脉管Ⅱ号胶囊对Ang-1/Tie2信号通路及人脐静脉内皮细胞的影响

Effect of Maiguan Ⅱ Capsule on Ang-1/Tie2 Signaling Pathway and Human Umbilical Vein Endothelial Cells

目的研究脉管Ⅱ号胶囊对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)的影响,探索其促进血管生成的可能机制。方法按照随机数表法将20只SPF级雌性SD大鼠随机分为高剂量血清组与低剂量血清组,每组10只,分别以90 mg·kg^-1·d^-1与30 mg·kg^-1·d^-1的剂量喂服脉管Ⅱ号胶囊,用于制备含药血清;将HUVECs随机分为空白组、空载组、低剂量组、低剂量+Ang-1干扰组、高剂量组及高剂量+Ang-1干扰组,其中空白组与空载组HUVECs置于含有10%胎牛血清、1%丙酮酸钠、0.1 mg/mL肝素和0.05 mg/mL血管内皮生长因子的F12K培养基内,并分别接种于96孔培养板中放在37℃、5%CO2环境内培养,每3d更换1次培养基,待HUVECs培养至融合度达到60%~80%时,按每孔102μL将空载质粒转染液加入空载组培养板中,充分混匀,放回培养箱中培养4h,去上清,再加入完全培养基,在37℃、5%CO2环境内继续培养;低剂量组与低剂量+Ang-1干扰组HUVECs培养时将空白组培养环境中的10%胎牛血清替换为10%低剂量含药血清(取自低剂量血清组大鼠),并于相同时间加入Ang-1-pReceiver-M98转染液培养;高剂量组与高剂量+Ang-1干扰组HUVECs培养时将空白组培养环境中的10%胎牛血清替换为10%高剂量含药血清(取自高剂量血清组大鼠),并于相同时间加入Ang-1-pReceiver-M98转染液培养。分别用MTT法检测HUVECs的增殖情况,Hoechst33258染色法观察HUVECs形态,Elisa法及Western blotting法检测血管生成素1(angiopoietin-1,Ang-1)和酪氨酸激酶受体2(tyrosine kinase receptors 2,Tie2)的表达水平。结果与空白组对比,空载组HUVECs的相对增殖率及Ang-1、Tie2表达水平均无明显变化(P均>0.05);高剂量组和低剂量组HUVECs的相对增殖率及Ang-1、Tie2表达水平均显著增加(P均<0.05),且高剂量组显著高于低剂量组(P均<0.05);高剂量+Ang-1干扰组和低剂量+Ang-1干扰组HUVECs的增殖率及Ang-1、Tie2表达水平均显著降低(P均<0.05),且低剂量+Ang-1干扰组显著低于高剂量+Ang-1干扰组(P均<0.05)。空白组、空载组、高剂量组和低剂量组细胞均维持原有形态,细胞凋亡和细胞形态变化较少;高剂量+Ang-1干扰组和低剂量+Ang-1干扰组细胞凋亡增加,且以低剂量+Ang-1干扰组细胞凋亡数量较大。结论调节Ang-1/Tie2信号通路的表达可能是脉管Ⅱ号胶囊促进血管生成的作用机制,有待进一步深入研究证实。

Objective To study the effect of MaiguanⅡcapsule on human umbilical vein endothelial cells(HUVECs)and explore its mechanism in promoting angiogenesis.Methods Twenty Specific Pathogen Free(SPF),Sprague-Dawley(SD)female rats were divided,according to the random number table,into a high-dose serum group(10 rats)and a low-dose serum group(10 rats).MaiguanⅡcapsules were administered to two groups of rats at the dose of 90 mg·kg^-1·d^-1 and 30 mg·kg^-1·d^-1 respectively to prepare drug-containing serum.The HUVECs were randomly divided into blank group,unloaded group,low-dose group,low-dose+Ang-1 interference group,high-dose group and high-dose+Ang-1 interference group.The HUVECs in the blank group and unloaded group were placed in F12 K medium containing 10%fetal bovine serum,1%sodium pyruvate,0.1 mg/mL heparin,and 0.05 mg/mL vascular endothelial growth factors and inoculated in a 96-well culture plate and then cultured in an environment with a temperature of 37℃,5%CO2 concentration.The medium was changed every 3 days.When the HUVECs were cultured to a fusion degree of 60%to 80%,the unloaded plasmid transfection reagent was added to the culture plate in unloaded group(102μL per well).After fully mixing,the obtained mixture was cultured in incubator for another 4 h,the supernatant was removed,and complete medium was added for further culture in the same environment.During the culture of HUVECs in low-dose group and low-dose+Ang-1 interference group,10%fetal bovine serum in the blank group was replaced with 10%low-dose drug-containing serum(taken from rats in low-dose serum group),and at the same time,Ang-1-pReceiver-M98 transfection reagent was added for further culture.During the culture of the HUVECs in the high-dose group and high-dose+Ang-1 interference group,10%fetal bovine serum in the blank group was replaced with 10%high-dose drug-containing serum(taken from the rats in high-dose serum group)and Ang-1-pReceiver-M98 transfection reagent was added for further culture.MTT colorimetry and Hoechst 33258 staining was used to detect the proliferation of HUVECs and the morphology of HUVECs respectively,Elisa and Western blotting were adopted to measure the expression levels of Ang-1 and Tie2.Results Compared with the blank group,no significant difference was observed in the relative proliferation rate of HUVECs and the expression levels of Ang-1 and Tie2 in the unloaded group(all P>0.05);the relative proliferation rate of HUVECs and the expression levels of Ang-1 and Tie2 in the high-dose and low-dose groups increased significantly(all P<0.05),and they were significantly higher in the high-dose group than in the low-dose group(all P<0.05);the proliferation rate of HUVECs and the expression levels of Ang-1 and Tie2 in the high-dose+Ang-1 interference group and the low-dose+Ang-1 interference group decreased significantly(all P<0.05),and they were significantly lower in the low-dose+Ang-1 interference group than in the high-dose+Ang-1 interference group(all P<0.05).All cells in the blank group,unloaded group,high-dose group,and low-dose group maintained their original morphology,with little apoptosis;cell apoptosis increased in the high-dose+Ang-1 interference group and low-dose+Ang-1 interference group and more cell apoptosis was found in the former group.Conclusion The mechanism of action of MaiguanⅡcapsule for angiogenesis is probably that it can regulate the expression of Ang-1/Tie2 signaling pathway,but further research is needed to verify that.