Construction of recombinant adenovirus vector of Antisense BTEB2 and its expression in vascular smooth muscle cells / 医学研究生学报

Objective: To construct recombinant adenovirus vector of Antisense BTEB2 cDNA and study the effect of antisense BTEB2 on proliferation of vascular smooth muscle cells and neointimal hyperplasia.Methods:BTEB2 cDNA was prepared by RT PCR technique and was subcloned reversedly into shuttle plasmid pDC315 to constructed recombinant shuttle plasmid pDC315AS BTEB2.Then the recombinant shuttle plasmid and adenovirus genomic plasmid pBHGlox?E1,3Cre were cotransfected into 293 cell to obtain recombinant adenovirus. The PCR technique was used to detect target gene fragment and adenovirus genomic characteristic fragment. After the recombinant adenovirus infected vascular smooth muscle cells, antisense RNA expression of BTEB2 was detected by RT PCR. Results:There was recombinant adenovirus containing BTEB2 cDNA in the lysate of cotransfected 293 cells.The recombinant adenovirus infected 293 cells and replicated in 293 cells. The expression of BTEB2 antisense RNA was very obvious in vascular smooth muscle cells after infected by recombinant adenovirus. Conclusion:The construction of recombinant adenovirus vector of Antisense BTEB2 has been achieved, and Antisense RNA of BTEB2 can express in vascular smooth muscle cells. This study has paved the way for furthur research.

Inhibitory effect of BTEB2 antisense RNA mediated by recombinant adenoviral vector on proliferation of vascular smooth muscle cells / 第三军医大学学报

Objective To construct recombinant adenoviral vector expressing basic transcription element binding protein 2 (BTEB2) antisense RNA and study the effects of BTEB2 antisense RNA on the proliferation of vascular smooth muscle cells (VSMCS). Methods BTEB2 cDNA was prepared by RT-PCR technique and was subcloned reversedly into the shuttle plasmid to construct recombinant shuttle plasmid, and then the recombinant shuttle plasmid and adenovirus genomic plasmid pBHG were cotransfected into 293 cells to obtain the recombinant adenovirus. The PCR technique was used to detect the target gene fragment and adenovirus genomic characteristic fragment. After transfection of the recombinant adenovirus into VSMCs, BTEB2 antisense RNA was detected by RT-PCR. The effects of BTEB2 antisense RNA on BTEB2 protein expression, proliferation, and cell cycles of VSMCs were analyzed by Western blot, MTT test, and flow cytometry, respectively. Results The recombinant adenovirus was proved correct by PCR. The expression of BTEB2 antisense RNA was very obvious in VSMCs after infection by recombinant adenovirus. Recombinant adenovirus infection significantly inhibited BTEB2 protein expression and proliferation of VSMCs and resulted in G 0/G 1 block. Conclusion The recombinant adenoviral vector expressing BTEB2 antisense RNA has been constructed. BTEB2 antisense RNA can significantly inhibit the proliferation of VSMCs.