sCD44分子對POAG小梁網(wǎng)細(xì)胞凋亡的影響及相關(guān)凋亡信號通路的研究
發(fā)布時(shí)間：2019-01-06 15:38
【摘要】：目的探討不同濃度的可溶性CD44分子(soluble CD44,sCD44)對原發(fā)性開角型青光眼(primary open-angle glaucoma,POAG)小梁網(wǎng)細(xì)胞凋亡及其凋亡調(diào)節(jié)蛋白Bcl-2相關(guān)死亡因子Bad蛋白表達(dá)的影響,研究sCD44與POAG發(fā)病的關(guān)系。 方法采用組織塊培養(yǎng)法原代培養(yǎng)POAG小梁網(wǎng)細(xì)胞,運(yùn)用透射電鏡,免疫細(xì)胞化學(xué)等方法對細(xì)胞進(jìn)行鑒定,觀察其生物學(xué)特性。取傳3代的小梁網(wǎng)細(xì)胞分別加入含sCD44終濃度為0ng/ml(對照組),1 ng/ml,5 ng/ml,10 ng/ml,25 ng/ml,50 ng/ml的無血清培養(yǎng)基,分別培養(yǎng)24小時(shí)后收集細(xì)胞,采用CCK-8、熒光顯微鏡、流式細(xì)胞儀和ELISA,研究sCD44對POAG小梁網(wǎng)細(xì)胞凋亡及其凋亡調(diào)節(jié)蛋白Bcl-2相關(guān)死亡因子Bad蛋白表達(dá)的影響。 結(jié)果1.組織塊經(jīng)培養(yǎng)7天左右,可見細(xì)胞從其邊緣開始向外生長,倒置顯微鏡下可見細(xì)胞形態(tài)多樣,呈梭形、圓形、橢圓形等。電鏡見細(xì)胞呈圓形或橢圓形,表面較多微絨毛,細(xì)胞之間的連接主要為縫隙連接,胞質(zhì)內(nèi)多見次級溶酶體、粗面內(nèi)質(zhì)網(wǎng)、線粒體;2.免疫細(xì)胞化學(xué)法檢測纖維粘連蛋白(fibronectin,FN)、層粘連蛋白(laminin,LN)和神經(jīng)元烯醇化酶(neuron specificenolase,NSE)染色陽性,而陰性對照不著色;3.通過CCK-8法檢測發(fā)現(xiàn):隨著sCD44終濃度的增加,sCD44對POAG小梁網(wǎng)細(xì)胞的抑制作用增強(qiáng),且各實(shí)驗(yàn)組之間以及實(shí)驗(yàn)組與對照組相比差異有統(tǒng)計(jì)學(xué)意義(P0.05);4.通過熒光顯微鏡觀察顯示隨著sCD44終濃度的增加,早期和晚期凋亡細(xì)胞量明顯增多;5.通過流式細(xì)胞儀法檢測sCD44終濃度為1ng/ml,5 ng/ml,10ng/ml,25ng/ml,50 ng/ml干預(yù)的實(shí)驗(yàn)組小梁網(wǎng)細(xì)胞凋亡率分別為(10.7283±0.0223),(17.3267±0.0250),(21.1483±0.0248),(25.1267±0.0281),(29.9900±0.0335),均高于對照組小梁網(wǎng)細(xì)胞凋亡率(2.5550±0.0187),且各實(shí)驗(yàn)組之間以及實(shí)驗(yàn)組與對照組相比差異有統(tǒng)計(jì)學(xué)意義(P0.05);6.ELISA法檢測sCD44終濃度為1 ng/ml,5 ng/ml,10 ng/ml,25ng/ml,50ng/ml干預(yù)的實(shí)驗(yàn)組小梁網(wǎng)細(xì)胞Bad蛋白濃度值分別為(89.9392±2.5995)pg/ml,(109.7353±3.9992) pg/ml,(120.1332±3.3993)pg/ml,(160.1252±5.3989)pg/ml, (220.5131±4.7990)pg/ml,均高于對照組(71.1430±0.3999)pg/ml,且各實(shí)驗(yàn)組之間以及實(shí)驗(yàn)組與對照組相比差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論1.采用組織塊體外培養(yǎng)法能成功培養(yǎng)出原發(fā)性開角型青光眼小梁網(wǎng)細(xì)胞;2. sCD44可以抑制POAG小梁網(wǎng)細(xì)胞的增殖,促進(jìn)細(xì)胞凋亡,且在一定范圍內(nèi)呈現(xiàn)一定的劑量依賴性;3. sCD44可以促進(jìn)細(xì)胞凋亡調(diào)節(jié)蛋白Bcl-2相關(guān)死亡因子Bad蛋白的表達(dá),對小梁網(wǎng)細(xì)胞凋亡可以通過內(nèi)源性線粒體途徑起作用。
[Abstract]:Objective to investigate the effects of soluble CD44 (soluble CD44,sCD44) at different concentrations on trabecular reticulum cell apoptosis and the expression of Bcl-2 related death factor Bad in primary open-angle glaucoma (primary open-angle glaucoma,POAG). To study the relationship between sCD44 and POAG. Methods the primary POAG trabecular meshwork cells were cultured by tissue mass culture. The cells were identified by transmission electron microscopy and immunocytochemistry, and their biological characteristics were observed. Trabecular meshwork cells were cultured for 24 hours in a serum-free medium containing sCD44 final concentration of 0ng/ml (control group) and 1 ng/ml,5 ng/ml,10 ng/ml,25 ng/ml,50 ng/ml. The effects of sCD44 on the apoptosis of POAG trabecular reticulum cells and the expression of Bcl-2 related death factor Bad were studied by CCK-8, fluorescence microscope, flow cytometry and ELISA,. Result 1. When the tissue mass was cultured for about 7 days, the cells began to grow outwards from the edge of the tissue. Under inverted microscope, the cells were found to be fusiform, round, oval and so on. Electron microscope showed that the cells were round or oval, with more microvilli on the surface. The connections between the cells were mainly gap junctions, and the secondary lysosomes, rough endoplasmic reticulum and mitochondria were found in the cytoplasm. 2. The positive staining of fibronectin (fibronectin,FN), laminin (laminin,LN) and neuronal enolase (neuron specificenolase,NSE) was detected by immunocytochemistry, but not by negative control. CCK-8 assay showed that: with the increase of sCD44 final concentration, the inhibitory effect of sCD44 on POAG trabecular meshwork cells was enhanced, and the difference between each experimental group and control group was statistically significant (P0.05); 4. Fluorescence microscopy showed that with the increase of sCD44 final concentration, the number of apoptotic cells increased significantly in the early and late stages. The apoptosis rate of trabecular meshwork reticulum cells in experimental group was (10.7283 鹵0.0223), () 17.3267 鹵0.0250), (21.1483 鹵0.0248) when the final concentration of sCD44 was 1 ng / ml ~ 5 ng/ml,10ng/ml,25ng/ml,50 ng/ml by flow cytometry. (25.1267 鹵0.0281), (29.9900 鹵0.0335), which was higher than that of the control group (2.5550 鹵0.0187), and the difference between each experimental group and the control group was statistically significant (P0.05). The concentration of Bad protein in trabecular meshwork reticulum cells treated with 1 ng/ml,5 ng/ml,10 ng/ml,25ng/ml,50ng/ml sCD44 final concentration by 6.ELISA assay was (89.9392 鹵2.5995) pg/ml, (109.7353 鹵3.9992) pg/ml, respectively. (120.1332 鹵3.3993) pg/ml, (160.1252 鹵5.3989) pg/ml, (220.5131 鹵4.7990) pg/ml, was higher than the control group (71.1430 鹵0.3999) pg/ml,. The difference between each experimental group and the control group was statistically significant (P0.05). Conclusion 1. The trabecular meshwork cells of primary open angle glaucoma could be successfully cultured by tissue mass culture in vitro. 2. SCD44 could inhibit the proliferation of POAG trabecular meshwork cells and promote cell apoptosis in a dose-dependent manner. 3. SCD44 can promote the expression of apoptosis regulating protein Bcl-2 related death factor Bad protein, and the apoptosis of trabecular meshwork cells can be mediated by endogenous mitochondrial pathway.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R775
本文編號：2402987
[Abstract]:Objective to investigate the effects of soluble CD44 (soluble CD44,sCD44) at different concentrations on trabecular reticulum cell apoptosis and the expression of Bcl-2 related death factor Bad in primary open-angle glaucoma (primary open-angle glaucoma,POAG). To study the relationship between sCD44 and POAG. Methods the primary POAG trabecular meshwork cells were cultured by tissue mass culture. The cells were identified by transmission electron microscopy and immunocytochemistry, and their biological characteristics were observed. Trabecular meshwork cells were cultured for 24 hours in a serum-free medium containing sCD44 final concentration of 0ng/ml (control group) and 1 ng/ml,5 ng/ml,10 ng/ml,25 ng/ml,50 ng/ml. The effects of sCD44 on the apoptosis of POAG trabecular reticulum cells and the expression of Bcl-2 related death factor Bad were studied by CCK-8, fluorescence microscope, flow cytometry and ELISA,. Result 1. When the tissue mass was cultured for about 7 days, the cells began to grow outwards from the edge of the tissue. Under inverted microscope, the cells were found to be fusiform, round, oval and so on. Electron microscope showed that the cells were round or oval, with more microvilli on the surface. The connections between the cells were mainly gap junctions, and the secondary lysosomes, rough endoplasmic reticulum and mitochondria were found in the cytoplasm. 2. The positive staining of fibronectin (fibronectin,FN), laminin (laminin,LN) and neuronal enolase (neuron specificenolase,NSE) was detected by immunocytochemistry, but not by negative control. CCK-8 assay showed that: with the increase of sCD44 final concentration, the inhibitory effect of sCD44 on POAG trabecular meshwork cells was enhanced, and the difference between each experimental group and control group was statistically significant (P0.05); 4. Fluorescence microscopy showed that with the increase of sCD44 final concentration, the number of apoptotic cells increased significantly in the early and late stages. The apoptosis rate of trabecular meshwork reticulum cells in experimental group was (10.7283 鹵0.0223), () 17.3267 鹵0.0250), (21.1483 鹵0.0248) when the final concentration of sCD44 was 1 ng / ml ~ 5 ng/ml,10ng/ml,25ng/ml,50 ng/ml by flow cytometry. (25.1267 鹵0.0281), (29.9900 鹵0.0335), which was higher than that of the control group (2.5550 鹵0.0187), and the difference between each experimental group and the control group was statistically significant (P0.05). The concentration of Bad protein in trabecular meshwork reticulum cells treated with 1 ng/ml,5 ng/ml,10 ng/ml,25ng/ml,50ng/ml sCD44 final concentration by 6.ELISA assay was (89.9392 鹵2.5995) pg/ml, (109.7353 鹵3.9992) pg/ml, respectively. (120.1332 鹵3.3993) pg/ml, (160.1252 鹵5.3989) pg/ml, (220.5131 鹵4.7990) pg/ml, was higher than the control group (71.1430 鹵0.3999) pg/ml,. The difference between each experimental group and the control group was statistically significant (P0.05). Conclusion 1. The trabecular meshwork cells of primary open angle glaucoma could be successfully cultured by tissue mass culture in vitro. 2. SCD44 could inhibit the proliferation of POAG trabecular meshwork cells and promote cell apoptosis in a dose-dependent manner. 3. SCD44 can promote the expression of apoptosis regulating protein Bcl-2 related death factor Bad protein, and the apoptosis of trabecular meshwork cells can be mediated by endogenous mitochondrial pathway.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R775
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 李 珍,郭 華,李冬梅,杜玉敏,張海燕,林躍生;重組人表皮生長因子對胬肉切除-羊膜移植后眼表上皮愈合速度的影響[J];第一軍醫(yī)大學(xué)學(xué)報(bào);2002年05期
2 陳書揚(yáng),吳瑜瑜,朱益華,林玲,李貴州;體外培養(yǎng)人眼小梁細(xì)胞黏附分子CD_(44s)的表達(dá)[J];福建醫(yī)科大學(xué)學(xué)報(bào);2005年03期
3 李超;吳瑜瑜;;白細(xì)胞介素-1β對人眼小梁細(xì)胞基質(zhì)金屬蛋白酶-3表達(dá)的影響[J];臨床眼科雜志;2008年04期
4 李中國;王林農(nóng);;可溶性CD44分子在不同類型青光眼房水中的表達(dá)特征及意義[J];實(shí)用醫(yī)學(xué)雜志;2008年19期
5 王溪,陳彬川,帖紅艷;術(shù)中應(yīng)用5-FU抑制后發(fā)性白內(nèi)障的實(shí)驗(yàn)研究[J];眼科新進(jìn)展;2004年04期
6 葛堅(jiān),卓業(yè)鴻;人眼小梁細(xì)胞體外培養(yǎng)及生物學(xué)特性研究[J];眼科學(xué)報(bào);1996年02期
7 牛嗣云,高福祿,龐小靜,李秀華,張玉娟,王瑞學(xué);人胎兒晶狀體上皮細(xì)胞增殖及相關(guān)因子的動態(tài)研究[J];眼科研究;2002年01期
8 郝玉華;馬景學(xué);;羊膜對體外培養(yǎng)的兔視網(wǎng)膜Müller細(xì)胞表皮生長因子受體表達(dá)的影響[J];眼科研究;2009年12期
9 黃秀榕;祁明信;胡艷紅;嚴(yán)京;吳正正;馬蘭;;欖香烯抑制表皮生長因子誘導(dǎo)的晶狀體上皮細(xì)胞增殖的信號轉(zhuǎn)導(dǎo)機(jī)制[J];中國臨床藥理學(xué)與治療學(xué);2007年01期
10 班勝剛,李永均,盧榮強(qiáng),蔣林志,黃明漢;重組人表皮生長因子治療外傷性角膜上皮缺損[J];眼外傷職業(yè)眼病雜志.附眼科手術(shù);2005年01期
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