【作者】 谷化剑；

【导师】 冯贤松；

【作者基本信息】 华中科技大学 ， 肝胆外科， 2013， 博士

【摘要】 第一部分Fractalkine/CX3CL1/CX3CR1在梗阻性黄疸大鼠肝肺综合征发病机制中作用的实验研究目的研究发现大鼠肝肺综合征(HPS)模型肺血管内巨噬细胞(PIM)大量粘附与聚积,并被激活后分泌大量iNOS、VEGF、PDGF等生长因子,促进肺微小血管异常过度增生,导致HPS的发生。基于肺血管内皮细胞可分泌趋化因子Fractalkine/CX3CL1,其特异性专一受体CX3CR1主要表达于单核巨噬细胞,假设表达CX3CR1的单核巨噬细胞与肺内皮细胞分泌的CX3CL1特异性结合是PIM聚积的关键信号通路,如果抑制肺组织CX3CL1/CX3CR1信号通路,能否减少PIM聚积、肺微血管再生及改善HPS?因此本研究目的是通过观察HPS模型中肺组织CX3CL1/CX3CR1表达变化,干预其表达水平后观察肺组织CX3CL1/CX3CR1水平及PIM聚积与肺微血管增生程度的变化,探讨Fractalkine/CX3CR1在梗阻性黄疸大鼠HPS发病机制中的作用。方法结扎大鼠胆总管(CBDL)建立HPS模型,设立假手术组(Sham,n=5),将成功模型随机分为实验对照(EC)组(n=15)、CX3CR1抗体干预(CX3CR1-Ab)组(n=15),于术后15d-28d予CX3CR1-Ab组大鼠腹腔注射多克隆CX3CR1中和抗体,Sham组与EC组腹腔注射相等剂量生理盐水,于术后29d检测大鼠肺泡动脉氧分压差(A-aDO2)、PIM聚积程度、微血管密度(MVD)及肺组织CX3CL1与CX3CR1的蛋白与mRNA表达水平。结果CBDL后4w,EC组大鼠血清总胆红素水平升高,伴有肝功能损害,A-aDO2水平升高,与Sham组比较,差异有统计学意义(P<0.001)；EC组肺组织CX3CL1-(mRNA、蛋白)与CX3CR1-(mRNA、蛋白)表达水平均明显升高,PIM显著聚积,MVD增加,与Sham组比较,差异有统计学意义(P<0.001)；CX3CR1-Ab组与EC组比较,肺组织CX3CL1-(mRNA、蛋白)与CX3CR1-(mRNA、蛋白)表达水平均明显下降,PIM聚积减少,MVD降低,A-aDO2正常,低氧血症得以纠正,肺组织形态学明显改善,其差异性有统计学意义(P<0.001)。结论CBDL后大鼠PIM聚积致肺组织微血管病理性再生是HPS的重要发病机制,趋化因子CX3CL1/CX3CR1在肺组织中表达增加并特异性结合是单核巨噬细胞在肺血管内聚积的关键信号通路,抑制该信号通路的表达可明显减少PIM聚积,改善HPS。第二部分甲硫氨酸维B1与丹参酮ⅡA磺酸钠对梗阻性黄疸后肝肺综合征大鼠肝、肺、肾结构与功能的影响目的梗阻性黄疸(OJ)在围手术期可导致内毒素血症(ETM)和多脏器功能损害,围手术期并发症和死亡率高,其中以肝、肺、肾的内毒素性损害严重,患者多因休克、肝、肺、肾功能衰竭死亡。本研究拟采用CBDL大鼠OJ模型,探讨HPS的发生及大鼠肝、肾功能损害的可能机制,及经甲硫氨酸维Bi(MVBi)与丹参酮IIA磺酸钠(STS)干预后CBDL大鼠内毒素水平与肝、肺、肾组织结构和功能的变化。方法经CBDL建立大鼠OJ模型,设立假手术组(sham,n=5),将成功模型随机分为实验对照(EC)组、MVB1组、STS组及MVB1与STS联合(M-S)组,每组n--20,于术后1d-13d给予CBDL大鼠腹腔内注射相应剂量药物,于2,6,10和14d测定血清ET、TNF-α、ALT、AST、ALP、TBIL、DBIL、Bun、Crea水平,于14d处死大鼠,作动脉血气分析,观察肝、肺、肾组织病理学改变及肺组织微血管再生程度。结果CBDL后大鼠死亡率为10%。与Sham组比较,CBDL后大鼠血清ET、TNF-α、 ALT、AST、ALP、TBIL、DBIL、BUN、Crea水平进行性升高。STS、MVBi与M-S组分别与EC组比较,血清ET、TNF-α水平不同程度下降,肝肾功能有一定改善,其差异有统计学意义(P<0.05),其中M-S组对肝肾功能改善作用明显比STS与MVBi组显著(P<0.001)。14d时EC组A-aDO2>20mmHg,肝、肺、肾MDA与SOD及肺MAD增加；M-S组与STS组A-aDO2下降,微血管再生程度下降(P<0.001)；MVB1组与EC组其差异无统计学意义(P>0.05)。结论OJ大鼠血清ET、TNF-α水平进行性升高与组织脂质过氧化作用是导致肝、肾功能损害的重要因素,MVB1与STS对OJ大鼠血浆TNF-α水平升高有抑制作用,STS可减少OJ时氧自由基损害作用,STS与MVB1联合应用具有叠加效应。CBDL后2w出现微血管异常再生,发生HPS,MVB1对HPS无改善作用,STS不能阻止HPS的发生,但具有改善HPS的作用。第三部分医源性胆道损伤的原因、防治策略及淤胆型肝硬化并肝肺综合征的外科治疗目的医源性胆道损伤(IBDI)经各种初期修复手术治疗后,仍可出现胆道狭窄、胆道感染、肝内胆管结石,胆汁淤积性肝硬化及HPS,是复杂棘手的临床疑难问题。再手术方式缺乏定式,复杂多变,术后死亡率高。本部分临床研究旨在探讨IBDI的原因、防治策略及伴HPS的再手术修复胆道的方式选择与疗效。方法对我院2004年1月～2010年9月期间所收治的术后发生IBDI56例病例作回顾性分析,探讨IBDI的原因与防治策略：分析2011年3月-2012年9月期间所收治6例IBDI伴HPS患者,评估IBDI伴HPS再次胆道修复手术方式与路径的选择、术后恢复情况及并发症。结果56例IBDI病例中1例胆总管下段后壁穿孔致感染性休克死亡；IBDI一次修复优良率75.0%(42/56),2次修复优良率53.8%(7/13),6例患者经3次修复1例预后良好,2例术后肝衰竭死亡,死亡率33.3%(2/6),3例胆肠吻合口狭窄,仍需手术治疗。术后随访优良率为89.3%,疗效差占10.7%。对6例IBDI患者均成功施行胆道修复手术,术式没有统一的定式,手术时间4.9±3.6h,术后发生呼吸衰竭3例,其中死亡1例,胆漏伴腹腔局部感染1例,治愈出院5例。术后随访0.5-2年,优良率为100%。结论IBDI的原因与局部解剖变异因素、病理因素、术者技术因素有关,IBDI后胆汁淤积性肝硬化伴HPS患者在施行胆道再次修复手术时,手术难度大、剥离面广、出血多、手术时间长,手术方式根据IBDI发生的原因、初次或多次修复的术式、肝硬化程度、肝内胆管结石的分布、第一肝门解剖特点及患者全身情况等综合因素选择个体化胆道修复术式,手术过程应力求简单、快速、有效,术后多需要ICU等多学科综合治疗。胆道修复手术并未改善A-aDO2的升高,IBDI并HPS患者术后呼吸功能发生率与死亡率高。更多还原

【Abstract】 Part one An experimental research about role of fractalkine/CX3CL1/CX3CR1in the pathogenesis ofhepatopulmonary syndrome rats with obstructive jaundiceBackground:Common bile duct ligation (CBDL) for rat is a model system for the research of HPS.Prior studies have discovered that pulmonary intravascular macrophages(PIM) adhere and accumulation occurs as experimental HPS develops.The activated CD68(+) macrophages generate angiogenic and proliferative growth factors,including inducible nitric oxide synthase(iNOS),vascular endothelial growth factor(VEGF),and platelet-derived growth factors(PDGF).That induces an occult proliferative pulmonary vasculopathy.Intravascular accumulation of activated macrophages is central to the pathogenesis of HPS.Fractalkine is known to be expressed on pulmonary vascular endothelial cells(PVEC).It binds highly specifically to its receptor CX3CR1which is expressed on monocytes and lymphocytes.Thus, we hypothesized that the specific binding of fractalkine expressed on PVEC to CX3CR1expressed on monocytes was crucial signal pathway of PIM adhere and accumulation.Could degree of PIM accumulation and pulmonary microvascular angiogenesis be decreased after CBDL if CX3CL1/CX3CR1expression and signaling were altered?Could HPS be improved?Therefore, the aim of this part was to define if pulmonary CX3CL1and CX3CR1expression are altered after CBDL and influence macrophages accumulation,angiogenesis and the development of HPS,to research the role of fractalkine/CX3CR1in the mechanisms of HPS rats.Material and methods:Establishing sham-operation group(n=5).The rats after CBDL were randomly divided into experimental control group(EC group, n=20) and anti-CX3CR1-neutralizing polyclonal antibody group(CX3CR1-Ab group,n=20).From15to28day after operation,the rats in CX3CR1-Ab group were intraperitoneal injected respectively with anti-CX3CR1-neutralizing polyclonal antibody each day,and he rats in EC and sham group received identical volume of normal saline.All rats were died on29day,and A-aDO2was immediately detected.Expression levels of ALT,ALP,TBIL in serum were determined with radioimmunoassay.Meanwhile,accumulation degree of pulmonary intravascular macrophages(PIM) and microvessel density (MVD) were tested by immunity fluorescence method.Furthermore,protein expression and mRNA level of CX3CR1and CX3CL1were measured with western blot and real time-PCR.Results:In the rats after CBDL,the serum levels of TBIL,ALT,ALP and A-aDO2were rapidly elevated,accompanied by liver function damage.There was statistical difference in the changes (P<0.001vs sham group).In EC group,PIM showed a larger accumulation,MVD was markedly elevated,protein and mRNA expression of CX3CL1and CX3CR1were significantly increased (P<0.001vs sham group). Compared to EC group,in CX3CR1-Ab group A-aD02showed the normal range,hypoxemia was improved,protein and mRNA expression levels of CX3CL1and CX3CR1were remarkably decreased,likewise,degree of PIM accumulation and pulmonary microvascular angiogenesis were reduced to some extent.There was significant difference between the two groups (P<0.001)Conclusion:Pulmonary intravascular macrophage aggregation increase after CBDL and contribute to pathological proliferation of pulmonary microvascular and the development of experimental HPS.Pulmonary CX3CL1/CX3CR1expression elevated and specific binding is a vital signaling pathway by which mononuclear macrophage adhere and accumulate in pulmonary intravascular.Macrophage depletion is greatly obvious though blocking the signaling pathway.Moreover,HPS could be clearly improved. Part towAn experimental study for effects of Methionine and Vitamin B1and Sodium Tanshinone IIA Sulfonate on liver,lung and kidney function of rats with hepatopulmonary syndrome and obstructive jaundiceBackground:Obstructive jaundice(OJ) could lead to endotoxemia(ETM) and multiple organ dysfunction during perioperative period.There are great high complication rate and mortality.Especially,the function injuries of liver,lung and kidney are extraordinary serious.More patients were dead due to shocking and hepatic, kidney and respiratory function failure.The purpose of this part is to research the mechanisms of HPS and pathogenesis of liver and kidney function injury through observing level changes of endotoxin(ET) and A-aD02and liver,lung,kidney function and tissue structure of rats with HPS after CBDL,to investigate effects of Sodium Tanshinone IIA Sulfonate(STS) and Methionine and Vitamin B1(MVB1) on the changes.Material and methods:The rats after CBDL were randomly divided into experimental control(EC)group,MVB1group,STS group and combined group with MVB1and STS(M-S)group.There were20rats in each one group,and establishing sham-operation group(n=5).From1to13day after operation EC,MVB1and STS group rats were intraperitoneally injected respectively with identical volume of normal saline,MVB1and STS.M-S group rats received MVB1and STS together.Expression levels of ET,TNF-a,ALT,AST,ALP,TBIL,BUN and Crea in serum on days2,6,10and14after operation were respectively determined with radioimmunoassay.All rats were died on14day,and A-aDO2was immediately detected.Meanwhile,histopathological changes of liver,lung,kidney tissues were observed,and CD31(+)angiogenesis were measured.Result:The mortality rate of rats with OJ was10%.In the groups after BDL,levels of ET,TNF-a,ALT,AST,ALP,TBIL,BUN and Crea in serum were rapidly elevated(P<0.001vs sham group). Compared to EC group,levels of serum ET,TNF-a were declined in STS,MVB1and M-S groups,and liver and kidney functions were improved to some extent.There was statistical difference in the changes(P<0.05).Compared respectively to STS and MVB1groups,level changes of the above tested indexes in M-S group were significant(P<0.001).A-aDO2was enhanced (≥20mmHg) in EC group rats on14d after CBDL.Meanwhile,Degree of pulmonary intravascular macrophages(PIM) accumulation and angiogenesis were markedly increased. In STS and M-S groups,A-aDO2was declined (≤15mmHg),and angiogenesis was reduced.There was statistical difference in the changes(P<0.001vs EC group).The histological changes were not significant between EC and MVB1group (P>0.05) Conclusion:The levels increased of TNF-a and ET in serum are ones of the most important factors which induce angiogenesis in HPS and damaging of liver and renal function in OJ rats.MVB1and STS could restrain the increase of plasmic TNF-a and ET of rats with OJ.There is more significant inhibiting effect on ET in STS than MVBi.The anti-endotoxin effect and protective effect on liver and kidney tissue of OJ rats could be clearly elevated when being injected with together MVB1and STS.Pulmonary angiogenesis occurs as experimental HPS on two weeks after CBDL.MVB1has not effect on lung tissue structure and function of HPS rats.STS could not prevent the occurrence of experimental HPS.Nevertheless,it could improve HPS to some extent through decreasing pulmonary microvascular angiogenesis. Part threeCause and prevention and surgical treatment of iatrogenic bile duct injury complicated with hepatopulmonary syndromeBackground:Long-term complications that occur for some patients,such as biliary stricture,intrahepatic stones,cirrhosis and HPS after initial repair surgery for iatrogenic bile duct injury(IBDI) patients.The events are hard problems in clinical treatment.The reoperation procedures are complicated, changing and different from traditional operation.There is a higher mortality rate after the reoperation.The purpose of this part is to research the causes,treatments of IBDI,to estimate the choice of reoperation methods and therapeutic effect on IBDI patients with HPS.Material and methods:A retrospective analysis was performed on56surgically treated cases with IBDI after operation in Wuhan Union Hospital from January2004to September2010.From March2011to September2012,6patients suffered from IBDI which complied with the standard diagnosis of HPS.We discussed the choice of reoperation methods and paths,estimated postoperative recovery,complication rate and mortality.Results:One case was dead for infectious shock that was caused by common bile duct segment back wall perforation in56ones.Fine rate was75percent for the first repair,53.8percent for the second repair.One case has better outcomes in6patients repaired for the third time,two ones dead,there ones needed further treatment.The excellent rate was89.3percent, poor rate10.7percent.The6cases were operated successfully. However,the procedures were painful after hours (4.9±3.6h) on average.The reoperation methods were different from traditional operation.After reoperation respiratory failure took place in3cases,one of them died.Bile leak with local intra-abdominal infection occured in1case.5cases obtained good cure rates.The excellent rate was100percent after0.5～2years follow-up.Conclusion:The reasons of IBDI are related to bile duct variable anatomy,lesion local pathological change and performer technology factors.The operation suffered from painful process,wide strip surface and more intraoperation bleeding during the reoperation.The reoperation methods are determined by a number of factors which are the causes,severity,repairing ways of primary IBDI,cirrhosis,distribution of intrahepatic stones,anatomy feature of the first porta and the general conditions of the patients.The procedure should be strived to simple, rapid and effective.Comprehensive treatments of ICU and other multi-disciplinary are needed for high incidence rate of complications.A-aDO2enhanced could not be improved after the reoperation of IBDI.Moreover,the incidence of respiratory failure and mortality rate were increased after the reoperation for patients with HPS.更多还原