SD大鼠角朊细胞分离培养及鉴定  

Cultivation and Identification of Epidermal Keratinocytes from the Skion of Newborn Sprague Dawley Rats

作　　者：杨亚冬[1] 张文元[1] 李颖[1] 房国坚[1] 张科技[1] 

机构地区：[1]浙江省医学科学院生物工程研究所,杭州310013

出　　处：《医学研究杂志》2013年第8期48-51,共4页Journal of Medical Research

基　　金：浙江省自然科学基金资助项目(Y2090404);浙江省医药卫生科学研究计划项目(2011KYA001)

摘　　要：目的探讨SD大鼠角朊细胞的分离培养技术并鉴定,观察细胞生长情况。方法采用中性蛋白酶(DispaseⅡ)和胰蛋白酶分离角朊细胞,加入含有10%胎牛血清的角质化细胞专用培养基(keratinocyte serum free,K-SFM)培养。用免疫组化法和反转录聚合酶链反应(RT-PCR)法进行鉴定。结果角朊细胞生长缓慢,原代培养第5天开始出现细胞克隆,核大而圆,培养11天连成片,培养24天长满单层,细胞传代培养比较困难,很难贴壁,经摸索在培养第15天,细胞长满80%左右时即可传代,但一般传3代后就不能再传。大鼠角朊细胞角蛋白19(cytokeratin 19,CK19)染色,细胞质呈棕褐色,提示培养的细胞CK19阳性。大鼠角朊细胞标志RT-PCR检测整合素β1、CK14(cytokeratin 14)、CK10(cytokeratin 10)的mRNA表达均阳性。结论用两种酶分离获得的大鼠角朊细胞活性好,降低成纤维细胞污染,用K-SFM培养基培养的细胞经鉴定具有角朊细胞特性,为皮肤组织工程的研究提供良好的种子细胞方面的实验数据。Objective To investigate the cultivation and identification of epidermal keratinocytes from newborn SD rats skin, and toobserve the cell growth. Methods Keratinocytes were harvested by sequential two - step enzyme digestion with dispase II and trypsin, and were cultured in K -SFM with 10% fetal bovine serum （FBS）. The expressions of CK19, CK10, CKlg, integrin hi were examined by immunohistochemistry and RT - PCR, respectively. Results Keratinocytes grew slowly. Cell clones appeared in the 5＇h day of primary culture and converged together in the 11th day. In the 24th day, a monolayer cell was observed. The cells were passaged when they grew to 80% confluence. However, keratinocytes were difficult to attach to the wall of the culture plate. It was difficult to maintain the ceils for more than three passages in culture. The results of immunohistochemical staining and RT - PCR analysis showed that the expression of CK19, in- tegrin β, CK14, CK10 were positive. Conclusion The sequential two - step enzyme digestion method and the culture in K - SFM with 10% FBS are advantageous to get high viability and purity keratinocytes with positive expression of CK19, integrin β1, CK14, CK10.

关 键 词：角朊细胞 CK19 CK10 CK14整合素β1 RT—PCR 

分 类 号：R446.5[医药卫生—诊断学]