过表达CXCL12基因的脂肪来源间充质干细胞的构建及其对内皮祖细胞趋化作用的实验研究

Construction of Lentiviral Vectors Carrying CXCL12 Gene and Exploration of Transfection Condition of Human Adipocyte-derived Mesenchymal Stem Cells

目的:本研究旨在构建携带间质细胞衍生因子1a(Stromal cell derived factor-1a,CXCL12/SDF-1a)基因慢病毒载体,探索CXCL12慢病毒转染脂肪间充质干细胞(ADSCs)及CXCL12的表达,并观察其对内皮祖细胞的趋化作用。方法:利用PCR的方法从基因库调取并扩增CXCL12基因,克隆到穿梭质粒p Len O-GTP的表达框中,将重组p Len O-GTP载体质粒和慢病毒包装质粒共转染293T细胞,收获并测定病毒滴度,以最适MOI值转染ADSCs,通过western-blot方法测定转染的ADSCs表达的CXCL12,将过表达CXCL12的脂肪干细胞移植到裸鼠背部,3天后流式细胞术鉴定外周血中的血管内皮祖细胞(EPC)的数量变化。结果:CXCL12基因PCR产物电泳与测序结果均正确。重组p Len O-GTP-CXCL12质粒酶切后产物电泳结果正确,含有CXCL12基因的穿梭质粒构建正确。穿梭质粒与慢病毒包装质粒共同感染293T细胞收获CXCL12慢病毒。测得病毒滴度为1.4×109TU/ml。重组慢病毒感染ADSCs的最佳感染复数(multiplicity of infection,MOI)值为50。western-blot检测结果慢病毒感染的ADSCs高表达CXCL12/SDF-1a,过表达CXCL12基因组裸鼠外周血EPC明显高于其余两组。结论:重组CXCL12慢病毒载体构建成功,包装得到高浓度病毒液,感染人ADSCs能稳定过表CXCL12蛋白,并可趋化骨髓内EPC进入外周血,为临床获取应用EPC提供了一种新的方法,为进一步研究过表达CXCL12的ADSCs,促进移植脂肪成活及血管化奠定基础。

Objective： The purpose of this study is to construct the stromal cell derived factor 1A （Stromal cell derived factor-la, CXCL12/SDF-la） gene lentiviral vector, explore CXCL12 lentivirus transfected adipose mesenchymal stem cells （ADSCs） and the expression of CXCLI2, and to observe the ehemotaxis for endothelial progenitor ceils. Methods： Using PCR method to retrieve and amplify CXCL12 gene fi＇om gene bank, the CXCL12 gene was cloned into the shuttle plasmid pLenO-GTP box, the recombinant pLenO-GTP plasmid and lentiviral packaging plasmids were transfeeted into 293T cells, harvest the virus and determine the titer of the virus, transfect the ADSCs with optimal MOI, the expression of CXCLI2 gene was measured by Western-blot method, the ADSCs of overexpressing CXCL12 gene were transplanted into the back of nude mice, the number of endothelial progenitor cells （EPC） in peripheral blood was determined by flow cytometry （FCM） after 3 days. Results： the results showed that the PCR product of CXCL12 gene was correct. The results showed that the recombinant plasmid pLenO-GTP-CXCLI2 was correct after the restriction enzyme digestion, and the shuttle plasmid containing CXCL12 gene was constructed correctly. Shuttle plasmid and lentiviral packaging plasmid were co infected with 293T cells to harvest CXCLI2 lentivirus. The virus titer was 1.4x l09 TO/mE. The optimal number of complex infections （multiplicity of infection, MOI） of recombinant lentivirus infected ADSCs was 50. The results of Western-blot detection showed that the ADSCs expression of CXCL12/SDF-1 a was higher than the other two groups, and the amount of EPC in the peripheral blood of the group of overexpression of CXCL12 gene was significantly more than the other two groups. Conclusion： The recombinant CXCL12 lentivirus vector was successfully constructed, and the high concentration of virus solution was obtained. The infection of human ADSCs could stabilize the expression of CXCL12 protein, and mobilize the EPC of bone marrow into the peripheral blood. It provides a new method for the clinical application of EPC, which lays the foundation for further study on the over expression of CXCL12 ADSCs and the promotion of the survival and vascularization of transplanted fat.