【作者】 张颖；

【导师】 王新颖；

【作者基本信息】 南京大学 ， 临床医学， 2019， 博士

【摘要】 癌性恶液质是一种多因素导致的临床综合征,以持续性骨骼肌丢失(伴有或不伴有脂肪组织丢失)为特征,不能被常规营养支持完全逆转。50%以上的肿瘤患者伴有恶液质状态,它是肿瘤患者生存期缩短、治疗失败和毒性反应增加的独立危险因素,严重降低患者的生活质量,并直接导致20%肿瘤患者的死亡。持续性骨骼肌丢失导致的骨骼肌减少症,也称肌少症,是癌性恶液质的一个重要的特征,也是癌性恶液质的一项重要诊断指标,与肿瘤患者的不良预后密切相关。然而癌性恶液质患者骨骼肌减少的机制尚不明确,临床缺乏有效的治疗方法。有关肿瘤患者骨骼肌蛋白周转的研究显示肿瘤患者肌纤维合成未受到明显抑制,分解代谢增强可能起到更重要的作用。本研究以拟行胃癌根治术患者为研究对象,首先分析肌少症等机体成分指标与胃癌患者术后并发症和炎症及营养状态的关系,为临床提供判断患者预后更有效的预测指标,同时明确骨骼肌减少在胃癌患者不良预后中的重要作用,为进一步的研究提供了依据;接着,留取胃癌患者的肌肉组织,使用光镜、电镜、Western blot、q PCR、免疫荧光等技术,明确胃癌恶液质患者骨骼肌蛋白合成和分解代谢的情况,探讨可能的分子机制;最后,采用基于TMT的定量蛋白质组学方法检测分析胃癌恶液质骨骼肌减少患者骨骼肌蛋白质组学特征,从整体上分析和揭示发病的过程和本质,进一步锁定重要的分子和信号通路,探寻癌症恶液质时引起骨骼肌分解增强、骨骼肌减少的可能触发和调控机制。研究结果将为临床治疗癌性恶液质骨骼肌减少提供理论依据和治疗思路,为进一步研究奠定实验基础,指明新的方向。第一部分:肌少症等机体成分指标对胃癌患者术后早期结局的预测价值目的:胃癌患者营养不良的发病率高,是影响临床预后的主要负面因素。机体成分反映患者的代谢状态和生理储备情况,可能是影响患者预后的决定因素。本研究通过分析基于CT检查获得的机体成分指标(肌少症、肌肉脂肪变性和腹型肥胖)与胃癌根治术患者早期结局的关系,明确与患者不良预后密切相关的机体组成,旨在为临床评估患者营养状况、判断预后提供新的更有价值的预测指标。方法:选取行胃癌根治术的患者,根据患者术前2周的腹部CT影像资料,计算患者骨骼肌指数(skeletal muscle index,SMI)、肌肉衰减值(muscle attenuation,MA)、内脏脂肪与皮下脂肪面积比值(visceral to subcutaneous fat area ratios,VSR)。低SMI、低MA、高VSR分别称为肌少症、肌肉脂肪变性和腹型肥胖。检测患者的血清学营养指标及炎症指标,记录患者术后并发症、术后住院时间等指标,分析三种机体成分指标与患者术后并发症及血清营养指标和炎症指标的关系。结果:该研究共前瞻性纳入156例胃癌患者。伴有肌少症或肌肉脂肪变性的患者术后总并发症的发生率显著高于非肌少症(62.5%vs.27.3%,p=0.001)或非肌肉脂肪变性患者(38.2%vs.4%,p=0.002),而腹型肥胖患者感染并发症的发生率则显著高于对照患者(20.3%vs.6.5%,p=0.01)。Logistic回归分析显示肌少症(p=0.013)、肌肉脂肪变性(p=0.017)和血清视黄醇结合蛋白水平减低(p=0.013)是术后总并发症的独立危险因素,腹型肥胖与感染并发症的发生密切相关(p=0.013)。肌少症患者术后血清视黄醇结合蛋白的水平显著低于非肌少症患者(p=0.007),而腹型肥胖患者术后血清C反应蛋白水平则显著高于对照患者(p=0.026)。结论:肌少症、肌肉脂肪变性和腹型肥胖与胃癌患者术后并发症发生率增高显著相关,并与患者术后不良的营养和炎症状态密切相关,是判断胃癌根治术患者临床结局的有价值的预测指标。第二部分:胃癌恶液质患者骨骼肌减少的分子机制研究目的:骨骼肌分解和合成代谢的不平衡将导致骨骼肌的减少。检测胃癌患者腹壁肌肉自噬-溶酶体系统(autophagy-lysosome system,ALS)、泛素-蛋白酶系统(ubiquitin-proteasome system,UPS)和雷帕霉素受体蛋白(mammalian target of rapamycin,m TOR)通路标记物的表达情况,明确骨骼肌分解和合成代谢的活动情况,探讨胃癌恶液质患者发生骨骼肌减少的可能分子机制。方法:本研究选取39名可手术切除的胃癌患者,根据癌性恶液质的诊断标准(近6月体重减轻大于5%)和肌少症诊断标准(腰3平面CT获取的骨骼肌指数:男≤40.8 cm2/m2,女≤34.9 cm2/m2)将其分为四组:N组(非恶液质非肌少症组),CS组(恶液质合并肌少症组),C组(恶液质非肌少症组),S组(肌少症非恶液质组)。术中经腹壁正中切口留取骨骼肌标本。使用HE染色和电镜检测明确各组肌纤维横截面和亚细胞结构的变化。使用Western blot、q PCR和免疫荧光的方法检测各组骨骼肌ALS和UPS标记物、m TOR和AMP依赖的蛋白激酶(Adenosine 5‘-monophosphate(AMP)-activated protein kinase,AMPK)的表达情况。结果:HE染色显示CS组和S组肌纤维横截面积显著低于N组和C组。电镜结果显示N组肌纤维形态基本正常,线粒体呈卵圆形,排列在z线两侧;其他三组肌纤维结构均存在不同程度的破坏,肌丝排列紊乱、断裂,Z线紊乱、溶解,线粒体变大、肿胀,局部可见肌纤维溶解及自噬体形成。CS组,C组和S组骨骼肌自噬标记物Beclin-1,LC3B和P62/SQSTM1的蛋白表达显著高于N组,而且CS组和S组的表达高于C组。与N组相比,泛素蛋白酶系统的标记物MURF1和泛素化蛋白的表达在CS组、C组和S组显著上调,且CS组和S组的MURF1表达显著高于C组。而另一泛素蛋白酶系统的标记物Atrogin-1的蛋白表达四组间无显著差异。各组患者p-m TOR蛋白水平无明显差异。而CS组、C组和S组AMPK及p-AMPK的表达均显著高于N组。结论:胃癌恶液质患者骨骼肌肌纤维结构存在不同程度的破坏,骨骼肌ALS和UPS均受到激活,此效应与患者骨骼肌显著减少的临床表征密切相关,而非单纯的体重下降,是导致癌性恶液质骨骼肌减少的重要机制。第三部分:胃癌恶液质患者骨骼肌减少的蛋白质组学研究目的:通过定量蛋白组学技术分析胃癌恶液质骨骼肌减少患者与对照胃癌患者骨骼肌蛋白质表达的差异,采用生物信息学分析,筛选出相关的“关键”蛋白和通路,探寻癌症恶液质时引起骨骼肌分解增强、骨骼肌减少的可能触发和调控机制,为进一步研究提供思路和靶点。方法:选择N组(非恶液质非肌少症组)和CS组(恶液质肌少症组)胃癌患者(每组各3例),提取肌肉总蛋白,采用基于串联质谱标签(tandem mass tag,TMT)的定量蛋白质组学方法,鉴定并定量多肽和蛋白,获得两组患者差异表达的蛋白质。通过生物信息学方法(层次聚类热力图分析、GO功能分类分析、KEGG通路分析、网络互作分析)分析差异表达的蛋白。结果:共鉴定出2621个可信蛋白质,两组间差异表达的蛋白质114个,其中CS组较N组表达下调的41个,上调的73个。采用生物信息学工具对所鉴定的差异表达蛋白质进行功能分类,发现这些蛋白主要参与的生物过程为肌肉收缩及调节方面、物质/能量代谢方面、细胞分解代谢和死亡方面。CS组患者肌肉收缩和调控蛋白上调,提示肌纤维结构的破坏和降解加速,是对癌性恶液质时骨骼肌分解消耗状态的一个反映。癌性恶液质时骨骼肌物质/能量代谢发生显著调控改变,以糖代谢改变,线粒体能量生成障碍为主要特征,细胞物质和能量代谢紊乱,氧化应激增强,可能是引起肌纤维结构破坏、降解增强的重要触发因素,是引起肌肉萎缩的重要原因之一。同时,研究结果显示CS组骨骼肌存在自噬通路、溶酶体通路、过氧化物酶体通路等细胞内降解系统的激活,骨骼肌蛋白降解的增强,以及坏死性凋亡和铁死亡等程序性死亡增加,可能是导致骨骼肌减少的重要机制。结论:胃癌恶液质患者骨骼肌物质/能量代谢异常,氧化应激增强是肌纤维结构破坏、降解增强的触发因素,而肌纤维降解系统的激活以及不同形式的肌细胞程序性死亡的增加可能是导致骨骼肌减少的重要分子机制。蛋白组学研究为进一步阐明癌性恶液质骨骼肌减少的分子机制奠定实验基础并指出新的方向。更多还原

【Abstract】 Cancer cachexia is defined as a multifactorial syndrome characterised by an ongoing loss of skeletal muscle mass(with or without loss of fat mass)that cannot be fully reversed by conventional nutritional support and leads to progressive functional impairment.Approximately more than 50% cancer patients develop the syndrome,which associated with reduced physical function,reduced tolerance to anticancer therapy,reduced survival and accounts for nearly 20% of all cancer deaths.The muscle loss,with the other name sarcopenia,is the key characteristic of cancer cachexia and is a very powerful prognostic factor,independent of the actual body weight loss.Despite a significant increase in research on cancer cachexia,limited therapeutic options are available and the underlying mechanisms are still poorly defined.Several studies measuring muscle protein turnover in cancer patients showed that muscle protein synthesis was not decreased and increase in protein breakdown might be the main mechanism accounting for muscle wasting in patients with cancer.In the present study,we firstly investigated the association between computed tomograph-quantified body composition and short-term outcomes after gastrectomy for gastric cancer,aiming to identify valuable new prognostic indicators.Then,we determined the synthesis and catabolism in skeletal muscle of gastric cancer patients using a series of techniques including light microscopy,electron microscopy,Western blot,q PCR,immunohistochemistry,exploring the molecular mechanism of the muscle loss in cancer cachexia.At last,we examined the differentially expressed proteins in the skeletal muscles of gastric cancer patients with or without cancer cachexia by quantitative proteomics technology and explored the biological function of those proteins,which contributed to systemically reveal the pathogenesis and mechanism of muscle loss in cancer cachexia.The results of this study will provide theoretical basis and therapeutic ideas for the clinical treatment of cancer cachexia.PART I Computed tomography-quantified body composition predicts short-term outcomes after gastrectomy in gastric cancerObjective: Malnutrition is a common and critical problem that influences outcome in cancer patients.Body composition reflects a patient’s metabolic profile and physiologic reserves,which might be the true determinant of prognosis.In the present study,which aimed to identify valuable new prognostic indicators,we investigated the association between computed tomography-quantified body composition and short-term outcomes after gastrectomy for gastric cancer.Methods: Skeletal muscle index,mean muscle attenuation,and ratio of visceral-to-subcutaneous fat area(VSR)were calculated from preoperative computed tomography images.Low skeletal muscle index,low mean muscle attenuation and high VSR were respectively termed “sarcopenia”,“myosteatosis” and “visceral obesity”.The association of body composition with postoperative complications and serum markers of nutrition and inflammation after radical gastrectomy were analyzed.Results: The overall complication rate was significantly higher in the sarcopenia(62.5% vs.27.3%,p = 0.001)and myosteatosis groups(38.2% vs.4%,p = 0.002).Patients with visceral obesity had a higher incidence of inflammatory complications(20.3% vs.6.5%,p = 0.01).Multivariate logistic regression analysis demonstrated that sarcopenia(p = 0.013),myosteatosis(p = 0.017)and low serum retinol-binding protein(p = 0.019)were independent risk factors for overall complications.Compared with control subjects,patients with sarcopenia had lower postoperative levels of serum retinol-binding protein(p = 0.007),and patients with visceral obesity had higher levels of C-reactive protein(p = 0.026).Conclusions: Sarcopenia,myosteatosis,and visceral obesity were significantly associated with increased rates of postoperative complications and affected the postoperative nutrition and inflammation status of patients with gastric cancer.PART II The molecular mechanism of muscle loss in cachexia induced by gastric cancerObjective: An impaired balance between muscle synthesis and breakdown leads to the development of muscle loss.In this study,we detemined the activity of autophagy-lysosome system(ALS),ubiquitin-protease system(UPS)and m TOR pathway in the rectus muscle of gastric cancer patients,which will help to elucidated the molecular mechanism of cancer cachexia.Method: 39 patients with resectable gastric cancer were divided into four groups based on the presence of cachexia(weight-loss)and/or sarcopenia(low musclularity)including non-cachexia and non-sarcopenia group(N group),cachexia and sarcopenia group(CS group),cachexia and non-sarcopenia group(C group),sarcopenia and non-cachexia group(S group).The biopsies of rectus muscle were obtained intraoperatively.The muscle fiber size and ultrastructural architecture,muscle expression of markers of ALS,UPS and m TOR pathway were analyzed between groups.Results: The muscle fiber cross-sectional areas were significantly decreased in CS and S groups compared to N group or C group.Compared with N group,the muscle ultrastructure exhibit tissue disorganization and autophagosomes formed in the other three groups.The expressions of autophagic markers including Beclin-1,LC3 B and P62/SQSTM1,were significantly increased in CS,C and S groups compared with N group and it was even higher in CS and S groups than C group.As for the expressions of UPS markers,the level of MURF1 and Ub proteins were upregulated in CS,especially in CS group compared with N group.However,there was no difference in Atrogin-1 level among the four groups.The expression of p-m TOR,which reflects the protein synthesis in muscle,has no significant difference among those four groups.The expression of p-AMPK,which plays as energy sensor in cell,was significantly upregulated in CS,C and S groups comparing to N group.Conclusion: ALS and UPS were activated in muscle of cachexia patients especially those with severe muscle loss but not those with obvious weight loss,which account for the muscle loss of cancer cachexia.PRAT III Comprehensive proteome analysis of skeletal muscle in gastric cancer patients with cachexia and sarcopeniaObjective: In this study,we analyzed the differentially expressed proteins in skeletal muscle of gastric caner patients with cachexia and sarcopenia(CS group)compared with non-cachexia and non-sarcopenia(N group)patients by quantitative proteomics technology and bioinformatics analysis to screen out the key protein and pathway contributing to muscle loss induced by cancer cachexia.Methods: The biopsies of rectus muscle of 3 CS patients and 3 N pateins were obtained and the total protein were extracted and quantitfied.Then,TMT proteomic experiments were used to identify and quantify the peptides and proteins.The differentially expressed proteins were analyzed by GO,KEGG pathway and STRING interaction analysis.Results: 2621 proteins were detected and the total of differentially expressed proteins were 114,among which there were 73 up-regulated and 41 down-regulated in CS group compared to N group.The functional groups of the differentially expressed proteins were analyzed by bioinformatics.It showed that the three most pronounced functional groups were muscle contraction and its regulation,substate/energy metabolism,cell catabolism and death.The upregulation of protein involved in muscle contraction and its regulation reflected the increased breakdown of muscle filaments and the enhanced catabolism of cancer cachexia.The distinct modification of substate/energy metabolism in muscle of cancer cachexia is characterized by carbohydrate metabolism change and mitochondrial dysfunction.The disturbance of energy metabolism in muscle is one of the important causes of muscle breakdown and loss.It also showed in the study that cell catabolism system such as autophagy-lysosome pathway and the programmed cell death including necroptosis and ferroptosis were activated,which might lead to the muscle loss in cancer cachexia.Conclusion: Our proteome analysis showed that the abnormal energy metabolism and the inceased catabolism and programed cell death of muscle might be the important leading cause of muscle loss in cancer cachexia.This study provides a basis and new direction for future mechanistic investigations of muscle loss in cancer cachexia.更多还原