PDGF-B慢病毒载体的构建及其对SGC7901胃癌细胞侵袭能力的影响

Construction of lentiviral vector pLeno-DCE-PDGF-B and its effect on invasion ability of SGC7901 gastric carcinoma cells

目的构建血小板源性生长因子B(PDGF-B)慢病毒载体,建立PDGF-B稳定过表达的SGC7901胃癌细胞株并观察PDGF-B对SGC7901胃癌细胞侵袭能力的影响。方法采用四质粒系统构建PDGF-B慢病毒载体,转染293T细胞包装获得高滴度病毒颗粒;以病毒颗粒转染SGC7901细胞,通过荧光显微镜观察转染效果,并用免疫印迹法检测PDGF-B的蛋白表达;用Transwell试验观察SGC7901细胞侵袭能力的变化。结果通过PCR及酶切鉴定成功构建PDGF-B载体;包装并得到高滴度的病毒颗粒;成功转染SGC7901细胞后,免疫印迹法检测到PDGF-B蛋白表达明显升高;Transwell试验证明PDGF-B过表达增强SGC7901细胞侵袭能力。结论成功构建并包装获得PDGF-B慢病毒颗粒,建立PDGF-B稳定过表达SGC7901胃癌细胞株,证明PDGF-B过表达可增强胃癌细胞侵袭转移能力。

Objective To construct a lentiviral vector that stably express platelet-derived growth factor B （PDGF-B）and to in-vestigate effect of PDGF-B overexpression on invasion of SGC7901 gastric carcinoma cell.Methods The lentiviral vector pLeno-DCE-PDGF-B was constructed and transfected into 293T cells.The supernatant containing the lentivirus particles was harvested to determine the virus titer and high titer lentivirus particles was gathered.Then PDGF-B lentiviral vector was transfected into SGC7901 cells for construction of stable PDGF-B overexpression SGC7901 cells.Western-blot and immunofluorescence were used for evaluation of the construction of stable PDGF-B overexpression SGC7901 cells.Transwell test was used for detection the inva-sion of SGC7901 cells.Results The lentiviral vector was correctly constructed and verified by sequencing.High titer PDGF-B lenti-viral particles were acquired successfully.After transfection,SGC7901 cells were green color by detection of fluorescence micro-scope;and the expression of PDGF-B protein in transfected SGC7901 cells were much higher than that in normal SGC7901 cells.Al-so,PDGF-B overexpression increased the invasion ability of SGC7901 cells.Conclusion The PDGF-B lentiviral particles are ob-tained after successful construction and package.The stable PDGF-B overexpression SGC7901 gastric carcinoma cell line is estab-lished,which proves that the PDGF-B overexpression might increase the invasion ability of gastric carcinoma cells.