胸导管淋巴液对重症急性胰腺炎并发肺损伤影响的实验研究
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摘要
目的:重症急性胰腺炎(Severe Acute Pancreatitis, SAP)临床过程凶险,其病死率高。目前研究证实,SAP并发急性肺损伤(acute lung injury,ALI)高达60～70%,终致并发急性呼吸窘迫综合征(acute respiratory distress syndrome ,ARDS),而成为SAP的主要死亡原因之一,SAP病死患者中65%以上与急性呼吸窘迫综合症(ARDS)有关,而ARDS是急性肺损伤的一个终末结局。因此,明确SAP时ALI的发病机制,早期预防和治疗ALI对降低SAP的死亡率及改善疾病的预后具有重要意义。
SAP时ALI是机体过度炎症反应导致肺血管内皮细胞和肺泡上皮细胞广泛破坏的结果,而中性粒细胞(polymorphonuclear neutrophils, PMN)能触发和放大炎症反应,在炎症反应中起着关键作用,是炎性反应的重要标志。有研究结果显示SAP时,PMN在肺内粘附聚集,导致肺血管内皮细胞、肺泡上皮细胞广泛损伤,致通透性增加、肺水肿及微血栓形成,进而发展成ALI。PMN在肺内聚集和激活是SAP时ALI发病的最初环节,如果能采取干预PMN在肺内的聚集激活,就能早期预防ALI。但是关于SAP时PMN在肺内聚集和激活的机制并不十分清楚,仍有待进一步研究。目前,肠系膜-胸导管淋巴液在重度烧伤、感染中毒性休克等中的作用得到重视,并认为其可以介导严重感染/创伤等引起的肺脏损伤,并进而导致肺和全身的炎症反应。临床及动物实验发现各种原因引起的多器官功能障碍综合症(multiple organs dysfunction syndromes, MODS)常常首先出现ARDS,而肺是首个接纳肠系膜淋巴液的器官,肠系膜淋巴液可能起着介导肺损伤的作用;又有研究显示了休克后收集到的肠系膜淋巴液能损伤或致内皮细胞死亡,增加内皮细胞的渗透性,结扎肠系膜淋巴管能减轻失血性休克后或烧伤后肺损害。这些研究都提示肠系膜淋巴液含有能够介导全身炎症反应时肺损伤的物质。而且在创伤性休克的动物模型或病人的门静脉血的研究中,并没有发现细菌或毒素。同时,休克时门静脉血浆对于血管内皮细胞的损伤作用很轻。这些研究表明经肠道微血管回流的门静脉途径在休克致器官损伤的发病学意义有一定的局限性。Louis报道失血性休克所致的肺损伤和内皮细胞通透性增高是由肠道有害物质经淋巴液而非经门静脉血液所引起的。据此,我们推测这种作用在类似烧伤和严重感染的SAP中可能也存在,并且有可能通过诱导PMN在肺脏聚集和激活来介导。
本研究应用SD大鼠建立颈部胸导管引流模型,并在此基础上制备SAP合并ALI模型,观察肺脏病理变化,检测血气分析变化,从而了解胸导管结扎/引流对ALI的影响;并检测胸导管淋巴液中抗炎和促炎因子、胰酶、内毒素的含量变化,以及其对动脉血中PMN功能的影响预后,观察SAP继发ALI时上述指标的变化。旨在探讨SAP时胸导管淋巴液的成分变化在激活肺脏中PMN、介导SAP时ALI发生中的作用,以及可行的干预途径,进一步阐明SAP合并ALI的发病机制,为临床治疗提供新的思路、途径及理论和实验依据。
方法:
第一部分:大鼠颈部胸导管引流动物模型的建立
选用成年清洁级雄性SD大鼠60只。随机将大鼠分为3组,每组20只:第1组:颈部胸导管直接组,第2组:颈部胸导管间接组,第3组:腹部胸导管组。在戊巴比妥钠麻醉并固定后,应用显微手术器械分别解剖颈部胸导管、左颈干、腹部胸导管,应用24G留置针分别穿刺保留颈部胸导管、左颈干、腹部胸导管,引流淋巴液。记录三种胸导管淋巴液引流模型之间淋巴流量、制模成功情况。
第二部分:胸导管结扎/引流对重症急性胰腺炎并发肺损伤早期干预及动脉中性粒细胞功能变化的实验研究
选取健康雄性SD大鼠96只,随机分为12组,每组8只,分别为颈部假手术组+腹部假手术2小时组(SO2h)、颈部假手术组+腹部假手术6小时组(SO6h)、颈部假手术组+腹部假手术12小时组(SO12h);颈部假手术组+SAP2小时组(SAP2h)、颈部假手术组+SAP6小时组(SAP6h)、颈部假手术组+SAP12小时组(SAP12h);颈部胸导管结扎+SAP2小时组(LC2h)、颈部胸导管结扎+SAP6小时组(LC6h)、颈部胸导管结扎+SAP12小时组(LC12h);颈部胸导管引流+SAP2小时组(DC2h)、颈部胸导管引流+SAP6小时组(DC6h)、颈部胸导管引流+SAP12小时组(DC12h)。按分组要求制备颈部假手术组+腹部假手术组(SOA)、颈部假手术组+SAP组(SAP)、颈部胸导管结扎+SAP组(LC)和颈部胸导管引流+SAP组(DC)动物模型。观察胰腺、肺脏组织病理切片、肺脏扫描电镜;应用生化法检测血清胰淀粉酶(AMY)、胰脂肪酶(LIP);应用自动血气分析仪检测动脉血气分析。
第三部分:胸导管结扎/引流对重症急性胰腺炎时动脉血中性粒细胞功能影响的实验研究
取样来自第二部分腹主动脉血(取动脉血气同时)应用流式细胞术(Flow Cytometry,FCM)检测动脉中中性粒细胞功能变化。
第四部分:重症急性胰腺炎并发肺损伤时胸导管淋巴液中TNF-α、IL-10、AMY、LIP及内毒素浓度变化的实验研究
选择健康SD大鼠20只,随机分为2组,每组10只。分别为颈部胸导管引流组+腹部假手术组(Sham)和颈部胸导管引流+SAP组(DC)。建立SD大鼠颈部胸导管引流情况下腹部手术组、SAP组模型,持续引流并分时段及时低温保存淋巴液。应用生化法检测淋巴液/血清中胰淀粉酶(AMY)、胰脂肪酶(LIP);酶联免疫法检测淋巴液/血清中TNF-α、IL-10,反应时间法检测淋巴液中内毒素浓度。
结果:
第一部分:大鼠颈部胸导管淋巴引流动物模型的建立
1各组动物模型均成功引出浅乳白色液,外观性状无差异,均为淋巴液。
2颈部胸导管直接组制模成功率明显低于腹部胸导管直接组和颈部胸导管间接组(P<0.05);
3腹部胸导管组的淋巴流量较颈部胸导管直接组和颈部胸导管间接组多且差异明显(P<0.05)。
第二部分:胸导管结扎/引流对SAP并发ALI早期干预的实验研究
1与同时点实验对照组(SO组)相比,SAP各时点腹水量、胰腺病理评分、肺脏病理评分、血清AMY、血清LIP明显升高(P<0.01); SAP2h、6h的PaO2与同时点SO组相比明显升高(P<0.01),且SAP2h组的PaO2明显高于SAP6h(P<0.01),而SAP12h组的PaO2明显高于同时点SO组(P<0.01);SAP2h、6h的PaCO2与同时点SO组相比明显降低(P<0.01),而SAP12h组的PaCO2明显高于同时点SO组(P<0.01);、
2与同时点SAP组(干预对照组)相比, LC6h组、LC12h组的腹水量、LC6h组的肺脏病理评分、LC2h组血清AMY和血清LIP明显降低(P<0.01或P<0.05);胸导管结扎组的LC2h组的腹水量、LC2h12h的肺脏病理评分有降低趋势,但无显著差异;LC2h、6h的胰腺病理评分、LC12h的PaO2有升高趋势,但无显著差异,LC12h的胰腺病理评分明显升高(P<0.01);LC6h的PaO2明显升高(P<0.01)。
3与同时点SAP组(干预对照组)相比,胸导管引流组的DC12h组的腹水量、DC6h、12h的肺脏病理评分、DC2h、6h、12h血清AMY和血清LIP明显降低(P<0.01或P<0.05);DC2h组的肺脏病理评分有降低趋势,但无显著差异;DC6h、12h的PaO2明显升高(P<0.01)。
第三部分:胸导管结扎/引流对重症急性胰腺炎时动脉血中性粒细胞功能影响的实验研究
1与同时点对照组(SO组)相比,SAP组各时点CD11b表达阳性PMN比例、PMN呼吸爆发作用(RB)平均荧光强度均明显升高(P<0.01);SAP2h的CD11b平均荧光强度与同时点SO组相比有升高趋势,但无显著性差异(P>0.05),SAP6h、12h的CD11b平均荧光强度与同时点SO组相比明显升高(P<0.01或P<0.05),且SAP12h组的CD11b平均荧光强度明显高于SAP6h;
2与同时点干预对照组(SAP组)相比,各时点LC的PMN呼吸爆发作用(RB)平均荧光强度、CD11b表达阳性PMN比例、CD11b平均荧光强度均有降低趋势,但无显著差异。
3与同时点干预对照组(SAP组)相比,各时点DC的PMN呼吸爆发作用(RB)平均荧光强度、CD11b平均荧光强度、DC6h、12h的CD11b表达阳性PMN比例均有明显降低(P<0.01或P<0.05)。第四部分:重症急性胰腺炎并发肺损伤时胸导管淋巴液中TNF-α、IL-10、AMY、LIP及内毒素浓度变化的实验研究
1 TNF-α,IL-10
1.1淋巴液中、血清中TNF-α
淋巴液中TNF-α水平:腹部假手术各组间Sham(2h)、Sham(6h)、Sham(12h)的TNF-α水平均无明显差异;与同时腹部假手术各组相比,SAP各时点组的淋巴液中TNF-α均明显升高(P<0.01);SAP各时点组相比:SAP6h、12h组的TNF-α较SAP2h组均明显升高(P<0.01),SAP6h、12h两组之间相比TNF-α水平均无明显差异。
血清中TNF-α水平:腹部假手术各组间Sham(2h)、Sham(6h)、Sham(12h)的TNF-α水平均无明显差异;与同时腹部假手术各组相比,SAP各时点组的血清中TNF-α均明显升高(P<0.01);SAP各时点组相比:SAP6h、12h组的TNF-α较SAP2h组均明显升高(P<0.05),SAP6h、12h两组之间相比TNF-α水平均无明显差异。
正常状态淋巴液中TNF-α浓度与血清中TNF-α浓度在对应时点均无明显差异;DC组淋巴液中TNF-α浓度与血清中TNF-α浓度在对应时点亦均无明显差异。
1.2淋巴液中、血清中IL-10
淋巴液中IL-10水平:腹部假手术各组间Sham(2h)、Sham(6h)、Sham(12h)的IL-10水平均无明显差异;与同时点腹部假手术各组相比,SAP各时点组的淋巴液中IL-10水平均明显升高(P<0.01);SAP各时点组相比:SAP6h组的IL-10水平较SAP2h组均明显升高(P<0.01);SAP12h组的IL-10水平较SAP2h、6h组均明显升高(P<0.01)。
血液中IL-10水平:腹部假手术各组间Sham(2h)、Sham(6h)、Sham(12h)的IL-10水平均无明显差异;与同时腹部假手术各组相比,SAP各时点组的血清中IL-10水平均明显升高(P<0.01);SAP各时点组相比:SAP6h组的IL-10水平较SAP2h组均明显升高(P<0.01);SAP12h组的IL-10水平较SAP2h、6h组均明显升高(P<0.01)。
正常状态淋巴液中IL-10浓度与血清中IL-10浓度在对应时点均无明显差异;DC组淋巴液中IL-10浓度与血清中IL-10浓度在对应时点亦均无明显差异。
2 AMY ,LIP
2.1淋巴液中AMY
淋巴液中AMY水平:腹部假手术各组间Sham(2h)、Sham 6h)、Sham (12h)的AMY水平均无明显差异;与同时腹部假手术各组相比,SAP各时点组的淋巴液中AMY均明显升高(P<0.01);SAP各时点组相比:SAP6h、12h组的AMY较SAP2h组均明显升高(P<0.05),SAP6h、12h两组之间相比AMY水平均无明显差异。
2.2血清中AMY
腹部假手术各组间Sham(2h)、Sham(6h)、Sham(12h)的AMY水平均无明显差异;与同时点的腹部假手术各组Sham(2h)、Sham (6h)、Sham (12h)相比,SAP各组SAP 2h、6h、12h各组的淋巴液中AMY均明显升高(P<0.01);SAP各时点组相比:SAP6h、12h组的AMY较SAP2h组均明显升高(P<0.05),SAP6h、12h两组之间相比AMY水平均无明显差异。DC组淋巴液中AMY浓度在对应时点明显高于与血清中AMY浓度(P<0.01)。
2.3淋巴液中LIP
淋巴液中LIP水平:腹部假手术各组间Sham(2h)、Sham(6h)、Sham(12h)的LIP水平均无明显差异;与同时腹部假手术各组相比, DC组各时点的淋巴液中LIP均明显升高(P<0.01);DC组各时点相比:SAP6h、12h组的LIP较SAP2h组均明显升高(P<0.01),SAP6h、12h两组之间相比LIP水平均无明显差异。
2.4血清中LIP
血清中LIP水平:腹部假手术各组间Sham(2h)、Sham(6h)、Sham(12h)的LIP水平均无明显差异;与同时腹部假手术各组相比, DC组各时点的血清中LIP均明显升高(P<0.01);DC组各时点相比:SAP6h、12h组的LIP较SAP2h组均明显升高(P<0.01),SAP6h、12h两组之间相比LIP水平均无明显差异;
DC组淋巴液中LIP浓度在对应时点明显高于与血清中LIP浓度(P<0.01)。
3淋巴液中内毒素水平变化
Sham各时点(2h、6h、12h)间没有明显差异;DC 2h与Sham各时点(2h、6h、12h)间相比没有明显差异;DC6h、DC12h与Sham组对应时点相比明显升高,有明显差异(p<0.01);DC6h、12h与DC 2h相比明显升高,有明显差异(p<0.01);DC12h与DC6h相比明显升高,有明显差异
结论
第一部分:大鼠颈部胸导管淋巴瘘动物模型的建立
1颈部胸导管间接组制模成功率最高,其次是腹部胸导管组,颈部胸导管间接组与腹部胸导管组操作成功率差异不明显(P>0.05);颈部胸导管直接组较另外两组操作成功率低(P<0.01)。;
2三种方法引流的淋巴液无明显性状差异;腹部胸导管组在引流量方面明显多于颈部胸导管直接组、颈部胸导管间接组(P<0.01),这可能是由于腹腔淋巴循环对腹腔骚扰较敏感造成的,说明腹部胸导管引流技术明显影响腹腔淋巴循环。
3大鼠颈部间接胸导管引流法可稳定引流淋巴液,且不骚扰腹腔,是稳定、可靠、制模成功率高胸导管引流动物模型制作方法,能够用于SAP并发ALI时与淋巴循环有关的实验研究。
第二部分:胸导管结扎/引流对SAP并发ALI早期干预的实验研究
1 SAP早期即可引起肺脏损伤性病理形态学变化,随后可进一步出现其功能障碍。
2颈部胸导管引流能够减轻SAP引起的急性肺损伤病理形态学变化及其功能障碍,而颈部胸导管结扎有减轻SAP引起的急性肺损伤病理形态学变化及其功能障碍的趋势:在早期(2h,6h)颈部胸导管结扎、引流,两者相比对肺功能的改善差异不明显;到中后期(12h)颈部胸导管引流较结扎能够明显减轻SAP引起的急性肺损伤病理形态学变化及其功能障碍。
3颈部胸导管引流没能减轻SAP时胰腺病理形态学变化,而颈部胸导管结扎能加重SAP时胰腺病理形态学变化
第三部分:胸导管结扎/引流对重症急性胰腺炎时动脉血中性粒细胞功能影响的实验研究
1颈部胸导管引流能够降低SAP时主动脉血中PMN的呼吸爆发作用、CD11b表达阳性PMN比例、CD11b平均荧光强度;而颈部胸导管结扎有降低SAP主动脉PMN的呼吸爆发作用、CD11b表达阳性PMN比例、CD11b平均荧光强度的趋势。
2颈部胸导管结扎/引流能够影响SAP时主动脉血中PMN的呼吸爆发作用、CD11b表达阳性PMN比例、CD11b平均荧光强度,这可能是颈部胸导管引流能够减轻SAP引起的急性肺损伤病理形态学变化及其功能障碍的原因。
第四部分SAP合并ALI时胸导管淋巴液内AMY、LIP、TNF-α、IL-10、内毒素水平变化的实验研究
1 SAP合并ALI时发病过程中淋巴液中内毒素浓度、AMY、LIP、TNF-α、IL-10浓度明显上升。
2 SAP合并ALI时胸导管淋巴液中成分复杂,可能通过激活的胰酶、内毒素TNF-α、IL-10等直接或间接的用过活化中性粒细胞,损伤肺组织。
3 SAP合并ALI时AMY、LIP浓度在淋巴液中与血清中相比,两者之间差异明显,据此,淋巴液中的AMY、LIP是导致血清中该物质浓度上升的来源之一。
4 SAP合并ALI时TNF-α浓度在淋巴液中与血清中相比,两者之间无明显差异,IL-10也是如此;据此,淋巴液中的TNF-α、IL-10不是导致血清中该物质浓度上升的来源。
5反应时间法(鲎试剂法之一)是测定淋巴液中内毒素含量的可靠方法。
Objectives:
Severe acute pancreatitis (SAP), a dangerous clinical process with high mortality, will develop acute lung injury (ALI) as demonstrated by the current studies; and finally it will develop acute respiratory distress syndrome (ARDS), the outcome of ALI and one of the leading causes of SAP, with over 65% patients died associated with ARDS. Therefore, it is significantly important to clarify the mechanism of ALI during SAP and to determine the early-stage prevention and the treatment in order to decrease the mortality and to improve the diagnosis of SAP.
During SAP, over inflammatory reaction of the body causes pulmonary vessel endothelia and alveolus epithelia extensively destroyed, resulting in ALI. Polymorphonuclear neutrophil leukocytes (PMN) can trigger and amplify inflammatory reaction, so it plays a key role in and is the important mark of inflammatory reaction. As documented by some studies, during SAP, PMN coagulate and assemble in the lungs, leading to pulmonary vessel endothelia and alveolus epithelia extensively destroyed and accordingly permeability increased, pneumonedema and microthrombus developed, and finally ALI. Coagulation and activation of PMN in the lungs is the initial process of ALI during SAP. Therefore, if the coagulation and activation can be intervened, the early prevention of ALI can be performed. The mechanism of it, however, is not clear, wanting further study. Currently, mesentery-thoracic duct lymph is highly valued in the severe burns, infectious toxic shock and other diseases, and is regarded as the mediator of lung injury in severe infection/trauma, finally leading to inflammatory reaction in the lungs or systemically. Clinical and animal experiments showed that ARDS will first occur in the multiple organs dysfunction syndromes (MODS) caused by multiple reasons, for lungs are the first organ to accept mesentery lymph, the one which possibly mediates lung injury. Other studies displayed that mesentery lymph collected after shock caused endothelia injured or died, finally increasing the permeability of the endothelia, and that ligating the mesentery lymph ducts can decrease lung injury after heamorrhagic shock and burn. All these studies suggest that mesentery lymph contains some substances that can mediate lung injury during systemic inflammation. Furthermore, no bacteria or toxins were found in the portal vein blood of the animal models or patients with traumatic shock. Meanwhile, portal vein plasma only causes light injury to the vessel endothelia. All this shows that the portal vein blood coming back through enteric canal capillaries plays a minor role in the organ injury caused by shock. As Louis reported, lung injury and increased permeability of endothelia caused by heamorrhagic shock was caused by enteric toxic substances in lymph not in portal vein blood. Based on this, we presume that this kind of function also exists in SAP caused by burn and severe infection, and may mediate SAP by inducing PMN coagulation and activation in the lung.
SD rats were used in this experiment to establish cervical thoracic duct drainage models, and then to prepare models of SAP accompanied by ALI, with the aim to observe the pathogenesis of the lung, to detect the changes of blood gas analysis, thus to find out the effect of thoracic duct ligation /conduction on ALI. Meanwhile, to detect the changed content of anti-inflammatory and proinflammatory factors, pancreatin, and endotoxin, and to understand the prognosis effect of these factors on PMN functions in the arterial blood, and finally to observe the changes of the indicators mentioned above during ALI secondary to SAP. In short, this study is to explore the functions of the changed substances in the thoracic duct lymph in the activation of PMN in the lung, in the mediating of ALI during SAP, to find out the feasible interventions, and to explain the mechanism of AVI secondary to SAP, finally to provide theoretical and experimental supports for clinical practice.
Methods:
Part I: Establishing models of cervical thoracic duct drainage in rats
Sixty clean SD adult male rats were randomized into 3 groups, with 20 in each. Group 1: cervical thoracic duct direct group; group 2: cervical thoracic duct indirect group; group 3: abdominal thoracic duct group. After being anaesthetized with napental and being fixed, the models were performed with microsurgery instruments to dissect cervical thoracic duct, left jugular trunk, and abdominal thoracic duct; 24G remaining needles then were used to puncture the above parts to drain lymph. Meanwhile, the drained lymph volumes in 3 groups and model making condition were recorded.
Part II: Experimental study on the early stage intervention of thoracic duct ligation /conduction in the SAP accompanied by ALI and on the changed functions of PMN in arterial blood
Ninety-six healthy male SD rats were randomized into 12 groups, with 8 in each, and the following operations were performed respectively: cervical sham-operation + abdominal sham-operation 2 hours (SO2h), cervical sham-operation + abdominal sham-operation 6 hours (SO6h), cervical sham-operation + abdominal sham-operation 12 hours (SO12h); cervical sham-operation + SAP2 hours (SAP2h), cervical sham-operation + SAP6 hours (SAP6h), cervical sham-operation + SAP12 hours (SAP12h); cervical thoracic duct ligation + SAP2 hours (LC2h), cervical thoracic duct ligation + SAP6 hours (LC6h), cervical thoracic duct ligation + SAP12 hours (LC12h); cervical thoracic duct drainage + SAP2 hours (DC2h), cervical thoracic duct drainage + SAP6 hours (DC6h), cervical thoracic duct drainage + SAP12 hours (DC12h). According to the grouping requirement, SOA, SAP, LC, and DC models were prepared. Histopathological sections of pancreas and lung were observed, and the lung underwent light microscope scan; serum amylopsin (AMY) and pancreatic lipase were detected bio-chemically; and automatic blood gas system was employed to make arterial blood gas analysis. Part III: Experimental study on the effect of thoracic duct ligation /conduction in SAP on PMN functions in the arterial blood
The sample was collected from abdominal aorta blood (when the blood for blood gas analysis was collected). Flow Cytometry (FCM) was employed to detect the changed functions of PMN in the arteries.
Part IV: Experimental study on the concentration changes of TNF-α, IL-10, AMY, LIP, and endotoxins in thoracic duct lymph in SAP accompanied by ALI
Twenty healthy SD rats were randomized into 2 groups, with 10 in each, with the operations of cervical thoracic duct drainage + abdominal sham operation (Sham) and cervical thoracic duct drainage + SAP (DC) respectively. After the model making, the conduction was performed continually, the drained lymph being collected for a certain period and then kept in low tempreture in time. AMY and LIP in the lymph and blood were detected bio-chemically; ELISA was employed to detect TNF-αand IL-10 in the lymph and blood; the method of reaction time was used to detect endotoxin content in the lymph.
Results:
Part I: Model establishing of cervical thoracic duct lymphatic fistula in rats
1 Light milky white fluid was successfully conducted from the models of all the groups, no differences in appearance and properties, and all lymph.
2 The success rate of model establishing in cervical thoracic duct direct group was apparently lower than those in cervical thoracic duct indirect group and abdominal thoracic duct direct group (P<0.05).
3 The amount of lymph in abdominal thoracic duct group was greater than that in cervical thoracic duct direct and indirect groups, with significant difference (P<0.05).
Part II: Experimental study on the early stage intervention of thoracic duct ligation /conduction in the SAP accompanied by ALI
1 Compared with SO groups at the same time point, the amount of ascites, pancreatic pathogenesis score, lung pathogenesis score, and serum AMY and LIP all increased significantly (P<0.01);compared with SO groups at the same time point, PaO2 in SAP2h and SAP6h also increased significantly (P<0.01), with PaO2 in SAP2h obviously higher than that in SAP6h (P<0.01), and PaO2 in SAP12h obviously higher than that in SO12h (P<0.01); compared with SO groups at the same time point, PaCO2 in SAP2h and SAP6h decreased significantly (P<0.01), while PaCO2 in SAP12h obviously higher than that in SO12h (P<0.01).
2 Compared with SAP groups at the same time point, the amount of ascites in LC6h and LC12h, lung pathogenesis score in LC6h, and serum AMY and LIP in LC2h all decreased significantly (P<0.01 or P<0.05);the ascites in LC2h , lung pathogenesis scores in LC2h and LC12h tended to decrease but with no significant difference; pancreatic pathogenesis scores in LC2h and LC6h, and PaO2 in LC12h tended to increase but with no significant difference, and with pancreatic pathogenesis scores in LC12h increased significantly (P<0.01), and likely PaO2 in LC6h (P<0.01).
3 Compared with SAP groups at the same time point, the amount of ascites in DC12h, lung pathogenesis score in DC6h and DC12h, and serum AMY and LIP in DC2h, DC6h and DC12h all decreased significantly (P<0.01 or P<0.05);lung pathogenesis score in DC2h tended to decrease but with no significant difference; PaO2 in DC6h and DC12h increased significantly (P<0.01).
Part III: Experimental study on the effect of thoracic duct ligation /conduction in SAP on PMN functions in the arterial blood
1 Compared with SO groups at the same time point, the proportion of PMN with CD11b expression positive, the average fluorescence intensity of PMN respiratory burst in SAP groups all increased significantly (P<0.01); compared with SO groups at the same time point, average fluorescence intensity of CD11b in SAP2h tended to increase, with no significant difference (P>0.05), while that in SAP6h and SAP12h increased significantly (P<0.01 or P<0.05), with that in SAP12h apparently higher than that in SAP6h.
2 Compared with SAP groups at the same time point, the average fluorescence intensity of PMN respiratory burst, the proportion of PMN with CD11b expression positive, average fluorescence intensity of CD11b in LC all tended to decrease, with no significant difference.
3 Compared with SAP groups at the same time point, the average fluorescence intensity of PMN respiratory burst and that of CD11b in DC, and the proportion of PMN with CD11b expression positive in DC6h and DC12h all decreased significantly (P<0.01或P<0.05).
Part IV: Experimental study on the concentration changes of TNF-α, IL-10, AMY, LIP, and endotoxins in thoracic duct lymph in SAP accompanied by ALI
1 TNF-α,IL-10
1.1 TNF-αin lymph and serum
TNF-αlevel in lymph: No significant difference among abdominal sham operation 2h, 6h and 12h groups; significantly increased lymph TNF-αin SAP groups at all time points compared with the abdominal sham operation groups at the same time point(P<0.01). Comparison among SAP groups: TNF-αlevel in SAP6h and 12h was apparently higher than that in SAP2h (P<0.01), while no significant difference between the former two.
TNF-αlevel in serum: No significant difference among abdominal sham operation 2h, 6h and 12h groups; significantly increased serum TNF-αin SAP at all time points compared with the abdominal sham operation groups at the same time point(P<0.01). Comparison among SAP groups: TNF-αlevel in SAP6h and 12h was apparently higher than that in SAP2h (P<0.01), while no significant difference between the former two.
There is no significant difference between the lymth and serum TNF-αconcentration; and likely the lymth and serum TNF-αconcentration in DC groups.
1.2 IL-10 in lymph and serum
IL-10 level in lymph: No significant difference among abdominal sham operation 2h, 6h and 12h groups; significantly increased lymph IL-10 in SAP at all time points compared with the abdominal sham operation groups at the same time point (P<0.01). Comparison among SAP groups: the level of IL-10 in SAP6h was significantly higher than that in SAP2h, and IL-10 in SAP12h higher than those in SAP2h and SAP6h.
IL-10 level in blood: No significant difference among abdominal sham operation 2h, 6h and 12h groups; significantly increased blood IL-10 in SAP at all time points compared with the abdominal sham operation groups at the same time point (P<0.01). Comparison among SAP groups: the level of IL-10 in SAP6h was significantly higher than that in SAP2h, and IL-10 in SAP12h higher than those in SAP2h and SAP6h. There is no significant difference between the lymth and serum IL-10 concentration; and likely the lymth and serum IL-10 concentration in DC groups.
2 AMY and LIP
2.1 AMY in lymph:
Level of AMY in lymph: No significant difference among abdominal sham operation 2h, 6h and 12h groups; significantly increased lymph AMY in SAP at all time points compared with the abdominal sham operation groups at the same time point (P<0.01). Comparison among SAP groups: the levels of AMY in SAP6h and SAP12h were significantly higher than that in SAP2h (P<0.05), with no significant difference between those in SAP12h and SAP6h.
2.2 AMY in serum:
No significant difference among abdominal sham operation 2h, 6h and 12h groups; significantly increased blood AMY in SAP at all time points compared with the abdominal sham operation groups at the same time point (P<0.01). Comparison among SAP groups: the levels of AMY in SAP6h and SAP12h were significantly higher than that in SAP2h (P<0.05), with no significant difference between those in SAP12h and SAP6h.
In DC groups, AMY concentration in lymph was apparently higher than that in serum (P<0.01).
2.3 LIP in lymph:
Level of LIP in lymph: No significant difference among abdominal sham operation 2h, 6h and 12h groups; significantly increased lymph LIP in DC groups at all times compared with the abdominal sham operation groups at the same time point (P<0.01). Comparison among DC groups: the levels of LIP in SAP6h and SAP12h were significantly higher than that in SAP2h (P<0.05), with no significant difference between those in SAP12h and SAP6h.
2.4 LIP in serum:
Level of LIP in serum: No significant difference among abdominal sham operation 2h, 6h and 12h groups; significantly increased serum LIP in DC groups at all time points compared with the abdominal sham operation groups at the same time point (P<0.01). Comparison among DC groups: the levels of serum LIP in SAP6h and SAP12h were significantly higher than that in SAP2h (P<0.05), with no significant difference between those in SAP12h and SAP6h.
In DC groups, LIP concentration in lymph at certain time point was apparently higher than that in serum (P<0.01).
3 Changed levels of endotoxins in lymph
No significant difference among the same time point sham groups; compared with sham groups, DC2h showed no significant difference, while both DC6h and DC12h showed apparently significant increase (P<0.01), with the increase in the latter apparently higher than that in the former (P<0.01), and the increase in DC6h and DC12h higher than that in DC2h. Conclusions:
Part I: Model establishing of cervical thoracic duct lymphatic fistula in rats
1 The success rate of the cervical thoracic duct direct group is lower than that of the other two groups, with no significant difference in the cervical thoracic duct indirect group and abdominal thoracic duct group.
2 There is no significant difference in properties about the drained lymph by the three methods. Compared with the cervical thoracic duct direct and indirect groups, there is more drained lymph in abdominal thoracic duct group.
3 Cervical thoracic duct indirect fistula in rats, which can drain lymph stably and does not disturb abdominal cavity, is a stable and reliable method to make animal models of thoracic duct lymphatic fistula, and can be used in the experimental study of SAP accompanied by ALI associated with lymphatic circulation.
Part II: Experimental study on the early stage intervention of thoracic duct ligation /conduction in the SAP accompanied by ALI
1 Early stage SAP will cause damaging and pathomorphological changes of the lung, followed by functional disorder.
2 Cervical thoracic duct conduction can relieve pathomorphological changes and dysfunction of the lung caused by ALI, and cervical thoracic duct ligation tends to have the same effect as conduction: at the early phase (2h, 6h), ligation and conduction show no significant difference in improving the function of the lung; while at the middle or late phase (12), conduction can apparently relieve pathomorphological changes and dysfunction of the lung caused by ALI secondary to SAP.
3 Cervical thoracic duct conduction cannot relieve pathomorphological changes of the pancreas secondary to SAP, while cervical thoracic duct ligation will aggravate pathomorphological changes.
Part III: Experimental study on the effect of thoracic duct ligation /conduction in SAP on PMN functions in the arterial blood
1 Cervical thoracic duct conduction can lower the effect of respiratory burst of arterial PMN in SAP, the proportion of PMN with CD11b expression positive, and MFI of CD11b; while cervical thoracic duct ligation tends to have the same effects as conduction.
2 Cervical thoracic duct conduction/ligation can lower the effect of respiratory burst of arterial PMN in SAP, the proportion of PMN with CD11b expression positive, and MFI of CD11b, which may be attributed to the fact that cervical thoracic duct conduction can relieve the pathomorphological change and dysfunctions of ALI secondary to SAP.
Part IV: Experimental study on the concentration changes of TNF-α, IL-10, AMY, LIP, and endotoxins in thoracic duct lymph in SAP accompanied by ALI
1 The concentrations of endotoxin, AMY, LIP, TNF-α, and IL-10 in the lymph increase apparently during SAP.
2 The lymph components, complicated in the thoracic duct during SAP, may activate PMN directly or indirectly through the activated pancreatin, endotoxin, TNF-α, IL-10, etc., and finally injure the lung tissue.
3 During SAP, the concentrations of AMY and LIP in the lymph and in the serum show significant difference, thereby indicating that AMY and LIP is one of the origins causing the concentration of AMY and LIP increase in the serum.
4 During SAP, the concentrations of TNF-αin the lymph and that in the serum show no significant difference, so as IL-10, thereby indicating that TNF-αand IL-10 is not the origin causing the concentration of those increase in the serum.
5 Reaction time method (one of the Limulus Amebocyte Lysate assay) is a reliable method to determine the endotoxin content in the lymph.
SAP时ALI是机体过度炎症反应导致肺血管内皮细胞和肺泡上皮细胞广泛破坏的结果,而中性粒细胞(polymorphonuclear neutrophils, PMN)能触发和放大炎症反应,在炎症反应中起着关键作用,是炎性反应的重要标志。有研究结果显示SAP时,PMN在肺内粘附聚集,导致肺血管内皮细胞、肺泡上皮细胞广泛损伤,致通透性增加、肺水肿及微血栓形成,进而发展成ALI。PMN在肺内聚集和激活是SAP时ALI发病的最初环节,如果能采取干预PMN在肺内的聚集激活,就能早期预防ALI。但是关于SAP时PMN在肺内聚集和激活的机制并不十分清楚,仍有待进一步研究。目前,肠系膜-胸导管淋巴液在重度烧伤、感染中毒性休克等中的作用得到重视,并认为其可以介导严重感染/创伤等引起的肺脏损伤,并进而导致肺和全身的炎症反应。临床及动物实验发现各种原因引起的多器官功能障碍综合症(multiple organs dysfunction syndromes, MODS)常常首先出现ARDS,而肺是首个接纳肠系膜淋巴液的器官,肠系膜淋巴液可能起着介导肺损伤的作用;又有研究显示了休克后收集到的肠系膜淋巴液能损伤或致内皮细胞死亡,增加内皮细胞的渗透性,结扎肠系膜淋巴管能减轻失血性休克后或烧伤后肺损害。这些研究都提示肠系膜淋巴液含有能够介导全身炎症反应时肺损伤的物质。而且在创伤性休克的动物模型或病人的门静脉血的研究中,并没有发现细菌或毒素。同时,休克时门静脉血浆对于血管内皮细胞的损伤作用很轻。这些研究表明经肠道微血管回流的门静脉途径在休克致器官损伤的发病学意义有一定的局限性。Louis报道失血性休克所致的肺损伤和内皮细胞通透性增高是由肠道有害物质经淋巴液而非经门静脉血液所引起的。据此,我们推测这种作用在类似烧伤和严重感染的SAP中可能也存在,并且有可能通过诱导PMN在肺脏聚集和激活来介导。
本研究应用SD大鼠建立颈部胸导管引流模型,并在此基础上制备SAP合并ALI模型,观察肺脏病理变化,检测血气分析变化,从而了解胸导管结扎/引流对ALI的影响;并检测胸导管淋巴液中抗炎和促炎因子、胰酶、内毒素的含量变化,以及其对动脉血中PMN功能的影响预后,观察SAP继发ALI时上述指标的变化。旨在探讨SAP时胸导管淋巴液的成分变化在激活肺脏中PMN、介导SAP时ALI发生中的作用,以及可行的干预途径,进一步阐明SAP合并ALI的发病机制,为临床治疗提供新的思路、途径及理论和实验依据。
方法:
第一部分:大鼠颈部胸导管引流动物模型的建立
选用成年清洁级雄性SD大鼠60只。随机将大鼠分为3组,每组20只:第1组:颈部胸导管直接组,第2组:颈部胸导管间接组,第3组:腹部胸导管组。在戊巴比妥钠麻醉并固定后,应用显微手术器械分别解剖颈部胸导管、左颈干、腹部胸导管,应用24G留置针分别穿刺保留颈部胸导管、左颈干、腹部胸导管,引流淋巴液。记录三种胸导管淋巴液引流模型之间淋巴流量、制模成功情况。
第二部分:胸导管结扎/引流对重症急性胰腺炎并发肺损伤早期干预及动脉中性粒细胞功能变化的实验研究
选取健康雄性SD大鼠96只,随机分为12组,每组8只,分别为颈部假手术组+腹部假手术2小时组(SO2h)、颈部假手术组+腹部假手术6小时组(SO6h)、颈部假手术组+腹部假手术12小时组(SO12h);颈部假手术组+SAP2小时组(SAP2h)、颈部假手术组+SAP6小时组(SAP6h)、颈部假手术组+SAP12小时组(SAP12h);颈部胸导管结扎+SAP2小时组(LC2h)、颈部胸导管结扎+SAP6小时组(LC6h)、颈部胸导管结扎+SAP12小时组(LC12h);颈部胸导管引流+SAP2小时组(DC2h)、颈部胸导管引流+SAP6小时组(DC6h)、颈部胸导管引流+SAP12小时组(DC12h)。按分组要求制备颈部假手术组+腹部假手术组(SOA)、颈部假手术组+SAP组(SAP)、颈部胸导管结扎+SAP组(LC)和颈部胸导管引流+SAP组(DC)动物模型。观察胰腺、肺脏组织病理切片、肺脏扫描电镜;应用生化法检测血清胰淀粉酶(AMY)、胰脂肪酶(LIP);应用自动血气分析仪检测动脉血气分析。
第三部分:胸导管结扎/引流对重症急性胰腺炎时动脉血中性粒细胞功能影响的实验研究
取样来自第二部分腹主动脉血(取动脉血气同时)应用流式细胞术(Flow Cytometry,FCM)检测动脉中中性粒细胞功能变化。
第四部分:重症急性胰腺炎并发肺损伤时胸导管淋巴液中TNF-α、IL-10、AMY、LIP及内毒素浓度变化的实验研究
选择健康SD大鼠20只,随机分为2组,每组10只。分别为颈部胸导管引流组+腹部假手术组(Sham)和颈部胸导管引流+SAP组(DC)。建立SD大鼠颈部胸导管引流情况下腹部手术组、SAP组模型,持续引流并分时段及时低温保存淋巴液。应用生化法检测淋巴液/血清中胰淀粉酶(AMY)、胰脂肪酶(LIP);酶联免疫法检测淋巴液/血清中TNF-α、IL-10,反应时间法检测淋巴液中内毒素浓度。
结果:
第一部分:大鼠颈部胸导管淋巴引流动物模型的建立
1各组动物模型均成功引出浅乳白色液,外观性状无差异,均为淋巴液。
2颈部胸导管直接组制模成功率明显低于腹部胸导管直接组和颈部胸导管间接组(P<0.05);
3腹部胸导管组的淋巴流量较颈部胸导管直接组和颈部胸导管间接组多且差异明显(P<0.05)。
第二部分:胸导管结扎/引流对SAP并发ALI早期干预的实验研究
1与同时点实验对照组(SO组)相比,SAP各时点腹水量、胰腺病理评分、肺脏病理评分、血清AMY、血清LIP明显升高(P<0.01); SAP2h、6h的PaO2与同时点SO组相比明显升高(P<0.01),且SAP2h组的PaO2明显高于SAP6h(P<0.01),而SAP12h组的PaO2明显高于同时点SO组(P<0.01);SAP2h、6h的PaCO2与同时点SO组相比明显降低(P<0.01),而SAP12h组的PaCO2明显高于同时点SO组(P<0.01);、
2与同时点SAP组(干预对照组)相比, LC6h组、LC12h组的腹水量、LC6h组的肺脏病理评分、LC2h组血清AMY和血清LIP明显降低(P<0.01或P<0.05);胸导管结扎组的LC2h组的腹水量、LC2h12h的肺脏病理评分有降低趋势,但无显著差异;LC2h、6h的胰腺病理评分、LC12h的PaO2有升高趋势,但无显著差异,LC12h的胰腺病理评分明显升高(P<0.01);LC6h的PaO2明显升高(P<0.01)。
3与同时点SAP组(干预对照组)相比,胸导管引流组的DC12h组的腹水量、DC6h、12h的肺脏病理评分、DC2h、6h、12h血清AMY和血清LIP明显降低(P<0.01或P<0.05);DC2h组的肺脏病理评分有降低趋势,但无显著差异;DC6h、12h的PaO2明显升高(P<0.01)。
第三部分:胸导管结扎/引流对重症急性胰腺炎时动脉血中性粒细胞功能影响的实验研究
1与同时点对照组(SO组)相比,SAP组各时点CD11b表达阳性PMN比例、PMN呼吸爆发作用(RB)平均荧光强度均明显升高(P<0.01);SAP2h的CD11b平均荧光强度与同时点SO组相比有升高趋势,但无显著性差异(P>0.05),SAP6h、12h的CD11b平均荧光强度与同时点SO组相比明显升高(P<0.01或P<0.05),且SAP12h组的CD11b平均荧光强度明显高于SAP6h;
2与同时点干预对照组(SAP组)相比,各时点LC的PMN呼吸爆发作用(RB)平均荧光强度、CD11b表达阳性PMN比例、CD11b平均荧光强度均有降低趋势,但无显著差异。
3与同时点干预对照组(SAP组)相比,各时点DC的PMN呼吸爆发作用(RB)平均荧光强度、CD11b平均荧光强度、DC6h、12h的CD11b表达阳性PMN比例均有明显降低(P<0.01或P<0.05)。第四部分:重症急性胰腺炎并发肺损伤时胸导管淋巴液中TNF-α、IL-10、AMY、LIP及内毒素浓度变化的实验研究
1 TNF-α,IL-10
1.1淋巴液中、血清中TNF-α
淋巴液中TNF-α水平:腹部假手术各组间Sham(2h)、Sham(6h)、Sham(12h)的TNF-α水平均无明显差异;与同时腹部假手术各组相比,SAP各时点组的淋巴液中TNF-α均明显升高(P<0.01);SAP各时点组相比:SAP6h、12h组的TNF-α较SAP2h组均明显升高(P<0.01),SAP6h、12h两组之间相比TNF-α水平均无明显差异。
血清中TNF-α水平:腹部假手术各组间Sham(2h)、Sham(6h)、Sham(12h)的TNF-α水平均无明显差异;与同时腹部假手术各组相比,SAP各时点组的血清中TNF-α均明显升高(P<0.01);SAP各时点组相比:SAP6h、12h组的TNF-α较SAP2h组均明显升高(P<0.05),SAP6h、12h两组之间相比TNF-α水平均无明显差异。
正常状态淋巴液中TNF-α浓度与血清中TNF-α浓度在对应时点均无明显差异;DC组淋巴液中TNF-α浓度与血清中TNF-α浓度在对应时点亦均无明显差异。
1.2淋巴液中、血清中IL-10
淋巴液中IL-10水平:腹部假手术各组间Sham(2h)、Sham(6h)、Sham(12h)的IL-10水平均无明显差异;与同时点腹部假手术各组相比,SAP各时点组的淋巴液中IL-10水平均明显升高(P<0.01);SAP各时点组相比:SAP6h组的IL-10水平较SAP2h组均明显升高(P<0.01);SAP12h组的IL-10水平较SAP2h、6h组均明显升高(P<0.01)。
血液中IL-10水平:腹部假手术各组间Sham(2h)、Sham(6h)、Sham(12h)的IL-10水平均无明显差异;与同时腹部假手术各组相比,SAP各时点组的血清中IL-10水平均明显升高(P<0.01);SAP各时点组相比:SAP6h组的IL-10水平较SAP2h组均明显升高(P<0.01);SAP12h组的IL-10水平较SAP2h、6h组均明显升高(P<0.01)。
正常状态淋巴液中IL-10浓度与血清中IL-10浓度在对应时点均无明显差异;DC组淋巴液中IL-10浓度与血清中IL-10浓度在对应时点亦均无明显差异。
2 AMY ,LIP
2.1淋巴液中AMY
淋巴液中AMY水平:腹部假手术各组间Sham(2h)、Sham 6h)、Sham (12h)的AMY水平均无明显差异;与同时腹部假手术各组相比,SAP各时点组的淋巴液中AMY均明显升高(P<0.01);SAP各时点组相比:SAP6h、12h组的AMY较SAP2h组均明显升高(P<0.05),SAP6h、12h两组之间相比AMY水平均无明显差异。
2.2血清中AMY
腹部假手术各组间Sham(2h)、Sham(6h)、Sham(12h)的AMY水平均无明显差异;与同时点的腹部假手术各组Sham(2h)、Sham (6h)、Sham (12h)相比,SAP各组SAP 2h、6h、12h各组的淋巴液中AMY均明显升高(P<0.01);SAP各时点组相比:SAP6h、12h组的AMY较SAP2h组均明显升高(P<0.05),SAP6h、12h两组之间相比AMY水平均无明显差异。DC组淋巴液中AMY浓度在对应时点明显高于与血清中AMY浓度(P<0.01)。
2.3淋巴液中LIP
淋巴液中LIP水平:腹部假手术各组间Sham(2h)、Sham(6h)、Sham(12h)的LIP水平均无明显差异;与同时腹部假手术各组相比, DC组各时点的淋巴液中LIP均明显升高(P<0.01);DC组各时点相比:SAP6h、12h组的LIP较SAP2h组均明显升高(P<0.01),SAP6h、12h两组之间相比LIP水平均无明显差异。
2.4血清中LIP
血清中LIP水平:腹部假手术各组间Sham(2h)、Sham(6h)、Sham(12h)的LIP水平均无明显差异;与同时腹部假手术各组相比, DC组各时点的血清中LIP均明显升高(P<0.01);DC组各时点相比:SAP6h、12h组的LIP较SAP2h组均明显升高(P<0.01),SAP6h、12h两组之间相比LIP水平均无明显差异;
DC组淋巴液中LIP浓度在对应时点明显高于与血清中LIP浓度(P<0.01)。
3淋巴液中内毒素水平变化
Sham各时点(2h、6h、12h)间没有明显差异;DC 2h与Sham各时点(2h、6h、12h)间相比没有明显差异;DC6h、DC12h与Sham组对应时点相比明显升高,有明显差异(p<0.01);DC6h、12h与DC 2h相比明显升高,有明显差异(p<0.01);DC12h与DC6h相比明显升高,有明显差异
结论
第一部分:大鼠颈部胸导管淋巴瘘动物模型的建立
1颈部胸导管间接组制模成功率最高,其次是腹部胸导管组,颈部胸导管间接组与腹部胸导管组操作成功率差异不明显(P>0.05);颈部胸导管直接组较另外两组操作成功率低(P<0.01)。;
2三种方法引流的淋巴液无明显性状差异;腹部胸导管组在引流量方面明显多于颈部胸导管直接组、颈部胸导管间接组(P<0.01),这可能是由于腹腔淋巴循环对腹腔骚扰较敏感造成的,说明腹部胸导管引流技术明显影响腹腔淋巴循环。
3大鼠颈部间接胸导管引流法可稳定引流淋巴液,且不骚扰腹腔,是稳定、可靠、制模成功率高胸导管引流动物模型制作方法,能够用于SAP并发ALI时与淋巴循环有关的实验研究。
第二部分:胸导管结扎/引流对SAP并发ALI早期干预的实验研究
1 SAP早期即可引起肺脏损伤性病理形态学变化,随后可进一步出现其功能障碍。
2颈部胸导管引流能够减轻SAP引起的急性肺损伤病理形态学变化及其功能障碍,而颈部胸导管结扎有减轻SAP引起的急性肺损伤病理形态学变化及其功能障碍的趋势:在早期(2h,6h)颈部胸导管结扎、引流,两者相比对肺功能的改善差异不明显;到中后期(12h)颈部胸导管引流较结扎能够明显减轻SAP引起的急性肺损伤病理形态学变化及其功能障碍。
3颈部胸导管引流没能减轻SAP时胰腺病理形态学变化,而颈部胸导管结扎能加重SAP时胰腺病理形态学变化
第三部分:胸导管结扎/引流对重症急性胰腺炎时动脉血中性粒细胞功能影响的实验研究
1颈部胸导管引流能够降低SAP时主动脉血中PMN的呼吸爆发作用、CD11b表达阳性PMN比例、CD11b平均荧光强度;而颈部胸导管结扎有降低SAP主动脉PMN的呼吸爆发作用、CD11b表达阳性PMN比例、CD11b平均荧光强度的趋势。
2颈部胸导管结扎/引流能够影响SAP时主动脉血中PMN的呼吸爆发作用、CD11b表达阳性PMN比例、CD11b平均荧光强度,这可能是颈部胸导管引流能够减轻SAP引起的急性肺损伤病理形态学变化及其功能障碍的原因。
第四部分SAP合并ALI时胸导管淋巴液内AMY、LIP、TNF-α、IL-10、内毒素水平变化的实验研究
1 SAP合并ALI时发病过程中淋巴液中内毒素浓度、AMY、LIP、TNF-α、IL-10浓度明显上升。
2 SAP合并ALI时胸导管淋巴液中成分复杂,可能通过激活的胰酶、内毒素TNF-α、IL-10等直接或间接的用过活化中性粒细胞,损伤肺组织。
3 SAP合并ALI时AMY、LIP浓度在淋巴液中与血清中相比,两者之间差异明显,据此,淋巴液中的AMY、LIP是导致血清中该物质浓度上升的来源之一。
4 SAP合并ALI时TNF-α浓度在淋巴液中与血清中相比,两者之间无明显差异,IL-10也是如此;据此,淋巴液中的TNF-α、IL-10不是导致血清中该物质浓度上升的来源。
5反应时间法(鲎试剂法之一)是测定淋巴液中内毒素含量的可靠方法。
Objectives:
Severe acute pancreatitis (SAP), a dangerous clinical process with high mortality, will develop acute lung injury (ALI) as demonstrated by the current studies; and finally it will develop acute respiratory distress syndrome (ARDS), the outcome of ALI and one of the leading causes of SAP, with over 65% patients died associated with ARDS. Therefore, it is significantly important to clarify the mechanism of ALI during SAP and to determine the early-stage prevention and the treatment in order to decrease the mortality and to improve the diagnosis of SAP.
During SAP, over inflammatory reaction of the body causes pulmonary vessel endothelia and alveolus epithelia extensively destroyed, resulting in ALI. Polymorphonuclear neutrophil leukocytes (PMN) can trigger and amplify inflammatory reaction, so it plays a key role in and is the important mark of inflammatory reaction. As documented by some studies, during SAP, PMN coagulate and assemble in the lungs, leading to pulmonary vessel endothelia and alveolus epithelia extensively destroyed and accordingly permeability increased, pneumonedema and microthrombus developed, and finally ALI. Coagulation and activation of PMN in the lungs is the initial process of ALI during SAP. Therefore, if the coagulation and activation can be intervened, the early prevention of ALI can be performed. The mechanism of it, however, is not clear, wanting further study. Currently, mesentery-thoracic duct lymph is highly valued in the severe burns, infectious toxic shock and other diseases, and is regarded as the mediator of lung injury in severe infection/trauma, finally leading to inflammatory reaction in the lungs or systemically. Clinical and animal experiments showed that ARDS will first occur in the multiple organs dysfunction syndromes (MODS) caused by multiple reasons, for lungs are the first organ to accept mesentery lymph, the one which possibly mediates lung injury. Other studies displayed that mesentery lymph collected after shock caused endothelia injured or died, finally increasing the permeability of the endothelia, and that ligating the mesentery lymph ducts can decrease lung injury after heamorrhagic shock and burn. All these studies suggest that mesentery lymph contains some substances that can mediate lung injury during systemic inflammation. Furthermore, no bacteria or toxins were found in the portal vein blood of the animal models or patients with traumatic shock. Meanwhile, portal vein plasma only causes light injury to the vessel endothelia. All this shows that the portal vein blood coming back through enteric canal capillaries plays a minor role in the organ injury caused by shock. As Louis reported, lung injury and increased permeability of endothelia caused by heamorrhagic shock was caused by enteric toxic substances in lymph not in portal vein blood. Based on this, we presume that this kind of function also exists in SAP caused by burn and severe infection, and may mediate SAP by inducing PMN coagulation and activation in the lung.
SD rats were used in this experiment to establish cervical thoracic duct drainage models, and then to prepare models of SAP accompanied by ALI, with the aim to observe the pathogenesis of the lung, to detect the changes of blood gas analysis, thus to find out the effect of thoracic duct ligation /conduction on ALI. Meanwhile, to detect the changed content of anti-inflammatory and proinflammatory factors, pancreatin, and endotoxin, and to understand the prognosis effect of these factors on PMN functions in the arterial blood, and finally to observe the changes of the indicators mentioned above during ALI secondary to SAP. In short, this study is to explore the functions of the changed substances in the thoracic duct lymph in the activation of PMN in the lung, in the mediating of ALI during SAP, to find out the feasible interventions, and to explain the mechanism of AVI secondary to SAP, finally to provide theoretical and experimental supports for clinical practice.
Methods:
Part I: Establishing models of cervical thoracic duct drainage in rats
Sixty clean SD adult male rats were randomized into 3 groups, with 20 in each. Group 1: cervical thoracic duct direct group; group 2: cervical thoracic duct indirect group; group 3: abdominal thoracic duct group. After being anaesthetized with napental and being fixed, the models were performed with microsurgery instruments to dissect cervical thoracic duct, left jugular trunk, and abdominal thoracic duct; 24G remaining needles then were used to puncture the above parts to drain lymph. Meanwhile, the drained lymph volumes in 3 groups and model making condition were recorded.
Part II: Experimental study on the early stage intervention of thoracic duct ligation /conduction in the SAP accompanied by ALI and on the changed functions of PMN in arterial blood
Ninety-six healthy male SD rats were randomized into 12 groups, with 8 in each, and the following operations were performed respectively: cervical sham-operation + abdominal sham-operation 2 hours (SO2h), cervical sham-operation + abdominal sham-operation 6 hours (SO6h), cervical sham-operation + abdominal sham-operation 12 hours (SO12h); cervical sham-operation + SAP2 hours (SAP2h), cervical sham-operation + SAP6 hours (SAP6h), cervical sham-operation + SAP12 hours (SAP12h); cervical thoracic duct ligation + SAP2 hours (LC2h), cervical thoracic duct ligation + SAP6 hours (LC6h), cervical thoracic duct ligation + SAP12 hours (LC12h); cervical thoracic duct drainage + SAP2 hours (DC2h), cervical thoracic duct drainage + SAP6 hours (DC6h), cervical thoracic duct drainage + SAP12 hours (DC12h). According to the grouping requirement, SOA, SAP, LC, and DC models were prepared. Histopathological sections of pancreas and lung were observed, and the lung underwent light microscope scan; serum amylopsin (AMY) and pancreatic lipase were detected bio-chemically; and automatic blood gas system was employed to make arterial blood gas analysis. Part III: Experimental study on the effect of thoracic duct ligation /conduction in SAP on PMN functions in the arterial blood
The sample was collected from abdominal aorta blood (when the blood for blood gas analysis was collected). Flow Cytometry (FCM) was employed to detect the changed functions of PMN in the arteries.
Part IV: Experimental study on the concentration changes of TNF-α, IL-10, AMY, LIP, and endotoxins in thoracic duct lymph in SAP accompanied by ALI
Twenty healthy SD rats were randomized into 2 groups, with 10 in each, with the operations of cervical thoracic duct drainage + abdominal sham operation (Sham) and cervical thoracic duct drainage + SAP (DC) respectively. After the model making, the conduction was performed continually, the drained lymph being collected for a certain period and then kept in low tempreture in time. AMY and LIP in the lymph and blood were detected bio-chemically; ELISA was employed to detect TNF-αand IL-10 in the lymph and blood; the method of reaction time was used to detect endotoxin content in the lymph.
Results:
Part I: Model establishing of cervical thoracic duct lymphatic fistula in rats
1 Light milky white fluid was successfully conducted from the models of all the groups, no differences in appearance and properties, and all lymph.
2 The success rate of model establishing in cervical thoracic duct direct group was apparently lower than those in cervical thoracic duct indirect group and abdominal thoracic duct direct group (P<0.05).
3 The amount of lymph in abdominal thoracic duct group was greater than that in cervical thoracic duct direct and indirect groups, with significant difference (P<0.05).
Part II: Experimental study on the early stage intervention of thoracic duct ligation /conduction in the SAP accompanied by ALI
1 Compared with SO groups at the same time point, the amount of ascites, pancreatic pathogenesis score, lung pathogenesis score, and serum AMY and LIP all increased significantly (P<0.01);compared with SO groups at the same time point, PaO2 in SAP2h and SAP6h also increased significantly (P<0.01), with PaO2 in SAP2h obviously higher than that in SAP6h (P<0.01), and PaO2 in SAP12h obviously higher than that in SO12h (P<0.01); compared with SO groups at the same time point, PaCO2 in SAP2h and SAP6h decreased significantly (P<0.01), while PaCO2 in SAP12h obviously higher than that in SO12h (P<0.01).
2 Compared with SAP groups at the same time point, the amount of ascites in LC6h and LC12h, lung pathogenesis score in LC6h, and serum AMY and LIP in LC2h all decreased significantly (P<0.01 or P<0.05);the ascites in LC2h , lung pathogenesis scores in LC2h and LC12h tended to decrease but with no significant difference; pancreatic pathogenesis scores in LC2h and LC6h, and PaO2 in LC12h tended to increase but with no significant difference, and with pancreatic pathogenesis scores in LC12h increased significantly (P<0.01), and likely PaO2 in LC6h (P<0.01).
3 Compared with SAP groups at the same time point, the amount of ascites in DC12h, lung pathogenesis score in DC6h and DC12h, and serum AMY and LIP in DC2h, DC6h and DC12h all decreased significantly (P<0.01 or P<0.05);lung pathogenesis score in DC2h tended to decrease but with no significant difference; PaO2 in DC6h and DC12h increased significantly (P<0.01).
Part III: Experimental study on the effect of thoracic duct ligation /conduction in SAP on PMN functions in the arterial blood
1 Compared with SO groups at the same time point, the proportion of PMN with CD11b expression positive, the average fluorescence intensity of PMN respiratory burst in SAP groups all increased significantly (P<0.01); compared with SO groups at the same time point, average fluorescence intensity of CD11b in SAP2h tended to increase, with no significant difference (P>0.05), while that in SAP6h and SAP12h increased significantly (P<0.01 or P<0.05), with that in SAP12h apparently higher than that in SAP6h.
2 Compared with SAP groups at the same time point, the average fluorescence intensity of PMN respiratory burst, the proportion of PMN with CD11b expression positive, average fluorescence intensity of CD11b in LC all tended to decrease, with no significant difference.
3 Compared with SAP groups at the same time point, the average fluorescence intensity of PMN respiratory burst and that of CD11b in DC, and the proportion of PMN with CD11b expression positive in DC6h and DC12h all decreased significantly (P<0.01或P<0.05).
Part IV: Experimental study on the concentration changes of TNF-α, IL-10, AMY, LIP, and endotoxins in thoracic duct lymph in SAP accompanied by ALI
1 TNF-α,IL-10
1.1 TNF-αin lymph and serum
TNF-αlevel in lymph: No significant difference among abdominal sham operation 2h, 6h and 12h groups; significantly increased lymph TNF-αin SAP groups at all time points compared with the abdominal sham operation groups at the same time point(P<0.01). Comparison among SAP groups: TNF-αlevel in SAP6h and 12h was apparently higher than that in SAP2h (P<0.01), while no significant difference between the former two.
TNF-αlevel in serum: No significant difference among abdominal sham operation 2h, 6h and 12h groups; significantly increased serum TNF-αin SAP at all time points compared with the abdominal sham operation groups at the same time point(P<0.01). Comparison among SAP groups: TNF-αlevel in SAP6h and 12h was apparently higher than that in SAP2h (P<0.01), while no significant difference between the former two.
There is no significant difference between the lymth and serum TNF-αconcentration; and likely the lymth and serum TNF-αconcentration in DC groups.
1.2 IL-10 in lymph and serum
IL-10 level in lymph: No significant difference among abdominal sham operation 2h, 6h and 12h groups; significantly increased lymph IL-10 in SAP at all time points compared with the abdominal sham operation groups at the same time point (P<0.01). Comparison among SAP groups: the level of IL-10 in SAP6h was significantly higher than that in SAP2h, and IL-10 in SAP12h higher than those in SAP2h and SAP6h.
IL-10 level in blood: No significant difference among abdominal sham operation 2h, 6h and 12h groups; significantly increased blood IL-10 in SAP at all time points compared with the abdominal sham operation groups at the same time point (P<0.01). Comparison among SAP groups: the level of IL-10 in SAP6h was significantly higher than that in SAP2h, and IL-10 in SAP12h higher than those in SAP2h and SAP6h. There is no significant difference between the lymth and serum IL-10 concentration; and likely the lymth and serum IL-10 concentration in DC groups.
2 AMY and LIP
2.1 AMY in lymph:
Level of AMY in lymph: No significant difference among abdominal sham operation 2h, 6h and 12h groups; significantly increased lymph AMY in SAP at all time points compared with the abdominal sham operation groups at the same time point (P<0.01). Comparison among SAP groups: the levels of AMY in SAP6h and SAP12h were significantly higher than that in SAP2h (P<0.05), with no significant difference between those in SAP12h and SAP6h.
2.2 AMY in serum:
No significant difference among abdominal sham operation 2h, 6h and 12h groups; significantly increased blood AMY in SAP at all time points compared with the abdominal sham operation groups at the same time point (P<0.01). Comparison among SAP groups: the levels of AMY in SAP6h and SAP12h were significantly higher than that in SAP2h (P<0.05), with no significant difference between those in SAP12h and SAP6h.
In DC groups, AMY concentration in lymph was apparently higher than that in serum (P<0.01).
2.3 LIP in lymph:
Level of LIP in lymph: No significant difference among abdominal sham operation 2h, 6h and 12h groups; significantly increased lymph LIP in DC groups at all times compared with the abdominal sham operation groups at the same time point (P<0.01). Comparison among DC groups: the levels of LIP in SAP6h and SAP12h were significantly higher than that in SAP2h (P<0.05), with no significant difference between those in SAP12h and SAP6h.
2.4 LIP in serum:
Level of LIP in serum: No significant difference among abdominal sham operation 2h, 6h and 12h groups; significantly increased serum LIP in DC groups at all time points compared with the abdominal sham operation groups at the same time point (P<0.01). Comparison among DC groups: the levels of serum LIP in SAP6h and SAP12h were significantly higher than that in SAP2h (P<0.05), with no significant difference between those in SAP12h and SAP6h.
In DC groups, LIP concentration in lymph at certain time point was apparently higher than that in serum (P<0.01).
3 Changed levels of endotoxins in lymph
No significant difference among the same time point sham groups; compared with sham groups, DC2h showed no significant difference, while both DC6h and DC12h showed apparently significant increase (P<0.01), with the increase in the latter apparently higher than that in the former (P<0.01), and the increase in DC6h and DC12h higher than that in DC2h. Conclusions:
Part I: Model establishing of cervical thoracic duct lymphatic fistula in rats
1 The success rate of the cervical thoracic duct direct group is lower than that of the other two groups, with no significant difference in the cervical thoracic duct indirect group and abdominal thoracic duct group.
2 There is no significant difference in properties about the drained lymph by the three methods. Compared with the cervical thoracic duct direct and indirect groups, there is more drained lymph in abdominal thoracic duct group.
3 Cervical thoracic duct indirect fistula in rats, which can drain lymph stably and does not disturb abdominal cavity, is a stable and reliable method to make animal models of thoracic duct lymphatic fistula, and can be used in the experimental study of SAP accompanied by ALI associated with lymphatic circulation.
Part II: Experimental study on the early stage intervention of thoracic duct ligation /conduction in the SAP accompanied by ALI
1 Early stage SAP will cause damaging and pathomorphological changes of the lung, followed by functional disorder.
2 Cervical thoracic duct conduction can relieve pathomorphological changes and dysfunction of the lung caused by ALI, and cervical thoracic duct ligation tends to have the same effect as conduction: at the early phase (2h, 6h), ligation and conduction show no significant difference in improving the function of the lung; while at the middle or late phase (12), conduction can apparently relieve pathomorphological changes and dysfunction of the lung caused by ALI secondary to SAP.
3 Cervical thoracic duct conduction cannot relieve pathomorphological changes of the pancreas secondary to SAP, while cervical thoracic duct ligation will aggravate pathomorphological changes.
Part III: Experimental study on the effect of thoracic duct ligation /conduction in SAP on PMN functions in the arterial blood
1 Cervical thoracic duct conduction can lower the effect of respiratory burst of arterial PMN in SAP, the proportion of PMN with CD11b expression positive, and MFI of CD11b; while cervical thoracic duct ligation tends to have the same effects as conduction.
2 Cervical thoracic duct conduction/ligation can lower the effect of respiratory burst of arterial PMN in SAP, the proportion of PMN with CD11b expression positive, and MFI of CD11b, which may be attributed to the fact that cervical thoracic duct conduction can relieve the pathomorphological change and dysfunctions of ALI secondary to SAP.
Part IV: Experimental study on the concentration changes of TNF-α, IL-10, AMY, LIP, and endotoxins in thoracic duct lymph in SAP accompanied by ALI
1 The concentrations of endotoxin, AMY, LIP, TNF-α, and IL-10 in the lymph increase apparently during SAP.
2 The lymph components, complicated in the thoracic duct during SAP, may activate PMN directly or indirectly through the activated pancreatin, endotoxin, TNF-α, IL-10, etc., and finally injure the lung tissue.
3 During SAP, the concentrations of AMY and LIP in the lymph and in the serum show significant difference, thereby indicating that AMY and LIP is one of the origins causing the concentration of AMY and LIP increase in the serum.
4 During SAP, the concentrations of TNF-αin the lymph and that in the serum show no significant difference, so as IL-10, thereby indicating that TNF-αand IL-10 is not the origin causing the concentration of those increase in the serum.
5 Reaction time method (one of the Limulus Amebocyte Lysate assay) is a reliable method to determine the endotoxin content in the lymph.
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