黄芪总黄酮对终末期肾脏病患者血清诱导的血管内皮细胞凋亡的影响

发布时间：2018-05-30 13:42

  本文选题：终末期肾脏病 + 心血管疾病　； 参考：《兰州大学》2014年硕士论文

【摘要】：目的：体外建立终末期肾脏病(End stage renal disease, ESRD)患者内环境模型,诱导人脐静脉血管内皮细胞(Human umbilical vein endothelial cells, HUVECs)凋亡,观察黄芪总黄酮(Total flavonoids of astragalus, TFA)干预后氧化应激(Oxidative stress, OS)终产物4-羟基壬烯醛(4-hydroxy nonene aldehyde,4-HNE)及内质网应激(Endoplasmic reticulum stress, ERS)信号分子C/EBP同源蛋白(C/EBPhomologous protein, CHOP)勺表达变化,探讨TFA减少ESRD患者血清诱导的血管内皮细胞(Vein endothelial cell, VEC)凋亡的分子机制,为ESRD患者防治心血管疾病(Cardiovascular disease, CVD)提供新的理论依据和治疗靶点。 方法：HUVECs培养于含10%胎牛血清、100U/mL青霉素和10Oμg/mL链霉素的DMEM普通培养基中,置于37℃、5%CO2环境的培养箱中培养,根据细胞生长情况择期更换新鲜培养基,应用0.25%浓度的胰酶对贴壁生长的内皮细胞进行消化传代。于实验前24h更换为无血清培养基,使细胞完成同步化。随机将细胞分为6组：空白对照组(10%胎牛血清)、正常对照组(10%健康志愿者血清)、终末期肾脏病组(10%终末期肾脏病患者血清)、低剂量(TFA1)、中剂量(TFA2)、高剂量(TFA3)黄芪总黄酮组(在终末期肾脏病组基础上分别加入0.5mg/mL、1.0mg/mL、2.0mg/mL黄芪总黄酮)。培养24h后倒置显微镜下观察HUVECs形态,四甲基偶氮畔盐(Methyl thiazolyl tetrazolium, MTT)试验检测细胞生存率,彗星实验检测细胞凋亡率,酶联免疫吸附测定(Enzyme-linked immuno sorbent assay, ELISA)法检测4-HNE浓度,免疫细胞化学法检测CHOP的表达量。 结果：正常对照组与空白对照组比较,细胞生存率、凋亡率、4-HNE浓度和CHOP表达量,差异无统计学意义(P0.05)。ESRD患者血清诱导HUVECs培养24h后出现细胞体积缩小,结构紧密,胞浆浓缩,细胞器聚集,胞膜出芽脱落,细胞核固缩呈均一的致密物等凋亡形态学变化。与正常对照组比较,终末期肾脏病组HUVECs生存率降低(P0.01),凋亡率增高(P0.01),4-HNE浓度和CHOP表达量均升高(P0.05)。与终末期肾脏病组比较,TFA干预后各组HUVECs生存率提高(P0.05),凋亡率减少(P0.05),4-HNE浓度降低,呈剂量依赖性(P0.05),CHOP的表达量降低(P0.05),中剂量组优于低剂量组(P0.01),但高剂量组与中剂量组比较差异无统计学意义(P0.05)。相关性分析示：4-HNE浓度与CHOP的表达量呈正相关(r=0.913,P0.01)。 结论：ESD患者血清可诱导HUVECs凋亡；TFA对ESRD患者血清诱导的HUVECs凋亡具有抑制作用,其可能通过减轻OS和ESR发挥抗凋亡作用。
[Abstract]:Objective: to establish an in vitro model of end-stage renal disease (End stage renal disease, ESRD) patients and induce apoptosis of human umbilical vein endothelial cells (HUVECs) in vitro. To observe the changes of oxidative stress (OS) end product 4-hydroxy nonene aldehydededededededededededededededededededededededededededededededededededededededededededededededehne4-HNE( 4-HNE4) after the intervention of Total flavonoids of astragalus, TFA) of Astragalus membranaceus Total reticulum stress, ERS) and the expression of C/EBP homologous protein (CHOPP) in the endoplasmic reticulum (ER) signal molecule Endoplastic reticulum stress, ERS). To explore the molecular mechanism of TFA reducing the apoptosis of vascular endothelial cells (VEC) induced by serum in patients with ESRD, and to provide a new theoretical basis and therapeutic target for the prevention and treatment of cardiovascular disease (CVDs) in patients with ESRD. Methods 1: HUVECs were cultured on the DMEM medium containing 10% fetal bovine serum 100 U / mL penicillin and 10 O 渭 g/mL streptomycin, and cultured in a incubator with CO 2 at 37 鈩，

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