慢病毒介导NgR基因转染骨髓基质细胞的研究

Transfection of rat bone marrow stromal cells with NgR ecto genes by ientiviral vector

目的构建编码大鼠NgR（Nogoreceptor）细胞外功能区的慢病毒载体，转染大鼠骨髓基质细胞（BMSCs），使其能高表达NgR的细胞外功能区。方法体外分离培养大鼠BMSCs、293FT包装制备病毒，用包装好的慢病毒颗粒感染BMSCs，问接免疫荧光及Western blot方法检测BMSCs表达NgR的情况。结果筛选阳性重组载体plenti6-NgR，酶切后得到930bp的条带，与设计的NgR基因片段大小一致，表明载体构建成功。转染BMSCs48h后，间接免疫荧光法可见胞质内明显荧光表达，Westernblot得到相对分子质量为37×10^3的条带，与NgR蛋白相对分子质量一致。结论ViraPower^TM慢病毒表达系统可将NgR（310）eeto基因转染入骨髓基质细胞表达，后者可以作为种子细胞拮抗脊髓损伤局部形成的抑制性微环境。

Objective To transfect rat bone marrow stem cells （BMSCs） with a lentiviral vector encoding Nogo receptor （NgR） ecto genes. Methods Rat BMSCs were isolated and cultured in vitro. NgR ecto gene was tranduced into BMSCs by lentivirus vector. The indirect immunofluorescence and Westem blot assays were used to detect the expression of NgR protein. Results A DNA fragment of 930 bp was obtained following the enzyme-digesting of plenti6-NgR, which was coincident with the designed fragment and confirmed the plenti6-NgR. The expression of NgR was found in the cell plasma of BMSCs after transfection by indirect immunofluorescence. Westernblot assays obtained a fragment of 37 × 10^3 consistent with NgR ecto. Conclusion The reconstructed lentiviral vectors could transfer NgR ecto gene into BMSCs effectively, which can serve as a gene therapy to antagonize the inhibitive environment in the injuried spinal cord.