200521140 玫、發明說明： 【發明所屬之技術領域】 本發明相關於一種萃取幹細胞 蛋白之方法及其產物。 養液中何爾象和膠原 【先前技術】 一般化妝品中，生物有效成分添加劑來源有動物性及 植物性萃取物兩大方向，動 勿及 乳類分娩時所生胎盤之分解萃取物質，其内含物包括= 因子、細胞激素、荷爾蒙、凝血因子、紅▲球生成素、； 醣體、卵磷脂等；（2)膠原蛋白， 八醢莖街“ 為動物軟骨、結締組織之 、純化所得產品。 #夕“用植物有效部位加工 現有市場上膠原蛋白的取得來源多為牛、諸、烏類或 魚類等，而細胞激素則是利用在原核細胞中重組蛋白為主 要生產來源。這些蛋白來源對 · ，實難確定其在人體使用上之有1吕多為異種來源 版便用上之有效相容性及吸收性。另外 ’以現今培養細胞的技術而言，一般細胞於二至三天的時 間’為了提供較佳之細胞營養及生長環境，需要定期更換 :養細胞之培養液，故一般實驗室所消耗之培養液非常大 量，且用過之培養液經過滅g後即傾倒丢棄，因此若 效利用則可節省資源。 # 【發明内容】 本發明係相關於由幹細胞培養液中純化出細胞激素、 200521140 生長因子及膠原蛋白等有效成份。幹細胞經體外培養時， 於不同時期會分別表現出不同種類與產量的細胞激素及生 長因子；且幹細胞本身亦會分泌出許多基f，包括：纖維 連結蛋白（fibronectin)、板素（laminin)、膠原蛋白及蛋白聚 糖（proteoglyCan)等。 多種激素類物質具備之功能包括維持細胞代謝穩定， 提供活動能量、促進細胞分裂與成長且可延緩皮膚老化現 象而在真皮中膠原纖維會形成一個密集的織網，以維護 皮膚組織及抵抗力；彈性膠原蛋白則使皮膚柔軟而有彈性 。它們的數量易隨著皮膚的老化而減少，並在45歲後完 全消失。這些纖維浸在富含透明質酸的凝膠中，可把水份 鎖在分子裡，是肌膚滋潤相當重要的一環。膠原蛋白亦為 一種生醫材料，可應用到多項高附加價值之醫療產品上及 技術上，舉凡外科材料、人工器官、人工皮膚、人工血管 、製藥、醫學美容與化妝品等產品領域，都可見到膠原蛋 白的縱跡。 表皮生長因子（Epidermal Growth Factor，EGF) 1962年，Cohen教授在新生小鼠頷下腺的萃取液中分 離出具有活性之成分，並且將之加入培養液中培養表皮細 胞時’可直接促進表皮細胞的生長與分化，因此將其命名 為表皮生長因子（Epidermal Growth Factor，EGF) 〇 人類的EGF是由53個胺基酸組成，其生理活性很強 ’在組織和體液中含量極低，經過純化的EGF可以刺激細 200521140 胞的生長、促進細胞分化。臨床上可促使外胚層細胞 和毛細血管生長，促進燒傷、創傷及外科傷口的癒合、加 速移植表皮或角膜的生長、修復腸胃道、肝臟、眼角膜的 損傷、促進神經元細胞的再生及製造人體器官等。 膠原蛋白（collagen) ，原蛋白存在於身體的各個部位，人體正常骨胳中含 有80〇/❶的膠原蛋白，其功能主要是將鈣、_、礦物質等成 分黏著，然後構成骨質，而膠原蛋白佔乾皮膚淨重的77% 左右；纖維母細胞（fibroblast)位於真皮層下方的皮下組織 中，專門製造合成膠原蛋白，纖維母細胞主要製造第一型（ Type I )膠原蛋白及第三型（Type ΙΠ )膠原蛋白，而真皮層 的膠原蛋白以第一型的膠原蛋白（Type ! c〇Uagen)最重要。 各種膠原蛋白的比較表 魚類、植物膠 原蛋白 牛、豬膠原蛋 白 鳥類膠原蛋白 财溫性 < 20°C 約 37〇C 41°C 類型 第I型和第III 型 第I型和第ΠΙ 型 第I型和第III 型 細胞激素（cytokine) 細胞激素（MW· 15_30kD)為一群具有不同功能之可溶 性蛋白和胜肽的總稱，其係為體液的調控者（hum〇ral 200521140 regulator)且作用濃度為十億分 在正常情況或病理情況下，細 織’大部分的細胞激素為醣蛋 分泌。 之一至兆分之一莫耳濃度， 胞激素控制個別的細胞和組 白且經由細胞標準分泌途徑 在本發明之一較佳具體拿每 爭只中，本發明提供一種萃取 幹細胞培養液中荷爾蒙之方法，包括· (a) 培養幹細胞； (b) 收集幹細胞培養液；200521140 Description of the invention: [Technical field to which the invention belongs] The present invention relates to a method for extracting stem cell proteins and its products. Hormone and collagen in nutrient solution [Previous technology] In general cosmetics, the sources of bioactive ingredient additives come from animal and plant extracts in two directions, and do not touch the decomposed and extracted substances of the placenta during milk delivery. Contains: = factors, cytokines, hormones, coagulation factors, red spheroids, glycosides, lecithin, etc .; (2) collagen, Hachiman Street "for animal cartilage, connective tissue, purified products # Xi "Processing the available collagen from the active parts of plants on the existing market is mostly bovine, boiled, black catfish or fish, etc., while cytokines use the recombinant protein in prokaryotic cells as the main source of production. The source of these proteins is, it is difficult to determine whether they are used in humans. Luduo is a heterogeneous source, which is effective for compatibility and absorption. In addition, in terms of current cell culture technology, it takes two to three days for normal cells. In order to provide better cell nutrition and growth environment, it is necessary to periodically replace: the culture solution of the cultured cells, so the culture solution consumed by the general laboratory It is very large, and the used culture solution is dumped and discarded after being destroyed, so if it is effectively used, it can save resources. # [Summary of the Invention] The present invention relates to the purification of active ingredients such as cytokines, 200521140 growth factors, and collagen from stem cell culture broth. When stem cells are cultured in vitro, they will show different types and yields of cytokines and growth factors at different times; and the stem cells themselves will secrete many basic f, including: fibronectin, laminin, Collagen and proteoglycan. The functions of various hormones include maintaining the stability of cell metabolism, providing active energy, promoting cell division and growth, and delaying skin aging. Collagen fibers in the dermis form a dense weave network to maintain skin tissue and resistance; Elastic collagen makes the skin soft and elastic. Their numbers tend to decrease as the skin ages and disappear completely after the age of 45. These fibers are immersed in a gel that is rich in hyaluronic acid, which can lock water in the molecules, which is an important part of skin moisturization. Collagen is also a biomedical material, which can be applied to many high value-added medical products and technologies. It can be found in various fields such as surgical materials, artificial organs, artificial skin, artificial blood vessels, pharmaceuticals, medical beauty and cosmetics. The longitudinal trace of collagen. Epidermal Growth Factor (EGF) In 1962, Professor Cohen isolated active components from the extract of the submandibular glands of neonatal mice and added them to the culture medium to directly cultivate the epidermal cells. Growth and differentiation, so it is named Epidermal Growth Factor (EGF). Human EGF is composed of 53 amino acids, and its physiological activity is very strong. 'It is extremely low in tissues and body fluids and has been purified. EGF can stimulate cell growth and promote cell differentiation. Clinically, it can promote the growth of ectoderm cells and capillaries, promote the healing of burns, trauma and surgical wounds, accelerate the growth of epidermis or cornea transplantation, repair the damage of gastrointestinal tract, liver and cornea, promote the regeneration of neuronal cells and manufacture the human body Organs, etc. Collagen (collagen), the original protein exists in various parts of the body. The normal bones of the human body contain collagen of 80 / ❶. Its function is mainly to adhere calcium, minerals, and other components, and then constitute bone, and collagen Protein accounts for about 77% of the dry skin's net weight; fibroblasts are located in the subcutaneous tissue below the dermis layer, which specializes in the manufacture of synthetic collagen. Fibroblasts mainly produce type I collagen and type III collagen. Type III collagen), and the collagen of the dermis layer is the most important type collagen (Type! CoUagen). Comparison table of various collagens Fish, vegetable collagen, bovine collagen, bird collagen, and collagen properties <20 ° C about 37 ° C 41 ° C Types I and III Types I and III Type I and type III cytokine Cytokines (MW · 15_30kD) are a collective term for a group of soluble proteins and peptides with different functions. They are humoral regulators (humoral 200521140 regulator) and the concentration of action is Billions of points Under normal or pathological conditions, weaving 'most of the cytokines secreted by sugar eggs. One to one trillionth of a mole concentration, cytokines control individual cells and tissues and pass through the cell's standard secretion pathway. In one of the preferred embodiments of the present invention, the present invention provides an extract of hormones in stem cell culture fluid. A method comprising: (a) culturing stem cells; (b) collecting stem cell culture fluid;
⑷將處理過之培養液進行鹽類沉澱萃取；和 (d)純化出荷爾蒙。 車乂佳地’本發明所述之方法所製備的荷爾蒙可為蛋白 質種類之荷爾蒙。 較佳地，本發明所述之方法所製備的荷爾蒙可為細胞 激素或生長因子。 較佳地，本發明戶斤祕；> 士、+ I ^ 叮述之方法中，步驟（a)的幹細胞可以盐 The treated culture broth is subjected to salt precipitation extraction; and (d) hormones are purified. CHE JIA JIA 'The hormone prepared by the method of the present invention may be a hormone of a protein type. Preferably, the hormone prepared by the method of the present invention may be a cytokine or a growth factor. Preferably, in the method of the present invention; > In the method of +10, the stem cells of step (a) may be
由哺乳動物、動物或植物作為細胞來源。 較佳地，本發明所述之方法中，步驟（a)的幹細胞之來 源可以包括分離或未分離之組織、血液或體液。 較佳地，本發明所述之方法中，該哺乳動物可以是人 類0 較佳地，本發明所述之方法中，該步驟⑷的幹細胞之 來源係可選擇自骨髓、胚胎卵黃囊、胎盤、臍帶、胎兒、 月少年以及成年人之體液及組織所組成的族群中。 較佳地，本發明所述之方法中，該步驟的幹細胞係 7 200521140 可以為造血幹細胞、間挚枓赵^ 々 m 生幹細胞或多向性幹細胞。 較佳地，本發明所述之古 乩之方法中，該步驟（C)係將處理過 之培養液加入中性鹽類溶液中進行沉殿萃取。 k 較佳地，本發明所述之方 將沉殿物回溶於低鹽溶液中。 ^驟（C)後進-步可 較佳地，本發明所述之 體利用離子交換及特定專亥步驟⑷係將回溶液 。 抗體親和性管柱進行純化分離 較佳地，本發明所述之 _ 爾蒙而選擇該特定專一抗體， =所選疋純化之荷 特定生長因子抗體。 八了為特疋細胞激素抗體或 步包括（e)將所純化分 步包括⑺將製成粉狀 較佳地，本發明所述之方法進 離之荷爾蒙經由冷凍乾燥製成粉狀 較佳地，本發明所述之方法進 之荷爾蒙分別裝於小管中冷;東儲存 另一方面，在本發明之另 提供-種由上述方法所製備"佳八體事貫中，本發明 岍ι備之細胞激素。 另—方®，在本發明q 提供-種由上述方法所製備之生長:子、體事貫中’本發明 另—方面，在本發明 提供—種萃取幹細胞具體事實中，本發明 ⑷培養幹細胞；巾Μ蛋白之方法’包括： (b) 收集幹細胞培養液； (c) 將收集之培養液以酵 阵素進仃部分消化作用； 200521140 (d) 將處理過之拉I 、 ° 液進行鹽類沉澱萃取；和 (e) 純化出膠原蛋白。 較佳地，本發明戶斤 厅迷之方法中，步驟（a)的幹細胞3 是來自包括哺乳動物、動％ 勒物或植物的細胞來源。 較佳地，本發明所述 源可以包括分離或未分離 較佳地，本發明所述 類0 來 人 之方法中，步驟（a)的幹細胞之 之組織、血液或體液。 之方法中，該哺乳動物可以是 車父佳地，本發明所^ . 7 ：C之方法中，該步驟（a)的幹細胞之 來源係選擇自骨髓、胚胎印黃囊、胎盤，、胎兒、青 少年以及成年人之體液及組織所組成的族群中。 月 較佳地，本發明所述之方法中，該步驟的幹細胞係 可以是造血幹細胞、間葉性幹細胞或多向性幹細胞。〜 b較佳地，本發明所述之方法中，該步驟（c)的酵素可以 是胃蛋白酶（pepsin)、果膠酶（pectinase)、膠原酶 (collagenase)或其上之組合。 杈佳地，本發明所述之方法中，該步驟（d)係將處理過 之培養液加入中性鹽類溶液中進行沉澱萃取。 較佳地，本發明所述之方法中，於步驟後進一步包 括將沉澱物回溶於低鹽溶液中。 較佳地，本發明所述之方法中，該步驟（e)係將回溶液 體利用離子交換及特定專一膠原蛋白抗體親和性管柱進行 純化分離。 較佳地，本發明所述之方法進一步包括（f)將所純化分 200521140 離之膠原蛋白經由冷凍乾燥製成粉狀。 較佳地’本發明所述之方法進一步包括⑻將製成粉狀 之膠原蛋白分別裝於小管中冷凍儲存。 另一方面，在本發明之一較佳具體事實中，本發明提 供一種由上述方法所製備之膠原蛋白。 經由人類幹細胞培養液所萃取之細胞激素、生長因子 及膠原蛋白對於人類的皮膚為同種來源，若應用於人體上 較具有相容性及吸收性。From mammals, animals or plants as a source of cells. Preferably, in the method according to the present invention, the source of the stem cells in step (a) may include isolated or non-isolated tissue, blood or body fluid. Preferably, in the method according to the present invention, the mammal may be a human. Preferably, in the method according to the present invention, the source of the stem cells in step ⑷ may be selected from bone marrow, embryo yolk sac, placenta, Umbilical cord, fetus, juvenile, and adult body fluids and tissues. Preferably, in the method according to the present invention, the stem cell line 7 200521140 in this step may be a hematopoietic stem cell, a malignant stem cell, or a pluripotent stem cell. Preferably, in the method of ancient tincture according to the present invention, the step (C) is adding the treated culture solution to a neutral salt solution for immersion extraction. k Preferably, the method described in the present invention dissolves the sunken objects in a low-salt solution. ^ Step (C) may be followed by further steps. Preferably, the body according to the present invention uses ion exchange and specific specific steps to return the solution. Purification and separation of antibody affinity column Preferably, the specific specific antibody selected in the present invention is the selected specific purified growth factor antibody. Eighty-eight specific cytokine antibodies or steps include (e) purifying the purified product step by step, including powder, preferably, the hormones removed by the method described in the present invention are preferably powdered by freeze drying In the method described in the present invention, the hormones are separately packed in small tubes for cold storage; on the other hand, in another aspect of the present invention, a method prepared by the above method is used in the present invention. The present invention provides: Cytokines. On the other hand, in the present invention q provides a kind of growth prepared by the method described above: In another aspect of the present invention, in the specific fact that the present invention provides a kind of extracted stem cells, the present invention cultivates stem cells The method of tissue protein M includes: (b) collection of stem cell culture fluid; (c) partial digestion of the collected culture fluid with enzymes; 200521140 (d) salting of the treated La I, ° solution Precipitation-like extraction; and (e) purified collagen. Preferably, in the method of the present invention, the stem cells 3 of step (a) are derived from a cell source including mammals, animals or plants. Preferably, the source according to the present invention may include an isolated or non-isolated method. Preferably, in the method of class 0 to human according to the present invention, the tissue, blood or body fluid of the stem cells of step (a). In the method, the mammal may be a car parent. In the method of the present invention ^. 7: C, the source of the stem cells in step (a) is selected from bone marrow, embryo printed yellow sac, placenta, fetus, A group of adolescents and adults with body fluids and tissues. Preferably, in the method according to the present invention, the stem cell line in this step may be a hematopoietic stem cell, a mesenchymal stem cell, or a pluripotent stem cell. ~ B Preferably, in the method of the present invention, the enzyme in step (c) may be pepsin, pectinase, collagenase, or a combination thereof. Preferably, in the method of the present invention, step (d) is adding the treated culture solution to a neutral salt solution for precipitation extraction. Preferably, the method according to the present invention further comprises dissolving the precipitate back into a low-salt solution after the step. Preferably, in the method of the present invention, the step (e) is to purify and separate the return solution using ion exchange and a specific specific collagen antibody affinity column. Preferably, the method according to the present invention further comprises (f) lyophilizing the purified collagen 200521140 to obtain a powder form by freeze-drying. Preferably, the method according to the present invention further comprises: storing the powdered collagen in a small tube and storing it in a frozen state. On the other hand, in one preferred specific fact of the present invention, the present invention provides a collagen prepared by the above method. The cytokines, growth factors, and collagen extracted from human stem cell culture fluid are the same source for human skin. If applied to the human body, they are more compatible and absorbable.
【實施方式】[Embodiment]
在本發明之一較佳具體事實中，幹細胞來源包括人類 胚胎及臍帶血之幹細胞，並且以專用之細胞培養液培養幹 、、田I並疋期收取培養液進行成分分離。將細胞離心與培 養液刀離，若有需要則再利用特定酵素如：胃蛋白酶 (pepsm)、果膠酶（pectinase)、膠原酶處理培 、、力入中〖生鹽類萃取培養液後，再利用離子交換及特 定專-抗體親和性管柱分離細胞；敫素、i長因子及膠原蛋 白樣本。利用聚合丙醯胺凝膠電泳進行萃取物之蛋白分析 ’經無菌方式分離出萃取物。 本發明其他的特徵及優點將可明顯見於下列較佳具體 事實及申請專利範圍。 實例 下列實施例用於示範說明本發明。這些實施例不以任 何方式意欲限制本發明之範圍，但用於指示如何實施本發 10 200521140 明的材料及方法。 !施對_1:由幹細胞培養液中製備細胞激素、生長因子 及膠原I白 步驟 (1) 將間葉性幹細胞（MSC)種植於1 〇〇mm培養盤中， 培養於37°C，5% 002培養箱中。待MSC細胞長至80%， 用胰蛋白酶（trypSin)-EDTA將細胞由培養盤上打下來，放 置於50ml離心管中，於室溫下離心12〇〇Γριη，5分鐘。 (2) 分別取出上層培養液轉移置另一 5〇ml離心管中保 存備用。 (3) 取PBS清洗下層MSC細胞，混合均勻後於室溫 下離心1200rpm，5分鐘。 (4) 除去上清液，再加入PbS清洗下層MSC細胞， 於室溫下離心1200rpm，5分鐘。 (5) 除去上清液，加入新鮮的培養液打散Msc細胞， 混合均勻後以1 : 4的比例重新種植MSC於新的丨〇〇 mm 培養皿中，培養於37。〇/50/0(:()2培養箱中。 (6) 重複（1)〜（5)步驟，直到收集足夠的幹細胞培養液 (7) 將收集之幹細胞培養液，進行細胞激素、生長因 子及膠原蛋白之萃取。 (8) 若要進行膠原蛋白的萃取，則將培養液以胃蛋白 酶（pepsin)、果膠酶（pectinase)、膠原酶（c〇Ua^nase)處理 11 200521140 步加入不同濃度之中性鹽類進行沉澱萃取，若 之二：因子以及細胞激素的萃取，則直接加入不同濃度 低鹽溶液中。 卒取。離心將不同的沉殿物再回溶於 親/49^回溶之液體分別利用離子交換及特定專一抗體 進行細胞激素、生長因子及膠原蛋白之分離， 如出之液體回收。(例如：細胞激素之抗體親和性 ,1 ^柱)生長因子之抗體親和性管柱及膠原蛋白之抗體親和 以聚合丙醯胺凝膠電 (10)取一小部分的分離回收液 泳進行萃取物之蛋白分析。 \ )最後將經過無菌方式分離出之細胞激素、生長因 ’原蛋白利用冷凍乾燥法將之變粉狀 小管中於-2(rC冷凍儲存。 ⑴A於 根據本發明可作之不同修正及變化對於熟習該項技術 者而言均顯然不會偏離本發明的範圍與精神。雖然本發明 已敘述特定的較佳具體事實，必須瞭解的是本發明不庫被 不當地限制於該等特定具體事實上。事實上，在實施:發 明之已述模式方面’對於熟習該項技術者而言顯而易知之 不同修正亦被涵蓋於下列申請專利範圍之内。 【圖式簡單說明】 (一）圖式部分 12 200521140 無 (二）元件代表符號 無In a preferred specific fact of the present invention, the stem cell source includes stem cells of human embryos and umbilical cord blood, and the stem cell is cultured in a special cell culture solution, and the culture solution is collected at the same stage as the component separation. Centrifuge the cells and cut off the culture solution. If necessary, use specific enzymes such as pepsm, pectinase, collagenase, and force the medium to extract the culture solution. Ion exchange and specific specific-antibody affinity columns were used to isolate cells; halide, i-factor and collagen samples. Protein analysis of extracts by polymerized propylammonium gel electrophoresis ′ The extracts were separated in a sterile manner. Other features and advantages of the present invention will be apparent from the following preferred specific facts and the scope of patent applications. Examples The following examples serve to illustrate the invention. These examples are not intended to limit the scope of the invention in any way, but are used to indicate how to implement the materials and methods of the present invention. Shi Du_1: Preparation of cytokines, growth factors, and collagen I from stem cell culture step (1) Mesenchymal stem cells (MSC) were planted in a 100 mm culture plate and cultured at 37 ° C, 5 % 002 in the incubator. After the MSC cells grew to 80%, trypsin-EDTA was used to beat the cells from the culture plate, put them in a 50 ml centrifuge tube, and centrifuged at room temperature for 5 minutes at room temperature. (2) Remove the upper culture medium and transfer to another 50ml centrifuge tube for storage. (3) Take PBS to wash the lower MSC cells, mix them well and centrifuge at 1200 rpm for 5 minutes at room temperature. (4) Remove the supernatant, add PbS to wash the lower MSC cells, and centrifuge at 1200 rpm for 5 minutes at room temperature. (5) Remove the supernatant, add fresh culture medium to disperse the Msc cells, and mix the MSCs in a new 1000 mm petri dish at a ratio of 1: 4 to mix and culture at 37. 〇 / 50/0 (:() 2 in an incubator. (6) Repeat steps (1) ~ (5) until enough stem cell culture fluid is collected. (7) Collect the collected stem cell culture fluid for cytokines, growth factors. And collagen extraction. (8) For collagen extraction, treat the culture solution with pepsin, pectinase, and collagenase 11 200521140. Add different steps. Concentrated neutral salts are subjected to precipitation extraction. If the second is: extraction of factors and cytokines, directly add them to low-concentration solutions of different concentrations. Purge. Centrifuge and re-dissolve the different sinks in the pro / 49 ^ back The dissolved liquid uses ion exchange and specific specific antibodies to separate cytokines, growth factors and collagen, and the liquid is recovered. (Eg, antibody affinity of cytokines, 1 ^ column) antibody affinity tube for growth factors The affinity of the antibody between the column and the collagen was analyzed by polymerized promethamine gel electrophoresis (10). A small portion of the separated recovery liquid was analyzed for protein extraction of the extract. \) Finally, the cytokines and growth factors isolated in a sterile manner ' original The protein is freeze-dried in a powdery tube stored in -2 (rC). ⑴A. The various modifications and changes that can be made according to the present invention will obviously not depart from the scope of the present invention and Spirit. Although the present invention has described specific preferred specific facts, it must be understood that the present invention is not unduly limited to those specific specific facts. In fact, in terms of implementation: the described mode of the invention, Different amendments obvious to those skilled in the art are also covered by the following patent applications. [Simplified description of the drawings] (1) Schematic section 12 200521140 None (II) Symbols for components None
1313