血清饥饿对大鼠椎间盘髓核细胞凋亡的影响及其机制研究

Effects of serum-free culture on apoptosis in intervertebral disc nucleus pulposus cells of rat and its primary mechanism

目的研究血清饥饿对大鼠椎间盘髓核细胞凋亡的影响及其可能机制。方法培养大鼠原代腰椎间盘髓核细胞,取2代细胞进行实验,分为血清饥饿组和正常对照组。血清饥饿组培养基为DMEM,无血清培养24 h。正常对照组培养基为10%胎牛血清+DMEM,余培养条件同血清饥饿组。CCK-8(Cell Counting Kit-8)检测细胞活性;脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(terminal deoxynucleotidyl transferase mediated d UTP nick end labeling,TUNEL)检测细胞凋亡;免疫细胞化学法检测p38MAPK、cleaved Caspase-3蛋白表达;RT-PCR(reverse transcription-PCR)检测Bcl-2 mRNA、Caspase-3 mRNA、p38MAPK mRNA的表达。结果血清饥饿组与对照组比较:1细胞活性明显下降(P<0.01);2凋亡率明显增高(P<0.01);3p38MAPK蛋白阳性表达明显增强,Caspase-3蛋白阳性表达明显增强;4Bcl-2 mRNA表达明显降低,Caspase-3 mRNA表达明显升高,p38MAPK mRNA表达明显升高(P<0.01)。结论血清饥饿培养可诱导大鼠腰椎间盘髓核细胞发生凋亡,其发生机制可能与p38MAPK通路下调Bcl-2,激活Caspase-3有关。

Objective To investigate the effects of serum-free culture on the apoptosis in intervertebral disc nucleus pulposus cells of rat and its primary mechanism. Methods The intervertebral disc nucleus pulposus cells of second generation from SD rats were randomly divided into the starvation group and the control group. The cells of the control group were culture in DMEM contain 10% serum while starvation group in Serum-free DMEM for 24 h. The cells survival were detected by CCK-8（ Cell Counting Kit-8）; The cells apoptosis was detected by TUNEL（ terminal deoxynucleotidyl transferase mediated d UTP nick end labeling）; The expression of p38 MAPK protein and cleaved Caspase-3 protein was observed by immunocytochemistry; the changes of Bcl-2 mRNA,Caspase-3 mRNA and p38 MAPK mRNA was measured by RT-PCR（ reverse transcription-polymerase chain reaction）. Results Compared with control group,the starvation group shows： 1Cells survival was significantly decreased（ P 〈 0. 01）; 2rate of cells apoptosis was significantly increased（ P 〈 0. 01）; 3The expression of p38 MAPK and cleaved Caspase-3 protein was significantly increased; 4Bcl-2 mRNA was significantly decreased; Caspase-3 mRNA and p38 MAPK mRNA was significantly increased（ P 〈 0. 01）. Conclusion Serum-free culture may lead to the apoptosis of intervertebral disc nucleus pulposus cells of rat and its mechanism is related to the downregulation of Bcl-2 and activation of caspase 3 through the p38 MAPK pathway.