【作者】 肖倩；

【导师】 孙晓宁；

【作者基本信息】 南华大学 ， 内科（专业学位）， 2015， 硕士

【摘要】 目的:探讨Runx3、TGF-β1及树突状细胞与肠易激综合征(Irritable bowel syndrome,IBS)发病中的相关性,进而为IBS的发病机制提供线索。方法:通过乙酸灌肠加束缚应激方法建立肠易激综合征大鼠模型。从每只大鼠中提取一小段结肠在电镜观察树突状细胞的形态。然后运用密度梯度离心法分离出大鼠结肠粘膜固有层的树突状细胞,分离出来的树突状细胞在体外培养12天后再经过免疫磁珠分选器分选。再运用siRNA技术转染RUNX3基因启动子发卡样特异性干扰寡核苷酸序列(shRNA-RUNX3)至分离出的树突状细胞。继续培养7天。分别进行流式细胞仪检测DC表型、运用Western blotting检测TGF-β1及Runx3蛋白的表达情况。结果:1、在不同扩张容量下,比较两组大鼠的AWR评分及腹壁肌电活动,发现模型组大鼠AWR评分较对照组明显增加,提示其内脏敏感性增加(P<0.01)。2、大鼠派氏结DC表面OX62的表达率IBS模型组大鼠明显高于正常对照组的(P<0.05)。3、培养的树突状细胞通过倒置荧光镜观察符合DC的结构特点。4、正常组DC的表面分子OX62、CD80、CD86、MHC-II的表达率均明显低于IBS组,但转染组与未转染组之间无明显差异。5、Western blotting结果显示转染组Runx3蛋白的表达量较未转染组明显减少。转染组与未转染组TGF-β1蛋白表达量没有明显差异。结论:1、利用乙酸灌肠加束缚应激的方法来建成的IBS动物模型是符合IBS的内脏敏感性机制的,这非常适用于关于IBS的试验研究。2、IBS的发生与发展可能有树突状细胞的参与。3、TGF-β1和Runx3在肠易激综合征发病中存在的作用有待进一步研究。4、Runx3基因有可能通过其他传导途径来影响树突状细胞在肠易激综合征中的作用。更多还原

【Abstract】 Objective: We are for the purpose of studying the correlation among the Runx3, TGF-β1 and dendritic cells in the paroxysm of IBS and it provides clues to the pathogenesis of IBS.Methods:We set up the model of IBS through the method of intracolonic instillation of acetic acid and restraint stress. The morphology of dendritic cells in a piece of colon obtained from each rats were observed through transmission electron microscope(TEM). Afterwards, the dendritic cell of the rat colon mucosa lamina propria isolated by the means of density gradient centrifugation was cultivated in vitro for 12 days and chosen by MACS Separators. Then RUNX3 short hairpin RNAs(sh RNA-RUNX3) were transfected by si RNA technology and the dendritic cells were isolated. Finally, after the cells were cultivated for 7 days, the DC phenotype detection was performed by the flow cytometry and the expression of TGF-β1 and Runx3 protein were tested by western blotting.Results: 1. Under different expansion capacity, the AWR score and the electroactivity of abdominal muscle were compared between the model group and normal group. And the model group has remarkably higher AWR score. It demonstrates that the sensibility of viscera has enhanced. 2.The expression rate of OX62 on the surface of peyer’s pathes in the IBS model group is obviously higher than that of normal group. 3. The dendritic cell observed by inverted fluorescence mirror is accord with the structural characteristics of DC.4. The OX62, CD80, CD86, MHC-II on the surface of DC of normal group have lower expression rate than that of IBS group. However, there is no difference between transfection group and non-transfection group.5. The results of western blotting demonstrate that the expression amount of Runx3 protean in transfection group has remarkably reduced than that of non-transfection group. And there is no obvious difference between the TGF-β1 expression rate of transfection group and non-transfection group.Conclusions: 1. Rats model of IBS by intracolonic instillation of acid and restraint stress and single restraint stress methods are accord with visceral hypersensitivity of IBS, this is practicable for experimental study.2. The increase of abnormal dendritic cells might influence the occurrence and development of irritable bowel syndrome patients.3. Runx3 and TGF- β1 needs to be further studies in irritable bowel syndrome patients.4. TGF-β1 signal pathway might not be involved in Runx3-regulated maturation of dendritic cells in irritable bowel syndrome patients.更多还原