人表皮干细胞改良培养及其组织工程皮肤的构建

Improved culture method of Human epidermal stem cell and tissue engineering skin construction

目的:改良传统人表皮干细胞(hESCs)分离、培养方法,为组织工程皮肤构建提供产率更高、活力更好的种子细胞。方法:应用改良的酶消化法(改良法)及传统酶消化法(传统法)分离、培养hESCs,倒置显微镜观察细胞形态,MTT法绘制生长曲线、免疫组化法测定hESCs标志物角蛋白19(K19)和β1整合素的表达,并且进一步比较上述两种方法所获种子细胞构建的组织工程皮肤的形态学的特性。结果:台盼蓝染色显示改良法细胞消化分离数为92.25±15.61个,传统法细胞消化分离数为68.50±26.91个(P<0.01);改良法活细胞比率是(94±0.01)%,传统法活细胞比率是(75±0.04)%(P<0.05)。在8天时改良法细胞MTT法OD值为1.300,传统法为0.779,改良法较传统法活力好,增殖快;细胞免疫组织化学染色显示表皮干细胞标志物角蛋白19(K19)和β1整合素均为阳性染色;HE染色结果显示采用改良法hESCs构建的组织工程皮肤表皮细胞复层层数为5～6层、基底细胞排列整齐,传统法hESCs构建的组织工程皮肤表皮细胞复层层数为3～4层、基底细胞排列散乱。结论:利用改良的酶消化法可获得数量更多、活力更好的人表皮干细胞,为以hESCs为种子细胞构建组织工程皮肤奠定基础。

Objective To improve the traditional methods of dissociation and culture of the human epidermal stem cells（hESCs） and provide more productive and more active seed cells for tissue engineering skin construction.Methods Cell morphology was observed by inverted microscope,MTT law growth curve.Immunocytochemistry was used to observe the expression of β1 integrin and keratin 19（CK19） on epidermal stem cells.Compare the morphological features of tissue engineered skin constructed with the seed cells which were obtained by two methods.Results Trypan blue staining displays the number of hESCs dissociated by modified method is（92.25±15.61）significantly elevated compared with traditional method（68.50±26.91）（P0.01）,and the proportion of living cells 94%±0.01% increased significantly compared with traditional method（75%±0.04%）（P0.05）.The result of immunocytochemistry showed that the expression of β1 integrin and keratin 19（CK19） was positive,which were markers of hESCs.HE staining showed that,by using the improved method,the number of multiple epidermal layers was 5～6 on tissue engineering,and cells of epidermal in the basal part were arranged.In contrast,the number of multiple epidermal layers was 3～4,and the cells of epidermal in basal part arranged in scattered by using the traditional method.Conclusion Human epidermal stem cells in a larger amount and better vitality can be obtained by using a modified enzyme digestion method,which form basis of construction of skin tissue engineering.