慢病毒载体介导的TNF-α基因在脐血间充质干细胞中的表达的实验研究

发布时间：2018-03-29 06:39

本文选题：慢病毒载体　切入点：TNF-α基因　出处：《青岛大学》2010年硕士论文

【摘要】： [目的]：构建带有人TNF-a基因的慢病毒载体,并观察该基因在人脐血间质干细胞中的表达。 [方法]：通过逆转录聚合酶链反应从PCD DNA-TNF-a质粒中获得人TNF-α基因,利用Infusion技术重组构建慢病毒载体穿梭质粒pGC-FU-TNF-α,在脂质体lipofectamine 2000介导下与结构质粒pHelperl.0和包膜质粒pHelper2.0共转染293T细胞包装生产慢病毒。将人脐血间质干细胞分为实验组(pGC-FU-TNF-a),空载体对照组(pGC-FU-EGFP)及空白组(UCBMSCs),分别用重组慢病毒,空载慢病毒,PBS感染后,采用RT-PCR以及Elisa检测TNF-a表达情况。 [结果]：(1)所获TNF-a基因测序证明与Gene Bank中序列一致：(2)重组慢病毒载体质粒pGC-FU-TNF-a经鉴定正确；(3)三质粒共转染293T细胞成功,收集、浓缩病毒后测定其滴度为2×107TU/L。(4)重组慢病毒感染人脐血间质干细胞后行RT-PCR检测三组细胞电泳结果显示均有TNF-a mRNA表达,其中实验组光密度值(2.71±1.57),与空载对照组(4.63±1.32),空白组(6.03±0.88)比较差异有统计学意义(F=11.677,P0.01)；ELISA检测三组细胞TNF-a浓度结果显示实验组(1.32±0.23ug/ml)与空载对照组(0.70±0.25ug/ml),空白组(0.76±0.11ng/ml)比较差异有统计学意义(F=21.321,P0.01)。 [结论]：(1)构建带有TNF-a基因的慢病毒载体,包装生产高滴度重组慢病毒；(2)重组慢病毒成功感染脐血间质干细胞并实现TNF-a在脐血间质干细胞中的表达；(3)转基因TNF-a脐血间充质干细胞的构建为治疗胃癌的应用奠定理论基础。
[Abstract]:Objective: to construct a lentivirus vector containing human TNF-a gene and observe its expression in human umbilical cord blood mesenchymal stem cells. [methods] Human TNF- 伪 gene was obtained from PCD DNA-TNF-a plasmid by reverse transcription polymerase chain reaction (RT-PCR). Lentivirus shuttle plasmid pGC-FU-TNF- 伪 was constructed by Infusion technique and co-transfected with structural plasmid pHelperl.0 and encapsulated plasmid pHelper2.0 into 293T cells to produce lentivirus under liposome lipofectamine 2000. Human umbilical cord blood mesenchymal stem cells were divided into experimental group pGC-FU-TNF-a- 伪 and empty. The vector control group (pGC-FU-EGFP) and the blank group (UCB MSC) were treated with recombinant lentivirus, respectively. RT-PCR and Elisa were used to detect the expression of TNF-a. [results] the sequence of TNF-a gene obtained from Gene Bank confirmed that the recombinant lentivirus vector pGC-FU-TNF-a was cotransfected into 293T cells successfully, and the recombinant lentivirus vector pGC-FU-TNF-a was identified as correct. The recombinant lentivirus was infected with human umbilical cord blood mesenchymal stem cells. The results of RT-PCR analysis showed that there was TNF-a mRNA expression in all three groups. The optical density of the experimental group (2.71 卤1.57) was significantly higher than that of the no-load control group (4.63 卤1.32) and the blank group (6.03 卤0.88). The TNF-a concentration of the three groups was detected by Elisa. The results showed that the concentration of TNF-a in the experimental group was 1.32 卤0.23ugrmlml) and that in the blank group was 0.76 卤0.11ng / ml. [conclusion] A lentivirus vector containing TNF-a gene was constructed. Packaging production of High titer Recombinant lentivirus (lentivirus 2) Recombinant lentivirus successfully infects umbilical cord blood mesenchymal stem cells and realizes the expression of TNF-a in umbilical cord blood mesenchymal stem cells. The construction of transgenic TNF-a umbilical cord blood mesenchymal stem cells lays a theoretical foundation for the treatment of gastric cancer.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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