VFA对亚急性瘤胃酸中毒肉牛瘤胃上皮细胞炎性信号通路的影响

发布时间：2018-08-10 18:54

【摘要】：亚急性瘤胃酸中毒（SARA）是由于反刍动物摄取大量快速发酵的碳水化合物，使瘤胃VFA蓄积，pH降低，瘤胃菌群紊乱所引发的一种以发病隐袭为临床特征的群发性消化障碍性疾病。瘤胃上皮是机体防御病原微生物感染的第一道防线也是营养物质吸收转化的重要器官。虽然临床研究表明，SARA与瘤胃炎、肝脓肿、蹄叶炎和后腔静脉栓塞综合征等炎性疾病的发生有密切关系。但瘤胃内容物对瘤胃壁的局部炎性作用和全身的炎性作用还不清楚，其炎性信号通的研究还未见报道。本实验采用体内体外实验相结合的研究策略。体内实验：检测瘤胃液VFA和血液相关炎性指标IL-1β、IL-6、TNF-α含量，结合瘤胃组织学形态学，结果显示SARA与瘤胃组织的炎性损伤相关。体外实验：建立体外犊牛瘤胃上皮细胞培养方法。用不同低pH培养基处理细胞，分别添加不同浓度的VFA处理细胞。运用Western Blot和RT-PCR技术研究其对NF-κB、p38炎性通路及其下游炎性因子的影响。建立瘤胃上皮细胞培养方法，使用连续胰酶消化法分离细胞。然后对细胞进行生化和形态学的鉴定。细胞阳性表达细胞角蛋白CK-18，形态学上呈典型的铺路石状，超微结构形态具有典型的微绒毛、桥粒、紧密连接等上皮特征，在第4天细胞增殖活性和乳酸脱氢酶活性最强。可以作为细胞模型研究瘤胃的生理、病理分子生物学。体外培养犊牛瘤胃上皮细胞,研究低pH环境对瘤胃上皮细胞炎性因子表达的影响。在不同pH（5.5和7.2）的培养环境下处理细胞，检测NF-κB p65和IκB蛋白的表达与细胞炎性因子IL-1β、IL-6、TNF-α和NF-κB p65mRNA表达。结果显示，在低pH环境（pH5.5）处理组，NF-κB p65和IκB蛋白的表达显著升高；IL-1β、IL-6、TNF-α和NF-κBp65的mRNA表达水平均显著或极显著高于对照组(P0.05/P0.01)。为了进一步验证不同浓度VFA对瘤胃上皮细胞炎性信号通路的作用。设置处理组：0，×1倍浓度，，×5倍浓度VFA，PDTC抑制剂20mM，PDTC抑制剂20mM+×5倍浓度VFA，SB203580，SB203580+×5倍浓度。结果显示，高浓度VFA活化NF-κB、p38炎性通路及其下游炎性因子。牛SARA对瘤胃上皮细胞炎性信号通路的活化，不仅受低pH的影响，还受VFA浓度的影响，说明SARA的致炎作用是低pH和高浓度VFA两因素共同作用的结果。
[Abstract]:Subacute rumen acidosis (SARA) is a group of digestive disorders caused by rapid uptake of carbohydrates by ruminants, which reduces rumen VFA accumulation and pH, and caused by rumen microbial disturbance. Rumen epithelium is not only the first line of defense against pathogenic microorganism infection, but also an important organ for nutrient absorption and transformation. Although clinical studies have shown that sara is closely associated with inflammatory diseases such as rumen gastritis, liver abscess, hoofleaf disease and posterior vena cava embolism syndrome. However, the local and systemic inflammatory effects of rumen contents on rumen wall are not clear, and the study of inflammatory signaling has not been reported. In this experiment, the research strategy of in vivo and in vitro experiment was adopted. In vivo experiment: the contents of VFA in rumen fluid and IL-1 尾 -IL-6 TNF- 伪 in blood were detected. The results showed that SARA was related to the inflammatory injury of rumen tissue. In vitro experiment: to establish the culture method of rumen epithelial cells of calf in vitro. The cells were treated with different low pH medium and different concentrations of VFA. The effects of Western Blot and RT-PCR on NF- 魏 B p38 inflammatory pathway and its downstream inflammatory factors were studied. The culture method of rumen epithelial cells was established and the cells were isolated by trypsin digestion. Then the cells were identified by biochemical and morphological methods. The cytokeratin (CK-18) expressed in the cells was characterized by typical paving stone morphology and typical microvilli desmosomes and tight junctions. On the 4th day the cell proliferation activity and lactate dehydrogenase activity were the highest. It can be used as a cell model to study the physiological and pathological molecular biology of rumen. To study the effect of low pH on the expression of inflammatory factors in rumen epithelial cells of calf in vitro. The expression of NF- 魏 B p65 and I 魏 B protein and the expression of cytokine IL-1 尾 -IL-6, TNF- 伪 and NF- 魏 B p65mRNA were detected under different pH (5.5 and 7.2) culture conditions. The results showed that the expression of NF- 魏 B p65 and I 魏 B protein in low pH environment (pH5.5) was significantly higher than that in control group (P0.05/P0.01). To further verify the effects of different concentrations of VFA on the inflammatory signaling pathway of rumen epithelial cells. In the treatment group, the concentration of VFAPDTC inhibitor 20mM 脳 5 times, the concentration of 1: 1, and the concentration of VFA-PDTC inhibitor 20mMmMtc were 20 mMTC 脳 5, VFASB203580, SB203580 脳 5 脳 5 times, respectively. The results showed that high concentration of VFA activated NF- 魏 B p38 inflammatory pathway and its downstream inflammatory factors. The activation of inflammatory signaling pathway in rumen epithelial cells by bovine SARA was affected not only by low pH but also by the concentration of VFA, indicating that the inflammatory effect of SARA was the result of both low pH and high concentration of VFA.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.23

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