Tumstatin活性片段-T7肽抑制血管生成的实验研究

Experimental study on anti-angiogenic activity of T7 peptide derived from active fragment of Tumstatin

目的:探讨肿瘤抑素(Tumstatin)的活性片段T7肽在体外对血管生成的影响及相关机制。方法:选取人脐静脉内皮细胞(HUVECs)作为研究对象,在模拟肿瘤缺氧微环境的缺氧培养箱内进行相关实验。分为缺氧对照组、缺氧+T7肽(1.0μmol/L)处理组、缺氧+T7肽(2.0μmol/L)处理组。体外小管形成实验观察T7肽在缺氧环境下对血管生成的影响;CCK-8法检测T7肽对HUVECs活力的影响;Annexin V-FITC法检测细胞凋亡情况;Western blot检测Bax、Bcl-2蛋白的表达变化;免疫荧光及Western blot检测HUVECs中VE-钙黏蛋白表达变化。结果 :在缺氧环境下,T7肽可明显抑制体外血管生成(P<0.05),并显著降低HUVECs活力,促进其凋亡(P<0.05);T7肽可诱导促凋亡蛋白BAX表达增加(P<0.05),抗凋亡蛋白Bcl-2表达降低(P<0.01);T7肽可降低HUVECs内VE-钙黏蛋白的表达(P<0.05)。结论:T7肽可通过抑制内皮细胞活力、促进内皮细胞凋亡及降低VE-钙黏蛋白表达发挥其抗血管生成的作用。

Objective： To investigate the influence and related mechanisms of T7 peptide de- rived from tumstatin on angiogenesis in vitro. Methods： HUVECs were incubated in hypoxia chamber（37℃, 1% 02, 5% CO2, 94% N2） to simulate the hypoxic microenvironment in tumors, and grouped into： hypoxia group, hypoxia＋T7 peptide （1.0 μmol/L） group, and hypoxia＋T7 peptide（2.0μmol/L） group. The tube formation assay was applied to analyze the influence of T7 peptide on an- giogenesis under hypoxia. Cell Counting Kit-8 was applied to observe the cell viability. The apoptosis rate was detected with Annexin V-FITC, and the expression levels of pro-apoptotic protein BAX and anti-apoptotic protein Bcl-2 were observed with western btotl Immunofluorescence and western blot were used to detect the expression of VE-cadherin. Results： Under hypoxic condition, T7 peptide significantly inhibited capillary-like tube formation （P〈0.05）, inhibited ECs viability（P〈0.05）, and increased the ECs apoptosis rate in vitro; T7 peptide resulted in the downregulation of an- ti-apoptotic protein Bcl-2 （P〈0.01）and upregulation of pro-apoptotic protein BAX（P〈0.05）;the ex- pression of VE-cadherin was downregulated by T7 peptide significantly（P〈0.05）. Conclusions： T7 peptide could execute its anti-angiogenic activity via inhibition of ECs viability, induction of ECs apoptosis rate, and downregulation of VE-cadherin expression.