逆转录病毒载体ex vivo途径表达TH和GDNF基因

为了通过逆转录病毒载体 ex vivo途径高效表达 TH和 GDNF基因。本实验构建有 TH和 GDNF基因的重组逆转录病毒载体 p LHCX/TH和 p L NCX2 /GDNF,分别转染包装细胞 PA3 17和 PT67中 ;经筛选、RT-PCR、免疫组化等鉴定得到产毒细胞 PA3 17/TH和 PT67/GDNF。培养 COS-7细胞 ,以两种产毒细胞的上清分别感染 COS-7细胞 ,筛选后 ,免疫组化、原位杂交检测基因的表达量 ,将基因改造的细胞移植到大鼠纹状体内 ,免疫组化检测体内移植的 TH、GDNF基因的表达。结果表明 :TH和GDNF基因可在靶细胞表达 ,且筛选后基因表达阳性细胞显著增加 ;TH阳性率达 5 0 % ,GDNF阳性率达 70 %。在体内实验中 ,可以观察到这两种外源基因可同时在大鼠脑内移植区表达。以上结果提示 ,TH和 GDNF基因改造细胞 ,可通过 ex vivo途径在脑内移植区高效表达。这一实验结果将对 Parkinson病复合性基因治疗提供依据

EX VIVO EXPRESSION OF TH AND GDNF GENES WITH RETROVIRUS VECTOR

The replacing TH gene or GDNF gene has been investigated well for gene therapy of Parkinson's disease(PD). The present study was to examine the expression efficiency of recombined TH and GDNF genes with retrovirus via ex vivo. TH or GDNF gene was reconstructed into retroviral vectors of pLHCX and pLNCX2, respectively. Then pLNCX2/GDNF and pLHCX/TH were transfected into packaging cell line PT67 and PA317 individually. The transfected PA317/TH and PT67/GDNF cells were selected by the corresponding antibiotics and examined by using RT-PCR and immunohistochemistry. Then the medium containing virus particles was harvested and used for infecting COS-7 cells, which were examined by immunohistochemistry and in situ hybridization. Those genetically modified COS-7 cells were grafted into the rat brain. TH and GDNF were expressed in COS-7 cells and much higher efficiency of gene expression was obtained after selection. 50% of cells expressed TH gene and 70% of cells expressed GDNF gene.In vivo, expression of TH and GDNF genes was found in the grafted area of rat brain by using immunohistochemistry of double labeling method. Therefore, it is feasible and valuable for further study of PD gene therapy with combined TH and GDNF genes via ex vivo way.