角质细胞无血清培养基促进大鼠毛囊干细胞的增殖

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。

Keratinocyte serum-free medium promotes the growth of rat hair follicle stem cells

BACKGROUND: The hair follicle stem cells are usual cultured in Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 containing 10% fetal bovine serum. Recently, keratinocyte serum-free medium has been used to culture hair follicle stem cells. OBJECTIVE:To observe the effects of three different culture media on the proliferation and purity of hair follicle stem cells. METHODS: After sterling, the skin of the rat’s beard was cut off, and the vibrissa follicles were dissected under a stereocroscope. The follicles were digested by Dispase Ⅱ firstly and by the mixture of trypsin and EDTA secondly. Cell suspension was divided into three pieces based on the average number of cells and respectively cultured by keratinocyte serum-free medium, Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 containing 10% fetal bovine serum and keratinocyte serum-free medium containing 10% fetal bovine serum. Hair follicle stem cells were selected by rapid adherence on collagen type Ⅳ, and passaged. RESULTS AND CONCLUSION:Cultured hair follicle stem cells spread to the third generation, and trypan blue assay showed there was no difference in cell survival rate among the three groups (P > 0.05). Cell Counting Kit 8 assay showed that the cells grew slowly in the three groups in the first 2 days. At day 4, the hair follicle stem cells grew into the growth logarithmic phase, and the proliferative activity was ranked as follows: keratinocyte serum-free medium+10% fetal bovine serum > Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12+10% fetal bovine serum > keratinocyte serum-free medium with a significant difference between the three groups (P < 0.05). Flow cytometric examination showed the expression of CD34 in the keratinocyte serum-free medium+10% fetal bovine serum group was lower than that in the keratinocyte serum free medium group (P < 0.05). The expressions of CD34, CK15 and β1-integrin (CD29) in the Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12+10% fetal bovine serum were lower than those in the other two groups (P < 0.05). These findings indicate that we can obtain higher purity hair follicle stem cells in the keratinocyte serum-free medium+10% fetal bovine serum than in the Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12+10% fetal bovine serum.