过表达NDRG2基因促进人髓核细胞衰老的体外实验研究

发布时间：2018-07-27 14:47

【摘要】：[目的]研究NDRG2(N-Myc downstream-regulated gene 2)对人髓核细胞衰老的影响,进而探讨NDRG2在椎间盘退变中的作用。[方法]通过慢病毒过表达NDRG2基因稳定感染髓核细胞。设立正常髓核细胞组(空白组)、红色荧光蛋白标记的慢病毒感染髓核细胞组(对照组)及过表达NDRG2慢病毒感染髓核细胞组(实验组)。Western blot检测NDRG2蛋白表达水平,细胞衰老相关β半乳糖苷酶(senescence associated-β-galactosidase,SA-β-ga1)染色检测髓核细胞衰老变化,流式细胞仪检测髓核细胞周期变化,MTT法绘制生长曲线,并分析髓核细胞的增殖情况。[结果]正常髓核细胞感染过表达NDRG2慢病毒48 h后荧光显微镜下观察基因感染效率在95%以上;感染72 h后Western blot检测示NDRG2表达明显升高(P0.01);实验组SA-β-gal染色阳性率明显高于对照组和空白组(P0.01);MTT生长曲线显示慢病毒感染后髓核细胞增殖速度较对照组和空白组均减慢,潜伏期延长,缓慢进入对数期;细胞周期结果示实验组G0/G1期百分比明显高于对照组和空白组,S期明显低于对照组和空白组,差异有统计学意义(P0.05)。[结论]NDRG2基因过表达的髓核细胞衰老速度明显加快,提示NDRG2可能通过调控髓核细胞的衰老参与椎间盘退变过程,为椎间盘的退变生物学治疗提供依据。
[Abstract]:[objective] to study the effect of NDRG2 (N-Myc downstream-regulated gene 2) on the aging of human nucleus pulposus cells, and to explore the role of NDRG2 in the degeneration of intervertebral disc. Methods NDRG2 gene was overexpressed by lentivirus to infect nucleus pulposus cells stably. Normal nucleus pulposus cells group (blank group), red fluorescent protein labeled lentivirus infected nucleus pulposus group (control group) and overexpression of NDRG2 lentivirus infected nucleus pulposus cell group (experimental group). Western blot was used to detect the expression of NDRG2 protein. Senescence associated 尾 -galactosidase (SA- 尾 -ga1) staining was used to detect the senescence of nucleus pulposus cells. Flow cytometry was used to detect the cell cycle changes of nucleus pulposus cells. The growth curve was plotted by flow cytometry, and the proliferation of nucleus pulposus cells was analyzed. [results] after 48 hours of overexpression of NDRG2 lentivirus in normal nucleus pulposus cells, the efficiency of gene infection was over 95% under fluorescence microscope. The positive rate of SA- 尾 -gal staining in experimental group was significantly higher than that in control group and blank group (P0.01). The proliferation rate of nucleus pulposus cells after lentivirus infection was slower and the incubation period was longer than that in control group and blank group. The results of cell cycle showed that the percentage of G0/G1 phase in the experimental group was significantly higher than that in the control group and the blank group compared with the control group and the blank group (P0.05). [conclusion] the aging rate of nucleus pulposus cells with overexpression of NDRG2 gene was significantly accelerated, suggesting that NDRG2 may participate in the degeneration of intervertebral disc by regulating the aging of nucleus pulposus cells, and provide the basis for the biological treatment of degeneration of intervertebral disc.
【作者单位】：第四军医大学唐都医院骨科;第四军医大学药学系;
【基金】：国家自然科学基金资助(项目编号:81201633)
【分类号】：R329.2

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