人血管内皮生长因子165慢病毒载体的构建及其抗放射所致细胞凋亡的作用

 【目的】 构建人血管内皮生长因子165(hVEGF165)慢病毒表达载体pCDH-CMV-MCS-EF1-copGFPhVEGF165,并转染至HT22 细胞,并进一步观察其对放射线损伤所致的细胞凋亡的保护作用。【方法】 PCR扩增hVEGF165基因,克隆到pCDH-CMV-MCS-EF1-copGFP载体;通过酶切电泳,菌落PCR初步筛选和测序鉴定构建的pCDH-CMV-MCS-EF-GFP1-hVEGF165重组载体;采用磷酸钙共转染法转染293T细胞包装制备慢病毒液,并感染HT22细胞,采用实时RT-PCR及Western Blot检测hVEGF165基因在HT22细胞中的表达情况。进一步对单纯HT22细胞、空质粒转染组以及hVEGF165转染组HT22细胞进行0、5、10 Gy的X线照射,流式细胞仪检测细胞凋亡情况。【结果】 hVEGF165基因片段重组到pCDH-CMV-MCS-EF1-copGFP载体,酶切后电泳结果显示均能得到与理论大小相符的片段,测序结果与GenBank序列完全一致,转染HT22后hVEGF mRNA及蛋白表达水平明显增高。而且同各对照组相比,hVEGF165可以降低细胞放疗后的凋亡率。【结论】 pCDH-CMV-MCS-EF1-copGFP-hVEGF165 慢病毒表达载体构建成功,其转染HT22细胞后可获得高水平hVEGF165 mRNA和蛋白的表达,hVEGF165具有抗放射所致细胞凋亡的作用。

Construction of Lentivirus Vector of hVEGF165 Gene and Its Protective Function of Irradiation-induced Apoptosis

【Objective】 To construct human vascular endothelial growth factor-165 (hVEGF165) gene lentivirus expression vector pCDH-CMV-MCS-EF1-copGFP-hVEGF165, and detect its expression after transfected into HT22 cells and further observe its effect of anti-apopotosis induced by irradiation. 【Methods】 hVEGF165 gene cDNA was amplicated by polymerase chain reaction (PCR) and cloned into the pCDHCMVMCSEF vector. Reconstructed pCDH-CMV-MCS-EF1-hVEGF165 plasmid was identified by electromigratory analysis, colony PCR, and sequencing analysis. 293T cells were transfected with recombinate vector using the way of calcium phosphate cotransfection to get lentiviral particles. Lentiviral particles were then transfect them into HT22 cells. Real-time RT-PCR and Western blot were used to detect the expression of VEGF165 gene in HT22 cells. Furthermore, 0 Gy, 5 Gy, and 10 Gy X-ray irradiation were given to HT22 cells, empty vector transfected HT22 cells and hVEGF165 gene transfected HT22 cells, respectively. Flow cytometry was then used to compare different rates of apoptosis.【Results】 hVEGF165 gene fragment was constructed into the pCDH-CMV-MCS-EF1-copGFP vector successfully. Enzyme digestion got correct length of hVEGF165 gene, DNA sequencing analysis confirmed that hVEGF165 gene sequence was exactly the same with that reported by GenBank. There were significant increased expression of hVEGF165 mRNA and protein in the HT22 cells after transfected. Compared with the control group, hVEGF165 could reduce apoptotic rate after cell radiation. 【Conclusions】 pCDH-CMV-MCS-EF1-copGFP-hVEGF165 lentivirus vector has been successfully constructed. High level of hVEGF165 mRNA and protein expression could be obtained in HT22 cells after transfected with pCDH-CMV-MCS-EF1-copGFP-hVEGF165. hVEGF165 gene can protect the cells from irradiation induced apoptosis.