低氧诱导因子1α基因在大鼠终板软骨细胞中的表达及意义研究

发布时间：2018-03-12 12:39

  本文选题：低氧诱导因子α　切入点：终板软骨细胞　出处：《中国修复重建外科杂志》2017年03期 　论文类型：期刊论文

【摘要】：目的探讨低氧诱导因子1α(hypoxia-inducible factor 1α,HIF-1α)基因在大鼠终板软骨细胞中的表达情况及其与终板软骨细胞凋亡的关系。方法取8只10周龄SPF级雄性SD大鼠L1~5腰椎终板软骨组织,采用酶消化法分离培养终板软骨细胞并传代,取第3代细胞进行实验。首先将终板软骨细胞分为两组,分别于20%O_2常氧条件下(A组)以及0.5%O_2低氧下条件(B组)培养;培养24 h,倒置相差显微镜观察细胞形态,流式细胞仪检测细胞凋亡率,实时荧光定量PCR检测HIF-1α基因表达,Western blot检测凋亡蛋白HIF-1α、Bax、Bcl-2表达。然后,构建Lipofectamin~(TM)2000载体试剂和HIF-1αsi RNA/Lipofectamin~(TM)2000载体混合剂分别转染终板软骨细胞后,于0.5%O_2低氧条件下培养(分别为C、D组)。培养24 h后,倒置相差显微镜下观察细胞形态,行HIF-1α免疫荧光染色观察,流式细胞仪检测细胞凋亡率,实时荧光定量PCR检测HIF-1α、Ⅱ型胶原、蛋白多糖、SOX9基因表达,Western blot检测凋亡蛋白HIF-1α、Bax、Bcl-2表达。结果培养24 h后,A、B组均见少量空泡坏死细胞;细胞凋亡率比较差异无统计学意义(t=1.026,P=0.471);B组HIF-1α基因及蛋白相对表达量显著高于A组(t=22.672,P=0.015;t=18.396,P=0.013);两组Bax、Bcl-2蛋白表达比较,差异均无统计学意义(t=0.594,P=0.781;t=1.251,P=0.342)。低氧培养24 h后,D组空泡坏死细胞较C组增多,且可见大量HIF-1α阳性终板软骨细胞;与C组相比,D组细胞凋亡率显著增高(t=27.143,P=0.002),D组HIF-1α、Ⅱ型胶原、蛋白多糖、SOX9基因表达较C组显著降低(t=21.097,P=0.015;t=34.829,P=0.002;t=18.673,P=0.022;t=31.949,P=0.007),HIF-1α、Bcl-2蛋白相对表达量较C组均显著降低(t=37.648,P=0.006;t=16.729,P=0.036),而Bax蛋白表达较C组提高(t=25.583,P=0.011)。结论缺氧条件下,终板软骨细胞中HIF-1α表达上调,以提高细胞耐氧性;HIF-1α可抑制终板软骨细胞在低氧环境中发生凋亡。
[Abstract]:Objective to investigate the expression of hypoxia-inducible factor 1 伪 (HIF-1 伪) gene in rat endplate chondrocytes and its relationship with endplate chondrocyte apoptosis. The endplate chondrocytes were isolated and subcultured by enzyme digestion method. The third passage of chondrocytes was carried out. Firstly, the endplate chondrocytes were divided into two groups: group A (group A) and group B (group B under hypoxia). After 24 h culture, cell morphology was observed by inverted phase contrast microscope, apoptosis rate was detected by flow cytometry, HIF-1 伪 gene expression was detected by real-time fluorescence quantitative PCR and the apoptotic protein HIF-1 伪 Baxbumin Bcl-2 was detected by Western blot. Lipofectamin~(TM)2000 vector reagent and HIF-1 伪 si RNA/Lipofectamin~(TM)2000 vector mixture were constructed and transfected into endplate chondrocytes respectively, and cultured under 0. 5% O\ -2 hypoxia (group C ~ (2) D respectively). After 24 h culture, cell morphology was observed under inverted phase contrast microscope and observed by HIF-1 伪 immunofluorescence staining. Apoptosis rate was detected by flow cytometry, HIF-1 伪, type 鈪，

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