| 赵婷,石琴.双调控肿瘤特异性溶瘤腺病毒对卵巢癌靶向治疗的研究[J].安徽医药,2021,25(1):4-8. |
| 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 |
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| DOI：10.3969/j.issn.1009?6469.2021.01.002. |
| 中文关键词: 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 |
| 英文关键词: Adenovirus The excision repair cross?complementation group 1(ERCC1) Transcription initiation site Hypox? ia?inducible factor Transfection Cisplatin Plasmid construction |
| 基金项目:上海市嘉定区卫健委青年项目(2018?QN?01) |
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| 中文摘要: |
| 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)。结示，当病毒接触卵巢癌细胞 24 h后发挥出对卵巢癌细胞较明显的抑瘤作用，且 AD?ERCC1?siRNA组与 AD?NC?siRNA组无明显差异，在感染强度(MOI)＝30时，卵巢癌 SKOV3细胞增殖抑制率下降到 50％以下。结论双调控腺病毒成功构建且具备高转染效率，该病毒对卵巢癌 SKOV3细胞株有明显生长抑制及促凋亡作用，可能与 JAK/STAT通路激活有关，而且该病毒能增强顺铂的用药效果，其机制可能与该病毒成功屏蔽 ER? 果提，CC1耐药基因有关。 |
| 英文摘要: |
| Objective To construct a dual?regulated dual?effect tumor?specific oncolytic adenovirus(RSOAds?hTERT/HIF?ER? CC1，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.Meth? ods January to December 2018，The human telomerase reverse transcriptase promoter(hTERT)was used to regulate the adenovi? rus Ela gene，the hypoxia regulatory element sequence(HRE)regulates the E1b gene，and the recombinant hTERT/HIF double?reg? ulated double?effect tumor?specific oncolytic adenovirus vector；The ERCC1 gene sequence was designed into the E1 region to de?sign a specific gene to construct a double regulatory adenovirus.MTT was used to detect the inhibition of cell proliferation afterSKOV3 cells were treated with different concentrations of virus.Flow cytometry to detect cancer cell apoptosis in different groups.The expression of JAK/STAT and PI3K/Akt pathway proteins in SKOV3 cells was detected by Western blot.Results A double?reg? ulated 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 groupand 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?ER? CC1?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 expression of cells(0.567±0.016)was signifi? cantly lower than that of the control group(1.612±0.072)(P＜0.01).The expression of JAK2，STAT3 in the cells after treatment was increased compared with the control group［(0.860±0.010)vs.(0.694±0.011)，(0.826±0.032)vs.(0.651±0.037)］(P＜0.01). The expressions of PI3K and AKT were not statistically different from those of the control group［(0.292±0.013) vs.(0.291± |
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