外源性SDF-1α和GDNF基因在恒河猴骨髓基质细胞内的稳定表达  被引量:1

Construction and expression of pBudCE4.1-SDF-1α-GDNF in bone marrow stromal cells of rhesus monkey

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作  者:徐强[1] 许百男[1] 刘柯[2] 潘力[3] 饶军华[4] 徐如祥[5] 

机构地区:[1]解放军总医院神经外科,北京100853 [2]南方医科大学附属花都医院,广州510800 [3]复旦大学华山医院神经外科,上海200040 [4]广东省昆虫研究所,广州510262 [5]北京军区总医院神经外科,北京100700

出  处:《中华神经医学杂志》2010年第1期43-46,50,共5页Chinese Journal of Neuromedicine

基  金:基金项目:国家自然科学基金(30600203);中国博士后科学基金(2005037124)

摘  要:目的构建携带人基质细胞衍生因子-1α(SDF-100和胶质细胞源神经营养因子(GDNF)基因的真核表达质粒pBudCE4.1-SDF-1α—GDNF,转染恒河猴骨髓基质细胞(BMSCs),观察外源性SDF-Jd和GDNF基因在BMSCs中的表达情况。方法应用基因重组技术,从cDNA文库获得GDNF和SDF-1α基因,重组到pBudCE4.1双表达载体上。pBudCE4.1-SDF-1α—GDNF经脂质体转染培养的恒河猴BMSCs,48h后行GDNF和SDF-1αWestern blot和免疫组化检测。结果酶切、PCR和DNA序列鉴定均证实插入基因片段的正确性:48h后行West blot和免疫组化证实GDNF和SDF-1α在细胞内能有效表达。pBudCE4.1-GDNF—SDF-1仅转染BMSCs表达的GDNF和SDF—1α蛋白的量是阴性对照细胞的10倍和6倍。结论双基因真核表达质粒pBudCE4.1-SDF-1α-GDNF转染至恒河猴BMSCs后.SDF-1α和GDNF基因在BMSCs内能有效表达,为BMSCs携带SDF-1α和GDNF双基因自体移植治疗相关疾病奠定了基础。Objective To observe the expression ofstromal cell-derived factor-1α (SDF-1α) and glial cell line-derived neurotrophic factor (GDNF) genes in bone marrow stromal cells (BMSCs) of rhesus monkey. Methods With gene recombination technique, SDF-1α and GDNF genes obtained from cDNA were subcloned into pBudce4.1 vector to get pBudCE4.1-SDF-1-GDNF, evaluated by restriction enzyme analysis and sequencing analysis. The pBudCE4.1-SDF-1α-GDNF was transfected into BMSCs with lipofectamine2000. After 48 h, the expression of SDF-1α and GDNF was measured by RT-PCR, Western blotting and immunohistochemistry. Results Correct construction of pBudCE4.1 -SDF-1α-GDNF was identified by enzyme restriction analysis and sequencing analysis. Western blotting and immunohistochemistry confirmed the expressions of SDF-1α and GDNF genes in the transfected cells. The protein expressions of GDNF and SDF-1α in the GDNF and SDF-1α transfected BMSCs were 6 times higher than those in the negative control cells. Conclusion The pBudCE4.1-SDF-1 α-GDNF can successfully express SDF-1α and GDNF in BMSCs of rhesus monkey, which may be used in gene therapy for Parkinson's disease.

关 键 词:基质细胞衍生因子-1Α 胶质细胞源神经营养因子 骨髓基质细胞 基因治疗 

分 类 号:Q78[生物学—分子生物学]

 

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