携带HGFK1基因溶瘤腺病毒的构建及其对胃癌细胞的体外杀伤作用

发布时间：2018-04-26 15:27

本文选题：胃癌 + HGFK1　；参考：《南京医科大学》2017年硕士论文

【摘要】：研究背景:胃癌在亚洲地区的发生率和死亡率都很高。传统胃癌患者的治疗方法有手术切除、化疗以及放疗等等,然而进展期的胃癌患者接受传统治疗之后的五年生存率仍就不高。近年来,随着分子生物学的进一步发展,胃癌的各种生物治疗临床试验也广泛研究。溶瘤病毒疗法是利用天然或改造的病毒靶向性溶解肿瘤细胞来治疗肿瘤,而抗血管生成治疗则通过抗肿瘤血管生成来抑制肿瘤的生长。因此,我们设想将具有抗肿瘤细胞和抗血管生成作用的人肝细胞生长因子第一结构域(HGFK1)基因与溶瘤腺病毒相结合,研究其抗胃癌作用。方法:人端粒酶逆转录酶启动子(hTERT)和缺氧调控元件序列(HRE)分别调控溶瘤病毒的E1a和E1b基因,基因组插入HGFK1基因,构建双调控溶瘤腺病毒(TH-Ad-HGFK1-EGFP),空斑计数法滴定病毒浓度;病毒分为三组:实验A组(TH-Ad-HGFI1-EGFP)、实验 B 组(TH-Ad-EGFP)和对照 C 组(Ad-EGFP),分别感染胃癌细胞SGC7901和正常人成纤维细胞HF;荧光显微镜下观察病毒复制增殖情况,半数细胞培养物感染量法(TCID50)检测病毒在细胞中的扩增倍数;噻唑蓝(MTT)法检测病毒对胃癌细胞的杀伤抑制作用;双重荧光染色检测病毒对胃癌细胞的促凋亡作用。结果:溶瘤腺病毒TH-Ad-HGFK1-EGFP、TH-Ad-EGFP经测序及PCR鉴定确认构建成功,滴度都为2× 1010pfu/ml;病毒扩增结果显示:A、B组病毒在SGC7901细胞中的扩增倍数分别为1.47× 104倍、1.6× 104倍,远大于对照C组病毒,在HF细胞中为110倍和124倍,远小于对照C组病毒;MTT结果显示:当感染复数(MOI)=100时,,A组病毒对SGC7901细胞72h、96h后的抑制率为(71.34±8.27)%、(74.56±1.78)%,B 组为(30.58±3.83)%、(31.84±6.62)%,C 组为(23.40±2.29)%、(21.18±2.32)%。A组与B组两时间点分别比较有显著差异(P=0.002、P=0.003),A组与C组分别比较也有显著差异(P=0.003、P0.001)。A、B组病毒对HF细胞在96h后的抑制率为(18.94±0.88)%、(22.84±3.34)%,C组为(53.25±3.50)%,A、B两组与C组分别比较有较大差异(P=0.003、P=0.001);凋亡结果显示:A组SGC7901细胞的凋亡率为(18.66±4.04)%,远大于其余各组。结论:溶瘤腺病毒TH-Ad-HGFK1-EGFP能在胃癌细胞SGC7901中复制增殖并对其有杀伤抑制、促凋亡作用。
[Abstract]:Background: gastric cancer has a high incidence and mortality in Asia. Surgical resection, chemotherapy, and radiotherapy are the traditional treatments for patients with gastric cancer. However, the 5-year survival rate of advanced gastric cancer patients after conventional treatment is still low. In recent years, with the further development of molecular biology, various biotherapy clinical trials of gastric cancer have been widely studied. Antiangiogenic therapy uses natural or modified virus-targeted tumor cells to treat tumors, while anti-angiogenesis therapy inhibits tumor growth by anti-angiogenesis. Therefore, we propose to combine the human hepatocyte growth factor (HGFK1) gene, which has anti-tumor and anti-angiogenic effects, with adenovirus to study its anti-gastric cancer effect. Methods: human telomerase reverse transcriptase promoter (hTERT) and hypoxia regulatory element sequence (hREE) were used to control the E1a and E1b genes of oncolytic virus respectively. The HGFK1 gene was inserted into the genome to construct TH-Ad-HGFK1-EGFPV, and the viral concentration was determined by plaque counting. The virus was divided into three groups: group A (TH-Ad-HGFI1-EGFPN), group B (TH-Ad-EGFPN) and control group C (Ad-EGFPN) infected with SGC7901 and normal human fibroblasts, respectively. TCID50) was used to detect the multiples of virus amplification in gastric cancer cells; thiazolyl methacrylate (MTT) method was used to detect the cytotoxicity of the virus to gastric cancer cells; and double fluorescent staining was used to detect the apoptotic effect of viruses on gastric cancer cells. Results: TH-Ad-HGFK1-EGFP-TH-Ad-EGFP was successfully constructed by sequencing and PCR identification, the titer of TH-Ad-EGFP was 2 脳 10 ~ (10) pfur / ml, the results of virus amplification showed that the amplification times of TH-Ad-HGFK1-EGFP-TH-EGFP in SGC7901 cells were 1.47 脳 10 ~ 4 times and 1.6 脳 10 ~ 4 times respectively, which were much higher than those of C group virus. In HF cells, they were 110 and 124 times, The inhibitory rate of group A virus on SGC7901 cells was 71.34 卤8.27g / 96 h after infection with plural moi = 100. The results showed that the inhibitory rate of group A on SGC7901 cells was significantly higher than that of group B (74.56 卤1.78) and group B (31.58 卤3.83C vs 23.40 卤2.29 卤21.18 卤2.321.32), respectively. There were significant differences between group A and group B at two time points. There was also a significant difference in the inhibition rate of SGC7901 cells in group B to HF cells at 96 hours after 96 hours. The inhibitory rate of group A on HF cells was 18.94 卤0.88 + 0.84 卤3.34%, which was significantly higher in group C than in group C (53.25 卤3.50) and group C, respectively. The apoptotic results showed that the apoptosis rate of SGC7901 cells in group A was 18.66 卤4.04%, which was much higher than that in group C (P 0.003 + P 0.001), which was significantly higher than that in group C (P 0.003 P 0.001), and the apoptosis rate of SGC7901 cells in group A was 18.66 卤4.04%, which was much higher than that in group C (P 0.003). Conclusion: adenovirus TH-Ad-HGFK1-EGFP can replicate and proliferate in SGC7901 of gastric cancer cells and can kill and inhibit apoptosis.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

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