腰退行性病变与TRALL基因之间关系的研究

A research on the relationship between TRAIL gene and of lumbar degenerative disease

[目的]探讨腰椎间盘退行性病变与肿瘤坏死因子相关凋亡诱导配体(tumor necrosis factor-related apoptosis-inducing ligand, TRAIL)基因之间的相关性及临床意义。[方法]取60例腰椎间盘突出症患者(退变组)和同期60例因创伤手术患者(未退变组)髓核组织。行组织化学染色。体外实验方面,分离细胞培养后,分为4组,分别为空白对照、TRAIL siRNA转染、TNF-α处理和TNF-α处理+TRAIL-siRNA转染组。采用MTT法检测细胞增殖,流式细胞仪检测细胞凋亡,Western blot法检测髓核细胞Caspase-3的活性和表达。[结果]Tunel染色显示凋亡阳性细胞率退变组为48.92%,未退变组为5.23%;TRAIL蛋白表达阳性率退变组为49.11%,未退变组为12.25%,两组间的差异均有统计学意义(P<0.05)。体外试验方面,相较于空白对照细胞,TNF-α处理细胞的增殖显著下降(P<0.05),而凋亡率显著升高(P<0.05),Caspase-3的表达显著升高(P<0.05)。TRAIL siRNA转染对细胞增殖、凋亡、Caspase-3的表达无明显影响(P>0.05),TNF-α处理后再用TRAIL-siRNA转染可显著逆转TNF-α对细胞增殖、凋亡和Caspase-3表现的影响(P<0.05)。[结论]髓核TRAIL阳性表达细胞率与凋亡细胞率呈正相关,TRAIL沉默能显著逆转TNF-α诱导的髓核细胞增殖抑制、细胞凋亡及Caspase-3的表达。

[Objective] To explore the relationship between TRAIL gene and lumbar degenerative disease. [Methods] The nucleus pulposus tissues were harvested from 60 patients who underwent discectomy for lumbar disc herniation(degeneration group), and 60 patients who received surgical treatment for spinal trauma(non-degeneration group) in the same period. Histochemical stains for cell apoptosis and TRAIL was compared between the degeneration and non-degeneration groups. In addition, a study was conducted in vitro. After the cells were separated from the degenerative nucleus pulposus tissue and cultured, they were divided into 4 groups, namely blank control group, TRAIL-siRNA transfection group, TNF-α treatment group, and TNF-α treatment + TRAIL-siRNA transfection group. MTT method was used to detect cell proliferation, flow cytometry was used to detect cell apoptosis, and Western blot method was used to detect the activity and expression of Caspase-3 in nucleus pulposus cells. [Results] The Tunel staining showed that the rate of apoptosis-positive cells was48.92% in the degeneration group, whereas 5.23% in the non-degeneration group;the positive rate of TRAIL protein expression was49.11% in the degeneration group, whereas 12.25% in the non-degeneration group, which all proved statistically significant(P<0.05). In term of experiments in vitro, the proliferation of TNF-α-treated cells was significantly decreased(P<0.05), while the apoptotic rate was significantly increased(P<0.05), and the expression of Caspase-3 was significantly increased compared with blank control cells(P<0.05).However, TRAIL-siRNA transfection had no significant effect on cell proliferation, apoptosis, and Caspase-3 expression(P>0.05).TRAIL-siRNA transfection after TNF-α treatment did significantly reverse the effects of TNF-α on cell proliferation, apoptosis and expression of Caspase-3(P<0.05). [Conclusion] The rate of TRAIL positive expression cells in the nucleus pulposus is positively correlated with the rate of apoptotic cells. TRAIL silencing does significantly reverse the TNF-α-induced nucleus pulposus cell proliferation inhibition,apoptosis and Caspase-3 expression.