脂质体介导转染神经干细胞行糖尿病大鼠视网膜下移植的研究

Research of lipofectamine on neural stem cells transplanted into the subretinal space of rats with streptozotocin-induced diabetes

目的观察大鼠胚胎神经干细胞(NSCs)阳离子脂质体法转染增强型绿色荧光蛋白(EGFP)的效果;研究阳离子脂质体Lipofectamine介导转染对大鼠胚胎NSCs糖尿病大鼠视网膜下移植的影响。方法将报告基因pEGFP-N1经Lipofectamine介导转染NSCs(EGFP组),观察EGFP的表达;以同期培养未转染的细胞作为对照组,测定细胞活力、生长曲线和转染效率。将两组NSCs分别行糖尿病大鼠视网膜下移植,RT-PCR、Westernblot分别检测TOLL受体4(TLR4)、NF-κB的mRNA及蛋白质表达。结果被转染的体外培养大鼠胚胎NSCs长期表达EGFP,两组细胞具有相似的形态学变化和生长曲线,流式细胞仪结果显示转染率最高可达35.2%。行视网膜下移植后,TLR4、NF-κBmRNA及蛋白质的表达差异无统计学意义。结论阳离子脂质体Lipofectamine介导转染NSCs效率较高,报告基因表达时间长,不影响糖尿病大鼠NSCs视网膜下移植。

Objective To observe the expression of enhanced green fluorescent protein （EGFP） transferred by lipofectamine-mediated cultured neural stem cells （NSCs） and investigate the effects of lipofectamine on NSCs transplanted into the subretinal space of rats with streptozotocin-induced diabetes. Methods NSCs from Wistar rat embryo brains were cultured in vitro and passaged. Cultured NSCs were transferred with pEGFP-NI by lipofectamine （ EGFP group） or uninfected （ control group）. The expression of EGFP in NSCs was detected by fluoresecent microscope. The cellular viability and growth curves of labeled cells were respectively analyzed and compared. Transfer efficiencies were evaluated by flow cytometry. The NSCs were transplanted into the subretinal space of diabetic male Wistar rats. Toll-like receptor 4 （TLR4） were measured by RT-PCR ; the protein expression of TLR4 and activation of NF-KB were detected by Western blot. Results Passaged NSCs showed positive reaction for Nestin,NSE and GFAP. EGFP was expressed in transferred NSCs for a long-term,showing a green fluorescence. The cellular viability reduced from 96% before transfer to 81% in 24 after transfer with a similar morphological development and growth curves in two groups. And flow cytometry revealed that the highest transfer rate was up to 35.2% in 8 mL/L of lipofectamine. No statistically significant difference was seen in the expressions of TLR4 mRNA （ F = 1.6, P 〉 0. 05 ） , the protein of TLR4（F=3.0,P〉0.05）and activation of NF-κB（F =2.8,P 〉0.05）between two groups. Conclusion The transfer of EGFP shows no significant effect on the proliferation of NSCs, The transfer of NSCs mediated by lipofectamine is effective and its report gene has a long-term expression. Lipofectamine has no obvious effect on the NSCs subretinal transplantation in rats with diabetes. This method can be further applied for the transplantation study of NSCs.