血浆高分子量激肽原在炎症性肠病发病中的作用及机制的初步研究

发布时间：2018-05-20 06:04

本文选题：高分子量激肽原 + 缓激肽　；参考：《苏州大学》2014年硕士论文

【摘要】：【研究背景和意义】血浆高分子量激肽原（High-molecular-weight kininogen，HK）是血浆激肽释放酶-激肽系统（kallikrein-kinin system，KKS）的主要成员之一。临床研究表明，IBD（inflammatory bowel disease）患者体内血浆KKS被活化，HK被裂解，说明HK参与IBD的发病过程。但是，迄今对于HK在IBD发病中的确切作用和机制并不清楚。【研究目的】研究血浆高分子量激肽原（HK）在葡聚糖硫酸钠（Dextran sulfate sodium，DSS）诱导的急性结肠炎中的作用，并初步探究其作用机制。【研究方法】用含2.5%DSS的饮用水饲喂小鼠，构建急性结肠炎模型。通过检测体重百分比，疾病活动指数，结肠长度与肠系膜淋巴结的湿重比较野生型小鼠和Kng1基因敲除小鼠发病程度。利用ELISA方法检测血浆缓激肽（BK），结肠组织中炎症因子与髓过氧化物酶（MPO）的含量。应用实时定量PCR检测结肠组织炎症因子的mRNA水平。分离小鼠结肠粘膜固有层细胞，通过特异性的抗体标记后用流式细胞仪检测细胞悬液中中性粒细胞所占的比例。制作末端结肠的石蜡切片，通过HE染色观察结肠组织病理变化。【研究结果】（1）Kng1基因敲除小鼠肝脏中没有Kng1mRNA的表达，并且血浆中检测不到HK蛋白，说明Kng1基因敲除小鼠模型建立成功。（2）DSS处理的WT小鼠血浆BK水平升高。（3）从day0起，给予小鼠DSS处理。与对照组小鼠相比，DSS处理组小鼠从day4开始体重逐渐下降。与WT小鼠相比，DSS处理的Kng1-/-小鼠体重下降程度（day5: p=0.0343; day6: p=0.024; day7: p=0.0248; day8: p=0.019）和疾病活动指数（day7: p=0.0331; day8：p=0.0039）明显降低，其结肠缩短程度明显减轻（p=0.021），而且其肠系膜淋巴结的湿重明显低于WT小鼠（p=0.0002）。（4）与野生型发病小鼠相比，DSS处理的Kng1-/-小鼠结肠组织中炎症因子的蛋白水平明显降低（TNF-α：p=0.032；IL-6：p=0.0083；IL-1β：p=0.0143；INF-γ：p=0.0012)，其结肠组织中炎症因子的mRNA水平明显降低（TNF-α：p=0.083；IL-6：p=0.0031；IL-1β：p=0.0036；INF-γ：p=0.024）（5）与野生型发病小鼠相比，DSS处理的Kng1-/-小鼠结肠组织研磨液中髓过氧化酶的含量显著降低（p=0.001），，其结肠粘膜固有层中性粒细胞的数目显著降低（p=0.002）。（6）野生型发病小鼠结肠粘膜上皮溃疡，大量炎性细胞浸润，隐窝脓肿，杯状细胞消失。与之相比，DSS处理的Kng1基因敲除小鼠结肠组织损伤与病变程度明显缓解，结肠粘膜上皮仅可见部分破损，少量炎症细胞浸润，部分杯状细胞破损，组织切片的病理评分低于WT小鼠（p=0.0026）。【研究结论】血浆高分子量激肽原在炎症性肠病的发病中起重要的促进作用。
[Abstract]:[background and significance of the study] High-molecular-weight kininogeny (HKK) is one of the major members of the plasma kallikrein-kinin system (KKS). Clinical studies showed that plasma KKS was activated by HK in patients with inflammatory bowel disease, which indicated that HK was involved in the pathogenesis of IBD. However, the exact role and mechanism of HK in the pathogenesis of IBD is unclear. [purpose of research] To study the role of plasma high molecular weight kallikrein (HKHK) in acute colitis induced by dextran sodium sulfate (Dextran sulfate sodium), and to explore its mechanism. [research methods] Acute colitis model was established by feeding mice with drinking water containing 2.5%DSS. The percentage of body weight disease activity index colon length and wet weight of mesenteric lymph nodes were compared between wild-type mice and Kng1 knockout mice. The content of inflammatory factors and myeloperoxidase (MPO) in colon tissue and plasma bradykinin (BK) were determined by ELISA method. Real-time quantitative PCR was used to detect the mRNA level of inflammatory factors in colon tissue. Murine colonic mucosal lamina propria cells were isolated and the proportion of neutrophils in suspension was detected by flow cytometry after specific antibody labeling. The paraffin sections of the terminal colon were made and the pathological changes of the colon were observed by HE staining. [results of research] There was no Kng1mRNA expression in the liver of Kng1 knockout mice, and no HK protein was detected in plasma, indicating that the plasma BK level of WT mice treated with Kng1 gene knockout mice was higher than that of WT mice treated with DSS. Compared with the control group, the mice in DSS group gradually lost weight from day4. Compared with WT mice, the weight loss degree of Kng1-r- mice treated with DSS was significantly lower than that of WT mice (day5: p0. 034; day6: p0. 024; day7: p0. 024; day8: p0. 019), and the disease activity index was 0. 033 1; day8: p0. 0039). Compared with wild-type mice treated with DSS, the level of inflammatory factor protein in colonic tissue of DSS treated with DSS significantly decreased the protein level of inflammatory factor in colonic tissue of TNF- 伪 -03032 IL-6: p0.0083IL-1 尾: p0.0143INF- 纬 -P0.0012.The colonic shortening degree of TNF- 伪 decreased significantly the wet weight of mesenteric lymph nodes in WT mice compared with that of wild-type mice treated with DSS, and the protein level of inflammatory factors in colonic tissue of mice treated with DSS was significantly lower than that of wild-type mice. MRNA level of inflammatory cytokines in intestinal tissue decreased significantly (TNF- 伪: 0. 083) IL-6: P0. 0031, IL-1 尾: p0. 0036 + INF- 纬: p0. 024. 5) compared with wild-type mice, the content of myeloperoxidase in the colonic tissue grinding fluid treated with DSSs significantly decreased the number of neutrophils in the mucosal lamina propria of the colonic mucosa, and the number of neutrophilic granulocytes in the mucosal lamina propria of the colonic mucosa. The colonic mucosal epithelium ulcer of wild-type mice was decreased significantly. A large number of inflammatory cell infiltration, crypt abscess, goblet cells disappear. Compared with the Kng1 gene knockout mice treated with DSS, the degree of colonic tissue injury and lesion was significantly alleviated. The colonic mucosal epithelium was only partially damaged, a small number of inflammatory cells infiltrated, and some goblet cells were damaged. The pathological score of histopathological sections was lower than that of WT mice. [conclusions of the study] Plasma high molecular weight kallikrein plays an important role in the pathogenesis of inflammatory bowel disease.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R574.62

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