摘要
目的研究水通道蛋白3(aquaporin-3,AQP-3)在高渗透压诱导的大鼠髓核细胞(rat nucleus pulposus)凋亡中的作用。方法体外培养的大鼠髓核细胞,按330、550 mOsm/kg的渗透压分组,CCK-8检测髓核细胞增殖,流式细胞分析细胞凋亡率,Western blot和定量PCR检测凋亡相关分子Bcl-2、Bax和caspase3表达情况;使用免疫细胞化学、Western blot和定量PCR检测AQP-3的表达。利用过表达慢病毒提高AQP-3的表达,进一步观察AQP-3对高渗环境诱导凋亡的影响。结果相比于330 mOsm/kg渗透压,550 mOsm/kg高渗环境中髓核细胞培养12、24、48、72 h细胞增殖显著降低,髓核细胞凋亡率显著增加(P<0.05),蛋白和mRNA表达结果显示Bcl-2表达明显降低(P<0.05),Bax和cleaved caspase 3/caspase 3表达明显增加(P<0.05)。相比于高渗环境中的空白对照髓核细胞,AQP-3过表达髓核细胞的凋亡率显著降低(P<0.05),蛋白和mRNA表达结果显示Bcl-2表达明显增加(P<0.05),Bax和cleaved caspase 3/caspase 3表达明显降低(P<0.05)。结论高渗透压显著促进髓核细胞的凋亡并降低AQP-3的表达;过表达AQP-3可缓解高渗透压环境下髓核细胞凋亡。
Objective To investigate the role of aquaporin-3(AQP-3)in hyperosmolarity-induced apoptosis of cultured rat nucleus pulposus(NP)cells.Methods Rat NP cells were cultured under different osmotic pressures(330 and 550 mOsm/kg),and the proliferation and apoptotic rate of the cells were determined using CCK-8 assay and flow cytometry,respectively.Western blotting and real-time quantitative PCR were used to detect the expression levels of apoptosis-related molecules(Bcl-2,Bax,cleaved caspase 3/caspase 3),and immunocytochemistry,Western blotting and real-time quantitative PCR were used to detect the expression of AQP-3 in the cells.The effect of lentivirus-mediated AQP-3 overexpression on apoptosis of NP cells was investigated after hyperosmolar exposure.Results An osmotic pressure of 550 mOsm/kg,as compared with 330 mOsm/kg,significantly decreased NP cell proliferation,increased the cell apoptotic rate,upregulated the expression of the pro-apoptotic molecules Bax and cleaved caspase 3/caspase 3(P<0.05),and down-regulated the expression of the anti-apoptotic molecule Bcl-2(P<0.05).The cells with lentivirus-mediated AQP-3 overexpression showed an obviously lowered apoptotic rate following the hyperosmolar exposure and significantly reduced expression of Bax and cleaved caspase 3/caspase 3(P<0.05)with an enhanced expression of Bcl-2(P<0.05).Conclusion Hyperosmolar exposure significantly promotes apoptosis of NP cells and reduce the expression of AQP-3.AQP-3 overexpression can partly attenuate apoptosis of NP cells following a hyperosmolar exposure.
作者
张泽桐
房朗
甘翼博
李培
叶吉星
赵晨
周强
ZHANG Zetong;FANG Lang;GAN Yibo;LI Pei;YE Jixing;ZHAO Chen;ZHOU Qiang(Department of Orthopedics,First Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400038;Department of Orthopedics,the Third Affiliated Hospital of Chongqing Medical University,Chongqing,401120,China)
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2020年第9期915-922,共8页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(81772378)
陆军军医大学第一附属医院基础研究军民融合项目(SWH2017YBXM-20)。
关键词
水通道蛋白
渗透压
凋亡
髓核细胞
椎间盘退变
aquaporin
osmolarity
apoptosis
nucleus pulposus
intervertebral disc degeneration
