本文選題：肝切除 + 肝衰竭　； 參考：《中國人民解放軍醫(yī)學院》2015年博士論文

【摘要】：背景和目的肝切除術是治療肝臟良惡性腫瘤的首選方法。隨著圍手術期管理水平的不斷提高、手術技術的不斷進步,尤其是精準外科理念的不斷深入,肝切除術的手術適應證及切除范圍不斷擴大,以往認為無法切除的肝臟巨大腫瘤、多發(fā)腫瘤以及肝門區(qū)腫瘤在今天得以切除,使患者長期無瘤生存成為可能。然而,盡管擴大切除范圍能夠完整的切除腫瘤并獲得陰性切緣,但大范圍切除術后由于預留的功能性肝體積不足,常常導致術后肝功能衰竭的發(fā)生,這是患者術后死亡的主要原因,嚴重限制了大范圍肝切除在臨床的應用。盡管大范圍肝切除術后肝功能衰竭是一種嚴重的臨床綜合征,但其具體的病理生理機制仍未完全明了。術后肝衰發(fā)生的本質在于殘余肝臟再生與損傷的不協(xié)調導致的功能性肝細胞簇數量不足。門脈高灌注、Kupffer細胞激活、腸道菌群異位、過度的炎癥反應以及氧化應激反應是造成實質損傷的主要原因,對于這些因素進行相應的干預能在一定程度上改善大范圍肝切除術的預后。然而殘余肝臟的再生在術后肝衰的病理生理過程中所起的作用,目前研究尚未達成統(tǒng)一觀點。有些研究認為,由于實質細胞與非實質細胞的再生進程不同步,大范圍肝切除術后早期過快的肝再生反而促進門脈壓力的進一步升高,無益于殘余肝臟組織結構的重建。但是另有研究也發(fā)現(xiàn)大范圍肝切除術后殘余肝臟的再生表現(xiàn)為明顯抑制,同時,促進肝再生的措施也能顯著提高術后生存率。因此,明確殘余肝臟組織再生進程的變化,對于進一步認識大范圍肝切除術后肝功能衰竭的病理生理機制及發(fā)掘潛在的治療策略意義重大。肝切除術后殘余肝臟的再生是受到精確調控的復雜生理過程,細胞因子、生長因子及代謝產物共同構成肝再生的調控網絡。肝臟是機體的代謝中樞,肝切除術后殘余肝臟組織一方面要維持肝臟特異性功能,如糖異生、尿素合成等,另一方面也要產生大量ATP,不僅用于蛋白、核酸及其他細胞成分的合成,也參與重要的再生相關信號通路的活化。肝切除術會嚴重影響肝臟的代謝功能,尤其是能量代謝。因此,能量代謝的異�？赡芡ㄟ^再生進程的調控參與術后肝衰的病理生理過程。mTOR信號通路通過整合胞內能量狀態(tài)、氧含量以及胞外生長因子、營養(yǎng)物質等信息,調控細胞的代謝以及生長增殖過程。當能量及營養(yǎng)物質來源充足時,該信號通路能夠促進細胞的生長；反之,在能量狀態(tài)低下的情況下,細胞轉向能量恢復的過程。基于以上認識,我們設想大范圍肝切除術后由于剩余肝臟組織代謝功能的紊亂導致能量狀態(tài)的顯著低下,可能干擾了mTOR信號通路,繼而抑制殘余肝臟細胞的再生,導致或者促進肝功能衰竭的發(fā)生。本課題擬通過建立不同范圍肝切除的大鼠模型,研究不同范圍肝切除術,尤其是90%肝切除術后殘余肝臟的再生與損傷改變,探討能量代謝對mTOR信號通路以及殘余肝臟再生的影響及機制；以及改善能量狀態(tài)對大范圍肝切除術后肝再生的影響。為臨床改善大范圍肝切除術后患者的肝功能和預后,提高肝臟腫瘤的手術切除率提供理論基礎。方法本課題實驗分三部分進行：第一部分：基于精準外科理念,建立不同范圍肝切除的大鼠模型,初步觀察術后肝功能、血生化及組織病理學變化和術后肝衰的發(fā)生率及死亡率,選出合適的大范圍肝切除術后肝功能衰竭的標準化模型。第二部分：將大鼠分為假手術組、85%肝切除組(大范圍肝切除無肝功能衰竭組)和90%肝切除組(大范圍肝切除肝功能衰竭組)。對比85%肝切除組和90%肝切除組術后生存率、肝功能、肝再生及實質損傷的相關指標的變化。檢測mmTOR信號通路活性在兩組的差異。第三部分：進一步檢測mTOR信號通路的上下游分子的活性在85%和90%肝切除組的差異。用代謝組學的方法探討90%肝切除術后大鼠全身代謝水平的改變。我們選擇葡萄糖作為治療組干預藥物,探討改善能量狀態(tài)對90%肝切除術后mTOR信號通路及殘余肝臟再生和預后的影響。結果第一部分：成功建立了大鼠不同范圍肝切除的標準化模型。與其它組相比,90%肝切除術后大鼠肝功能障礙更加明顯,表現(xiàn)為血清ALT、AST、TBIL水平的明顯升高,ALB水平明顯降低。70%和85%肝切除術后大鼠的生存率均為100%,而90%肝切除術的生存率明顯降低,僅為23%。對死亡大鼠進行尸檢,見腹腔內少量至中等量淡黃色腹水,尾葉腫脹,表面顏色略有蒼白,并可見擴張血管,未發(fā)現(xiàn)出血、膽漏、腔靜脈狹窄等并發(fā)癥,證實死于肝功能衰竭。病理學檢測發(fā)現(xiàn)肝切除術后24小時殘余肝臟表現(xiàn)為肝小葉基本結構存在,肝血竇擁塞,肝細胞表現(xiàn)為明顯的空泡樣變,但在70%、85%和90%肝切除組之間未發(fā)現(xiàn)明顯的差異,同時,三組也未出現(xiàn)明顯的肝細胞壞死表現(xiàn)。第二部分：1.與假手術組相比,85%肝切除組與90%肝切除組術后大鼠血清ALT、AST水平及HMGB-1的mRNA水平均顯著升高,但兩組之間的差異無統(tǒng)計學意義；組織病理學檢測顯示術后24小時兩組殘余肝葉組織肝小葉結構基本正常,肝細胞空泡變明顯,無炎性細胞浸潤、壞死等病理改變,中性粒細胞標志物-MPO染色進一步證實兩組無明顯的中性粒細胞浸潤；TUNEL染色顯示兩組僅有少量TUNEL陽性細胞，Western blot結果顯示caspase-3蛋白表達在兩組之間無差異。2與假手術組相比,85%肝切除組與90%肝切除組術后殘肝都表現(xiàn)出明顯的再生反應,但后者的再生率明顯低于前者,術后24小時有絲分裂指數表現(xiàn)出相同的趨勢；肝組織再生相關因子的nRNA表達情況：TNF-α、myc和HGF的mRNA表達在兩組之間無明顯差異,IL-6的mRNA表達水平在90%肝切除組明顯上調；細胞周期相關因子(cyclinD、cyclinE、cyclinA、cyclin B)的1nRNA的表達,在90%肝切除組明顯低于85%肝切除組；肝組織Ki-67結果顯示,兩組術后均有不同程度的細胞增殖,且90%肝切除組的陽性率明顯低于85%肝切除組。肝組織PCNA蛋白的VVesternb1ot結果顯示出類似的變化趨勢。3Western blot檢測各組mTOR及其下游分子p70s6k、4EBP1的磷酸化水平,結果發(fā)現(xiàn)90%肝切除術后mTOR、p70s6k和4EBP1的磷酸化水平明顯下調,提示該通路的激活出現(xiàn)異常。第三部分：1.Western blot結果顯示,與85%肝切除組相比,90%肝切除組AMPKa的磷酸化水平明顯上調,而cyclin D蛋白的表達顯著降低。Akt的磷酸化水平兩組之間無明顯差異。磷鉬酸比色法檢測肝組織ATP含量,結果顯示：術后早期90%肝切除組殘余肝組織ATP含量明顯低于85%肝切除組。2.代謝組學結果顯示90%肝切除術嚴重干擾了大鼠的代謝,尤其是能量相關的代謝通路發(fā)生了明顯改變。3.葡萄糖干預顯著提高90%肝切除術后殘余肝組織ATP水平,殘余肝臟的再生率及有絲分裂指數顯著提高,組織病理學檢測顯示Ki-67陽性率明顯升高,Western blot的結果PCNA、cyclin D的蛋白表達水平及mTOR信號通路活性明顯提高。結論：1.基于實驗室前期的工作,在充分了解大鼠肝臟及管道系統(tǒng)解剖的基礎上,規(guī)范手術步驟和方法,精確地處理大鼠肝臟實質內入肝血流及肝靜脈等重要結構,成功建立了穩(wěn)定的不同范圍肝切除的標準化模型。2.利用我們的方法建立的大鼠90%肝切除模型術后早期發(fā)生急性肝功能衰竭,表現(xiàn)為肝臟排泄、合成及代謝功能的明顯障礙,死亡率高但有一定的自愈率等特點,能夠很好的模擬肝切除術后肝功能衰竭的臨床表現(xiàn),是一個符合外科臨床實際的理想模型。3.再生早期啟動因子的明顯上調說明90%肝切除誘導了強烈的再生信號,但細胞增殖周期相關因子的結果提示殘余肝臟并未表現(xiàn)出同樣強烈的增殖能力。殘余肝臟的再生抑制是導致90%肝切除術后肝功能衰竭的主要原因。4.代謝組學的結果證實90%肝切除造成嚴重的代謝紊亂,嚴重影響了能量的產生,表現(xiàn)為90%肝切除術后殘余肝臟的ATP含量的明顯降低。低能量狀態(tài)通過AMPK的激活抑制了mTOR信號通路,繼而造成殘余肝臟的再生抑制。5.提高90%肝切除術后的能量狀態(tài)能夠促進殘余肝臟的再生,并延長大鼠術后生存時間及生存率,改善大范圍肝切除術的預后。6.能量的產生涉及多個方面,比如線粒體是否能夠維持正常的氧化磷酸化及電子傳遞的功能,是否足量恰當的能量代謝底物的供應等。因此需要進一步探討大范圍肝切除術后能量代謝異常的原因,以更加有效的改善圍手術期能量狀態(tài),改
[Abstract]:Background and objective hepatectomy is the first choice for the treatment of benign and malignant liver tumors. With the continuous improvement of the management level in the perioperative period, the continuous progress of the surgical technique, especially the deepening of the concept of precision surgery, the surgical indications of hepatectomy and the enlargement of the resection range, and the previous unresectable huge tumor of the liver Tumor and hilar tumor can be excised today to make it possible for the patient to live without tumor for a long time. However, although the enlarged excision range can complete the resection of the tumor and obtain a negative margin, the postoperative liver failure often leads to postoperative liver failure due to the reserved functional liver volume after the large resection, which is the postoperative patient's operation. The main cause of death severely restricts the clinical application of large range hepatectomy. Although liver failure after extensive hepatectomy is a serious clinical syndrome, its specific pathophysiological mechanism is still not fully understood. The essence of postoperative liver failure lies in the functional liver caused by the incongruity of residual liver regeneration and injury. The number of cell clusters is insufficient. High portal perfusion, activation of Kupffer cells, ectopia of intestinal flora, excessive inflammatory response and oxidative stress are the main causes of substantial damage. The corresponding intervention for these factors can improve the prognosis of large range hepatectomy to a certain extent. However, the regeneration of residual liver is in the postoperative liver failure. There is no unified view on the role of pathophysiology. Some studies suggest that the rapid regeneration of the liver in the early stage of large hepatectomy can promote the further increase of portal pressure, but it is not beneficial to the reconstruction of the residual liver tissue because the process of regeneration of the parenchymal cells and the non parenchymal cells is not synchronized. Other studies also found that the regenerated liver regeneration after large hepatectomy was obviously inhibited, and the measures to promote liver regeneration could also significantly increase the postoperative survival rate. Therefore, the changes of the process of regenerated liver tissue regeneration were clear, and the pathophysiological mechanism and potential of the liver failure after extensive hepatectomy were further recognized. The treatment strategy is of great significance. The regeneration of residual liver after hepatectomy is a complex physiological process which is regulated accurately. Cytokines, growth factors and metabolites constitute the regulatory network of liver regeneration. The liver is the metabolic center of the body, and the residual liver after hepatectomy should maintain the liver specific function, such as sugar, such as sugar. On the other hand, a large number of ATP are produced, not only in the synthesis of proteins, nucleic acids, and other cell components, but also in the activation of important regeneration related signaling pathways. Hepatectomy will seriously affect the metabolic function of the liver, especially energy metabolism. Therefore, the abnormality of energy metabolism may be regulated by the regulation of the process of regeneration. The pathophysiological process of liver failure after operation,.MTOR signaling pathway regulates cell metabolism and growth and proliferation by integrating intracellular energy status, oxygen content and extracellular growth factors, nutrients and other information. When the source of energy and nutrients is sufficient, the signal pathway can promote cell growth; conversely, the energy state is low. Under the circumstances, the cells turn to the process of energy recovery. Based on the above understanding, we envisage a significant low energy state due to the disturbance of the metabolic function of the remaining liver tissue after large hepatectomy, which may interfere with the mTOR signaling pathway and then inhibit the regeneration of residual liver cells, leading to or promoting the occurrence of liver failure. The aim of this study is to investigate the effects and mechanisms of energy metabolism on mTOR signaling pathway and residual liver regeneration after hepatectomy in different range of hepatectomy, especially after 90% hepatectomy, and to investigate the effect of energy metabolism on the liver regeneration after hepatectomy. It provides a theoretical basis for improving the liver function and prognosis of patients after large hepatectomy and improving the surgical resection rate of liver tumors. Methods the experiment was carried out in three parts: first part: Based on the concept of precision surgery, a rat model with different range of hepatectomy was established, and the liver function, blood biochemistry and blood biochemistry were preliminarily observed. The histopathological changes and the incidence and mortality of postoperative liver failure were selected to select a standardized model for liver failure after large hepatectomy. The second part: the rats were divided into sham operation group, 85% hepatectomy group (large liver resection without liver failure group) and 90% liver resection group (large liver resection group). 85% Changes in the survival rate, liver function, liver regeneration and parenchymal injury in the hepatectomy group and 90% hepatectomy group. The difference in the activity of mmTOR signaling pathway in the two groups was detected. The third part: further detection of the differences in the activity of the upper and lower reaches of the mTOR signaling pathway in the 85% and 90% hepatectomy groups. The 90% hepatectomy was explored by the metabonomics method. In addition to the changes in the overall metabolic level of the rats after the operation, we chose glucose as the treatment group, and discussed the effect of improving the energy status on the mTOR signaling pathway and the residual liver regeneration and prognosis after 90% hepatectomy. Results the first part: a standardized model of rat liver resection was successfully established. Compared with the other groups, 90% The liver dysfunction in rats after hepatectomy was more obvious, showing a significant increase in serum ALT, AST and TBIL levels. The survival rate of rats after.70% and 85% hepatectomy was 100%, while the survival rate of 90% hepatectomy was significantly reduced, only 23%. was performed to the dead rats, and a small amount to moderate yellowish ascites in the abdominal cavity was found. The caudal lobe was swollen, the surface color was slightly pale, and the blood vessels were dilated, no bleeding, bile leakage, and stenosis of the vena cava were found, which proved to die of liver failure. Pathological examination found that the residual liver in the 24 hours after hepatectomy showed the basic structure of hepatic lobule, the congestion of the hepatic sinusoids, and the obvious vacuolation of the liver cells, but 70%, There was no significant difference between the 85% and 90% hepatectomy groups. At the same time, there was no obvious hepatocyte necrosis in the three groups. Second: 1. compared with the sham group, the serum ALT, AST level and the mRNA level of HMGB-1 in the 85% hepatectomy and 90% hepatectomy groups were significantly higher, but the difference between the two groups was not statistically significant; Pathological examination showed that the hepatic lobule structure of residual hepatic lobes in two groups after 24 hours was basically normal, the vacuoles of liver cells became obvious, no inflammatory cell infiltration, necrosis and other pathological changes, neutrophilic granulocyte marker -MPO staining further confirmed that there was no significant neutrophils infiltration in two groups; TUNEL staining showed that only a small amount of TUNEL was thin in the two groups. Western blot results showed that the expression of caspase-3 protein in the two groups was not different from that of the sham group. Compared with the sham group, the 85% hepatectomy group and the 90% hepatectomy group showed significant regenerative response, but the regenerative rate of the latter was significantly lower than that in the former. The number of mitotic fingers in the 24 hours after the operation showed the same trend; the related causes of liver tissue regeneration were related. The expression of nRNA: the expression of mRNA in TNF- a, myc and HGF was not significantly different between the two groups. The expression level of mRNA in the 90% hepatectomy group was obviously up-regulated, and the expression of cell cycle related factors (cyclinD, cyclinE, cyclinA, cyclin B) in the 90% hepatectomy group was lower than that in the 85% hepatectomy group; the hepatic tissue results showed that two groups were found. There were different degrees of cell proliferation after operation, and the positive rate of the 90% hepatectomy group was significantly lower than that in the 85% hepatectomy group. The VVesternb1ot results of the PCNA protein in the liver tissue showed a similar trend of changes in.3Western blot in each group of mTOR and its downstream molecules P70S6K, the phosphorylation level of 4EBP1, and the results were found in mTOR, P70S6K and 4EBP1 after 90% hepatectomy. The phosphorylation level was obviously down, suggesting that the activation of the pathway was abnormal. Third part: 1.Western blot results showed that the phosphorylation level of AMPKa in the 90% hepatectomy group was significantly higher than that of the 85% hepatectomy group, while the expression of cyclin D protein significantly decreased the level of phosphorylation of.Akt, and the phospho molybdate colorimetric assay was not significant. The results of ATP content showed that the content of ATP in the residual liver tissue in the 90% hepatectomy group was significantly lower than that of the 85% hepatectomy group. The results of.2. metabolism in the 85% hepatectomy group showed that 90% hepatectomy seriously interfered with the metabolism of rats, especially the energy related metabolic pathways changed significantly by.3. glucose intervention significantly increased the residual liver group after 90% hepatectomy. ATP level, regenerated liver regeneration rate and mitotic index increased significantly. Histopathological detection showed a significant increase in Ki-67 positive rate, Western blot results in PCNA, cyclin D protein expression level and mTOR signaling pathway activity. Conclusion: 1. based on the work in the pre laboratory period, the liver and pipeline system in rats are fully understood. On the basis of the dissection, the procedures and methods of operation were standardized to accurately deal with the important structures of liver blood flow and hepatic vein in the rat liver. A stable and stable hepatectomy standardized model.2. was successfully established. Acute hepatic failure in the early stage of 90% hepatectomy model of rats established by our method was manifested as liver. The obvious obstacle of dirty excretion, synthetic and metabolic function, high mortality but a certain self healing rate, can well simulate the clinical manifestation of liver failure after hepatectomy. It is a clear indication of the early start factor of.3. regeneration, an ideal model conforming to the clinical practice of surgery, that the 90% hepatectomy induces a strong regenerative signal. However, the results of cell proliferation cycle related factors suggest that residual liver does not exhibit the same strong proliferation ability. Regenerative inhibition of residual liver is the main cause of liver failure after 90% hepatectomy. The results of.4. metabolomics have confirmed that 90% hepatectomy causes severe metabolic disorder, which seriously affects the production of energy, which is 90. The residual liver ATP content decreased significantly after hepatectomy. The low energy state inhibited the mTOR signaling through the activation of the AMPK, and then resulted in the regeneration of the remnant liver to inhibit the regeneration of the liver after 90% hepatectomy, which could promote the regeneration of the residual liver, prolong the survival time and survival rate of the rats, and improve the large range liver resection. In addition to the outcome of the operation, the generation of.6. energy involves many aspects, such as whether mitochondria can maintain normal oxidative phosphorylation and electron transfer function, whether adequate adequate energy metabolism is supplied, and so on. Therefore, further exploration of the causes of abnormal energy metabolism after large hepatectomy is needed to improve the perioperative management. Phase energy state, change
【學位授予單位】：中國人民解放軍醫(yī)學院
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R735.7

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