自制血清替代物行无血清条件培养人表皮干细胞

Human epidermal stem cells cultured under serum-free culture conditions by using self-made serum replacer

目的:建立一种人表皮干细胞的无血清培养方法,并与目前表皮干细胞常用的有血清培养方法进行比较,探讨该条件是否更适合表皮干细胞的生长和有利于实施表皮干细胞的调控,提高实验的准确性。方法:实验于2002-05/2004-07在江西医学院第二附属医院医学分子中心完成。①从幼儿包皮中分离表皮细胞、用胶原Ⅳ分别黏附表皮细胞10～15min,收集黏附细胞,即为纯化的表皮干细胞。②分3组进行培养,3组培养条件均有无钙低糖Dulbecco改良的Eagle培养基,0.05mmol/LCaCl2,10μg/L表皮细胞生长因子,1×10-6mol/L氢化可的松;有血清培养组还含有100g/L螯合钙的干细胞专用血清;无血清培养组1还含有100g/L无血清培养基血清替代物。无血清培养组2还含有100g/L无血清培养基血清替代物和50μg/L牛垂体提取物。③培养20d后,在倒置显微镜下对所培养细胞进行形态学观察;同时计数细胞数(大于100个细胞团为1个集落称克隆),计数各组克隆数;采用胰酶消化传代,计数传代数及多次传代后所得细胞总数;流式细胞术分析α6,CD71(人表皮干细胞表面标记)的表达情况,计算人表皮干细胞α6briCD71dim细胞百分率;采用流式细胞术进行细胞周期分析处于静止期/DNA合成前期人表皮干细胞百分率,采用免疫组化法鉴定细胞角蛋白19及5/8(均表明人表皮干细胞生长情况,且以角蛋白19更能明确表明人表皮干细胞生成情况)和角蛋白10表达情况。结果:①人表皮干细胞细胞形态学观察结果:应用倒置显微镜下观察检测,无血清培养组1与有血清培养组人表皮干细胞细胞形态学基本相同,无血清培养组1集落形成时间稍晚于有血清培养组。②形成的克隆数及经胰酶消化可传代数、细胞总得数:胰酶消化传代后,形成的克隆数、细胞总得数及细胞传代数无血清培养组1与有血清培养组相近(P>0.05),两组形成的克隆数、细胞总得数明显低于无血清培养组2(P<0.05)而细胞传代数组明显高于无血清培养组2(P<0.05)。③细胞周期分析及α6,CD71鉴定结果:采用流式细胞术检测,有血清培养组与无血清培养组1中人表皮生长干细胞α6briCD71dim细胞百分率及处于静止期/DNA合成前期细胞(即人表皮干细胞)百分率相近(P>0.05),且均高于无血清培养组2(P<0.05)。④细胞角蛋白19和5/8及10表达情况:免疫细胞化学鉴定结果,有血清培养组与无血清培养组1角蛋白19和5/8及10阳性细胞百分率相近(P>0.05),而两组角蛋白19阳性细胞百分率明显高于无血清培养组2(P<0.05),角蛋白5/8和10阳性细胞百分率明显低于无血清培养组2(P<0.05)。结论:①利用自制的无血清培养基血清替代物,添加一些辅助因子(0.05mmol/LCaCl2,10μg/L表皮细胞生长因子,1×10-6mol/L氢化可的松)能较好的培养表皮干细胞,传代次数多,培养时间长,形成克隆数较多,说明此类细胞具有较强的自我复制能力,结合所培养细胞中人表皮干细胞α6briCD71dim细胞及角蛋白19和5/8阳性细胞比例较高等特点,表明该无血清培养条件适合表皮干细胞的生长。②自制的无血清培养基血清替代物添加一些辅助因子(0.05mmol/LCaCl2,10μg/L表皮细胞生长因子,1×10-6mol/L氢化可的松,50μg/L牛垂体提取物)的无血清培养组2能较快获得大量人表皮细胞,当组织工程需要大量细胞时可用此种培养扩增方法。

AIM: To establish a method of culturing human epidermal stem cells under serum-free culture conditions in vitro and compare with serum containing culture, so as to investigate whether it is helpful to investigate the modulation mechanism of epidermal stem cells and improve the assurance of study. METHODS: The study was completed in the Medical Molecular Center of the Second Hospital Affiliated to Jiangxi Medical College from May 2002 to July 2004. ①Epidermal cells were isolated from children foreskin and purify them by adhering with collagen Ⅳfor 10-15 minutes, then the adherence cells were collected, i.e., the purified epidermal stem cells. ②Them were divided into three groups to culture during Dulbecco's modified Eagle's medium including 0.05 mmol/L CaCl2, 103 μg/L epidermal growth factor, 1×10-6 mmol/L hydrocortisone; except that above substances, serum containing group including 100 g/L special serum with Chelated calcium; serum-free culture group 1 including 100 g/L serum substitutes; serum-free culture group 2 including 100 g/L serum substitutes and 50 μg/L bovine pituitary extract. ③After 20 days of culture, cells morphology was observed and numbers of colony was counted (a cluster including more than 100 cells as a colony) under microscope. Meanwhile, passage time was counted and the total cell numbers after passage by trypsin digesting. The expression of surface phenotype α6 and CD71 (human epidermal stem cells) were analyzed by flow cytometry, and the percentage of α6bri CD71dim cells was calculated. Cell cycle at the static phase/the former phase of DNA-synthesis was analyzed with flow cytometry and the expressions of cytokeratin 19 (the phenotype of epidermal stem cell), 5/8 and 10 were assayed with immunocytochemical stain. RESULTS: ①Cell morphological results of human human epidermal stem cells: There were no significant differences in morphology between serum containing group and serum-free culture group 1 by the usage of invert micorscope, but the colony appearing time of serum-free culture group 1 was later than that of serum containing group. ②Clonal numbers, passage number digested by trypsin and total number: After passage by trypsin digest, there were no significant differences in the colony numbers, the total cell numbers and the passage time between serum containing group and serum-free culture group 1 (P >0.05), but the colony numbers and the total cell numbers of two groups were lower than those of serum-free culture group 2 (P< 0.05), but the passage time of two groups was higher than that of serum-free culture group 2 (P< 0.05). ③Analysis of cell cycle and assay of α6 CD71: There were no significant differences in the percentage of α6bri CD71dim cells and the percentage of cells at the static phase/the former phase of DNA-synthesis between serum containing group and serum-free culture group 1 (P >0.05), but higher than those of serum-free culture group 2 (P< 0.05). ④Expression of cytokeratin 19, 5/8 and 10: There were no significant differences in the percentage of cytokeratin 19, 5/8 and 10 positive cells between serum containing group and serum-free culture group 1 (P >0.05), but the percentage of cytokeratin 19 positive cells of two groups was higher than that of serum-free culture group 2 (P< 0.05), but the percentage of cytokeratin 5/8 and 10 positive cells were lower than those of serum-free culture group 2 (P< 0.05). CONCLUSION: ①The serum free medium made by ourselves is appropriate to culture epidermal stem cells supplemented with some factors such as 0.05 mmol/L CaCl2,10 μg/L epidermal growth factor, 1×10-6 mmol/L hydrocortisone, because the cells under this culture condition can be cultured for a long time and get more passage times and more colonies, at that time, the percentage of α6bri CD71dim cells and cytokeratin 19 positive cells are high in culturing process. ②The serum free medium made by ourselves is appropriate to culture epidermal stem cells supplemented with some factors such as 0.05 mmol/L CaCl2,10 μg/L epidermal growth factor, 1×10-6 mmol/L hydroco