Inhibitory effect of vascular endothelial growth factor on transforming growth factor β1-induced epithelial-mesenchymal transition of HK2 cells and its relationship with connective tissue growth factor and PI3K-Akt pathway

Objective To examine the relationship of the inhibitory effect of vascular endothelial growth factor(VEGF) on epithelial-mesenchymal transition (EMT) induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor (CTGF) and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups: normal control group, TGF-β1 (5 μg/L) group, VEGF (100 μg/L) group, TGF-β1 plus VEGF group. LY294002 (25 μmol/L), the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study, α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin (FN) and collagen Ⅰ (Col Ⅰ) in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control (P<0.05), while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone (P<0.05, respectively). The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF co-treated cells, the expressions of α-SMA, CTGF, FN and Col Ⅰ were markedly up-regulated, when compared with those without LY294002 treatment (P<0.05). Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 ceils in vitro may be related to down-regulation of CTGF expression and reduction of FN and Col Ⅰ, which may be partly dependent on PI3K-Akt pathway.     

Inhibitory effect of vascular endothelial growth factor on transforming growth factor β1-induced epithelial-mesenchymal transition of HK2 cells and its relationship with connective tissue growth factor and PI3K-Akt pathway

Objective To examine the relationship of the inhibitory effect of vascular endothelial growth factor(VEGF) on epithelial-mesenchymal transition (EMT) induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor (CTGF) and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups: normal control group, TGF-β1 (5 μg/L) group, VEGF (100 μg/L) group, TGF-β1 plus VEGF group. LY294002 (25 μmol/L), the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study, α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin (FN) and collagen Ⅰ (Col Ⅰ) in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control (P<0.05), while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone (P<0.05, respectively). The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF co-treated cells, the expressions of α-SMA, CTGF, FN and Col Ⅰ were markedly up-regulated, when compared with those without LY294002 treatment (P<0.05). Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 ceils in vitro may be related to down-regulation of CTGF expression and reduction of FN and Col Ⅰ, which may be partly dependent on PI3K-Akt pathway.     

Inhibitory effect of vascular endothelial growth factor on transforming growth factor β1-induced epithelial-mesenchymal transition of HK2 cells and its relationship with connective tissue growth factor and PI3K-Akt pathway

Objective To examine the relationship of the inhibitory effect of vascular endothelial growth factor(VEGF) on epithelial-mesenchymal transition (EMT) induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor (CTGF) and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups: normal control group, TGF-β1 (5 μg/L) group, VEGF (100 μg/L) group, TGF-β1 plus VEGF group. LY294002 (25 μmol/L), the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study, α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin (FN) and collagen Ⅰ (Col Ⅰ) in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control (P<0.05), while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone (P<0.05, respectively). The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF co-treated cells, the expressions of α-SMA, CTGF, FN and Col Ⅰ were markedly up-regulated, when compared with those without LY294002 treatment (P<0.05). Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 ceils in vitro may be related to down-regulation of CTGF expression and reduction of FN and Col Ⅰ, which may be partly dependent on PI3K-Akt pathway.     

Inhibitory effect of vascular endothelial growth factor on transforming growth factor β1-induced epithelial-mesenchymal transition of HK2 cells and its relationship with connective tissue growth factor and PI3K-Akt pathway

Objective To examine the relationship of the inhibitory effect of vascular endothelial growth factor(VEGF) on epithelial-mesenchymal transition (EMT) induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor (CTGF) and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups: normal control group, TGF-β1 (5 μg/L) group, VEGF (100 μg/L) group, TGF-β1 plus VEGF group. LY294002 (25 μmol/L), the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study, α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin (FN) and collagen Ⅰ (Col Ⅰ) in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control (P<0.05), while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone (P<0.05, respectively). The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF co-treated cells, the expressions of α-SMA, CTGF, FN and Col Ⅰ were markedly up-regulated, when compared with those without LY294002 treatment (P<0.05). Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 ceils in vitro may be related to down-regulation of CTGF expression and reduction of FN and Col Ⅰ, which may be partly dependent on PI3K-Akt pathway.     

Inhibitory effect of vascular endothelial growth factor on transforming growth factor β1-induced epithelial-mesenchymal transition of HK2 cells and its relationship with connective tissue growth factor and PI3K-Akt pathway

Objective To examine the relationship of the inhibitory effect of vascular endothelial growth factor(VEGF) on epithelial-mesenchymal transition (EMT) induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor (CTGF) and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups: normal control group, TGF-β1 (5 μg/L) group, VEGF (100 μg/L) group, TGF-β1 plus VEGF group. LY294002 (25 μmol/L), the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study, α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin (FN) and collagen Ⅰ (Col Ⅰ) in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control (P<0.05), while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone (P<0.05, respectively). The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF co-treated cells, the expressions of α-SMA, CTGF, FN and Col Ⅰ were markedly up-regulated, when compared with those without LY294002 treatment (P<0.05). Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 ceils in vitro may be related to down-regulation of CTGF expression and reduction of FN and Col Ⅰ, which may be partly dependent on PI3K-Akt pathway.     

Inhibitory effect of vascular endothelial growth factor on transforming growth factor β1-induced epithelial-mesenchymal transition of HK2 cells and its relationship with connective tissue growth factor and PI3K-Akt pathway

Objective To examine the relationship of the inhibitory effect of vascular endothelial growth factor(VEGF) on epithelial-mesenchymal transition (EMT) induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor (CTGF) and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups: normal control group, TGF-β1 (5 μg/L) group, VEGF (100 μg/L) group, TGF-β1 plus VEGF group. LY294002 (25 μmol/L), the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study, α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin (FN) and collagen Ⅰ (Col Ⅰ) in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control (P<0.05), while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone (P<0.05, respectively). The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF co-treated cells, the expressions of α-SMA, CTGF, FN and Col Ⅰ were markedly up-regulated, when compared with those without LY294002 treatment (P<0.05). Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 ceils in vitro may be related to down-regulation of CTGF expression and reduction of FN and Col Ⅰ, which may be partly dependent on PI3K-Akt pathway.     

Inhibitory effect of vascular endothelial growth factor on transforming growth factor β1-induced epithelial-mesenchymal transition of HK2 cells and its relationship with connective tissue growth factor and PI3K-Akt pathway

Objective To examine the relationship of the inhibitory effect of vascular endothelial growth factor(VEGF) on epithelial-mesenchymal transition (EMT) induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor (CTGF) and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups: normal control group, TGF-β1 (5 μg/L) group, VEGF (100 μg/L) group, TGF-β1 plus VEGF group. LY294002 (25 μmol/L), the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study, α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin (FN) and collagen Ⅰ (Col Ⅰ) in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control (P<0.05), while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone (P<0.05, respectively). The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF co-treated cells, the expressions of α-SMA, CTGF, FN and Col Ⅰ were markedly up-regulated, when compared with those without LY294002 treatment (P<0.05). Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 ceils in vitro may be related to down-regulation of CTGF expression and reduction of FN and Col Ⅰ, which may be partly dependent on PI3K-Akt pathway.     

Inhibitory effect of vascular endothelial growth factor on transforming growth factor β1-induced epithelial-mesenchymal transition of HK2 cells and its relationship with connective tissue growth factor and PI3K-Akt pathway

Objective To examine the relationship of the inhibitory effect of vascular endothelial growth factor(VEGF) on epithelial-mesenchymal transition (EMT) induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor (CTGF) and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups: normal control group, TGF-β1 (5 μg/L) group, VEGF (100 μg/L) group, TGF-β1 plus VEGF group. LY294002 (25 μmol/L), the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study, α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin (FN) and collagen Ⅰ (Col Ⅰ) in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control (P<0.05), while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone (P<0.05, respectively). The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF co-treated cells, the expressions of α-SMA, CTGF, FN and Col Ⅰ were markedly up-regulated, when compared with those without LY294002 treatment (P<0.05). Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 ceils in vitro may be related to down-regulation of CTGF expression and reduction of FN and Col Ⅰ, which may be partly dependent on PI3K-Akt pathway.     

Inhibitory effect of vascular endothelial growth factor on transforming growth factor β1-induced epithelial-mesenchymal transition of HK2 cells and its relationship with connective tissue growth factor and PI3K-Akt pathway

Objective To examine the relationship of the inhibitory effect of vascular endothelial growth factor(VEGF) on epithelial-mesenchymal transition (EMT) induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor (CTGF) and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups: normal control group, TGF-β1 (5 μg/L) group, VEGF (100 μg/L) group, TGF-β1 plus VEGF group. LY294002 (25 μmol/L), the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study, α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin (FN) and collagen Ⅰ (Col Ⅰ) in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control (P<0.05), while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone (P<0.05, respectively). The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF co-treated cells, the expressions of α-SMA, CTGF, FN and Col Ⅰ were markedly up-regulated, when compared with those without LY294002 treatment (P<0.05). Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 ceils in vitro may be related to down-regulation of CTGF expression and reduction of FN and Col Ⅰ, which may be partly dependent on PI3K-Akt pathway.     

Inhibitory effect of vascular endothelial growth factor on transforming growth factor β1-induced epithelial-mesenchymal transition of HK2 cells and its relationship with connective tissue growth factor and PI3K-Akt pathway

Objective To examine the relationship of the inhibitory effect of vascular endothelial growth factor(VEGF) on epithelial-mesenchymal transition (EMT) induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor (CTGF) and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups: normal control group, TGF-β1 (5 μg/L) group, VEGF (100 μg/L) group, TGF-β1 plus VEGF group. LY294002 (25 μmol/L), the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study, α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin (FN) and collagen Ⅰ (Col Ⅰ) in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control (P<0.05), while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone (P<0.05, respectively). The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF co-treated cells, the expressions of α-SMA, CTGF, FN and Col Ⅰ were markedly up-regulated, when compared with those without LY294002 treatment (P<0.05). Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 ceils in vitro may be related to down-regulation of CTGF expression and reduction of FN and Col Ⅰ, which may be partly dependent on PI3K-Akt pathway.     

丹参抑制转化生长因子-β1诱导的肌源性干细胞向肌成纤维母细胞分化

目的 观察丹参对失神经骨骼肌肌源性干细胞(MDSCs)向肌成纤维母细胞分化的抑制作用.方法 采用差速贴壁法分离出大鼠失神经骨骼肌MDSCs,加入转化生长因子-β1(TGF-β1)和丹参进行干预,将细胞分为3组:A:对照组;B:10 μg/L TGF-β1组;C:10 μg/L TGF-β1+ 150 mg/L丹参组.荧光实时定量聚合酶链反应(qRT-PCR)和Western blot检测各组细胞在干预后5个时间点α-平滑肌肌动蛋白(α-SMA)和波形蛋白(Vimentin) mRNA和蛋白表达.结果 与A组比较,B组和C组细胞α-SMA、Vimentin的mRNA在干预后第2、3、5、7天均明显升高(P＜0.05),其蛋白表达在干预后第3、4、6、8天亦显著升高(P＜0.05);与B组比较,C组细胞α-SMA、Vimentin的mRNA和蛋白在对应时间点均明显降低(P＜0.05).结论 丹参能抑制TGF-β1诱导的失神经骨骼肌MDSCs向肌成纤维母细胞的分化.     

转化生长因子β1诱导肾间质成纤维细胞表型转化及其对结缔组织生长因子基因表达的影响

目的 研究转化生长因子β1（transforming growth factor beta-1,TGF-β1）诱导大鼠正常肾间质成纤维细胞（NRK-49F）表型转化过程中结缔组织生长因子（connective tissue growth factor,CTGF）基因表达的变化.方法 以不同浓度的TGF-β1（0、0.5、1、2、5、10 ng/ml）刺激NRK-49F细胞,分别应用MTT比色法、Western Blot、Northern Blot方法,检测TGF-β1刺激后肾间质成纤维细胞的增殖、Ⅰ型、Ⅲ型前胶原mRAN的表达、细胞表型标志物α-SMA mRNA及蛋白质表达、CTGFmRNA的表达变化.结果 1 ng/ml浓度以上TGF-β1能显著促进NRK-49F细胞增殖,上调Ⅰ、Ⅲ型前胶原mRNA的表达水平,诱导肌成纤维细胞表型标志α-SMA mRNA及蛋白质的表达,显著上调CTGF mRNA的表达水平（P＜0.01或P＜0.05）;以上效应均呈浓度依赖性.同时CTGF mRNA表达水平的增高与细胞表型转化的程度相一致.结论 TGF β1能诱导肾间质成纤维细胞发生表型转化,并促进了细胞增殖及细胞外基质的合成;该效应与TGF β1显著上调CTGF的基因表达一致.     

TGF-β1对UVA照射皮肤成纤维细胞Ⅰ型，Ⅲ型胶原合成和表达的影响

目的：探讨转化生长因子-β1（tansforming gowth factor—beta 1,TGF—β1）对长波紫外线（ultraviolet A,UVA）照射皮肤成纤维细胞Ⅰ型、Ⅲ型胶原合成和表达的影响。方法：通过MTT法检测TGF-β1干预后成纤维细胞增殖活性,选择UVA照射剂量为15J／cm^2,联免疫法（ELISA）测定不同剂量即TGF—β1小剂量组（UVA＋TGF—β1 0．1ng／ml）、中剂量组（UVA＋TGF—β1 1ng／ml）、大剂量组（UVA＋TGF—β1 10ng／ml）处理后成纤维细胞清夜中Ⅰ型、Ⅲ型胶原含量,半定量RT—PCR检测成纤维细胞Ⅰ型、Ⅲ型胶原mRNA表达。结果：UVA照射体外培养的皮肤成纤维细胞,导致成纤维细胞增殖活性下降,给予不同剂量TGF-β1干预后,成纤维细胞增殖活性与UVA照射组比较,明显提高;成纤维细胞上清液中Ⅰ型、Ⅲ型胶原含量增加,成纤维细胞内Ⅰ型、Ⅲ型胶原mRNA表达增强。结论：TGF-β1可提高UVA照射体外培养成纤维细胞增殖活性;增加UVA照射体外培养的皮肤成纤维细胞Ⅰ型、Ⅲ型胶原含量,增强成纤维细胞Ⅰ型、Ⅲ型胶原mRNA表达,对皮肤成纤维细胞起保护作用。     

CTGF shRNA表达质粒的构建及对大鼠肌源性干细胞CTGF表达的影响

目的 观察针对结缔组织生长因子(CTGF)所构建的短发卡RNA(shRNA)对转化生长因子β1(TGF-β1)诱导大鼠肌源性干细胞(MDSCs)表达CTGF的影响.方法 针对CTGF mRNA上的序列,体外合成表达shRNA的DNA质粒载体pGenesil-3-shRNA并行测序鉴定;从新生SD大鼠骨骼肌中分离培养MDSCs并鉴定.实验分成对照组(MDSCs+TGF-β1培养),实验组(MDSCs+ TGF-β1+pGenesil-3-shRNA培养).观察shRNA质粒转染结果.利用Real-Time PCR及Western Blot检测CTGF mRNA和CTGF蛋白表达.结果 正确构建了靶向CTG,F基因的质粒载体pCenesil-3-shCTGF;大鼠MDSCs 48 h、72h和96h时实验组的CTGF mRNA及蛋白质水平均低于对照组(P＜0.05).结论 针对CTGF构建的pGenesil-3-shCTGF能够转染MDSCs,并在48h、72h和96h时能明显降低TGF-β1刺激MDSCs所产生CTGF的表达.     

Erk和p38信号转导通路对大鼠肌源性干细胞内结缔组织生长因子表达的调控作用

目的 探讨肌源性干细胞(muscle derived stem cells,MDSC)受转化生长因子β1(transforming growth factor-1,TCF-β1)刺激产生结缔组织生长因子(connective tissue growth factor,CTGF)的过程中,Erk信号转导通路和p38信号转导通路是否发挥作用.方法 采用细胞差速贴壁法,从新生大鼠骨骼肌中分离提取肌源性干细胞,进行细胞表型鉴定后传代培养.实验设空白组、对照组和实验组共8组:空白组,添加液为基础培养基;对照组,基础培养基中加TGF-β1;PD98059实验组,分别用20、40、80μM的PD98059预处理;SB203580实验组,分别用0.2、0.5、1.0μM的SB203580预处理.用Real Time-PCR和Western Blot分别检测细胞中CTGF mRNA和蛋白质的水平.方果 空白组、对照组和PD98059实验组间CTGF mRNA和蛋白质的相对表达量差异均有统计学意义(P<0.05),对照组和SB203580实验组间差异均无统计学意义(P>0.05).方论 TGF-β1诱导NDSC产生CTCF的过程中,存在着Erk信号转导途径,不存在p38信号转导途径.     

神经纤维瘤组织中致纤维化生长因子和胶原的表达

目的：观察结缔组织生长因子（CTGF）在神经纤维瘤组织中的表达情况及与其上、下游效应基因表达的关系,探讨CTGF在神经纤维瘤组织中的促纤维化机制。方法：收集神经纤维瘤组织、瘢痕疙瘩组织及正常皮肤组织标本,免疫组化检测CTGF的表达,逆转录-聚合酶链式反应（RT-PCR）方法检测组织CTGF、TGF-β1、Ⅰ型胶原（ColⅠ）及Ⅲ型胶原（ColⅢ）mRNA表达,Western blotting检测组织CTGF蛋白的表达。结果：免疫组织化学染色发现,CTGF在瘤细胞中有强阳性表达,在汗腺和皮脂腺细胞中有阳性表达。神经纤维瘤组织及瘢痕疙瘩组织中CTGF、TGF-β1、ColⅠ和ColⅢ的mRNA过表达,而正常皮肤组织无或仅有极弱表达。Western blotting结果显示神经纤维瘤组织有明显的CTGF蛋白表达条带。结论：在神经纤维瘤发病过程中,CTGF可能是重要的调控因子之一,与TGF-β1协同促进细胞外基质合成。     

腹膜透析液诱导结缔组织生长因子在大鼠腹膜间皮细胞表达

目的 研究应用不同腹膜透析液对大鼠腹膜间皮细胞(RPMCs)结缔组织生长因子(CTGF)合成的影响。方法 分离培养的RPMC分为6组，分别以不同腹膜透析液[1.5% Dextrose(低糖组)、2.5% Dextrose(中糖组)、4.25% Dextrose(高糖组)、7.5% Icodextrin(糊精组) ]进行刺激培养，而无血清DMEM为阴性对照(对照组)， TGF-β1(2.5 ng/ml)为阳性对照(阳性对照组)。刺激培养24 h后，RT-PCR法检测RPMCs的CTGF mRNA、胶原Ⅰ mRNA、α-SMA mRNA表达。Western印迹法检测RPMCs的CTGF、胶原Ⅰ、α-SMA蛋白表达以及培养上清中的CTGF蛋白表达。结果 各组均见CTGF mRNA表达，高糖组、阳性对照组CTGF mRNA 表达水平显著高于中糖组、低糖组及对照组(P < 0.05)；中糖组与糊精组CTGF mRNA 表达亦显著上调(P < 0.05)。各组细胞均检测到CTGF蛋白质表达，为相对分子质量38 000及 25 000的2种亚型，与RT-PCR结果一致。高糖组、阳性对照组CTGF 蛋白表达水平显著高于糊精组、中糖组、低糖组及对照组(P < 0.05)。RPMCs培养上清液中检测出CTGF 38 000亚型的表达，其表达强弱趋势与CTGF在细胞中表达一致。高糖组、阳性对照组胶原I mRNA、蛋白质的表达水平显著高于对照组(P < 0.05)，其余各组间无显著性差异。各组α-SMA mRNA、蛋白质表达未见显著性差异(P > 0.05)。结论 正常培养的RPMCs表达低水平的CTGF。腹膜透析液、尤其是高浓度葡萄糖透析液，能明显上调CTGF表达的水平，这可能是导致长期腹膜透析过程中腹膜结构改变的机制之一。糊精腹膜透析液生物相容性可能优于高浓度葡萄糖透析液。     

结缔组织生长因子介导转化生长因子β1促进瘢痕成纤维细胞转分化的研究

目的 了解结缔组织生长因子(CTGF)介导TGF-β1发挥促人增生性瘢痕Fb(HSFb)转分化的作用.方法 体外培养人HSFb,取5份细胞标本分别加入不同浓度TGF-β1(0、2.5、5.0、7.5、10.0 ng/mL),作用48 h后待测.余下标本分为:空白对照组;CTGF刺激组,培养液中加入终浓度10.0 ng/mL重组人CTGF;TGF-β1刺激组,培养液中加入终浓度10.0 ng/mL重组人TGF-β1;CTGF反义寡脱氧核苷酸(ASODN)转染组,细胞转染CTGF ASODN后,加入培养液;CTGF ASODN转染+TGF-β1刺激组,细胞转染CTGF ASODN后2 h,加人含终浓度10.0 ng/mL重组人TGF-β1的培养液.蛋白质印迹法分析不同浓度TGF-β1刺激对细胞CTGF表达的影响,比较各组α-平滑肌肌动蛋白(α-SMA)表达变化;流式细胞仪检测α-SMA阳性细胞百分率.结果 TGF-β1浓度为10.0 ng/mL时,CTGF的表达明显高于未受刺激的细胞(P<0.05).CTGF刺激组与TGF-β1刺激组α-SMA表达明显高于空白对照组(P<0.01).CTGF ASODN转染组以及CTGF ASODN转染+TGF-β1刺激组α-SMA表达与空白对照组接近(P>0.05).上述各组细胞α-SMA阳性细胞百分率依次为(10.8±2.8)%、(29.1±4.0)%、(28.7±4.8)%、(10.7±2.3)%、(14.3±2.9)%,统计学分析结果类似于α-SMA表达.结论 CTGF是TGF-β1发挥促人HSFb转分化的重要下游效应分子之一.     

CCN3 inhibits deposition of extracellular matrix of glomerular mesangial cells via promoting the expression of microRNA-29a

Objective To investigate the effects of nephroblastoma over-expressed protein (CCN3) on the formation of extracellular matrix (ECM) induced by transforming growth factor -β1 (TGF-β1) in human mesangial cells (HMCs) and its underlying signal transduction mechanism related with microRNA-29(miRNA-29). Methods HMCs were pretreated with different doses of exogenous CCN3 (5 μg/L, 50 μg/L and 500 μg/L) or transfected with pcDNA3.1(+)-CCN3 before exposed to TGF-β1(2 μg/L), to observe the expression of fibronectin (FN), type Ⅰ collagen (COLⅠ) and miRNA-29a, b and c. The mimics or inhibitor of the miRNA-29a were transfected into HMCs to analyze whether miRNA-29a affect CCN3. The expressions of FN mRNA, COLⅠmRNA and miRNA-29 family were detected by real time PCR. The protein expressions of FN and COLⅠ were detected by Western blotting and cell immunofluorescence. Results (1) Compared with the normal control group, the expressions of FN and COLⅠ were up-regulated in TGF-β1 group, while the expressions of miRNA-29a, b, c were down-regulated in TGF-β1 group (all P＜0.05). (2) Compared with the TGF-β1 group, the expressions of FN and COLⅠ were decreased when pretreated with the different doses of exogenous of CCN3 or transfected with pcDNA3.1(+)-CCN3 (all P＜0.05). Meanwhile, the expression of miRNA-29a was significantly increased when pretreated with 50 μg/L and 500 μg/L CCN3 or transfected with pcDNA3.1(+)-CCN3 (all P＜0.05); whereas miRNA-29b and c had no statistical difference (all P＞0.05). (3) Compared with TGF-β1+CCN3 group, the expressions of FN and COLⅠ were decreased in CCN3+TGF-β1+miRNA-29a mimics group (all P＜0.05), whereas the expressions of FN and COLⅠ in CCN3+TGF-β1+miRNA-29a inhibitors group were increased (all P＜0.05). Conclusions CCN3 reduces the TGF-β1-induced production of ECM by the up-regulation of miRNA-29a.     

血管内皮生长因子抑制肾小管上皮间充质转化及其与结缔组织生长因子和PI3K-Akt信号通路的关系

目的　探讨血管内皮生长因子（VEGF）对转化生长因子β1（TGF-β1）诱导的肾小管上皮间充质转化（EMT）的作用,及其与结缔组织生长因子（CTGF）、PI3K-Akt信号通路的关系。 方法 （1）将体外培养的HK2细胞分为正常对照组、TGF-β1（5 μg/L,下同）组、VEGF组（100 μg/L,下同）、TGF-β1＋VEGF组。HK2细胞体外培养48 h,用免疫组化双染方法检测肾小管上皮细胞α平滑肌肌动蛋白（α-SMA）和E钙黏蛋白的表达。（2）将体外培养的HK2细胞分为正常对照组、TGF-β1组、VEGF组、TGF-β1＋VEGF组、PI3K-Akt信号通路阻断剂LY294002组（25 μmol/L,下同）、TGF-β1＋LY294002组、VEGF＋LY294002组、TGF-β1＋VEGF＋LY294002组。HK2细胞体外培养48 h,用Western印迹和RT-PCR方法检测α-SMA和CTGF的表达;用ELISA方法检测培养上清中纤连蛋白（FN）和I型胶原（ColⅠ）的表达。 结果 免疫组化结果显示,TGF-β1组α-SMA表达比正常对照组增强,而E钙黏蛋白表达减弱; TGF-β1＋VEGF组α-SMA表达比TGF-β1组显著减弱,而E钙黏蛋白表达增强。 TGF-β1组α-SMA、CTGF蛋白和 mRNA及FN、ColⅠ表达比正常对照组显著增强（均P < 0.05）;TGF-β1＋VEGF组α-SMA、CTGF蛋白和mRNA及FN、ColⅠ表达比TGF-β1组显著减弱（均P < 0.05）;TGF-β1＋VEGF＋LY294002组α-SMA、CTGF蛋白和 mRNA及FN、ColⅠ表达比TGF-β1＋VEGF组显著增强（均P < 0.05）。 结论　VEGF能抑制TGF-β1诱导的体外培养的HK2细胞发生EMT,其机制可能与VEGF下调HK2细胞CTGF表达及减少细胞外FN、ColⅠ合成有关。VEGF的这种作用可能部分通过PI3K-Akt信号转导通路实现,其确切机制有待进一步研究。