人同源盒基因HOXB4慢病毒载体的构建和鉴定及其在脐带间充质干细胞中的表达

发布时间：2018-06-27 20:28

本文选题：HOXB4 + 人脐带间充质干细胞　；参考：《青岛大学》2012年硕士论文

【摘要】：目的本实验旨在构建人同源盒基因(Homeobox Gene) HOXB4的慢病毒载体,探讨慢病毒介导的HOXB4感染人脐带间充质干细胞后的变化。方法利用PCR扩增获得HOXB4,并克隆入慢病毒穿梭载体Lenti-shuttle。运用四质粒慢病毒包装系统转染HEK293T细胞,48h收获慢病毒Lenti-HOXB4,并测定慢病毒滴度。将Lenti-HOXB4惑染人脐带间充质干细胞,倒置荧光显微镜观察细胞感染效果,确定最佳的病毒感染复数(MOI)。同时,利用RT-PCR、免疫荧光染色、流式细胞术检测HOXB4的表达情况,CCK-8检测HOXB4对细胞生长的影响。结果利用四质粒共转染293T,成功获得Lenti-HOXB4,测定的病毒滴度为3×108TU/ml；当MOI为20时,Lenti-HOXB4对人脐带间充质干细胞具有较高的转染效率,达80%以上。感染Lenti-HOXB4的人脐带间充质干细胞,在nRNA、蛋白水平上均检测到了目的基因的表达,其能促进脐带间充质干细胞增殖一倍,流式结果表明：CD340.79%CD451.48%CD10592.68%CD4497.41%与转染前比较CD340.13%CD450.14%.CD10599.95%CD9099.88%,造血细胞表面标志略有增高,间充质干细胞的表面标志无明显改变。结论1.成功构建了HOXB4慢病毒载体并获得了HUCMSCs-HOXB4基因工程细胞。2.HOXB4基因能促进脐带间充质干细胞的增殖3.HOXB4基因转染并不影响脐带间充质干细胞表面标志的表达。
[Abstract]:Objective to construct the lentivirus vector of Homeobox Gene (Homeobox Gene) HOXB4 and to investigate the changes of Homeobox Gene HOXB4 infected with human umbilical cord mesenchymal stem cells. Methods HOXB4 was amplified by PCR and cloned into lentivirus shuttle vector Lenti-shuttle. Lentivirus Lenti-HOXB4 was harvested from HEK293T cells transfected with four plasmids lentivirus packaging system for 48 h and the titer of lentivirus was measured. Lenti-HOXB4 was used to infect human umbilical cord mesenchymal stem cells, and the effect of cell infection was observed by inverted fluorescence microscope, and the optimal number of virus infection (moi) was determined. At the same time, RT-PCR, immunofluorescence staining and flow cytometry were used to detect the expression of HOXB4. CCK-8 was used to detect the effect of HOXB4 on cell growth. Results Lenti-HOXB4 was successfully transfected into 293T by four plasmids, and the titer of the virus was 3 脳 108TU / ml. When moi was 20:00, Lenti-HOXB4 had a higher transfection efficiency to human umbilical cord mesenchymal stem cells (more than 80%). Human umbilical cord mesenchymal stem cells infected with Lenti-HOXB4 detected the expression of the target gene at the level of nRNA and protein, which could double the proliferation of umbilical cord mesenchymal stem cells. The flow rate showed that CD340.79451.4810592.684497.41% was higher than that before transfection. CD340.13450.14.CD10599.959099.88, the surface markers of hematopoietic cells increased slightly, but the surface markers of mesenchymal stem cells did not change significantly. Conclusion 1. HOXB4 lentivirus vector was successfully constructed and HUCMSCs-HOXB4 gene was obtained. 2. HOXB4 gene could promote the proliferation of umbilical cord mesenchymal stem cells 3.HOXB4 gene transfection did not affect the expression of surface markers of umbilical cord mesenchymal stem cells.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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