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背景:以往研究培养毛囊干细胞多使用DMEM/F12+体积分数10%胎牛血清培养基,而进年来开发出的角质细胞无血清培养基也可应用于毛囊干细胞的培养。目的:观察3种不同培养基对大鼠毛囊干细胞增殖情况及干细胞纯度的影响。方法:取大鼠触须部皮肤组织,体式显微镜下分离出毛囊组织,中性蛋白酶Ⅱ与胰蛋白酶和乙二胺四乙酸混合液“两步酶法”消化。所得细胞悬液按细胞数平均分为3份,分别使用角质细胞无血清培养基、DMEM/F12+体积分数10%胎牛血清及角质细胞无血清培养基+体积分数10%胎牛血清共3种培养基培养,Ⅳ型胶原差速贴壁法筛选毛囊干细胞,进行传代培养。结果与结论:培养毛囊干细胞传至第3代,锥虫蓝染色计数法检测结果显示,此3组间细胞活率差异无显著性意义(P>0.05)。CCK8比色法检测细胞生长曲线显示,培养前2 d,此3组细胞生长均较缓慢;培养4 d,细胞生长进入对数生长期,3种培养基培养的细胞增殖活性:角质细胞无血清培养基+10%胎牛血清组>DMEM/F12+10%胎牛血清>角质细胞无血清培养基(P<0.05)。流式细胞仪检测显示,角质细胞无血清培养基组CD34的表达高于角质细胞无血清培养基+10%胎牛血清组(P<0.05)。DMEM/F12+10%胎牛血清组中CD34、β1-整合素(CD29)及CK15标记物的表达低于其他2组(P<0.05)。结果表明,角质细胞无血清培养基较DMEM/F12+体积分数10%胎牛血清能培养出纯度更高的毛囊干细胞,并且在此培养基培养的基础上加入血清,能够促进毛囊干细胞的增殖。
BACKGROUND: In the past, more hair follicle stem cells were cultured using DMEM / F12 + 10% fetal bovine serum. However, keratinocyte serum-free medium developed in recent years can also be applied to the culture of hair follicle stem cells. OBJECTIVE: To observe the effects of three different media on the proliferation of stem cells and the purity of stem cells in rats. Methods: The skin tissue of the rat tentacles was taken. The hair follicle tissue was isolated under a stereomicroscope. The neutral protease Ⅱ and trypsin and ethylenediaminetetraacetic acid mixed solution were digested by two-step enzymatic method. The cell suspension was divided equally into 3 parts according to the number of cells, respectively using keratinocyte serum-free medium, DMEM / F12 + 10% fetal bovine serum and keratinocyte serum-free medium + 10% Culture medium, type Ⅳ collagen differential adherence method screening hair follicle stem cells, subculture. RESULTS AND CONCLUSION: The hair follicle stem cells were passaged to the third generation. The results of trypan blue staining showed that there was no significant difference in cell viability among the three groups (P> 0.05). CCK8 colorimetric detection of cell growth curve showed that 2 d before culture, the three groups of cells were relatively slow growth; cultured 4 d, cell growth into the logarithmic growth phase, three kinds of culture medium cell proliferation activity: keratinocyte serum-free Medium + 10% fetal bovine serum> DMEM / F12 + 10% fetal bovine serum> keratinocyte serum-free medium (P <0.05). Flow cytometry showed that the expression of CD34 in keratinocyte serum-free medium was higher than that in keratinocyte-free serum + 10% fetal bovine serum (P <0.05). The expression of CD34, β1-integrin (CD29) and CK15 in DMEM / F12 + 10% fetal bovine serum was lower than the other two groups (P <0.05). The results showed that keratinocyte serum-free medium can produce hair follicle stem cells with higher purity than DMEM / F12 + 10% fetal bovine serum, and the addition of serum on the basis of this culture medium can promote the proliferation of hair follicle stem cells.