共转染EGFP和hMnSOD基因对猴骨髓源神经干细胞抗氧化能力的影响

Effect of cotransfection of hMnSOD and EGFP gene on the anti-oxidation of rhesus monkey neural stem cells derived from bone marrow

目的:人正义、反义MnSOD基因和EGFP共转染恒河猴骨髓源神经干细胞,观察MnSOD基因对猴骨髓源神经干细胞抗氧化能力和报告基因表达的影响。方法:应用NucleofectorTM核转染仪将人正义、反义MnSOD基因和荧光真核表达载体共转染体外培养的恒河猴骨髓源神经干细胞,荧光显微镜下观察荧光蛋白在细胞内的表达,并检测转染细胞的超氧化物歧化酶(SOD)活性。结果:①转染24h后,共转染细胞可见荧光蛋白在骨髓源神经干细胞内有效表达,荧光显微镜下发强绿色荧光,其转染率约为80%。②转染人正义MnSOD基因的细胞的总SOD活性犤(8.03±0.74)NU/mg犦与转染人反义MnSOD基因细胞的总SOD活性犤7.19±0.86)NU/mg犦差异无显著性意义,但MnSOD活性前者明显高于后者1.89倍。结论:MnSOD基因与EGFP基因共转染骨髓源神经干细胞,均有效表达,提高了细胞内MnSOD活性,为多基因联合治疗帕金森病提供了可行的技术平台。

AIM:To observe the effects of hMnSOD gene on the anti-oxidation activity and the expression of report gene-EGFP in neural stem cells derived from bone marro w(NSEs-BM) of rhesus monkey by sense and antisense cotransfection of hMnSOD gen e and pEGFP-C2. METHODS:The sense and antisense hMnSOD gene and pEGFP-C2 were evaluated and e xtracted and cotransfected into the NSEs-BM cultured in vitro by using the Nucl eofectorTM technology.The expression of green fluorescent protein was measured w ith the fluorescence microscope, and the SOD activities of transfected cells wer e detected. RESULTS:①After 24 hours,EGFP genes were effectively expressed in NSCs-BM,and strong green fluorescence in NSCs-BM cotransfected with MnSOD cDNA and pEGFP- C2 was observed under fluorescence microscope, the transfection rate was about 8 0%.②There was no significant difference between the total SOD activities in NS Cs-BM transfected with sense MnSOD cDNA[(8.03±0.74) NU/mg] and in NSCs-BM tra nsfected with antisense MnSOD cDNA[(7.19±0.86) NU/mg],but the MnSOD activity in the former was 1.89 times higher than that in the latter. CONCLUSION:MnSOD and EGFP genes can be cotransfected into rhesus monkey NSCs- BM and be expressed effectively,which increase the MnSOD activity in NSCs-BM an d provide a feasible technological platform for the polygene therapy of Parkinso n's disease.