腺相关病毒介导的仓鼠δ-SG基因在TO-2型仓鼠骨髓间充干细胞的表达

Expression of Hamster δ-SG Gene after Adeno-associated virus Infection in TO-2 Hamster Bone Marrow-derived Mesenchymal Stem Cells

目的构建含有δ-SG基因的腺相关病毒载体并检测经腺相关病毒介导基因转染的TO-2型仓鼠骨髓间充质干细胞(MSCs)中目的基因的表达。方法用EcoRI酶和XhoI酶酶切含有δ-SG基因的pUC57-SG质粒,获得δ-SG基因。片段回收后定向插入腺相关病毒载体质粒pITR-GFP中重组成pITR-GFP-SG,并同pXX680、pHelper三质粒经钙磷沉淀法共转染HEK293细胞,斑点杂交法测定重组病毒颗粒滴度,再将重组病毒颗粒转染TO-2型仓鼠的MSCs,利用RT-PCR法,免疫荧光检测目的基因的表达,MTT检测转染细胞增殖能力。结果成功构建了δ-SG基因的腺相关病毒载体。含δ-SG基因的腺相关病毒感染TO-2型仓鼠MSCs48h后,RT-PCR产物经琼脂糖凝胶电泳出现884bp-DNA片段;免疫荧光检测显示TO-2型仓鼠MSCs中呈现绿色颗粒,大小深浅不一;MTT证实转染后细胞的增殖能力未受明显影响。结论采用腺相关病毒介导的方法,可以将δ-SG基因导入TO-2型仓鼠的MSCs并成功表达。

Objective To construct the adeno-associated virus（AAV） vector containing hamster δ-SG gene and detect the exprdssion of hamster δ-SG gene in TO-2 hamster bone marrow-derived mesenchymal stem cells transferred （MSCs） after adeno-associated virus infection. Methods δ-SG cDNA was obtained from recombinant plasmid pUC57-SG digested with endonuclease EcoRI and XhoI ,the production was inserted directionally into AAV vector plasmid pITR-GFP to be plasmid pITR-GFP-SG, and transferred into the packing cell HEK293 together with plasmid pXX680 and pHelper mediated by phosphate-calcium deposit method . The titer of recombinant AAV viral particles were determined by dot hybridization and used to infect MSCs of TO-2 hamster. δ-SG expression was examined by methods of immunofluorescence staninig , reverse transcriptase-polymerase chain reaction. MTT was used to see the proliferation of the infected MSCs. Results The adeno-associated virus was successfully constructed and transferred into HEK293 cells, and 48h after in- fection with the recombinant adeno-associated virus in TO-2 hamster MSCs ,884 bp fragment could be detected by electrophoresis in the RT-PCR products. The green particles could be detected in some infected TO-2 hamster MSCs with immunofluorescence staining. MTT test give an evidence that the recombinant adeno-assoeiated virus do not influence the cell＇ s proliferation. Conclusion TO-2 hamster MSCs infected with adenoassociated virus containing δ-SG gene can express δ-SG protein.