肠梗阻导致肠功能障碍的中西医结合实验研究Ⅱ
摘要
第一部分
肠梗阻导致肠功能障碍中肠上皮、肠免疫屏障及微生态的时相性研究
实验一
家兔可复性肠梗阻造模方式及材料的改良
目的：在前期实验建立的家兔机械性肠梗阻的基础上进行梗阻方式和材料的改良，使模型操作更简便、更经济、效果更稳定。方法：改用“肠管外压迫”的方式，将输液器部件改造成体外牵拉式锁扣，在末段回肠肠系膜无血管区戳孔，套住肠管，引出切口旁腹壁外。手术1～2天后由体外牵拉锁扣固定，造成该部位肠管的闭塞梗阻。解除梗阻时只需剪断锁扣后抽出装置。同时对体外水囊充注式梗阻装置、塑封钢缆圈套器等方法也进行了尝试。结果：该方法在造成梗阻的安全性、确切性等方面较原模型更优，避免了原方法所存在的腹腔感染、肠粘连、肠套叠以及肠穿孔等意外情况，并且不存在装置脱落等不稳定因素。结论：在获得更确切稳定的梗阻效果的基础上，改良后的方法创伤更小、造模周期更短，操作简单且材料经济，是较为理想的可复性机械性完全性肠梗阻模型。
实验二
肠梗阻对小肠上皮细胞的损害及其增殖修复能力的影响
目的：观察急性机械性低位小肠梗阻对于小肠上皮损害程度以及其自身再增殖、修复能力的影响。方法：选用健康大耳白兔，随机分为正常对照组(Norm)、假手术组(Sham)、梗阻1h组(H_1)、梗阻6h组(H_6)、梗阻12h组(H_(12))、梗阻24h组(H_(24))、梗阻48h组(H_(48))、梗阻72h组(H_(72))，每组6只。分别对各组动物血清瓜氨酸水平、生化、小肠组织鸟氨酸脱羧酶活性，以及小肠上皮内杯状细胞形态、数量变化进行检测和观察。结果：肠梗阻后早期肠上皮细胞存在保护性的增殖加速过程，表现为ODC活性在梗阻后6h开始增高(p＜0．001)，随着梗阻未解除，损害因素不断加重，ODC活性在梗阻24h下降，至72h甚至低于正常水平。对小肠上皮形态学观察中发现杯状细胞增多趋势，且形态发生时相性改变。以上指标在小肠不同部位发生变化的时相和程度并不一致，回肠较之空肠变化更显著。至梗阻48h-72h阶段，血清瓜氨酸水平低于正常水平(p＜0．05)。另外在对生化指标的检测中发现CK发生显著变化较早，相对其它指标较为敏感(24h vs 48h-72h)。结论：①肠梗阻发生后的早期就已经存在上皮的修复，表现为小肠组织ODC活性增高；②另外杯状细胞可能参与了小肠粘膜的修复，并且可能是上皮修复的关键细胞；③在梗阻后期随着上皮增殖活性的降低，血清瓜氨酸水平这一间接指标也随之降低，说明其对判断小肠功能障碍严重程度具有一定参考价值；④在肠梗阻发生后，CK相对其他生化指标可能对肠实质性损害更具有敏感的诊断提示意义。
实验三
小肠梗阻后肠腔内环境及微生态变化
目的：观察急性机械性低位小肠梗阻发生后肠道内环境及微生态的改变，以了解肠梗阻发生后肠道自稳态破坏与肠屏障破坏的时相性关系。方法：选用健康大耳白兔，随机分为正常对照组(Norm)、假手术组(Sham)、梗阻1h组(H_1)、梗阻6h组(H_6)、梗阻12h组(H_(12))、梗阻24h组(H_(24))、梗阻48h组(H_(48))、梗阻72h组(H_(72))，每组6只。观察梗阻后每一时段小肠内PH、含水量及采用平板计数法了解肠杆菌科与双歧杆菌菌群数量变化。另外在不同时段进行血内毒素水平、各脏器细菌移位阳性率等进行观察。结果：结果显示肠梗阻发生后末段回肠的PH值逐渐下降，肠内容物含水量进行性增加，两者均在梗阻12h-24h阶段出现显著变化(p＜0．05，p＜0．01)。在对该部位肠杆菌科和双歧杆菌的培养发现，在梗阻12h-24h阶段二者出现比例倒置，肠杆菌科成为优势菌种。另外，对脏器组织的培养和对血浆内毒素检测发现，同样也是在12h-24h阶段二者出现显著性变化。结论：肠梗阻后肠功能障碍是对方面因素的综合结果，其中肠道自稳态的破坏为之后的细菌大量增殖、细菌和内毒素移位提供了环境基础。肠梗阻较早期的12h-24h阶段可能是肠内环境、微生态发生剧烈转变的阶段，同时也可能是细菌和内毒素移位的开始。而此时肠道机械屏障往往并没有出现广泛破坏，这更说明机械屏障、生物屏障、化学屏障的破坏都是肠功能障碍的危险因素。
实验四
肠梗阻对肠上皮Claudin1 mRNA表达的影响
目的：了解急性肠梗阻发生后肠道上皮细胞间紧密连接破坏的时相性特点。方法：选用健康大耳白兔，随机分为正常对照组(Norm)、假手术组(Sham)、梗阻1h组(H_1)、梗阻6h组(H_6)、梗阻12h组(H_(12))、梗阻24h组(H_(24))、梗阻48h组(H_(48))、梗阻72h组(H_(72))，每组6只。利用实时荧光定量RT-PCR技术，对动物末段小肠上皮的紧密连接相关蛋白Claudin_1的mRNA表达情况进行相对定量分析。结果：本实验显示，梗阻发生后6h已经发现Claudin_1 mRNA表达已经显著减少至正常表达量的一半左右(p＜0．05)，12h为18％(p＜0．01)，24h为11％，48h-72h阶段表达量很低。结论：肠梗阻发生后早期(6h)已经发生机械屏障的破坏，表现为紧密连接蛋白上游基因的低表达，随着梗阻进展，这种低表达更加显著，至梗阻48h-72h阶段，这一基因的表达水平已经很低。提示这一阶段肠上皮的完整性已经完全失去，肠道机械屏障破坏严重。
实验五
肠梗阻家兔小肠免疫屏障功能的变化
目的：利用肠梗阻模型，观察急性机械性肠梗阻的发生对肠道免疫系统的影响。
方法：选用健康大耳白兔，随机分为正常对照组(Norm)、假手术组(Sham)、梗阻1h组(H_1)、梗阻6h组(H_6)、梗阻12h组(H_(12))、梗阻24h组(H_(24))、梗阻48h组(H_(48))、梗阻72h组(H_(72))，每组6只。采用流式细胞术对梗阻近端低位回肠、高位空肠两个不同部位小肠派伊尔淋巴结(PP)的CD3~+、CD4~+、CD8~+淋巴细胞亚群进行分析，利用HE染色切片计数上述两个不同部位IEL和LPL的百分比于梗阻不同阶段的变化。另外ELISA法检测小肠内分泌型免疫球蛋白A(S-IgA)含量。结果：本实验显示肠梗阻发生后12h-24h阶段，动物小肠的PP在数量上和总面积上均增加(p＜0．05)，随着梗阻进展又逐渐减少，至梗阻72h阶段则显著低于正常水平(p＜0．01)。流式细胞术对PP淋巴细胞CD3~+、CD4~+、CD8~+亚群的分析结果显示，肠梗阻解除后PP内CD3+亚群为增多趋势，低位回肠Sham组为29．44±2．54％，12h增加为35．92±2．08％(p＜0．05)，72h增至49．48±6．37％(p＜0．01)；CD4~+亚群同样在12h阶段明显高于Sham组(84．68±2．80％vs 80．5 1±2．22％，p＜0．05)，此后逐渐下降至72h则迅速减少显著低于Sham组(61．62±6．96％，p＜0．01)；CD8~+亚群变化趋势与CD4~+相反，在12h-24h阶段较Sham组下降(8．47±1．41％vs 11．52±1．04％，p＜0．05)，之后逐渐增高至72h达到最高(22．97±4．83％，p＜0．05)；CD4~+／CD8~+比值的变化趋势为早期阶段先增高，24h阶段明显高于Sham组(12．10±2．17 vs 7．97±1．40，p＜0．01)，后期逐渐下降至72h阶段则显著低于Sham组(3．35±1．03，p＜0．01)。上述几项在高位空肠中观察到的结果要明显晚于低位回肠，并且变化不剧烈。通过HE染色对IEL和LPL的百分比计数结果显示，不同部位的IEL百分比随着梗阻时间变化未发现明显差异，而LPL百分比在梗阻6h阶段明显增多，其中空肠部位的较Sham组有统计学差异(p＜0．05)。随着梗阻时间延长，两个部位的LPL百分比均呈下降趋势。肠腔内s-IgA的ELISA结果显示，梗阻发生后其肠内含量增高，24h阶段高于Sham组(p＜0．05)，随后下降至72h的最低水平，与24h水平有差异，但与Sham组间未发现统计学差异。结论：①肠梗阻发生后，小肠PP数量和总面积均增加，这可能与梗阻后受到更多炎性因素刺激有关，另外其内淋巴细胞亚群也出现相应变化，CD3~+呈不断增多趋势，CD4~+亚群比例、CD4~+／CD8~+均在早期增多，而后随着梗阻进展逐渐下降。反之，CD8~+亚群比例早期降低，后期增高，这些都预示着肠道免疫的一过性增强和之后的减弱；②通过对上皮内IEL、固有层LPL占总细胞数量百分比的统计，可以看出，IEL变化存在一定改变，但本实验尚未发现统计学的差异。而LPL百分比特点也是梗阻早期增大，后期下降，这可能是淋巴细胞归巢至固有层这一效应部位增多有关，后期随着淋巴细胞凋亡增多和其它大量炎性细胞浸润而至其比例下降；③s-IgA的量的变化进一步印证了上述现象，在早期肠腔内增多，至后期逐渐低于正常水平；④肠道免疫系统的激活是较早出现的，而且肠梗阻引发的肠免疫屏障改变可能存在部位上的差异，回肠更早、更强烈的出现免疫改变。而固有层效应部位上淋巴细胞可能这种差异并不明显，有待印证。
第二部分
四君子汤对肠梗阻解除后肠功能障碍的调节作用的实验研究
实验六
四君子汤对肠梗阻解除后小肠上皮修复的调理作用
目的：利用可复性机械性完全性肠梗阻模型，观察比较肠梗阻梗阻解除后自然恢复与中药四君子汤对小肠上皮细胞增殖与修复的影响。方法：选用健康大耳白兔，随机分为假手术组(Sham)、梗阻48模型组(m_(48))、自然恢复48h组(S_(48))、中药治疗48h组(T_(48))、自然恢复96h组(S_(96))、中药治疗96h组(T_(96))，每组6只。对动物模型梗阻解除后48h、96h时肠杯状细胞形态学、小肠组织鸟氨酸脱羧酶活性、血清瓜氨酸水平、紧密连接相关蛋白Claudin_1 mRNA表达等分别进行检测和观察。结果：本实验显示，在肠梗阻解除后小肠上皮的杯状细胞数量逐渐减少，48h后已经出现明显差异，96h基本恢复正常中药治疗组较自然恢复组差异更明显。血清瓜氨酸水平逐渐恢复至正常水平，中药组要早于自然恢复组(48h，p＜0．05)，鸟氨酸脱羧酶活性明显增高，尤其是而中药治疗组明显高于自然恢复组(P＜0．05)。另外发现中药四君子汤可以上调Claudin_1 mRNA的表达，高于自然恢复组水平。结论：①中药四君子汤可以促进肠梗阻解除后肠上皮细胞的再增殖和上皮完整性的修复过程，表现为其可以加速肠上皮内杯状细胞对肠上皮的重建的完成进度；②可以提高小肠组织ODC活性，提示四君子汤能提高上皮再生能力；③而上皮再生后的结果体现为血清瓜氨酸水平的提高；④四君子汤可以上调肠上皮紧密连接相关蛋白Claudin_1的上游基因表达，促进肠上皮完整性的修复⑤通过结果说明肠梗阻解除后的肠功能障碍在辨证上存在脾胃虚的情况，邪实已祛，正气受损的实质可能应该包括肠上皮的损害，而四君子汤的作用靶点之一可能是促进肠隐窝干细胞的增殖从而达到上皮完整性的修复。
实验七
四君子汤对肠梗阻解除后小肠免疫的调理作用
目的：利用可复性机械性完全性肠梗阻模型，观察比较肠梗阻解除后自然恢复及中药四君子汤对肠道免疫屏障的影响。方法：选用健康大耳白兔，随机分为假手术组(Sham)、梗阻48模型组(M_(48))、自然恢复48h组(S_(48))、中药治疗48h组(T_(48))、自然恢复96h组(S_(96))、中药治疗96h组(T_(96))，每组6只。通过流式细胞术对动物模型梗阻解除后48h、96h时PP淋巴细胞亚群(CD3~+、CD4~+、CD8~+、CD4~+／CD8~+)进行分析、利用ELISA法对肠道内s-IgA含量进行检测以及通过病理切片对IEL、LPL百分比进行计数。结果：本实验显示，肠梗阻解除后中药干预的T48组、T96组较自然恢复的S48、S96组的PP淋巴细胞CD3+、CD4+、CD8+亚群及CD4+／CD8+比值具有调节作用，使之更快恢复或接近梗阻前水平；在对iIEL、LPL相对数量的观察中，四君子汤干预组的LPL更早的接近正常比例；各处理组s-IgA水平之间未发现统计学差异。结论：肠梗阻解除后小肠的免疫屏障功能紊乱并没有立即恢复，自然恢复48h后CD4+、CD4+／CD8+比值等指标并没有随之恢复，而却较M48组更趋下降，这可能提示肠功能的免疫状态并没有因肠梗阻解除而立即恢复，这可能是肠道内环境紊乱、肠道菌群失调以及肠上皮完整性的破坏不可能马上改善有关。而应用四君子汤可以明显促进小肠在梗阻解除之后存在的免疫屏障功能障碍。表现为，四君子汤干预组较自然恢复组的PP淋巴亚群更快恢复正常，并可能对肠效应部位的淋巴细胞分布或数量具有调节作用。肠梗阻解除后的脾胃功能受损的实质可能涵盖了肠上皮功能低下以及肠免疫系统的紊乱，而四君子汤健脾作用的靶点很可能包括对肠道免疫系统的调理作用。
PARTⅠ
Phasic research of enterocyte,intestinal mucosal barrier andlumen flora of intestinal dysfunction induced by ileus
ExperimentⅠ
Improved methods and materials of reusable ileus model of rabbits
Objects:In order to make models easier to operate,more economical and morestable results.The improved the methods and materials were taken on the basis ofEstablished in the pre-experimental rabbit's mechanical ileus.
Methods:taking the methods of“Intestinal extrinsic oppression”,infusion set partswill be transformed into In vitro pull-type locking,made a hole in the Avascular zonemesenteric of the terminal ileum,trap tube,Leads to the incision of the abdominalwall next to the outside.1～2 days after operation was fixed by in vitro pull locking,resulting in the obstruction of the bowel.When the need to lift the obstruction onlyneed to cut the lock and withdrawing the device.At the same time,other methodssuch as in vitro-filled cystic obstruction device,plastic cable trap also have been tried.Results:This method is better than the original model in the safety,accuracy,etc.avoiding the existence of intra-abdominal infections,intestinal adhesion,intussusceptions,as well as intestinal perforation,and there is no Factors of instabilitysuch as Device off.
Conclusion:on the basis of getting certainly more stable obstruction effect.theimproved methods has the advantage of trauma smaller,shorter-cycle model,simpleoperation and material economy,is a more satisfactory reusable mechanical model ofcomplete intestinal obstruction.
ExperimentⅡ
Influence of acute intestinal obstruction on damage and proliferativeand repair ability of intestinal epithelial cells
Objective:To observe the acute mechanical low small bowel obstruction for extentof intestinal epithelial damage,as well as the effects of their own re-proliferation,repair capacity.
Methods:Healthy rabbits were randomly divided into normal control group(Norm),sham-operated group(Sham),obstruction lh group(H1),obstruction 6h group(H6),obstruction 12h group(H12),obstruction 24h group(H24),obstruction 48h group(H48),obstruction 72h group(H72),n = 6.Animals of each group were detected andanalyzed in serum citrulline levels,biochemical,intestinal tissue ornithinedecarboxylase activity,as well as in intestinal epithelial goblet cell morphology,changes in the number.
Results:Early intestinal obstruction after the existence of the protection of intestinalepithelial cells to accelerate the process of proliferation,ODC activity showedincreased when 6h after the beginning of obstruction(p＜0.001),the damage factorsincreasing with the obstruction is not lifted,ODC activity decreased in obstructive24h,to 72h or even lower than normal level.Morphological observation of intestinalepithelial goblet cells found an increase in the trend,and patterns of phase changeoccured.Indicators over different parts of the small intestine changes in the degree ofphase and is not consistent,ileum more significant than changes in the jejunum.48h-72h to the obstructive phase,lower than the normal level of serum citrullinelevels(p＜0.05).In addition to biochemical indicators in detection of CK found in asignificant change in the earlier,more sensitive than other indicators(24h vs48h-72h).
Conclusion:①Intestine epithelial repair has existed early after the obstructionoccurred,manifested as increased activity of intestinal tissue ODC;②Goblet cells may be involved in the repair of small intestinal mucosa,and may be the key cell toepithelial cell repair;③Late in the obstruction With the reduction of epithelialproliferative activity,serum citrulline levels which is a indirect indicators decreasedon their dysfunction,and it has a certain reference value on determining the severityof the small intestine;④After the intestinal obstruction,CK may be more sensitiveto the meaning of the diagnosis tips compared with other biochemical indicators ofmaterial injury to the intestine.
ExperimentⅢ
Phase relationship among Intestinal Environment andmicro-ecological changes and bacteria and endotoxin translocationafter intestinal obstruction
Objective:To observe the changes of Intestinal Environment and Microecologyafter the occurrence of the acute mechanical low small bowel obstruction in order tounderstand homeostasis after intestinal damage and intestinal barrier damage phase ofthe relationship.
Methods:Healthy rabbits were randomly divided into normal control group(Norm),sham-operated group(Sham),obstruction l h group(H1),obstruction 6h group(H6),obstruction 12h group(H12),obstruction 24h group(H24),obstruction 48h group(H48),obstruction 72h group(H72),n = 6.Observation of small bowel obstructionwithin hours of each PH,water content and understanding of the use of plate countand Enterobacteriaceae bacteria change in the number of Bifidobacteria.Also bloodendotoxin levels,the organs of the rate of bacterial translocation was observed atdifferent times.
Results:The results showed that the PH value of terminal ileum was graduallydeclined while the water content in intestinal contents was gradually increases afterthe intestinal obstruction,both groups showed significant changes in the 12h-24hphase obstruction(p＜0.05,p＜0.01).Enterobacteriaceae in the site and found the cultivation of bifidobacteria,in the 12h-24h stage of obstruction there the ratio of thetwo inverted,Enterobacteriaceae become dominant bacteria.In addition,thecultivation of tissues and found that plasma endotoxin detection,also showedsignificant changes in the 12h-24h phase of the obstruction.
Conclusion:Intestinal dysfunction after intestinal obstruction is the result of manyfactors,the destruction of intestinal homeostasis provides the basis for theenvironment for a large number of bacteria proliferation and of bacteria andendotoxin translocation.Obstruction compared to the earlier stages of 12h-24h maybe the intestinal environment,drastic changes microecological stage,but also may bestart of bacterial and endotoxin translocation.And gastro-intestinal mechanical barrierat this time often did not show extensive damage,it has also said that,mechanicalbarriers,biological barriers,chemical barriers intestinal damage are risk factors fordysfunction.
ExperimentⅣ
The changes of intestinal epithelial Claudin1 mRNA expression inthe occurrence after intestinal obstruction
Objective:To understand the characteristics of phase of destruction of tightjunctions in intestinal epithelial cells after the occurrence of acute intestinalobstruction.
Methods:Healthy rabbits were randomly divided into normal control group(Norm),sham-operated group(Sham),obstruction l h group(H1),obstruction 6h group(H6),obstruction 12h group(H12),obstruction 24h group(H24),obstruction 48h group(H48),obstruction 72h group(H72),n = 6.the relative quantitative analysis of theanimals of the last paragraph of the intestinal epithelial tight junction-associatedprotein Claudinl's mRNA's expression were made through the real-time fluorescencequantitative RT-PCR technology.
Results:The experiments showed that after 6h obstruction Claudinl mRNA expression has been found to have significantly reduced levels to normal about half(p＜0.05),12h to 18%(p＜0.01),24h to 11%,48h-72h phase expression was very low.
Conclusions:mechanical barriers have been destroyed at Early stage after theoccurrence of intestinal obstruction(6h),the manifestation was lowly expressedgenes for the tight junction protein upstream gene expression,with the obstruction ofprogressing,the low expression was more prominent at 48h-72h phase of obstruction.Indicated that this stage of intestinal epithelial integrity is completely lost,seriouslydestruction of intestinal mechanical barrier.
ExperimentⅤ
Changes of the immune barrier function in small bowel obstructionin rabbits
Objective:To observe the impact of the immune system when occurrence of acutemechanical intestinal obstruction by making use of intestinal obstruction model
Methods:Healthy rabbits were randomly divided into normal control group(Norm),sham-operated group(Sham),obstruction lh group(H1),obstruction 6h group(H6),obstruction 12h group(H12),obstruction 24h group(H24),obstruction 48h group(H48),obstruction 72h group(H72),n = 6.By flow cytometry of low obstruction ofproximal ileum,two different parts of high jejunal Peyer intestinal lymph nodes(PP)of CD3+,CD4+,CD8+lymphocyte subsets in the analysis,using HE stainingsections in different parts of the two counts the percentage of IEL and LPL indifferent stages of obstructive changes.Another ELISA assay endocrine intestinalimmunoglobulin A(S-IgA)content.
Results:The experiments showed that intestinal obstruction after 12h-24h stage,thesmall intestine of animals in the number of PP and the total area increased(p＜0.05),with the progress and gradually reduce obstruction to obstruction 72h significantlylower than the normal stage level(p＜0.01).PP lymphocytes by flow cytometry onCD3+,CD4+,CD8+sub-group analysis showed that after the lifting of PP within intestinal obstruction CD3+subpopulations for the increase in the trend of low ilealSham group was 29.44±2.54%,12h increased to 35.92±2.08%(p＜0.05),72h to49.48±6.37%(p＜0.01);CD4+subsets in the 12h stage of the same wassignificantly higher than Sham group(84.68±2.80% vs 80.51±2.22%,p＜0.05),Been steadily declining since then to a rapid decline in 72h was significantly lowerthan Sham group(61.62±6.96%,p＜0.01);CD8+subsets and CD4+Trends Incontrast,12h-24h phase of decline than the Sham group(8.47±1.41% vs 11.52±1.04%,p＜0.05),after 72h was gradually increased to the highest(22.97±4.83%,p＜0.05);CD4+/ CD8+ratio of the change in the trend of first increase for the earlystages,24h stage was significantly higher than Sham group(12.10±2.17 vs 7.97±1.40,p＜0.01),the late phase of gradual decline to 72h was significantly lower thanSham group(3.35±1.03,p＜0.01).Campylobacter high above in the results observedsignificantly later than the low ileum,and the changes are not dramatic.Through theHE staining of the percentage of IEL and LPL counting results showed that thepercentage of IEL in different parts of the time with the obstruction found nosignificant difference,and LPL in the percentage of obstruction increased 6h stage,inwhich parts of the jejunum than the Sham group,significant difference(p＜0.05).With prolonged obstruction,the percentage of the two sites decreased LPL.Mesocaval s-IgA in the ELISA results showed that after the occurrence of theintestinal obstruction content increased,24h stage higher than the Sham group(p＜0.05),then dropped to the lowest level in 72h,and 24h are different levels,but withthe Sham group found no significant difference between.
Conclusions:①when obstruction occurred,the number of the small intestine andthe total area of PP increased,which may be related to obstruction by inflammatoryfactors stimulate more relevant,while its lymphocyte subsets is also a correspondingchange,CD3+showed increasing trend,the proportion of CD4+subsets,CD4+/CD8+were increased in the early days,and then gradually decreased with theprogress obstruction.In contrast,CD8+subsets to reduce the proportion of earlystage,the latter increased,which indicates that intestinal immunity and transientenhanced after the decline;②By intraepithelial IEL,lamina propria LPL percentage of the total cell number of statistics,can be seen,IEL changes in a certain change,butthis experiment has not yet found the difference statistically.LPL is characterized bythe percentage of early obstruction increased,the latter decline,this may belymphocyte homing to the lamina part of the effect of the increase,and the latter withthe increase in lymphocyte apoptosis and other inflammatory cell infiltration anddecreased to its;③s-IgA changes in the amount of further verify theabove-mentioned phenomenon,with an increase in the early mesocaval to graduallylower than the level of the latter;④intestinal immune system activation occursearlier,and intestinal obstruction caused by changes in the immune barrier there maybe differences between sites,ileum earlier,the emergence of a stronger immune tochange.The effect of position on the lamina propria lymphocytes Such differencesmay not be apparent,to be confirmed.
PARTⅡ
Phasic research of enterocyte,intestinal mucosal barrier andlumen flora of intestinal dysfunction induced by ileus
Experiment VI
Repair of intestinal epithelial role of Sijunzi tang After theobstruction was removed
Objective:To use reusable mechanical model of complete intestinal obstruction,tocompare and observe the natural recovery and influence of traditional Chinesemedicine Sijunzitang on the impact of intestinal epithelial cell proliferation andrepair.
Methods:Healthy rabbits were randomly divided into sham operation group(Sham),obstruction of 48 model group(M48),natural recovery 48h group(S48),Chinese medicine treatment of 48h group(T48),natural recovery 96h group(S96),Chinese medicine treatment of 96h group(T96),each group 6.The lifting of ananimal model of obstruction after 48h,96h.Making intestinal goblet cell morphology,organization of small intestinal ornithine decarboxylase activity and serum citrullinelevels of tight junction-associated protein expression Claudinl mRNA's detection andobservation,respectively.
Results:The experiments showed that the number of goblet cells of small intestineepithelium was gradually decline after the lifting of the obstruction,the significantdifferences have emerged 48h later,96h returned to normal level,traditional Chinesemedicine treatment group was more effectively.Serum citrulline levels graduallyreturned to normal levels,the Chinese Medicines Board was earlier than the natural recovery group(48h,p＜0.05),omithine decarboxylase activity was significantlyincreased,especially in Chinese medicine the treatment group was significantlyhigher than the natural recovery group(P＜0.05).Also found in traditional Chinesemedicine Sijunzitang Claudinl mRNA can increase the expression level higher thanthe natural recovery group.
Conclusions:①Sijunzi tang medicine can promote intestinal obstruction intestinalepithelial cells after the lifting of the re-proliferation and epithelial integrity of therepair process,can accelerate the performance of their intestinal intraepithelial gobletcells of the intestinal epithelium of the completion of the reconstruction progress;②itcan improve ODC activity in intestinal tissue,suggesting Sijunzitang the ability toincrease epithelial regeneration;③epithelial regeneration and the results reflected inserum citrulline levels,④Sijunzitang can increase intestinal epithelial tightjunction-associated protein gene expression in the upper reaches of the Claudinl,andpromote the integrity of intestinal epithelial repair⑤The results showed throughafter the lifting of the intestinal obstruction dysfunction exist in the differentiation ofthe spleen and stomach empty,the evil is already Qu,the essence of righteousnessmay be damaged,including damage to intestinal epithelium,and the role ofSijunzitang one target may be the promotion of intestinal crypt stem cell proliferationso as to achieve the integrity of epithelial repair.
ExperimentⅦ
Intestinal immune opsonization role of Sijunzi tang After theobstruction was removed
Objective:To use reusable mechanical model of complete intestinal obstruction,tocompare and observe the natural recovery and influence of traditional Chinesemedicine Sijunzitang on the impact of intestinal immune barrier.
Methods:Healthy rabbits were randomly divided into sham operation group(Sham),obstruction of 48 model group(M48),natural recovery 48h group(S48), Chinese medicine treatment of 48h group(T48),natural recovery 96h group(S96),Chinese medicine treatment of 96h group(T96),each group 6.after the lifting of ananimal model of obstructive 48h,96h,getting PP-lymphocyte subsets(CD3+,CD4+,CD8+,CD4+/ CD8+)for analysis Through flow cytometry,s-IgA in intestinalcontent were detected by using ELISA method as well as through the biopsy of IEL,LPL percentage count.
Results:The experiments showed that after the Chinese intervention in intestinalobstruction to lift the T48 group,T96 group than in the natural restoration of the S48,S96 Group PP lymphocytes CD3+,CD4+,CD8+subsets and CD4+/ CD8+ratiohas a regulatory role,so that faster restoration of or close to the former level ofobstruction;in iIEL,LPL relative number of observation,Sijunzitang LPL earlierintervention group the proportion of the near normal;the treatment group the level ofs-IgA did not find significant difference among them.
Conclusion:the barrier function of the immune disorder was not restored withoutdelay,after 48h natural CD4+,CD4+/CD8+ratio and other indicators have notrecovered,and has declined more than the M48 group,which may indicate theimmune status of intestinal function has not been affected by intestinal obstructionand an immediate resumption of disarmament,it may be gastro-intestinal disorderwithin the environment,imbalance of intestinal flora,as well as the integrity ofintestinal epithelial damage can not be immediately improved.Sijunzitang andapplications can be in the promotion of small bowel obstruction after the lifting of theexistence of dysfunction of the immune barrier.Reflected in the intervention groupSijunzitang more natural restoration of the PP group Vallarta leaching faster return tonormal group,and may be part of the intestinal effects of the distribution or thenumber of lymphocytes with a regulatory role.Obstruction after the lifting of thesubstance of the spleen and stomach function may be covered by the intestinalepithelial dysfunction,as well as the immune system,intestinal disorders,and the roleof Sijunzitang strengthening spleen's target is likely to include the intestinal immunesystem opsonization.
肠梗阻导致肠功能障碍中肠上皮、肠免疫屏障及微生态的时相性研究
实验一
家兔可复性肠梗阻造模方式及材料的改良
目的：在前期实验建立的家兔机械性肠梗阻的基础上进行梗阻方式和材料的改良，使模型操作更简便、更经济、效果更稳定。方法：改用“肠管外压迫”的方式，将输液器部件改造成体外牵拉式锁扣，在末段回肠肠系膜无血管区戳孔，套住肠管，引出切口旁腹壁外。手术1～2天后由体外牵拉锁扣固定，造成该部位肠管的闭塞梗阻。解除梗阻时只需剪断锁扣后抽出装置。同时对体外水囊充注式梗阻装置、塑封钢缆圈套器等方法也进行了尝试。结果：该方法在造成梗阻的安全性、确切性等方面较原模型更优，避免了原方法所存在的腹腔感染、肠粘连、肠套叠以及肠穿孔等意外情况，并且不存在装置脱落等不稳定因素。结论：在获得更确切稳定的梗阻效果的基础上，改良后的方法创伤更小、造模周期更短，操作简单且材料经济，是较为理想的可复性机械性完全性肠梗阻模型。
实验二
肠梗阻对小肠上皮细胞的损害及其增殖修复能力的影响
目的：观察急性机械性低位小肠梗阻对于小肠上皮损害程度以及其自身再增殖、修复能力的影响。方法：选用健康大耳白兔，随机分为正常对照组(Norm)、假手术组(Sham)、梗阻1h组(H_1)、梗阻6h组(H_6)、梗阻12h组(H_(12))、梗阻24h组(H_(24))、梗阻48h组(H_(48))、梗阻72h组(H_(72))，每组6只。分别对各组动物血清瓜氨酸水平、生化、小肠组织鸟氨酸脱羧酶活性，以及小肠上皮内杯状细胞形态、数量变化进行检测和观察。结果：肠梗阻后早期肠上皮细胞存在保护性的增殖加速过程，表现为ODC活性在梗阻后6h开始增高(p＜0．001)，随着梗阻未解除，损害因素不断加重，ODC活性在梗阻24h下降，至72h甚至低于正常水平。对小肠上皮形态学观察中发现杯状细胞增多趋势，且形态发生时相性改变。以上指标在小肠不同部位发生变化的时相和程度并不一致，回肠较之空肠变化更显著。至梗阻48h-72h阶段，血清瓜氨酸水平低于正常水平(p＜0．05)。另外在对生化指标的检测中发现CK发生显著变化较早，相对其它指标较为敏感(24h vs 48h-72h)。结论：①肠梗阻发生后的早期就已经存在上皮的修复，表现为小肠组织ODC活性增高；②另外杯状细胞可能参与了小肠粘膜的修复，并且可能是上皮修复的关键细胞；③在梗阻后期随着上皮增殖活性的降低，血清瓜氨酸水平这一间接指标也随之降低，说明其对判断小肠功能障碍严重程度具有一定参考价值；④在肠梗阻发生后，CK相对其他生化指标可能对肠实质性损害更具有敏感的诊断提示意义。
实验三
小肠梗阻后肠腔内环境及微生态变化
目的：观察急性机械性低位小肠梗阻发生后肠道内环境及微生态的改变，以了解肠梗阻发生后肠道自稳态破坏与肠屏障破坏的时相性关系。方法：选用健康大耳白兔，随机分为正常对照组(Norm)、假手术组(Sham)、梗阻1h组(H_1)、梗阻6h组(H_6)、梗阻12h组(H_(12))、梗阻24h组(H_(24))、梗阻48h组(H_(48))、梗阻72h组(H_(72))，每组6只。观察梗阻后每一时段小肠内PH、含水量及采用平板计数法了解肠杆菌科与双歧杆菌菌群数量变化。另外在不同时段进行血内毒素水平、各脏器细菌移位阳性率等进行观察。结果：结果显示肠梗阻发生后末段回肠的PH值逐渐下降，肠内容物含水量进行性增加，两者均在梗阻12h-24h阶段出现显著变化(p＜0．05，p＜0．01)。在对该部位肠杆菌科和双歧杆菌的培养发现，在梗阻12h-24h阶段二者出现比例倒置，肠杆菌科成为优势菌种。另外，对脏器组织的培养和对血浆内毒素检测发现，同样也是在12h-24h阶段二者出现显著性变化。结论：肠梗阻后肠功能障碍是对方面因素的综合结果，其中肠道自稳态的破坏为之后的细菌大量增殖、细菌和内毒素移位提供了环境基础。肠梗阻较早期的12h-24h阶段可能是肠内环境、微生态发生剧烈转变的阶段，同时也可能是细菌和内毒素移位的开始。而此时肠道机械屏障往往并没有出现广泛破坏，这更说明机械屏障、生物屏障、化学屏障的破坏都是肠功能障碍的危险因素。
实验四
肠梗阻对肠上皮Claudin1 mRNA表达的影响
目的：了解急性肠梗阻发生后肠道上皮细胞间紧密连接破坏的时相性特点。方法：选用健康大耳白兔，随机分为正常对照组(Norm)、假手术组(Sham)、梗阻1h组(H_1)、梗阻6h组(H_6)、梗阻12h组(H_(12))、梗阻24h组(H_(24))、梗阻48h组(H_(48))、梗阻72h组(H_(72))，每组6只。利用实时荧光定量RT-PCR技术，对动物末段小肠上皮的紧密连接相关蛋白Claudin_1的mRNA表达情况进行相对定量分析。结果：本实验显示，梗阻发生后6h已经发现Claudin_1 mRNA表达已经显著减少至正常表达量的一半左右(p＜0．05)，12h为18％(p＜0．01)，24h为11％，48h-72h阶段表达量很低。结论：肠梗阻发生后早期(6h)已经发生机械屏障的破坏，表现为紧密连接蛋白上游基因的低表达，随着梗阻进展，这种低表达更加显著，至梗阻48h-72h阶段，这一基因的表达水平已经很低。提示这一阶段肠上皮的完整性已经完全失去，肠道机械屏障破坏严重。
实验五
肠梗阻家兔小肠免疫屏障功能的变化
目的：利用肠梗阻模型，观察急性机械性肠梗阻的发生对肠道免疫系统的影响。
方法：选用健康大耳白兔，随机分为正常对照组(Norm)、假手术组(Sham)、梗阻1h组(H_1)、梗阻6h组(H_6)、梗阻12h组(H_(12))、梗阻24h组(H_(24))、梗阻48h组(H_(48))、梗阻72h组(H_(72))，每组6只。采用流式细胞术对梗阻近端低位回肠、高位空肠两个不同部位小肠派伊尔淋巴结(PP)的CD3~+、CD4~+、CD8~+淋巴细胞亚群进行分析，利用HE染色切片计数上述两个不同部位IEL和LPL的百分比于梗阻不同阶段的变化。另外ELISA法检测小肠内分泌型免疫球蛋白A(S-IgA)含量。结果：本实验显示肠梗阻发生后12h-24h阶段，动物小肠的PP在数量上和总面积上均增加(p＜0．05)，随着梗阻进展又逐渐减少，至梗阻72h阶段则显著低于正常水平(p＜0．01)。流式细胞术对PP淋巴细胞CD3~+、CD4~+、CD8~+亚群的分析结果显示，肠梗阻解除后PP内CD3+亚群为增多趋势，低位回肠Sham组为29．44±2．54％，12h增加为35．92±2．08％(p＜0．05)，72h增至49．48±6．37％(p＜0．01)；CD4~+亚群同样在12h阶段明显高于Sham组(84．68±2．80％vs 80．5 1±2．22％，p＜0．05)，此后逐渐下降至72h则迅速减少显著低于Sham组(61．62±6．96％，p＜0．01)；CD8~+亚群变化趋势与CD4~+相反，在12h-24h阶段较Sham组下降(8．47±1．41％vs 11．52±1．04％，p＜0．05)，之后逐渐增高至72h达到最高(22．97±4．83％，p＜0．05)；CD4~+／CD8~+比值的变化趋势为早期阶段先增高，24h阶段明显高于Sham组(12．10±2．17 vs 7．97±1．40，p＜0．01)，后期逐渐下降至72h阶段则显著低于Sham组(3．35±1．03，p＜0．01)。上述几项在高位空肠中观察到的结果要明显晚于低位回肠，并且变化不剧烈。通过HE染色对IEL和LPL的百分比计数结果显示，不同部位的IEL百分比随着梗阻时间变化未发现明显差异，而LPL百分比在梗阻6h阶段明显增多，其中空肠部位的较Sham组有统计学差异(p＜0．05)。随着梗阻时间延长，两个部位的LPL百分比均呈下降趋势。肠腔内s-IgA的ELISA结果显示，梗阻发生后其肠内含量增高，24h阶段高于Sham组(p＜0．05)，随后下降至72h的最低水平，与24h水平有差异，但与Sham组间未发现统计学差异。结论：①肠梗阻发生后，小肠PP数量和总面积均增加，这可能与梗阻后受到更多炎性因素刺激有关，另外其内淋巴细胞亚群也出现相应变化，CD3~+呈不断增多趋势，CD4~+亚群比例、CD4~+／CD8~+均在早期增多，而后随着梗阻进展逐渐下降。反之，CD8~+亚群比例早期降低，后期增高，这些都预示着肠道免疫的一过性增强和之后的减弱；②通过对上皮内IEL、固有层LPL占总细胞数量百分比的统计，可以看出，IEL变化存在一定改变，但本实验尚未发现统计学的差异。而LPL百分比特点也是梗阻早期增大，后期下降，这可能是淋巴细胞归巢至固有层这一效应部位增多有关，后期随着淋巴细胞凋亡增多和其它大量炎性细胞浸润而至其比例下降；③s-IgA的量的变化进一步印证了上述现象，在早期肠腔内增多，至后期逐渐低于正常水平；④肠道免疫系统的激活是较早出现的，而且肠梗阻引发的肠免疫屏障改变可能存在部位上的差异，回肠更早、更强烈的出现免疫改变。而固有层效应部位上淋巴细胞可能这种差异并不明显，有待印证。
第二部分
四君子汤对肠梗阻解除后肠功能障碍的调节作用的实验研究
实验六
四君子汤对肠梗阻解除后小肠上皮修复的调理作用
目的：利用可复性机械性完全性肠梗阻模型，观察比较肠梗阻梗阻解除后自然恢复与中药四君子汤对小肠上皮细胞增殖与修复的影响。方法：选用健康大耳白兔，随机分为假手术组(Sham)、梗阻48模型组(m_(48))、自然恢复48h组(S_(48))、中药治疗48h组(T_(48))、自然恢复96h组(S_(96))、中药治疗96h组(T_(96))，每组6只。对动物模型梗阻解除后48h、96h时肠杯状细胞形态学、小肠组织鸟氨酸脱羧酶活性、血清瓜氨酸水平、紧密连接相关蛋白Claudin_1 mRNA表达等分别进行检测和观察。结果：本实验显示，在肠梗阻解除后小肠上皮的杯状细胞数量逐渐减少，48h后已经出现明显差异，96h基本恢复正常中药治疗组较自然恢复组差异更明显。血清瓜氨酸水平逐渐恢复至正常水平，中药组要早于自然恢复组(48h，p＜0．05)，鸟氨酸脱羧酶活性明显增高，尤其是而中药治疗组明显高于自然恢复组(P＜0．05)。另外发现中药四君子汤可以上调Claudin_1 mRNA的表达，高于自然恢复组水平。结论：①中药四君子汤可以促进肠梗阻解除后肠上皮细胞的再增殖和上皮完整性的修复过程，表现为其可以加速肠上皮内杯状细胞对肠上皮的重建的完成进度；②可以提高小肠组织ODC活性，提示四君子汤能提高上皮再生能力；③而上皮再生后的结果体现为血清瓜氨酸水平的提高；④四君子汤可以上调肠上皮紧密连接相关蛋白Claudin_1的上游基因表达，促进肠上皮完整性的修复⑤通过结果说明肠梗阻解除后的肠功能障碍在辨证上存在脾胃虚的情况，邪实已祛，正气受损的实质可能应该包括肠上皮的损害，而四君子汤的作用靶点之一可能是促进肠隐窝干细胞的增殖从而达到上皮完整性的修复。
实验七
四君子汤对肠梗阻解除后小肠免疫的调理作用
目的：利用可复性机械性完全性肠梗阻模型，观察比较肠梗阻解除后自然恢复及中药四君子汤对肠道免疫屏障的影响。方法：选用健康大耳白兔，随机分为假手术组(Sham)、梗阻48模型组(M_(48))、自然恢复48h组(S_(48))、中药治疗48h组(T_(48))、自然恢复96h组(S_(96))、中药治疗96h组(T_(96))，每组6只。通过流式细胞术对动物模型梗阻解除后48h、96h时PP淋巴细胞亚群(CD3~+、CD4~+、CD8~+、CD4~+／CD8~+)进行分析、利用ELISA法对肠道内s-IgA含量进行检测以及通过病理切片对IEL、LPL百分比进行计数。结果：本实验显示，肠梗阻解除后中药干预的T48组、T96组较自然恢复的S48、S96组的PP淋巴细胞CD3+、CD4+、CD8+亚群及CD4+／CD8+比值具有调节作用，使之更快恢复或接近梗阻前水平；在对iIEL、LPL相对数量的观察中，四君子汤干预组的LPL更早的接近正常比例；各处理组s-IgA水平之间未发现统计学差异。结论：肠梗阻解除后小肠的免疫屏障功能紊乱并没有立即恢复，自然恢复48h后CD4+、CD4+／CD8+比值等指标并没有随之恢复，而却较M48组更趋下降，这可能提示肠功能的免疫状态并没有因肠梗阻解除而立即恢复，这可能是肠道内环境紊乱、肠道菌群失调以及肠上皮完整性的破坏不可能马上改善有关。而应用四君子汤可以明显促进小肠在梗阻解除之后存在的免疫屏障功能障碍。表现为，四君子汤干预组较自然恢复组的PP淋巴亚群更快恢复正常，并可能对肠效应部位的淋巴细胞分布或数量具有调节作用。肠梗阻解除后的脾胃功能受损的实质可能涵盖了肠上皮功能低下以及肠免疫系统的紊乱，而四君子汤健脾作用的靶点很可能包括对肠道免疫系统的调理作用。
PARTⅠ
Phasic research of enterocyte,intestinal mucosal barrier andlumen flora of intestinal dysfunction induced by ileus
ExperimentⅠ
Improved methods and materials of reusable ileus model of rabbits
Objects:In order to make models easier to operate,more economical and morestable results.The improved the methods and materials were taken on the basis ofEstablished in the pre-experimental rabbit's mechanical ileus.
Methods:taking the methods of“Intestinal extrinsic oppression”,infusion set partswill be transformed into In vitro pull-type locking,made a hole in the Avascular zonemesenteric of the terminal ileum,trap tube,Leads to the incision of the abdominalwall next to the outside.1～2 days after operation was fixed by in vitro pull locking,resulting in the obstruction of the bowel.When the need to lift the obstruction onlyneed to cut the lock and withdrawing the device.At the same time,other methodssuch as in vitro-filled cystic obstruction device,plastic cable trap also have been tried.Results:This method is better than the original model in the safety,accuracy,etc.avoiding the existence of intra-abdominal infections,intestinal adhesion,intussusceptions,as well as intestinal perforation,and there is no Factors of instabilitysuch as Device off.
Conclusion:on the basis of getting certainly more stable obstruction effect.theimproved methods has the advantage of trauma smaller,shorter-cycle model,simpleoperation and material economy,is a more satisfactory reusable mechanical model ofcomplete intestinal obstruction.
ExperimentⅡ
Influence of acute intestinal obstruction on damage and proliferativeand repair ability of intestinal epithelial cells
Objective:To observe the acute mechanical low small bowel obstruction for extentof intestinal epithelial damage,as well as the effects of their own re-proliferation,repair capacity.
Methods:Healthy rabbits were randomly divided into normal control group(Norm),sham-operated group(Sham),obstruction lh group(H1),obstruction 6h group(H6),obstruction 12h group(H12),obstruction 24h group(H24),obstruction 48h group(H48),obstruction 72h group(H72),n = 6.Animals of each group were detected andanalyzed in serum citrulline levels,biochemical,intestinal tissue ornithinedecarboxylase activity,as well as in intestinal epithelial goblet cell morphology,changes in the number.
Results:Early intestinal obstruction after the existence of the protection of intestinalepithelial cells to accelerate the process of proliferation,ODC activity showedincreased when 6h after the beginning of obstruction(p＜0.001),the damage factorsincreasing with the obstruction is not lifted,ODC activity decreased in obstructive24h,to 72h or even lower than normal level.Morphological observation of intestinalepithelial goblet cells found an increase in the trend,and patterns of phase changeoccured.Indicators over different parts of the small intestine changes in the degree ofphase and is not consistent,ileum more significant than changes in the jejunum.48h-72h to the obstructive phase,lower than the normal level of serum citrullinelevels(p＜0.05).In addition to biochemical indicators in detection of CK found in asignificant change in the earlier,more sensitive than other indicators(24h vs48h-72h).
Conclusion:①Intestine epithelial repair has existed early after the obstructionoccurred,manifested as increased activity of intestinal tissue ODC;②Goblet cells may be involved in the repair of small intestinal mucosa,and may be the key cell toepithelial cell repair;③Late in the obstruction With the reduction of epithelialproliferative activity,serum citrulline levels which is a indirect indicators decreasedon their dysfunction,and it has a certain reference value on determining the severityof the small intestine;④After the intestinal obstruction,CK may be more sensitiveto the meaning of the diagnosis tips compared with other biochemical indicators ofmaterial injury to the intestine.
ExperimentⅢ
Phase relationship among Intestinal Environment andmicro-ecological changes and bacteria and endotoxin translocationafter intestinal obstruction
Objective:To observe the changes of Intestinal Environment and Microecologyafter the occurrence of the acute mechanical low small bowel obstruction in order tounderstand homeostasis after intestinal damage and intestinal barrier damage phase ofthe relationship.
Methods:Healthy rabbits were randomly divided into normal control group(Norm),sham-operated group(Sham),obstruction l h group(H1),obstruction 6h group(H6),obstruction 12h group(H12),obstruction 24h group(H24),obstruction 48h group(H48),obstruction 72h group(H72),n = 6.Observation of small bowel obstructionwithin hours of each PH,water content and understanding of the use of plate countand Enterobacteriaceae bacteria change in the number of Bifidobacteria.Also bloodendotoxin levels,the organs of the rate of bacterial translocation was observed atdifferent times.
Results:The results showed that the PH value of terminal ileum was graduallydeclined while the water content in intestinal contents was gradually increases afterthe intestinal obstruction,both groups showed significant changes in the 12h-24hphase obstruction(p＜0.05,p＜0.01).Enterobacteriaceae in the site and found the cultivation of bifidobacteria,in the 12h-24h stage of obstruction there the ratio of thetwo inverted,Enterobacteriaceae become dominant bacteria.In addition,thecultivation of tissues and found that plasma endotoxin detection,also showedsignificant changes in the 12h-24h phase of the obstruction.
Conclusion:Intestinal dysfunction after intestinal obstruction is the result of manyfactors,the destruction of intestinal homeostasis provides the basis for theenvironment for a large number of bacteria proliferation and of bacteria andendotoxin translocation.Obstruction compared to the earlier stages of 12h-24h maybe the intestinal environment,drastic changes microecological stage,but also may bestart of bacterial and endotoxin translocation.And gastro-intestinal mechanical barrierat this time often did not show extensive damage,it has also said that,mechanicalbarriers,biological barriers,chemical barriers intestinal damage are risk factors fordysfunction.
ExperimentⅣ
The changes of intestinal epithelial Claudin1 mRNA expression inthe occurrence after intestinal obstruction
Objective:To understand the characteristics of phase of destruction of tightjunctions in intestinal epithelial cells after the occurrence of acute intestinalobstruction.
Methods:Healthy rabbits were randomly divided into normal control group(Norm),sham-operated group(Sham),obstruction l h group(H1),obstruction 6h group(H6),obstruction 12h group(H12),obstruction 24h group(H24),obstruction 48h group(H48),obstruction 72h group(H72),n = 6.the relative quantitative analysis of theanimals of the last paragraph of the intestinal epithelial tight junction-associatedprotein Claudinl's mRNA's expression were made through the real-time fluorescencequantitative RT-PCR technology.
Results:The experiments showed that after 6h obstruction Claudinl mRNA expression has been found to have significantly reduced levels to normal about half(p＜0.05),12h to 18%(p＜0.01),24h to 11%,48h-72h phase expression was very low.
Conclusions:mechanical barriers have been destroyed at Early stage after theoccurrence of intestinal obstruction(6h),the manifestation was lowly expressedgenes for the tight junction protein upstream gene expression,with the obstruction ofprogressing,the low expression was more prominent at 48h-72h phase of obstruction.Indicated that this stage of intestinal epithelial integrity is completely lost,seriouslydestruction of intestinal mechanical barrier.
ExperimentⅤ
Changes of the immune barrier function in small bowel obstructionin rabbits
Objective:To observe the impact of the immune system when occurrence of acutemechanical intestinal obstruction by making use of intestinal obstruction model
Methods:Healthy rabbits were randomly divided into normal control group(Norm),sham-operated group(Sham),obstruction lh group(H1),obstruction 6h group(H6),obstruction 12h group(H12),obstruction 24h group(H24),obstruction 48h group(H48),obstruction 72h group(H72),n = 6.By flow cytometry of low obstruction ofproximal ileum,two different parts of high jejunal Peyer intestinal lymph nodes(PP)of CD3+,CD4+,CD8+lymphocyte subsets in the analysis,using HE stainingsections in different parts of the two counts the percentage of IEL and LPL indifferent stages of obstructive changes.Another ELISA assay endocrine intestinalimmunoglobulin A(S-IgA)content.
Results:The experiments showed that intestinal obstruction after 12h-24h stage,thesmall intestine of animals in the number of PP and the total area increased(p＜0.05),with the progress and gradually reduce obstruction to obstruction 72h significantlylower than the normal stage level(p＜0.01).PP lymphocytes by flow cytometry onCD3+,CD4+,CD8+sub-group analysis showed that after the lifting of PP within intestinal obstruction CD3+subpopulations for the increase in the trend of low ilealSham group was 29.44±2.54%,12h increased to 35.92±2.08%(p＜0.05),72h to49.48±6.37%(p＜0.01);CD4+subsets in the 12h stage of the same wassignificantly higher than Sham group(84.68±2.80% vs 80.51±2.22%,p＜0.05),Been steadily declining since then to a rapid decline in 72h was significantly lowerthan Sham group(61.62±6.96%,p＜0.01);CD8+subsets and CD4+Trends Incontrast,12h-24h phase of decline than the Sham group(8.47±1.41% vs 11.52±1.04%,p＜0.05),after 72h was gradually increased to the highest(22.97±4.83%,p＜0.05);CD4+/ CD8+ratio of the change in the trend of first increase for the earlystages,24h stage was significantly higher than Sham group(12.10±2.17 vs 7.97±1.40,p＜0.01),the late phase of gradual decline to 72h was significantly lower thanSham group(3.35±1.03,p＜0.01).Campylobacter high above in the results observedsignificantly later than the low ileum,and the changes are not dramatic.Through theHE staining of the percentage of IEL and LPL counting results showed that thepercentage of IEL in different parts of the time with the obstruction found nosignificant difference,and LPL in the percentage of obstruction increased 6h stage,inwhich parts of the jejunum than the Sham group,significant difference(p＜0.05).With prolonged obstruction,the percentage of the two sites decreased LPL.Mesocaval s-IgA in the ELISA results showed that after the occurrence of theintestinal obstruction content increased,24h stage higher than the Sham group(p＜0.05),then dropped to the lowest level in 72h,and 24h are different levels,but withthe Sham group found no significant difference between.
Conclusions:①when obstruction occurred,the number of the small intestine andthe total area of PP increased,which may be related to obstruction by inflammatoryfactors stimulate more relevant,while its lymphocyte subsets is also a correspondingchange,CD3+showed increasing trend,the proportion of CD4+subsets,CD4+/CD8+were increased in the early days,and then gradually decreased with theprogress obstruction.In contrast,CD8+subsets to reduce the proportion of earlystage,the latter increased,which indicates that intestinal immunity and transientenhanced after the decline;②By intraepithelial IEL,lamina propria LPL percentage of the total cell number of statistics,can be seen,IEL changes in a certain change,butthis experiment has not yet found the difference statistically.LPL is characterized bythe percentage of early obstruction increased,the latter decline,this may belymphocyte homing to the lamina part of the effect of the increase,and the latter withthe increase in lymphocyte apoptosis and other inflammatory cell infiltration anddecreased to its;③s-IgA changes in the amount of further verify theabove-mentioned phenomenon,with an increase in the early mesocaval to graduallylower than the level of the latter;④intestinal immune system activation occursearlier,and intestinal obstruction caused by changes in the immune barrier there maybe differences between sites,ileum earlier,the emergence of a stronger immune tochange.The effect of position on the lamina propria lymphocytes Such differencesmay not be apparent,to be confirmed.
PARTⅡ
Phasic research of enterocyte,intestinal mucosal barrier andlumen flora of intestinal dysfunction induced by ileus
Experiment VI
Repair of intestinal epithelial role of Sijunzi tang After theobstruction was removed
Objective:To use reusable mechanical model of complete intestinal obstruction,tocompare and observe the natural recovery and influence of traditional Chinesemedicine Sijunzitang on the impact of intestinal epithelial cell proliferation andrepair.
Methods:Healthy rabbits were randomly divided into sham operation group(Sham),obstruction of 48 model group(M48),natural recovery 48h group(S48),Chinese medicine treatment of 48h group(T48),natural recovery 96h group(S96),Chinese medicine treatment of 96h group(T96),each group 6.The lifting of ananimal model of obstruction after 48h,96h.Making intestinal goblet cell morphology,organization of small intestinal ornithine decarboxylase activity and serum citrullinelevels of tight junction-associated protein expression Claudinl mRNA's detection andobservation,respectively.
Results:The experiments showed that the number of goblet cells of small intestineepithelium was gradually decline after the lifting of the obstruction,the significantdifferences have emerged 48h later,96h returned to normal level,traditional Chinesemedicine treatment group was more effectively.Serum citrulline levels graduallyreturned to normal levels,the Chinese Medicines Board was earlier than the natural recovery group(48h,p＜0.05),omithine decarboxylase activity was significantlyincreased,especially in Chinese medicine the treatment group was significantlyhigher than the natural recovery group(P＜0.05).Also found in traditional Chinesemedicine Sijunzitang Claudinl mRNA can increase the expression level higher thanthe natural recovery group.
Conclusions:①Sijunzi tang medicine can promote intestinal obstruction intestinalepithelial cells after the lifting of the re-proliferation and epithelial integrity of therepair process,can accelerate the performance of their intestinal intraepithelial gobletcells of the intestinal epithelium of the completion of the reconstruction progress;②itcan improve ODC activity in intestinal tissue,suggesting Sijunzitang the ability toincrease epithelial regeneration;③epithelial regeneration and the results reflected inserum citrulline levels,④Sijunzitang can increase intestinal epithelial tightjunction-associated protein gene expression in the upper reaches of the Claudinl,andpromote the integrity of intestinal epithelial repair⑤The results showed throughafter the lifting of the intestinal obstruction dysfunction exist in the differentiation ofthe spleen and stomach empty,the evil is already Qu,the essence of righteousnessmay be damaged,including damage to intestinal epithelium,and the role ofSijunzitang one target may be the promotion of intestinal crypt stem cell proliferationso as to achieve the integrity of epithelial repair.
ExperimentⅦ
Intestinal immune opsonization role of Sijunzi tang After theobstruction was removed
Objective:To use reusable mechanical model of complete intestinal obstruction,tocompare and observe the natural recovery and influence of traditional Chinesemedicine Sijunzitang on the impact of intestinal immune barrier.
Methods:Healthy rabbits were randomly divided into sham operation group(Sham),obstruction of 48 model group(M48),natural recovery 48h group(S48), Chinese medicine treatment of 48h group(T48),natural recovery 96h group(S96),Chinese medicine treatment of 96h group(T96),each group 6.after the lifting of ananimal model of obstructive 48h,96h,getting PP-lymphocyte subsets(CD3+,CD4+,CD8+,CD4+/ CD8+)for analysis Through flow cytometry,s-IgA in intestinalcontent were detected by using ELISA method as well as through the biopsy of IEL,LPL percentage count.
Results:The experiments showed that after the Chinese intervention in intestinalobstruction to lift the T48 group,T96 group than in the natural restoration of the S48,S96 Group PP lymphocytes CD3+,CD4+,CD8+subsets and CD4+/ CD8+ratiohas a regulatory role,so that faster restoration of or close to the former level ofobstruction;in iIEL,LPL relative number of observation,Sijunzitang LPL earlierintervention group the proportion of the near normal;the treatment group the level ofs-IgA did not find significant difference among them.
Conclusion:the barrier function of the immune disorder was not restored withoutdelay,after 48h natural CD4+,CD4+/CD8+ratio and other indicators have notrecovered,and has declined more than the M48 group,which may indicate theimmune status of intestinal function has not been affected by intestinal obstructionand an immediate resumption of disarmament,it may be gastro-intestinal disorderwithin the environment,imbalance of intestinal flora,as well as the integrity ofintestinal epithelial damage can not be immediately improved.Sijunzitang andapplications can be in the promotion of small bowel obstruction after the lifting of theexistence of dysfunction of the immune barrier.Reflected in the intervention groupSijunzitang more natural restoration of the PP group Vallarta leaching faster return tonormal group,and may be part of the intestinal effects of the distribution or thenumber of lymphocytes with a regulatory role.Obstruction after the lifting of thesubstance of the spleen and stomach function may be covered by the intestinalepithelial dysfunction,as well as the immune system,intestinal disorders,and the roleof Sijunzitang strengthening spleen's target is likely to include the intestinal immunesystem opsonization.
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