慢病毒转染人角质形成细胞高表达核纤层蛋白B第三军医大学学报.DOC

慢病毒转染人角质形成细胞高表达核纤层蛋白B1 （400038重庆，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） ［要］目的 通过对慢病毒稳定转染人角质形成细胞（HaCaT）株和未转染HaCaT细胞进行比较蛋白组学分析，找出其差异表达蛋白，寻找基因修饰组织工程表皮种子细胞生长特性改变的原因和可能存在的成瘤性安全隐患。方法 选择慢病毒稳定转染后生长发生明显改变的转染株细胞，采用二维电泳技术对该转染株和未转染株的总蛋白进行分离和比较，找出差异表达蛋白点，再进行串联质谱鉴定。从鉴定得到的蛋白中选择可能与细胞生长特性改变和肿瘤生成相关的核纤层蛋白B1（lamin B1）采用蛋白质印迹（WB）和实时荧光定量PCR技术（qPCR）对其表达差异进行验证。结果 与未转染株比较，转染株在二维电泳中存在11个差异表达蛋白点，质谱从其中鉴定出7个蛋白质。选取其中与细胞增殖和凋亡相关的核纤层蛋白B1通过WB和qPCR证实转染株蛋白和转录水平均存在明显的高表达。结论 慢病毒稳定转染株HaCaT细胞株核纤层蛋白B1。 ［关键词］人角质形成细胞；慢病毒；蛋白组学；核纤层蛋白B1 ［中图法分类号］R739.5；[R34] ［］The overexpression of lamin B1 in lentiviral vector-infected human immortal keratinocyte line Li Ruifu，Peng Daizhi, Qian Wei, Wang LiLua, He Bing, Liu Xiaoling, Shu Wenting, Liu Xiao, Zhou Xin, Liu Jing (Institute of Burn Research, Southwest Hospital, State Key Laboratory of Trauma, Burns and Combined Injury, Third Military Medical University, Chongqing, 40038, China) ［Abstract］Objective To find out the cause of altered growth characteristics and the potential threat of tumorigenicity in lentiviral vector-infected human immortal keratinocyte line (HaCaT). Methods Lentiviral vector-infected HaCaT cell with significant change of growth characteristics and uninfected HaCaT cell were chosen as experimental and control cell lines, respectively. The differentially expressed proteins between them were revealed by two-dimensional electrophoresis and further identified these proteins by LC-MS-MS. Lamin B1, which is closely related to tumorigenicity, cell growth and cell cycle, was picked out from these proteins and its differential expressions at protein and mRNA levels were validated by western blot (WB) and quantitative PCR(qPCR). Results Between these two cell lines, 11 differentially expressed proteins were found by two-dimensional electrophoresis and 7 proteins including lamin B1 were identified from them by LC-MS-MS. Furthermore, increased expression of lamin B1 protein in lentiviral vector-infected HaCaT cell was proved by WB as well as its mRNA expression by