200932909 九、發明說明： 【發明所屬之技術領域】 本發明是有關於一種用以促進表皮細胞再生之人類成 長因子組成及其製備方法。 【先前技術】 皮膚不但具有調節人體之體溫、水分等功能，也是人 體免疫系統的第一道防線，一旦失去表皮的保護就極容易 遭受外來病原感染。而經歷燒傷、潰瘍、發炎、放射線治 療、手術以及糖尿病之患者，長時間處於皮膚缺損及異常 之情形下，容易造成感染而對身體產生莫大的傷害，故傷 口癒合以及表皮細胞再生機制是一項重要的醫學研究領 域。 目前用於治療傷口以促進癒合之藥物多為殺菌劑、抗 生素、皮質類固醇激素等，對於傷口癒合皆各有其缺點， 且傷口癒合率差。因而發展出人工皮膚或其它皮膚替代 物、細胞治療等方法。 相關研究指出，在皮膚的傷口癒合過程中，有多種生 長因子扮演了重要的角色。皮膚受傷後，血小板會釋放出 其中所含之多種生長因子及細胞激素，包括表皮生長因子 (epidermal growth factor，EGF )、鹼性纖維母細胞生長因 子（basic fibroblast growth factor，bFGF)、血小板衍化生 長因子（platelet-derived growth factor，PDGF )、人類胰島 素生長因子（insulin-like growth factor，IGF-1 )、血管内皮 生長因子（vascular endothelial growth factor，VEGF)、轉 200932909 化生長因子 α ( transforming growth factor-cn，TGF-o:)、轉 化生長因子 j8 ( transforming growth factor-尽，TGF-尽）、介 白素-1( interleukin-1，IL-1 )、結締組織生長因子（connective tissue growth factor，CTGF )、激活素（activin )、腫瘤壞死 因子 α ( tumour necrosis factor，TNF-c〇 等。這些生長因子 可吸引嗜中性白血球（neutrophil )和巨嗤細胞（macrophage ) 朝向傷口處聚集，以清除受傷組織、細胞並吞噬感染物， 亦可活化附近的纖維母細胞及角質細胞以及誘導周圍血管 進行血管新生作用。 另一方面，傷口部位的纖維母細胞會在某些生長因子 (特別是PDGF以及TGF- /5 )的影響下，轉型為肌纖維母細 胞。這些肌纖維母細胞會大量表現α肌動蛋白（a -actin ) 以利收縮傷口，並合成膠原蛋白纖維。這些沈積於傷口處 之膠原蛋白纖維的排列方式受到肌纖維母細胞收縮之影響 而與原本正常組織之膠原蛋白纖維列方式不同，因而形成 疤痕組織。在受傷後第四星期開始一直到約六個月間，疤 痕組織中之膠原蛋白會重新排列，而使得疤痕組織之外形 及機能接近原先之正常組織。 近來有研究指出’ TGF-|33於傷口癒合的調節上，不僅 對巨噬細胞與纖維細胞有趨化性，並可刺激第I型及第π 型膠原蛋白之mRNA的轉錄作用，以促進傷口中膠原蛋白 的聚積，以提高傷口的癒合率。故TGF-j83經證實可產生無 疤的上皮再生作用，並加速癒合因放射線照射所形成之皮 膚傷口。研究指出，在哺乳動物之胚胎時期可分泌大量 TGF-/33，然而，成體哺乳動物的表皮於受傷時，所分泌的 200932909 TGF-幻的含量卻非常少，是以出生後之傷口，易有肌纖維 細胞收縮而造成之症痕。 由於生長因子對於傷口之再生及修復機制扮演了重要 的角色，包括 EGF、bFGF、IGF-l、PDGF、及 VEGF 等， 皆已廣泛運用於醫藥及美容保養產業。然、而，目前係利用 基因工程以大量製備上述生長因子，需將表現上述人類生 長因子之基因個別插入載體内以大量繁殖’並進一步純化 其表現之人類生長因子。雖然此種方法可降低成本，但其 缺點在於純化後之蛋白質的純度不一，且可能含有内毒素 或其他不良之蛋白質，而影響療效或併發過敏反應；此外， 個質體要同時插入三種以上的生長因子基因，且皆有活 陡表現，難度非常高，若欲同時操作多個選殖質體既費時 又費工’再者’以原核生物或真菌類來表現人類生長因子 蛋白質時’大部分所產生之蛋白質的活性會降低。 另一方面’幹細胞治療則是利用人類成體幹細胞，例 如位於骨髓、表皮、腸道等部位之幹細胞來治療各種疾病。 由人類骨趙分離的間葉幹細胞（human mesenchymal stem eell ’ hMSC )是一種成體幹細胞，其具有多種分化潛能， 在不同的培養環境下可分化成骨骼、軟骨、肌肉、皮膚等 、组織’又有易於分離、培養與體外增生速度快之特性，已 有多項研究證實其可強化皮膚傷口癒合機制。然而，以幹 '細胞注射或手術移植入人體，需要長期的臨床試驗以評估 異體幹細胞移植是否產生免疫排斥反應。再者，幹細胞治 療耗費之醫療資源以及金錢亦高於一般療法。 因此’需要提出一種方法，可以結合基因工程以及幹 200932909 細胞療法之優點並改進其缺點。亦 ^ 高要—種方法可同 ^供二種以上由人類細胞表現之生長因子，同時可透過 2外用或内服之途徑施㈣生長因子，以促進表皮細胞 【發明内容】 有鑑於上述問題，本發明提出一種於體外培養人體骨 〇 髓間葉幹細胞和/或製備人類生長因子的方法。首先，在初 代培養液中進行人體骨髓間葉幹細胞之初代培養。接著^ 去除初代培養液中之懸浮型細胞並選擇黏貼型細胞，並以 繼代培養液進行繼代培養。其後，選擇黏貼型細胞並以末 代培養液進行末代培養，上述末代培養液是_種無血清培 養液，且含有人類胰島素生長因子。末代培養可使得黏貼 型細胞能夠分泌多種生長因子至末代培養液中，以形成人 類生長因子溶液。亦即，上述人類生長因子溶液至少包含 Q 人類骨髓幹細胞所分泌之複數種人類生長因子的組合，即 為本發明所稱之人類生長因子組成。上述之人類生長因子 組成包含至少三種選自下列群組之生長因子：EGF、bFGF、 IGF-卜 PDGF、VEGF、TGF-/33、幹細胞生長因子（siem _ fact〇r ’ SCF )、干擾素（interferon，IFN-γ)、趨化因子 (RANTES)、血小板生長因子（th麵b〇p〇ietin，Tp〇)、金 屬蛋白酶組織抑制因子（tissue inh馳〇r 〇f metalloproteinase，TIMP )、瘦體素（lepHn )、介白素 _6 (interleukin-6，IL-6 )、以及介白素 _8( interleukin_8，IL_8 )。 根據本發明一實施例，可在上述末代培養液中選擇性 8 200932909 地加入一營養液。此一營養液可包含至少一種胺基酸和/或 至少一種維生素。 根據本發明一實施例，可將上述之人類生長因子溶液 取出並進行透析’以純化上述人類生長因子溶液。 根據本發明之又另一種態樣’提出一種用以促進表皮 細胞再生的組合物，上述組合物至少包含本發明實施例之 人類生長因子組成、營養成分、乳化劑溶液、以及油相液 ❹ 體。上述營養成分包含根據本發明實施例製備人類生長因 子組成時，所用之營養液的一部份。上述乳化劑溶液以及 油相液體可將人類生長因子組成及營養成分以油包水_水 包油（water in oil in water，w/o/w )之方式包埋成一種微 脂體包埋物。 根據本發明之一實施例，更可在上述用以促進表皮細 胞再生的組合物中加入一水溶性膠體，以增加上述微脂體 包埋物之安定性。 ©利用本發明實施例之體外培養人體骨趙間葉幹細胞的 方法’可輕易得到一種人類生長因子組成，其中所含之人 類生長因子的種類、活性、安全性皆高於利用基因工程所 產生者。且根據本發明實施例之方法所得到之生長因子組 成至少包含多種和表皮細胞再生相關之生長因子，可有效 提升表皮細胞再生之能力。 此外’根據本發明實施例之營養液，不但可在人類生 長因子組成製備過程中提供額外的養分給人髏骨髓間葉幹 細胞’亦有助於提升本發明實施例之用以促進表皮細胞再 生的組合物中所含之人類生長因子組成促進表皮細胞再生 9 200932909 之能力。 【實施方式】 下發明之上述目的、特徵、和優點能更明顯易懂， 、舉較佳具體實施例，詳細說明如下。 實施例（一） ❹於想外培養人想骨趙間葉幹細胞和/或製備人類生長因子 組成之方法 本發明之一實施例提出一種於體外培養人體骨髓間葉 幹細胞的方法’該方法包含下列步驟： (a) 在初代培養液中進行人體骨髓間葉幹細胞之初代 培養； (b) 去除初代培養液中之懸浮型細胞並選擇黏貼型細 胞’並以繼代培養液進行繼代培養； (c) 選擇繼代培養液中之黏貼型細胞，並以末代培養 液進行末代培養。上述末代培養液為無血清培養 液且含有人類騰島素生長因子，以使得黏貼型細 胞能夠分泌多種生長因子至末代培養液中，以形 成人類生長因子溶液 在本發明之具體實施例中，係將健康人體骨趙間葉幹 細胞（購自 The National Disease Research Interchange，NDRI， USA)以含10%胎牛血清之DMEM培養液（Dulbecco，s modified eagle medium，購自 Gibico，Cat. No.12100-061 ) 作為初代培養液，進行初代培養3日。接著去除初代培養 200932909 液中之懸浮型細胞並選擇黏貼型細胞，再以含腦胎牛血 清之DMEM培養液作為繼代培養液，進行繼代培養3日。 繼代培養之後，財代培養液進行末代培養3日，上述末 代培養液為-種無血清培養液，且含有約5〜25ng/mi人類 胰島素生長因子。 在末代培養步驟中，末代培養液中之黏貼型細胞可分 泌複數種人類生長因子至末代培養液中，以形成—種人類 生長口子/谷液。這些人類生長因子至少包含三種下列生長 因子：EGF、bFGF、IGF-卜 PDGF、VEGF、Τ(3]Μ3、SCF、 跡7、RANTES、Tp〇 ' 肅、瘦體素、il_6、以及 il_8。 這些人類生長因子的組合為本I明實施例所稱之「人 類生長因子組成」。此外，根據本發明實施例，人類生長因 子、卫成t所含的生長因子濃度為可^貞測的，其 <貞測方式以 及偵測極限將於下文詳述。 根據本發明之具體實施例，繼代培養步驟可視需求重 複至多2Gh根據本發明之具體實施例，可在繼代培養步 驟後將經培養之人體骨髓間葉幹細胞冷凍保存，且此一冷 凍保存之人體骨髓間葉幹細胞經解凍後可再度進行繼代培 養，但解凍後之人體骨髓間葉幹細胞的繼代培養次數最多 可重複3次。 在本發明之一具體實施例中，可於末代培養液中加入 營養液，上述營養液包含至少一種胺基酸和/或至少一種維 生素。上述之胺基酸例如可為丙胺酸、精胺酸、天門冬醯 胺酸、天冬胺酸、半胱胺酸、胱胺酸、麵胺酸、甘胺酸、 組胺酸、#白胺酸、白胺酸、離胺酸、甲硫胺酸、苯丙胺 200932909 酸、脯胺酸、絲胺酸、籙 -且所選之每種胺基酸的Z酸二胺胺酸， •素例如可為維生素a、m\b 00mg/L。上述之維生 維峰去W 維生素m、維生素B2、維生素B3、 D2、維生音Γ、生素I維生素B12、維生素C、維生素 、、維生素H、維生素K3、氣化膽鹼、葉酸、 或肌醇，且所選之每種維生素的濃度約為。.卜⑺球。 在本發明-實施例中，將利用本發明實施例製備之人 鲁 2生長因子溶液進行透析，以進一步純化該人類生長因子 溶液。根據本發明之一具體實施例，利用- 3.5 KD之透析 膜（購自 Pierce, SnakeSkinTM Pieated Dialysis Tubing，3 5〇〇 MWCO)來進行上述人類生長因子溶液之透析，透析可持 續進打約3〜5日。藉由透析步驟，可實質上移除人類生長 因子溶液中分子量小於3,500道爾吞（Dalt〇n，D)之物質， 因而達到純化人類生長因子溶液之目的。 由於根據本發明實施例欲製備之人類生長因子組成中 φ 所含之人類生長因子的分子量皆遠大於3_5 KD，例如EGF 之分子量約為6KD而TGF-/53之分子量更高達250 KD，故 而根據本發明實施例之方法所產生之人類生長因子大部分 會留存於透析膜之中。最後，收集透析膜中留存之溶液， 其至少包含三種人類生長因子。此種利用本發明實施例之 方法所製備之人類生長因子組成的特徵在於包含至少三種 可偵測之生長因子（偵測方法將於下文詳述），上述生長因 子可以是 EGF、bFGF、IGF-1、PDGF、VEGF、TGF-/33、 SCF、IFN-γ、RANTES、Τρο、TIMP、瘦體素、IL-6、或 IL-8。 12 200932909 實施例（二） 人類生長因子组成之定性分析 以人類細胞介素抗體微陣晶片（RayBio™ Human Cytokine Antibody Arrays)檢測該透析液中之生長因子組 成，檢測結果如第1圖之人類細胞介素抗體微陣圖所示。 由第1圖可以發現，根據本發明實施例製備之人類生長因 子組成所含之人類生長因子種類及其偵測極限（limit of detection，LoD)如下：EGF (债測極限 1 pg/ml )、bFGF (偵測極限 10000 pg/ml)、IGF-1 (偵測極限 10 pg/ml)、 PDGF (偵測極限1000 pg/ml )、VEGF (偵測極限100 pg/ml )、TGF-/33 (債測極限 100 pg/ml )、SCF (積測極限 10 pg/ml)、IFN-γ (偵測極限 100 pg/ml)、RANTES (偵測 極限 2000 pg/ml)、Τρο (偵測極限 100 pg/ml)、TIMP (偵 測極限1 pg/ml )、瘦體素（偵測極限100 pg/ml )、IL-6 (偵 測極限1 pg/ml)、以及IL-8 (偵測極限1 pg/ml)。 在本說明書中，「偵測極限」一詞係指利用本發明實施 例之方法進行人類生長因子定性分析時，所採用之檢測方 法的最低偵測濃度。換句話說，待測樣本中所含之特定人 類生長因子的濃度，必須至少等同於或大於檢測方法所用 之偵測極限，才可能被檢測出來，在此種情形下，亦可將 該特定人類生長因子稱為「可偵測的」人類生長因子。200932909 IX. Description of the Invention: [Technical Field of the Invention] The present invention relates to a human growth factor composition for promoting epidermal cell regeneration and a preparation method thereof. [Prior Art] The skin not only has the functions of regulating body temperature and moisture, but also the first line of defense of the human immune system. Once the skin is protected, it is extremely susceptible to foreign pathogen infection. Patients who have experienced burns, ulcers, inflammation, radiation therapy, surgery, and diabetes are prone to infection and cause great harm to the body in the case of skin defects and abnormalities for a long time. Therefore, wound healing and epidermal cell regeneration mechanism is a An important area of medical research. The drugs currently used to treat wounds to promote healing are mostly fungicides, antibiotics, corticosteroids, etc., which have their own shortcomings for wound healing, and the wound healing rate is poor. Thus, methods such as artificial skin or other skin substitutes, cell therapy, and the like have been developed. Related studies have pointed out that a variety of growth factors play an important role in the wound healing process of the skin. After skin injury, platelets release a variety of growth factors and cytokines, including epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and platelet-derived growth. Platelet-derived growth factor (PDGF), human insulin-like growth factor (IGF-1), vascular endothelial growth factor (VEGF), transform 200932909 transforming growth factor -cn, TGF-o:), transforming growth factor j8 (transformation growth factor - TGF-Europe), interleukin-1 (IL-1), connective tissue growth factor (connective tissue growth factor) CTGF), activin, tumour necrosis factor (TNF-c〇, etc. These growth factors can attract neutrophil and macrophage to accumulate toward the wound to clear Injured tissue, cells and phagocytosis of infectious agents can also activate nearby fibroblasts and Qualitative cells and induction of peripheral blood vessels for angiogenesis. On the other hand, fibroblasts at the wound site are transformed into myofibroblasts under the influence of certain growth factors (especially PDGF and TGF-/5). The cells will express a large amount of α-actin to shrink the wound and synthesize collagen fibers. The arrangement of these collagen fibers deposited on the wound is affected by the contraction of the myofibroblasts and the collagen of the original normal tissue. The protein fibers are arranged in different ways, thus forming scar tissue. The collagen in the scar tissue is rearranged from the fourth week after the injury to about six months, and the shape and function of the scar tissue are close to the original normal tissue. Studies have shown that 'TGF-|33 regulates wound healing, not only on macrophages and fibroblasts, but also stimulates the transcription of mRNA of type I and type π collagen to promote wounds. The accumulation of collagen to improve the healing rate of the wound. Therefore, TGF-j83 has been proven to produce innocent epithelial regeneration. It accelerates the healing of skin wounds formed by radiation. Studies have shown that a large amount of TGF-/33 can be secreted during the embryonic period of mammals. However, when the epidermis of adult mammals is injured, the 200932909 TGF-magic is secreted. The content is very small, it is the wound after birth, easy to have the scar of muscle fiber cells contraction. Growth factors play an important role in the regeneration and repair mechanisms of wounds, including EGF, bFGF, IGF-1, PDGF, and VEGF, which have been widely used in the pharmaceutical and beauty care industries. However, currently, genetic engineering is used to prepare a large amount of the above growth factors, and genes expressing the above human growth factors are individually inserted into a vector to mass-produce and further purify the human growth factor of the expression. Although this method can reduce the cost, the disadvantage is that the purified protein has different purity and may contain endotoxin or other undesirable proteins, which may affect the therapeutic effect or complicated allergic reaction; in addition, more than three types of plastids are inserted at the same time. The growth factor genes, all of which have alive and steep performance, are very difficult. If you want to operate multiple plastids at the same time, it takes time and labor. 'Well' is a prokaryote or fungus to express human growth factor protein. The activity of some of the produced proteins is reduced. On the other hand, stem cell therapy uses human adult stem cells, such as stem cells located in the bone marrow, epidermis, and intestines, to treat various diseases. Human mesenchymal stem eell 'hMSCs, which are human stem cells, have a variety of differentiation potentials and can differentiate into bones, cartilage, muscles, skin, etc. in different culture environments. There are also characteristics of easy separation, culture and rapid proliferation in vitro, and many studies have confirmed that it can strengthen the mechanism of skin wound healing. However, transplantation with stem cells or surgery into the human body requires long-term clinical trials to assess whether allogeneic stem cell transplantation produces immune rejection. Furthermore, the medical resources and money spent on stem cell treatment are also higher than general therapy. Therefore, there is a need to propose a method that combines the advantages of genetic engineering and stem cell therapy with 200932909 and improves its shortcomings. Also ^ Gao Yao - a method can be used to provide two or more growth factors expressed by human cells, and at the same time, through the external or oral administration of (four) growth factors to promote epidermal cells [invention content] In view of the above problems, this The invention proposes a method for culturing human bone marrow mesenchymal stem cells and/or preparing human growth factors in vitro. First, primary culture of human bone marrow mesenchymal stem cells is carried out in primary culture fluid. Next, the suspension cells in the primary culture medium are removed and the adherent cells are selected and subcultured in the subculture medium. Thereafter, the adherent cells are selected and cultured in the final culture medium, which is a serum-free culture solution containing human insulin growth factor. The last culture allows the adherent cells to secrete a variety of growth factors into the final culture to form a human growth factor solution. That is, the above human growth factor solution contains at least a combination of a plurality of human growth factors secreted by Q human bone marrow stem cells, which is a human growth factor component of the present invention. The above human growth factor composition comprises at least three growth factors selected from the group consisting of EGF, bFGF, IGF-PDGF, VEGF, TGF-/33, stem cell growth factor (siem_fact〇r 'SCF), interferon ( Interferon, IFN-γ), chemokine (RANTES), platelet growth factor (th-face b〇p〇ietin, Tp〇), tissue inhibitor of metalloproteinase (TIMP), lean body Prime (lepHn), interleukin-6 (IL-6), and interleukin_8 (IL_8). According to an embodiment of the present invention, a nutrient solution can be added to the above-mentioned last culture solution in a selective state of 200932909. The nutrient solution may comprise at least one amino acid and/or at least one vitamin. According to an embodiment of the present invention, the above human growth factor solution can be taken out and dialyzed to purify the above human growth factor solution. According to still another aspect of the present invention, a composition for promoting regeneration of epidermal cells is provided, the composition comprising at least the human growth factor composition, the nutrient component, the emulsifier solution, and the oil phase liquid steroid of the embodiment of the present invention. . The above nutrient component comprises a portion of the nutrient solution used in the preparation of the human growth factor component in accordance with an embodiment of the present invention. The emulsifier solution and the oil phase liquid can embed the human growth factor composition and nutrient components in a water-in-oil in water (w/o/w) manner into a micro-lipid body embedding material. . According to an embodiment of the present invention, a water-soluble colloid can be further added to the above composition for promoting epidermal cell regeneration to increase the stability of the above-mentioned liposome-embedded material. The method for in vitro culture of human bone mesenchymal stem cells by using the method of the present invention can easily obtain a human growth factor composition, wherein the type, activity and safety of human growth factors are higher than those produced by genetic engineering. . Further, the growth factor composition obtained by the method according to the embodiment of the present invention comprises at least a plurality of growth factors associated with epidermal cell regeneration, which can effectively enhance the ability of epidermal cells to regenerate. In addition, the nutrient solution according to the embodiment of the present invention not only provides additional nutrients to the human bone marrow mesenchymal stem cells during the preparation of the human growth factor component, but also contributes to the promotion of epidermal cell regeneration in the examples of the present invention. The human growth factor component contained in the composition contributes to the ability of epidermal cell regeneration 9 200932909. The above described objects, features, and advantages of the invention will be more apparent and understood in the preferred embodiments. EXAMPLES (I) Method for cultivating human bone marrow stem cells and/or preparing human growth factor composition according to an embodiment of the present invention A method for culturing human bone marrow mesenchymal stem cells in vitro is proposed. Steps: (a) Primary culture of human bone marrow mesenchymal stem cells in primary culture medium; (b) Removal of suspension cells in primary culture medium and selection of adherent cells' and subculture with subculture medium; c) Select the adherent cells in the subculture medium and carry out the final culture with the last culture medium. The above-mentioned last culture medium is a serum-free medium and contains a human growth factor to enable the adhesion cells to secrete various growth factors into the final culture solution to form a human growth factor solution in a specific embodiment of the present invention. Healthy human bone mesenchymal stem cells (purchased from The National Disease Research Interchange, NDRI, USA) in DMEM medium containing 10% fetal bovine serum (Dulbecco, s modified eagle medium, available from Gibico, Cat. No. 12100- 061) As a primary culture solution, the primary culture was carried out for 3 days. Then, the suspension cells in the primary culture 200932909 solution were removed and the adherent cells were selected, and the DMEM culture medium containing the fetal fetal bovine serum was used as the subculture medium, and subculture was carried out for 3 days. After subculture, the cultivar was cultured for 3 days, and the above-mentioned broth was a serum-free medium containing about 5 to 25 ng/mi of human insulin growth factor. In the final culture step, the adherent cells in the final culture solution can secrete a plurality of human growth factors into the final culture solution to form a human growth mouth/trough solution. These human growth factors comprise at least three of the following growth factors: EGF, bFGF, IGF-PDGF, VEGF, Τ(3)Μ3, SCF, Trace 7, RANTES, Tp〇'Su, Leptin, il_6, and il_8. The combination of the human growth factors is referred to as the "human growth factor composition" in the embodiment of the present invention. Further, according to the embodiment of the present invention, the concentration of the growth factor contained in the human growth factor and Weicheng t is measurable. <Measurement method and detection limit will be described in detail below. According to a specific embodiment of the present invention, the subculture step can be repeated up to 2 Gh depending on the requirements, according to a specific embodiment of the present invention, which can be cultured after the subculture step The human bone marrow mesenchymal stem cells are cryopreserved, and the cryopreserved human bone marrow mesenchymal stem cells can be subcultured after thawing, but the subculture of the human bone marrow mesenchymal stem cells after thawing can be repeated up to 3 times. In a specific embodiment of the present invention, a nutrient solution may be added to the last culture solution, the nutrient solution comprising at least one amino acid and/or at least one vitamin. The base acid can be, for example, alanine, arginine, aspartic acid, aspartic acid, cysteine, cysteine, face acid, glycine, histidine, #al6, white Amino acid, lysine, methionine, amphetamine 200932909 acid, valine, serine, hydrazine - and Z acid diamine acid of each amino acid selected, such as vitamin A , m\b 00mg/L. The above-mentioned vitamins and peaks go to W vitamins M, vitamin B2, vitamin B3, D2, vitamins, vitamins, vitamins, vitamins, vitamins, vitamins, vitamins, vitamins, vitamins Choline, folic acid, or inositol, and the concentration of each of the selected vitamins is about .. (b). In the present invention - the embodiment, the human Lu 2 growth factor solution prepared by using the embodiment of the present invention is used. Dialysis to further purify the human growth factor solution. According to one embodiment of the invention, the above human growth factor solution is performed using a -3.5 KD dialysis membrane (purchased from Pierce, Snake SkinTM Pieated Dialysis Tubing, 35 MWCO). Dialysis, dialysis can be carried out for about 3 to 5 days. By dialysis The substance having a molecular weight of less than 3,500 Dalc〇n (D) in the human growth factor solution can be substantially removed, thereby achieving the purpose of purifying the human growth factor solution. Due to the human growth factor to be prepared according to the embodiment of the present invention The molecular weight of the human growth factor contained in the composition φ is much larger than 3_5 KD, for example, the molecular weight of EGF is about 6 KD and the molecular weight of TGF-/53 is as high as 250 KD, so the human growth factor produced by the method according to the embodiment of the present invention is Most of it will remain in the dialysis membrane. Finally, the solution remaining in the dialysis membrane is collected, which contains at least three human growth factors. Such a human growth factor composition prepared by the method of the present invention is characterized by comprising at least three detectable growth factors (detection methods will be described in detail below), and the growth factors may be EGF, bFGF, IGF- 1. PDGF, VEGF, TGF-/33, SCF, IFN-γ, RANTES, Τρο, TIMP, leptin, IL-6, or IL-8. 12 200932909 Example (II) Qualitative analysis of human growth factor composition The growth factor composition in the dialysate was measured by RayBioTM Human Cytokine Antibody Arrays, and the results were as shown in Figure 1. The interleukin antibody microarray is shown. It can be found from Fig. 1 that the human growth factor composition and the limit of detection (LoD) contained in the human growth factor composition prepared according to the embodiment of the present invention are as follows: EGF (debt limit 1 pg/ml), bFGF (detection limit 10000 pg/ml), IGF-1 (detection limit 10 pg/ml), PDGF (detection limit 1000 pg/ml), VEGF (detection limit 100 pg/ml), TGF-/33 (debt limit 100 pg/ml), SCF (integration limit 10 pg/ml), IFN-γ (detection limit 100 pg/ml), RANTES (detection limit 2000 pg/ml), Τρο (detection limit 100 pg/ml), TIMP (detection limit 1 pg/ml), lean voxel (detection limit 100 pg/ml), IL-6 (detection limit 1 pg/ml), and IL-8 (detection) Limit 1 pg/ml). In the present specification, the term "detection limit" means the lowest detection concentration of the detection method used in the qualitative analysis of human growth factors by the method of the embodiment of the present invention. In other words, the concentration of a particular human growth factor contained in the sample to be tested must be at least equal to or greater than the detection limit used by the detection method before it can be detected. In this case, the specific human can also be Growth factors are called "detectable" human growth factors.
此外，分別利用SCF RayBioTM ELISA Kit套組以及 VEGF RayBioTM ELISA Kit 套組，針對 SCF 與 VEGF 二種 生長因子進行定量分析，其結果如第2A及2B圖所示。由 第2A及2B圖可以發現，以各套組内所附的SCF與VEGF 13 200932909 標準品做迴歸直線，可求得本發明實施例製備之人類生長 因子組成中，SCF之濃度約1380 pg/ml，且VEGF之濃度 約 25130 pg/ml。 相較於先前技藝所用之幹細胞培養方法，培養液中則 不含可偵測的TGF-/33 (偵測極限1〇〇 Pg/ml)以及SCF (偵 測極限10 pg/ml)，亦即利用先前技藝幹細胞培養方法，培 養液中TGF-/33含量小於1〇〇 pg/m卜且SCF含量小於10 pg/m卜此外，利用先前技藝幹細胞培養方法，培養液中 VEGF的濃度僅為約200-500 pg/ml。然而，本發明實施例 之人類生長因子溶液中’則含有可偵測的TGF-/S3，亦即 TGF-/33的含量大於等於1〇〇 pg/m卜且所含之sCF濃度 (1380 pg/ml)以及 VEGF 濃度（2513〇 pg/ml)皆遠高於 先前技藝。 實施例（三） 促進表皮細胞再生的組合物 在本發明之另一具體實施例中’提出一種用以促進表 皮細胞再生的組合物’其至少包含根據本發明實施例之人 類生長因子組成、-營養成分、—乳化劑溶液、以及一油 相液體。_L述營養成分包含根據本發明實施例製備人類生 長因子組成時所用之營養液的—部份，在較佳的情形中， 營養成分至少包含一或更多種胺基酸和/或維生素。上述之 乳化劑溶液及油相㈣可將人類生長时組成及營養成分 以油包水-水包油（w/0/w)之方式包埋成—微脂趙包埋物。 200932909 在本發明實施例中，上述乳化劑溶液包含約0.5-1%之 Tween 80、以及約之山梨糖醇半油酸脂（sorbitan sesquioleate，Arlacel 83 )。在本發明之實施例中，上述乳 化劑溶液可更包含約80-90%之純水、約1-10%之玻尿酸和/ 或約1-10%之葡萄糖。 在本發明實施例中，上述油相液體包含約1-10%之卵 磷脂、約1-10%之膽固醇以及約80-90%之磷脂質。在本發 明之實施例中，能夠以其它油脂成分取代上述之磷脂質， 例如上述油相液體可包含篦麻油和/或礦物油。 根據本發明實施例，更可在上述用以促進表皮細胞再 生的組合物中加入水溶性膠體，以增加微脂體包埋物之安 定性。水溶性膠體包含約1_10%之Tween 20、約1-10%之 丙二醇（propylene glyco卜 PG)、約 1-10%之 PEG-600、以 及約1-5%之膠體物質，其中上述膠體物質可以是三仙膠、 透明膠、或其組合物。在本發明一實施例中，水溶性膠體 可更包含約1-10%之玻尿酸。 根據本發明之一具體實施例，於約4°C下在約10ml純 水中加入約2-5公克Tween 80、約2-5公克Arlacel 83、約 3-5公克玻尿酸和約2-5公克葡萄糖加以溶解，以製備成總 體積約20 ml之乳化劑溶液。另一方面，將約5-10公克卵 磷脂、以及約2-5公克膽固醇加入約20-30ml篦麻油和約 10-20 ml礦物油中’以製備成總體積約40 ml之油相液體》 此外，在約60-80ml之純水中，加入約10-20公克Tween 20、 約10-20公克PG、約2-5公克PEG-600、約5-10公克三仙 膠、以及約10-20公克玻尿酸，以製備成總體積約120 ml 15 200932909 之水溶性膠體。 於約4°C下取約20 ml乳化劑溶液以及等體積（即，約 20 ml)之經3.5 KD透析膜透析之人類生長因子溶液混合。 接著，緩慢加入約40 ml油相液體，以8,〇〇〇_1〇 〇〇〇rpm的 高壓均質機於約4°C下擾拌均勻’攪拌持續約ι〇_2〇分鐘。 以形成微脂體奈米顆粒（w/o/w )。最後，加入約丨2〇 ml水 /谷性膠體’以2,500-3,000 rpm的均質機於約4°c下授掉均 ❹ 勻，攪拌持續約2〇分鐘，最終所得之乳糜狀組合物，即為 本發明實施例之用以促進表皮細胞再生的組合物。 在本實施例中，加入水溶性膠體之目的在於增加微脂 體奈米顆粒之安定性，可使微脂體奈米顆粒内的水溶性蛋 白質於室溫保持活性達2〜3年之久。 實施例（四） 小鼠皮膚傷口療合/表皮細胞再生試驗 在本發明具體實施例中，以小鼠皮膚傷口癒合試驗測 試本發明實施例之用以促進表皮細胞再生的組合物對傷口 癒合率之影響。本試驗共進行了二組試驗組（微脂體A、 以及微脂體B )及一組對照組（微脂體C ),每組各利用四 隻八週齡大之BalbC鼠，將每隻小鼠以滅菌處理之解剖刀 於小鼠刹毛後之背部剪下〇·5 X 0.5 cm2之傷口，隨即以顯 微鏡觀察小鼠皮膚傷口，並擷取及記錄第0日傷口影像， 並以軟體#算第〇日傷口面積；小鼠皮膚傷口療合試驗之 喊驗設計如下： 微月曰體A :以微脂體包埋本發明實施例所述之人類生 16 200932909 長因子溶液（即’實施例（三）之用以促進表皮細胞再生 的組合物） 微脂體B:以微脂體包埋本發明實施例所述之營養液 微脂體C :以微脂體包埋生理食鹽水 在傷口造成一小時候，於試驗用小鼠的傷口上均勻塗 抹約100 /d之微脂體A、B、或c。其後，每隔24小時， 分別以同樣之方式塗抹約1〇〇 之微脂體A、B、或c，一 〇 共塗抹三次。第三次塗抹後24小時，即傷口產生後之第3 曰’以顯微鏡觀察小鼠皮膚傷口’並掏取及記錄第3曰傷 口影像’並以軟體計算第3日傷口面積。 本實施例所用之顯微鏡係連接電荷耦合元件（Charge Coupled Device，CCD)以擷取小鼠皮膚傷口之影像，並於 擷取影像後，將該影像傳輸至電腦進行影像處理及傷口面 積計算、統計；影像處理及傷口面積計算所使用之軟體為 Northern Eclipse image system ;而數據統計則以軟體 _ SigmaStat program (2002)進行分析，係以單項變方分析 (One-way Analysis of Variance，ANOVA)的鄧肯氏多變域測 驗（Duncan’s New Multiple Range Test, DMRT)進行計算。 小鼠皮膚傷口影響如第3圖所示。第3A圖為小氣第〇 曰之傷口影像；第3Β圖為經微脂體Α處理之小鼠第3日 之傷口影像；第3C圖為經微脂體B處理之小鼠第3曰之傷 口影像；以及第3D圖為經微脂體C處理之小鼠第3曰之 傷口影像。比較第3A-D圖可以發現，經過本發明實施例之 用以促進表皮細胞再生的組合物（微脂體A)處理三天後 的小鼠皮膚傷口面積（如第3B圖所示），明顯小於另二組處 17 200932909 ㈣小鼠皮膚傷π面積（如第3c&3d圖所示）。 :處理軟體計算各組小鼠第0天及第3天之皮膚 傷口面積，以換算傷口癒合率，其計算方式如下. 傷口癒合率（％)=(第〇 膚傷口面積）/第〇曰皮廣傷 曰皮膚傷口面積_第3天之皮 口面積X 100%In addition, the SCF RayBioTM ELISA Kit kit and the VEGF RayBioTM ELISA Kit kit were used to quantify both SCF and VEGF growth factors, and the results are shown in Figures 2A and 2B. It can be found from Figures 2A and 2B that the concentration of SCF in the composition of the human growth factor prepared in the examples of the present invention can be determined by using the SCF and VEGF 13 200932909 standard attached to each set as a regression line. The concentration of SCF is about 1380 pg/ Ml, and the concentration of VEGF is about 25130 pg/ml. Compared to the stem cell culture method used in the prior art, the culture solution contains no detectable TGF-/33 (detection limit 1 〇〇 Pg/ml) and SCF (detection limit 10 pg/ml), that is, Using the prior art stem cell culture method, the TGF-/33 content in the culture solution is less than 1〇〇pg/mb and the SCF content is less than 10 pg/m. In addition, the concentration of VEGF in the culture solution is only about about the prior art stem cell culture method. 200-500 pg/ml. However, the human growth factor solution of the embodiment of the present invention contains detectable TGF-/S3, that is, the content of TGF-/33 is greater than or equal to 1 〇〇pg/m b and the concentration of sCF contained therein is (1380 pg). /ml) and VEGF concentration (2513〇pg/ml) are much higher than previous techniques. EXAMPLES (III) Compositions for Promoting Epidermal Cell Regeneration In another embodiment of the present invention, a composition for promoting epidermal cell regeneration is proposed which comprises at least a human growth factor composition according to an embodiment of the present invention, Nutritional ingredients, emulsifier solutions, and an oil phase liquid. The nutritional component comprises a portion of a nutrient solution used in the preparation of a human growth factor component according to an embodiment of the present invention. In a preferred embodiment, the nutrient component comprises at least one or more amino acids and/or vitamins. The emulsifier solution and the oil phase (4) described above can be used to embed the composition and nutrients of human growth in a water-in-oil-in-water (w/0/w) manner into a micro-lipid package. 200932909 In an embodiment of the invention, the emulsifier solution comprises from about 0.5% to about 1% Tween 80, and about sorbitan sesquioleate (Arlacel 83). In an embodiment of the invention, the emulsifier solution may further comprise from about 80% to about 90% pure water, from about 1-10% hyaluronic acid, and/or from about 1-10% glucose. In an embodiment of the invention, the oil phase liquid comprises from about 1% to about 10% lecithin, from about 1% to about 10% cholesterol, and from about 80% to about 90% phospholipids. In the examples of the present invention, the above phospholipids may be replaced by other fat and oil components, for example, the above oil phase liquid may comprise castor oil and/or mineral oil. According to an embodiment of the present invention, a water-soluble colloid can be added to the above composition for promoting epidermal cell regeneration to increase the stability of the liposome embedding. The water-soluble colloid comprises about 1-10% Tween 20, about 1-10% propylene glycol (PG), about 1-10% PEG-600, and about 1-5% of the colloidal substance, wherein the colloidal substance can be It is a Sanxian gum, a transparent gum, or a combination thereof. In one embodiment of the invention, the water soluble colloid may further comprise from about 1% to about 10% hyaluronic acid. According to one embodiment of the invention, about 2-5 grams of Tween 80, about 2-5 grams of Arlacel 83, about 3-5 grams of hyaluronic acid, and about 2-5 grams are added to about 10 ml of purified water at about 4 °C. The glucose was dissolved to prepare an emulsifier solution having a total volume of about 20 ml. In another aspect, about 5-10 grams of lecithin, and about 2-5 grams of cholesterol are added to about 20-30 ml of castor oil and about 10-20 ml of mineral oil to prepare an oil phase liquid having a total volume of about 40 ml. Further, in about 60-80 ml of pure water, about 10-20 grams of Tween 20, about 10-20 grams of PG, about 2-5 grams of PEG-600, about 5-10 grams of Sanxian gum, and about 10- 20 g of hyaluronic acid was prepared to prepare a water-soluble colloid having a total volume of about 120 ml 15 200932909. Approximately 20 ml of the emulsifier solution and an equal volume (i.e., about 20 ml) of the 3.5 KD dialyzed dialyzed human growth factor solution were mixed at about 4 °C. Next, about 40 ml of the oil phase liquid was slowly added, and the mixture was uniformly stirred at about 4 ° C with a high pressure homogenizer at 8, 〇〇〇 1 〇 rpm for about 〇 〇 2 〇 minutes. To form microlipid nanoparticles (w/o/w). Finally, about 2 ml of water/glutamate colloid was added to the homogenizer at 2,500-3,000 rpm, and the mixture was uniformly stirred at about 4 ° C for about 2 minutes, and the resulting chylomicron composition was It is a composition for promoting epidermal cell regeneration according to an embodiment of the present invention. In the present embodiment, the purpose of adding a water-soluble colloid is to increase the stability of the liposome nanoparticle, and the water-soluble protein in the liposome nanoparticle can be kept active at room temperature for 2 to 3 years. EXAMPLES (IV) Mouse Skin Wound Healing/Epidermal Cell Regeneration Test In a specific embodiment of the present invention, a wound healing rate of a composition for promoting epidermal cell regeneration in an embodiment of the present invention was tested in a mouse skin wound healing test. The impact. A total of two experimental groups (microlipid A, and liposome B) and one control group (lipid C) were used in this experiment. Each group used four eight-week-old BalbC mice, each of which was used. The mice were sterilized with a scalpel and the wound of 〇·5×0.5 cm2 was cut on the back of the mouse. Then the skin wound of the mouse was observed under a microscope, and the wound image of the 0th day was taken and recorded, and the software was used. #算〇日日 wound area; the mouse skin wound healing test call design is as follows: Micro Moon Body A: The human body 16 200932909 long factor solution (ie ' The composition for promoting the regeneration of epidermal cells in the embodiment (3)) The liposome B: the nutrient solution of the embodiment of the invention is embedded in the liposome C: the physiological saline is embedded in the liposome Approximately 100 / d of liposome A, B, or c was evenly applied to the wound of the test mouse one hour after the wound was caused. Thereafter, about 1 hour of the liposome A, B, or c was applied in the same manner every 24 hours, and a total of three times were applied. 24 hours after the third application, the third 曰 after the wound was produced, the mouse skin wound was observed under a microscope and the image of the third sputum was taken and recorded, and the wound area on the third day was calculated by software. The microscope used in this embodiment is connected to a charge coupled device (CCD) to capture the image of the mouse skin wound, and after capturing the image, the image is transmitted to a computer for image processing and wound area calculation and statistics. The software used for image processing and wound area calculation is the Northern Eclipse image system; the data is analyzed by the software _ SigmaStat program (2002), which is a one-way analysis of Variance (ANOVA) of Duncan The calculation is performed by Duncan's New Multiple Range Test (DMRT). The effect of mouse skin wounds is shown in Figure 3. Figure 3A shows the wound image of Xiaoqi Dihao; the third image shows the wound image of the third day of mice treated with liposome sputum; and the 3C is the wound of the third sputum of mice treated with lipophilic B. The image; and the 3D image is the wound image of the third sputum of mice treated with liposome C. Comparing Figures 3A-D, it was found that the wound area of the mouse after three days of treatment with the composition (microlipid A) for promoting epidermal cell regeneration in the examples of the present invention (as shown in Fig. 3B) was apparent. Less than the other two groups 17 200932909 (d) mouse skin injury π area (as shown in Figure 3c & 3d). : The software was used to calculate the skin wound area on day 0 and day 3 of each group of mice to convert the wound healing rate as follows. Wound healing rate (%) = (the skin area of the skin wound) / mink skin Wide wounded skin wound area _ 3rd day skin area X 100%
❹ 取各組小鼠傷口瘡合率平均值，結果如第4圖，其中微脂 體A組小鼠的傷口癒合率約為7〇%，而微脂體B小鼠的傷 口癒合率約為61%，微脂體C組小鼠的傷口癒合率約為52 % °傷口癒合率之統計分析結果如表-所示。由表-可以 發現，相對於對照_脂體C，試驗組微㈣A以及微脂 體B中’小鼠傷口癒合率皆具有顯著差異(p<〇 〇5)。 鼠皮膚率統計分析 微脂體A 傷 I 69 ( % ) 平均 值 標準 偏差 II — 68 — III IV 73 71 70.25 2.22 微脂體B 61 60 62 60 60.75 0.96 |微脂體C 52 Γ ' 一 —— 51 49 54 51.50 2.10 實施例（五） 人類皮膚試驗 以上述實施例三所製得微脂體A:以微脂體包埋本發明實 施例所述之人類生長因子溶液（用以促進表皮細胞再生的 18 200932909 組合物）進行人類皮膚試驗，試驗對象之資料如表二所示，於試驗前 拍照記錄各試驗對象之試驗部位，如第5A、5C、5E、及5G圖所示。 表二人類皮膚試驗試驗對象之資料 試驗者 性別 年齡 試驗部位 試驗前 試驗後 試驗者1 男 44 眼尾皺紋處 第5A圖 第5B圖 試驗者2 男 29 額頭青春痘 第5C圖 第5D圖 試驗者3 女 20 臉部青春癌· 第5E圖 第5F圖 試驗者4 女 25 臉部青春痘 第5G圖 第5H圖 將上述實施例三所製得微脂體A局部塗抹於各試驗者之試驗部 位，每曰塗抹2次，一次塗抹2 ml，連續塗抹3〇曰後，拍照記錄各試 驗對象之试驗部位’如第5B、5D、5F、及5H圖所示。由第5A及5B 圖可以看出，連續使用本發明所提供之促進傷口癒合之組合物3〇曰 後，可以使臉部歒紋淡化。此外，由第5D、5F、5H圖也可看出，本 發明所提供之促進傷口癒合之組合物亦可使臉部皮膚之疤痕(如：青春 痘)淡化。 由上述本發明較佳實施例可知，應用本發明具有下列 優點。 首先’藉由本發明實施例之體外培養人體骨髓間葉幹 細胞的方法，使得人體骨髓間葉幹細胞可產生人類生長因 19 200932909 子組成，上述人類生長因子組成至少包含複數種與傷口癒 合/表皮細胞再生相關之生長因子。此外，由於這些生長因 子是由人體骨髓間葉幹細胞所表現，其成分和人體自身表 現之生長因子相同且不含原核内毒素。另一方面，這些生 長因子之活性及種類亦優於人類成體細胞和/或基因重組 之異種細胞所表現者。 再者，根據本發明實施例之培養方法，可於末代培養 Ο 液中加入一營養液，其含有一或更多種胺基酸和/或維生 素，可在培#過程中提供額外的#分給人體骨髓間葉幹細 胞。此外，根據本發明製備用以促進表皮細胞再生的組合 物時，不需移除末代培養時額外加入之營養液，因而最終 用以促進表皮細胞再生的組合物中所含的營養液可額外提 供表皮、真皮組織再生、以及膠原纖維重整所需之養份， 而和本發明實施例之人類生長因子組成產生—種加乘效 果進步提升表皮及真皮組織之傷口癒合和/或膠原纖維 ^ 重整能力。 最後，上述用以促進表皮細胞再生的組合物不僅可透 過微脂體包埋法以形成一種可内用或外服之生長因子微脂 體更可力口人人工皮膚或一皮膚替代物中，以促進表皮 細胞再生。 雖然本發明已以較佳實施例揭露如上，然其並非用以 限定本發明，任何熟習此技藝者，在不脫離本發明之精神 和範_，當可作各種之更動與潤飾，因此本發明之保護 範圍當視後附之中請專利範圍所界定者為準。 20 200932909 【圖式簡單說明】 為讓本發明之上述和其他目的、特徵、優點與實施例 能更明顯易懂’所附圖式之詳細說明如下： 第1圖為一人類生長因子抗體微陣圖； 第2A圖闡明本發明之生長因子組成中幹細胞生長因 子（SCF)之濃度；平均值 The average wound healing rate of each group of mice was obtained. The results are shown in Fig. 4. The wound healing rate of mice in the liposome group A is about 7〇%, and the wound healing rate of mice in the liposome B is about 61%, the wound healing rate of the mice in the liposome group C was about 52%. The statistical analysis results of the wound healing rate are shown in Table--. From the table - it can be found that the wound healing rate of the mice in the test group micro (tetra) A and the liposome B was significantly different (p < 〇 〇 5) relative to the control _ lipid C. Statistical analysis of mouse skin rate Liposomal A injury I 69 (%) Mean standard deviation II — 68 — III IV 73 71 70.25 2.22 Microlipid B 61 60 62 60 60.75 0.96 | Microlipid C 52 Γ ' 51 49 54 51.50 2.10 Example (5) Human skin test The liposome A was prepared in the above Example 3: the human growth factor solution (for promoting epidermal cell regeneration) of the present invention was embedded in a liposome. 18 200932909 Composition) The human skin test was carried out. The data of the test subjects are shown in Table 2. The test sites of each test subject were photographed before the test, as shown in Figures 5A, 5C, 5E, and 5G. Table 2 Human skin test test data Tester Gender age test site Test before tester Tester 1 Male 44 Eye wrinkles 5A Figure 5B Figure 2 Male 29 Forehead pimples 5C Figure 5D Tester 3 Female 20 Face Youth Cancer · 5E Figure 5F Figure Tester 4 Female 25 Face Pimples 5G Figure 5H Figure The above-mentioned Example 3 was prepared by applying the liposome A to the test site of each tester. 2 times per smear, 2 ml at a time, 3 s after continuous application, and photographed the test site of each test subject as shown in Figures 5B, 5D, 5F, and 5H. As can be seen from Figures 5A and 5B, after continuous use of the composition for promoting wound healing provided by the present invention, facial crepe can be diluted. Further, as can be seen from the 5D, 5F, and 5H diagrams, the composition for promoting wound healing provided by the present invention can also dilute the scar of the skin of the face (e.g., acne). It will be apparent from the above-described preferred embodiments of the present invention that the application of the present invention has the following advantages. First, the method for in vitro culture of human bone marrow mesenchymal stem cells by the method of the present invention enables human bone marrow mesenchymal stem cells to produce human growth factor 19 200932909 sub-composition, wherein the human growth factor composition comprises at least a plurality of types and wound healing/epidermal cell regeneration. Related growth factors. In addition, since these growth factors are expressed by human mesenchymal stem cells, they have the same growth factors as the human body itself and do not contain prokaryotic endotoxin. On the other hand, the activity and species of these growth factors are also superior to those of human adult cells and/or genetically modified xenogenic cells. Furthermore, according to the culture method of the embodiment of the present invention, a nutrient solution containing one or more amino acids and/or vitamins may be added to the last culture sputum, and an additional #分分Give human bone marrow mesenchymal stem cells. Further, when the composition for promoting epidermal cell regeneration is prepared according to the present invention, it is not necessary to remove the nutrient solution additionally added in the last culture, and thus the nutrient solution contained in the composition for promoting epidermal cell regeneration can be additionally provided. The nutrients required for epidermal, dermal tissue regeneration, and collagen fiber reforming, and the human growth factor composition of the present invention are produced to enhance the wound healing and/or collagen fiber weight of the epidermis and dermal tissue. Whole ability. Finally, the above-mentioned composition for promoting epidermal cell regeneration can be formed not only by the liposome entrapment method to form an internal or external growth factor liposome, but also a human oral skin or a skin substitute. To promote epidermal cell regeneration. While the invention has been described above by way of a preferred embodiment, it is not intended to limit the invention, and the invention may be modified and modified without departing from the spirit and scope of the invention. The scope of protection shall be subject to the definition of patent scope in the attached annex. BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features, advantages and embodiments of the present invention will become more <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; Figure 2A illustrates the concentration of stem cell growth factor (SCF) in the growth factor composition of the present invention;
第2B圖闡明本發明之生長因子組成中血管内皮生長 因子（VEGF)之濃度； 阐明依照本發明 第3A-D為顯微照相圖 例，小鼠皮膚傷口癒合情形；以及 第：圖為長條圖，闌明依照本發明一具體實施例，以 各種微脂體成分處理小I## 影響。 ^皮膚傷σ對於皮膚傷Π癒合率之 第5A-Η為顯微照相圖，閣 例，人體皮廣傷口癒合與淡化 細 明依照本發明一具體實施 紋·情形。 ❹ 【主要元件符號說明】 21Figure 2B illustrates the concentration of vascular endothelial growth factor (VEGF) in the growth factor composition of the present invention; clarifies the condition of mouse skin wound healing in accordance with the photomicrographs 3A-D of the present invention; and the figure: the bar graph According to one embodiment of the present invention, the effect of small I## is treated with various liposome components. ^ Skin injury σ for the skin wound healing rate of the 5A-Η is a photomicrograph, the case, the human skin wide wound healing and desalination detail according to a specific embodiment of the invention. ❹ [Main component symbol description] 21