本文選題：17β-雌二醇　切入點(diǎn)：IL-1β　出處：《河北醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:椎間盤(pán)退行性病變是指隨著年齡的增長(zhǎng),椎間盤(pán)各組織發(fā)生的老化退變。是導(dǎo)致許多脊柱退行性疾病的前提和基礎(chǔ),如椎管狹窄、椎間盤(pán)突出、脊柱不穩(wěn)等。雖然目前導(dǎo)致椎間盤(pán)退變的確切機(jī)制還不清楚,但大多數(shù)學(xué)者認(rèn)為椎間盤(pán)的退變是多因素共同作用的結(jié)果。椎間盤(pán)細(xì)胞數(shù)量的減少以及細(xì)胞外基質(zhì)成分的改變是椎間盤(pán)退變的病理生理基礎(chǔ),而髓核及纖維環(huán)細(xì)胞的凋亡是椎間盤(pán)細(xì)胞減少的主要原因。研究發(fā)現(xiàn)退變的椎間盤(pán)組織能夠產(chǎn)生大量的炎癥因子,其中IL-1β是一種能導(dǎo)致炎癥反應(yīng)及細(xì)胞凋亡的多效性細(xì)胞因子,在椎間盤(pán)細(xì)胞凋亡中起重要作用。臨床觀察發(fā)現(xiàn)絕經(jīng)后婦女與同年齡男性相比,椎間盤(pán)退行性疾病的發(fā)病率較高,提示雌激素與椎間盤(pán)退變有關(guān)。近年來(lái),研究發(fā)現(xiàn)雌激素有抗軟骨細(xì)胞及胰腺β細(xì)胞凋亡的作用,雌激素是否有抗椎間盤(pán)細(xì)胞凋亡的作用未見(jiàn)報(bào)道。本文的目的是探討17β-雌二醇是否有抑制IL-1β誘導(dǎo)的大鼠纖維環(huán)細(xì)胞凋亡的作用及是否有濃度依賴性。方法:酶消化法原代培養(yǎng)大鼠纖維環(huán)細(xì)胞,細(xì)胞貼壁融為單層時(shí),用0.25%II型膠原酶及0.2%胰酶(含0.02%EDTA)消化,細(xì)胞傳代至第3代,用不含胎牛血清和酚紅的DMEM/F12培養(yǎng)液培養(yǎng),按以下分為六組:(1)組1,空白對(duì)照組;(2)組2,IL-1β誘導(dǎo)組;(3)組3,0.1μmol/L雌激素組;(4)組4,1μmol/L雌激素組;(5)組5,10μmol/L雌激素組;(6)組6,ICI182,780抑制劑組,每組均為6個(gè)樣本。細(xì)胞培養(yǎng)24 h使細(xì)胞生長(zhǎng)同步,加入相應(yīng)藥物培養(yǎng)24h后收獲細(xì)胞。應(yīng)用:(1)倒置顯微鏡下觀察凋亡細(xì)胞形態(tài);(2)流式細(xì)胞術(shù)及Caspase-3活性檢測(cè)細(xì)胞凋亡;(3)MTT法檢測(cè)細(xì)胞增殖活性。結(jié)果:(1)倒置顯微鏡下可見(jiàn)凋亡的纖維環(huán)細(xì)胞萎縮,包膜起泡及細(xì)胞核固縮。(2)流式細(xì)胞術(shù)檢測(cè)纖維環(huán)細(xì)胞凋亡率:組1為3.47%±0.25%;組2為18.80%±0.99%;組3為12.97%±0.61%;組4為8.67%±0.74%;組5為6.00%±0.61%;組6為18.57%±0.51%。組1、組2、組5、組6之間細(xì)胞凋亡率差異有統(tǒng)計(jì)學(xué)意義(F=237.21,P0.001),組5凋亡率低于組2(P0.001)和組6(P0.001),組2和組6之間差異無(wú)統(tǒng)計(jì)學(xué)差異;組1、組2、組3、組4、組5之間細(xì)胞凋亡率差異有統(tǒng)計(jì)學(xué)意義(F=474.78,P0.001),組3、組4、組5細(xì)胞凋亡率逐漸下降。Caspase-3活性檢測(cè)結(jié)果顯示:組2 Caspase-3活性明顯降低,組3、組4、組5 Caspase-3活性逐漸上升,組6 Caspase-3活性降低。(3)細(xì)胞增殖活性:各組OD值(吸光度):組1為71.31%±1.31%;組2為26.74%±1.85%;組3為42.13%±2.10%;組4為53.68%±2.71%;組5為62.89%±3.12%;組6為32.14%±2.25%。組1、組2、組5、組6之間細(xì)胞增殖活性差異有統(tǒng)計(jì)學(xué)意義(F=340.95,P0.001),組5增殖活性大于組2(P0.001)和組6(P0.001),組2和組6之間差異無(wú)統(tǒng)計(jì)學(xué)差異;組1、組2、組3、組4、組5之間細(xì)胞增殖活性差異有統(tǒng)計(jì)學(xué)意義(F=575.44,P0.001),組3、組4、組5細(xì)胞增殖活性逐漸上升。結(jié)論:17β-雌二醇可有效抑制IL-1β誘導(dǎo)的大鼠纖維環(huán)細(xì)胞凋亡,并且這種作用具有濃度依賴性。
[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% 鹵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% 鹵0. 25, 18.80% 鹵0. 99, 12.97% 鹵0. 61, 8.67% 鹵0. 74, 6.00% 鹵0. 61 and 18.57% 鹵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% 鹵1.31 in group 1; 26.74% 鹵1.85 in group 2; 42.13% 鹵2.10 in group 3; 53.68% 鹵2.71 in group 4; 62.89% 鹵3.12 in group 5; 32.14% 鹵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.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R681.5

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