【作者】 艾宇航；

【导师】 张阳德；

【作者基本信息】 中南大学 ， 外科学， 2007， 博士

【摘要】 目的：多器官功能障碍综合征（MODS）是全身感染、创伤、烧伤、重症胰腺炎、心肺复苏等疾病中存在的一个共同的难点问题。MODS中致炎因子的过度生成和中性粒细胞、单核／巨噬细胞凋亡障碍是导致疾病发生恶化的两个重要因子。而NF-κB的活化则是其中一个中心环节。在以往的研究中，许多学者试图通过阻断或封闭炎症因子来治疗MODS，但效果均不理想，甚至增加了死亡率。其主要原因可能为炎症因子及代谢产物数量众多，仅针对其中一种或几种进行干预往往达不到预期目的。NF-κB是一种重要的转录因子复合物，它通过激活细胞因子级联反应，生成促炎介质，抑制炎性细胞凋亡在MODS的病理生理过程中起重要作用。因此通过抑制上游转导通路NF-κB的活化，在较高水平上对产生炎症介质的总环节进行控制，是防治MODS新的曙光。本研究以腺病毒转载的IκB基因从中心静脉途径注入增加野生型IκB的表达，即通过直接增加细胞内NF-κB内源性抑制物水平以抑制其活化，从而有效阻断MODS发生发展的通路。方法：第一部分：成年SD大鼠28只，随机分为7个时相：注射病毒前，注射病毒后1d、3d、7d、14d、21d及28d。将重组腺病毒转载的AdLacZ基因经中心静脉途径导入SD大鼠体内，以X-gal染色法检测腺病毒介导的标识基因（LacZ）在大鼠体内的表达部位和时间。第二部分：30只大鼠随机分为三组：A组（正常对照组）；B组（MODS24小时组）；C组（MODS7天组），A组大鼠缓慢注入生理盐水0.25ml／kg，B、C组大鼠缓慢注入油酸0.25ml／kg，4小时后B、C组经中心静脉途径注入脂多糖3.5mg／kg（体积1ml）。观察三组大鼠从致伤开始后的生理状态、刺激反应、活动度、死亡率、各脏器功能的生化指标及形态学改变等情况。第三部分；50只大鼠随机分为五组：A组（正常对照组）；B组（MODS损伤1天组）；C组（MODS损伤7天组）；D组（腺病毒转载IκB治疗1天组）；E组（腺病毒转载IκB治疗7天组）。每组10只。A组大鼠缓慢注入生理盐水0.25ml／kg，B、C、D、E组大鼠缓慢注入油酸0.25ml／kg，4小时后颈部备皮消毒，经中心静脉途径A组缓慢注入生理盐水1ml，B、C、D、E组注入脂多糖3.5mg／kg（体积1ml）。同时D、E组注入滴度为1×10⁹空斑形成单位（pfu）的腺病毒转载的IκB基因1ml。观察五组大鼠从致伤开始后1天、7天的生理状态、刺激反应、活动度、死亡率、各脏器功能的生化指标及形态学改变、血浆肿瘤坏死因子α（TNF-α）、白介素—6（IL-6）含量、肺、肝组织中NF-κBP65蛋白、Fas蛋白、FasL蛋白的表达结果：1．注射AdLacZ（1×10⁹pfu／mL）后1d，在大鼠肺、肝、肾组织即有少量LacZ表达，3d后大鼠肺、肝、肾、脾组织表达明显，7d达高峰，到14d开始下降，28d基本消失。2．B、C组大鼠血肌酐、谷丙转氨酶、总胆红素、肌酸磷酸肌酶水平较A组明显升高（P＜0.05），D、E组血肌酐、谷丙转氨酶、总胆红素、肌酸磷酸肌酶水平与A组相比无显著差异（P＞0.05）；D组谷丙转氨酶、总胆红素、肌酸磷酸肌酶水平较B组降低（P＜0.05），而血肌酐水平无显著差异（P＞0.05）；E组血肌酐、谷丙转氨酶、总胆红素、肌酸磷酸肌酶水平较C组明显降低（P＜0.05）；B组与C组，D组与E组相比血肌酐、谷丙转氨酶、总胆红素、肌酸磷酸肌酶水平均无显著差异（P＞0.05）。B、C、D、E组动脉血氧分压均较A组有所降低（P＜0.05）；但E组动脉血氧分压较C组明显升高（P＜0.05）D组与B组、B组与C组、D组与E组相比动脉血氧分压水平均无显著差异（P＞0.05）PH值C组与A、B组相比有所降低（P＜0.05），其余各组相比均无差异（P＞0.05）；PaCO₂五组之间比较均无显著性差异（P＞0.05）；3．A、C、E三组大鼠血液中TNF-α、IL-6值比较：其中TNF-α浓度以C组最高，A组最低，E组居中，三组间比较均有显著的统计学差异（P＜0.05）。IL-6浓度以C组最高，A组最低，E组居中，三组间比较均有显著的统计学差异（P＜0.05）。见表3。4．在A组肺组织内可见NF-κBP65阳性细胞表达多分布于肺泡巨噬细胞、中性粒细胞和血管内皮细胞，胞浆阳性。C组在油酸—内毒素刺激后可见阳性细胞主要以细胞核为主，染色重，胞浆也有少量染色，但染色程度较正常组织浅。E组经IκB基因治疗后见阳性细胞分布于胞浆和胞核，胞核内阳性细胞较C组明显减少。免疫组化评分C组（9.38±1.06）最高，A组（0.9±0.88）最低，E组（3.78±1.20）居中，三组间比较有显著统计学差异（P＜0.05）。在A组肝组织内可见NF-κBP65阳性细胞表达于肝细胞胞浆。C组在油酸—内毒素刺激后可见阳性细胞主要以细胞核为主，染色重，胞浆也有少量染色，但染色程度较正常组织浅，E组经IκB基因治疗后见阳性细胞分布于胞浆和胞核，胞核内阳性细胞较C组明显减少。免疫组化评分C组（9.75±1.39）最高，A组（0.70±0.82）最低，E组（4.21±1.56）居中，三组间比较有显著统计学差异（P＜0.05）。5．A组大鼠肺、肝组织中可见NF-βBP65、Fas、FasL蛋白表达，B、C组肺、肝组织中NF-κBP65、Fas、FasL蛋白表达较A组明显升高，以肺组织升高幅度最大。D、E组肺、肝组织中NF-κBP65、Fas、FasL表达较B、C组明显下降结论：1．经中心静脉途径注入腺病毒转载的IκB基因，可在肺、肝、肾、脾内高效表达，可用于相关系统疾病的基因治疗。2．经中心静脉途径注入腺病毒转载的IκB基因在脑内始终无表达，不适于中枢神经系统疾病的治疗3．油酸／脂多糖序贯损伤可成功复制大鼠MODS模型。是研究以肺启动机制较好的MODS模型。4．经中心静脉途径导入腺病毒转载的IκB基因，可降低MODS大鼠血肌酐、谷丙转氨酶、总胆红素、肌酸磷酸肌酶水平，改善动脉血氧分压。5．经中心静脉途径注入腺病毒转载的IκB基因，可降低MODS大鼠血液中炎性因子TNF-α、IL-6的含量，抑制肝、肺组织NF-κB的活化（核转移），减轻NF-Kbp65蛋白表达。6．经中心静脉途径导入腺病毒转载的IκB基因，可明显降低肝、肺组织的Fas和FasL表达，减轻MODS大鼠器官组织过度的细胞凋亡。7．经中心静脉途径注入腺病毒转载的IκB基因治疗MODS的机制可能为直接增加野生型IκB的表达，抑制NF-κB活化，从而抑制炎症级联反应，抑制过度的细胞凋亡，治疗MODS。其具体的机制尚有待于进一步研究。更多还原

【Abstract】 Objective: multiple organ disfunction syndrome was a corporatedifficulty problem in systemic inflammation, trauma, burn, and diseases.overexpression of inflammation factors and handicap of monocyte andmacrophages apoptosis had deteriorated diseases .one of the focus tacheswas the activation of nuclear factor -κB .in the past studies,manyscholars wanted to therapy multiple organ disfunction syndrome byinterdiction expression of inflammation factors .but the effect was notperfect,some even increased mortality rate.the main cause was likely tointerventens a sort of inflammatorymediators and metabolizablesubstances could not carried anticipatable point, nuclear factor -κBwas a importmont transfer factor compound.it had importment functionin the pathophysiological process of multiple organ disfunctionsyndrome by activated series reactions of cytokines ,createdinflammatory mediators and controlled cells apoptosis.so it was a newmethods to therapy multiple organ dis function syndrome by modulatednuclear factor -κB activation .in the sudy , we want to interdictdevelopmental of multiple organ disfunction syndrome by directly increase endogenic inhibitor of NF-κB, inhibition NFκ-B activation andinflammation factor and enzyme Releasing by infusing IκB genetansported adenovirus through central vein.Methods:part one: pathogen-free rats were randomly divided intoseven scheduling: before inject adenovirus , afte inject adenovirus oneday, afte inject adenovirus three days, afte inject adenovirus seven days,afte inject adenovirus fourteen days, afte inject adenovirus twenty-onedays, afte inject adenovirus twenty-eight days,Recombinant adenovirusvector containing LacZ was transferred to SD rats by injecting intointernal jugular vein. To identify the sites and periods of LacZ geneexpression,X-gal staining was used to detectβ-gal level and period ofLacZ gene expression of different organs in the transfected andnon-transfected rats at different time intervals, part two: 30pathogen-free rats were randomly divided into three groups: A group(control group) , B group (MODS24 hours), C group(MODS72hours),The rats of A groups was infused Saline at the dose of 0.25ml/kg, Band C groups were infused oleic acid at the dose of 0.25ml/kg andlipopolysaccharide at the dose of 3.5ml/kg through central vein(4 hoursinterval), the stimulatory reaction, activity, the morality rate and thefunction of the heart, liver, kidney, and lung after injection wereobserved, part three: 50 pathogen-free rats were randomly divided intofive groups: A group (control group), B group (MODS24 hours), Cgroup (MODS72hours), D group (AdIκB therapy one day), E group (AdIκB therapy seven day). The rats of A groups was infused Saline atthe dose of 0.25ml/kg, B, C, D and E groups were infused oleic acid atthe dose of 0.25ml/kg and lipopolysaccharide at the dose of 3.5ml/kgthrough central vein(4 hours interval). At the same time, C and D groupwere infused 1ml IκB gene tansported adenovirus (tite:1×10~9pfu), thestimulatory reaction, activity, th morality rate and the function of theheart ,liver, kidney, and lung were observed after injection one day andsenven days. plasm content of TNF-α, IL-6, expression of NF-κBP65 inlung and liver tissue through immunohistochemical staining andwestblotting means, expression of Fas and Fas ligand in lung and livertissue through westblotting means were recorded after injection oneand seven 7 days.Result:1. The 1st day after the injection, the lungs, livers, kidneys ,and spleensexpressed few level ofβ-gal ; the 3rd day after the injection, the lungs,livers, kidneys and spleens expressedβ-gal obviously ;their peak levelswere on the 7th days;theβ-gal level decreased on the 14th day;.β-galexpression disappeared in most organs except lungs on the 28th days.Inall animals ,the brain did not appear anyβ-gal expression2. the level of creatinine, alanine aminotranferase ,total bilirubin ,creatinephosphokinase in group B and C were significantly higher than Agroup(P＜0.05).but its in D and E group had little differences than in Agroup(P＞0.05), the level of alanine aminotranferase, total bilirubin, creatine phosphokinase in group D was significantly lowerer than Bgroup (P＜0.05). but the level of creatinine had no difference(P＞0.05).the level of creatinine, alanine aminotranferase, total bilirubin, creatinephosphokinase in group E were significantly lower than C group(P＜0.05).there were no difference in B and C group, D and Egroup(P＞0.05).the lever of partial pressure of arterial oxygen(PaO2) inB, C, D, E group was significantly lower in group A (p＜0.05),but thelever of partial pressure of arterial oxygen(PaO2) in E group wassignificantly higher in group C(p＜0.05), there were no difference in Band D group, B and C group, D and E group(P＞0.05), the lever of PH inC group was lower than A and B group(p＜0.05).although PaCO2 in fivegroup had little differences.3. the serum content of TNF-αwas the highest in group C and the lowestin group A(group A vs C and group E vs C, P＜0.05,respectively). theserum content of IL-6 was the highest in group C and the lowest ingroup A(group A vs C and group E vs C, P＜0.05,respectively).4. in lung of group A,the male cells of NF-κBP65 were distribution inpulmonary alveolus macrophage ,neutrophilic granulocyte and vascularendothelial cell .cytoplasmic is male .in group C the male cells were innuclei mainly , a small quantity male cell were in cytoplasmic butstaining was superficial than the normal group, in group E. the malecells were in nuclei and cytoplasmic, the male cells in nuclei reducedobviously than group C. the scale of immunohistochemistry was the highest in group C and the lowest in group A(group A vs C group E vsAand group E vs C, P＜0.05, respectively). in liver of group A, the malecells of NF-κBP65 were distribution in hepatic cells , cytoplasmic ismale in group C the male cells were in nuclei mainly, a small quantitymale cell were in cytoplasmic but staining was superficial than thenormal group, in group E. the male cells were in nuclei and cytoplasmic.the male cells in nuclei reduced obviously than group C. the scale ofimmunohistochemistry was the highest in group C and the lowest ingroup A(group A vs C group E vs Aand group E vs C, P＜0.05,respectively).5.. the experssions of NF-κBP65、Fas and FasL protein in group B andC were significantly higher than A group.but its in D and E group hadlittle differences than in A group, the level of NF-κd3P65、Fas and FasLprotein in group E was significantly lowerer than C groupConclusions:1. The adenovirus-mediated IκB gene transfer in internal jugular veinmay be an effective approach of gene therapy in some diseases such aslungs,livers, and kidneys.2. The adenovirus-mediated IκB gene transfer in internal jugular veincan not be an approach of gene therapy in central nervous systemdiseases3.the model established by injection of oleic acid and lipopolysaccharidesubsequently is a suitable modle for the studies of the primordial role of lung in the pathogenesis of the multiple organ disfunction syndrome.4..Infusing IκB gene tansported adenovirus through central vein canreduced the lever of creatinine, alanine aminotranferase ,total bilirubin,creatine phosphokinase in multiple organ disfunction syndrome rats.andalso improved the lever of partial pressure of arterial oxygen.5. Infusing IκB gene tansported adenovirus through central vein canreduced the lever of the serum content of TNF-αand IL-6,and suppressthe activation of nuclear factor-κB in lung and liver tissues,accordinglyabated expression of NF-κBP65.6. Infusing IκB gene tansported adenovirus through central vein canreduced the expression of Fas and Fas ligand in lung and liver tissues,accordingly alleviate the excessive cells apoptosis in multiple organdisfunction syndrome rats.7..To increased expression of IκB gene can relieve rat multiple organdisfunction syndrome result in infuse by internal jugular vein.themechanism of therapy multiple organ disfunction syndrome was enhanceendogenic inhibitor of NFκ-B inhibition NF-κB activation andinflammation cascade reaction更多还原