hTERT基因修饰神经干细胞移植修复大鼠脊髓损伤

Transplantation of h TERT Gene Modified Neural Stem Cells for Spinal Cord Injury Treatment in Rats

目的对神经干细胞的生物学特性进行观察,探讨h TERT基因修饰神经干细胞移植对修复大鼠脊髓损伤的影响。方法体外培养的大鼠神经干细胞,用病毒PLXSN为载体介导h TERT基因反转录转染神经干细胞,分为阴性转染组、对照组与h TERT转染组3个组。经Western blot法检测分析h TERT蛋白的表达。运用细胞生长曲线、CCK-8比色法两种方法,分析细胞生长的优化作用。将造模成功的72只大鼠随机分为h TERT-NSCs、NSCs与对照组3组,每组各分24只,通过改良的Allen打击法来建立大鼠急性脊髓损伤模型。通过斜板试验,BBB评分给各组大鼠进行运动功能检测。通过HE染色及荧光显微镜研究,PKH-26标记的分布情况及NSC存活取材进行病理切片。术后72h通过RT-PCR分析脊髓损伤区周围MMP9/2、AQP/9基因的表达,运用UNEL法分析细胞凋亡情况。荧光显微镜观察PKH-26标记的分布情况及NSCs存活。结果 h TERT基因转染大鼠神经干细胞后,h TERT基因转染组蛋白水平有高表达、细胞的生长速度出现明显增快,相比与阴性h TERT转染组与对照组,各组间差异有统计学意义(P<0.05)。对照组的细胞凋亡指数略低于阴性转染组,阴性转染组略低于h TERT转染组大鼠下肢运动功能评价。与对照组相比h TERT转染组AQP4/9基因表达均较显著降低。PKH-26标记的阳性细胞数:对照组最少,h TERT转染组最多,阴性h TERT转染组次之,各组差异有统计学意义(P<0.05)。结论通过PLXSN病毒为载体介导h TERT基因逆转染神经干细胞,可以促进大鼠神经干细胞增殖。h TERT基因修饰神经干细胞移植可促进脊髓损伤大鼠神经突触的再生,降低脊髓损伤区周围AQP/9、神经细胞凋亡和MMP9/2基因的表达,且能够促进大鼠的电生理及肢体运动功能的恢复。

Objective To explore repair of spinal cord injury by neural stem cells （NSCs） modified with telomerase reverse transcriptase （hTERT） gene in rats. Methods In vitro cultured rat neural stem cells, with the virus PLXSN as the carrier mediated hTERT gene retroviral transfeetion neural stem cells. The expression of hTERT protein by Western blot analysis. Using the cell growth curve, CCK 8 colorimetrie method two kinds of methods, analyzing the optimization of cell growth. The successful model of 72 rats were randomly divided into three groups ： hTERT - NSCs group, NSCs group and the control group, 24 rats of each group. The model of acute spinal cord injury in rats was established by modified Allen＇s method. Respectively, before modeling, model after 1 days, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks by BBB score, inclined plane test for evaluation of motor function. Model after 4 weeks for pathological section HE staining and fluorescence microscope observation of PKH26 labeled NSCs the survival and distribution. After 72 h,The expression of MMP9/ 2 and AQP/9 gene of spinal cord injury was detected by RT - PCR. Results After transfeetion of hTERT gene into rat neural stem cells, the expression of hTERT gene was significantly increased, and the growth rate of cells was significantly increased, compared with the negative hTERT transfeetion group and control group, the difference was significant. The apoptosis index of the control group was slightly lower than that of the negative transfeetion group, and the function of the lower limb was slightly lower than that of the hTERT group. Compared with the control group, the expression of AQP4/9 gene was significantly decreased in hTERT group. Pkh - 26 labeled positive cells ： con- trol group at least, hTERT transfection group most negative hTERT transfection group, the difference of each group slgnificantly（P 〈 0.05. ）. Conclusion HTERT gene mediated by PLXSN gene can promote the proliferation of neural stem cells, which can promote the proliferation of neural stem cells in rats. HTERT gene modified neural stem cells transplantation can promote the regeneration of nerve syn- apses in spinal cord injury rats, reduce the expression of AQP/9 and MMP9/2 gene in the spinal cord injury area, and can promote the re- covery of physiological and motor function of rats.