AAV-TGFβ_1的构建及其与AV-TGFβ_1对髓核细胞蛋白多糖合成影响的比较

Construction of AAV-TGFβ_1 and Comparision of Its Biological Effects on Proteoglycan Synthesis of Nucleus Pulpous Cells with AV-TGFβ_1

目的探讨转化生长因子β1腺相关病毒表达系统(AAV-TGFβ1)的构建及其与转化生长因子β1腺病毒表达系统(AV-TGFβ1)对髓核细胞蛋白多糖合成影响的比较。方法采用PCR技术扩增转化生长因子β1(TGFβ1)基因,并于其两端分别连接EcoRI和SalI酶切位点。将TGFβ1基因亚克隆于腺相关病毒(AAV)载体中,并经酶切和测序分析进行鉴定。AAV-TGFβ1病毒被包装并转染H293细胞,通过免疫荧光检测AAV介导的目的基因表达。利用AAV-EGFP病毒检测AAV对髓核细胞的转染效率。AAV-TGFβ1和AV-TGFβ1分别转染髓核细胞后,通过Antonopulos法检测蛋白多糖的合成。结果测序分析证明TGFβ1序列与NCBI Gene Bank所报道的一致。经酶切鉴定证实AAV-TGFβ1重组质粒被成功构建。AAV可介导TGFβ1高效表达并高效转染髓核细胞。AV-TGFβ1可快速一过性地促进髓核细胞蛋白多糖合成,而AAV-TGFβ1可稳定促进蛋白多糖合成。结论AAV-TGFβ1病毒被成功构建并可稳定地促进髓核细胞蛋白多糖的合成。

Objective To construct AAV- TGFβ1 and compare its biological effects on proteoglycan synthesis of the rabbit lumbar disc NP cells with AV - TGFβ1. Methods TGFβ1 gene was obtained using PCR. The upstream of TGFβ1 contained restriction enzyme site EcoR I , and the downstream of TGFβ1 contained restriction enzyme site Sal I . Using the multiple cloning sites （MCS） in plasmid AAV and the corresponding contained restriction enzyme site in PCR product of TGFβ1 , TGFβ1 gene was subcloned into AAV. The recombinant plasmid AAV - TGFβ1 was detected by restriction enzyme digestion and DNA sequencing. Then, AAV - TGFβ1 virus was pack- aged and TGFβ1 expression mediated by AAV was detected using immunofluence analysis inH293 cells. AAV transfect rate to NP cells was detected with AAV - PEGF. After NP cells were respectively transfected by AAV - TGFβ1 virus or AV - TGFβ1 virus, proteoglycan synthesis was detected and compared using Antonopulos methods. Results DNA sequencing revealed that the PCR - amplified TGFβ1 gene was consistent with NCBI Gene Bank. The recombinant plasmid was proved to be right using restriction enzyme digestion. AAV could mediate TGFβ1 protein to be expressed efficiently and transfect the NP cells. AV - TGFβ1 virus could quickly enhance the proteoglycan synthesis of the NP cells, but its biological effect was transient. AAV - TGFβ1 virus could enhance stably proteoglycan synthesis. Conclusion AAV - TGFβ1 virus is successfully constructed and enhances stably proteoglycan synthesis of NP cells.