摘要
目的探讨血管内皮生长因子(VEGF)对肾小管上皮-间充质细胞转化(EMT)的作用及其与骨形成蛋白-7(BMP-7)、分化抑制因子(Id)2、Id3表达的关系。方法体外培养的人肾小管上皮细胞(HK-2)经转化生长因子-β1(TGF-β1,5ng/ml)与不同浓度VEGF165(0.1、1、10、100ng/ml)共同作用,或TGF-β1(5ng/ml)与血管内皮生长因子受体-1(VEGFR1)抗体(10μg/ml)共同作用48h后,免疫组织化学双染方法检测细胞α-平滑肌肌动蛋白(α-SMA)、E-钙黏素表达,实时荧光定量PCR法、Westernblot法检测细胞α-SMA、BMP-7、Id2和Id3的表达。TGF-β1(5ng/ml)、VEGF165(100ng/ml)与激活素受体样激酶-6/Fc嵌合体(Alk6/FcChimera)(2μg/ml,中和内源性BMP-7)共同作用48h后,Westernblot法检测细胞α-SMA、Id2的表达情况。结果TGF-β1作用后,α-SMA表达比正常对照显著增强(P<0.05),E-钙黏素、BMP-7、Id2及Id3的mRNA及蛋白质表达均显著减弱(P<0.05)。VEGF165以浓度依赖方式增强BMP-7及Id2蛋白表达,而显著降低TGF-β1上调α-SMA表达的作用(P<0.05)。加入VEGFR1抗体(10μg/ml)后,TGF-β1上调α-SMA表达的作用显著增强(P<0.05),而E-钙黏素、BMP-7、Id2表达显著减弱(P<0.05)。TGF-β1+VEGF165+Alk6/FcChimera共同作用后α-SMA蛋白表达比TGF-β1+VEGF165共同作用后显著增强(P<0.05),而Id2表达差异无显著性。结论VEGF165可抑制TGF-β1诱导HK-2细胞发生EMT;其机制可能与BMP-7和Id2表达上调有关,而与Id3表达变化无关。Id2表达增强可能与VEGF165的直接作用有关,而与BMP-7无关。
Objective To examine the relationship between effect of vascular endothelial growth factor （VEGF） on epithelial-myofibroblast transition （EMT） of HK-2 cells and changes in expressions of bone morphogenetic protein-7 （BMP-7） and inhibitor of DNA binding/differentiation （Id） 2, Id3. Methods The cultured HK-2 cells were co-treated with transforming growth factor-β1 （TGF-β1） （5 ng/ml） and VEGF165 （0.1, 1, 10, 100 ng/ml）, or with TGF-β1 （5 ng/ml） and VEGF receptor-1 neutralized antibody （ 10 μg/ ml）, and were also co-treated with TGF-β1 （5 ng/ml） and VEGF165 （ 100 ng/ml） with or without activin receptor-like kinase 6 （Alk6）/Fc Chimera （2 μg/ml, to neutralize endogenous BMP-7） for 48 hours, mRNA and protein expressions of α-smooth muscle actin （α-SMA） , E-cadherin, BMP-7, Id2 and Id3 of HK-2 cells were assessed with double-stain immunocytochemistry, real-time PCR and Western blot respectively. Results Compared with normal controls, a-SMA expression significantly increased, while E-cadherin, BMP-7, Id2, and Id3 mRNA and protein expressions markedly decreased in HK-2 cells treated with TGF-β1（5 ng/ml） （ P 〈 0.05 ）. VEGF165 interrupted TGF-131 induced α-SMA expression in a dose-dependent manner and upregulated BMP-7, Id2 mRNA and protein expressions of the cells （ P 〈 0.05 ）. α-SMA expression increased, while E-cadherin, BMP-7, and Id2 expressions decreased further in HK-2 cells co-treated with TGF-β1 and VEGFR1 antibody compared with normal controls （ P 〈 0.05 ）. When endogenous BMP-7 was neutralized with Alk6/Fc Chimera in the cells co-treated with TGF-β1 and VEGF165, α-SMA expression upregulated （P 〈 0.05 ） , while Id2 was not changed. Conclusions VEGF165 may partially inhibit TGF-β1-induced EMT of HK-2 cells in vitro. This effect is related to the upregulated expressions of BMP-7 and Id2. Id2 may be upregulated directly by VEGF165 , but not related to BMP-7.
出处
《中国医学科学院学报》
CAS
CSCD
北大核心
2008年第6期703-710,I0009,I0010,共10页
Acta Academiae Medicinae Sinicae
基金
国家自然科学基金(30570854)~~
关键词
血管内皮生长因子
上皮-间充质细胞转化
骨形成蛋白-7
分化抑制因子
vascular endothelial growth factor
epithelial-mesenchymal transition
bone morphogenetic protein-7
inhibitor of DNA binding/differentiation
