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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
引用本文:顾运涛,陈克伟,卞阳阳,傅鉴,袁伟,刑孔明,彭磊.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[J].中华全科医学,2018,16(1):14.
作者姓名:顾运涛  陈克伟  卞阳阳  傅鉴  袁伟  刑孔明  彭磊
作者单位: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;
基金项目:海南省重点科技计划项目(ZDXM-20110051)国际合作专项(GJXM201102)浙江省卫生厅项目(2009B-107)国家自然科学基金(81460339)海南省卫生厅项目(琼卫2013重点-06号)海南省科技厅项目(SF201416)海南省卫生厅项目(琼卫2014重点-06号)
摘    要:目的 探讨二氮嗪对H2O2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),H2O2损伤组(B组),H2O2+100 μmol/L二氮嗪组(C组),H2O2+200 μmol/L二氮嗪组(D组),H2O2+300 μmol/L二氮嗪组(E组),H2O2+400 μmol/L二氮嗪组(F组)。A组细胞不做特殊处理,B组用400 μmol/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、400 μmol/L的二氮嗪在37℃的恒温箱内预处理30 min,再用400 μmol/L的H2O2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RtPCR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 ①CCK8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;③RtPCR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;⑤Western 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H2O2诱导的大鼠软骨细胞的凋亡。 

关 键 词:二氮嗪    软骨细胞    内质网应激    凋亡
收稿时间:2017-05-02

Diazoxide prevents rat chondrocyte apoptosis via Inhibition of endoplasmic reticulum stress
Institution:Department of Orthopedics, the Second Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, China
Abstract: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 + 200 μmol/L DZ group(D), H2O2 + 300 μmol/L DZ group(E), H2O2 + 400 μmol/L(F). Group A had no special treatment, group B incubated with 400 μmol/L H2O2 for 8 hours in 37℃ incubator, the C, D, E group were pre-incubated with 100, 200, 300, 400 μmol/L DZ in a 37℃ incubator incubated 30 minutes, then incubated with 400 μmol/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 chondrocytes 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 ①Using the CCK8 assay, the order of the rate of cells activity:group A > group E > group D > group F > group B. ②The apoptosis rates of cells were detected by Flow cytometer. The order of apoptosis rates of chondrocyte as follow:group B > group F > group D > group E > group A. ③The Coll-Ⅱ protein expression of chondrocyte were detected by RTPCR analysis, as follow:group A > group E > group D > group F > group B. However, the aggrecanase expression Contrary to Coll-Ⅱ. ④the expression of CHOP in each chondrocytes group detected by immunofluorescence, as follow:group B > group F > group A. ⑤The 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. 
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