LYTAK1抑制人視網(wǎng)膜色素上皮細胞增殖、遷移和間質(zhì)化機制的研究
發(fā)布時間：2018-07-24 13:18
【摘要】：研究背景增殖性玻璃體視網(wǎng)膜病變(Proliferative vitreoretinopathy,PVR)是孔源性視網(wǎng)膜脫離(rhegmatogenous retinal detachment,RRD)以及行視網(wǎng)膜復位手術(shù)后的一種并發(fā)癥。PVR的病理變化涉及細胞去極化、遷移、黏附、增殖等一系列細胞活動過程,此外還與分泌膠原等細胞外基質(zhì)(extracellular matrix,ECM)的異常有關(guān);經(jīng)過上述病理過程之后,在視網(wǎng)膜前后表面以及玻璃體中將形成具有收縮能力的增殖膜,由于后者的收縮,從而造成牽拉性視網(wǎng)膜脫離(tractional retinal detachment,TRD)。在近年來的研究中,PVR的發(fā)生以及其機制一直是眼底病研究的熱點及難點。視網(wǎng)膜色素上皮細胞(retinal pigment epithelial,RPE)處于視網(wǎng)膜光感受器細胞層的外圍,其結(jié)構(gòu)由一層排列整齊的六角形細胞組成。目前的研究已證實,RPE細胞對PVR的發(fā)生和發(fā)展至關(guān)重要。上皮-間質(zhì)轉(zhuǎn)化(Epithelial-mesenchymal transition,EMT)為PVR的發(fā)生和發(fā)展過程中的重要環(huán)節(jié)。EMT是指具有上皮樣表型的細胞,當其失去極性后活動能力增強,在細胞間質(zhì)之間能夠自由移動并且表現(xiàn)出纖維樣表型的轉(zhuǎn)化過程。EMT通常分為三種亞型:其一,是原腸胚形成,機體中原始的上皮細胞通過EMT參與多種細胞的生物胚胎發(fā)育及器官的形成中;其二,為內(nèi)皮或者第二上皮轉(zhuǎn)化為組織成纖維細胞,從而在機體的傷口愈合以及器官的纖維化過程中發(fā)揮重要作用;其三,是上皮來源的細胞失去極性,從而轉(zhuǎn)化為具有侵襲能力的細胞。轉(zhuǎn)化生長因子β活化激酶-1(transforming growth factor-βactivated kinase-1,TAK1)為MAP3K家族中的主要成員之一,屬于色氨酸/蘇氨酸蛋白激酶,其活性受到TGF-β以及骨形態(tài)發(fā)生蛋白(bone morphogenetic proteins,BMPs)的調(diào)控。在生物機體中,TAK1經(jīng)由P38、JNK以及NF-κB通路活化實現(xiàn)對一系列特異性細胞轉(zhuǎn)錄因子的調(diào)控,從而影響細胞的生存、分化等生理病理過程,同時還對炎癥反應(yīng)具有調(diào)控作用。由于tak1功能具有多樣性,因此tak1在炎癥性疾病中成為了主要的靶激酶。除此之外,tak1還參與了mkk3/4-p38/jnk-ap-1以及ikk-nf-κb等重要信號轉(zhuǎn)導通路的活化,對細胞的存活、分化以及炎癥應(yīng)答具有十分重要的調(diào)控作用。轉(zhuǎn)化生長因子-β(transforminggrowthfactor-β,tgf-β)為一類新發(fā)現(xiàn)的具有對細胞分化和生長起調(diào)節(jié)作用的多功能細胞因子,大量研究證實,tgf-β是目前已知的具有促進組織纖維化的最重要的細胞因子,其在腎纖維化、肝纖維化和肺纖維化的患者血液中高表達。研究表明,tgf-β在pvr的發(fā)生和發(fā)展過程中發(fā)揮了重要作用,但其具體作用和機制并不十分清楚,尚需進一步深入研究。目的本課題旨在明確:1.tak1在tgf-β1誘導的人rpe細胞間質(zhì)化過程中的作用;2.探討tak1抑制劑lytak1對rpe細胞間質(zhì)化的作用;3.研究lytak1對rpe細胞間質(zhì)化的作用機制,旨在為pvr的治療奠定實驗基礎(chǔ)。方法1.采用elisa法檢測2014年6月~2014年12月期間在云南省第一人民醫(yī)院眼科30例pvr患者、20例imh患者以及同期15例正常志愿者,分別檢測玻璃體液和/或外周血中tgf-β1和tak1的表達水平。所有患者的年齡均在45~65歲之間。其中,pvr患者中有男性為18例,女性為12例;imh患者中男性為8例,女性為12例;健康志愿者中男性為9例,女性為6例。2.將0.01ng/mltgf-β1作用于人rpe細胞株arpe-19,培養(yǎng)24h后采用熒光定量pcr法檢測細胞中tak1mrna的表達;分別用不同濃度的lytak1(0μm、1μm、10μm、25μm、50μm)作用于0.01ng/mltgf-β1誘導后的arpe-19細胞,westernblot法檢測細胞中tak1蛋白的表達;transwell小室法檢測細胞的侵襲能力;流式細胞術(shù)(annexinv-fitc/pi雙染色法)檢測細胞的凋亡情況;細胞劃痕實驗檢測細胞的遷移能力。3.采用熒光定量pcr法分別檢測對照組、tgf-β1+dmso組以及tgf-β1+lytak1組arpe-19細胞中α-平滑肌肌動蛋白(α-smoothmuscleactin,α-sma)和纖維粘連蛋白(fibronectin)mrna的表達。采用westernblot法分別檢測對照組、tgf-β1+dmso組以及tgf-β1+lytak1各濃度組(0μm、1μm、10μm、25μm、50μm)arpe-19細胞中a-sma、fibronectin、p-smad2、p-smad3、ikkα、nf-κbp65蛋白的表達。結(jié)果1.pvr和imh兩組間tak1的濃度存在顯著差異(t=3.114,p=0.0350.05),即pvr患者玻璃體中tak1的濃度顯著高于imh組;對pvr和正常組兩組的間血清tak1濃度是否存在差異性進行比較分析,結(jié)果表明兩組間tak1的濃度存在顯著差異(t=3.156,p=0.0340.05),即pvr患者血清中tak1的濃度顯著高于imh組。上述結(jié)果表明,pvr患者玻璃體以及血清中tak1的含量均顯著高于imh患者,血清中的tak1含量也明顯高于健康人群,提示其可能對pvr的發(fā)生以及發(fā)展過程具有促進作用。pvr患者、imh患者以及健康志愿者的玻璃體液和/或血清中tgf-β1的濃度存在顯著差異(t=2.319,p=0.0330.05),即pvr患者玻璃體中tgf-β1的濃度顯著高于imh組。對pvr和正常組兩組間的血清tgf-β1濃度是否存在差異性采用t檢驗進行比較分析,結(jié)果表明兩組間tgf-β1的濃度存在顯著差異(t=3.312,p=0.0370.05),即pvr患者血清中tgf-β1的濃度顯著高于imh組。上述結(jié)果表明,pvr患者玻璃體和/或血清中tgf-β1的含量均顯著高于imh患者以及健康人群,提示其與pvr的發(fā)生以及發(fā)展過程有關(guān)。2.熒光定量pcr檢測結(jié)果表明,tgf-β1處理組arpe-19細胞中tak1mrna的表達顯著高于對照組,tgf-β1能夠明顯增高tak1蛋白的表達,而當用不同濃度的lytak1作用細胞后,細胞中tak1蛋白的表達量均發(fā)生不同程度的降低(p0.05),其中當lytak1的濃度為25μm時tak1蛋白的表達量最低。cck-8檢測表明,當lytak1作用24h時,各濃度組細胞的增殖活性均未見顯著變化(p0.05);而當lytak1作用48h以及72h之后,隨著lytak1作用濃度的增加,當lytak1作用濃度為50μm時對arpe-19細胞的增殖活性的抑制率最高。arpe-19細胞的增殖活性逐漸降低,表明lytak1對arpe-19細胞的活性呈劑量依賴性。transwell小室法檢測結(jié)果表明,lytak1各濃度組與對照組相比視野平均侵襲細胞數(shù)量顯著減少,且隨著lytak1濃度的增高其侵襲細胞數(shù)量逐漸降低,提示lytak1對arpe-19細胞的侵襲具有抑制作用,且呈劑量依賴性。流式細胞術(shù)檢測結(jié)果表明,隨著lytak1作用濃度的升高,arpe-19細胞的凋亡率也隨著上升,提示lytak1誘導arpe-19細胞凋亡呈現(xiàn)一定的劑量依賴性。細胞劃痕實驗結(jié)果表明,48 h后各濃度LYTAK1作用組ARPE-19細胞的遷移距離較對照組明顯縮短(P0.05);且隨著LYTAK1濃度的增高,細胞的遷移距離逐漸降低,提示LYTAK1抑制ARPE-19細胞遷移呈現(xiàn)一定的劑量依賴性。3.與對照組相比,TGF-β1+DMSO組以及TGF-β1+LYTAK1組中a-SMA和fibronectin mRNA的表達均明顯增高;而相較于TGF-β1+DMSO組,TGF-β1+LYTAK1組中a-SMA和fibronectin mRNA的表達量顯著降低。Western blot法檢測結(jié)果表明,TGF-β1+DMSO組細胞中a-SMA、fibronectin、p-Smad2、p-Smad3、IKKα、NF-κBp65蛋白的表達均顯著高于對照組。TGF-β1+LYTAK1各濃度組細胞中a-SMA、fibronectin、p-Smad2、IKKα、NF-κBp65蛋白的表達隨LYTAK1濃度的增高而逐漸降低,呈劑量依賴性,而LYTAK1對p-Smad3蛋白的表達無顯著影響。結(jié)論1.PVR患者血清以及玻璃體中TAK1和TGF-β1的表達量顯著增高,其表達上調(diào)可能與PVR的發(fā)生和發(fā)展有關(guān)。2.TGF-β1能夠顯著上調(diào)ARPE-19細胞中TAK1的表達;3.LYTAK1能夠明顯抑制ARPE-19細胞中TAK1的表達,且呈劑量依賴性;4.TGF-β1能夠通過上調(diào)ARPE-19細胞中TAK1、a-SMA、fibronectin、p-Smad2、p-Smad3、IKKα、NF-κBp65的表達,促進EMT的發(fā)生和發(fā)展;而LYTAK1能夠通過抑制TAK1、a-SMA、fibronectin、p-Smad2、IKKα、NF-κBp65的表達發(fā)揮抑制EMT的發(fā)生和發(fā)展。5.LYTAK1能夠明顯抑制ARPE-19細胞的增殖活性、細胞凋亡、侵襲及遷移,且呈劑量依賴性。
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R774.1
本文編號：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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R774.1
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