ICC与炎症细胞因子改变在PI-IBS中的作用

发布时间：2018-05-23 08:51

  本文选题：Cajal间质细胞 + 炎症细胞因子　； 参考：《重庆医科大学》2014年硕士论文

【摘要】：目的 本实验主要利用旋毛虫感染C57BL\6小鼠建立PI-IBS动物模型，对该模型的内脏敏感性和肠道动力进行评价，检测各肠段中c-kit蛋白、IL-1β、IL-10、IL-17和IFN-γ表达，分析c-kit蛋白与炎症因子的关系，初步探讨肠道ICC与炎症细胞因子改变在PI-IBS发病机制中的作用。 方法 1. PI-IBS小鼠模型的建立：34只雌性C57BL/6小鼠随机分为PI-IBS组（17只）和对照组（17只），PI-IBS组用0.2ml含400条旋毛虫幼虫的生理盐水悬液灌胃感染，对照组给予等量的生理盐水。 2.苏木精-伊红染色（hematoxylin-eosin staining, HE staining）：感染后第14天、28天和56天小鼠的十二指肠、空肠、末端回肠和近端结肠组织做病理学检查，观察炎症改变。 3.内脏敏感性和肠道动力评价:对感染第56天小鼠进行结直肠扩张（colorectal distention，CRD），观察小鼠的腹壁撤退反射（abdominalwithdrawal reflex，AWR）并进行评分；检测小鼠肠道传输时间（intestinetransmit time，ITT）并统计每2小时的粪便粒数、Bristol评分、湿/干重和含水百分数。 4.炎症细胞因子和c-kit蛋白表达的检测:用酶联免疫吸附测定（enzyme linked immunosobrent assay，ELISA）检测不同肠段组织蛋白匀浆液中IL-1β、IL-10、IL-17和IFN-γ的表达水平；用免疫组织化学染色检测不同肠道组织中c-kit蛋白表达的部位，并对c-kit蛋白染色程度和c-kit蛋白阳性细胞比例进行综合评分；用western blotting法对不同肠道组织的蛋白匀浆液中c-kit蛋白进行半定量分析。 5.炎症因子和c-kit信使核糖核酸(message ribonucleic acid, mRNA)检测：通过反转录聚合酶链式反应（reverse transcription polymerasechain reaction, RT-PCR）检测各肠道组织中c-kitmRNA、IL-1βmRNA、IL-10mRNA、IL-17mRNA和IFN-γmRNA的表达。 结果 1. PI-IBS组小鼠的十二指肠、空肠、末端回肠和近端结肠组织在旋毛虫感染后第14天存在显著的炎症细胞浸润和间质水肿，第14天到28天炎症和水肿逐渐减轻，至第56天基本恢复正常。 2.当CRD的空气容量为0.35ml和0.5ml时，PI-IBS组小鼠的AWR评分明显高于对照组小鼠，，且差异具有统计学意义（P0.01）。与对照组小鼠比较，PI-IBS组的ITT明显短于对照组（P0.01）；每2小时的粪便粒数、Bristol评分、湿重和含水百分数均高于对照组（P0.05）,两组小鼠粪便干重差别无统计学意义（P0.05）。 3.与对照组小鼠比较，PI-IBS小鼠的IFN-γ和IL-17在十二指肠和回肠组织中表达升高(P0.05);IL-10在空肠、回肠和结肠组织中的表达均降低（P0.05），c-kit在十二指肠、空肠、回肠和结肠组织中的表达均升高（P0.05），IL-1β在各肠段组织中表达的差异无统计学意义。 4.免疫组织化学染色提示c-kit阳性信号主要分布在肠道组织的固有层和肌肉层。PI-IBS小鼠各肠段组织染色程度和c-kit阳性细胞比例的综合评分明显高于对照小鼠（P0.05）。 结论 1.旋毛虫感染C57BL\6小鼠能较好的模拟PI-IBS内脏高敏感性和肠道动力改变，可作为PI-IBS动物模型。 2. PI-IBS小鼠肠道组织的ICC数量和c-kit蛋白表达量的改变可能与内脏敏感性和肠道动力改变相关。 3. PI-IBS小鼠肠道组织中炎症因子的改变可能与ICC改变有关，参与PI-IBS的发病机制。
[Abstract]:Purpose

In this experiment , we established a PI - IBS animal model by using Trichina spiralis . The visceral sensitivity and intestinal motility of the model were evaluated . The expression of c - kit protein , IL - 1尾 , IL - 10 , IL - 17 and IFN - 纬 in various intestinal segments was detected . The relationship between c - kit protein and inflammatory factor was analyzed .

method

1 . Establishment of the model of PI - IBS mice : 34 female C57BL / 6 mice were randomly divided into PI - IBS group ( 17 rats ) and control group ( 17 rats ) . The PI - IBS group was infected with 0.2 ml physiological saline suspension containing 400 spiralis larvae , and the control group gave equivalent normal saline .

2 . Hematoma - eosin staining ( HE staining ) : The duodenum , jejunum , distal ileum and proximal colon tissues of 14 , 28 , and 56 days post - infection were examined by pathology to observe the changes of inflammation .

3 . Visceral sensitivity and intestinal motility evaluation : colorectal distension ( CRD ) was performed on 56 day - infected mice , and the abdominal wall withdrawal reflex ( AWR ) of mice was observed and scored ;
Mice intestinal transit time ( ITT ) was tested and the number of fecal pellets , Bristol score , wet / dry weight , and water content per 2 hours were counted .

4 . Detection of inflammatory cytokines and c - kit protein expression : The levels of IL - 1尾 , IL - 10 , IL - 17 and IFN - 纬 were detected by enzyme linked immunoadsorption assay ( ELISA ) .
the expression of c - kit protein in different intestinal tissues was detected by immunohistochemical staining , and the degree of c - kit protein staining and the proportion of c - kit protein positive cells were scored ;
The c - kit protein was semi - quantitatively analyzed by western blotting .

5.鐐庣棁鍥犲瓙鍜宑-kit淇′娇鏍哥硸鏍搁吀(message ribonucleic acid, mRNA)妫

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