17β-雌二醇通过整联蛋白α1β1和α2β1抗大鼠髓核细胞凋亡的机制研究

发布时间：2018-01-18 11:34

本文关键词：17β-雌二醇通过整联蛋白α1β1和α2β1抗大鼠髓核细胞凋亡的机制研究　出处：《河北医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：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细胞凋亡的作用机制。方法：酶消化法原代培养大鼠髓核细胞，贴壁细胞融合为单层时，用含EDTA的0.2%的胰酶消化传代.细胞生长至第3代时，用不含胎牛血清、不含酚红的DMEM/F12培养液培养，按以下分组实验，每组均为6个样本。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（ER）抑制剂ICI182780干预。细胞培养48小时后，应用：（1）免疫细胞化学法行II型胶原染色鉴定髓核细胞；（2）行流式细胞术以及TUNEL法检测细胞凋亡；（3）细胞-胶原粘附实验检测细胞与I、II型胶原的粘附水平；（4）western印迹法对整联蛋白亚基α1，α2，β1定量检测。结果：（1）原代腰椎髓核细胞呈多角形或长梭形，轮廓清楚，细胞核呈圆形或椭圆形，，胞质内可见分泌颗粒,24～48h细胞贴壁,7～8d细胞进入对数生长期,可传6～8代,并分泌II型胶原。（2）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。（3）TUNEL法检测到雌激素可以有效地降低髓核细胞凋亡发生率；流式细胞术检测：髓核细胞早期凋亡率：对照组为4.00%0.16%；雌激素组为0.41%0.19%；雌激素+抑制剂组为3.20%0.05%。细胞晚期凋亡率：对照组为2.01%0.18%；雌激素组为0.50%0.10%；雌激素+抑制剂组为2.63%0.20%。各组细胞凋亡率的差异有统计学意义（F=24.20，P0.001）。雌激素组凋亡率低于对照组（P0.01）和雌激素+抑制剂组（P0.01），对照组与雌激素+抑制剂组差异无统计学意义。（4）细胞与II型胶原粘附实验:各组OD值（吸光度）：对照组为0.600.03，95%CI(0.53,0.68)；雌激素组为0.730.04，95%CI(0.63,0.83)，雌激素+抑制剂组为0.550.07，95%CI(0.37,0.73)。各组细胞对I型胶原的粘附水平差异无统计学意义（P0.05），对II型胶原的粘附水平差异有统计学意义（F=10.68，P0.05），雌激素组高于对照组（P0.05）和雌激素+抑制剂组（P0.001），对照组与雌激素+抑制剂组差异无统计学意义。（5）整联蛋白2亚基条带的相对灰度值（/β-actin）：对照组为0.230.005，95%CI (0.22,0.24)；雌激素组为0.510.019，95%CI (0.46,0.55)；雌激素+抑制剂组为0.210.009，95%CI (0.18,0.23)。整联蛋白β1亚基条带的相对灰度值（/β-actin）：对照组为0.260.011，95%CI (0.24,0.29)；雌激素组为0.500.031，95%CI (0.43,0.58)；雌激素+抑制剂组为0.250.018，95%CI (0.20,0.29)。整联蛋白α1亚基表达水平差异无统计学意义（P0.05）。α2和β1亚基表达水平均有统计学差异，雌激素组高于对照组（P0.01）和雌激素+抑制剂组（P0.01），对照组与雌激素+抑制剂组无统计学差异。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型胶原的粘附作用。
[Abstract]:Aim: to investigate the mechanism of 17 尾 -estradiol (17 尾 -estradiol) involved in cytoskeletal integrin 伪 1 尾 1 and 伪 2 尾 1 against apoptosis of rat lumbar disc cells. Methods: the primary cultured rat nucleus pulposus cells were cultured by enzyme digestion. When the adherent cells were fused into monolayers, the cells were digested and subcultured with 0.2% trypsin containing EDTA. When the cells grew to the third generation, they were treated with fetal bovine serum. The DMEM/F12 medium without phenolic red was cultured in the following groups: each group was divided into 6 samples. Control group: a small amount of ethanol was added as control group. Estrogen group: 17 尾 -estradiol was added; Estrogen inhibitor group: ICI182780 (17 尾 -estradiol and estrogen receptor) inhibitor ICI182780 was added. The cells were cultured for 48 hours. Type II collagen staining was used to identify the nucleus pulposus cells by immunocytochemistry. (2) flow cytometry and TUNEL assay were used to detect apoptosis. (3) Cell-collagen adhesion assay was used to detect the adhesion level of cells to type II collagen. The integrin subunits 伪 1, 伪 2, 尾 1 were detected quantitatively by western blotting. Results (1) the primary lumbar spinal nucleus cells were polygonal or fusiform, with clear outline, round or elliptical nucleus, and the secretory granules could be seen in the cytoplasm for 48 hours. At 7 ~ 8 days, the cells entered logarithmic growth stage, which could be passed through 6 to 8 passages and secreted type II collagen. (2) Immunocytochemistry showed that type II collagen was positive in nucleus pulposus cells. The high purity of nucleus pulposus cells detected by Tunel method showed that estrogen could effectively reduce the incidence of apoptosis of nucleus pulposus cells. Flow cytometry: early apoptosis rate of nucleus pulposus cells: 4.000.16 in control group; The estrogen group was 0.41 and 0.19. The rate of late cell apoptosis in the estrogen inhibitor group was 3.200.05.The rate of late apoptosis in the control group was 2.010.18. The estrogen group was 0.50 and 0.10. The percentage of apoptosis in estrogen inhibitor group was 2.63 0.20.The difference of apoptosis rate in each group was statistically significant (P < 0.05). The apoptosis rate of estrogen group was lower than that of control group (P 0.01) and estrogen inhibitor group (P 0.01). There was no significant difference between the control group and estrogen inhibitor group (P < 0.05). The adhesion test of cells to collagen II: OD value of each group (absorbance: 0.600.03). (95) CII 0.53 (0.68); The estrogen group was 0.730.04 / 95 and the estrogen inhibitor group was 0.550.07 / 95 / 0.37. There was no significant difference in the adhesion level of the cells to type I collagen in each group (P 0.05), but there was significant difference in the adhesion level of type II collagen in each group (P < 0.05). P0.05, estrogen group was higher than control group (P0.05) and estrogen inhibitor group (P0.001). There was no significant difference between the control group and the estrogen inhibitor group. 5) the relative gray value of the integrin 2 subunit band was 0.230.005 in the control group and 0.230.005 in the control group. 95 CI 0.22 ~ 0.24; The estrogen group was 0.510.01995% CI 0.46U 0.55; The estrogen inhibitor group was 0.210.009% CI 0.18. The relative gray value of integrin 尾 1 subunit band was 0.260.01195 CI 0.240.29 in the control group. The estrogen group was 0.500.031 and 95% CI 0.43 ~ 0.58; The estrogen inhibitor group was 0.250.01895% CI 0.20. There was no significant difference in the expression level of integrin 伪 1 subunit. The expression levels of 伪 2 and 尾 1 subunits were significantly different. Estrogen group was higher than control group (P 0.01) and estrogen inhibitor group (P 0.01). There was no significant difference between control group and estrogen inhibitor group. Conclusion the anti-apoptotic mechanism of W17 尾 -estradiol on rat nucleus pulposus cells is to up-regulate the expression of integrin 伪 _ 2 尾 _ 1 and enhance the adhesion of the cells to extracellular type II collagen.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965

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