摘要
目的观察外源性转化生长因子-β1(transforming growth factor-β1,TGF-β1)对人脐静脉内皮细胞系EA.hy926细胞迁移和间质标志蛋白表达的影响及其发生的机制。方法体外培养EA.hy926,随机分为2组:(1)对照组:培养液中添加PBS;(2)TGF-β1组:培养液中添加10μg.L-1TGF-β1。倒置相差显微镜观察细胞形态变化,MTT法检测TGF-β1对细胞增殖的影响,划痕实验检测细胞迁移能力的变化,免疫荧光方法检测VE-钙黏蛋白、β-连环蛋白、波形蛋白和Twist表达变化,RT-PCR检测VE-钙黏蛋白、波形蛋白和Twist基因表达变化。结果对照组内皮细胞呈铺路石样生长,TGF-β1组内皮细胞由铺路石样变为梭形。对照组培养24h、48h和72h时吸光度A值分别为0.4483±0.0172、0.5633±0.0087、0.7005±0.0479,TGF-β1组分别为0.3792±0.0231、0.4708±0.0185、0.5580±0.0159,2组不同时间点间比较差异均有统计学意义(t=4.779、9.058、5.648,P均<0.05);划痕实验检测对照组和TGF-β1组内皮细胞迁移个数分别为每视野(47.17±8.28)个、(63.00±11.33)个,2组比较差异有统计学意义(t=2.763,P=0.02)。免疫荧光检测可见TGF-β1组VE-钙黏蛋白和β-连环蛋白在细胞连接处表达减少,胞浆内游离的β-连环蛋白增多,波形蛋白和Twist表达增多。RT-PCR检测结果显示,对照组和TGF-β1组VE-钙黏蛋白目的基因灰度值与内参GAPDH灰度值的比值分别为0.8344±0.1933、0.4821±0.0909,波形蛋白分别为0.9687±0.1632、1.4404±0.1502,Twist分别为0.2316±0.0785、2.1626±0.2956,2组比较差异均有统计学意义(t=4.038、9.933、15.465,P均<0.05)。结论TGF-β1促进内皮细胞迁移,诱导内皮细胞表达间质细胞标志蛋白,同时抑制VE-钙黏蛋白表达。TGF-β1介导的Twist表达增多,可能在内皮细胞表达间质标志蛋白中起着重要作用。
Objective To investigate the effects of exogenous transforming growth factor-β1 （ TGF-β1 ） on cell migration and expression of mesenchymal marker protein in human umbilical vein endothelial cell line EA. hy926, and to explore its mechanisms. Methods EA. hy926 cells were cultured in vitro, and divided into two groups randomly. PBS was added to culture medium in control group;10 μg · L^-1 TGF-β1 was added to culture medium in TGF-β1 group. The changes of cell morphology were observed with inverted phase contrast microscope. The effects of TGF-β1 on cell proliferation were assessed by MTr assay. The cell migrative ability was examined with scratch test. The expression changes of VE-cadherin, β-catenin,vimentin and Twist protein were examined with immunofluorescence method, and the gene expression changes of VEcadherin, vimentin and Twist were evaluated with RT-PCR. Results Endothelial cells showed cobblestone-like morphology in control group, while changed to spindle-shaped from cobblestone-like morphology in TGF-β1 group. At culturing for 24 hours,48 hours and 72 hours,the absorbance A values of control group were 0.448 3 ±10. 017 2,0. 553 3 ± 0. 008 7,0. 700 5 ±0. 047 9 ,and those of TGF-β1 group were 0. 379 2 ± 0. 023 1,0. 470 8 ±0. 018 5,0. 558 0 ±0. 015 9, respectively. There were significant differences between two groups in different times （ t = 4.779,9.058,5.648 ;all P 〈 0.05 ）. The number of endothelial ceil migration per visual field in control group and TGF-β1 group were 47. 17 ±8.28,53.00 ±11.33, respectively（ t = 2. 763 ,P = 0.02 ）. Immunofluorescence assay showed that the expression of VE-cadherin and β1-catenin decreased at cell conjunction, free β1-catenin increased in the intracytoplasm, and the expression of vimentin and Twist increased in the TGF-β1 group. RT-PCR study showed that the ratios of gray value of the target gene to that of inner reference GAPDH for VE-cadherin in the control group and TGF-β1 group were 0.834 4 ±0. 193 3,0. 482 1 ±0. 090 9, those o
出处
《眼科新进展》
CAS
北大核心
2009年第9期653-658,共6页
Recent Advances in Ophthalmology
基金
国家自然科学基金资助(编号:30772386)
山东省自然科学基金资助(编号:Y2006C94)~~
