| 王世军,张美吉,杨尾莲,陈福伟,林欣欣.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[J].中国烧伤创疡杂志,2020,(1):15~21. |
| DOI： |
| 中文关键词: 脉管Ⅱ号胶囊 人脐静脉内皮细胞 Ang-1/Tie2 信号通路 血管新生 细胞活力 凋亡 |
| 英文关键词:aiguan Ⅱ capsule Human umbilical vein endothelial cells Ang-1/Tie2 signaling pathway Angio- genesis Cell viability Apoptosis |
| 基金项目:福建省自然科学基金 (2017J01311) |
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| 中文摘要: |
| 【摘要】 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 3 d 更换 1 次培养基，待 HUVECs 培养至融合度达到 60%～80% 时，按每孔 102 μL 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 4 h，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。 |
| 英文摘要: |
| 【Abstract】 Objective To study the effect of Maiguan Ⅱ capsule 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). Maiguan Ⅱ capsules 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 F12K 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 (102 μL per well). Af- ter 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 colorime- try 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 expres- sion levels of Ang-1 and Tie2 in the high-dose＋Ang-1 interference group and the low-dose＋Ang-1 interference group de- creased 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 Maiguan Ⅱ capsule for angiogenesis is probably that it can regulate the expression of Ang-1/Tie2 signaling pathway，but further research is needed to verify that. |
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