推測以 有效成分的促進傷口癒合藥劑 降至最低的效果。 可能藉由前述之釋放因子組 0 前述之釋放因子組成物作為 ，可提供使過敏與交又感染 實施例3 以具免疫力之ICR舆CS7小鼠測試釋玫因子 组成物之傷口癒合促進效果 ❹ 纟本實施例中所採用的釋放因子組成物與實施例i所 採用的完全相同。 一在本實施例令所採用的實驗動物均為來自財團法人國 家動物中〜、實驗鼠齡為8週大、具有免疫力的心與 C57BL/6 (簡稱 C57)小鼠。 ’ 在本實施例中所採用的實驗方法是先將前述兩種小鼠 各分為相同隻數的釋放因子組成物治療組與控制組。將所 彳小鼠背部以穿刺刀製造直徑約5毫米之㈣皮下組織的 參傷口。傷口建立完成後，每隻釋放因子組成物治療組的小 鼠傷口每天各滴人2G微升的釋放因子組成物，每隻控制組 的小鼠傷口每天各“ 2G微升新鮮的前述培養基；所有小 鼠的傷口皆以透明薄膜狀的貼布(〇 p, 貼附。 在本實施例令以ICR小鼠測試的實驗共進行17天； 實驗觀察結果發現’在第17天時釋放因子組成物治療組小 、傷已# &癒合為直徑约為2〇 $米；控制組小鼠的 傷口在第17天直徑約為4〇毫米。 25 201012928 在本實施例中以C57小鼠測試本實驗共進行9天；實 驗觀察結果發現，在第9天時釋放因子組成物治療組小鼠 的傷口已部份癒合為直徑約為10毫米；控制組小鼠的傷 口在第9天直徑約為2.0毫米。 本實施例中的兩組實驗結果均顯示前述之釋放因子組 成物具有促進傷口癒合之效果。 實施例4 參以C3HT小鼠測試釋放因子组成物之增生毛髮促進效 果 在本實施例中所採用的釋放因子組成物與實施例1所 採用的完全相同。 在本實施例中所採用的實驗動物均為來自財團法人國 家動物中心、實驗鼠齡為8週大的C3HT小鼠；此種C3HT小 鼠在7周大時會自然脫毛。在本實施例中所採用的實驗方 法是先將三隻C3HT小鼠的背部予以剃毛（約為2·5χ5 〇公 參 分的區塊），分別依序指定為釋放因子組成物治療組、 1%釋放因子組成物治療組、與控制組。每天早晚（相距8 至10小時）各一次分別塗抹i毫升的5%釋放因子組成物、 1%釋放因子組成物、與前述標準磷酸鹽緩衝液於三組小 鼠的剃毛區塊。 在本實施例中本實驗共進行4週’觀察各組小鼠剃毛 區塊毛髮生長的面積；實驗觀察結果發現5%釋放因子組 成物組毛髮生長約覆蓋70%的剃毛區塊，1%釋放因子組 26 201012928 成物組毛髮生長約覆蓋50%的剃毛區塊，控制組毛髮生 長約覆蓋20°/❶的剃毛區塊。 此實驗證明前述之釋放因子組成物可促進毛髮生長， 並進一步證明促進的效果與釋放因子組成物的濃為正向關 係。此實驗證明臨床上可利用前述之釋放因子組成物來應 用於促進毛髮生長；又根據前述實驗觀察之促進毛髮生長 結果’所屬領域中具有通常知識者可合理推知包括禿髮、 掉髮、毛髮稀疏、髮線後移、毛囊細胞老化、及一切未提 ® 及所有可能與毛髮生長相關之應用需求，均可能藉由前述 之釋放因子組成物達到促進毛髮生長之效果。 實施例5 以B ALB/c小鼠測試釋放因子组成物之 活化黑色素細胞效果 在本實施例中所採用的釋放因子組成物與實施例1所 採用的完全相同。 ® 纟本實施例中所採用的實驗動物均為來自財團法人國 家動物中心、實驗鼠齡18個月大的BALB/e小鼠。鼠齡約 為！5到18個月的BALB/C小鼠會因年齡的衰老而明顯喪 失黑色素細胞的活性，因而一般會長出約佔全身表皮面積 20-30%比例的白毛’並且有明顯脫毛的跡象。在本實施例 中所採㈣實驗方法是先將前述18則大的W小鼠 6隻各分為釋放因子組成物治療組與控制組各3隻·每天 於每隻釋放因子組成物治療組小鼠的背部（約為2·5χ5·〇公 27 201012928 分的區塊）各-次塗抹i毫升的5% #放因子組成物，同 時於每隻控制組小鼠的背部各—次塗#丨毫升的前述 磷酸鹽緩衝液。 在本實施例中本實驗共進行12週，觀察各組小氣背部 白毛與脫毛的改善情形(觀察時實驗鼠齡已為…固 鲁 ❹ 發現釋放因子组成物治療组3隻小鼠的背部已無明顯的白 毛區塊’同時亦有大量黑色的新毛生長 '黑色毛所佔背部 =均約為8。%;而控制组的3隻小鼠的 為：續=脫毛（小鼠年齡的3個月約等於人類 9年以造成大面積表皮無毛之裸露’因此白毛所 面積的比例已經無從計算。 反 此實驗證明前述之釋放因子組成物可促進深色化已存 在的白色或灰色毛髮，並且促 進衣色化已存 長。此實驗證明臨床上可利用 S ’衣#新毛髮生 用於深色化淡色毛髮；又根據二釋放因子組成物來應 淡色毛髮的結果，所屬領域中：有：觀察之促進深色化 包括白斑、以及一切未提及所有：通常知識者可合理推知 胞活性降低或死亡相關之應I能與治療皮膚黑色素細 放因子組成物達到效果。需求，均可能藉由前述之釋 【主要元件符號說明】 無 28The aforementioned medium; all mouse wounds were attached with a transparent film-like patch (〇.p site). The results of the experimental observations were obtained after the complete cortical injury on the 14th day. In this example, the experiment was performed in 14 cases. The wound healing of the human adult stem cell treatment group's epidermis layer completely covered the wound. Because of the true wound on the 14th day, there was no more suede and the obvious dermis was scarred. The control group's mice's epidermis still did not completely cover the wound, and the depression was trapped. This experiment proves that the aforementioned human adult stem cells can be clinically applied to accelerate the healing of skin wounds; and according to the results of wound healing observed in the foregoing experiments, those having ordinary knowledge in the art can reasonably infer that the wounds are poorly healed, burned, Acne, electro-acoustic post-operative wounds, and all possible skin wounds are not mentioned, and the effect of promoting wound healing can be achieved by the aforementioned human adult stem cell composition. From the present embodiment, it is possible to reasonably speculate that the wound healing agent which uses the aforementioned human adult stem cells as an active ingredient can provide an effect of minimizing allergy and cross-infection. 23 201012928 (II) Effect of testing money release factor composition for promoting wound healing The release factor compositions used in this example were prepared by in vitro culture of the aforementioned human squeezed mesenchymal stem cells within ten times of subculture. The experimental animals used in this example were all from the National Animal Center of the consortium and the 8-week-old immunocompromised mice (Nude mice: BLAB/CAnN-Cg-F〇xninu/CrlNarl). In a sterile environment.实验 The experimental method used in this example is to first divide the aforementioned mice into the same number of release factor composition treatment groups and control groups. A wound of about 5 mm in diameter and subcutaneous tissue was made with a puncture knife on the back of all mice. After the wound was established, 20 μl of the release factor composition was added to each of the mice in the treatment group of the release factor composition, and 20 μl of fresh medium was added to each wound of the control group. The wounds of all mice were attached with a transparent film patch (Op site). In the present example, the experiment was carried out for a total of 14 days; the experimental observations were made. The wounds of the mice in the treatment group were partially healed to a diameter of about 1.5 to 2.0 mm on the 14th day, and the epidermis was partially The wound was covered; the wounds of the control group mice were all larger than 3.0 mm on the 14th day, and the epidermis was still not covered with scars. This experiment proves that the above-mentioned release factor composition can be used clinically to accelerate the healing of skin wounds; and the wound healing result observed according to the foregoing experiment can be reasonably inferred to include poor wound healing, burns, and youth. Acne, electro-acoustic post-operative wounds, and everything is not 24 201012928 All possible skin wounds are mentioned, and the effect of achieving the wound healing is determined by the present embodiment. It can be reasonably presumed that the effect of promoting the wound healing agent with the active ingredient is minimized. . It is possible to provide the wound healing promoting effect of the immune-competent ICR 舆CS7 mouse test release composition by using the aforementioned release factor group 0 as the release factor composition described above. The release factor composition employed in this example is identical to that employed in Example i. The experimental animals used in the present embodiment were all from the national animals of the corporation, and the experimental mice were 8-week-old, immunogenic, and C57BL/6 (C57) mice. The experimental method employed in this example was to divide the aforementioned two mice into the same number of release factor composition treatment groups and control groups. A ginseng wound of a subcutaneous tissue having a diameter of about 5 mm was made with a puncture knife on the back of the mouse. After the wound was established, each of the mice in the treatment group of the release factor composition was treated with 2 G of microliters of release factor per day, and each control group of mice had "2 G microliters of fresh medium" per day; The wounds of the mice were all covered with a transparent film (〇p, attached. In the present example, the experiment tested with ICR mice was carried out for 17 days; the experimental observation found that the release of the factor composition on the 17th day The treatment group was small, wounded # & healed to a diameter of about 2 〇 $ m; the wound of the control group of mice was about 4 mm in diameter on day 17. 25 201012928 In this example, the experiment was tested with C57 mice. A total of 9 days were carried out; experimental observations showed that the wounds of the mice in the treatment group on the 9th day had partially healed to a diameter of about 10 mm; the wounds of the control group had a diameter of about 2.0 on the 9th day. The results of the two sets of experiments in this example show that the aforementioned release factor composition has an effect of promoting wound healing. Example 4 The proliferative hair promoting effect of the C3HT mouse test release factor composition is in this embodiment. The release factor composition used was exactly the same as that used in Example 1. The experimental animals used in this example were all C3HT mice from the National Animal Center of the consortium and were 8 weeks old. C3HT mice naturally depilate at 7 weeks of age. The experimental method used in this example is to shave the back of three C3HT mice (approximately 2.5·5 〇 5 〇 参 )), depending on The order was designated as the release factor composition treatment group, the 1% release factor composition treatment group, and the control group. Each morning and evening (8 to 10 hours apart) each applied 1 ml of 5% release factor composition and 1% release factor. The composition and the aforementioned standard phosphate buffer solution were shaved in three groups of mice. In this example, the experiment was carried out for 4 weeks to observe the area of hair growth of the shaved blocks of each group of mice; experimental observation results The 5% release factor composition group was found to cover about 70% of the shaved area, and the 1% release factor group 26 201012928. The hair growth of the adult group covered about 50% of the shaved area, and the hair growth of the control group covered about 20°. /❶ shaving This experiment demonstrates that the aforementioned release factor composition promotes hair growth and further demonstrates that the promoted effect is positively proportional to the concentration of the release factor composition. This experiment demonstrates that the release factor composition described above can be clinically applied. Promote hair growth; and promote hair growth results according to the aforementioned experimental observations. Those having ordinary knowledge in the art can reasonably infer that including baldness, hair loss, hair thinning, hairline migration, hair follicle cell aging, and all unrecognized and All application requirements related to hair growth may achieve the effect of promoting hair growth by the aforementioned release factor composition. Example 5 Activated melanocyte effect of releasing release factor composition in B ALB/c mice in this embodiment The release factor composition employed in the examples was identical to that employed in Example 1. ® The experimental animals used in this example were BALB/e mice from the National Animal Center of the consortium and 18 months old. The age of the rat is about! BALB/C mice from 5 to 18 months will significantly lose the activity of melanocytes due to age aging, and thus generally produce white hairs that account for about 20-30% of the total epidermal area and have signs of significant hair loss. In the present embodiment, the experimental method is as follows: the 18 large W mice are divided into the release factor composition treatment group and the control group, respectively, 3 per day, each of the release factor composition treatment group is small. The back of the mouse (approximately 2·5χ5·〇公27 201012928 points) were coated with 5% of the milliliters of the composition of each factor, and simultaneously on the back of each control group of mice. ML of the aforementioned phosphate buffer. In this example, the experiment was carried out for a total of 12 weeks, and the improvement of white hair and hair removal in each group of small gas was observed (the experimental mouse age was observed... Gu Luzhen found that the release factor composition of the treatment group 3 mice back There was no obvious white hair block 'there was also a large amount of black new hair growth' black hair accounted for the back = about 8.%; and the control group of 3 mice was: continued = hair removal (mouse age) 3 months is about equal to human beings for 9 years to cause a large area of skinless nudity. Therefore, the proportion of white hair area has not been calculated. The experiment proves that the aforementioned release factor composition can promote the darkening of existing white or gray. Hair, and promote the colorization of the clothing has been long. This experiment proves that the clinical use of S 'clothing #新毛 occurs for darkening pale hair; and according to the two release factor composition to light hair, in the field : There are: observation of the promotion of darkening, including white spots, and everything is not mentioned: usually the knowledge can reasonably infer that the decrease in cell activity or death should be related to the treatment of skin melanin fine factor composition Fruit. Demand, are likely by reference numerals DESCRIPTION Main the release of free 28

Claims (1)

.201012928 十、申請專利範圍： 1. 一種組成物，其係以源自一人體組織之人類非造血 型成體幹細胞’使用液態培養基進行體外培養增殖所製得 者。 2·如申請專利範圍第1項所述之組成物，前述人體組 織係選自由骨髓、脂肪、臍帶以及乳牙所構成的群組。 3. 如申請專利範圍第1項所述之組成物，前述液態培 養基至少包括有：alpha_最小必需培養基（MEM)、1%抗生 φ 素、10%胎牛血清、ImM L-麩醯胺、ImM非必需胺基酸及 ImM丙酮酸納。 4. 如申請專利範圍第1項所述之組成物，其進一步包 括有最小必需培養基、抗生素、胎牛血清、胺基酸以及丙 酮酸鈉。 5. 如申請專利範圍第1至4項中任一項所述之組成 物’其進一步包括有： 將前述人類非造血型成體幹細胞進行體外培養增殖所 ❹ 獲取的一人類非造血型成體幹細胞；以及將前述人類非造 血型成體幹細胞進行體外培養增殖所獲取的一釋放因子。 6. 如申請專利範圍第5項所述之組成物，前述成體幹 細胞係經進一步分離者。 7. 如申請專利範圍第6項所述之組成物，前述人類非 造血型成體幹細胞係處於由過濾、沉降、離心、層析、萃 取、及其它物理性分離純化所構成的群組中至少一種程序 處理過的狀態。 29 201012928 8. 如申請專利範圍第5項所述之組成物，前述釋放因 子組成物具有促進周圍細胞活化與增生的功能。 9. 如申請專利範圍第5項所述之組成物，前述釋放因 子係選自由生長因子、胞核嘧啶、訊息核苷酸、調節性蛋 白質及其組合所構成的群組。 10. 如申請專利範圍第5項所述之組成物’其狀態係 處於選自由液體狀態、冷凍乾燥狀態、與回溶於生理緩衝 液狀態所構成之群組。 11.如申請專利範圍第i項所述之組成物.201012928 X. Patent Application Range: 1. A composition obtained by in vitro culture and proliferation of human non-hematopoietic adult stem cells derived from a human tissue using liquid medium. 2. The composition according to claim 1, wherein the human body tissue is selected from the group consisting of bone marrow, fat, umbilical cord and deciduous teeth. 3. The composition according to claim 1, wherein the liquid medium comprises at least: alpha_minimum essential medium (MEM), 1% antibiotic oxime, 10% fetal bovine serum, 1 mM L-glutamine, ImM non-essential amino acid and 1 mM sodium pyruvate. 4. The composition of claim 1, further comprising a minimum essential medium, an antibiotic, fetal calf serum, an amino acid, and sodium ketone. 5. The composition according to any one of claims 1 to 4, which further comprises: a human non-hematopoietic adult obtained by inoculating the aforementioned human non-hematopoietic adult stem cells in vitro; Stem cells; and a release factor obtained by in vitro culture and proliferation of the aforementioned human non-hematopoietic adult stem cells. 6. The composition according to claim 5, wherein the adult stem cell line is further separated. 7. The composition according to claim 6, wherein the human non-hematopoietic adult stem cell line is in a group consisting of filtration, sedimentation, centrifugation, chromatography, extraction, and other physical separation and purification. A state that has been processed by a program. 29 201012928 8. The composition according to claim 5, wherein the release factor composition has a function of promoting activation and proliferation of surrounding cells. 9. The composition of claim 5, wherein the release factor is selected from the group consisting of growth factors, cytosine, message nucleotides, regulatory proteins, and combinations thereof. 10. The composition as described in claim 5, wherein the state is selected from the group consisting of a liquid state, a freeze-dried state, and a state of being dissolved back into a physiological buffer. 11. The composition as claimed in claim i - w建人類 非造金型成體幹細胞係與—種或—種以上的另種人類非造 企型成體幹細胞共培相得者，前述另種人_造血型成 體幹細胞係源於選自由骨髓、膪帶血、上皮組織、乳牙以 及脂肪組織所構成之群組。 有： 12. -種獲取人類非造血型成體幹細胞之方法，包括 提供一人體組織； 以胰蛋白5解酵素及膠原蛋白分解酵素水解前述人體 體幹細胞； I雜Μ離並獲取單核 將前述單核成體幹細胞植入含培養基之培養撒； 移除Μ小時後’以—新鮮培養基更換前述培養基 附於培4皿之單核成體幹細胞，· 將貼附於培養皿之單核成體幹細胞留置於該培養皿 30 201012928 使之於新鮮培養基中增殖至一定數量； 將增殖的幹細胞自前述培養皿脫附； 以流式細胞儀確認幹細胞之表面抗原標記特徵. 獲取一人類非造血型成體幹細胞。 13. —種人類非造血型成體幹細胞，包括有源自不门 人體組織之非造血型成體幹細胞進行體外共培養、再將細 胞增殖分離後的所獲取之混合細胞。 14. 如申請專利範圍第13項所述之人類非造血型成體 〇 幹細胞，其係以一獲取方法所獲取者，該方法包括有： 提供一人體組織； 以胰蛋白分解酵素及膠原蛋白分解酵素水解前述人體 組織； 以離心方式自水解之前述人體組織分離並獲取單核成 體幹細胞； 將前述單核成體幹細胞植入含培養基之培養孤； 經過24小時後，以一新鮮培養基更換前述培養基並 © 移除未貼附於培養皿之單核成體幹細胞； 將貼附於培養孤之單核成體幹細胞留置於該培養皿， 使之於新鮮培養基中增殖至一定數量； 將增殖的幹細胞自前述培養皿脫附； 以流式細胞儀確認幹細胞之表面抗原標記特徵； 獲取一人類非造血型成體幹細胞。 15. 如申請專利範圍第13項所述之人類非造血_型成體 幹細胞’其係將複數種人類非造企型成體幹細胞共培養所 31 201012928 得者，前述複數種人_造血型成體幹細胞係源 骨髓、臍帶血、上皮組锉、澍'遷自由 參 反，、且織孔牙以及脂肪組織所構成之蛑 項所述之 16. 一種如申請專利範圍第1至11項中任_ 組成物於製備促進皮膚傷口修復之藥物的應用。 前迷皮膚傷 美容術後傷 17.如申請專利範圍第16項所述之應用， 口係選自由手術傷口、燒燙傷、青春痘及電射 口所組成的群組。- Constructing a human non-gold-forming adult stem cell line with a variety of human or non-artificial adult stem cells, and the above-mentioned alternative human _ hematopoietic adult stem cell line A group of free bone marrow, sputum blood, epithelial tissue, deciduous teeth, and adipose tissue. There are: 12. A method for obtaining human non-hematopoietic adult stem cells, comprising providing a human tissue; hydrolyzing the aforementioned human body stem cells with trypsin 5 dehydrogenase and collagen decomposing enzyme; Mononuclear adult stem cells are implanted with culture medium containing culture medium; after removing the Μ hours, the medium is attached to the mononuclear adult stem cells of the culture dish with the fresh medium, and the mononuclear adult body attached to the culture dish is attached. The stem cells are left in the culture dish 30 201012928 to proliferate to a certain amount in the fresh medium; the proliferated stem cells are desorbed from the culture dish; the surface antigen labeling characteristics of the stem cells are confirmed by flow cytometry. Obtaining a human non-hematopoietic type Somatic stem cells. 13. A human non-hematopoietic adult stem cell, comprising a mixed cell obtained by in vitro co-culture of non-hematopoietic adult stem cells derived from a human tissue, and then separating the proliferation of the cells. 14. The human non-hematopoietic adult stem cell as described in claim 13 of the patent application, which is obtained by an acquisition method, the method comprising: providing a human tissue; decomposing the trypsin and collagen The enzyme hydrolyzes the aforementioned human tissue; separates and obtains mononuclear adult stem cells from the aforementioned human body tissue by hydrolysis; and implants the aforementioned mononuclear adult stem cells into the cultured orphan containing the medium; after 24 hours, replaces the aforementioned with a fresh medium Medium and © remove mononuclear adult stem cells that are not attached to the culture dish; leave the mononuclear adult stem cells attached to the culture in the culture dish, and proliferate to a certain amount in the fresh medium; The stem cells were desorbed from the culture dish; the surface antigen labeling characteristics of the stem cells were confirmed by flow cytometry; and a human non-hematopoietic adult stem cell was obtained. 15. The human non-hematopoietic _ type adult stem cell described in claim 13 of the patent application is a plurality of human non-artificial adult stem cell co-cultures, 31 201012928, the aforementioned plurality of human _ hematopoietic The body stem cell line is derived from the bone marrow, the cord blood, the epithelial group, the sputum, and the sputum, and the perforation and adipose tissue are as described in the item 16. One of the claims 1 to 11 _ The use of the composition for the preparation of a medicament for promoting skin wound repair. Former skin injury Cosmetic surgery injury 17. As applied in the scope of claim 16, the mouth is selected from the group consisting of surgical wounds, burns, acne and electric shots. 18. 一種如申請專利範圍第1至11項中任一項所述 組成物於製備加速皮膚傷口癒合之藥物的應用。 a 19. 一種如申請專利範圍第1至11項中任一項所 組成物於製備降低傷口感染機率之藥物的應用。 之 20· 一種如申請專利範圍第1至11項中任一項所述之 組成物於製備抑制傷口發炎的機率之藥物的應用。a 21. 一種如申請專利範圍第1至11項中任一項所述之 組成物於製備減少疤痕之藥物的應用。 ^之 22. 一種如申請專利範圍第1至11項中任一項所述之 組成物於製備活化毛囊細胞之藥物的應用。 a 23. 一種如申請專利範圍第1至11項中任一項所述之 組成物於製備促進毛囊細胞生長之藥物的應用。 α 24. —種如申請專利範圍第1至u項中任—項所述之 組成物於製備增生毛髮之藥物的應用。 L 25. 一種如申請專利範圍第1至11項中任一項所述之 組成物於製備治療禿髮之藥物的應用。 a 32 Ο 201012928 26.—種如申請專利範圍第1至11項中任. 組成物於製備活化黑色素細胞之藥物的應用。 27·—種如申請專利範圍第1至11項中任-組成物於製備促進黑色素細胞生長之藥物的應用 28. 一種如申請專利範圍第1至11項中任-組成物於製備深色化淡色毛髮之藥物的應用。 29·—種如申請專利範圍第1至11項中任- 組成物於製借、、A , 攝…療白斑之藥物的應用。 十一、圖式： 無 項所述之 項所述之 ) 項所述之 項所述之 3318. Use of a composition according to any one of claims 1 to 11 for the preparation of a medicament for accelerating healing of a skin wound. a 19. Use of a composition according to any one of claims 1 to 11 for the preparation of a medicament for reducing the risk of wound infection. 20. The use of a composition according to any one of claims 1 to 11 for the preparation of a medicament for inhibiting the risk of inflammation of a wound. a 21. The use of a composition according to any one of claims 1 to 11 for the manufacture of a medicament for reducing scarring. The use of the composition according to any one of claims 1 to 11 for the preparation of a medicament for activating hair follicle cells. A 23. Use of a composition according to any one of claims 1 to 11 for the preparation of a medicament for promoting growth of hair follicle cells. 24. 24. The use of a composition according to any one of claims 1 to 5 for the preparation of a medicament for proliferating hair. L 25. Use of a composition according to any one of claims 1 to 11 for the preparation of a medicament for treating alopecia. a 32 Ο 201012928 26. The application of the composition as claimed in claims 1 to 11 for the preparation of a drug for activating melanocytes. 27. The use of a composition as claimed in claims 1 to 11 - for the preparation of a medicament for promoting melanocyte growth. 28. A composition as claimed in claims 1 to 11 - for darkening The application of light hair medicine. 29·—As for the application of patent scopes 1 to 11 - the application of the composition in the manufacture of drugs, leucorrhea, leukoplakia. XI. Schema: None of the items mentioned in the item mentioned in the item.