与CIK细胞结合的肿瘤特异性增殖腺病毒KGHV400的构建

发布时间：2018-03-27 12:01

本文选题：肿瘤特异性增殖腺病毒　切入点：CIK细胞　出处：《病毒学报》2017年03期

【摘要】：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。
[Abstract]:In order to construct a tumor-specific adenovirus that binds to Cytokine induced killer cell line of CIK cells, we used the chemically synthesized adenovirus type 35 (Ad35) ciliated gene to replace the cytoskeleton plasmid of adenovirus type 5 (Ad5), and obtained the cytoskeleton plasmid of 5 / 35 chimeric adenovirus. Then the shuttle plasmid carrying tumor specific promoter hTERT (telomerase reverse transcriptase gene promoter) and hRE1 (hypoxia response element promoter) were homologous recombined in HEK293 cells. Tumor-specific adenovirus KGHV400. human peripheral blood mononuclear cells (PBMC) were isolated and cultured into CIK cells. CIK cells loaded with KGHV400 were co-cultured with KGHV400 to establish a breast cancer xenograft model in nude mice. CIK cells loaded with KGHV400 were injected into caudal vein and dissected on the 1st day, 2nd day, 3d, 5d, 7d, 14d after injection. The expression of adenovirus hexahedron protein in transplanted tumor, heart, liver, spleen and kidney was detected by immunohistochemical technique, the results showed that the expression of Hexon protein in transplanted tumor, heart, liver, spleen and kidney was detected by immunohistochemistry. The recombinant adenovirus KGHV400 could infect CIK cells efficiently, the expression of Hexon protein was found in the tumor cells on the 1st day after KGHV400 was injected into the tail vein of the tumor-bearing mice, and then increased gradually, and was still expressed on the 14th day. At day 1 ~ 14, a few lymphocytes were detected for adenovirus Hexon protein, but not in heart, liver and kidney. The control group (KGHV400) was injected with KGHV400 on the 1st day after injection, and the liver, spleen, liver, spleen, liver, spleen, liver, spleen, liver, spleen, liver, spleen, liver, spleen, liver, spleen, liver, spleen, liver, spleen, liver, spleen, liver and spleen, respectively. The presence of adenovirus Hexon protein was detected in renal tissue, but turned negative after 7 days. No adenovirus Hexon protein was detected in tumor cells and cardiac myocytes. Recombinant adenovirus KGHV400 is a useful gene therapy vector for cancer.
【作者单位】：昆明理工大学医学院;昆明医科大学昆明总医院临床学院;解放军昆明总医院病理科;
【基金】：国家自然科学基金(项目号:81460464),题目:CIK细胞和肿瘤特异性增殖腺病毒共同介导p21Ras单链抗体治疗恶性肿瘤的实验研究云南省应用基础研究重点项目(项目号:2013FA059),题目:野生型腺病毒介导p21Ras单链抗体靶向治疗乳腺癌的实验研究~~
【分类号】：R730.5

【相似文献】