【摘要】：目的:研究缺氧對胎羊肝細(xì)胞發(fā)育的影響。 方法:采用臍靜脈膠原酶灌注法和Percoll密度梯度離心法分離胎羊肝細(xì)胞并原代培養(yǎng),建立肝細(xì)胞缺氧損傷模型,即為缺氧組,以原代正常培養(yǎng)的肝細(xì)胞為對照組,電鏡下觀察細(xì)胞超微結(jié)構(gòu)的改變,用全自動生化分析儀檢測細(xì)胞培養(yǎng)液中總蛋白(Total protein,TP)、白蛋白(Albumin,ALB)、肌酐(Creatinine,CREA)、尿素氮(Urea nitrogen,BUN)、天冬氨酸轉(zhuǎn)氨酶(Aspartate transferase,AST)、丙氨酸轉(zhuǎn)氨酶(Alanine transaminase,ALT)、乳酸脫氫酶(Lactic acid dehydrogenase,LDH)的濃度,用流式細(xì)胞儀檢測肝細(xì)胞的細(xì)胞周期及凋亡的變化。 結(jié)果:肝細(xì)胞缺氧后電鏡下可見胞質(zhì)內(nèi)空泡狀改變,內(nèi)質(zhì)網(wǎng)擴張,線粒體腫脹變形,線粒體嵴腫脹斷裂,細(xì)胞內(nèi)可見退行性變;細(xì)胞培養(yǎng)上清液中TP、ALB濃度降低(p0.05),AST、LDH濃度顯著升高(p0.01),處于S期的細(xì)胞比例降低(p0.05),凋亡細(xì)胞比例升高,與對照組比較差異顯著(p0.05),但CREA、BUN及ALT濃度與對照組比較無明顯改變。 結(jié)論:缺氧可導(dǎo)致胎羊肝細(xì)胞超微結(jié)構(gòu)發(fā)生一定程度的改變;肝細(xì)胞功能顯著降低;肝細(xì)胞增殖減少而凋亡增多。 第二部分缺氧對胎羊肝細(xì)胞RAS機制的影響 目的:研究缺氧對胎羊肝細(xì)胞RAS機制的影響。 方法:取正常培養(yǎng)3天(d)的肝細(xì)胞,隨機分為六組,分別給予以下處理:1)正常對照組;2)對照+AngⅡ(血管緊張素Ⅱ)組;3)缺氧組;4)缺氧+AngⅡ組;5)Losartan+AngⅡ組;6)PD123319+AngⅡ組;(所加藥物終濃度均為10~(-6)mol/L)。用流式細(xì)胞儀檢測肝細(xì)胞的細(xì)胞周期變化。取正常培養(yǎng)3d的肝細(xì)胞,隨機分為四組:1)正常對照組;2)對照+AⅡ(10~(-6)mol/L)組;3)缺氧組;4)缺氧+AⅡ(10~(-6)mol/L)組。用流式細(xì)胞儀檢測肝細(xì)胞凋亡比例,用Western blot檢測肝細(xì)胞AT1R及AT2R蛋白的表達(dá)。 結(jié)果:與正常對照組相比,處于S期的肝細(xì)胞比例在缺氧組、對照+AngⅡ組及缺氧+AngⅡ組均明顯下降(p0.05,p0.01,p0.01),而凋亡比例均明顯上升(p0.05);缺氧+AngⅡ組處于S期的肝細(xì)胞比例較之缺氧組明顯下降(p0.05)但與對照+AngⅡ組之間無顯著差異(p0.05),而其凋亡比例與缺氧組無明顯差異(p0.05),但較之對照+AngⅡ組明顯上升(p0.05);與正常對照組相比,處于S期的肝細(xì)胞比例在對照+AngⅡ及Losartan+AngⅡ組均明顯下降(p0.01),在PD123319+AngⅡ組無顯著差異(p0.05),與對照+AngⅡ組相比,處于S期的肝細(xì)胞比例在Losartan+AngⅡ組不明顯差異(p0.05)而在PD123319+AngⅡ組明顯上升(p0.05);AT1R蛋白表達(dá)在缺氧組及對照+AngⅡ組肝細(xì)胞中較之正常組均無顯著改變(p0.05),而AT2R蛋白在缺氧組肝細(xì)胞中較之正常組顯著上調(diào)(p0.05),對照+AngⅡ組肝細(xì)胞中較之正常組無明顯改變(p0.05)。 結(jié)論:缺氧和AngⅡ刺激均可使胎羊肝細(xì)胞增殖減少而凋亡增多,且與肝細(xì)胞AT1受體無關(guān)而主要通過與AT2受體結(jié)合產(chǎn)生其抑制增殖和促凋亡效應(yīng)。
[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，

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