【摘要】：目的肝癌、肝移植等肝臟外科手術(shù)往往面臨肝臟缺血再灌注損傷的難題,明顯增加了肝臟外科手術(shù)的難度和風(fēng)險。本實(shí)驗(yàn)在建立大鼠肝臟缺血再灌注模型的基礎(chǔ)上,術(shù)前予黃芪甲苷預(yù)處理。通過對術(shù)后4 h、8 h、16 h血清轉(zhuǎn)氨酶、炎性相關(guān)因子的檢測及肝組織顯微結(jié)構(gòu)的觀察,判斷黃芪甲苷預(yù)處理對大鼠肝臟缺血再灌注損傷是否具有保護(hù)作用。用Western Blot及免疫組化染色分析各組大鼠肝組織中自噬相關(guān)蛋白Beclin-1的表達(dá);電鏡下觀察各組大鼠肝臟細(xì)胞內(nèi)自噬小體數(shù)量的變化。探討缺血再灌注后大鼠肝臟自噬水平的變化,以及黃芪甲苷預(yù)處理對大鼠肝臟缺血再灌注損傷可能存在的作用機(jī)制,為減輕患者肝臟缺血再灌注損傷提供潛在的理論依據(jù)。方法實(shí)驗(yàn)分組:90只純系SD大鼠,隨機(jī)分為假手術(shù)組(Sham組),肝臟缺血再灌注組(HIRI組)、黃芪甲苷組(干預(yù)組),每組30只。再按術(shù)后4h、8h、16h三個時間段,每個時間段各10只。Sham組大鼠行腹腔注射麻醉后,腹部正中縱形切口,分離肝蒂,不建立肝臟缺血再灌注模型。HIRI組和干預(yù)組大鼠麻醉后,建立肝臟缺血再灌注模型。干預(yù)組大鼠術(shù)前予黃芪甲苷溶液腹腔注射,1次/天,連續(xù)7天。觀察指標(biāo):各組大鼠分別在術(shù)后4 h、8 h、16 h采取血液和肝臟組織標(biāo)本。用全自動生化分析儀檢測各組大鼠血清中谷丙轉(zhuǎn)氨酶(ALT)、谷草轉(zhuǎn)氨酶(AST)水平;用ELISA法測定大鼠血清炎性相關(guān)因子TNF-α,IL-6水平;使用蘇木精-尹紅(HE)染色后,觀察各組肝臟組織病理學(xué)變化;Western Blot及免疫組化染色分析肝組織中自噬相關(guān)蛋白Beclin-1的表達(dá);電鏡下觀察肝臟細(xì)胞內(nèi)自噬小體數(shù)量的變化。結(jié)果1.血清中轉(zhuǎn)氨酶水平的變化:同Sham組大鼠比較,HIRI組大鼠血清中ALT和AST水平在術(shù)后4 h、8 h、16 h均明顯升高(P0.05),于再灌注后8 h時達(dá)到頂峰。與HIRI組大鼠比較,干預(yù)組大鼠血清ALT和AST水平在術(shù)后4 h、8 h、16 h均明顯降低(P0.05)。2.血清中炎性相關(guān)因子水平的變化:同Sham組大鼠比較,HIRI組大鼠血清中TNF-α,IL-6水平在術(shù)后4h、8h、16h均明顯升高(P0.05),于再灌注后8h達(dá)到頂峰。與HIRI組大鼠比較,干預(yù)組大鼠血清TNF-α,IL-6水平在術(shù)后4h、8h、16h均明顯降低(P0.05)。3.肝組織HE染色后觀察:Sham組大鼠肝細(xì)胞索排列整齊、肝小葉結(jié)構(gòu)清晰、中央靜脈區(qū)無淤血、無炎性細(xì)胞浸潤。HIRI組大鼠在再灌注后4 h、8 h、16 h,出現(xiàn)了不同程度的肝細(xì)胞索排列紊亂、中央靜脈區(qū)淤血、肝小葉結(jié)構(gòu)模糊、炎性細(xì)胞浸潤和肝細(xì)胞的水腫、壞死,其中以再灌注后8h的損傷最為嚴(yán)重。與HIRI組大鼠相比,干預(yù)組大鼠的肝臟組織病理變化在術(shù)后4 h、8 h、16 h均有明顯改善。4.肝組織自噬相關(guān)蛋白Beclin-1表達(dá)水平的變化:同Sham組大鼠比較,HIRI組大鼠Beclin-1蛋白的灰度比在術(shù)后4h、8h、16h均明顯升高(P0.05),于再灌注后8h達(dá)到頂峰。與HIRI組大鼠比較,干預(yù)組大鼠Beclin-1蛋白的灰度比在術(shù)后4h、8h、16h均明顯降低(P0.05)。5.免疫組化染色分析肝組織中Beclin-I的表達(dá):與Sham組大鼠相比,HIRI組大鼠肝臟組織Beclin-1的平均標(biāo)記指數(shù)(LI)在術(shù)后8 h明顯升高(P0.05)。與HIRI組大鼠相比,干預(yù)組大鼠肝臟組織Beclin-1平均LI在術(shù)后8 h明顯降低(P0.05)。6.肝臟細(xì)胞自噬小體數(shù)量的變化:與Sham組大鼠比較,HIRI組大鼠肝臟細(xì)胞的自噬小體數(shù)量在術(shù)后8 h明顯上升(P0.05);與HIRI組大鼠比較,干預(yù)組大鼠肝臟細(xì)胞的自噬小體在術(shù)后8 h明顯減少(P0.05)。結(jié)論本實(shí)驗(yàn)結(jié)果提示黃芪甲苷預(yù)處理可以降低大鼠肝臟缺血再灌注后轉(zhuǎn)氨酶水平并減輕炎癥反應(yīng),同時肝組織HE染色觀察結(jié)果也顯示黃芪甲苷預(yù)處理能明顯減輕大鼠肝臟的病理損傷程度,由此推斷黃芪甲苷預(yù)處理能夠減輕大鼠肝臟缺血再灌注損傷。本研究還發(fā)現(xiàn)缺血再灌注后的大鼠肝臟組織Beclin-1呈高表達(dá)狀態(tài),且肝細(xì)胞內(nèi)自噬小體數(shù)量也明顯升高,而黃芪甲苷預(yù)處理可以明顯降低自噬相關(guān)蛋白Beclin-1在蛋白水平和組織水平的表達(dá),并且也減少了肝臟細(xì)胞內(nèi)自噬小體的數(shù)量,表明黃芪甲苷可能通過降低大鼠肝臟自噬水平來保護(hù)大鼠肝缺血再灌注損傷。本研究為黃芪甲苷減輕肝臟缺血再灌注損傷提供了新的理論基礎(chǔ),具有潛在的臨床應(yīng)用前景。
[Abstract]:Objective liver surgery, such as liver cancer and liver transplantation, often faces the problem of liver ischemia reperfusion injury, which obviously increases the difficulty and risk of liver surgery. On the basis of establishing rat liver ischemia-reperfusion model, this experiment was pretreated by Astragalus glucoside preoperatively. By 4 h, 8 h, 16 h serum aminotransferase, inflammatory related cause On the basis of Western Blot and immunohistochemical staining, the expression of autophagy related protein Beclin-1 in liver tissues of rats was analyzed by using Western Blot and immunohistochemistry. The number of autophagic bodies in the liver cells of each group was observed under electron microscope. Changes of autophagy in rat liver after ischemia and reperfusion, and the possible mechanism of astragalin preconditioning on hepatic ischemia reperfusion injury in rats to provide a potential theoretical basis for alleviating liver ischemia reperfusion injury in the patients. Methods the experimental group: 90 pure SD rats were randomly divided into sham operation group (group Sham). Liver ischemia reperfusion group (HIRI group), astragalin group (intervention group), 30 rats in each group, and then three time periods of 4h, 8h, 16h after operation, 10 rats in each time period, each of the.Sham groups were injected into the abdominal cavity, the abdominal median longitudinal incision, the liver pedicle, the liver ischemia reperfusion model.HIRI group and the intervention group were not established, and the liver deficiency was established. Blood reperfusion model. The rats in the intervention group were given Astragalus methoside solution intraperitoneally before operation, 1 times / day for 7 days. The indexes of blood and liver tissue were taken at 4 h, 8 h and 16 h after operation. The serum alanine transaminase (ALT), cereal transaminase (AST) level in the serum of each group was detected by automatic biochemical analyzer, and the level of glutarine transaminase (AST) was measured by the total automatic biochemical analyzer. The ELISA method was used to determine the level of the rats. Rat serum inflammatory related factor TNF- alpha, IL-6 level; after hematoxylin Yin Hong (HE) staining, the pathological changes of liver tissues were observed; Western Blot and immunohistochemical staining were used to analyze the expression of autophagic protein Beclin-1 in liver tissue; the changes in the number of autophagic corpuscles in liver cells were observed under electron microscopy. Results 1. serum transaminase water was observed. Compared with the Sham group, the level of ALT and AST in the serum of HIRI rats was 4 h, 8 h and 16 h significantly increased (P0.05), and reached the peak at 8 h after reperfusion. Compared with the HIRI group, the serum ALT and AST levels in the intervention group were 4, 8 and 16. The levels of inflammatory related factors in the serum were significantly decreased. Compared with the Sham group, the level of TNF- alpha and IL-6 in the serum of HIRI rats increased significantly (P0.05) after the operation (P0.05), and the 8h reached the peak after reperfusion. Compared with the HIRI group, the serum TNF- alpha was observed in the intervention group, and the IL-6 level was obviously decreased after the operation. The structure of hepatic lobule was clear and there was no blood stasis in the central venous area. The rats in the.HIRI group without inflammatory cell infiltration were 4 h, 8 h and 16 h after reperfusion. There were different degrees of disorder of hepatic cell cord arrangement, the congestion of the central venous area, the blurred structure of hepatic lobule, the infiltration of inflammatory cells and the edema and necrosis of the hepatocytes, among which the most serious injury was 8h after reperfusion. Compared with the HIRI group, the pathological changes in the liver tissue of the rats in the intervention group were 4 h, 8 h and 16 h significantly improved the level of the expression of autophagy related protein Beclin-1 expression in.4. liver tissue. Compared with the Sham group, the gray level of Beclin-1 protein in HIRI group rats was significantly higher than that in 4h, 8h, and reached the peak after reperfusion. Compared with the HIRI group, the gray level of Beclin-1 protein in the intervention group was significantly lower than that of 4h, 8h and 16h after the operation (P0.05).5. immunohistochemical staining analysis of the expression of Beclin-I in the liver tissue: compared with the Sham group, the Beclin-1 average marker index (LI) in the liver tissue of the HIRI group was significantly higher than the 8 after the operation. The average LI of Beclin-1 in the liver tissue of the rats in the intervention group decreased significantly at 8 h after operation (P0.05), the number of autophagic corpuscles in.6. liver cells: compared with the Sham group, the number of autophagic corpuscles in the liver cells of the HIRI group increased significantly (P0.05) after the operation (P0.05). Compared with the HIRI group, the autophagic corpuscle of the liver cells in the intervention group was 8 h after the operation. P0.05. Conclusion the results of this experiment suggest that astragaloside pretreatment can reduce the level of aminotransferase and reduce the inflammatory response after liver ischemia and reperfusion in rats. The results of HE staining in liver tissue also show that astragaloside preconditioning can significantly reduce the degree of pathological injury in the liver of rats. The liver ischemia-reperfusion injury in rats was alleviated. The high expression of Beclin-1 in the liver tissues of rats after ischemia-reperfusion was also found, and the number of autophagic corpuscles in the liver cells was also significantly increased, and the expression of autophagy related protein Beclin-1 at protein level and tissue level could be significantly reduced, and the expression of autophagic related protein was decreased. The number of autophagic bodies in the liver cells indicates that astragaloside may protect the rat liver from ischemia reperfusion injury by lowering the level of autophagy in the rat liver. This study provides a new theoretical basis for alleviating liver ischemia reperfusion injury by Astragalus membranaceus, and has potential clinical application prospects.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R657.3
，

本文編號：2145969