WO2011116636A1 - 用于治疗皮肤创伤之医药组合物 - Google Patents

用于治疗皮肤创伤之医药组合物 Download PDF

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Publication number
WO2011116636A1
WO2011116636A1 PCT/CN2011/000502 CN2011000502W WO2011116636A1 WO 2011116636 A1 WO2011116636 A1 WO 2011116636A1 CN 2011000502 W CN2011000502 W CN 2011000502W WO 2011116636 A1 WO2011116636 A1 WO 2011116636A1
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Prior art keywords
skin
pharmaceutical composition
wound
stem cells
umbilical cord
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PCT/CN2011/000502
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English (en)
French (fr)
Inventor
傅毓秀
施养性
Original Assignee
Fu Yu-Show
Shih Yang-Hsin
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Priority to US13/637,608 priority Critical patent/US20130243882A1/en
Publication of WO2011116636A1 publication Critical patent/WO2011116636A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood

Definitions

  • the present invention relates to the technical field of skin wound treatment.
  • the present invention provides a pharmaceutical composition for treating skin wounds comprising umbilical cord mesenchymal stem cells.
  • the skin is the first defense level for the body in the face of external threats, microbes, etc. It has important functions in physiology. Trauma, burns, chronic ulcers, etc., may cause serious damage to the skin structure, affecting the primary immune function of the membrane barrier, which may be associated with a systemic difficulty. The healing of skin wounds must mainly undergo inflammatory response, epidermal regeneration, granulation tissue
  • stem cell production can help regenerate skin tissue after trauma.
  • researchers have used different sources of stem cells to treat traumatic skin. It is expected that stem cells can help the regeneration and reconstruction of the entire skin wound in various aspects, and has achieved good results (Yaojiong et al., Mesenchymal stem cells) Enhance wound healing through differentiation and angiogenesis. Stem Cells, 25(10): 2648-59, 2007).
  • stem cell sources are not easy to obtain, and some even have moral or safety concerns. There are still many problems to be solved.
  • the human umbilical cord is a post-production waste.
  • the present invention provides a pharmaceutical composition for treating skin wounds comprising umbilical mesenchymal stem cells.
  • the pharmaceutical composition promotes wound healing.
  • the invention is directed to the use of umbilical cord mesenchymal stem cells for the manufacture of a medicament for the treatment of skin wounds.
  • the umbilical cord mesenchymal stem cells are from a human
  • Figure 1 shows that HUMSCs were co-cultured with dermal fibroblasts, and the creep retraction gap was observed at 0, 24, 48, and 72 hours. It was found that the rat dermal fibroblasts co-cultured with HUMSCs had better crawling ability. Good (Fig. 1A), it was found that there was a significant difference between 24 hours and 48 hours (p ⁇ 0.05, Fig. IB), and it was found that the amount of collagen secreted into the culture medium by fibroblasts co-cultured with HUMSC was significantly higher (p ⁇ 0.05). , Figure 1C).
  • Figure 2 shows the history of traumatic changes in wounds treated with HUMSCs, showing that the graft group was healed faster (Fig. 2A), and the percentage of wound area in the transplant group was significantly lower than that in the control group (p ⁇ 0.05, Figure 2B).
  • Figure 3 shows the results of HE staining of the serial sections of the control group and the HUMSCs treatment group on day 4, 8 and 14 after treatment of wounded skin by HUMSCs (Fig. 3A).
  • the volume of wounds treated with HUMSCs was smaller than that of the control group (p ⁇ 0.05, 3 B), the distance between the hair follicles on both sides of the wound in the transplantation group was relatively close (P ⁇ 0.05, Figure 3C).
  • Figure 4 shows the results of HUMSCs treatment of traumatic skin during the healing of skin wounds.
  • the results of neutrophil-aspirating macrophage aggregation were observed.
  • the cells with MPO response on the second and fourth days of control group skin tissue showed extremely low performance (Fig. 4 A, C), and the cells in the transplanted group had an increased MPO response on the second and fourth days (B, D in Figure 4).
  • Figure 5 shows the use of ED1 to identify neutrophils and macrophages after HUMSCs have treated wound skin.
  • the control group had cells with ED1 reaction entering the wound site on the second and fourth days (A, C in Fig. 5); the transplant group had more ED1 response on the second and fourth days.
  • Cell infiltration B, D in Figure 5
  • E, F in Fig. 5 the distribution of cells with ED1 reaction
  • the control group and the transplantation group have no divisions (E in Figure 5) El, F in F1)
  • cells with ED1 response in the unconstructed area of the skin structure are distributed (E2 in E in Fig. 5); in the case where the infiltration phenomenon has slowed down, the transplantation group remains There are more cells with an ED1 reaction in it (F2 in F in Fig. 5).
  • FIGS 6A-6B show the collagen folding after HUMSCs treatment of wounded skin (eight, B, C, D in Figure 6A). On the eighth day, the control group still failed to accumulate the collagen necessary for the construction of the dermis (Fig. 6A). E:).
  • Figure 7 shows that HUMSCs are still present in the skin tissue after treatment of the wounded skin (Figure 7, K).
  • the D and E in Figure 7 also have the ability to crawl during wound healing and can be moved to the skin wound.
  • B, C in Fig. 7 DETAILED DESCRIPTION OF THE INVENTION All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, unless otherwise defined.
  • the article “a” refers to one or more (i.e., at least one) of the grammatical objects of the article.
  • a component means one component or more than one component.
  • the invention provides a pharmaceutical composition for treating skin wounds comprising umbilical mesenchymal stem cells.
  • the invention provides the use of umbilical cord mesenchymal stem cells for the manufacture of a medicament for treating skin wounds.
  • the term "umbilical cord mesenchymal stem cells” refers to stem cells located in the umbilical cord of a mammal, preferably in human umbilical cord mesenchymal tissue, and may be unpurified cell cultures or purified cells.
  • the following examples illustrate the procedure for obtaining umbilical cord mesenchymal stem cells from individual tissues.
  • the umbilical cord is a waste after production. The source is easy to obtain, there is no ethical concern, the treatment method is simple, the quantity is very large, and the reproduction is rapid. And the laboratory has previously found that transplantation of human umbilical cord mesenchymal stem cells does not cause host immune rejection. Therefore, human umbilical cord mesenchymal stem cells are suitable for allogeneic A good source of stem cells for transplantation.
  • pharmaceutical composition refers to a mixture of pharmaceuticals which typically contains a carrier, such as a pharmaceutically acceptable carrier or excipient, which is well known to those skilled in the art and suitable for administration to a subject. For therapeutic, diagnostic or prophylactic purposes, it may also include cell cultures or cells.
  • the pharmaceutical composition may be in the form of a solution, suspension, lozenge, pill, capsule or powder, which is preferably administered by injection.
  • pharmaceutically acceptable carrier refers to any conventional type of non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation adjuvant or excipient.
  • the pharmaceutically acceptable carrier is non-toxic to the recipient at the dosages and concentrations employed and is compatible with the other ingredients of the formulation.
  • Pharmaceutically acceptable carriers are generally readily available to the public.
  • pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, osmotic pressure adjusting agents, stabilizers, wetting agents and the like are also available to the public.
  • Suitable carriers include, but are not limited to, water, glucose, glycerol, saline, ethanol, and combinations thereof.
  • the carrier may contain additional agents, such as wetting and emulsifying agents, pH buffering agents or adjuvants, which may enhance the effectiveness of the formulation.
  • Local carriers include liquid petroleum, isopropyl palmitate, polyethylene glycol, ethanol (95%), polyoxyethylene monolaurate (5%) dissolved in water, or dodecyl sulfate in water. Sodium (5%). Other materials such as antioxidants, humectants, viscosity stabilizers and the like may be added as needed.
  • the pharmaceutical composition of the present invention can be used for the treatment of skin wounds, and the individual is preferably a mammal, more preferably a human.
  • the pharmaceutical composition can be implanted into a skin wound, or placed around a skin wound, or on a skin wound. Or applied to the dressing on the skin wound.
  • the human umbilical cord was collected aseptically and stored at HBC (Biochrom L201-10) for no more than 24 hours at 4 °C.
  • the umbilical cord was first soaked in 75% ethanol for 30 seconds to disinfect.
  • the sterilized umbilical cord is placed in a buffer solution (CMF, Gibco 14185-052) with no calcium and no magnesium ions in a sterile operating table, and the umbilical cord is slit by a disinfected instrument, and the blood vessels and mesenchymal tissues are Watson's gel) removed.
  • CMF buffer solution
  • the mesenchymal tissue was cut into small pieces of 0.5 cm 3 and centrifuged at 250 x g for 5 minutes. The supernatant was removed, and the pellet was washed twice with an appropriate amount of serum-free DMEM (Gibco 12100-046) and centrifuged at 250 xg for 5 minutes.
  • the mesenchymal tissue was treated with collagenase (collagnase) for 14 to 18 hours at 37 ° C, washed, and treated with 2.5% trypsin at 37 ° C for 30 minutes under shaking.
  • FBS Hyclone SH30071.03
  • Mesenchymal cells were dispersed in 10% FBS-DMEM and counted, and then directly used for culture and subsequent experiments.
  • Example 2 In vitro culture of rat dermal fib rob lasts
  • the monolayer dermal fibroblasts were scraped off an equidistant gap using a blue tip tip and co-cultured with human umbilical cord mesenchymal stem cells.
  • the crawling of the light microscopy and the retraction of the gap showed that the rat dermal fibroblasts co-cultured with human umbilical cord mesenchymal stem cells had better crawling ability ( Figure 1A).
  • significant differences were found between 24 hours and 48 hours (P ⁇ 0.05, Figure 1B).
  • Example 3 Establishment of a rat model of skin trauma
  • the experimental animals were divided into two groups.
  • the first group was the control group. After the skin wounds were established, 20 ⁇ physiological saline (saline) was immediately given at four points around the wound.
  • the second group was the experimental group. Immediately after the skin wound was established, 5*10 5 /20 ⁇ human umbilical cord mesenchymal stem cells (HUMSCs) were transplanted at four points around the wound.
  • HUMSCs human umbilical cord mesenchymal stem cells
  • the experimental animals were divided into two groups. The first group was the control group. After the skin wounds were established, 20 ⁇ of physiological saline was administered immediately at four points around the wound. The second group was the experimental group, which was divided into rat dermal fibroblasts and cultured separately with fibroblasts and stem cells. After three days of culture, the cell culture medium was collected to measure the soluble collagen fibers (SircolTM Soluble Collagen Assay). Kit) Measure the content of soluble collagen fibers in the skin tissue of the rats or in the cell culture medium.
  • the primary antibodies used in this experiment were rabbit anti-MPO antibody, mouse anti-EDl antibody, mouse anti-human specific nuclear antigen antibody (Mouse anti-human) Specific nuclei antigen antibody ). After the action, the appropriate secondary antibody is ligated, followed by color development with DAB.
  • Example 6 Effect of HUMSCs on skin wound volume reduction and skin tissue reconstruction
  • HE staining showed that on the fourth day, there was already cell infiltration (blue-purple:) at the skin wound.
  • the skin removed from the control group still has many voids, and the cells that help the wound repair still do not fully enter the wound (Fig. 3A, Day 4 control group:).
  • Many cells in the transplant group have entered the wound to help repair, and the wound volume is small (Fig. 3A, day 4 transplant group:).
  • the number of cells infiltrated in the control group had filled the wound site and the HE staining was blue-violet (Fig. 3A, day 8 control group:).
  • the cells infiltrating the wound site in the transplanted group have begun to release the extracellular matrix, making the red staining of the HE part obvious (Fig. 3A, day 8 transplantation group).
  • the wounds On the fourteenth day, the wounds have been significantly reduced, and the hair follicles and other tissues are obviously restored or regenerated.
  • the skin thickness of the post-traumatic regeneration in the transplantation group is closer to the surrounding normal skin structure, and the distance between the hair follicles on both sides of the residual wound is also closer than that of the control group. 3 A, day 14).
  • the statistical results showed that the wounds treated with HUMSCs were smaller than the control group during the healing of the fourth, eighth and fourteenth days (p ⁇ 0.05, Figure 3 B:).
  • the distance between the two sides of the wound was statistically analyzed.
  • the transplant group was closer (P ⁇ 0.05, Fig. 3C).
  • the ability of hair follicle regeneration or wound contraction in the transplant group was stronger than that in the control group.
  • Example 7 Effect of HUMSCs on neutrophils and macrophages in wound skin
  • ED1 is used to identify neutrophils and macrophages. The results show that the control group is in the second and fourth The EDI-positive cells of the day have entered the wound site (A in Figure 5,
  • the collagen in the peritoneal tissue of the parietal layer i.e., the red region
  • the results showed that on the second and fourth days, the expression of collagen fibers in the skin tissues of the control group and the transplant group was lower than that of the surrounding normal structured skin, and the collagen fibers and the underlying fascia of the raw skin were The boundary is difficult to distinguish, so it is impossible to determine the collagen folding situation (A, B, C, D in Figure 6A.
  • the control group still cannot accumulate the collagen necessary for the dermis in the wound site (Fig. 6A. Transplantation).
  • the group clearly deposited a lot of collagen (F in Fig. 6A).
  • Example 9 Reconstruction of HUMSCs in traumatic skin
  • the nucleus of human umbilical cord mesenchymal stem cells which is labeled with anti-human specific nuclei antigen, is still present in the skin tissue (D in Figure 7, E in Figure 7).
  • the process of wound healing it also has the ability to crawl and move to the skin wound (B in Figure 7, C in Figure 7), which continues to help rebuild the skin tissue. It is shown that HUMSCs have an excellent effect on the treatment of skin wounds.

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Description

用于治疗皮肤创伤之医药组合物 技术领域 本发明系关于皮肤创伤治疗之技术领域。 具体而言， 本发明提供一种 治疗皮肤创伤之医药组合物， 其包括脐带间质干细胞。
说 背景技术
书
皮肤是身体在面对外在威胁性伤害、 微生物等， 的第一道防御关卡。 在生理上具有重要功能。 创伤、 烧烫伤、 慢性溃疡等， 可能会造成皮肤构 造严重损害， 影响其皮膜屏障的初级免疫功能， 进而可能伴随全身性难以 估计之危险。 皮肤创伤的愈合主要必须历经发炎反应、 表皮再生、 肉芽组织
(granulation tissue)生成、 血管增生， 创伤收缩以及细胞外基质重新构建等 过程， 才能帮助创伤后再生的皮肤组织。 近年来随着干细胞研究的兴起， 研究人员使用不同来源的干细胞给予创伤皮肤治疗， 期望干细胞能够在各 方面帮助整个皮肤创伤的再生与重建， 且已达不错成效 (Yaojiong et al.， Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells, 25(10)： 2648-59, 2007)。惟大部分干细胞来源不易 取得， 有些甚至有道德或安全上的疑虑， 仍有许多问题待解决。 人类脐带是一种生产后的废物， 来源取得容易， 没有道德顾虑， 处理 方式简单， 而且人类脐带干细胞数量极多、 繁殖快速， 是很好的干细胞来 源。 过去研究出移植人类脐带间质干细胞于大白鼠紋状体中， 植入的细胞 可以存活在大白鼠脑中四个月， 表示人类脐带间质干细胞， 不会引起宿主 的免疫及排斥反应。 因此， 人类脐带间质干细胞为一株适合用于进行异体 移植的良好干细胞来源。 发明内容 在一方面， 本发明提供一种用于治疗皮肤创伤之医药组合物， 其包括 挤带间质干细胞 (umbilical mesenchymal stem cells 特定而言， 所述医药 组合物可促进创伤愈合。
再另一方面， 本发明是针对脐带间质干细胞之用途， 其用以制备供治 疗治疗皮肤创伤之医药。在一具体实施例中， 所述脐带间质干细胞来自人
附图说明 前文之所述以及实施方式可通过说明书附图达到更好的说明效果。为 了加强本发明之说明， 将适当的实施例之附图列举于此。
图 1是 HUMSCs与真皮纤维母细胞共同培养， 于时间点 0小时、 24 小时、 48小时、 72小时观察其爬行补回缺口的情形， 发现与 HUMSCs共 同培养的大鼠真皮纤维母细胞爬行能力较好 （图 1A), 发现在 24小时和 48小时有显著差异 （p < 0.05， 图 IB) , 发现与 HUMSC共同培养的纤维 母细胞分泌到培养液中的胶原蛋白量明显较多 （p<0.05， 图 1C)。
图 2为 HUMSCs处理创伤皮肤之创伤变化情形纪录， 显示移植组的 愈合速度较快 (图 2A), 移植组创伤面积的百分比和控制组相比显著较低 (p < 0.05， 图 2B)。
图 3为 HUMSCs处理创伤皮肤后第 4、 8及 14天控制组及 HUMSCs 治疗组连续切片 HE染色的结果 (图 3A), 给予 HUMSCs治疗的创伤剩于 体积较控制组小 （p < 0.05， 图 3 B)， 移植组之创伤两侧毛囊间距离较近 (P < 0.05， 图 3C)。
图 4为 HUMSCs处理创伤皮肤在皮肤创伤愈合的过程中观察嗜中性 球吸引巨噬细胞聚集的结果， 控制组皮肤组织第二天和第四天有 MPO反 应之细胞表现极低 (图 4中的 A、 C), 而移植组皮肤组织第二天和第四天 有 MPO反应之细胞表现有增多的趋势 (图 4中的 B、 D
图 5为 HUMSCs处理创伤皮肤后， ED1用以标识嗜中性球与巨噬细 胞之结果，控制组在第二、第四天已有有 ED1反应之细胞进入创伤部位 (图 5中的 A、 C); 移植组在第二、第四天有较多的有 ED1反应之细胞浸润 (图 5中的 B、 D)；。在第十四天时， 有 ED1反应之细胞分布情形 (图 5中的 E、 F), 在皮肤构造已经构建完成的区域， 控制组和移植组皆没有其分部 (图 5 中的 E中的 El、 F中的 F1), 而在皮肤构造上未构建完成区域仍有 ED1 反应之细胞分布于内（图 5中的 E中的 E2); 在浸润现象已减缓的情况下， 移植组仍留有较多有 ED1反应之细胞在其中 (图 5中的 F中的 F2)。
图 6A-6B为 HUMSCs处理创伤皮肤后， 胶原蛋白折叠情形 (图 6A中 的八、 B、 C、 D) ， 第八天控制组在创伤部位仍无法累积构建真皮所必须 之胶原蛋白（图 6A中的 E：)。
图 7 为 HUMSCs 处理创伤皮肤后与控制组织比较 (图 7 中的 K), HUMSCs仍然存在于皮肤组织 (图 7中的 D、 E 在创伤愈合的过程中也 具有爬行能力， 可移动到皮肤创伤处 (图 7中的 B、 C)。 具体实施方式 除非另外定义，本文中所用的所有技术及科学词汇具有所属技术领域 的技术人员所通常明了之相同意义。
在本文中， 冠词 "一"指一个或一个以上 （亦即， 至少一个） 之该冠 词之文法客体。 举例而言， "一元件"意谓一个组件或一个以上之元件。
在一方面， 本发明提供一种用于治疗皮肤创伤之医药组合物， 其包括 脐带间质干细胞（umbilical mesenchymal stem cells )。另一方面， 本发明提 供脐带间质干细胞之用途， 其用以制备供治疗皮肤创伤之医药。
本文所使用的 "脐带间质干细胞"一词指位于哺乳类动物脐带， 较佳 为人类脐带间质组织中之干细胞，可为未经纯化的细胞培养物或纯化后的 细胞。 以下实施例说明从个体组织取得脐带间质干细胞之流程。 脐带为生 产后废弃物， 来源取得容易， 没有道德顾虑， 处理方式简单， 数量极多、 繁殖快速。 且本实验室先前发现， 移植人类脐带间质干细胞， 不会引起宿 主产生免疫排斥反应。所以人类脐带间质干细胞为一种适合用来进行异体 移植的良好干细胞来源。
本文所使用的 "医药组合物"是指一种作为医药之混合物， 其通常含 有载体， 诸如， 医药可接受之载体或赋形剂， 其是所属技术领域的技术人 员公知且适合投药至对象中， 以作为治疗性、 诊断性或预防性之目的， 其 亦可包括细胞培养物或细胞。医药组合物之形式可为溶液、悬浮液、锭剂、 丸剂、 胶囊或粉末， 其给药方式较佳为注射。
本文所使用的 "医药可接受之载体"指任何习知类型之无毒固体、 半 固体或液体之填充剂、 稀释剂、 包胶囊材料、 调配物辅助剂或赋形剂。 医 药可接受之载体系在所用之剂量及浓度下， 对接受者无毒性， 且可与该调 配物之其它成分兼容。医药可接受之载体一般均可由公众轻易取得。此外， 医药可接受之辅助物质， 诸如， pH调节及缓冲剂、 渗透压调节剂、 安定 剂、 湿润剂及类似物， 亦皆可由公众取得。
适当之载体包括， 但不限于， 水、 葡萄糖、 甘油、 盐水、 乙醇及其组 合。 载体可含有额外之试剂， 诸如， 湿润及乳化剂、 pH缓冲剂或是佐剂， 其可增强该调配物之有效性。 局部性载体包括液体石油、 棕榈酸异丙酯、 聚乙二醇、 乙醇 （95%)、 溶于水中的聚氧乙烯单月桂酸酯 （5%) 或是溶 于水中的十二垸基硫酸钠 （5%)。 可视需要加入其它材料， 诸如， 抗氧化 剂、 保湿剂、 黏度稳定剂及类似试剂。
本发明之医药组合物可用于治疗皮肤创伤， 个体较佳为哺乳动物， 更 佳为人类。 在一具体实施例中， 所述医药组合物可植入皮肤创伤， 或置于 皮肤创伤周围， 或皮肤创伤上。 或施加于敷料在置于皮肤创伤上。
本发明之各个具体实施例的细节说明如后。本发明之其它特征将会通 过以下各个具体实施例中的详细说明及权利要求而清楚呈现。所属技术领 域的技术人员可了解本发明具有各种实施例方式， 下述具体实施例仅用以 说明而非作为本发明之限制。
除非另外定义，本文中所用之所有技术及科学词汇具有所属技术领域 的技术人员所通常明了之相同意义。
本发明之各个具体实施例的细节说明如后。本发明之其它特征将会经 由以下各个具体实施例中的详细说明及申请专利范围而清楚呈现。所属技 术领域的技术人员可了解本发明具有各种实施方式，下述具体实施例仅用 以说明而非作为本发明之限制。
实施例 1： 人类脐带间叶细胞之制备
人类脐带以无菌操作方式收集并在 4°C下储存于 HBSS ( Biochrom L201-10) 不超过 24小时。
脐带先泡在 75%之乙醇中 30秒以消毒。 在无菌操作台中将消毒过之 脐带置于无钙及无镁离子之缓冲溶液（CMF, Gibco 14185-052 )， 以消毒过 之器具将脐带纵切， 并将其中之血管及间叶组织 （瓦顿氏凝胶） 去除。 将 间叶组织切成 0.5立方厘米之小块， 以 250xg离心 5分钟。 去除上清液， 并以适量之无血清 DMEM (Gibco 12100-046)清洗沉淀物二次， 再以 250 xg离心 5分钟。 间叶组织在 37°C下以胶原酶 （collagnase) 处理 14至 18 小时， 清洗后， 再以 2.5%之胰蛋白酶 （trypsin) 于 37°C及震荡下处理 30 分钟。 加入 FBS (Hyclone SH30071.03 ) 至间叶组织以终止胰蛋白反应。 此时间叶组织已变成间叶细胞。 间叶细胞以 10% FBS-DMEM使其分散并 计算其数量后， 便可直接用于培养及进行后续实验。
实施例 2: 大白鼠真皮纤维母细胞 (Rat dermal fib rob lasts)之体外培养
初生 3~5天的大白鼠， 取其皮肤， 加入胰蛋白酶 （trypsin) 以剔除表 皮， 再以胶原酶处理真皮组织， 此时真皮组织内的纤维母细胞便可分离出 来， 再以 10%FBS DMEM进行培养。
利用 blue tip尖端， 将培养单层 （monolayer) 的真皮纤维母细胞刮出 一道等距离的缺口， 同时与人类脐带间质干细胞共同培养。 于时间点 0小 时、 24小时、 48小时、 72小时， 由光学显微镜观察其爬行， 补回缺口的 情形，可以发现与人类脐带间质干细胞共同培养的大鼠真皮纤维母细胞爬 行能力较好（图 1A)。统计后， 发现在 24小时和 48小时有显著差异（P < 0.05 , 图 1B)。
实施例 3: 皮肤创伤的大白鼠模式建立
取 7周大， 250g重之雄性大白鼠，剔除其背部毛，于两耳联机下 1.5 cm, 利用组织切割器（biopsy punch)移除直径 8 mm的皮肤， 盖上防水透气贴 布， 以防止抓伤。
本研究将实验动物分成两组， 第一组为控制组， 皮肤创伤建立后， 立 即于创伤周围四个点分别给予 20λ生理食盐水 （saline) 。 第二组为实验 组， 皮肤创伤建立后立即于创伤周围四点分别移植 5*105/20λ人类脐带间 质干细胞 （HUMSCs)。
实施例 4: HUMSCs对创伤皮肤产生胶原蛋白之影响
将实验动物分成两组， 第一组为控制组， 皮肤创伤建立后， 立即于创 伤周围四个点分别给予 20λ之生理食盐水。 第二组为实验组， 分为大鼠真 皮纤维母细胞单独培养， 与纤维母细胞与干细胞共同培养两组， 培养三天 后收集细胞培养液， 以可溶性胶原纤维测量套组 (Sircol™ Soluble Collagen Assay kit)测量大白鼠皮肤组织内或细胞培养液中可溶性胶原纤维的含量。
检测控制组及实验组之细胞培养液中之胶原蛋白含量结果显示， 与干 细胞共同培养之纤维母细胞的培养液中， 每毫升含有 (1263.73±52.24) μ^, 较单独培养纤维母细胞组，每毫升 （724.83±78.91) μ8的胶原蛋白相比， 明 显高出许多 (ρ<0.05， 图 1C：)。 证实在体外培养系统中， 与 HUMSC共同培 养后， 纤维母细胞分泌到培养液中的胶原蛋白量明显较多。
实施例 5： HUMSCs对皮肤创伤愈合之影响
利用抗原抗体结合的原理， 标定细胞内蛋白质所在位置的方法。 本实 验所用的初级抗体为兔抗 - MPO 抗体 （Rabbit anti-MPO antibody) , 小鼠 抗 - MPO 抗体 （Mouse anti-EDl antibody) , 小鼠抗-人类专一性细胞核抗 原抗体 (Mouse anti-human specific nuclei antigen antibody )。作用后连接适 当二级抗体， 接着以 DAB进行成色。
由巨观型态定义其出血或湿润乃至形成血栓纤维蛋白 （blood clotting fibrin)是为创伤伤口， 持续关注创伤变化情形， 测量面积且持续记录其数 值到第十四天为止。 第四天， 在移植 HUMSCs之实验组 （移植组）， 观察 到创伤有收缩现象， 创伤面积较仅给予生理食盐水的控制组小， 显示移植 组的愈合速度较快 (图 2A：)。经由统计发现自第四天起， 移植组创伤面积的 百分比和控制组相比显著较低 (p<0.05， 图 2B：)。 另外， 组内前后每两天进 统计分析， 移植组第二天到第四天的愈合面积比例有显著差异 (p<0.05， 图 2B), 而控制组在此阶段并无差异， 因此， 我们推测此时间点为 HUMSCs 影响创伤愈合最剧的时机，后续便于此时观察创伤愈合微观变化与可能机 制。
实施例 6: HUMSCs对皮肤创伤体积缩小及皮肤组织重建之效果
连续切片后 HE染色的结果显示， 第四天， 已经有细胞浸润 (蓝紫色:) 于皮肤创伤处。 控制组被移除的皮肤仍有许多空洞， 帮助创伤修复的细胞 仍未完全进入创部 (图 3A, 第 4天控制组：)。移植组已经有许多细胞进入创 伤帮助修复， 且创伤体积较小 (图 3A, 第 4天移植组：)。 第八天， 控制组的 细胞浸润数量已将创伤部位填满 HE染色呈现蓝紫色 (图 3A， 第 8天控制 组：)。移植组浸润创伤部位的细胞已经开始释放细胞外基质， 使 HE染色红 色部分明显 (图 3A, 第 8天移植组)。 第十四天， 创伤已经明显缩小， 且毛 囊等组织明显复原或再生，移植组的创伤后再生的皮肤厚度较接近周边正 常皮肤构造，残余创伤两侧的毛囊距离也较控制组为近 (图 3 A, 第 14天)。 统计结果显示第四天、 第八天以及第十四天愈合过程中， 给予 HUMSCs 治疗的创伤剩于体积较控制组小 （p<0.05， 图 3 B：)。 同时统计分析创伤两 侧毛囊间距离， 移植组较近 (P<0.05， 图 3C), 第十四天， 移植组的毛囊再 生或是创伤收缩的能力较控制组为强。
实施例 7： HUMSCs对创伤皮肤聚集嗜中性球与巨噬细胞之效果
皮肤创伤愈合的过程中， 嗜中性球（neutrophil)会自血管浸润到创伤 部位， MPO用以标识嗜中性球。 结果显示， 控制组皮肤组织中， 第二天 和第四天有 MPO反应之细胞（MPO-positive cells)表现极低 (图 4中的 A、
C：)。 移植组皮肤组织中， 第二天和第四天有 MPO反应之细胞表现， 从低 倍到高倍的染色图观察到明显有增多的趋势 (图 4中的 B、 D 嗜中性球 会吸引巨噬细胞的聚集，而巨噬细胞所分泌的细胞激素在皮肤创伤愈合过 程中扮演关键角色。
ED1用以标识嗜中性球与巨噬细胞。 结果显示， 控制组在第二、 第四 天已有 EDI反应之细胞 （EDI -positive cells) 进入创伤部位 (图 5中的 A、
C：)。移植组在第二、第四天有较多的有 ED1反应之细胞浸润 (图 5中的 B、 D：)。 在第十四天时， 有 ED1反应之细胞分布情形 (图 5中的 E、 F), 可由 两部分观察。 第一， 在皮肤构造已经构建完成的区域， 控制组和移植组皆 没有其分部 (图 5中的 E中的 El、 F中的 Fl)。第二， 在皮肤构造上未构建 完成区域仍有有 ED1反应之细胞分布于内（图 5中的 E中的 E2), 在浸润 现象已减缓的情况下， 移植组仍留有较多有 ED1反应之细胞在其中 (图 5 中的 F中的 F2), 可持续改善其最后阶段的组织再生。
实施例 8: HUMSCs对创伤皮肤胶原蛋白之构建
以 sirius red标定壁层腹膜组织中的胶原蛋白， 即红色区域，用以定量 真皮胶原蛋白折叠构建在创伤皮肤部位的比例。 结果显示， 第二天和第四 天， 控制组和移植组的皮肤组织中， 胶原纤维的表现量都跟周围正常构造 皮肤相比都较低， 且在生皮肤的胶原纤维与下层筋膜的界线难以分辨， 故 无法确实判定胶原蛋白折叠情形 (图 6A中的 A、 B、 C、 D 第八天， 控 制组在创伤部位仍无法累积构建真皮所必须之胶原蛋白 (图 6A中的 。 移植组则明显堆栈许多胶原蛋白 (图 6A中的 F)。在第十四天时移植组的胶 原蛋白在创伤处的分部比例（80.27±5.19)%， 明显高于控制组 (33.22±1.18)% , 其创伤皮肤组织内的胶原蛋白分部， 明显优于控制组 (p<0.05, 图 6A中的 1)。
实施例 9: HUMSCs于创伤皮肤之再构建
移植后第二周，以抗人类专一性细胞核抗原（anti-human specific nuclei antigen)标定的人类脐带间质干细胞之细胞核， 仍然存在于皮肤组织 (图 7 中的 D、 图 7中的 E)。 在创伤愈合的过程中， 其也具有爬行能力， 可移动 到皮肤创伤处 (图 7中的 B、 图 7中的 C)， 持续帮助皮肤组织的再构建。 显示 HUMSCs对于治疗皮肤创伤有极佳之效果。
统计分析
所有数据以 Mean ± SEM表示。各组之平均值以 One-Way ANOVA或 Two-Way ANOVA分析， 再以 LSD test进行多重比较。 实验数据 p<0.05 判定为具有显著差异。 所属技术领域的技术人员将可在不背离本发明精神之下，根据实施 进行改变和修改。 要注意的是， 本发明并不受限于说明书中实施例所揭 之范围， 而涵盖于其它根据权利要求所揭示的所有变化之形式。

Claims

权 利 要 求 书
1. 一种用于治疗皮肤创伤之医药组合物， 其包括脐带间质干细胞。
2. 根据权利要求 1所述之医药组合物，其特征在于，可促进创伤愈合、 皮肤再生和毛囊再生。
3. 根据权利要求 1或 2所述之医药组合物，其特征在于，脐带间质干 细胞为植入皮肤创伤。
4. 根据权利要求 1或 2所述之医药组合物，其特征在于，脐带间质干 细胞为置于皮肤创伤周围。
5. 根据权利要求 1或 2所述之医药组合物，其特征在于，脐带间质干 细胞为置于皮肤创伤上。
6. 根据权利要求 1或 2所述之医药组合物，其特征在于，脐带间质干 细胞系来自人类。
7. 根据权利要求 1或 2所述之医药组合物，其特征在于，该医药组合 物置于敷料上。
8. 一种脐带间质干细胞之用途， 其用于制备供治疗皮肤创伤之医药。
9. 根据权利要求 8所述之用途，其特征在于，该医药可促进创伤愈合、 皮肤再生和毛囊再生。
10. 根据权利要求 8所述之用途， 其特征在于， 该脐带间质干细胞系 来自人类。
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