【作者】 付妤；

【导师】 侯晓华；

【作者基本信息】 华中科技大学 ， 内科学， 2009， 博士

【摘要】 第一部分：溃疡性结肠炎患者肠粘膜和外周血中Th细胞表型的改变目的：探讨Th细胞各亚群比例在溃疡性结肠炎发病中的变化及意义。方法：按照2007年济南标准收集UC患者20例，对照组16例。利用细胞内细胞因子染色和四色荧光抗体流式细胞术对UC组患者及对照组中肠粘膜和外周血淋巴细胞作表型分析，比较各组肠粘膜和外周血中Th1、Th2、Th17比例改变，Western blot检测各组肠粘膜IL-4、IL-12、IL-17表达，ELISA检测各组外周血IL-4、IL-12、IL-17水平。结果：（1）UC组患者肠粘膜中Th17比例和IL-17表达较对照组明显增加：Th17比例在UC组和对照组分别为3．75（6．93）vs．1．25（3．70），P＜0．05；IL-17表达在UC组和对照组分别为0．20（0．15）vs．0．10（0．03），P＜0．05。UC组患者肠粘膜中IL-17表达与疾病评分呈正相关（r=0．503，P=0．024）。UC中、重度患者外周血中Th17比例和IL-17表达增加：外周血中Th17比例在UC中、重度患者和对照组中分别为1．40（2．15）vs．0．70（0．33），P＜0．05；外周血中IL-17含量在UC中、重度患者和对照组中分别为1．58（3．37）pg·ml^-1vs．0．19（0．64）pg·ml^-1，P＜0．05。（2）肠粘膜中Th1的比例在UC和对照组中分别为13．6（16．88）vs．9．10（17．53）；外周血中Th1的比例在UC和对照组中分别为12．85（9．28）vs．10．15（7．48），UC组与对照组相比肠粘膜和外周血中Th1比例差异均无统计学意义，且不同活动度患者间无明显差异。肠粘膜中IL-12的表达在UC组和对照组中分别是0．22（0．16）vs．0．16（0．13），表达无明显改变。外周血中IL-12水平在UC组与对照组中分别是1．59（2．26）pg·ml^-1vs．1．66（5．79）pg·ml^-1，两组之间差异无统计学意义。（3）肠粘膜中Th2的比例在UC和对照组中分别为1．10（1．53）vs．1．15（1．50）；外周血中Th2的比例在UC和对照组中分别为1．20（0．55）vs．1．10（0．45），肠粘膜和外周血中差异均无统计学意义，不同活动度患者间差异无显著性。肠粘膜IL-4在UC组和对照组中分别是0．29（0．14）vs．0．23（0．11），表达无明显改变。外周血IL-4水平在UC组与对照组分别是1．27（2．48）pg·ml^-1vs．0（2．5）pg·ml^-1，两组之间差异无统计学意义。结论：在Th细胞各亚群中Th17细胞是介导UC肠道局部和外周免疫应答的优势细胞，它可能成为UC治疗的有效靶点。第二部分：腹泻型IBS患者肠粘膜和外周血中Th细胞表型的改变目的：探讨Th细胞各亚群在腹泻型IBS患者中的变化，揭示IBS粘膜免疫激活具体免疫学机制。方法：按照罗马Ⅲ标准收集腹泻型IBS患者27例，另设对照组16例。采用细胞内细胞因子染色和四色荧光抗体流式细胞术检测各组肠粘膜和外周血中Th1／Th2／Th17比例，Western blot检测肠粘膜IL-4、IL-12、IL-17表达，ELISA检测外周血IL-4、IL-12、IL-17水平。结果：（1）腹泻型IBS患者部分肠粘膜病理学表现为非特异性炎症，归为IBS-A组，肠粘膜无非特异性炎症者归为IBS组。（2）IBS-A组患者肠粘膜Th17比例较对照组明显增加，为3．60（4．05）vs．1．25（3．70），P=0．045，而Th1、Th2比例无明显改变；肠粘膜中相关细胞因子IL-4、IL-12、IL-17表达与对照组比较差异无统计学意义。IBS-A组外周血中Th1、Th2、Th17比例和对照组比较差异无统计学意义。外周血中相关细胞因子IL-4、IL-12、IL-17水平与对照组比较也无明显改变。（3）IBS组患者肠粘膜及外周血Th细胞比例和相关细胞因子表达均无明显改变。结论：腹泻型IBS患者部分存在肠粘膜非特性炎症，提示肠粘膜存在免疫激活，该免疫状态表现为Th细胞向Th17偏移。第三部分：Th17细胞在旋毛虫感染小鼠内脏高敏感性中的作用目的：研究发现旋毛虫感染小鼠在急性感染期会出现肠道功能紊乱，在肠道炎症消退后肠道功能紊乱症状仍可持续存在。本实验旨在阐明Th17细胞及相关细胞因子在旋毛虫感染小鼠急性和慢性感染期期出现的内脏高敏感性中的作用。方法：实验分为对照组、感染后2周组、8周组和12周组。用含有旋毛虫的混悬液灌胃方法感染NIH小鼠，剂量为300条／只；结直肠扩张诱导的腹部回撤反射（AWR）评估内脏敏感性；HE染色观察空肠和结肠粘膜炎症状态；细胞内细胞因子染色检测空肠和结肠粘膜固有层Th17细胞比例；Wentern blot检测空肠和结肠粘膜IL-17、IL-23和TGF-β1蛋白表达。结果：旋毛虫感染造成的空肠和结肠一过性炎症在感染后2周时最明显，感染后8周时肠粘膜破坏和炎症反应基本消失，感染后12w时空肠和结肠粘膜无明显炎症改变。AWR评分在感染后2周时最高，感染后8周和12周时仍高于对照组水平（P＜0．05）。空肠和结肠粘膜固有层Th17细胞比例和IL-17表达在感染后2周时增高（P＜0．05），感染后8周和12周降至正常水平。感染后2周，感染组和对照组小鼠空肠粘膜Th17细胞比例分别是9．13±2．73 vs．3．78±1．97，P＜0．01；结肠粘膜中Th17细胞比例分别是8．00±2．43 vs．3．6±1．80，P＜0．01。感染后2周，感染组和对照组空肠中IL-17含量分别是1．70±0．47和0．68±0．26，P＜0．01；结肠中IL-17含量分别是0．59±0．12和0．44±0．09，P=0．041。2周时空肠和结肠粘膜IL-17表达与AWR评分呈正相关，相关系数分别是r=0．658，P=0．032和r=0．532，P=0．04。空肠和结肠粘膜TGF-β1表达在感染后2周时增加，8周和12周降至正常水平。感染后2周，空肠中TGF-β1含量在感染组和对照组分别是0．31±0．03和0．17±0．05，P＜0．01；结肠中TGF-β1含量在感染组和对照组分别是0．11±0．03和0．08±0．02，P=0．035。急、慢性感染期间空肠和结肠粘膜IL-23表达无明显改变。结论：旋毛虫感染NIH小鼠造成肠道一过性炎症，炎症急性期和慢性期小鼠内脏敏感性增加。Th17细胞的主要效应因子IL-17可能影响了急性期感染期小鼠内脏敏感性，TGF-β1可能诱导了Th17细胞的改变。更多还原

【Abstract】 PartⅠPhenotypic analysis of Th cells in the colon and peripheralblood in patients with ulcerative colitisObjective To analyse the proportion of Th1/Th2/Th17 in colonic mucosa and peripheralblood in ulcerative colitis in relation to healthy controls.Methods Colonic biopsy specimens and peripheral blood were obtained from controls(n=16) and patients with UC (n=20) .We used flow cytometric detection of intracellularIFN-γ/IL-4/ IL-17 cytokine production to investigate Th1,Th2 and Th17 cells in thecolonic lamina propria and peripheral blood.The levels of IL-12,IL-4 and IL-17 inintestinal mucosa and peripheral blood were explored by Western blot and ELISA,respectively.Results In colonic mucosa,the proportion of Th17 and the expression of IL-17increased in UC compared with controls (P＜0.05).The expression of IL-17 in the colonwas correlated with the disease related clinical parameters in UC patients (r=0.503,P=0.024).The frequency of Th17 in severely active UC was higher than in slightly activeUC in colonic mucosa (P＜0.05).In peripheral blood,the proportion of Th17 and the expression of IL-17 was upregulated in moderately and severely active UC (P＜0.05) Nodifferences were found in the proportions of Th1 or Th2 in colonic mucosa or peripheralblood between UC and controls.The contents of IL-4 and IL-12 in colonic mucosa orperipheral blood showed no differences between UC and controls.Conclusions In UC patients,Th17 is predominant of three Helper T-Cells both in colonicmucosa and peripheral blood.Th17 would be an effective target for treatment.PartⅡPhenotypic analysis of Th cells in the colon and peripheralblood in patients with diarrhea-predominant IBSObjective We analyse the change of Th1/Th2/Th17 in colonic mucosa and peripheralblood in diarrhea-predominant IBS (D-IBS) to uncover the mechanism underlying theactivation of mucosal immune system.Methods Colonic biopsy specimens and peripheral blood were obtained from controls(n=16) and patients with D-IBS (n = 27) .We used flow cytometric detection of intracellularIFN-γ/IL-4/ IL-17 cytokine production to investigate Th1,Th2 and Th17 cells in thecolonic lamina propria and peripheral blood.Western blot was used to determine theexpression of IL-12,IL-4 and IL-17 in colonic mucosa.The level of IL-12,IL-4 and IL-17in peripheral blood was detected by ELISA.Results Histological assessment of biopsy specimens from D-IBS patients indicated 2 different groups.One group (14 of 27) had normal conventional histology (IBS).Anothergroup (13 of 27) had nonspecific microscopic inflammation (IBS-A).In colonic mucosa,the proportion of Th17 increased in IBS-A compared with controls (P＜0.05),but not in IBS.In peripheral blood,a similar tendency was found although differences were not significant.No differences could be observed in the frequencies of Th1 and Th2 in colon mucosa andperipheral blood.The level of IL-12,IL-4 and IL-17 in IBS and IBS-A showed nodifferences both in colonic mucosa and in peripheral blood.Conclusions Subgroup of D-IBS showed abnormal conventional histology,implicating theactivation of mucosal immune system in pathogenesis.The shift of Th1/ Th2 / Th17balance in colon mucosa showed enhanced Th17 activity.PartⅢThe role of Th17 in visceral hypersensitivity in mice infectedwith Trichinella spiralisObjective Trichinella spiralis infection in rodents is a well-known model of intestinalinflammation associated with hypermotility.Our aim was to use this experimental model toelucidate if Th17 cells were involved in the development of gastrointestinal hypermotility.Methods Mice were divided into four groups;control,2 weeks post infection,8 weekspost infection and 12 weeks post infection.Inflammation in jejunum and colon was judgedby hematoxylin-eosin (H&E) staining.Visceral hypersensitivity was measured byabdominal withdraw reflex (AWR).The levels of IL- 17,IL-23 and TGF-β1 in jejunum and colon were detected by Western blot.Flow cytometric detection of intracellular IL-17cytokine production was used to analysis the proportions of Th17 cells subset in jejunumand colon.Results Our results showed that visceral hypersensitivity was increased at 2 weeks postinfection (PI) and kept higher at 8 and 12 weeks PI when no discernible inflammationpresented in the gut.The proportion of Th17 cells and the expression of IL-17 wereupregulated in jejunum and colon at 2 weeks PI and normalized at 8 weeks PI.The contentof IL-17 in jejunum and colon was correlated with the AWR at 2 weeks PI.Meanwhile,injejunum and colon TGF-β1 was increased at 2 weeks PI in jejunum and colon,while IL-23was in the normal condition.Conclusions Our data suggest that Th17 cells affected the visceral hypersensitivity inmice infected with Trichinella spiralis at intestine stage in support of TGF-β1,but not atmuscle stage when other cytokines might involved in the visceral hypersensitivity.更多还原