摘要
目的探讨恒河猴骨髓基质源神经干细胞进行基因修饰的可行性。方法分离恒河猴骨髓基质细胞(BMSCs),体外培养,bFGF诱导增殖,细胞生长至神经干细胞期时以NucleofectorTM核转染仪行pEGFP—C2转染,倒置荧光显微镜观察基因表达情况,并检测细胞的活力。另外,对同期培养的未转染细胞进行神经干细胞特异性抗原—nestin和CD133抗原的免疫细胞化学检测。结果分离得到的BMSCs能在体外培养液中进行增殖和分化,而在bFGF诱导的情况下,细胞的增殖更为明显:转染pEGFP—C2的细胞于转染后24 h后即表达强绿色荧光蛋白,转染率达30％以上,且细胞的活力基本不受影响:免疫细胞化学检测可见有nestin和CD133抗原在同期培养的未转染细胞内表达。结论灵长类骨髓基质细胞具有向神经干细胞分化、增殖的能力,bFGF能促进细胞的增殖,在神经干细胞培养条件下,可发育分化成神经干细胞;在神经干细胞阶段,应用电转染技术进行神经干细胞基因修饰是可行的,可应用于骨髓基质源神经干细胞的基因治疗领域。
Objective To explore the feasibility of pEGFP-C2 gene transfection into the neural stem cells (NSCs) derived from the bone marrow stromal cells (BMSCs) of rhesus monkey. Methods The BMSCs isolated from adult rhesus monkey were cultured with DMEM/F12 and induced to proliferate with bFGF. When BMSCs were differentiated into NSCs,pEGFP-C2 gene was transfected into nucleus by NucleofectorTM device. Then the cells were observed under an inverted fluorescence microscope to examine the gene expression, and finally the cellular vitality was detected. In addition, the synchronously cultured but non-transfected cells were evaluated whether they could present Nestin and CD133 antigen by double -labelled immunohistochemistrical method. Results The BMSCs isolated from bone marrow could proliferate and generate clone when they were cultured in vitro. Moreover, these cells could grow more rapidly while adding bFGF into the culture medium. The cells transferred with pEGFP-C2 by Nucleofector?device after 24 hours could strongly express green fluorescence under a fluorescence microscope, their transfection efficiency up to 30 %, and the primary cellular vitality was almost maintained. The expression of nestin and CD 133 antigen were found in the synchronously cultured but non-transfected cells. Conclusion BMSCs derived from primate have the ability of differentiation into NSCs and proliferation while bFGF can accelerate the proliferation. It is feasible to transfect therapeutic gene into NSCs derived from the BMSCs of rhesus monkey with Nucleofector?device, which can be applied in the gene therapy of NSCs from BMSCs.
出处
《中华神经医学杂志》
CAS
CSCD
2004年第2期85-88,共4页
Chinese Journal of Neuromedicine
基金
广东省科技厅[粤科基办(2000)25
粤财企(2001)367]
国家自然科学基金(30270491)
