能量状态介导的mTOR信号通路在大鼠大范围肝切除术后肝功能衰竭中的作用研究

发布时间：2018-05-27 22:05

本文选题：肝切除 + 肝衰竭　；参考：《中国人民解放军医学院》2015年博士论文

【摘要】：背景和目的肝切除术是治疗肝脏良恶性肿瘤的首选方法。随着围手术期管理水平的不断提高、手术技术的不断进步,尤其是精准外科理念的不断深入,肝切除术的手术适应证及切除范围不断扩大,以往认为无法切除的肝脏巨大肿瘤、多发肿瘤以及肝门区肿瘤在今天得以切除,使患者长期无瘤生存成为可能。然而,尽管扩大切除范围能够完整的切除肿瘤并获得阴性切缘,但大范围切除术后由于预留的功能性肝体积不足,常常导致术后肝功能衰竭的发生,这是患者术后死亡的主要原因,严重限制了大范围肝切除在临床的应用。尽管大范围肝切除术后肝功能衰竭是一种严重的临床综合征,但其具体的病理生理机制仍未完全明了。术后肝衰发生的本质在于残余肝脏再生与损伤的不协调导致的功能性肝细胞簇数量不足。门脉高灌注、Kupffer细胞激活、肠道菌群异位、过度的炎症反应以及氧化应激反应是造成实质损伤的主要原因,对于这些因素进行相应的干预能在一定程度上改善大范围肝切除术的预后。然而残余肝脏的再生在术后肝衰的病理生理过程中所起的作用,目前研究尚未达成统一观点。有些研究认为,由于实质细胞与非实质细胞的再生进程不同步,大范围肝切除术后早期过快的肝再生反而促进门脉压力的进一步升高,无益于残余肝脏组织结构的重建。但是另有研究也发现大范围肝切除术后残余肝脏的再生表现为明显抑制,同时,促进肝再生的措施也能显著提高术后生存率。因此,明确残余肝脏组织再生进程的变化,对于进一步认识大范围肝切除术后肝功能衰竭的病理生理机制及发掘潜在的治疗策略意义重大。肝切除术后残余肝脏的再生是受到精确调控的复杂生理过程,细胞因子、生长因子及代谢产物共同构成肝再生的调控网络。肝脏是机体的代谢中枢,肝切除术后残余肝脏组织一方面要维持肝脏特异性功能,如糖异生、尿素合成等,另一方面也要产生大量ATP,不仅用于蛋白、核酸及其他细胞成分的合成,也参与重要的再生相关信号通路的活化。肝切除术会严重影响肝脏的代谢功能,尤其是能量代谢。因此,能量代谢的异常可能通过再生进程的调控参与术后肝衰的病理生理过程。mTOR信号通路通过整合胞内能量状态、氧含量以及胞外生长因子、营养物质等信息,调控细胞的代谢以及生长增殖过程。当能量及营养物质来源充足时,该信号通路能够促进细胞的生长；反之,在能量状态低下的情况下,细胞转向能量恢复的过程。基于以上认识,我们设想大范围肝切除术后由于剩余肝脏组织代谢功能的紊乱导致能量状态的显著低下,可能干扰了mTOR信号通路,继而抑制残余肝脏细胞的再生,导致或者促进肝功能衰竭的发生。本课题拟通过建立不同范围肝切除的大鼠模型,研究不同范围肝切除术,尤其是90%肝切除术后残余肝脏的再生与损伤改变,探讨能量代谢对mTOR信号通路以及残余肝脏再生的影响及机制；以及改善能量状态对大范围肝切除术后肝再生的影响。为临床改善大范围肝切除术后患者的肝功能和预后,提高肝脏肿瘤的手术切除率提供理论基础。方法本课题实验分三部分进行：第一部分：基于精准外科理念,建立不同范围肝切除的大鼠模型,初步观察术后肝功能、血生化及组织病理学变化和术后肝衰的发生率及死亡率,选出合适的大范围肝切除术后肝功能衰竭的标准化模型。第二部分：将大鼠分为假手术组、85%肝切除组(大范围肝切除无肝功能衰竭组)和90%肝切除组(大范围肝切除肝功能衰竭组)。对比85%肝切除组和90%肝切除组术后生存率、肝功能、肝再生及实质损伤的相关指标的变化。检测mmTOR信号通路活性在两组的差异。第三部分：进一步检测mTOR信号通路的上下游分子的活性在85%和90%肝切除组的差异。用代谢组学的方法探讨90%肝切除术后大鼠全身代谢水平的改变。我们选择葡萄糖作为治疗组干预药物,探讨改善能量状态对90%肝切除术后mTOR信号通路及残余肝脏再生和预后的影响。结果第一部分：成功建立了大鼠不同范围肝切除的标准化模型。与其它组相比,90%肝切除术后大鼠肝功能障碍更加明显,表现为血清ALT、AST、TBIL水平的明显升高,ALB水平明显降低。70%和85%肝切除术后大鼠的生存率均为100%,而90%肝切除术的生存率明显降低,仅为23%。对死亡大鼠进行尸检,见腹腔内少量至中等量淡黄色腹水,尾叶肿胀,表面颜色略有苍白,并可见扩张血管,未发现出血、胆漏、腔静脉狭窄等并发症,证实死于肝功能衰竭。病理学检测发现肝切除术后24小时残余肝脏表现为肝小叶基本结构存在,肝血窦拥塞,肝细胞表现为明显的空泡样变,但在70%、85%和90%肝切除组之间未发现明显的差异,同时,三组也未出现明显的肝细胞坏死表现。第二部分：1.与假手术组相比,85%肝切除组与90%肝切除组术后大鼠血清ALT、AST水平及HMGB-1的mRNA水平均显著升高,但两组之间的差异无统计学意义；组织病理学检测显示术后24小时两组残余肝叶组织肝小叶结构基本正常,肝细胞空泡变明显,无炎性细胞浸润、坏死等病理改变,中性粒细胞标志物-MPO染色进一步证实两组无明显的中性粒细胞浸润；TUNEL染色显示两组仅有少量TUNEL阳性细胞，Western blot结果显示caspase-3蛋白表达在两组之间无差异。2与假手术组相比,85%肝切除组与90%肝切除组术后残肝都表现出明显的再生反应,但后者的再生率明显低于前者,术后24小时有丝分裂指数表现出相同的趋势；肝组织再生相关因子的nRNA表达情况：TNF-α、myc和HGF的mRNA表达在两组之间无明显差异,IL-6的mRNA表达水平在90%肝切除组明显上调；细胞周期相关因子(cyclinD、cyclinE、cyclinA、cyclin B)的1nRNA的表达,在90%肝切除组明显低于85%肝切除组；肝组织Ki-67结果显示,两组术后均有不同程度的细胞增殖,且90%肝切除组的阳性率明显低于85%肝切除组。肝组织PCNA蛋白的VVesternb1ot结果显示出类似的变化趋势。3Western blot检测各组mTOR及其下游分子p70s6k、4EBP1的磷酸化水平,结果发现90%肝切除术后mTOR、p70s6k和4EBP1的磷酸化水平明显下调,提示该通路的激活出现异常。第三部分：1.Western blot结果显示,与85%肝切除组相比,90%肝切除组AMPKa的磷酸化水平明显上调,而cyclin D蛋白的表达显著降低。Akt的磷酸化水平两组之间无明显差异。磷钼酸比色法检测肝组织ATP含量,结果显示：术后早期90%肝切除组残余肝组织ATP含量明显低于85%肝切除组。2.代谢组学结果显示90%肝切除术严重干扰了大鼠的代谢,尤其是能量相关的代谢通路发生了明显改变。3.葡萄糖干预显著提高90%肝切除术后残余肝组织ATP水平,残余肝脏的再生率及有丝分裂指数显著提高,组织病理学检测显示Ki-67阳性率明显升高,Western blot的结果PCNA、cyclin D的蛋白表达水平及mTOR信号通路活性明显提高。结论：1.基于实验室前期的工作,在充分了解大鼠肝脏及管道系统解剖的基础上,规范手术步骤和方法,精确地处理大鼠肝脏实质内入肝血流及肝静脉等重要结构,成功建立了稳定的不同范围肝切除的标准化模型。2.利用我们的方法建立的大鼠90%肝切除模型术后早期发生急性肝功能衰竭,表现为肝脏排泄、合成及代谢功能的明显障碍,死亡率高但有一定的自愈率等特点,能够很好的模拟肝切除术后肝功能衰竭的临床表现,是一个符合外科临床实际的理想模型。3.再生早期启动因子的明显上调说明90%肝切除诱导了强烈的再生信号,但细胞增殖周期相关因子的结果提示残余肝脏并未表现出同样强烈的增殖能力。残余肝脏的再生抑制是导致90%肝切除术后肝功能衰竭的主要原因。4.代谢组学的结果证实90%肝切除造成严重的代谢紊乱,严重影响了能量的产生,表现为90%肝切除术后残余肝脏的ATP含量的明显降低。低能量状态通过AMPK的激活抑制了mTOR信号通路,继而造成残余肝脏的再生抑制。5.提高90%肝切除术后的能量状态能够促进残余肝脏的再生,并延长大鼠术后生存时间及生存率,改善大范围肝切除术的预后。6.能量的产生涉及多个方面,比如线粒体是否能够维持正常的氧化磷酸化及电子传递的功能,是否足量恰当的能量代谢底物的供应等。因此需要进一步探讨大范围肝切除术后能量代谢异常的原因,以更加有效的改善围手术期能量状态,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
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7

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