本文關鍵詞：17β-雌二醇通過整聯(lián)蛋白α1β1和α2β1抗大鼠髓核細胞凋亡的機制研究　出處：《河北醫(yī)科大學》2014年碩士論文　論文類型：學位論文

  更多相關文章： 17β-雌二醇 腰椎退變 凋亡 整聯(lián)蛋白 膠原

【摘要】：目的：探索胞膜整聯(lián)蛋白α1β1、α2β1參與的17β-雌二醇抗大鼠腰椎間盤細胞凋亡的作用機制。 方法：酶消化法原代培養(yǎng)大鼠髓核細胞，貼壁細胞融合為單層時，用含EDTA的0.2%的胰酶消化傳代.細胞生長至第3代時，用不含胎牛血清、不含酚紅的DMEM/F12培養(yǎng)液培養(yǎng)，按以下分組實驗，每組均為6個樣本。對照組：加入少量乙醇作為對照；雌激素組：加入17β-雌二醇干預；雌激素+抑制劑組：加入17β-雌二醇和雌激素受體（ER）抑制劑ICI182780干預。細胞培養(yǎng)48小時后，應用：（1）免疫細胞化學法行II型膠原染色鑒定髓核細胞；（2）行流式細胞術以及TUNEL法檢測細胞凋亡；（3）細胞-膠原粘附實驗檢測細胞與I、II型膠原的粘附水平；（4）western印跡法對整聯(lián)蛋白亞基α1，α2，β1定量檢測。 結(jié)果：（1）原代腰椎髓核細胞呈多角形或長梭形，輪廓清楚，細胞核呈圓形或橢圓形，，胞質(zhì)內(nèi)可見分泌顆粒,24～48h細胞貼壁,7～8d細胞進入對數(shù)生長期,可傳6～8代,并分泌II型膠原。（2）免疫細胞化學檢測到髓核細胞II型膠原呈陽性，髓核細胞純度較高。（3）TUNEL法檢測到雌激素可以有效地降低髓核細胞凋亡發(fā)生率；流式細胞術檢測：髓核細胞早期凋亡率：對照組為4.00%0.16%；雌激素組為0.41%0.19%；雌激素+抑制劑組為3.20%0.05%。細胞晚期凋亡率：對照組為2.01%0.18%；雌激素組為0.50%0.10%；雌激素+抑制劑組為2.63%0.20%。各組細胞凋亡率的差異有統(tǒng)計學意義（F=24.20，P0.001）。雌激素組凋亡率低于對照組（P0.01）和雌激素+抑制劑組（P0.01），對照組與雌激素+抑制劑組差異無統(tǒng)計學意義。（4）細胞與II型膠原粘附實驗:各組OD值（吸光度）：對照組為0.600.03，95%CI(0.53,0.68)；雌激素組為0.730.04，95%CI(0.63,0.83)，雌激素+抑制劑組為0.550.07，95%CI(0.37,0.73)。各組細胞對I型膠原的粘附水平差異無統(tǒng)計學意義（P0.05），對II型膠原的粘附水平差異有統(tǒng)計學意義（F=10.68，P0.05），雌激素組高于對照組（P0.05）和雌激素+抑制劑組（P0.001），對照組與雌激素+抑制劑組差異無統(tǒng)計學意義。（5）整聯(lián)蛋白2亞基條帶的相對灰度值（/β-actin）：對照組為0.230.005，95%CI (0.22,0.24)；雌激素組為0.510.019，95%CI (0.46,0.55)；雌激素+抑制劑組為0.210.009，95%CI (0.18,0.23)。整聯(lián)蛋白β1亞基條帶的相對灰度值（/β-actin）：對照組為0.260.011，95%CI (0.24,0.29)；雌激素組為0.500.031，95%CI (0.43,0.58)；雌激素+抑制劑組為0.250.018，95%CI (0.20,0.29)。整聯(lián)蛋白α1亞基表達水平差異無統(tǒng)計學意義（P0.05）。α2和β1亞基表達水平均有統(tǒng)計學差異，雌激素組高于對照組（P0.01）和雌激素+抑制劑組（P0.01），對照組與雌激素+抑制劑組無統(tǒng)計學差異。 結(jié)論：17β-雌二醇抗大鼠髓核細胞凋亡，其作用機制為上調(diào)了整聯(lián)蛋白α2β1的表達，進而增強了細胞與胞外II型膠原的粘附作用。
[Abstract]:Aim: to investigate the mechanism of 17 尾 -estradiol (17 尾 -estradiol) involved in cytoskeletal integrin 偽 1 尾 1 and 偽 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 偽 1, 偽 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 偽 1 subunit. The expression levels of 偽 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 偽 _ 2 尾 _ 1 and enhance the adhesion of the cells to extracellular type II collagen.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R965

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