miR-155对人椎间盘退变髓核细胞凋亡的作用及机制研究

Effect and Mechanism of miR-155 on the Apoptosis of Degenerative Nucleus Pulposus Cells in the Human Intervertebral Disc

目的:探讨mi R-155对人椎间盘退变髓核细胞凋亡的影响及其作用机制。方法:首先构建慢病毒表达载体,在293T细胞中获得重组慢病毒,然后感染椎间盘退变髓核细胞得到稳定过表达细胞系,同时设置空载体和空白细胞组对照。用荧光显微镜观察慢病毒载体的标签蛋白GFP的表达,分别提取三组细胞总RNA,采用RT-q PCR方法检测mi R-155的表达;通过流式细胞术检测细胞凋亡,Western-Blot检测细胞中凋亡相关蛋白FADD、Caspase-3、Bcl-2及Bax的表达,JC-1试剂盒检测细胞线粒体膜电位的变化情况。结果:在荧光显微镜下,经慢病毒感染的过表达细胞系和空载体细胞系均出现绿色荧光,而空白细胞组未见绿色荧光;RT-qPCR结果显示构建的稳定过表达细胞系(GV369-miR-155-NP)中mi R-155的表达水平较高,且与空载体细胞系及空白细胞对照组均呈显著性差异(P<0.05);与空载体组(GV369-NP)及空白细胞对照组相比,过表达组(GV369-miR-155-NP)细胞凋亡率显著降低(P<0.05),FADD、Caspase-3、Bax的表达水平均明显下降,而Bcl-2表达水平显著增加(P<0.05)。结论:mi R-155可能通过靶向结合Caspase-3和FADD阻止FasL-Fas途径或通过线粒体途径抑制人椎间盘退变髓核细胞凋亡。

Objective: To establish a intervertebral disc degeneration of nucleus pulposus cell line(NP) with stable overexpression of mi R-155, and to explore the effect and mechanism of mi R-155 in apoptosis of NP. Methods: The lentivirus expression vector was first constructed and the recombinant lentivirus was rescued in 293 T cells. Then the stable overexpression cell lines were obtained by infecting the degenerated nucleus pulposus cells of the intervertebral disc. At the same time the empty vector and the blank cell group were set up as controls. The expression of lentivirus label protein GFP was observed by fluorescence microscope. The total RNA was extracted respectively from three groups of cells and the expression of mi R-155 was detected by RT-q PCR method. Apoptosis of cells was detected by flow cytometry and the expression of apoptosis-related proteins(FADD, Caspase-3, Bcl-2 and Bax) was detected by Western-Blot.The change of cell mitochondrial membrane potential was detected with the JC-1 kit. Results: Using fluorescence microscope, green fluorescence was observed in the over-expressed cell lines and empty vector cell lines infected by lentivirus, but green fluorescence was not found in the blank cell group. RT-qPCR results showed that the expression of mi R-155 in stable overexpression cell line(GV369-miR-155-NP) was highly higher than that in empty vector cell line and blank cell control group(P<0.05). Compared with the empty vector group(GV369-NP) and the blank cell control group, the apoptosis rate of overexpressed group(GV369-miR-155-NP) was significantly lower(P<0.05). The expressions of FADD, caspase-3 and Bax were significantly decreased, while the expression of bcl-2 was significantly increased(P<0.05). Conclusion: Mi R-155 may inhibit the apoptosis of degenerative nucleus pulposus cells through preventing FasL-Fas pathway targeting Caspase-3 and FADD or mitochondrial pathway.