腺病毒介导LRIG1过表达联合顺铂对膀胱癌细胞株EJ的抑制作用

李凡 叶章群 杨为民 摘 要：目的 观察腺病毒介导的LRIG1过表达是否能增强顺铂对膀胱癌细胞株EJ的损伤作用.方法 构建重组腺病毒载体包装pLRIG1-GFP质粒,采用逆转录-聚合酶链反应（RT-PCR）及Western blot法鉴定其是否能在膀胱癌细胞株EJ中上调LRIG1表达,并下调表皮生长因子（EGFR）的表达水平.通过采用单细胞凝胶电泳实验、流式细胞技术、免疫细胞化学染色及Matrigel侵袭实验,对比对照组、顺铂单药组、顺铂联合腺病毒载体组和顺铂联合腺病毒介导的LRIG1过表达组间细胞DNA损伤,细胞凋亡、增殖及侵袭能力.结果与对照组比较,顺铂在联合腺病毒介导的LRIG1过表达后,能下调细胞EGFR表达水平,同时显著增强肿瘤细胞DNA损伤程度（OTM值,对照组：2.54±0.54;CDDP组：4.57±0.79;CDDP/Ad-GFP组：5.38±1.16;CDDP/Ad-LRIG1组：9.45±2.64）;引起细胞周期抑制[S期抑制,对照组：（40.82±2.11）%;CDDP组：（37.31±1.12）%,CDDP/Ad-GFP组：（37.57±2.52）%,CDDP/Ad-LRIG1组：（55.04±4.28）%];诱导细胞凋亡[细胞凋亡率,对照组：（2.63±2.49）%,CDDP组（3.49±1.94）%,CDDP/Ad-GFP组：（3.96±4.68）%,CDDP/Ad-LRIG1组：（12.56±0.77）%];抑制细胞增殖[细胞计数,对照组：（371.33±16.17）个;CDDP组：（224.67±88.06）个;CDDP/Ad-GFP组：（176.33±69.79）个;CDDP/Ad-LRIG1组：（138.33±8.39）个]并逆转细胞侵袭性[细胞侵袭数量,对照组：（259.40±9.21）个;CDDP组：（175.00±25.78）个;CDDP/Ad-GFP组：（157.20±22.79）个;CDDP/Ad-LRIG1组：（114.20±25.11）个].结论 腺病毒介导LRIG1过表达能增强顺铂对膀胱肿瘤细胞株EJ的损伤作用.关键词：膀胱癌;EGFR;顺铂;化疗Inhibitory effects of adenovirus-vector-mediated LRIG1 over-expression in combination with cisplatin on EJ bladder cancer cellsLI Fan（Department of Urology,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China） YE Zhang-qun（Department of Urology,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China） YANG Wei-min（Department of Urology,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China） Abstract：Objective To investigate whether LRIG1 can facilitate cisplatin-induced bladder cancer cell lesions.Methods EJ bladder cancer cells were transfected with LRIG1 mediated by adenovirus vector.LRIG1 and epidermal growth factor receptor （EGFR） expression levels were detected in the transfected cells and compared to the untransfected cells.DNA damage,cell apoptosis,proliferation and invasion abilities were examined by single cell gel electrophoresis,flow cytometry,immunohistochemical staining and matrigel invasion assays,respectively.Results LRIG1 was up-regulated by Ad-LRIG1 transfection and EGFR was down-regulated in EJ cells.LRIG1 combined with cisplatin was responsible for severer DNA damage （OTM value,Control： 2.54 ±0.54; CDDP： 4.57 ±0.79; CDDP/Ad-GFP： 5.38 ± 1.16;CDDP/Ad-LRIGI： 9.45 ±2.64）,apoptosis [apoptosis rate,control： （2.6 ±2.49）%,CDDP： （3.49 ±1.94）%,CDDP/Ad-GFP： （3.96 ± 4.68 ）%,CDDP/Ad-LRIG1： （ 12.56 ± 0.77 ）%],growth inhibition （cell number,control： 371.33 ± 16.17; CDDP： 224.67 ± 88.06; CDDP/Ad-GFP： 176.33 ± 69.79;CDDP/Ad-LRIG1： 138.33 ± 8.39 ） and invasion reversal （ matrigel invasion,control： 259.40 ± 9.21;CDDP： 175.00 ±25.78; CDDP/Ad-GFP： 157.20 ±22.79; CDDP/Ad-LRIG1： 114.20 ±25.11 ）.Conclusion LRIG1 reduced EGFR expession and facilitated cisplatin-induced DNA damage in vitro,which might represent a novel therapeutic approach to improve the response to chemotherapy in bladder cancer.Keywords：Bladder carcinoma;Epidermal growth factor receptor;Cisplatin;