LYTAK1抑制人视网膜色素上皮细胞增殖、迁移和间质化机制的研究
发布时间：2018-07-24 13:18
【摘要】：研究背景增殖性玻璃体视网膜病变(Proliferative vitreoretinopathy,PVR)是孔源性视网膜脱离(rhegmatogenous retinal detachment,RRD)以及行视网膜复位手术后的一种并发症。PVR的病理变化涉及细胞去极化、迁移、黏附、增殖等一系列细胞活动过程,此外还与分泌胶原等细胞外基质(extracellular matrix,ECM)的异常有关;经过上述病理过程之后,在视网膜前后表面以及玻璃体中将形成具有收缩能力的增殖膜,由于后者的收缩,从而造成牵拉性视网膜脱离(tractional retinal detachment,TRD)。在近年来的研究中,PVR的发生以及其机制一直是眼底病研究的热点及难点。视网膜色素上皮细胞(retinal pigment epithelial,RPE)处于视网膜光感受器细胞层的外围,其结构由一层排列整齐的六角形细胞组成。目前的研究已证实,RPE细胞对PVR的发生和发展至关重要。上皮-间质转化(Epithelial-mesenchymal transition,EMT)为PVR的发生和发展过程中的重要环节。EMT是指具有上皮样表型的细胞,当其失去极性后活动能力增强,在细胞间质之间能够自由移动并且表现出纤维样表型的转化过程。EMT通常分为三种亚型:其一,是原肠胚形成,机体中原始的上皮细胞通过EMT参与多种细胞的生物胚胎发育及器官的形成中;其二,为内皮或者第二上皮转化为组织成纤维细胞,从而在机体的伤口愈合以及器官的纤维化过程中发挥重要作用;其三,是上皮来源的细胞失去极性,从而转化为具有侵袭能力的细胞。转化生长因子β活化激酶-1(transforming growth factor-βactivated kinase-1,TAK1)为MAP3K家族中的主要成员之一,属于色氨酸/苏氨酸蛋白激酶,其活性受到TGF-β以及骨形态发生蛋白(bone morphogenetic proteins,BMPs)的调控。在生物机体中,TAK1经由P38、JNK以及NF-κB通路活化实现对一系列特异性细胞转录因子的调控,从而影响细胞的生存、分化等生理病理过程,同时还对炎症反应具有调控作用。由于tak1功能具有多样性,因此tak1在炎症性疾病中成为了主要的靶激酶。除此之外,tak1还参与了mkk3/4-p38/jnk-ap-1以及ikk-nf-κb等重要信号转导通路的活化,对细胞的存活、分化以及炎症应答具有十分重要的调控作用。转化生长因子-β(transforminggrowthfactor-β,tgf-β)为一类新发现的具有对细胞分化和生长起调节作用的多功能细胞因子,大量研究证实,tgf-β是目前已知的具有促进组织纤维化的最重要的细胞因子,其在肾纤维化、肝纤维化和肺纤维化的患者血液中高表达。研究表明,tgf-β在pvr的发生和发展过程中发挥了重要作用,但其具体作用和机制并不十分清楚,尚需进一步深入研究。目的本课题旨在明确:1.tak1在tgf-β1诱导的人rpe细胞间质化过程中的作用;2.探讨tak1抑制剂lytak1对rpe细胞间质化的作用;3.研究lytak1对rpe细胞间质化的作用机制,旨在为pvr的治疗奠定实验基础。方法1.采用elisa法检测2014年6月~2014年12月期间在云南省第一人民医院眼科30例pvr患者、20例imh患者以及同期15例正常志愿者,分别检测玻璃体液和/或外周血中tgf-β1和tak1的表达水平。所有患者的年龄均在45~65岁之间。其中,pvr患者中有男性为18例,女性为12例;imh患者中男性为8例,女性为12例;健康志愿者中男性为9例,女性为6例。2.将0.01ng/mltgf-β1作用于人rpe细胞株arpe-19,培养24h后采用荧光定量pcr法检测细胞中tak1mrna的表达;分别用不同浓度的lytak1(0μm、1μm、10μm、25μm、50μm)作用于0.01ng/mltgf-β1诱导后的arpe-19细胞,westernblot法检测细胞中tak1蛋白的表达;transwell小室法检测细胞的侵袭能力;流式细胞术(annexinv-fitc/pi双染色法)检测细胞的凋亡情况;细胞划痕实验检测细胞的迁移能力。3.采用荧光定量pcr法分别检测对照组、tgf-β1+dmso组以及tgf-β1+lytak1组arpe-19细胞中α-平滑肌肌动蛋白(α-smoothmuscleactin,α-sma)和纤维粘连蛋白(fibronectin)mrna的表达。采用westernblot法分别检测对照组、tgf-β1+dmso组以及tgf-β1+lytak1各浓度组(0μm、1μm、10μm、25μm、50μm)arpe-19细胞中a-sma、fibronectin、p-smad2、p-smad3、ikkα、nf-κbp65蛋白的表达。结果1.pvr和imh两组间tak1的浓度存在显著差异(t=3.114,p=0.0350.05),即pvr患者玻璃体中tak1的浓度显著高于imh组;对pvr和正常组两组的间血清tak1浓度是否存在差异性进行比较分析,结果表明两组间tak1的浓度存在显著差异(t=3.156,p=0.0340.05),即pvr患者血清中tak1的浓度显著高于imh组。上述结果表明,pvr患者玻璃体以及血清中tak1的含量均显著高于imh患者,血清中的tak1含量也明显高于健康人群,提示其可能对pvr的发生以及发展过程具有促进作用。pvr患者、imh患者以及健康志愿者的玻璃体液和/或血清中tgf-β1的浓度存在显著差异(t=2.319,p=0.0330.05),即pvr患者玻璃体中tgf-β1的浓度显著高于imh组。对pvr和正常组两组间的血清tgf-β1浓度是否存在差异性采用t检验进行比较分析,结果表明两组间tgf-β1的浓度存在显著差异(t=3.312,p=0.0370.05),即pvr患者血清中tgf-β1的浓度显著高于imh组。上述结果表明,pvr患者玻璃体和/或血清中tgf-β1的含量均显著高于imh患者以及健康人群,提示其与pvr的发生以及发展过程有关。2.荧光定量pcr检测结果表明,tgf-β1处理组arpe-19细胞中tak1mrna的表达显著高于对照组,tgf-β1能够明显增高tak1蛋白的表达,而当用不同浓度的lytak1作用细胞后,细胞中tak1蛋白的表达量均发生不同程度的降低(p0.05),其中当lytak1的浓度为25μm时tak1蛋白的表达量最低。cck-8检测表明,当lytak1作用24h时,各浓度组细胞的增殖活性均未见显著变化(p0.05);而当lytak1作用48h以及72h之后,随着lytak1作用浓度的增加,当lytak1作用浓度为50μm时对arpe-19细胞的增殖活性的抑制率最高。arpe-19细胞的增殖活性逐渐降低,表明lytak1对arpe-19细胞的活性呈剂量依赖性。transwell小室法检测结果表明,lytak1各浓度组与对照组相比视野平均侵袭细胞数量显著减少,且随着lytak1浓度的增高其侵袭细胞数量逐渐降低,提示lytak1对arpe-19细胞的侵袭具有抑制作用,且呈剂量依赖性。流式细胞术检测结果表明,随着lytak1作用浓度的升高,arpe-19细胞的凋亡率也随着上升,提示lytak1诱导arpe-19细胞凋亡呈现一定的剂量依赖性。细胞划痕实验结果表明,48 h后各浓度LYTAK1作用组ARPE-19细胞的迁移距离较对照组明显缩短(P0.05);且随着LYTAK1浓度的增高,细胞的迁移距离逐渐降低,提示LYTAK1抑制ARPE-19细胞迁移呈现一定的剂量依赖性。3.与对照组相比,TGF-β1+DMSO组以及TGF-β1+LYTAK1组中a-SMA和fibronectin mRNA的表达均明显增高;而相较于TGF-β1+DMSO组,TGF-β1+LYTAK1组中a-SMA和fibronectin mRNA的表达量显著降低。Western blot法检测结果表明,TGF-β1+DMSO组细胞中a-SMA、fibronectin、p-Smad2、p-Smad3、IKKα、NF-κBp65蛋白的表达均显著高于对照组。TGF-β1+LYTAK1各浓度组细胞中a-SMA、fibronectin、p-Smad2、IKKα、NF-κBp65蛋白的表达随LYTAK1浓度的增高而逐渐降低,呈剂量依赖性,而LYTAK1对p-Smad3蛋白的表达无显著影响。结论1.PVR患者血清以及玻璃体中TAK1和TGF-β1的表达量显著增高,其表达上调可能与PVR的发生和发展有关。2.TGF-β1能够显著上调ARPE-19细胞中TAK1的表达;3.LYTAK1能够明显抑制ARPE-19细胞中TAK1的表达,且呈剂量依赖性;4.TGF-β1能够通过上调ARPE-19细胞中TAK1、a-SMA、fibronectin、p-Smad2、p-Smad3、IKKα、NF-κBp65的表达,促进EMT的发生和发展;而LYTAK1能够通过抑制TAK1、a-SMA、fibronectin、p-Smad2、IKKα、NF-κBp65的表达发挥抑制EMT的发生和发展。5.LYTAK1能够明显抑制ARPE-19细胞的增殖活性、细胞凋亡、侵袭及迁移,且呈剂量依赖性。
[Abstract]:Background proliferative vitreoretinopathy (Proliferative vitreoretinopathy, PVR) is a complication of retinal detachment (rhegmatogenous retinal detachment, RRD) and a complication of retinal reposition surgery. The pathological changes of.PVR involve cell depolarization, migration, adhesion and proliferation, in addition to a series of cell activities. It is also associated with the abnormality of the extracellular matrix (extracellular matrix, ECM) that secretes collagen and so on. After the above pathological process, a proliferating membrane with contractile ability in the front and back of the retina and in the vitreous body is formed, resulting in traction retinal detachment (tractional retinal detachment, TRD) in recent years. In the study, the occurrence of PVR and its mechanism have been a hot and difficult point in the study of fundus disease. The retinal pigment epithelial (RPE) is located on the periphery of the retinal photoreceptor cell layer, and its structure is composed of a layer of neat hexagonal cells. The present study has confirmed that the occurrence and development of PVR in RPE cells is confirmed. Epithelial-mesenchymal transition (EMT) is an important part of the development and development of PVR..EMT is a cell with an epithelioid phenotype. When it loses its polarity, the activity is enhanced, and it can move freely between the cells and shows the transformation of the fibrous phenotype, usually.EMT. It is divided into three subtypes: one is the formation of the gastrula, the original epithelial cells in the body participate in the development of biological embryos and the formation of organs through EMT; secondly, the transformation of the endothelial or second epithelium into tissue fibroblasts, which plays an important role in the healing of the body's wounds and the fibrosis of the organs. The epithelial derived cells lose polarity and turn into invasive cells. Transforming growth factor beta activated kinase -1 (transforming growth factor- beta activated kinase-1, TAK1) is one of the main members of the MAP3K family, belonging to tryptophan / threonine protein kinase, and its activity is subject to TGF- beta and bone morphogenetic protein (bone). Regulation of morphogenetic proteins, BMPs). In biological organisms, TAK1 is activated by P38, JNK, and NF- kappa B pathways to regulate a series of specific cell transcription factors, which affect cell survival, differentiation and other physiological and pathological processes, and also regulate the inflammatory response. As TAK1 function is diverse, TAK1 is In addition to this, TAK1 also participates in the activation of important signal transduction pathways such as mkk3/4-p38/jnk-ap-1 and ikk-nf- kappa B, which plays a very important role in the regulation of cell survival, differentiation and inflammatory responses. Transforming growth factor beta (tgf- beta) is a new type of discovery. A large number of studies have proved that tgf- beta is the most important cytokine that is known to promote tissue fibrosis and is highly expressed in the blood of patients with renal fibrosis, liver fibrosis and pulmonary fibrosis. The study shows that tgf- beta is in the process of the development and development of PVR. It plays an important role, but its specific role and mechanism are not very clear. The purpose of this study is to clarify the role of 1.tak1 in the interstitial process of human RPE cells induced by tgf- beta 1; 2. to explore the effect of TAK1 inhibitor lytak1 on the interstitial cells of RPE cells; and 3. to study the mechanism of lytak1 on the interstitial cells of RPE cells, Objective to establish the experimental basis for the treatment of PVR. Method 1. the expression level of tgf- beta 1 and TAK1 in vitreous fluid and / or peripheral blood was detected by ELISA in 30 PVR patients in the first people's Hospital of Yunnan Province, 20 cases of IMH and 15 normal volunteers during the period of ~2014 June 2014. The age of all patients was 45~. Among the 65 years old, there were 18 males and 12 females in PVR patients, 8 men and 12 women in IMH, 9 in the healthy volunteers and 6 in.2. by 0.01ng/mltgf- beta 1 to the human RPE cell line. The expression of tak1mrna in the cells was detected by the fluorescence quantitative PCR method after the cultivation of 24h, and ly of different concentrations was used respectively. TAK1 (0 mu m, 1 mu m, 10 mu m, 25 mu m, 50 micron m) acted on ARPE-19 cells induced by 0.01ng/mltgf- beta 1. Westernblot method was used to detect the expression of TAK1 protein in cells; Transwell chamber method was used to detect the invasion ability of cells; flow cytometry (annexinv-fitc/pi double staining) was used to detect the apoptosis of cells; cell scratch test was used to detect cell migration ability The expression of alpha smooth muscle actin (alpha -smoothmuscleactin, alpha -sma) and fibronectin (fibronectin) mRNA were detected in the control group, the tgf- beta 1+dmso group and the ARPE-19 cells of the tgf- beta 1+lytak1 group by fluorescence quantitative PCR, respectively. The Westernblot method was used to detect the control group, the tgf- beta group and the concentration group (0 mu, 1) respectively. The concentrations of a-SMA, fibronectin, p-Smad2, p-smad3, IKK a, nf- kappa bp65 protein in the ARPE-19 cells of M, 10, 25, 25, and 50 m were significantly different from those of the two groups. The comparative analysis showed that the concentration of TAK1 in the two groups was significantly different (t=3.156, p=0.0340.05), that is, the concentration of TAK1 in the serum of PVR patients was significantly higher than that of the IMH group. The results showed that the content of TAK1 in the vitreous body and serum of the PVR patients was significantly higher than that of the IMH patients, and the TAK1 content in the serum was significantly higher than that of the healthy population. The concentration of tgf- beta 1 in the vitreous fluid and / or serum of the healthy volunteers was significantly different (t=2.319, p=0.0330.05), that is, the concentration of tgf- beta 1 in the vitreous body of the PVR patients was significantly higher than that in the IMH group. The concentration of tgf- beta 1 between the PVR and the two groups of the normal group was the concentration of the tgf- beta 1 in the two groups of the normal group, and the concentration of the serum tgf- beta 1 between the PVR and the two groups of the normal group. T test was used to analyze the difference. The results showed that there was a significant difference in the concentration of tgf- beta 1 between the two groups (t=3.312, p=0.0370.05), that is, the concentration of tgf- beta 1 in the serum of PVR patients was significantly higher than that in the IMH group. The results showed that the content of tgf- beta 1 in the vitreous body and / or serum of PVR patients was significantly higher than that of the IMH patients and the healthy population. The results of.2. fluorescence quantitative PCR detection showed that the expression of tak1mrna in ARPE-19 cells of tgf- beta 1 treated group was significantly higher than that of the control group. Tgf- beta 1 could significantly increase the expression of TAK1 protein, while the expression of TAK1 protein in the cells of the cells in the cells of different concentrations were all different degrees after using different concentrations of lytak1 cells. Decrease (P0.05), in which the lowest.Cck-8 expression of TAK1 protein was detected when the concentration of lytak1 was 25 u m. The proliferation activity of cells in each concentration group was not significantly changed when lytak1 acted 24h, but when lytak1 action 48h and 72h, with the increase of the concentration of lytak1 action, the cell concentration was 50 mu. The inhibitory rate of proliferation activity was highest in.Arpe-19 cells, and the proliferation activity of lytak1 cells decreased gradually. It showed that the activity of ARPE-19 cells in a dose dependent.Transwell cell method showed that the average number of invasive cells in each concentration group of lytak1 decreased significantly compared with the control group, and the number of cells invaded with the increase of lytak1 concentration. Gradually decreased, suggesting that lytak1 has a inhibitory effect on the invasion of ARPE-19 cells and is dose-dependent. Flow cytometry results showed that the apoptosis rate of ARPE-19 cells increased with the increase of lytak1 concentration, suggesting that lytak1 induced apoptosis in ARPE-19 cells showed a dose dependence. The results of cell scratch test showed that After 48 h, the migration distance of ARPE-19 cells in each concentration LYTAK1 group was significantly shorter than that in the control group (P0.05), and with the increase of LYTAK1 concentration, the migration distance of cells decreased gradually. It suggested that LYTAK1 inhibit the migration of ARPE-19 cells to present a dose dependent.3., compared with the control group, TGF- beta 1+DMSO group and TGF- beta 1+LYTAK1 group. The expression of nectin mRNA increased significantly, while the expression of a-SMA and fibronectin mRNA in the group of TGF- beta 1+LYTAK1 was significantly lower than that in the group of TGF- beta 1+DMSO. The expression of.Western blot method in the TGF- beta group was significantly higher than that of the control group. The expression of a-SMA, fibronectin, p-Smad2, IKK a, NF- kappa Bp65 protein in the concentration group gradually decreased with the increase of LYTAK1 concentration, which was dose-dependent, but LYTAK1 had no significant effect on the expression of p-Smad3 protein. Conclusion the expression of TAK1 and TGF- beta 1 in the serum and vitreous body of 1.PVR patients was significantly higher than that in the vitreous body. And the development of.2.TGF- beta 1 significantly up-regulated the expression of TAK1 in ARPE-19 cells; 3.LYTAK1 can obviously inhibit the expression of TAK1 in ARPE-19 cells, and is dose-dependent; 4.TGF- beta 1 can promote the occurrence and development of ARPE-19 cells by up-regulation the expression of TAK1, a-SMA, fibronectin, p-Smad2, p-Smad2, alpha, etc. By inhibiting the expression of TAK1, a-SMA, fibronectin, p-Smad2, IKK a, and NF- kappa Bp65, the inhibition of the occurrence and development of EMT can inhibit the proliferation activity, apoptosis, invasion and migration of ARPE-19 cells in a dose-dependent manner.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R774.1
本文编号：2141508
[Abstract]:Background proliferative vitreoretinopathy (Proliferative vitreoretinopathy, PVR) is a complication of retinal detachment (rhegmatogenous retinal detachment, RRD) and a complication of retinal reposition surgery. The pathological changes of.PVR involve cell depolarization, migration, adhesion and proliferation, in addition to a series of cell activities. It is also associated with the abnormality of the extracellular matrix (extracellular matrix, ECM) that secretes collagen and so on. After the above pathological process, a proliferating membrane with contractile ability in the front and back of the retina and in the vitreous body is formed, resulting in traction retinal detachment (tractional retinal detachment, TRD) in recent years. In the study, the occurrence of PVR and its mechanism have been a hot and difficult point in the study of fundus disease. The retinal pigment epithelial (RPE) is located on the periphery of the retinal photoreceptor cell layer, and its structure is composed of a layer of neat hexagonal cells. The present study has confirmed that the occurrence and development of PVR in RPE cells is confirmed. Epithelial-mesenchymal transition (EMT) is an important part of the development and development of PVR..EMT is a cell with an epithelioid phenotype. When it loses its polarity, the activity is enhanced, and it can move freely between the cells and shows the transformation of the fibrous phenotype, usually.EMT. It is divided into three subtypes: one is the formation of the gastrula, the original epithelial cells in the body participate in the development of biological embryos and the formation of organs through EMT; secondly, the transformation of the endothelial or second epithelium into tissue fibroblasts, which plays an important role in the healing of the body's wounds and the fibrosis of the organs. The epithelial derived cells lose polarity and turn into invasive cells. Transforming growth factor beta activated kinase -1 (transforming growth factor- beta activated kinase-1, TAK1) is one of the main members of the MAP3K family, belonging to tryptophan / threonine protein kinase, and its activity is subject to TGF- beta and bone morphogenetic protein (bone). Regulation of morphogenetic proteins, BMPs). In biological organisms, TAK1 is activated by P38, JNK, and NF- kappa B pathways to regulate a series of specific cell transcription factors, which affect cell survival, differentiation and other physiological and pathological processes, and also regulate the inflammatory response. As TAK1 function is diverse, TAK1 is In addition to this, TAK1 also participates in the activation of important signal transduction pathways such as mkk3/4-p38/jnk-ap-1 and ikk-nf- kappa B, which plays a very important role in the regulation of cell survival, differentiation and inflammatory responses. Transforming growth factor beta (tgf- beta) is a new type of discovery. A large number of studies have proved that tgf- beta is the most important cytokine that is known to promote tissue fibrosis and is highly expressed in the blood of patients with renal fibrosis, liver fibrosis and pulmonary fibrosis. The study shows that tgf- beta is in the process of the development and development of PVR. It plays an important role, but its specific role and mechanism are not very clear. The purpose of this study is to clarify the role of 1.tak1 in the interstitial process of human RPE cells induced by tgf- beta 1; 2. to explore the effect of TAK1 inhibitor lytak1 on the interstitial cells of RPE cells; and 3. to study the mechanism of lytak1 on the interstitial cells of RPE cells, Objective to establish the experimental basis for the treatment of PVR. Method 1. the expression level of tgf- beta 1 and TAK1 in vitreous fluid and / or peripheral blood was detected by ELISA in 30 PVR patients in the first people's Hospital of Yunnan Province, 20 cases of IMH and 15 normal volunteers during the period of ~2014 June 2014. The age of all patients was 45~. Among the 65 years old, there were 18 males and 12 females in PVR patients, 8 men and 12 women in IMH, 9 in the healthy volunteers and 6 in.2. by 0.01ng/mltgf- beta 1 to the human RPE cell line. The expression of tak1mrna in the cells was detected by the fluorescence quantitative PCR method after the cultivation of 24h, and ly of different concentrations was used respectively. TAK1 (0 mu m, 1 mu m, 10 mu m, 25 mu m, 50 micron m) acted on ARPE-19 cells induced by 0.01ng/mltgf- beta 1. Westernblot method was used to detect the expression of TAK1 protein in cells; Transwell chamber method was used to detect the invasion ability of cells; flow cytometry (annexinv-fitc/pi double staining) was used to detect the apoptosis of cells; cell scratch test was used to detect cell migration ability The expression of alpha smooth muscle actin (alpha -smoothmuscleactin, alpha -sma) and fibronectin (fibronectin) mRNA were detected in the control group, the tgf- beta 1+dmso group and the ARPE-19 cells of the tgf- beta 1+lytak1 group by fluorescence quantitative PCR, respectively. The Westernblot method was used to detect the control group, the tgf- beta group and the concentration group (0 mu, 1) respectively. The concentrations of a-SMA, fibronectin, p-Smad2, p-smad3, IKK a, nf- kappa bp65 protein in the ARPE-19 cells of M, 10, 25, 25, and 50 m were significantly different from those of the two groups. The comparative analysis showed that the concentration of TAK1 in the two groups was significantly different (t=3.156, p=0.0340.05), that is, the concentration of TAK1 in the serum of PVR patients was significantly higher than that of the IMH group. The results showed that the content of TAK1 in the vitreous body and serum of the PVR patients was significantly higher than that of the IMH patients, and the TAK1 content in the serum was significantly higher than that of the healthy population. The concentration of tgf- beta 1 in the vitreous fluid and / or serum of the healthy volunteers was significantly different (t=2.319, p=0.0330.05), that is, the concentration of tgf- beta 1 in the vitreous body of the PVR patients was significantly higher than that in the IMH group. The concentration of tgf- beta 1 between the PVR and the two groups of the normal group was the concentration of the tgf- beta 1 in the two groups of the normal group, and the concentration of the serum tgf- beta 1 between the PVR and the two groups of the normal group. T test was used to analyze the difference. The results showed that there was a significant difference in the concentration of tgf- beta 1 between the two groups (t=3.312, p=0.0370.05), that is, the concentration of tgf- beta 1 in the serum of PVR patients was significantly higher than that in the IMH group. The results showed that the content of tgf- beta 1 in the vitreous body and / or serum of PVR patients was significantly higher than that of the IMH patients and the healthy population. The results of.2. fluorescence quantitative PCR detection showed that the expression of tak1mrna in ARPE-19 cells of tgf- beta 1 treated group was significantly higher than that of the control group. Tgf- beta 1 could significantly increase the expression of TAK1 protein, while the expression of TAK1 protein in the cells of the cells in the cells of different concentrations were all different degrees after using different concentrations of lytak1 cells. Decrease (P0.05), in which the lowest.Cck-8 expression of TAK1 protein was detected when the concentration of lytak1 was 25 u m. The proliferation activity of cells in each concentration group was not significantly changed when lytak1 acted 24h, but when lytak1 action 48h and 72h, with the increase of the concentration of lytak1 action, the cell concentration was 50 mu. The inhibitory rate of proliferation activity was highest in.Arpe-19 cells, and the proliferation activity of lytak1 cells decreased gradually. It showed that the activity of ARPE-19 cells in a dose dependent.Transwell cell method showed that the average number of invasive cells in each concentration group of lytak1 decreased significantly compared with the control group, and the number of cells invaded with the increase of lytak1 concentration. Gradually decreased, suggesting that lytak1 has a inhibitory effect on the invasion of ARPE-19 cells and is dose-dependent. Flow cytometry results showed that the apoptosis rate of ARPE-19 cells increased with the increase of lytak1 concentration, suggesting that lytak1 induced apoptosis in ARPE-19 cells showed a dose dependence. The results of cell scratch test showed that After 48 h, the migration distance of ARPE-19 cells in each concentration LYTAK1 group was significantly shorter than that in the control group (P0.05), and with the increase of LYTAK1 concentration, the migration distance of cells decreased gradually. It suggested that LYTAK1 inhibit the migration of ARPE-19 cells to present a dose dependent.3., compared with the control group, TGF- beta 1+DMSO group and TGF- beta 1+LYTAK1 group. The expression of nectin mRNA increased significantly, while the expression of a-SMA and fibronectin mRNA in the group of TGF- beta 1+LYTAK1 was significantly lower than that in the group of TGF- beta 1+DMSO. The expression of.Western blot method in the TGF- beta group was significantly higher than that of the control group. The expression of a-SMA, fibronectin, p-Smad2, IKK a, NF- kappa Bp65 protein in the concentration group gradually decreased with the increase of LYTAK1 concentration, which was dose-dependent, but LYTAK1 had no significant effect on the expression of p-Smad3 protein. Conclusion the expression of TAK1 and TGF- beta 1 in the serum and vitreous body of 1.PVR patients was significantly higher than that in the vitreous body. And the development of.2.TGF- beta 1 significantly up-regulated the expression of TAK1 in ARPE-19 cells; 3.LYTAK1 can obviously inhibit the expression of TAK1 in ARPE-19 cells, and is dose-dependent; 4.TGF- beta 1 can promote the occurrence and development of ARPE-19 cells by up-regulation the expression of TAK1, a-SMA, fibronectin, p-Smad2, p-Smad2, alpha, etc. By inhibiting the expression of TAK1, a-SMA, fibronectin, p-Smad2, IKK a, and NF- kappa Bp65, the inhibition of the occurrence and development of EMT can inhibit the proliferation activity, apoptosis, invasion and migration of ARPE-19 cells in a dose-dependent manner.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R774.1
【相似文献】
相关期刊论文 前10条
1 ;Effect of fibronectin and leukaemia inhibitory factor on matrix metalloproteinases in mouse blastocyst[J];Chinese Science Bulletin;2001年15期
2 Jintang Wang;Ling Yin;Zheng Chen;;Neuroprotective role of fibronectin in neuron-glial extrasynaptic transmission[J];Neural Regeneration Research;2013年04期
3 Yan-Yun Wu;Huan-Huan Cao;Ning Kang;Ping Gong;Guo-Min Ou;;Expression of cellular fibronectin mRNA in adult periodontitis and peri-implantitis: a real-time polymerase chain reaction study[J];International Journal of Oral Science;2013年04期
4 刘建平;向居正;李奇芬;;A Clinical Study of Plasma Fibronectin in Patients with Severe Viral Hepatitis[J];Journal of Medical Colleges of PLA;1987年02期
5 李文革;徐莘香;;The expression of N-cadherin,fibronectin during chondrogenic differentiation of MSC induced by TGF-β_1[J];Chinese Journal of Traumatology;2005年06期
6 孙庆斌;徐印坎;张文明;孔宪涛;周志华;谢映华;;Experimental Study on Changes of Fibronectin in Wound Fluid and Plasma after Fracture[J];Journal of Medical Colleges of PLA;1988年01期
7 孙庆斌;刘植珊;高建章;杨瑞和;蒋美英;刘清乐;季品芳;陈文珍;;Relationship of Hyperbaric Oxygen Therapy of Acute Compartment Syndrome with Changes of Plasma Fibronectin Concentration[J];Journal of Medical Colleges of PLA;1989年01期
8 简华刚 ,林水金 ,冯素珍;Protective effects of fibronectin on vascular endothelial cells during trauma in rats[J];Chinese Journal of Traumatology;1998年01期
9 ;Study on using cryoprecipitate fibronectin ocusilla to treat the corneal epithelial wounds[J];中国输血杂志;2001年S1期
10 赵寅涛,李立新,戴建础;Schwann cells and fibronectin treating lesioned spinal cord of adult rats[J];Chinese Journal of Traumatology;1999年02期
相关会议论文 前5条
1 娄阁;钱素娟;高庆玉;宁小明;隋丽华;马宝璋;;子宫颈鳞状细胞癌Fibronectin、Laminin及Ⅳ型胶原的异常表达[A];2000全国肿瘤学术大会论文集[C];2000年
2 ;Inhibition of phosphoinositide 3-kinase suppresses the transforming grouth factor-pi-induced transformation of cardiac fibroblast[A];中华医学会心电生理和起搏分会第十次全国学术年会会议汇编[C];2012年
3 Liang Chen;Limeng Zhang;Jian Dai;Jianxin Zhang;Xuan Yang;Daolong Liu;Xijing Yang;Chunyu Tong;Liquan Yu;Zhanbo Zhu;Fanze Piao;Yudong Cui;;Comparative evaluation of immunogenicity and protective efficacy of clumping factors, fibronectin-binding protein A and their combination against Staphylococcus aureus[A];中国畜牧兽医学会家畜传染病学分会第八届全国会员代表大会暨第十五次学术研讨会论文集[C];2013年
4 ;Characterization of Fibronectin-mediated FAK Signaling Pathways in Lung Cancer Cell Migration and Invasion[A];第八次全国医学遗传学学术会议(中华医学会2009年医学遗传学年会)论文摘要汇编[C];2009年
5 于生金;樊建慧;柳林华;张丽君;李楠洋;张嘉宁;汪淑晶;;Caveolin-1上调小鼠肝癌细胞与fibronectin的粘附依赖于α-2,6连接唾液酸结构的增加[A];第三届泛环渤海（七省二市）生物化学与分子生物学会——2012年学术交流会论文集[C];2012年
相关博士学位论文 前1条
1 陈臻;LYTAK1抑制人视网膜色素上皮细胞增殖、迁移和间质化机制的研究[D];重庆医科大学;2016年
，本文编号：2141508
本文链接：https://www.wllwen.com/yixuelunwen/wuguanyixuelunwen/2141508.html
最近更新
教材专著
