反义寡核苷酸对视神经少突胶质细胞Nogo-A mRNA表达调控的实验研究

Regulation of antisense oligodeoxynucleotides on expression of Nogo-A in oligodendrocytes in vitro

目的 研究反义寡核苷酸对体外培养的视神经少突胶质细胞Nogo-A表达的影响,为进一步研究视神经损伤修复奠定基础。方法 使用新生2d的Wistar大鼠视神经,采用组织块接种法获得少突胶质细胞,并通过GC抗体免疫染色对培养的细胞进行鉴定。实验分为对照组(A)、随机序列组(B)和2μmol/L(C)、5μmol/L(D)、10μmol/L(E)三种Nogo-A反义寡核苷酸浓度组。细胞培养24h后,提取总RNA,使用RT-PCR检测Nogo-A mRNA的表达变化。结果 视神经组织接种后3d,有圆形或梭形的细胞自视神经迁出;11d左右,细胞基本铺满盖玻片;GC抗体免疫组化显示获得的细胞为少突胶质细胞。RT-PCR结果显示三种浓度的反义寡核苷酸组,Nogo-A mRNA的表达均显著降低(P<0.01),随机序列对Nogo-A mRNA的表达无影响(P>0.05)。结论 Nogo-A反义寡核苷酸可有效地、特异性地抑制靶基因的表达。

Objective To investigate the effect of antisense oligodeoxynucleotides ( ODN ) on Nogo-A mRNA expression in oligodendrocytes. Methods ( 1) Oligodendrocytes were obtained by inoculating the optic nerve of 2 day of newborn Wistar rats and were identified as oligodendrocytes by using immunocytochemical staining. (2)2umol/L,5umol/L,10 umol/L of Nogo-A ODN and a random sequence ODN were added to medium to observe the effects of Nogo-A ODN on cultured cells. Effects of ODN on the expression of Nogo-A in oligodendrocytes were studied using RT-PCR. Results (1) In day 3 after inoculation, a few of round or fusiform cells with GC antibody immunocytochemical stain positive migrated from optic nerve tissue. 11 days later, the coverlips were completely covered by the cells. (2) The result of RT-PCR showed that Nogo-A ODN could significantly and specifically inhibit the expression of Nogo-A after 24 hours exposure (P < 0. 01). Random sequence ODN has no noticeable effect on Nogo-A expresson. Conclusion Nogo-A ODN can effectively and specifically inhibit the expression of Nogo-A. This finding establishes the base for further research of repair of optic nerve injury.