本文關鍵詞： 缺血再灌注損傷 線粒體融合蛋白2 飽和氫氣鹽水 肝臟 大鼠　出處：《暨南大學》2015年碩士論文　論文類型：學位論文

【摘要】：目的利用大鼠肝臟部分缺血再灌注損傷模型,觀察氫氣鹽水對肝缺血再灌注損傷的保護作用,從分子水平和超微結(jié)構(gòu)上研究肝缺血再灌注損傷和線粒體損傷的關系,并探討其可能的機制,為臨床肝臟外科手術(shù)后缺血再灌注損傷的防護提供理論基礎。方法將健康雄性SD大鼠56只,隨機取8只為假手術(shù)組(N組),另48只隨機分為對照組(NS組)和實驗組(HS組),其中再按缺血時間分為NS1組和HS1組(缺血20min),NS2組和HS2組(缺血40min),NS3組和HS3組(60min),每組8只。假手術(shù)組術(shù)前10min腹腔注射生理鹽水10ml/kg,對照組、實驗組術(shù)前10min分別腹腔注入生理鹽水10ml/kg、飽和氫氣鹽水10ml/kg。動物模型為經(jīng)典肝臟缺血再灌注模型。分別以各組大鼠肝臟門靜脈復流后24h取肝臟標本及靜脈血。1.運用自動生化分析儀檢測血清ALT、AST水平。2.病理切片觀察肝組織形態(tài)學改變及炎癥細胞浸潤。3.透射電鏡觀察肝組織中線粒體形態(tài)結(jié)構(gòu)改變。4.實時定量PCR、Western blot檢測在肝組織中Mfn2表達變化。結(jié)果 1.大鼠肝臟缺血20、40、60min恢復再灌注24h后,各組血清ALT、AST水平逐漸升高,以60min升高最明顯(P0.01)。與NS組比較,相應的HS組ALT、AST水平均明顯降低(P0.05)。2.HE染色生理NS1、NS2組可見肝細胞明顯腫脹,脂肪變性;NS3組可見少量肝細胞嗜酸性壞死及點狀壞死,匯管區(qū)有大量炎性細胞浸潤。與NS組比較,相應的HS組肝臟細胞水腫、炎性細胞浸潤顯著減輕。3.透射電鏡可以看出NS1、NS2組缺血再灌注大鼠肝細胞部分線粒體腫脹,數(shù)量減少,線粒體嵴稍減少,少數(shù)線粒體膜不完整,基質(zhì)密度降低,NS3組則線粒體高度腫脹、形狀不規(guī)則,空泡形成,而且線粒體脊紊亂、短小,排列紊亂,甚至崩解,基質(zhì)凝聚。內(nèi)質(zhì)網(wǎng)明顯擴張。而HS組的大鼠肝細胞線粒體呈長柱狀或網(wǎng)狀,大小、形態(tài)較一致,線粒體膜完整,線粒體嵴呈板層狀分布、清晰可見,較規(guī)則,基質(zhì)結(jié)構(gòu)較清晰。4.NS3組較N組Mfn2蛋白及m RNA表達量明顯降低(P0.01)。HS組Mfn2蛋白及m RNA表達較相應的NS組均有升高(P0.05)。結(jié)論 1.氫氣鹽水對肝臟缺血再灌注的肝功能損害、肝組織及線粒體的功能形態(tài)有一定保護作用。2.氫氣鹽水可促使Mfn2蛋白及RNA的表達上調(diào),可能通過促進線粒體的融合來逆轉(zhuǎn)線粒體結(jié)構(gòu)與功能的改變,從而減輕對肝臟的缺血再灌注損傷。
[Abstract]:Objective to observe the protective effect of hydrogen saline on hepatic ischemia-reperfusion injury in rats. The relationship between hepatic ischemia-reperfusion injury and mitochondrial injury was studied at molecular level and ultrastructure, and the possible mechanism was discussed. Methods 56 healthy male Sprague-Dawley rats were randomly selected as sham operation group (n = 8). The other 48 rats were randomly divided into control group (NS group) and experimental group (HS group). According to the time of ischemia, they were divided into NS1 group and HS1 group (20 min ischemia). NS2 group and HS2 group (NS2 group and HS3 group, n = 8) were injected intraperitoneally with normal saline 10 ml / kg 10 minutes before operation. In the control group, 10 min before operation, 10 ml / kg normal saline was injected intraperitoneally in the experimental group. 10 ml / kg saturated hydrogen salt water. The animal model was a classical liver ischemia reperfusion model. The liver samples and venous blood were taken 24 hours after reflow of portal vein in each group. 1. The automatic biochemical analyzer was used. Serum ALT was detected. AST level. 2. Pathological sections were used to observe the morphologic changes of liver tissue and inflammatory cell infiltration. 3. Transmission electron microscope was used to observe the changes of mitochondrial morphology in liver tissue. 4. Real-time quantitative PCR. The expression of Mfn2 in liver tissue was detected by Western blot. Results 1.The serum ALT of each group was detected after reperfusion for 24 hours after 20 minutes of hepatic ischemia. The level of AST increased gradually, especially in 60 minutes. Compared with NS group, the ALT of HS group was higher than that of NS group. The levels of AST were significantly decreased in NS1 NS2 group. 2. In the NS1 NS2 group, the hepatocytes were swollen and steatosis was observed. A small amount of eosinophilic necrosis and punctate necrosis were observed in NS3 group, and a large number of inflammatory cells were infiltrated in the portal area. Compared with NS group, hepatic cell edema was observed in HS group. Transmission electron microscope showed that the mitochondria of NS1 + NS2 group were swollen, the number of mitochondria decreased, the mitochondrial cristae decreased slightly, and a few of mitochondria membrane was incomplete in NS1 + NS2 group. In NS3 group, the mitochondria were highly swollen, irregular in shape, vacuolated, and the mitochondria ridges were disordered, short, disarranged, and even disintegrated. In HS group, the mitochondria of hepatocytes were long columnar or reticular, the size of mitochondria was the same, the membrane of mitochondria was intact, and the mitochondrial crest was lamellar distribution, which was clearly visible and regular. Clear matrix structure .4.The expression of Mfn2 protein and m RNA in NS3 group was significantly lower than that in N group (P 0.01). The expression of Mfn2 protein and m RNA in HS group were higher than those in NS group. The functional morphology of liver tissue and mitochondria has some protective effect. 2. Hydrogen saline can promote the expression of Mfn2 protein and RNA up-regulated. It is possible to reverse the structural and functional changes of mitochondria by promoting the fusion of mitochondria so as to alleviate the ischemia-reperfusion injury to the liver.
【學位授予單位】：暨南大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R657.3

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