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目的通过对慢病毒载体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。方法选择慢病毒载体稳定转染后生长特性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。从鉴定得到的差异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Western blot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证实其在转染株中蛋白和转录水平均存在明显的高表达。结论慢病毒载体稳定转染株HaCaT细胞株相对未转染株核纤层蛋白B1高表达。这种高表达可能与转染株细胞生长特性改变和潜在的成瘤性隐患相关。
OBJECTIVE: To compare proteomic analysis of HaCaT cells stably transfected with lentiviral vector and untransfected HaCaT cells, identify the differentially expressed proteins and find out the reason for the change of cell growth characteristics in genetically engineered epidermal cells And possible tumorigenic safety hazard. Methods Transfectant cells with significant changes in growth characteristics after stable transfection of lentiviral vector were selected. The total protein of the transfectant and non-transfectant strains was isolated and compared by two-dimensional electrophoresis to find the differentially expressed protein spots, Then conduct tandem mass spectrometry identification. From the identified differentially expressed proteins, lamin B1, which may be related to the change of cell growth characteristics and tumorigenesis, was verified by Western blot and real-time quantitative PCR (qPCR). Results Compared with the untransfected cells, there were eleven differentially expressed protein spots in the two-dimensional electrophoresis. Seven proteins were identified by mass spectrometry. Among them, lamin B1, which is related to cell proliferation and apoptosis, was confirmed by WB and qPCR to confirm that there was a significant high expression of protein and transcription in the transfectants. Conclusion The lentiviral vector-stable HaCaT cell line is highly expressed relative to the non-transfected line lamina B1. This high expression may be associated with altered cell growth characteristics of transfectants and potential tumorigenic potential.