重组逆转录病毒pLNCX2-GDNF转染许旺细胞及其表达

Transfection of Schwann cells with retrovirus pLNCX2-GDNF and its expression

目的 利用基因转染技术将胶质细胞源性神经营养因子 (GDNF)基因转入许旺细胞 (SC)以提高其分泌该因子的水平。 方法 应用逆转录聚合酶链反应 (RT PCR)方法合成GDNFcDNA片段 ,以逆转录病毒pLNCX2为载体导入许旺细胞内 ,并运用RT PCR、免疫细胞化学染色、ELISA及运动神经元联合培养法检测外源性基因在mRNA和蛋白水平的表达及其产物的活性。 结果 RT PCR检测结果示GDNF SCs表达GDNFmRNA的水平明显优于SCs ;免疫细胞化学检测发现CDNF SCs抗GDNF蛋白染色呈强阳性 ,正常SCs呈弱阳性 ;ELISA法检测结果示转染后 4周CDNF SCs分泌GDNF蛋白的水平升至正常SCs的 5 1倍 ;运动神经元联合培养法结果显示GDNF SCs分泌的外源性基因产物具有生物学活性。 结论 GDNF基因转染SC在mRNA和蛋白水平表达GDNF明显优于正常SCs ,外源性基因表达产物具有神经生物学活性。

Objective To examine the effectiveness of a gene transfer of rat glial cell-line derived neurotrophic factor(GDNF)into Schwann cells Methods Rat GDNFcDNA was amplified from newly-born rat sciatic nerve by RT-PCR and ligated into the retroviral vector pLNCX-2 A fter being packaged by the amphotropic packaging cell line PT67,the viral supernatants from these anti-clonal producing lines were titred on fibroblast N IH3T3 The highest tire recombinant retrovirus was used to infect deviding populations of newly-born rat SCs The level of GDNF mRNA in GDNF-SCs and normal SCs was tested by RT-PCR The GDNF protein in genetically modified SCs and normal SCs was assayed by immunocytochemistry The amounts of GDNF in conditioned medium of GDNF-SCs in different phrase and normal SCs were detected by enzyme-linked immunoassay sensitive assay Biological activity of the secreted GDNF was tested using motoneuron bioassay by MTT method Results (1)The GDNF cDNA band of GDNF-SCs was more prominent than that of SCs (2)Cells in both groups were immunohistochemically positive for GDNF expression Staining for GDNF was more prominent in the Scs infected with pLNCX2-GDNF (3)The rate of GDNF secreted by GDNF-Scs was 5 1-fold compared with normal SCs (4)The bioassay comfirmed that the secreted GDNF from GDNF-Scs was biologically active,and more motoneurons survived than normal SCs group Conclusion With the help of retrovirus,GDNF gene can be transferred and stably expressed in SCs,and it′s may be a better way to graft SCs promoing regeneration of PNS injuries.