摘要
目的探讨缺氧条件下大鼠髓核细胞中缺氧诱导因子1α(hypoxia inducible factor 1α,HIF-1α)与自噬相关分子Beclin1、LC3B的表达及其相关性。方法取健康成年SD大鼠髓核组织,分离培养髓核细胞并传代。取第3代髓核细胞行HE染色和Ⅱ型胶原酶免疫荧光染色鉴定后,随机分为4组。A组细胞于常氧条件下(37℃、5%CO2、20%O2)培养,B组细胞于缺氧条件下(37℃、5%CO2、1%O2)培养,C组细胞转染HIF-1α-小干扰RNA后于缺氧条件下培养,D组细胞加入自噬抑制剂3-MA后于缺氧条件下培养。各组细胞培养8 h后,采用Western blot和实时荧光定量PCR(real-time fluorescence quantitative PCR,qRT-PCR)检测HIF-1α与自噬相关分子Beclin1、LC3B的表达情况。结果经分离纯化的第3代大鼠髓核细胞HE染色后细胞质呈淡粉色,细胞核呈蓝黑色;Ⅱ型胶原酶免疫荧光染色为阳性。Western blot和qRT-PCR检测示,B组HIF-1α、Beclin1和LC3B蛋白及mRNA相对表达量均显著高于A组(P<0.05),C组均显著低于B组,差异均有统计学意义(P<0.05)。D组HIF-1α蛋白和mRNA相对表达量与B组比较差异无统计学意义(P>0.05),Beclin1和LC3B蛋白和mRNA相对表达量均较B组显著降低(P<0.05)。结论缺氧条件能诱导大鼠髓核细胞中HIF-1α和自噬相关分子Beclin1、LC3B的表达,且HIF-1α与自噬相关分子的表达具有相关性,即HIF-1α下调能降低自噬相关分子的表达,而缺氧条件下自噬水平下调对HIF-1α的表达无明显影响。
Objective To investigate the expression and correlation of hypoxia inducible factor 1α(HIF-1α)and autophagy related molecules(Beclin1 and LC3 B)in rat nucleus pulposus cells under hypoxia in vitro.Methods The nucleus pulposus cells were extracted from the nucleus pulposus of healthy adult Sprague Dawley rats and passaged.The3 rd generation cells were identified by HE staining and collagenase typeⅡimmunofluorescence staining and randomly divided into 4 groups.The cells in group A were cultured for 8 hours under normal oxygen condition(37℃,5%CO2,20%O2);the cells in group B were cultured for 8 hours under hypoxia condition(37℃,5%CO2,1%O2);the cells in group C were transfected with HIF-1α-small interfering RNA and cultured for 8 hours under hypoxia condition;and the cells in group D were cultured with autophagy inhibitor 3-MA for 8 hours under hypoxia condition.Western blot and real-time fluorescence quantitative PCR(qRT-PCR)were used to detect the expressions of HIF-1αand autophagy related molecules(Beclin1 and LC3 B)in all groups.Results HE staining of the 3 rd generation nucleus pulposus cells showed that the cytoplasm was light pink and the nucleus was blue black,and the collagenase typeⅡimmunofluorescence staining was positive.Western blot and qRT-PCR results showed that the relative expressions of HIF-1α,Beclin1,and LC3 B proteins and genes in group B were significantly higher than those in group A(P<0.05);the relative expressions of HIF-1α,Beclin1,and LC3 B proteins and genes in group C were significantly lower than those in group B(P<0.05).There was no significant difference in the relative expression of HIF-1αprotein and gene between groups B and D(P>0.05);while the relative expressions of Beclin1 and LC3 B proteins and genes in group D were significant lower than those in group B(P<0.05).Conclusion Hypoxia can induce the expressions of HIF-1αand autophagy related molecules(Beclin1 and LC3 B)in rat nucleus pulposus cells,and HIF-1αin rat nucleus pulposus cells under hypoxia is related to the expression of autophagy related molecules,that is,down-regulation of HIF-1αcan significantly reduce the expression of autophagy related molecules,while the down-regulation of autophagy levels under hypoxia has no or little effect on the expression of HIF-1α.
作者
程傲
何鹏杰
张俊宇
郑伟伟
杨民
CHENG Ao;HE Pengjie;ZHANG Junyu;ZHENG Weiwei;YANG Min(Department of Orthopedics and Traum atology,Yijishan Hospital Affiliated to Wannan Medical College,Wuhu Anhui,241001,P.R.C hina)
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2020年第3期318-322,共5页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家自然科学基金资助项目(81341054、81171732)
皖南医学院人才引进基金(YR201411)。
关键词
髓核细胞
HIF-1Α
自噬
缺氧
大鼠
Nucleus pulposus cells
hypoxia inducible factor 1α
autophagy
hypoxia
rat
作者简介
通信作者:杨民,Email:pkuyang@hotmail.com。
