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为了构建能与CIK细胞(Cytokine induced killer cell,CIK)结合的肿瘤特异性增殖腺病毒,我们用化学合成的35型腺病毒(Ad35)纤毛基因替换5型腺病毒(Ad5)骨架质粒的纤毛基因,获得5/35嵌合型腺病毒骨架质粒,然后与携带肿瘤特异性启动子hTERT(端粒酶逆转录酶基因启动子)和HRE(缺氧反应元件启动子)的穿梭质粒在HEK293细胞中同源重组,获得能与CIK细胞结合的肿瘤特异性增殖腺病毒KGHV400。分离人外周血单个核细胞,诱导培养成CIK细胞,与KGHV400共培养获得荷载KGHV400的CIK细胞。建立乳腺癌裸鼠移植瘤模型,经尾静脉注射荷载KGHV400的CIK细胞,在注射后第1d、2d、3d、5d、7d、14d解剖实验鼠,用免疫组化技术检测腺病毒六邻体蛋白在移植瘤、心、肝、脾、肾组织的表达。结果显示,构建的重组腺病毒KGHV400能高效率感染CIK细胞;荷瘤鼠尾静脉注射荷载KGHV400的CIK细胞后第1d,肿瘤细胞中即有腺病毒六邻体蛋白表达,之后表达逐渐增多,第14d仍有表达;脾脏在第1~14d检测到少量淋巴细胞中存在腺病毒六邻体蛋白;心、肝、肾组织未检测到该蛋白。对照组(尾静脉注射KGHV400)在注射后第1d、2d、3d、5d,肝、脾、肾组织中检测到存在腺病毒六邻体蛋白,但第7d后为转阴性;肿瘤细胞和心肌细胞未检测到腺病毒六邻体蛋白。我们的研究结果表明,重组腺病毒KGHV400是一种有用的肿瘤基因治疗载体。
To construct a tumor-specific adenovirus that binds to CIK cells (CIK), we replaced the ciliated gene of adenovirus type 5 adenovirus (Ad5) with the chemically synthesized adenovirus type 35 adenovirus (Ad35) , A 5/35 chimeric adenovirus backbone plasmid was obtained and then ligated with a shuttle plasmid carrying the tumor-specific promoter hTERT (telomerase reverse transcriptase gene promoter) and HRE (hypoxia response element promoter) in HEK293 cells Homologous recombination to obtain CIK cells can be combined with the tumor-specific adenovirus KGHV400. Peripheral blood mononuclear cells were isolated and induced to grow into CIK cells, which were co-cultured with KGHV400 to obtain CIK cells loaded with KGHV400. The nude mice model of transplanted breast cancer was established. The CIK cells loaded with KGHV400 were injected through the caudal vein, and the mice were dissected on the 1st, 2nd, 3rd, 5th, 7th and 14th day after injection, and the adenovirus hexon protein was detected by immunohistochemistry In the xenografts, heart, liver, spleen, kidney tissue expression. The results showed that the constructed recombinant adenovirus KGHV400 could efficiently infect CIK cells. The adenovirus hexon protein expression in the tumor cells was detected on day 1 after KGHV400 injection into the tail vein of tumor-bearing mice, 14d was still expressed; spleen in the first ~ 14d detected a small amount of lymphocytes in the presence of adenovirus hexon protein; heart, liver, kidney tissue did not detect the protein. Adenovirus hexon protein was detected in the control group (tail vein injection of KGHV400) on the 1st day, the 2nd day, the 3rd day, the 5th day, the liver, the spleen and the kidney, but negative on the 7th day. The tumor cells and cardiomyocytes Adenovirus hexon protein was not detected. Our results show that the recombinant adenovirus KGHV400 is a useful vector for gene therapy of tumors.