本文選題：胃癌 + HGFK1�。� 參考：《南京醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究背景:胃癌在亞洲地區(qū)的發(fā)生率和死亡率都很高。傳統(tǒng)胃癌患者的治療方法有手術(shù)切除、化療以及放療等等,然而進(jìn)展期的胃癌患者接受傳統(tǒng)治療之后的五年生存率仍就不高。近年來(lái),隨著分子生物學(xué)的進(jìn)一步發(fā)展,胃癌的各種生物治療臨床試驗(yàn)也廣泛研究。溶瘤病毒療法是利用天然或改造的病毒靶向性溶解腫瘤細(xì)胞來(lái)治療腫瘤,而抗血管生成治療則通過(guò)抗腫瘤血管生成來(lái)抑制腫瘤的生長(zhǎng)。因此,我們?cè)O(shè)想將具有抗腫瘤細(xì)胞和抗血管生成作用的人肝細(xì)胞生長(zhǎng)因子第一結(jié)構(gòu)域(HGFK1)基因與溶瘤腺病毒相結(jié)合,研究其抗胃癌作用。方法:人端粒酶逆轉(zhuǎn)錄酶啟動(dòng)子(hTERT)和缺氧調(diào)控元件序列(HRE)分別調(diào)控溶瘤病毒的E1a和E1b基因,基因組插入HGFK1基因,構(gòu)建雙調(diào)控溶瘤腺病毒(TH-Ad-HGFK1-EGFP),空斑計(jì)數(shù)法滴定病毒濃度;病毒分為三組:實(shí)驗(yàn)A組(TH-Ad-HGFI1-EGFP)、實(shí)驗(yàn) B 組(TH-Ad-EGFP)和對(duì)照 C 組(Ad-EGFP),分別感染胃癌細(xì)胞SGC7901和正常人成纖維細(xì)胞HF;熒光顯微鏡下觀察病毒復(fù)制增殖情況,半數(shù)細(xì)胞培養(yǎng)物感染量法(TCID50)檢測(cè)病毒在細(xì)胞中的擴(kuò)增倍數(shù);噻唑藍(lán)(MTT)法檢測(cè)病毒對(duì)胃癌細(xì)胞的殺傷抑制作用;雙重?zé)晒馊旧珯z測(cè)病毒對(duì)胃癌細(xì)胞的促凋亡作用。結(jié)果:溶瘤腺病毒TH-Ad-HGFK1-EGFP、TH-Ad-EGFP經(jīng)測(cè)序及PCR鑒定確認(rèn)構(gòu)建成功,滴度都為2× 1010pfu/ml;病毒擴(kuò)增結(jié)果顯示:A、B組病毒在SGC7901細(xì)胞中的擴(kuò)增倍數(shù)分別為1.47× 104倍、1.6× 104倍,遠(yuǎn)大于對(duì)照C組病毒,在HF細(xì)胞中為110倍和124倍,遠(yuǎn)小于對(duì)照C組病毒;MTT結(jié)果顯示:當(dāng)感染復(fù)數(shù)(MOI)=100時(shí),,A組病毒對(duì)SGC7901細(xì)胞72h、96h后的抑制率為(71.34±8.27)%、(74.56±1.78)%,B 組為(30.58±3.83)%、(31.84±6.62)%,C 組為(23.40±2.29)%、(21.18±2.32)%。A組與B組兩時(shí)間點(diǎn)分別比較有顯著差異(P=0.002、P=0.003),A組與C組分別比較也有顯著差異(P=0.003、P0.001)。A、B組病毒對(duì)HF細(xì)胞在96h后的抑制率為(18.94±0.88)%、(22.84±3.34)%,C組為(53.25±3.50)%,A、B兩組與C組分別比較有較大差異(P=0.003、P=0.001);凋亡結(jié)果顯示:A組SGC7901細(xì)胞的凋亡率為(18.66±4.04)%,遠(yuǎn)大于其余各組。結(jié)論:溶瘤腺病毒TH-Ad-HGFK1-EGFP能在胃癌細(xì)胞SGC7901中復(fù)制增殖并對(duì)其有殺傷抑制、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 鹵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 鹵1.78) and group B (31.58 鹵3.83C vs 23.40 鹵2.29 鹵21.18 鹵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 鹵0.88 + 0.84 鹵3.34%, which was significantly higher in group C than in group C (53.25 鹵3.50) and group C, respectively. The apoptotic results showed that the apoptosis rate of SGC7901 cells in group A was 18.66 鹵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 鹵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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.2

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本文編號(hào)：1806593