【摘要】 目的探讨应用hVEGF165与TGFβ1基因逆转椎间盘退变的可行性。方法用分子克隆技术,从pcDNA3(+)-hVEGF165获得hVEGF165cDNA,并克隆至AAV包装质粒pSNAV上,构建成pSNAV-hVEGF165的AAV重组质粒。经酶切及测序鉴定后,用脂质体介导pSNAV-hVEGF165转染HEK293细胞和血管内皮细胞。荧光免疫组化方法检测hVEGF165蛋白,并用MTT法测定hVEGF165对血管内皮细胞增殖的影响。由本元正阳公司对pSNAV-hVEGF165进行AAV-hVEGF165包装。AAV-hVEGF165与AAV-TGFβ1联合转染退变的椎间盘纤维环细胞,Westernblot检测hVEGF165与TGFβ1的表达以及纤维环细胞Ⅰ型胶原表达量的变化。结果经酶切鉴定及基因测序证实AAV包装质粒pSNAV-hVEGF165构建完成,荧光免疫组化显示hVEGF165蛋白表达,MTT法显示hVEGF165蛋白对血管内皮细胞增殖有促进作用。本元正阳公司包装出具有生物学功能的AAV-hVEGF165,AAV-hVEGF165构建成功。Westernblot显示hVEGF165与TGFβ1在纤维环细胞中表达,AAV-hVEGF165与AAV-TGFβ1双基因联合转染的纤维环细胞Ⅰ型胶原表达量较hVEGF165与TGFβ1基因各自单独转染的细胞Ⅰ型胶原表达量明显增多。结论hVEGF165在体外能够协同TGFβ1促进退变纤维环细胞Ⅰ型胶原表达增加。更多还原

【Abstract】 Objective To evaluate the reversion possibility of hVEGF165 and TGFβ1 to intervertebral disc degeneration by gene method. Methods The hVEGF165cDNA obtained from plasmid pcDNA3(+)-hVEGF165 was subcloned into the packaging plasmid pSNAV of AAV by molecular clone ways. The recombinant plasmid pSNAV-hVEGF165 was identified by restriction enzymes analysis and sequencing analysis, and then transfered to the HEK293 cell and VEC by lipofectamine mediated gene transfer method. The protein hVEGF165 was detected by immunofluorescence for immunocytochemistry and explored the influence to the proliferation of vascular endothelial cell by MTT. Whereafter the AAV-hVEGF165 was packaged by Benyuan Zhengyang Company. AAV-hVEGF165 and AAV-TGFβ1 were cotransfected into annulus fibrosus cell of intervertebral disc, then the expression of hVEGF165 and TGFβ1, and the change of collagen Ⅰin annulus fibrosus cell were detected by Western blot. Results The recombinant pSNAV-hVEGF165 was completely constructed and confirmed by restriction enzymes analysis and sequencing analysis. The protein hVEGF165 was detected by immunofluorescence for immunocytochemistry in experimental group, and hVEGF165 could promote the proliferation of vascular endothelial cell. The bioactive AAV-hVEGF165 was successfully constructed. The expression of AAV-hVEGF165 and AAV-TGFβ1 were manifested in degenerative annulus fibrosus cell by Western blot, and the expression of collagen Ⅰin annulus fibrosus cell cotransfected by AAV-hVEGF165 and AAV-TGFβ1 was markedly more than that of the monogenic transfected cell. Conclusion hVEGF165 could cooperate with TGFβ1 to promote the expression of collagen Ⅰ.更多还原