氢气饱和生理盐水对大鼠胆管缺血再灌注损伤的保护作用及机制

发布时间：2018-05-07 07:14

本文选题：氢气 + 饱和　；参考：《泸州医学院》2014年硕士论文

【摘要】：目的：本实验通过建立大鼠肝内胆管缺血再灌注损伤(IR)动物模型，探讨静脉注射氢气饱和生理盐水是否对大鼠肝内胆管缺血再灌注损伤具有保护作用，观察其病理学改变，并从胆管上皮细胞Bcl-2/Fas基因表达、凋亡等方面探讨氢气饱和生理盐水可能的作用机制。方法：将32只雄性SD大鼠随机分成4组：假手术组（SO组），缺血再灌注组（IR组），缺血再灌注+生理盐水组（IR+NS组），缺血再灌注+氢气饱和生理盐水组（IR+HS组），每各8只。用1％戊巴比妥钠以每100g体重0.6ml的剂量注射进入腹腔，等待麻醉药物效果显效后，用碘伏对大鼠的腹部进行消毒，然后沿着大鼠的腹正中线打开腹部，分离至胆总管及十二指肠，并于二者汇合处经十二指肠外用硬膜外导管逆行置管于胆总管内，固定并引流胆汁；分离肝门部，用无创血管夹夹闭肝左、中叶血管（包括动、静脉和胆管），阻断血流，并保持肝右、尾状叶血流通畅，按规定时间撤夹，恢复血供，从而制成70％肝缺血再灌注损伤模型。各组SD大鼠均于缺血60min，经尾静脉注射氢气饱和生理盐水，再灌注120min后统一取左肝组织并收集胆汁（均为I/R前后两组）。肝组织分别用10%甲醛固定和冻存在-80℃冰箱内，HE染色后光镜病理学观察，观察肝组织和肝内胆管上皮细胞的病理形态变化；采用免疫组织化学法（PV二步法）观察肝组织中Bcl-2/Fas蛋白在肝内胆管上皮细胞的表达情况；Tunel法检测细胞凋亡情况；分光光度法检测丙二醛(mal0ndia1dehyde，MDA)和谷胱甘肽(glutathione， GSH)胡含量，超氧化物歧化酶(superoxidedismutase，SOD)和过氧化氢酶(catalase，CAT)的活力。将上面所有得到的数据用均数±标准差（X±s）表示，，采用方差分析对样本均数进行比较；采用snk法对两两进行比较,使用SPSS17.0统计软件进行统计学处理；统计检验P0.05被认为差异具有统计学意义。结果：假手术组（SO组），缺血再灌注组（IR组），缺血再灌注+生理盐水组（IR+NS组）相比较，术后缺血再灌注+富氢水组（IR+HS组）的肝组织病理情况有所改善；胆汁中葡萄糖（Glu）的含量在IR前后升高的幅度明显降低（IR后值均大于IR前值）；胆汁中γ-谷酰转移酶（GGT）的活性在IR前后升高的幅度明显明显降低（IR后值均大于IR前值）；IR组、IR+NS组、IR+HS组动物于建模术后肝组织内MDA含量明显升高，GSH含量降低，SOD活力和CAT活力下降;与IR+NS组相比较，IR+HS组术后肝组织内MDA含量升高较少（P0.01）.，GSH含量下降程度降低（P0.01），SOD活力和CAT活力下降程度也降低（P0.01）。免疫组化法测bcl-2基因和Fas基因的表达，IR+HS组肝内胆管上皮细胞阳性细胞数明显比IR+NS组减少。Tunel检测肝内胆管上皮细胞凋亡，IR+HS组肝内胆管上皮细胞凋亡明显比IR+NS组减轻。结论:氢气饱和生理盐水可以减轻肝缺血再灌注后肝内胆道损伤，可以抑制肝脏缺血再灌注损伤所引发的氧化应激反应，减轻氧化损伤，可以抑制肝缺血再灌注后肝内胆管上皮细胞Bcl-2／Fas蛋白的表达，减少细胞凋亡。
[Abstract]:Objective : To study the possible mechanism of hydrogen saturated physiological saline by establishing an animal model of intra - hepatic bile duct ischemia - reperfusion injury ( IR ) in rats . The mechanism of possible mechanism of hydrogen saturated physiological saline was investigated in the following aspects : sham operation group ( SO group ) , ischemia / reperfusion group ( IR group ) , ischemia / reperfusion + physiological saline group ( IR + NS group ) , ischemia / reperfusion + hydrogen saturated physiological saline group ( IR + HS group ) .
The liver tissues were isolated from left and middle lobe vessels ( including dynamic , venous and bile duct ) , the blood flow was blocked and the blood supply was recovered after reperfusion for 120 min after reperfusion for 120 min . The pathological changes of liver and intrahepatic bile duct epithelial cells were observed .
The expression of Bcl - 2 / Fas protein in liver tissues was observed by immunohistochemical method ( PV - 2 ) .
Tunel method was used to detect the apoptosis of cells .
The activity of malondialdehyde ( MDA ) and glutathione ( GSH ) , superoxide dismutase ( SOD ) and catalase ( CAT ) were determined by spectrophotometry . All the data obtained were expressed by mean 卤 standard deviation ( X 卤 s ) , and the mean number of samples was compared by variance analysis .
Using the snk method , the two methods were compared , and the SPSS 17.0 statistical software was used for statistical processing ;
The results were as follows : sham operation group ( SO group ) , ischemia / reperfusion group ( IR group ) , ischemia / reperfusion + physiological saline group ( IR + NS group ) .
The content of Glu in bile was significantly decreased before and after IR ( both IR and IR values were higher than those before IR ) .
The activity of 纬 - glutamyltransferase ( GGT ) in bile was significantly lower than that before and after IR ( both IR and IR values were higher than those before IR ) .
Compared with IR + NS group , the content of MDA in the liver tissues of IR + HS group decreased significantly ( P0.01 ) . The expression of bcl - 2 gene and Fas gene was decreased in IR + HS group .

【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R657.3

【参考文献】