甲基乙二醛、晚期糖基化產(chǎn)物對(duì)人腹膜間皮細(xì)胞生物學(xué)作用的體外研究
發(fā)布時(shí)間：2018-07-29 16:30
【摘要】： 目的：1．研究葡萄糖降解產(chǎn)物甲基乙二醛(MGO)、晚期糖基化產(chǎn)物(AGE-HSA)對(duì)人腹膜間皮細(xì)胞(HPMC)分泌血管內(nèi)皮細(xì)胞生長(zhǎng)因子(VEGF)、單核細(xì)胞趨化因子-1(MCP-1)的影響，參與的信號(hào)傳導(dǎo)通路及Simvastatin的干預(yù)作用。探討MGO和AGE-HSA對(duì)腹膜血管生成的影響及Simvastatin的干預(yù)作用。2．研究MGO和AGE-HSA對(duì)HPMC細(xì)胞毒性、細(xì)胞增殖的影響及參與信號(hào)傳導(dǎo)通路。探討MGO和AGE-HSA對(duì)腹膜間皮細(xì)胞的損傷的影響3．研究AGE-HSA對(duì)HPMC合成細(xì)胞外基質(zhì)的作用及參與信號(hào)傳導(dǎo)通路。探討AGE-HSA對(duì)腹膜纖維化的影響。 方法：分別用不同濃度的MGO、AGE-HSA、細(xì)胞內(nèi)信號(hào)傳導(dǎo)通路抑制劑、抗氧化劑(NAC)及Simvastatin作用于HPMC。1．采用半定量逆轉(zhuǎn)錄聚合酶鏈反應(yīng)(RT-PCR)、酶聯(lián)免疫吸附試驗(yàn)(ELISA)檢測(cè)HPMC的VEGF、MCP-1、纖維連接蛋白(FN)的基因和蛋白的表達(dá)。2．采用流式細(xì)胞術(shù)檢測(cè)細(xì)胞內(nèi)活性氧(ROS)水平。3．采用免疫印跡(Western blot)檢測(cè)MAPK信號(hào)傳導(dǎo)通路的激活。4．采用相差顯微鏡下觀察HPMC細(xì)胞形態(tài)的改變。5．采用MTT法檢測(cè)細(xì)胞活力。6．采用~3H-胸腺嘧啶核苷滲入法檢測(cè)細(xì)胞增殖。 結(jié)果：1．MGO和AGE-HSA以時(shí)間和濃度依賴的方式刺激HPMC的VEGF mRNA和蛋白的表達(dá)，二者有協(xié)同作用。2．AGE-HSA以時(shí)間和濃度依賴的方式刺激HPMC的MCP-1 mRNA和蛋白的表達(dá)，MGO對(duì)HPMC的MCP-1 mRNA的表達(dá)則沒(méi)有明顯的影響。3．MGO在刺激HPMC表達(dá)VEGF的同時(shí)，誘導(dǎo)細(xì)胞內(nèi)ROS的產(chǎn)生、同時(shí)以時(shí)間依賴方式促進(jìn)P-38MAPK磷酸化�？寡趸瘎㎞AC抑制細(xì)胞內(nèi)ROS后，P-38MAPK磷酸化減少，同時(shí)VEGF表達(dá)也減少。P-38MAPK特異性阻斷劑SB203580阻斷P-38MAPK磷酸化后，VEGF表達(dá)也減少。4．AGE-HSA在刺激HPMC表達(dá)VEGF、MCP-1的同時(shí)，誘導(dǎo)細(xì)胞內(nèi)ROS的產(chǎn)生、同時(shí)以時(shí)間依賴方式促進(jìn)P-42／44MAPK、P-38MAPK磷酸化，，用抗氧化劑NAC抑制細(xì)胞內(nèi)ROS后，P-42／44MAPK、P-38MAPK磷酸化減少，同時(shí)VEGF和MCP-1表達(dá)也減少。MEK特異抑制劑PD98059抑制P-42／44MAPK磷酸化后、或者P-38MAPK特異性阻斷劑SB203580抑制P-38MAPK磷酸化后，VEGF和MCP-1表達(dá)也減少了。AGE-HSA不激活JNK信號(hào)通路。5．Simvastatin能夠以劑量依賴的方式抑制AGE-HSA誘導(dǎo)的MCP-1表達(dá)，F(xiàn)PP和GGPP能夠逆轉(zhuǎn)Simvastatin的作用，而Simvastatin對(duì)VEGF表達(dá)則沒(méi)有明顯的作用。6．高濃度的MGO(160～320μM)以濃度依賴的方式使細(xì)胞活力下降，形態(tài)發(fā)生改變和抑制細(xì)胞增殖，而低濃度的MGO(0～80μM)以及AGE-HSA對(duì)細(xì)胞活力、形態(tài)和細(xì)胞增殖沒(méi)有明顯的影響。7．AGE-HSA以時(shí)間和濃度依賴的方式刺激HPMC合成FN。8．PKC激動(dòng)劑佛波酯PMA也能刺激HPMC合成FN，先用PMA耗竭細(xì)胞內(nèi)PKC或加用PKC抑制劑Calphostin C后，可以抑制AGE-HSA誘導(dǎo)的FN分泌。 結(jié)論：1．MGO和AGE-HSA部分通過(guò)誘導(dǎo)細(xì)胞內(nèi)ROS、激活P-42／44MAPK、P-38MAPK信號(hào)通路，促進(jìn)VEGF、MCP-1表達(dá)，參與腹膜血管生成。二者有協(xié)同作用。2．高濃度的MGO具有明顯細(xì)胞毒性和抑制細(xì)胞增殖作用，參與長(zhǎng)期腹透腹膜間皮細(xì)胞的損傷。3．AGE-HSA部分通過(guò)活化PKC誘導(dǎo)細(xì)胞內(nèi)ROS的表達(dá)增加，促進(jìn)HPMC合成和分泌FN。參與腹膜纖維化。4．Simvastatin可以通過(guò)非降脂作用抑制AGE-HSA誘導(dǎo)的MCP-1表達(dá)，這提示他汀類藥物在防治CAPD患者長(zhǎng)期腹透引起的腹膜血管生成、腹膜纖維化、腹膜失超濾方面有潛在的治療價(jià)值。
[Abstract]:Objective: 1. to study the effect of glucose degradation product methyl ethyl two aldehyde (MGO) and advanced glycosylation product (AGE-HSA) on the secretion of vascular endothelial growth factor (VEGF) and monocyte chemoattractant factor -1 (MCP-1) in human peritoneal mesothelial cells (HPMC), the intervention of signal transduction pathway and Simvastatin. The study of MGO and AGE-HSA on peritoneal vessels Effects of generation and intervention of Simvastatin.2. study on the toxicity of MGO and AGE-HSA on HPMC cytotoxicity, cell proliferation and involvement of signal transduction pathways. The effect of MGO and AGE-HSA on the damage of peritoneal mesothelial cells 3. research on the effect and signal transduction pathway of AGE-HSA on HPMC synthesis of extracellular matrix. The study of AGE-HSA on peritoneum The effect of fibrosis.
Methods: different concentrations of MGO, AGE-HSA, intracellular signal transduction pathway inhibitors, antioxidants (NAC) and Simvastatin were used to use semi quantitative reverse transcription polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA) to detect the expression of genes and proteins of HPMC in VEGF, MCP-1, and fibronectin (FN) in HPMC.1.. The intracellular reactive oxygen species (ROS) level of.3. was detected by flow cytometry. The activation of MAPK signal transduction pathway was detected by immunoblotting (Western blot). The morphological changes of HPMC cells were observed under phase contrast microscope. MTT method was used to detect the cell viability of.5., and MTT method was used to detect cell viability.6. using ~3H- thymidine infiltration method to detect cell proliferation.
Results: 1.MGO and AGE-HSA stimulated the expression of VEGF mRNA and protein in HPMC in time and concentration dependent manner. The two had synergistic effect of.2.AGE-HSA to stimulate the HPMC MCP-1 mRNA and protein expression in a time and concentration dependent manner. MGO did not significantly affect the expression of HPMC MCP-1. At the same time, it induces the production of ROS in cells, and promotes the phosphorylation of P-38MAPK in time dependent manner. Antioxidant NAC inhibits ROS, P-38MAPK phosphorylation decreases, and VEGF expression reduces.P-38MAPK specific blocking agent SB203580 blocking P-38MAPK phosphorylation, VEGF expression also reduces.4.AGE-HSA in stimulus HPMC expression At the same time, it induces the production of ROS in cell, while promoting P-42 / 44MAPK, P-38MAPK phosphorylation in time dependent manner, P-42 / 44MAPK, P-38MAPK phosphorylation, P-42 / 44MAPK, P-38MAPK phosphorylation with anti oxidant NAC, and VEGF and MCP-1 expression. After SB203580 inhibition of P-38MAPK phosphorylation, the expression of VEGF and MCP-1 also reduced the.AGE-HSA inactivation of JNK signaling pathway,.5.Simvastatin could inhibit AGE-HSA induced MCP-1 expression in a dose-dependent manner, FPP and GGPP could reverse the action of Simvastatin. To 320 micron M) in a concentration dependent manner, cell viability decreased, morphogenesis and cell proliferation inhibited, while low concentration of MGO (0~80 M) and AGE-HSA had no obvious effects on cell viability, morphology and cell proliferation..7.AGE-HSA was stimulated by time and concentration of HPMC synthesis FN.8.PKC agonist, phorbol ester PMA also spines The synthesis of FN by HPMC can inhibit AGE-HSA induced FN secretion after depletion of intracellular PKC or PMA inhibitor Calphostin C with PMA.
Conclusion: 1.MGO and AGE-HSA partially induce intracellular ROS, activate P-42 / 44MAPK, P-38MAPK signaling pathway, promote VEGF, MCP-1 expression, and participate in the angiogenesis of peritoneum. The two has synergistic effect of.2. high MGO with obvious cytotoxicity and inhibition of cell proliferation, and participates in the.3.AGE-HSA part of the long-term peritoneal mesothelial cells. The increased expression of ROS in cells induced by activation of PKC, promoting HPMC synthesis and secretion of FN. involved in peritoneal fibrosis.4.Simvastatin can inhibit AGE-HSA induced MCP-1 expression by non lipid-lowering effect, which suggests that statins can prevent peritoneal blood Guan Shengcheng, peritoneal fibrosis and peritoneal ultrafiltration in the long-term peritoneal dialysis of CAPD patients. There is a potential therapeutic value.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R329.2
本文編號(hào)：2153295
[Abstract]:Objective: 1. to study the effect of glucose degradation product methyl ethyl two aldehyde (MGO) and advanced glycosylation product (AGE-HSA) on the secretion of vascular endothelial growth factor (VEGF) and monocyte chemoattractant factor -1 (MCP-1) in human peritoneal mesothelial cells (HPMC), the intervention of signal transduction pathway and Simvastatin. The study of MGO and AGE-HSA on peritoneal vessels Effects of generation and intervention of Simvastatin.2. study on the toxicity of MGO and AGE-HSA on HPMC cytotoxicity, cell proliferation and involvement of signal transduction pathways. The effect of MGO and AGE-HSA on the damage of peritoneal mesothelial cells 3. research on the effect and signal transduction pathway of AGE-HSA on HPMC synthesis of extracellular matrix. The study of AGE-HSA on peritoneum The effect of fibrosis.
Methods: different concentrations of MGO, AGE-HSA, intracellular signal transduction pathway inhibitors, antioxidants (NAC) and Simvastatin were used to use semi quantitative reverse transcription polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA) to detect the expression of genes and proteins of HPMC in VEGF, MCP-1, and fibronectin (FN) in HPMC.1.. The intracellular reactive oxygen species (ROS) level of.3. was detected by flow cytometry. The activation of MAPK signal transduction pathway was detected by immunoblotting (Western blot). The morphological changes of HPMC cells were observed under phase contrast microscope. MTT method was used to detect the cell viability of.5., and MTT method was used to detect cell viability.6. using ~3H- thymidine infiltration method to detect cell proliferation.
Results: 1.MGO and AGE-HSA stimulated the expression of VEGF mRNA and protein in HPMC in time and concentration dependent manner. The two had synergistic effect of.2.AGE-HSA to stimulate the HPMC MCP-1 mRNA and protein expression in a time and concentration dependent manner. MGO did not significantly affect the expression of HPMC MCP-1. At the same time, it induces the production of ROS in cells, and promotes the phosphorylation of P-38MAPK in time dependent manner. Antioxidant NAC inhibits ROS, P-38MAPK phosphorylation decreases, and VEGF expression reduces.P-38MAPK specific blocking agent SB203580 blocking P-38MAPK phosphorylation, VEGF expression also reduces.4.AGE-HSA in stimulus HPMC expression At the same time, it induces the production of ROS in cell, while promoting P-42 / 44MAPK, P-38MAPK phosphorylation in time dependent manner, P-42 / 44MAPK, P-38MAPK phosphorylation, P-42 / 44MAPK, P-38MAPK phosphorylation with anti oxidant NAC, and VEGF and MCP-1 expression. After SB203580 inhibition of P-38MAPK phosphorylation, the expression of VEGF and MCP-1 also reduced the.AGE-HSA inactivation of JNK signaling pathway,.5.Simvastatin could inhibit AGE-HSA induced MCP-1 expression in a dose-dependent manner, FPP and GGPP could reverse the action of Simvastatin. To 320 micron M) in a concentration dependent manner, cell viability decreased, morphogenesis and cell proliferation inhibited, while low concentration of MGO (0~80 M) and AGE-HSA had no obvious effects on cell viability, morphology and cell proliferation..7.AGE-HSA was stimulated by time and concentration of HPMC synthesis FN.8.PKC agonist, phorbol ester PMA also spines The synthesis of FN by HPMC can inhibit AGE-HSA induced FN secretion after depletion of intracellular PKC or PMA inhibitor Calphostin C with PMA.
Conclusion: 1.MGO and AGE-HSA partially induce intracellular ROS, activate P-42 / 44MAPK, P-38MAPK signaling pathway, promote VEGF, MCP-1 expression, and participate in the angiogenesis of peritoneum. The two has synergistic effect of.2. high MGO with obvious cytotoxicity and inhibition of cell proliferation, and participates in the.3.AGE-HSA part of the long-term peritoneal mesothelial cells. The increased expression of ROS in cells induced by activation of PKC, promoting HPMC synthesis and secretion of FN. involved in peritoneal fibrosis.4.Simvastatin can inhibit AGE-HSA induced MCP-1 expression by non lipid-lowering effect, which suggests that statins can prevent peritoneal blood Guan Shengcheng, peritoneal fibrosis and peritoneal ultrafiltration in the long-term peritoneal dialysis of CAPD patients. There is a potential therapeutic value.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R329.2
【參考文獻(xiàn)】
相關(guān)期刊論文 前4條
1 汪濤,葉任高;腹膜透析應(yīng)作為腎臟病替代治療的首選方法[J];中國(guó)中西醫(yī)結(jié)合腎病雜志;2002年03期
2 洪富源,孫芳,劉軍,姚建,黃一新,唐知還;細(xì)胞內(nèi)活性氧在甲基乙二醛促人腹膜間皮細(xì)胞分泌血管內(nèi)皮生長(zhǎng)因子中的作用[J];腎臟病與透析腎移植雜志;2005年04期
3 田莉;晚期糖基化終末產(chǎn)物與腹膜透析相關(guān)性腹膜硬化[J];透析與人工器官;2005年01期
4 于青,趙東杰,姚建;腹膜血管形成對(duì)腹膜透析失超濾的影響[J];中國(guó)血液凈化;2005年03期
本文編號(hào)：2153295
本文鏈接：http://sikaile.net/yixuelunwen/binglixuelunwen/2153295.html
最近更新
教材專著
