CCL20基因敲低型人永生化角质形成细胞克隆的体外生长特性观察  

The Growth Characteristics of Human Immortalized Keratinocyte Clones with CCL20 Gene Knockdown in Vitro

作　　者：何斌[1] 彭代智[1] 何升东[1] 周新[1] 刘敬[1] 王勇[1] 王丽华[1] 郑必祥[1] 左海斌[1] 

机构地区：[1]第三军医大学西南医院全军烧伤研究所,创伤、烧伤与复合伤国家重点实验室,重庆400038

出　　处：《细胞生物学杂志》2009年第6期863-866,共4页Chinese Journal of Cell Biology

基　　金：国家重点基础研究发展计划(973计划)(No.2005CB522605);国家高技术研究发展计划(863计划)(No.2006AA02A121)资助项目~~

摘　　要：采用MTT比色法、碘化丙啶染色法和直接免疫荧光双标记法分别观察4个CCL20基因敲低型人永生化角质形成细胞(HaCaT细胞)克隆(代号分别为Ⅱ～Ⅴ)的生长曲线、细胞周期以及表皮干细胞相关表型,预期筛选出体外生物学性状良好的表皮种子细胞克隆。结果显示,与未转染的HaCaT细胞(代号为Ⅰ)比较,Ⅲ～Ⅴ在体外培养第5、6天的光密度值显著降低;Ⅲ和Ⅳ的G2+M期显著增多;Ⅳ和Ⅴ表达CD49f^+CD71-的百分率显著升高,而表达CD49f^+CD71^+、CD71^+的百分率显著降低。可见,4个CCL20基因敲低型HaCaT细胞克隆中仅有Ⅱ表现出与未转染HaCaT类似的体外生长特性,该克隆有可能成为构建低免疫原性组织工程皮肤的表皮种子细胞。Four human immortalized keratinocyte （HaCaT） clones with CCL20 gene knockdown （Clone Ⅱ- Ⅴ） and untransfected HaCaT cells （clone Ⅰ） were served as experimental groups and control group, respectively. Their cell growth curves, cell cycles and the expressions of epidermal stem cell phenotypes were determined by MTT colorimetry, propidium iodide staining, and double-labeling direct immunofluorescence, respectively. Compared to untransfected HaCaT cells, the optical density values of clone Ⅲ-Ⅴsignificantly reduced at 5 and 6 days after culture in vitro. The G2＋M phase proportion of clone Ⅲ and Ⅳobviously increased than that of control group. Both clone Ⅳ and Ⅴ had higher percentages of CD49f ^＋CD71^- cells and lower percentages of CD49f^＋CD71^＋ and CD71^＋ phenotypes than that in control group. One clone （clone Ⅱ） with almost normal growth properties in vitro might be selected from these four HaCaT clones with CCL20 gene knockdown. This clone might have the potential to become a candidate of epidermal seed cells for constructing a novel tissue engineered skin with lower immunogenicity.

关 键 词：角质形成细胞 基因敲低 生长曲线 细胞周期 干细胞表型 

分 类 号：Q78[生物学—分子生物学]