甲基乙二醛、晚期糖基化产物对人腹膜间皮细胞生物学作用的体外研究
发布时间：2018-07-29 16:30
【摘要】： 目的：1．研究葡萄糖降解产物甲基乙二醛(MGO)、晚期糖基化产物(AGE-HSA)对人腹膜间皮细胞(HPMC)分泌血管内皮细胞生长因子(VEGF)、单核细胞趋化因子-1(MCP-1)的影响，参与的信号传导通路及Simvastatin的干预作用。探讨MGO和AGE-HSA对腹膜血管生成的影响及Simvastatin的干预作用。2．研究MGO和AGE-HSA对HPMC细胞毒性、细胞增殖的影响及参与信号传导通路。探讨MGO和AGE-HSA对腹膜间皮细胞的损伤的影响3．研究AGE-HSA对HPMC合成细胞外基质的作用及参与信号传导通路。探讨AGE-HSA对腹膜纤维化的影响。 方法：分别用不同浓度的MGO、AGE-HSA、细胞内信号传导通路抑制剂、抗氧化剂(NAC)及Simvastatin作用于HPMC。1．采用半定量逆转录聚合酶链反应(RT-PCR)、酶联免疫吸附试验(ELISA)检测HPMC的VEGF、MCP-1、纤维连接蛋白(FN)的基因和蛋白的表达。2．采用流式细胞术检测细胞内活性氧(ROS)水平。3．采用免疫印迹(Western blot)检测MAPK信号传导通路的激活。4．采用相差显微镜下观察HPMC细胞形态的改变。5．采用MTT法检测细胞活力。6．采用~3H-胸腺嘧啶核苷渗入法检测细胞增殖。 结果：1．MGO和AGE-HSA以时间和浓度依赖的方式刺激HPMC的VEGF mRNA和蛋白的表达，二者有协同作用。2．AGE-HSA以时间和浓度依赖的方式刺激HPMC的MCP-1 mRNA和蛋白的表达，MGO对HPMC的MCP-1 mRNA的表达则没有明显的影响。3．MGO在刺激HPMC表达VEGF的同时，诱导细胞内ROS的产生、同时以时间依赖方式促进P-38MAPK磷酸化。抗氧化剂NAC抑制细胞内ROS后，P-38MAPK磷酸化减少，同时VEGF表达也减少。P-38MAPK特异性阻断剂SB203580阻断P-38MAPK磷酸化后，VEGF表达也减少。4．AGE-HSA在刺激HPMC表达VEGF、MCP-1的同时，诱导细胞内ROS的产生、同时以时间依赖方式促进P-42／44MAPK、P-38MAPK磷酸化，，用抗氧化剂NAC抑制细胞内ROS后，P-42／44MAPK、P-38MAPK磷酸化减少，同时VEGF和MCP-1表达也减少。MEK特异抑制剂PD98059抑制P-42／44MAPK磷酸化后、或者P-38MAPK特异性阻断剂SB203580抑制P-38MAPK磷酸化后，VEGF和MCP-1表达也减少了。AGE-HSA不激活JNK信号通路。5．Simvastatin能够以剂量依赖的方式抑制AGE-HSA诱导的MCP-1表达，FPP和GGPP能够逆转Simvastatin的作用，而Simvastatin对VEGF表达则没有明显的作用。6．高浓度的MGO(160～320μM)以浓度依赖的方式使细胞活力下降，形态发生改变和抑制细胞增殖，而低浓度的MGO(0～80μM)以及AGE-HSA对细胞活力、形态和细胞增殖没有明显的影响。7．AGE-HSA以时间和浓度依赖的方式刺激HPMC合成FN。8．PKC激动剂佛波酯PMA也能刺激HPMC合成FN，先用PMA耗竭细胞内PKC或加用PKC抑制剂Calphostin C后，可以抑制AGE-HSA诱导的FN分泌。 结论：1．MGO和AGE-HSA部分通过诱导细胞内ROS、激活P-42／44MAPK、P-38MAPK信号通路，促进VEGF、MCP-1表达，参与腹膜血管生成。二者有协同作用。2．高浓度的MGO具有明显细胞毒性和抑制细胞增殖作用，参与长期腹透腹膜间皮细胞的损伤。3．AGE-HSA部分通过活化PKC诱导细胞内ROS的表达增加，促进HPMC合成和分泌FN。参与腹膜纤维化。4．Simvastatin可以通过非降脂作用抑制AGE-HSA诱导的MCP-1表达，这提示他汀类药物在防治CAPD患者长期腹透引起的腹膜血管生成、腹膜纤维化、腹膜失超滤方面有潜在的治疗价值。
[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.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R329.2
本文编号：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.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R329.2
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