反义细胞外信号调节激酶对移植物血管的保护作用

目的探讨反义细胞外信号调节激酶（ERK1／2）基因治疗对移植物血管的保护作用及可能的保护机制。方法建立BN至Lewis大鼠的腹主动脉移植模型。反义ERK1／2治疗组移植前取供者动脉血管段给予经脂质体包装的反义ERK1／2基因转染；腹主动脉移植术后1个月内受者每日从尾静脉或阴茎背静脉注入经脂质体包装的反义ERK1／2寡核苷酸100μl。对照组移植未经任何处理的血管段，移植后也无特殊处理。移植术后60d取移植段主动脉进行组织病理学观察内膜和胶原纤维变化；免疫组织化学法观察移植段血管ERK1／2基因的表达和CD4^＋、CD8^＋T淋巴细胞的浸润情况；ELISA法检测血清中细胞间粘附分子（ICAM-1）的变化。结果移植术后60d，对照组的移植动脉呈慢性移植物血管病表现，血管内膜显著增厚，移植血管中ERK1／2基因高表达，CD4^＋、CD8^＋T淋巴细胞大量浸润；反义ERK1／2基因治疗组移植动脉呈血管内膜炎改变，ERK1／2基因表达不明显，内膜有少量CD4^＋、CD8^＋T淋巴细胞；对照组ICAM-1表达显著高于反义ERK1／2治疗组（P〈0．05）。结论反义ERK1／2基因治疗对移植物血管具有保护作用，可以减缓慢性移植物血管病的发生，这种保护机制可能和减少ICAM-1的表达以及减少移植血管T淋巴细胞的浸润有关。     

阻断ERK2信号转导通路对血小板源生长因子-BB诱导血管平滑肌细胞增殖、迁移和表达转化生长因子-β1的影响

目的 观察ERK2信号转导通路在血小板源生长因子(PDGF-BB)诱导血管平滑肌细胞(VSMC)增殖、迁移和表达转化生长因子(TGF)-β1中的作用.方法 将原代培养的大鼠VSMC分为4组:(1)对照组;(2)PDGF刺激组;(3)Ad-LacZ组;(4)Adanti-ERK2组.用Westernblot检测细胞磷酸化ERK2蛋白水平;噻唑蓝(MTT)比色法测定细胞增殖率;流式细胞仪检测细胞细胞周期;Boyden小室测定细胞的跨膜迁移能力;酶联免疫吸附试验(ELISA)法检测细胞培养液上清中TGF-β1的浓度.结果 Adanti-ERK2组和对照组细胞增殖率、S期细胞百分比及跨膜迁移细胞数目明显低于PDGF刺激组和Ad-LacZ组(细胞增殖率:2.75%、0.00%比64.45%、61.88%;s期细胞百分比:14.18%、13.58%比38.14%、32.99%;跨膜迁移细胞数:8.2±3.2、6.3±2.6比24.8±6.1、23.3±5.8,均P<0.05);(2)Adanti-ERK2组和对照组细胞培养上清液中TGF-β1含量明显低于PDGF刺激组和Ad-LacZ组(P<0.05);而Adanti-ERK2组明显高于对照组(P<0.05).结论 ERK2信号转导通路参与调控PDGF-BB诱导的VSMC增殖、迁移和基因表达.反义ERK2基因预先转染阻断ERK信号转导能显著抑制PDGF-BB刺激的VSMC增殖、迁移,阻断细胞周期由G1期进入S期,并且部分下调TGF-β1的合成分泌.     

结缔组织生长因子诱导人肾小管上皮细胞向间充质细胞转化的实验研究

目的 探讨结缔组织生长因子(CTGF)对肾小管上皮细胞向间充质细胞转化的影响.方法 体外培养的人肾小管上皮细胞(HK-2),按是否用CTGF处理将其分为实验组和对照组.直接免疫荧光法、间接免疫化学检测培养72 h后肾小管上皮细胞E-cadherin、波形蛋白、α-平滑肌肌动蛋白((α-SMA)、细胞外信号调节激酶2(ERK2)的表达;Western blot方法 在72 h检测E-cadherin、波形蛋白和ERK2的表达;Boyden小室在1、3、5 d检测细胞迁移能力的变化.结果 (1)实验组上皮细胞表型标志蛋白E-cadherin随着时间的延长而表达递减,波形蛋白表达渐增,ERK2表达上调;(2)在第1天,实验组与对照组细胞迁移能力差异无统计学意义,第3天实验组迁移至滤膜下面细胞多于对照组,第5天实验组细胞迁移数明显高于对照组[(45.0±1.1):(14.0±1.2),P<0.05].结论 在体外,人肾小管上皮细胞由CTGF刺激表现出向间充质细胞转化的特性,并且ERK2信号转导通路可能参与了对这一过程的调控效应.     

结缔组织生长因子诱导人肾小管上皮细胞向间充质细胞转化的实验研究

Objective To investigate the role of connective tissue growth factor ( CTGF) in epithelial mesenchymal transition of HK-2 cells in vitro.Methods HK-2 cells were randomly divided into two groups; (1) control group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum only; and (2) experimental group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum and recombinant CTGF at a final concentration of 5 p-g/L The cells were collected at 72 h time points.Direct immunoiluorescence staining and immunohistochemistry were used to evaluate the E-cadherin,Vimentin,α-SMA and ERK2 in cells.Western-blotting was used to detect the E-cadherin,Vimentin and ERK2 protein expression.Boyden Chamber was used to detect the migration of tubular endothelium at 1 d,3 d and S d.Results There were less E-cadherin but more Vimentin expressed in cells of the experimental group.The presence of α-SMA was detected at 48 h with peak at 72 h in the cells of the experimental group.On the first day,the cellular migration in the two groups showed no difference.However,after 3 days,the transformed cells migrated surpassed the control group with peak at the 5th day [ (45.0±1.1) : (14.0±1.2),P < 0.05 ) ].Conclusion Connective tissue growth factor induces mesenchymal transformation of HK-2 cells,in which the ERK2 signaling pathway may play an important role.     

结缔组织生长因子诱导人肾小管上皮细胞向间充质细胞转化的实验研究

Objective To investigate the role of connective tissue growth factor ( CTGF) in epithelial mesenchymal transition of HK-2 cells in vitro.Methods HK-2 cells were randomly divided into two groups; (1) control group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum only; and (2) experimental group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum and recombinant CTGF at a final concentration of 5 p-g/L The cells were collected at 72 h time points.Direct immunoiluorescence staining and immunohistochemistry were used to evaluate the E-cadherin,Vimentin,α-SMA and ERK2 in cells.Western-blotting was used to detect the E-cadherin,Vimentin and ERK2 protein expression.Boyden Chamber was used to detect the migration of tubular endothelium at 1 d,3 d and S d.Results There were less E-cadherin but more Vimentin expressed in cells of the experimental group.The presence of α-SMA was detected at 48 h with peak at 72 h in the cells of the experimental group.On the first day,the cellular migration in the two groups showed no difference.However,after 3 days,the transformed cells migrated surpassed the control group with peak at the 5th day [ (45.0±1.1) : (14.0±1.2),P < 0.05 ) ].Conclusion Connective tissue growth factor induces mesenchymal transformation of HK-2 cells,in which the ERK2 signaling pathway may play an important role.     

结缔组织生长因子诱导人肾小管上皮细胞向间充质细胞转化的实验研究

Objective To investigate the role of connective tissue growth factor ( CTGF) in epithelial mesenchymal transition of HK-2 cells in vitro.Methods HK-2 cells were randomly divided into two groups; (1) control group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum only; and (2) experimental group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum and recombinant CTGF at a final concentration of 5 p-g/L The cells were collected at 72 h time points.Direct immunoiluorescence staining and immunohistochemistry were used to evaluate the E-cadherin,Vimentin,α-SMA and ERK2 in cells.Western-blotting was used to detect the E-cadherin,Vimentin and ERK2 protein expression.Boyden Chamber was used to detect the migration of tubular endothelium at 1 d,3 d and S d.Results There were less E-cadherin but more Vimentin expressed in cells of the experimental group.The presence of α-SMA was detected at 48 h with peak at 72 h in the cells of the experimental group.On the first day,the cellular migration in the two groups showed no difference.However,after 3 days,the transformed cells migrated surpassed the control group with peak at the 5th day [ (45.0±1.1) : (14.0±1.2),P < 0.05 ) ].Conclusion Connective tissue growth factor induces mesenchymal transformation of HK-2 cells,in which the ERK2 signaling pathway may play an important role.     

MAPK反义寡核苷酸诱导大鼠成纤维细胞凋亡的研究

目的 探讨MAPK反义寡核苷酸（antisense oligodeoxynucleotide，ASO）对大鼠肾成纤维细胞凋亡的影响。方法 脂质体转染法将MAPK反义寡核苷酸导入NRK-49F细胞中，Western—blotting测定细胞内MAPK蛋白的表达。DAPI染色观察细胞形态，流式细胞术检测细胞凋亡比率。结果 MAPKASO降低细胞MAPK蛋白含量；且可促进细胞凋亡，转染细胞出现凋亡特征性改变，凋亡率为35．8％，与其余各组有显著差异（P〈0．05）。结论 MAPK反义寡核苷酸通过阻断信号转导分子MAPK诱导大鼠成纤维细胞细胞凋亡，为防治肾移植术后间质纤维化提供一定的理论依据。     

结缔组织生长因子诱导人肾小管上皮细胞向间充质细胞转化的实验研究

Objective To investigate the role of connective tissue growth factor ( CTGF) in epithelial mesenchymal transition of HK-2 cells in vitro.Methods HK-2 cells were randomly divided into two groups; (1) control group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum only; and (2) experimental group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum and recombinant CTGF at a final concentration of 5 p-g/L The cells were collected at 72 h time points.Direct immunoiluorescence staining and immunohistochemistry were used to evaluate the E-cadherin,Vimentin,α-SMA and ERK2 in cells.Western-blotting was used to detect the E-cadherin,Vimentin and ERK2 protein expression.Boyden Chamber was used to detect the migration of tubular endothelium at 1 d,3 d and S d.Results There were less E-cadherin but more Vimentin expressed in cells of the experimental group.The presence of α-SMA was detected at 48 h with peak at 72 h in the cells of the experimental group.On the first day,the cellular migration in the two groups showed no difference.However,after 3 days,the transformed cells migrated surpassed the control group with peak at the 5th day [ (45.0±1.1) : (14.0±1.2),P < 0.05 ) ].Conclusion Connective tissue growth factor induces mesenchymal transformation of HK-2 cells,in which the ERK2 signaling pathway may play an important role.     

结缔组织生长因子诱导人肾小管上皮细胞向间充质细胞转化的实验研究

Objective To investigate the role of connective tissue growth factor ( CTGF) in epithelial mesenchymal transition of HK-2 cells in vitro.Methods HK-2 cells were randomly divided into two groups; (1) control group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum only; and (2) experimental group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum and recombinant CTGF at a final concentration of 5 p-g/L The cells were collected at 72 h time points.Direct immunoiluorescence staining and immunohistochemistry were used to evaluate the E-cadherin,Vimentin,α-SMA and ERK2 in cells.Western-blotting was used to detect the E-cadherin,Vimentin and ERK2 protein expression.Boyden Chamber was used to detect the migration of tubular endothelium at 1 d,3 d and S d.Results There were less E-cadherin but more Vimentin expressed in cells of the experimental group.The presence of α-SMA was detected at 48 h with peak at 72 h in the cells of the experimental group.On the first day,the cellular migration in the two groups showed no difference.However,after 3 days,the transformed cells migrated surpassed the control group with peak at the 5th day [ (45.0±1.1) : (14.0±1.2),P < 0.05 ) ].Conclusion Connective tissue growth factor induces mesenchymal transformation of HK-2 cells,in which the ERK2 signaling pathway may play an important role.     

结缔组织生长因子诱导人肾小管上皮细胞向间充质细胞转化的实验研究

Objective To investigate the role of connective tissue growth factor ( CTGF) in epithelial mesenchymal transition of HK-2 cells in vitro.Methods HK-2 cells were randomly divided into two groups; (1) control group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum only; and (2) experimental group including cells cultured in DMEM medium supplemented with 10% fetal bovine serum and recombinant CTGF at a final concentration of 5 p-g/L The cells were collected at 72 h time points.Direct immunoiluorescence staining and immunohistochemistry were used to evaluate the E-cadherin,Vimentin,α-SMA and ERK2 in cells.Western-blotting was used to detect the E-cadherin,Vimentin and ERK2 protein expression.Boyden Chamber was used to detect the migration of tubular endothelium at 1 d,3 d and S d.Results There were less E-cadherin but more Vimentin expressed in cells of the experimental group.The presence of α-SMA was detected at 48 h with peak at 72 h in the cells of the experimental group.On the first day,the cellular migration in the two groups showed no difference.However,after 3 days,the transformed cells migrated surpassed the control group with peak at the 5th day [ (45.0±1.1) : (14.0±1.2),P < 0.05 ) ].Conclusion Connective tissue growth factor induces mesenchymal transformation of HK-2 cells,in which the ERK2 signaling pathway may play an important role.