不同浓度17β-雌二醇对IL-1β诱导的大鼠纤维环细胞凋亡的影响

发布时间：2018-03-16 02:10

本文选题：17β-雌二醇　切入点：IL-1β　出处：《河北医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:椎间盘退行性病变是指随着年龄的增长,椎间盘各组织发生的老化退变。是导致许多脊柱退行性疾病的前提和基础,如椎管狭窄、椎间盘突出、脊柱不稳等。虽然目前导致椎间盘退变的确切机制还不清楚,但大多数学者认为椎间盘的退变是多因素共同作用的结果。椎间盘细胞数量的减少以及细胞外基质成分的改变是椎间盘退变的病理生理基础,而髓核及纤维环细胞的凋亡是椎间盘细胞减少的主要原因。研究发现退变的椎间盘组织能够产生大量的炎症因子,其中IL-1β是一种能导致炎症反应及细胞凋亡的多效性细胞因子,在椎间盘细胞凋亡中起重要作用。临床观察发现绝经后妇女与同年龄男性相比,椎间盘退行性疾病的发病率较高,提示雌激素与椎间盘退变有关。近年来,研究发现雌激素有抗软骨细胞及胰腺β细胞凋亡的作用,雌激素是否有抗椎间盘细胞凋亡的作用未见报道。本文的目的是探讨17β-雌二醇是否有抑制IL-1β诱导的大鼠纤维环细胞凋亡的作用及是否有浓度依赖性。方法:酶消化法原代培养大鼠纤维环细胞,细胞贴壁融为单层时,用0.25%II型胶原酶及0.2%胰酶(含0.02%EDTA)消化,细胞传代至第3代,用不含胎牛血清和酚红的DMEM/F12培养液培养,按以下分为六组:(1)组1,空白对照组;(2)组2,IL-1β诱导组;(3)组3,0.1μmol/L雌激素组;(4)组4,1μmol/L雌激素组;(5)组5,10μmol/L雌激素组;(6)组6,ICI182,780抑制剂组,每组均为6个样本。细胞培养24 h使细胞生长同步,加入相应药物培养24h后收获细胞。应用:(1)倒置显微镜下观察凋亡细胞形态;(2)流式细胞术及Caspase-3活性检测细胞凋亡;(3)MTT法检测细胞增殖活性。结果:(1)倒置显微镜下可见凋亡的纤维环细胞萎缩,包膜起泡及细胞核固缩。(2)流式细胞术检测纤维环细胞凋亡率:组1为3.47%±0.25%;组2为18.80%±0.99%;组3为12.97%±0.61%;组4为8.67%±0.74%;组5为6.00%±0.61%;组6为18.57%±0.51%。组1、组2、组5、组6之间细胞凋亡率差异有统计学意义(F=237.21,P0.001),组5凋亡率低于组2(P0.001)和组6(P0.001),组2和组6之间差异无统计学差异;组1、组2、组3、组4、组5之间细胞凋亡率差异有统计学意义(F=474.78,P0.001),组3、组4、组5细胞凋亡率逐渐下降。Caspase-3活性检测结果显示:组2 Caspase-3活性明显降低,组3、组4、组5 Caspase-3活性逐渐上升,组6 Caspase-3活性降低。(3)细胞增殖活性:各组OD值(吸光度):组1为71.31%±1.31%;组2为26.74%±1.85%;组3为42.13%±2.10%;组4为53.68%±2.71%;组5为62.89%±3.12%;组6为32.14%±2.25%。组1、组2、组5、组6之间细胞增殖活性差异有统计学意义(F=340.95,P0.001),组5增殖活性大于组2(P0.001)和组6(P0.001),组2和组6之间差异无统计学差异;组1、组2、组3、组4、组5之间细胞增殖活性差异有统计学意义(F=575.44,P0.001),组3、组4、组5细胞增殖活性逐渐上升。结论:17β-雌二醇可有效抑制IL-1β诱导的大鼠纤维环细胞凋亡,并且这种作用具有浓度依赖性。
[Abstract]:Objective: intervertebral disc degeneration refers to the aging degeneration of intervertebral disc tissues with age. It is the premise and foundation of many degenerative diseases of spine, such as spinal stenosis, disc herniation. Spinal instability, etc. Although the exact mechanism leading to disc degeneration is unclear, However, most scholars believe that the degeneration of intervertebral disc is the result of multiple factors. The decrease of the number of intervertebral disc cells and the change of extracellular matrix are the pathophysiological basis of disc degeneration. The apoptosis of nucleus pulposus and fibroannular cells was the main cause of the decrease of intervertebral disc cells. It was found that degenerated intervertebral disc tissue can produce a large number of inflammatory factors, among which IL-1 尾 is a multifunctional cytokine that can induce inflammatory reaction and apoptosis. Clinical observation showed that the incidence of intervertebral disc degeneration in postmenopausal women was higher than that in men of the same age, suggesting that estrogen was associated with disc degeneration. It was found that estrogen could inhibit the apoptosis of chondrocytes and pancreatic 尾 cells. Whether estrogen has the effect of inhibiting apoptosis of intervertebral disc cells has not been reported. The purpose of this paper is to investigate whether 17 尾 -estradiol can inhibit the apoptosis of rat fibrocyclic cells induced by IL-1 尾 and whether it has a concentration-dependent effect. Primary culture of rat fibrous annulus cells by chemical method, When the cells were fused into monolayers, they were digested with 0.25% type II collagenase and 0.2% trypsin (containing 0.02 TA), and the cells were subcultured to the third generation. The cells were cultured in DMEM/F12 medium without fetal bovine serum and phenolic red. They were divided into six groups: 1) group (1), 1) group (1), 1) group (control group)) 2) IL-1 尾 -induced group (3)) group (3) 0. 1 渭 mol/L estrogen group (4 渭 mol/L estrogen group)) group (5 ~ 10 渭 mol/L estrogen group)) group (6) ICI182780 inhibitor group, 6 samples in each group. Cell growth was synchronized after 24 hours of cell culture. The apoptotic cells were observed by flow cytometry and the Caspase-3 activity was detected by flow cytometry. The proliferative activity was detected by MTT assay. Results the cell proliferation activity was detected under the inverted microscope. Apoptotic fibrous ring cells atrophy, The apoptotic rate of fibrous ring cells in group 1, group 2, group 2, group 2, group 3, group 3, group 3, 12.97% 卤0. 61, group 4, group 5, group 5, group 5, group 1, group 2, group 2, group 2 and group 6, respectively, were 3.47% 卤0. 25, 18.80% 卤0. 99, 12.97% 卤0. 61, 8.67% 卤0. 74, 6.00% 卤0. 61 and 18.57% 卤0. 51, respectively. The apoptotic rate of group 5 was lower than that of group 2 (P 0.001) and group 6 (P 0.001). There was no significant difference between group 2 and group 6. The apoptosis rate of group 1, group 2, group 3, group 4 and group 5 were significantly different. The apoptosis rate of group 3, group 4 and group 5 decreased gradually. The activity of Caspase-3 decreased significantly in group 2, group 3, group 4 and group 5. The activity of Caspase-3 increased gradually in group 3, group 4 and group 3, respectively. Cell proliferation activity decreased in group 6 (absorbance: 71.31% 卤1.31 in group 1; 26.74% 卤1.85 in group 2; 42.13% 卤2.10 in group 3; 53.68% 卤2.71 in group 4; 62.89% 卤3.12 in group 5; 32.14% 卤2.25 in group 6; in group 2, 5 and between group 6 and group 6 respectively). The proliferative activity of group 5 was higher than that of group 2 (P 0.001) and group 6 (P 0.001), but there was no significant difference between group 2 and group 6. The cell proliferation activity of group 1, group 2, group 3, group 4, group 5 was significantly different from that of group 5. The proliferative activity of group 3, group 4 and group 5 increased gradually. Conclusion: 1: 17 尾-estradiol can effectively inhibit the apoptosis of rat fibroculoid cells induced by IL-1 尾. And this effect is concentration dependent.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R681.5

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