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

Expression and significance of hypoxia-inducible factor 1α in endplate chondrocytes of rats

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。

Objective To explore the expression and significance ofhypoxia-inducible factor 1α （HIF-1α） in endplate chondrocytes, and to study the relations between HIF-1α expression and endplate chondrocytes apoptosis. Methods Eight Sprague Dawley rats were selected to obtain the L1.5 intervertebral disc endplate; the endplate chondrocytes were isolated by enzyme digestion method, and the endplate chondrocytes at passage 3 were cultured under 20% O2 condition （group A）, and under 0.5% O2 condition （group B）. Cell morphology was observed by inverted phasecontrast microscope and cell apoptosis was detected using flow cytometry after cultured for 24 hours; the mRNA expression of HIF-la was detected by real-time fluorescent quantitative PCR, the protein expressions of HIF-l a, Bax, and Bcl-2 by Western blot. Gene clone technology to design and synthesize two siRNAs based on the sequence of HIF-lct mRNA. HIF-lct specific RNAi sequence compound was constructed and transfected into cells. The transfected endplatechondrocytes at passage 3 were cultured under 0.5% O2 condition in group C and group D （HIF-lu gene was silenced）. After cultured for 24 hours, cells were observed via immunofluorescence staining of HIF-la, and cell apoptosis was detected using flow cytometry. Meanwhile, the mRNA expressions of HIF-lu, collagen type II （COL II）, Aggrecan, and SOX9 were detected by real-time fluorescent quantitative PCR, and the protein expressions of HIF-lct, Bax, and Bcl-2 by Western blot. Results At 24 hours after culture, small amount of vacuoles necrotic cells could be observed in group Aand group B; there was no significant difference in apoptosis rate between groups A and B （t=1.026, P=0.471）, and HIF-la mRNA and protein expressions in group B were significantly higher than those in group A （t=22.672, P=0.015; t=18.396, P=0.013）, but, there was no significant difference in protein expressions of Bax and Bcl-2 between groups A and B （t=0.594, P=0.781; t= 1.251, P=0.342）. The number of vacuolar necrosis cells in group D was significantly higher than that in group C, and HIF-1 a positive cells were observed in group D. The apoptosis rate of group D was significantly higher than that of group C （t=27.143, P=0.002）. The mRNA expressions of HIF-1α, COL II, Aggrecan, and SOX9 in group D were significantly lower than those in group C （t=21.097, P=0.015; t=34.829, P=O.002; t=18.673, P=0.022; t=31.949, P=0.007）. The protein expressions of HIF-1α and Bcl-2 in group D were significantly lower than those in group C （t=37.648, P=0.006; t=16.729, P=0.036）, but the protein expression of Bax in group D was significantly higher than that in group C （t=25.583, P=0.011）. Conclusion HIF-1α mRNA expression is up-regulated under hypoxia condition, which will increase the hypoxia tolerance of endplate chondrocytes. Cell apoptosis is suppressed by the activation of HIF-1α in endplate chondrocytes under hypoxia condition.