LINGO-1在脊髓神经干细胞分化中的表达及影响

Expression and influence of LINGO-1 in differentiation of spinal cord neural stem cells

目的 :观察LINGO-1在大鼠脊髓神经干细胞(spinal cord derived neural stem cells,SpNSCs)分化过程中的表达特征及其对SpNSCs分化的影响。方法:分别在体外分离培养3只10周龄Fischer 344大鼠SpNSCs,培养至第3代对其进行干性Nestin表达鉴定和观察在其分化过程中LINGO-1的表达量(分化第0、1、3、5天)。随后用包装好的LINGO-1 shRNA慢病毒及Scramble shRNA慢病毒感染SpNSCs,观察感染效率及对比感染后下调LINGO-1蛋白表达的效果。在确定LINGO-1 shRNA的下调效果后,将第4代SpNSCs分为干扰组及对照组,干扰组感染LINGO-1 shRNA慢病毒,对照组感染Scramble-sh RNA慢病毒,于分化5d后使用免疫荧光染色检测神经元及星形胶质细胞的比例。结果:体外分离培养的SpNSCs表达Nestin阳性。LINGO-1蛋白的积分光密度(integrated option density,IOD)在第0、1、3、5天分别为514±45、715±68、1017±92、654±61,LINGO-1从SpNSCs分化第1天起表达量上升,为第0天表达量的1.39倍,至分化第3天为第0天表达量的1.98倍,随后表达下降,至第5天为第0天表达量的1.36倍,以上4个时间点LINGO-1蛋白表达量有统计学差异(P<0.05)。LINGO-1 shRNA慢病毒感染效率>90%,感染LINGO-1 shRNA慢病毒的SpNSCs的LINGO-1蛋白表达量为感染Scramble-sh RNA慢病毒Sp NSCs的26%。干扰组神经元分化比例为对照组的3.85倍,星形胶质细胞分化比例为对照组的0.65倍,两组神经元分化比例及星形胶质细胞分化比例的差异均有统计学意义(P<0.05)。结论 :LINGO-1在大鼠SpNSCs分化初期表达上升,抑制LINGO-1表达可促进Sp NSCs向神经细胞分化,减少其向星形胶质细胞分化。

Objectives： To investigate the expression and effect of LINGO-1 in differentiation of spinal cord derived neural stem cells（SpNSCs）. Methods： SpNSCs were isolated from spinal cord in three 10-week-old Fisher 344 rats and cultured in vitro, stained with Nestin, and the expression of LINGO-1 on P3（on differen-tiation day 0, 1, 3, 5） was observed. SpNSCs were transfected with LINGO-1 sh RNA lentiviral vector and Scramble-sh RNA lentiviral vector to investigate the infection efficiency and RNA interference effect. SpNSCs of P4 were divided into RNAi group and control group. The RNAi group was transfected with LINGO-1 sh RNA lentiviral vector and control group was transfected with Scramble-sh RNA lentiviral vector. The propor-tions of neurons and astrocyte were measured by immunofluorescence histochemistry. Results： SpNSCs were Nestin positive. The expression of LINGO-1 IOD was 514 ±45, 715±68, 1017±92, 654±61 on the 0 th, 1 st,3 rd, 5 th differentiation day respectively. The expression of LINGO-1 at the very 1 st differentiation day in-creased and was 1.39 times of expression on the day 0, 1.98 times on the 3 rd day, and then decreased to1.36 times on the 5 th day, the differences of all expressions on the 4 days were statistically significant（ P〈0.05）. The rate of LINGO-1 shRNA lentivirus infection was higher than 90%, which reduced the protein ex-pression of LINGO-1 to 26% expression of SpNSCs with Scramble-sh RNA lentiviral vector infection. The proportion of neurons in RNAi group was 3.85 times of that of the control group, and the differentiation of astrocytes was 0.65 times of the control group, the proportions of neurons and astrocytes in two groups were significantly different（P〈0.05）. Conclusions： The expression of LINGO-1 increases in the early days of differentiation. Inhibition of LINGO-1 gene expression in spinal cord derived neural stem cell, can promote differentiation into neurons and reduce into astrocyte.