双调控肿瘤特异性溶瘤腺病毒对卵巢癌靶向治疗的研究  

Study on dual-regulated tumor-specific oncolytic adenovirus for targeted therapy of ovarian cancer

作　　者：赵婷 石琴 ZHAO Ting;SHI Qin(Department of gynecology and obstetrics,Jiading District Central Hospital Affiliated Shanghai University of Medicine&Health Sciences,Shanghai,201800,China)

机构地区：[1]上海市嘉定区中心医院妇产科,上海201800

出　　处：《安徽医药》2021年第1期4-8,I0001,共6页Anhui Medical and Pharmaceutical Journal

基　　金：上海市嘉定区卫健委青年项目(2018⁃QN⁃01)。

摘　　要：目的构建表达携带切除修复交叉互补基因1(ERCC1)siRNA的人端粒酶反转录酶启动子(hTERT)及缺氧诱导因子(HIF)的双调控肿瘤特异性溶瘤腺病毒(RSOAds-hTERT/HIF-ERCC1,以下简称双调控腺病毒),作用于卵巢癌细胞后研究其抑瘤作用及相关分子机制,为卵巢癌的基因治疗奠定基础。方法2018年1—12月,以人端粒酶逆转录酶启动子(hTERT)调控腺病毒Ela基因,缺氧调控元件序列(HRE)调控E1b基因,重组hTERT/HIF双调控双功效的肿瘤特异性溶瘤腺病毒载体;于E1区插入ERCC1基因序列设计特异基因构建双调控腺病毒。将构建好的病毒增加荧光载体,荧光显微镜下观察SKOV3人卵巢癌细胞中病毒转染情况。以噻唑蓝(MTT)实验检测不同组别癌细胞增殖活性,流式细胞术检测不同组别癌细胞凋亡情况。蛋白质印迹法检测SKOV3细胞中ERCC1蛋白、JAK/STAT、PI3K/Akt通路蛋白表达情况。结果成功构建了双调控腺病毒,病毒能够在卵巢癌中高效转染并快速增殖,屏蔽ERCC1耐药基因,并具明显的抑瘤作用。MTT结果显示,24 h后双调控腺病毒对SKOV3细胞株具有明显增殖抑制,随着病毒数目增加抑制率逐渐上升,感染强度(MOI)=30时,卵巢癌SKOV3细胞增殖率下降到50%以下,且AD-ERCC1-siRNA组与AD-NC-siRNA组肿瘤抑制差异无统计学意义(P>0.05),AD-ERCC1-siRNA联合顺铂(74.19±3.04)%抑瘤作用较AD-NC-siRNA+DDP组(56.85±2.51)%更强。流式细胞术结果显示:AD-ERCC1-siRNA组细胞凋亡率(5.5±0.75)%较对照组(0.5±0.24)%明显升高。我们对病毒作用后的SKOV3细胞运用WB方法进行检测,结果显示作用后的细胞ERCC1蛋白表达(0.567±0.016)较对照组(1.612±0.072)下降(P<0.01),JAK2[(0.860±0.010)比(0.694±0.011)]、STAT3[(0.826±0.032)比(0.651±0.037)]显著高于对照组,(P<0.01),而PI3K[(0.292±0.013)比(0.291±0.015)]、AKT[(0.810±0.018)比(0.813±0.024)]较对照组差异无统计学意义(P>0.05)。结果提示,当病毒接触卵巢癌细胞24 hObjective To construct a dual-regulated dual-effect tumor-specific oncolytic adenovirus(RSOAds-hTERT/HIF-ERCC1,hereinafter referred to as double-regulated adenovirus),which expresses hTERT/HIF carrying ERCC1 gene siRNA,then To study the anti-tumor effect and related molecular mechanisms of it which lays a foundation for gene therapy of ovarian cancer.Methods January to December 2018,The human telomerase reverse transcriptase promoter(hTERT)was used to regulate the adenovirus Ela gene,the hypoxia regulatory element sequence(HRE)regulates the E1 b gene,and the recombinant hTERT/HIF double-regulated double-effect tumor-specific oncolytic adenovirus vector;The ERCC1 gene sequence was designed into the E1 region to design a specific gene to construct a double regulatory adenovirus.MTT was used to detect the inhibition of cell proliferation after SKOV3 cells were treated with different concentrations of virus.Flow cytometry to detect cancer cell apoptosis in different groups.The expression of JAK/STAT and PI3 K/Akt pathway proteins in SKOV3 cells was detected by Western blot.Results A double-regulated adenovirus was successfully constructed,and the virus which could proliferate in ovarian cancer had obvious antitumor effect.The results of MTT showed that after 24 hours,the double-regulated adenovirus had obvious proliferation inhibition on SKOV3 cell line,and the inhibition rate increased with the increase of concentration(P<0.05),When the intensity of infection(MOI)=30,the proliferation rate of ovarian cancer SKOV3 cells dropped below 50%.And there was no difference in AD-ERCC1-siRNA group and AD-NC-siRNA group(P>0.05).The anti-tumor effect of AD-ERCC1-siRNA+DDP group(74.19±3.04)%is stronger than that of AD-NC-siRNA+DDP group(56.85±2.51)%(P<0.05).The results of flow cytometry showed that the apoptosis rate of AD-ERCC1-siRNA group(5.5±0.75)%was significantly higher than that of the control group(0.5±0.24)%(P<0.01).We performed WB assay on SKOV3 cells after viral action.The results showed that the ERCC1 protein expr

关 键 词：腺病毒 切除修复交叉互补基因1(ERCC1) 转录启动子 缺氧诱导因子 转染 顺铂 质粒构建 

分 类 号：R737.31[医药卫生—肿瘤]