【作者】 陈胜；

【导师】 邹开芳；

【作者基本信息】 华中科技大学 ， 内科学， 2007， 硕士

【摘要】 第一部分实验性结肠炎大鼠结肠黏膜TLR2、TLR4、TLR9的表达目的:观察TLR2、TLR4、TLR9在实验性结肠炎大鼠结肠组织中的表达,探讨TLR2、TLR4、TLR9在IBD发病机制中的作用。方法:三硝基苯磺酸(TNBS)+乙醇灌肠制备实验性结肠炎大鼠模型,肉眼及光镜下观察黏膜损伤程度及组织学变化;免疫组化S-P法观察TLR2、TLR4、TLR9的表达,RT-PCR检测其mRNA相对含量;紫外分光光度法、黄嘌呤氧化酶法分别检测组织中MPO、SOD含量的变化。结果:造模后结肠固有层及黏膜下层可见大量中性粒细胞、部分淋巴细胞和少许嗜酸性粒细胞浸润,并可见隐窝脓肿、肉牙组织及黏膜肌层增厚;与正常组相比,模型组肠黏膜MPO含量显著上升(P<0.01),SOD含量明显下降(P<0.01);正常组TLR2、TLR4在黏膜下层及固有层炎性细胞胞膜和胞浆上有少量表达,TLR9未见表达,造模后肠组织TLR2、TLR4、TLR9表达明显增加(P均<0.01),且见TLR2表达于肠上皮近肠腔侧胞膜,TLR4表达于肠上皮近肠腔侧胞膜及腺上皮近腺腔侧胞膜;RT-PCR检测三者mRNA相对含量增高,而正常对照组三者均未能检出;TLR2、TLR4、TLR9表达与MPO呈正相关,与SOD呈负相关(P均<0.05)。结论:TNBS+乙醇模型实验性结肠炎大鼠TLR2、TLR4、TLR9表达增强,可能与肠道的自我免疫损伤有关。第二部分不同微生态制剂应用于实验性结肠炎大鼠TLR2、TLR4、TLR9表达变化目的:观察美常安、丽珠肠乐应用于实验性结肠炎大鼠后TLR2、TLR4、TLR9表达的变化,探讨其作用机制,并与柳氮磺胺吡啶(SASP)比较。方法:三硝基苯磺酸(TNBS)+乙醇灌肠制备实验性结肠炎大鼠模型后,美常安、丽珠肠乐及SASP每日灌胃一次,10天后处死,肉眼及光镜下观察黏膜损伤程度及组织学变化;免疫组化S-P法观察TLR2、TLR4、TLR9的表达,RT-PCR检测其mRNA相对含量;紫外分光光度法、黄嘌呤氧化酶法分别检测组织中MPO、SOD含量的变化。结果:美常安及SASP治疗后肉眼及组织学变化好转,与模型组相比差异具有统计学意义(P均<0.01),TLR2、TLR4、TLR9阳性率下降,其mRNA含量降低,与模型组相比差异具有统计学意义(P均<0.05),肠黏膜MPO含量下降,SOD含量上升,与模型对照组相比差异均具有统计学意义(P均<0.01)。美常安及SASP两组之间上述指标均无统计学意义。丽珠肠乐治疗后上述改变与模型组相比均不具有统计学意义。TLR2、TLR4、TLR9表达与MPO呈正相关,与SOD呈负相关,(MPO: r = 0.515, 0.386, 0.537; P均< 0.01; SOD: r =-0.523, -0.474, -0.566; P均< 0.01)。结论:美常安及SASP应用于实验性结肠炎模型大鼠有效,可能于下调TLR2、TLR4、TLR9的表达有关。更多还原

【Abstract】 PARTⅠThe expression of TLR2、TLR4、TLR9 in rats with colitisObjective To investigate the expression of TLR2、TLR4 and TLR9 in the colon mucosa of rats with colitis.Methods The rat colitis model was induced by the combined enema of trinitrobenzene sulphonic acids (TNBS) and ethanol. The disease activity index (DAI)、colon mucosa damage index (CMDI) and histological change of the rats were observed and evaluated. The superoxide dimutase (SOD) and activity of myeloperoxidase (MPO) were measured by biochemical method. The expression of TLR2、TLR4 and TLR9 were detected by immunohistochemistry, and the mRNA were detected by RT-PCR.Results As compared with the normal group, the DAI, CMDI, and the activity of MPO in the colon tissues of the rats in the model group was significantly increased(all P<0.01), while the activity of SOD was decreased(P<0.01). The expression of TLR2、TLR4 and TLR9 in the rat colon mucosa were significantly increased(all P<0.01), and the mRNA were significantly increased (all P<0.01). The expression of TLR2、TLR4 and TLR9 in rat colon mucosa were positively related with MPO and SOD(all P<0.05).Conclusion The expression of TLR2、TLR4 and TLR9 were significantly increased in the colon mucosa of rats induced by TNBS and ethanol. This may be related with the autoimmune impair of the gut. PARTⅡEffects of different probiotics on TLR2、TLR4 and TLR9 in rats with colitisObjective To investigate the effect of different probiotics on the expression of TLR2、TLR4 and TLR9 in the colon mucosa of rats with colitis.Methods The rat colitis model was induced by the combined enema of trinitrobenzene sulphonic acids(TNBS) and ethanol. The experimental animals were randomly divided into five group: normal group, model group, Meichang’an (praeparatum of viable organism of enterococci and bacillus subtilis)group, sulfasalazine(SASP)group and Lizhuchangle (praeparatum of viable organism of bacillus bifidus)group. The Saline, Meichang’an , SASP and Lizhuchangle were administered by gastric irrigation daily, Respectively, from 24 hours after the establishment of model to the end of experiment. The disease activity index (DAI)、colon mucosa damage index (CMDI) and histological change of the rats were observed and evaluated. The superoxide dimutase (SOD) and activity of myeloperoxidase (MPO) were measured by biochemical method. The expression of Toll-like receptor 2(TLR2)、Toll-like receptor 4(TLR4) and Toll-like receptor 9(TLR9) were detected by immunohistochemistry, and their mRNA were detected by RT-PCR.Results As compared with the model group, the DAI, CMDI, histological change and the activity of MPO in the colon tissues of the rats in the Meichang’an group and SASP group were significantly decreased(all P<0.01), while the activity of SOD was significantly increased(all P<0.01). The expression of TLR2、TLR4、TLR9 in the rat colon mucosa were significantly increased , either their mRNA(all P<0.05). And compared with the model group, all these items in Lizhuchangle group didn’t change significantly. The expression of TLR2、TLR4 and TLR9 in rat colon mucosa was positively related with MPO and SOD(MPO: r = 0.515, 0.386, 0.537; all P< 0.01; SOD: r =-0.523, -0.474, -0.566; all P< 0.01).Conclusion Meichang’an and SASP has protective effect against rat model with colitis, and their mechanism may be related with the inhibition of TLR2、TLR4 and TLR9.更多还原