Mfn2在氢气盐水抗肝脏缺血再灌注损伤中的作用和机制

发布时间：2018-01-27 07:43

本文关键词： 缺血再灌注损伤线粒体融合蛋白2 饱和氢气盐水肝脏大鼠　出处：《暨南大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的利用大鼠肝脏部分缺血再灌注损伤模型,观察氢气盐水对肝缺血再灌注损伤的保护作用,从分子水平和超微结构上研究肝缺血再灌注损伤和线粒体损伤的关系,并探讨其可能的机制,为临床肝脏外科手术后缺血再灌注损伤的防护提供理论基础。方法将健康雄性SD大鼠56只,随机取8只为假手术组(N组),另48只随机分为对照组(NS组)和实验组(HS组),其中再按缺血时间分为NS1组和HS1组(缺血20min),NS2组和HS2组(缺血40min),NS3组和HS3组(60min),每组8只。假手术组术前10min腹腔注射生理盐水10ml/kg,对照组、实验组术前10min分别腹腔注入生理盐水10ml/kg、饱和氢气盐水10ml/kg。动物模型为经典肝脏缺血再灌注模型。分别以各组大鼠肝脏门静脉复流后24h取肝脏标本及静脉血。1.运用自动生化分析仪检测血清ALT、AST水平。2.病理切片观察肝组织形态学改变及炎症细胞浸润。3.透射电镜观察肝组织中线粒体形态结构改变。4.实时定量PCR、Western blot检测在肝组织中Mfn2表达变化。结果 1.大鼠肝脏缺血20、40、60min恢复再灌注24h后,各组血清ALT、AST水平逐渐升高,以60min升高最明显(P0.01)。与NS组比较,相应的HS组ALT、AST水平均明显降低(P0.05)。2.HE染色生理NS1、NS2组可见肝细胞明显肿胀,脂肪变性;NS3组可见少量肝细胞嗜酸性坏死及点状坏死,汇管区有大量炎性细胞浸润。与NS组比较,相应的HS组肝脏细胞水肿、炎性细胞浸润显著减轻。3.透射电镜可以看出NS1、NS2组缺血再灌注大鼠肝细胞部分线粒体肿胀,数量减少,线粒体嵴稍减少,少数线粒体膜不完整,基质密度降低,NS3组则线粒体高度肿胀、形状不规则,空泡形成,而且线粒体脊紊乱、短小,排列紊乱,甚至崩解,基质凝聚。内质网明显扩张。而HS组的大鼠肝细胞线粒体呈长柱状或网状,大小、形态较一致,线粒体膜完整,线粒体嵴呈板层状分布、清晰可见,较规则,基质结构较清晰。4.NS3组较N组Mfn2蛋白及m RNA表达量明显降低(P0.01)。HS组Mfn2蛋白及m RNA表达较相应的NS组均有升高(P0.05)。结论 1.氢气盐水对肝脏缺血再灌注的肝功能损害、肝组织及线粒体的功能形态有一定保护作用。2.氢气盐水可促使Mfn2蛋白及RNA的表达上调,可能通过促进线粒体的融合来逆转线粒体结构与功能的改变,从而减轻对肝脏的缺血再灌注损伤。
[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.
【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R657.3

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