二氮嗪通过抑制内质网应激作用降低软骨细胞凋亡的研究  

Diazoxide prevents rat chondrocyte apoptosis via Inhibition of endoplasmic reticulum stress

作　　者：顾运涛 陈克伟[1] 卞阳阳 傅鉴 袁伟[2] 刑孔明 彭磊[1] GU Yun-tao;CHEN Ke-wei;BIAN Yang-yang;et al(Department of Orthopedics, the Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, Chin)

机构地区：[1]温州医科大学附属第二医院育英儿童医院骨科,浙江温州325000 [2]海南医学院第一附属医院创伤外科中心,海南海口570102

出　　处：《中华全科医学》2018年第1期14-18,37,共6页Chinese Journal of General Practice

基　　金：国家自然科学基金(81460339);国际合作专项(GJXM201102);海南省卫生厅项目(琼卫2014重点-06号);海南省卫生厅项目(琼卫2013重点-06号);海南省科技厅项目(SF201416);浙江省卫生厅项目(2009B-107);海南省重点科技计划项目(ZDXM-20110051)

摘　　要：目的探讨二氮嗪对H_2O_2诱导的软骨细胞凋亡的机制研究。方法每组取3只SD大鼠膝关节软骨细胞进行原代培养,将软骨细胞分成6组,分别为对照组(A组),H_2O_2损伤组(B组),H_2O_2+100μmol/L二氮嗪组(C组),H_2O_2+200μmol/L二氮嗪组(D组),H_2O_2+300μmol/L二氮嗪组(E组),H_2O_2+400μmol/L二氮嗪组(F组)。A组细胞不做特殊处理,B组用400μmol/L双氧水在37℃恒温箱内孵育8 h,C、D、E、F组分别用100、200、300、400μmol/L的二氮嗪在37℃的恒温箱内预处理30 min,再用400μmol/L的H_2O_2孵育8 h,用CCK8法检测各组软骨细胞的活性,用流式细胞仪检测各组软骨细胞凋亡情况,用Rt PCR检测软骨细胞功能情况,用免疫荧光、Western Blot检测凋亡和内质网应激相关蛋白的表达情况。结果 (1)CCK8法检测各组细胞的活性率从大至小依次为A组>E组>D组>F组>B组;(2)流式细胞仪检测各组软骨细胞凋亡率由大至小依次为B组>F组>D组>E组>A组;(3)Rt PCR检测软骨细胞二型胶原蛋白(Coll-Ⅱ)、Caspase-3和聚集蛋白聚糖酶(aggrecanase)表达量,表达量大至小依次为A组>E组>D组>F组>B组;各组聚集蛋白聚糖酶表达量大至小依次为B组>F组>D组>E组>A组;(4)免疫荧光检测软骨细胞中CHOP蛋白的表达量,各组表达量依次是B组>F组>A组;(5)Western bolt检测各组软骨细胞中Caspase-3、Bax、CHOP蛋白的表达情况,表达量依次为B组>F组>A组。结论二氮嗪通过抑制内质网应激作用,从而减少H_2O_2诱导的大鼠软骨细胞的凋亡。Objective To research the mechanism of diazoxide on hydrogen peroxide induced chondrocyte apoptosis. Methods We obtain the carilage cells from the 3 SD rat articular cartilages. The cartilage cells were divided into six groups, namely the control group（A）, the H2O2 -injured group（B）, H2O2 ＋ 100 μmol/L DZ group（C）, H2O2 ＋ 200μmol/L DZ group（D）, H2O2 ＋300μmol/L DZ group（E）, H2O2 ＋400μmol/L（F）. Group A had no special treat- ment, group B incubated with 400μmol/L H202 for 8 hours in 37℃ incubator, the C, D, E group were pre-incubated with 100, 200, 300,400μmol/L DZ in a 37℃ incubator incubated 30 minutes, then incubated with 400μmol/L H2O2 solution in 37℃ incubator. The activity of chondrocytes in group A, B, C, D, E and F were detected by CCK8 method. The apoptosis of ehondrocytes were detected by flow cytometry. The function of chondrocytes were detected by RT-PCR and immunofluorescence, Western blot were used to detect the expression of endoplasmic reticulum stress proteins. Results （1）Using the CCK8 assay, the order of the rate of cells activity ： group A 〉 group E 〉 group D 〉 group F 〉 group B. （2）The apoptosis rates of ceils were detected by Flow eytometer. The order of apoptosis rates of chondrocyte as follow： group B 〉 group F 〉 group D 〉 group E 〉 group A. （3）The Coilil protein expression of chondrocyte were detected by RT- PCR analysis, as follow ： group A 〉 group E 〉 group D 〉 group F 〉 group B. However, the aggrecanase expression Contrary to Coll- Ⅱ. （4）the expression of CHOP in each chondrocytes group detected by immunofiuorescence, as follow： group B 〉 group F 〉 group A. （5）The Caspase-3, Bax, CHOP protein expression of chondrocyte were detected by Westernblot analysis, as follow ： group B 〉 group F 〉 group A. Conclusion Diazoxide prevents H2O2-induced rat chondrocyte apoptosis via inhibition of endoplasmic reticulum stress.

关 键 词：二氮嗪 软骨细胞 内质网应激 凋亡 

分 类 号：R684.3[医药卫生—骨科学] R329.25[医药卫生—外科学]