缺氧对胎羊肝细胞发育及其RAS机制的影响
发布时间：2018-10-08 19:58
【摘要】：目的:研究缺氧对胎羊肝细胞发育的影响。 方法:采用脐静脉胶原酶灌注法和Percoll密度梯度离心法分离胎羊肝细胞并原代培养,建立肝细胞缺氧损伤模型,即为缺氧组,以原代正常培养的肝细胞为对照组,电镜下观察细胞超微结构的改变,用全自动生化分析仪检测细胞培养液中总蛋白(Total protein,TP)、白蛋白(Albumin,ALB)、肌酐(Creatinine,CREA)、尿素氮(Urea nitrogen,BUN)、天冬氨酸转氨酶(Aspartate transferase,AST)、丙氨酸转氨酶(Alanine transaminase,ALT)、乳酸脱氢酶(Lactic acid dehydrogenase,LDH)的浓度,用流式细胞仪检测肝细胞的细胞周期及凋亡的变化。 结果:肝细胞缺氧后电镜下可见胞质内空泡状改变,内质网扩张,线粒体肿胀变形,线粒体嵴肿胀断裂,细胞内可见退行性变;细胞培养上清液中TP、ALB浓度降低(p0.05),AST、LDH浓度显著升高(p0.01),处于S期的细胞比例降低(p0.05),凋亡细胞比例升高,与对照组比较差异显著(p0.05),但CREA、BUN及ALT浓度与对照组比较无明显改变。 结论:缺氧可导致胎羊肝细胞超微结构发生一定程度的改变;肝细胞功能显著降低;肝细胞增殖减少而凋亡增多。 第二部分缺氧对胎羊肝细胞RAS机制的影响 目的:研究缺氧对胎羊肝细胞RAS机制的影响。 方法:取正常培养3天(d)的肝细胞,随机分为六组,分别给予以下处理:1)正常对照组;2)对照+AngⅡ(血管紧张素Ⅱ)组;3)缺氧组;4)缺氧+AngⅡ组;5)Losartan+AngⅡ组;6)PD123319+AngⅡ组;(所加药物终浓度均为10~(-6)mol/L)。用流式细胞仪检测肝细胞的细胞周期变化。取正常培养3d的肝细胞,随机分为四组:1)正常对照组;2)对照+AⅡ(10~(-6)mol/L)组;3)缺氧组;4)缺氧+AⅡ(10~(-6)mol/L)组。用流式细胞仪检测肝细胞凋亡比例,用Western blot检测肝细胞AT1R及AT2R蛋白的表达。 结果:与正常对照组相比,处于S期的肝细胞比例在缺氧组、对照+AngⅡ组及缺氧+AngⅡ组均明显下降(p0.05,p0.01,p0.01),而凋亡比例均明显上升(p0.05);缺氧+AngⅡ组处于S期的肝细胞比例较之缺氧组明显下降(p0.05)但与对照+AngⅡ组之间无显著差异(p0.05),而其凋亡比例与缺氧组无明显差异(p0.05),但较之对照+AngⅡ组明显上升(p0.05);与正常对照组相比,处于S期的肝细胞比例在对照+AngⅡ及Losartan+AngⅡ组均明显下降(p0.01),在PD123319+AngⅡ组无显著差异(p0.05),与对照+AngⅡ组相比,处于S期的肝细胞比例在Losartan+AngⅡ组不明显差异(p0.05)而在PD123319+AngⅡ组明显上升(p0.05);AT1R蛋白表达在缺氧组及对照+AngⅡ组肝细胞中较之正常组均无显著改变(p0.05),而AT2R蛋白在缺氧组肝细胞中较之正常组显著上调(p0.05),对照+AngⅡ组肝细胞中较之正常组无明显改变(p0.05)。 结论:缺氧和AngⅡ刺激均可使胎羊肝细胞增殖减少而凋亡增多,且与肝细胞AT1受体无关而主要通过与AT2受体结合产生其抑制增殖和促凋亡效应。
[Abstract]:Objective: to study the effect of hypoxia on the development of fetal sheep hepatocytes. Methods: fetal goat hepatocytes were isolated and cultured by umbilical vein collagenase perfusion and Percoll density gradient centrifugation. The model of hypoxia injury was established, namely hypoxia group, and the primary normal cultured hepatocytes were used as control group. The ultrastructural changes of cells were observed under electron microscope. The concentrations of total protein (Total protein,TP), albumin (Albumin,ALB), creatinine (Creatinine,CREA), urea nitrogen (Urea nitrogen,BUN), aspartate aminotransferase (Aspartate transferase,AST), alanine aminotransferase (Alanine transaminase,ALT) and lactate dehydrogenase (Lactic acid dehydrogenase,LDH) in cell culture medium were detected by automatic biochemical analyzer. Cell cycle and apoptosis of hepatocytes were detected by flow cytometry. Results: the cytoplasmic vacuolar changes, endoplasmic reticulum dilatation, mitochondrial swelling and deformation, mitochondrial ridge swelling and rupture, degenerative changes were observed under electron microscope after hypoxia. In the supernatant of cell culture, the concentration of TP,ALB in the supernatant was significantly increased (p0.01), the proportion of cells in S phase was decreased (p0.05), the proportion of apoptotic cells was increased, the difference was significant (p0.05), but the concentration of CREA,BUN and ALT did not change significantly compared with the control group. Conclusion: hypoxia can induce the ultrastructure of fetal sheep hepatocytes to a certain extent, decrease the function of hepatocytes, decrease the proliferation of hepatocytes and increase the apoptosis of hepatocytes. Part two effects of hypoxia on RAS mechanism of fetal sheep hepatocytes objective: to study the effect of hypoxia on RAS mechanism of fetal goat hepatocytes. Methods: the hepatocytes of (d) cultured for 3 days were randomly divided into six groups. Ang 鈪，
本文编号：2258072
[Abstract]:Objective: to study the effect of hypoxia on the development of fetal sheep hepatocytes. Methods: fetal goat hepatocytes were isolated and cultured by umbilical vein collagenase perfusion and Percoll density gradient centrifugation. The model of hypoxia injury was established, namely hypoxia group, and the primary normal cultured hepatocytes were used as control group. The ultrastructural changes of cells were observed under electron microscope. The concentrations of total protein (Total protein,TP), albumin (Albumin,ALB), creatinine (Creatinine,CREA), urea nitrogen (Urea nitrogen,BUN), aspartate aminotransferase (Aspartate transferase,AST), alanine aminotransferase (Alanine transaminase,ALT) and lactate dehydrogenase (Lactic acid dehydrogenase,LDH) in cell culture medium were detected by automatic biochemical analyzer. Cell cycle and apoptosis of hepatocytes were detected by flow cytometry. Results: the cytoplasmic vacuolar changes, endoplasmic reticulum dilatation, mitochondrial swelling and deformation, mitochondrial ridge swelling and rupture, degenerative changes were observed under electron microscope after hypoxia. In the supernatant of cell culture, the concentration of TP,ALB in the supernatant was significantly increased (p0.01), the proportion of cells in S phase was decreased (p0.05), the proportion of apoptotic cells was increased, the difference was significant (p0.05), but the concentration of CREA,BUN and ALT did not change significantly compared with the control group. Conclusion: hypoxia can induce the ultrastructure of fetal sheep hepatocytes to a certain extent, decrease the function of hepatocytes, decrease the proliferation of hepatocytes and increase the apoptosis of hepatocytes. Part two effects of hypoxia on RAS mechanism of fetal sheep hepatocytes objective: to study the effect of hypoxia on RAS mechanism of fetal goat hepatocytes. Methods: the hepatocytes of (d) cultured for 3 days were randomly divided into six groups. Ang 鈪，
本文编号：2258072
本文链接：https://www.wllwen.com/xiyixuelunwen/2258072.html
最近更新
教材专著
