sCD44分子对POAG小梁网细胞凋亡的影响及相关凋亡信号通路的研究
发布时间：2019-01-06 15:38
【摘要】：目的探讨不同浓度的可溶性CD44分子(soluble CD44,sCD44)对原发性开角型青光眼(primary open-angle glaucoma,POAG)小梁网细胞凋亡及其凋亡调节蛋白Bcl-2相关死亡因子Bad蛋白表达的影响,研究sCD44与POAG发病的关系。 方法采用组织块培养法原代培养POAG小梁网细胞,运用透射电镜,免疫细胞化学等方法对细胞进行鉴定,观察其生物学特性。取传3代的小梁网细胞分别加入含sCD44终浓度为0ng/ml(对照组),1 ng/ml,5 ng/ml,10 ng/ml,25 ng/ml,50 ng/ml的无血清培养基,分别培养24小时后收集细胞,采用CCK-8、荧光显微镜、流式细胞仪和ELISA,研究sCD44对POAG小梁网细胞凋亡及其凋亡调节蛋白Bcl-2相关死亡因子Bad蛋白表达的影响。 结果1.组织块经培养7天左右,可见细胞从其边缘开始向外生长,倒置显微镜下可见细胞形态多样,呈梭形、圆形、椭圆形等。电镜见细胞呈圆形或椭圆形,表面较多微绒毛,细胞之间的连接主要为缝隙连接,胞质内多见次级溶酶体、粗面内质网、线粒体;2.免疫细胞化学法检测纤维粘连蛋白(fibronectin,FN)、层粘连蛋白(laminin,LN)和神经元烯醇化酶(neuron specificenolase,NSE)染色阳性,而阴性对照不着色;3.通过CCK-8法检测发现:随着sCD44终浓度的增加,sCD44对POAG小梁网细胞的抑制作用增强,且各实验组之间以及实验组与对照组相比差异有统计学意义(P0.05);4.通过荧光显微镜观察显示随着sCD44终浓度的增加,早期和晚期凋亡细胞量明显增多;5.通过流式细胞仪法检测sCD44终浓度为1ng/ml,5 ng/ml,10ng/ml,25ng/ml,50 ng/ml干预的实验组小梁网细胞凋亡率分别为(10.7283±0.0223),(17.3267±0.0250),(21.1483±0.0248),(25.1267±0.0281),(29.9900±0.0335),均高于对照组小梁网细胞凋亡率(2.5550±0.0187),且各实验组之间以及实验组与对照组相比差异有统计学意义(P0.05);6.ELISA法检测sCD44终浓度为1 ng/ml,5 ng/ml,10 ng/ml,25ng/ml,50ng/ml干预的实验组小梁网细胞Bad蛋白浓度值分别为(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,均高于对照组(71.1430±0.3999)pg/ml,且各实验组之间以及实验组与对照组相比差异有统计学意义(P0.05)。 结论1.采用组织块体外培养法能成功培养出原发性开角型青光眼小梁网细胞;2. sCD44可以抑制POAG小梁网细胞的增殖,促进细胞凋亡,且在一定范围内呈现一定的剂量依赖性;3. sCD44可以促进细胞凋亡调节蛋白Bcl-2相关死亡因子Bad蛋白的表达,对小梁网细胞凋亡可以通过内源性线粒体途径起作用。
[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 卤0.0223), () 17.3267 卤0.0250), (21.1483 卤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 卤0.0281), (29.9900 卤0.0335), which was higher than that of the control group (2.5550 卤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 卤2.5995) pg/ml, (109.7353 卤3.9992) pg/ml, respectively. (120.1332 卤3.3993) pg/ml, (160.1252 卤5.3989) pg/ml, (220.5131 卤4.7990) pg/ml, was higher than the control group (71.1430 卤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.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R775
本文编号：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 卤0.0223), () 17.3267 卤0.0250), (21.1483 卤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 卤0.0281), (29.9900 卤0.0335), which was higher than that of the control group (2.5550 卤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 卤2.5995) pg/ml, (109.7353 卤3.9992) pg/ml, respectively. (120.1332 卤3.3993) pg/ml, (160.1252 卤5.3989) pg/ml, (220.5131 卤4.7990) pg/ml, was higher than the control group (71.1430 卤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.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R775
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