Objective To explore the mechanism of 17β-estradiol in protecting rat nucleus pulposus (NP) cell from apoptosis via α11β1 and α2β1 integrins.Methods Rat NP cells were primarily cultured and the third generation were cultured in complete DMEM/F12 without fetal bovine serum (FBS) and phenol red.Then experiments were performed in three groups as follows:control group administrated with ethanol,estrogen group administrated with 17β-estradiol and estrogen plus inhibitor group administrated with 17β-estradiol and ICI 182780.Forty-eight hours later,cell apoptosis was determined by flow cytometry and TUNEL assay; cell attachment to type Ⅰ and Ⅱ collagen was detected by cell adhesion assay; and the expression levels of subunits (α1,α2,β1) of integrins were determined by western blotting.Results The immunocytochemistry examination displayed that the cultured NP cells had a high level of purity.There were statistically significant differences in cell apoptosis between groups (F=24.20,P＜0.001).The apoptotic rate in estrogen group was lower than those in control group and estrogen plus inhibitor group.There was no statistical difference in apoptotic rate between control group and estrogen plus inhibitor group.There was no statistical difference in cell adhesion to type Ⅰ collagen between groups,while a statistical difference was found in cell adhesion to type Ⅱ collagen between groups (F=10.68,P＜0.05).The OD value in estrogen group was significantly higher than those in control group and estrogen plus inhibitor group.There was no statistical difference in expression level of αl subunit,while the expression levels of α2 and β1 subunits were higher in estrogen group than those in control group and estrogen plus inhibitor group.Conclusion The up-regulated expression of integrin α2β1 maybe the mechanism of 17β-estradiol in protecting rat NP cells from apoptosis.Moreover,the integrin α2β1 can enhance cell attachment to type Ⅱ collagen.%目的 探索胞膜整联蛋白α1β1、α2β1参与的17β-雌二醇抗大鼠腰椎间盘髓核细胞凋亡的作用机制.方法 原代培养大鼠髓核细胞,细胞传代至第3代,用不含胎牛血清和酚红的培养液培养,按以下分组实验:对照组,加入少量乙醇作为对照；雌激素组,加入17β-雌二醇干预；雌激素+抑制剂组,加入17β-雌二醇和雌激素受体抑制剂ICI 182780干预.细胞培养48 h后,行流式细胞术以及TUNEL法检测细胞凋亡；细胞-胶原黏附实验检测细胞与Ⅰ、Ⅱ型胶原的粘附水平；western印迹法对整联蛋白亚基α1、α2、β1定量检测.结果 免疫细胞化学检测到髓核细胞Ⅱ型胶原呈阳性,髓核细胞纯度较高.各组细胞凋亡率的差异有统计学意义(F=24.20,P＜0.001).雌激素组凋亡率低于对照组和雌激素+抑制剂组,对照组与雌激素+抑制剂组差异无统计学意义.各组细胞对Ⅰ型胶原的黏附水平差异无统计学意义,对Ⅱ型胶原的黏附水平差异有统计学意义(F=10.68,P＜0.05),雌激素组高于对照组和雌激素+抑制剂组,对照组与雌激素+抑制剂组差异无统计学意义.整联蛋白α1亚基表达水平差异无统计学意义.α2和β1亚基表达水平差异均有统计学意义,雌激素组高于对照组和雌激素+抑制剂组,对照组与雌激素+抑制剂组差异无统计学意义.结论 17β-雌二醇抗大鼠髓核细胞凋亡,其作用机制为上调了整联蛋白α2β1的表达,进而增强了细胞与胞外Ⅱ型胶原的黏附作用.
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