携HBV PreS1重组腺病毒基因治疗载体对肝细胞嗜向性的研究
发布时间：2019-06-04 01:49
【摘要】：目的：扩增纯化鉴定重组腺病毒基因治疗载体rAd-preS1，，体外多种细胞实验检测其对肝细胞的嗜向性。 方法：同源重组法构建了含HBV嗜肝性基因PreS1的重组腺病毒质粒prAd-PreS1，酶切鉴定后293细胞包装生成腺病毒rAd-preS1。聚合酶链反应PCR检测目的基因片段Pres1。经反复感染293细胞扩增获得较高滴度重组腺病毒，氯化铯经典法纯化病毒。用rAd-preS1体外分别感染人正常肝细胞（L02）、人肺癌细胞(A549)、人喉癌细胞（Hep2）检测其肝嗜向性，同时野生腺病毒rAd作为对照，观察各细胞中荧光蛋白GFP表达情况，比较各细胞内的病毒滴度值，同时用流式细胞术检测各细胞重组腺病毒的感染率，并进行统计学分析。 结果：酶切鉴定证实rAd-preS1载体构建成功，扩增后病毒滴度1.15×10~9efu/ml。PCR结果示大小750bp目的基因片段。rAd-preS1转染多种细胞示L02细胞中绿色荧光蛋白表达明显强于A549、Hep2细胞，L02细胞的病毒滴度值高于A549、Hep2，有显著差异(P值均0．01)，而A549、Hep2细胞间表达差异无统计学意义(P值0．05)；对照组各细胞间表达差异无统计学意义(P值0．05)。流式细胞术结果示人肝细胞感染率显著高于非肝细胞组。 结论：携HBV PreS1重组腺病毒载体rAd-preS1对人肝细胞相对较强嗜向性，而对非肝细胞无明显效果，野生腺病毒无明显细胞嗜向性，为进一步研究肝脏疾病靶向基因治疗奠定了基础。
[Abstract]:Aim: to detect the tropism of recombinant adenoviral gene therapy vector rAd-preS1, to hepatocytes by amplification and purification in vitro. Methods: the recombinant adenoviral plasmid PreS1 containing HBV hepatophilic gene was constructed by homologous recombination method and identified by restriction endonuclease digestion. The adenoviral rAd-preS1. was generated in the packaging of 293cells. Detection of target gene fragment Pres1. by polymerase chain reaction PCR The recombinant adenovirus with high titer was obtained by repeated infection of 293cells, and the virus was purified by cesium chloride classical method. Human normal hepatocytes (L02), human lung cancer cells (A549) and human laryngeal cancer cells (Hep2) were infected with rAd-preS1 in vitro to detect their hepatic tropism. At the same time, wild adenovirus rAd was used as control to observe the expression of fluorescent protein GFP in each cell. The virus drop values in each cell were compared, and the infection rate of recombinant adenoviruses in each cell was detected by flow cytometry and statistically analyzed. Results: enzyme digestion confirmed that the rAd-preS1 vector was successfully constructed. The titer of the virus was 1.15 脳 10 ~ 9efu/ml.PCR. The expression of green fluorescent protein in L02 cells was significantly stronger than that in A549 cells. The results showed that the expression of green fluorescent protein in L02 cells was significantly stronger than that in A549. the expression of green fluorescent protein in L02 cells was significantly stronger than that in A549 cells. The viral drop values of Hep2 cells and L02 cells were significantly higher than those of A549 and Hep2 cells, but there was no significant difference in the expression of A549 and Hep 2 cells. There was no significant difference in the expression of each cell in the control group (P 0.05). The results of flow cytometry showed that the infection rate of human hepatocytes was significantly higher than that of non-hepatocytes. Conclusion: the recombinant adenoviral vector rAd-preS1 carrying HBV PreS1 has relatively strong tropism to human hepatocytes, but no obvious effect on non-hepatocytes, and no obvious cell tropism for wild adenoviruses, which lays a foundation for further study of targeted gene therapy for liver diseases.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R450;R512.6
本文编号：2492385
[Abstract]:Aim: to detect the tropism of recombinant adenoviral gene therapy vector rAd-preS1, to hepatocytes by amplification and purification in vitro. Methods: the recombinant adenoviral plasmid PreS1 containing HBV hepatophilic gene was constructed by homologous recombination method and identified by restriction endonuclease digestion. The adenoviral rAd-preS1. was generated in the packaging of 293cells. Detection of target gene fragment Pres1. by polymerase chain reaction PCR The recombinant adenovirus with high titer was obtained by repeated infection of 293cells, and the virus was purified by cesium chloride classical method. Human normal hepatocytes (L02), human lung cancer cells (A549) and human laryngeal cancer cells (Hep2) were infected with rAd-preS1 in vitro to detect their hepatic tropism. At the same time, wild adenovirus rAd was used as control to observe the expression of fluorescent protein GFP in each cell. The virus drop values in each cell were compared, and the infection rate of recombinant adenoviruses in each cell was detected by flow cytometry and statistically analyzed. Results: enzyme digestion confirmed that the rAd-preS1 vector was successfully constructed. The titer of the virus was 1.15 脳 10 ~ 9efu/ml.PCR. The expression of green fluorescent protein in L02 cells was significantly stronger than that in A549 cells. The results showed that the expression of green fluorescent protein in L02 cells was significantly stronger than that in A549. the expression of green fluorescent protein in L02 cells was significantly stronger than that in A549 cells. The viral drop values of Hep2 cells and L02 cells were significantly higher than those of A549 and Hep2 cells, but there was no significant difference in the expression of A549 and Hep 2 cells. There was no significant difference in the expression of each cell in the control group (P 0.05). The results of flow cytometry showed that the infection rate of human hepatocytes was significantly higher than that of non-hepatocytes. Conclusion: the recombinant adenoviral vector rAd-preS1 carrying HBV PreS1 has relatively strong tropism to human hepatocytes, but no obvious effect on non-hepatocytes, and no obvious cell tropism for wild adenoviruses, which lays a foundation for further study of targeted gene therapy for liver diseases.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R450;R512.6
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相关期刊论文 前3条
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本文编号：2492385
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