rhBD3通过Rho-ROCK信号通路干预新生儿坏死性小肠结肠炎的研究

发布时间：2018-05-06 12:39

本文选题：坏死性小肠结肠炎 + 人β-防御素3　；参考：《上海交通大学》2014年博士论文

【摘要】：目的评价重组人β-防御素3（rhBD3）对新生大鼠坏死性小肠结肠炎（NEC）模型的干预作用，并探讨Rho-ROCK信号通路和细胞内钙离子在rhBD3干预NEC中可能的分子机制。方法体外实验以人和大鼠肠上皮细胞Caco-2和IEC-6为研究对象，观察rhBD3对肠上皮细胞的毒性作用、诱导肠上皮细胞迁移和增生的作用（CCK-8、BrdU掺入实验、细胞周期检测、划痕实验和RTCA）。检测趋化因子受体CCR6在大鼠小肠和结肠中的表达情况。通过中和抗体、抑制剂和siRNA预处理观察CCR6介导rhBD3诱导肠上皮细胞迁移中的作用。并通过沉淀实验、细胞免疫荧光染色、Western Blot等方法检测rhBD3处理后细胞内钙离子浓度、RhoA的活性水平、MLC2磷酸化、以及F-actin的表达。然后联合人工喂养+缺氧+冷刺激作用于新生SD大鼠建立NEC模型，并通过肠道组织病理检查证实模型的可靠性和成功率。体内实验以新生大鼠NEC模型为研究对象，实验分组：A组为正常组（经胃管注入生理盐水），B组为正常rhBD3干预组（经胃管注入rhBD3），，C组为NEC模型组（经胃管注入生理盐水），D组为NEC模型rhBD3干预组（经胃管注入rhBD3），收集肠管和血液标本。利用real-time PCR、ELISA和免疫组织化学染色等方法检测肠壁组织和血清中炎症因子的表达、肠壁粘膜屏障的完整性、以及肠上皮细胞迁移和增生的情况。结果rhBD3在一定的pH范围内（pH5.8-pH8.5）仍保持抗菌活性，对肠上皮细胞无明显毒性作用，在体内和体外均能诱导肠上皮细胞的迁移，但是对增生无明显促进作用。趋化因子受体CCR6在新生大鼠和成年大鼠的小肠和结肠中稳定表达。rhBD3通过细胞跨膜受体CCR6，激活细胞内小分子GTP结合蛋白Rho（由Rho-GDP转变为Rho-GTP），通过下游效应分子Rho激酶磷酸化肌球蛋白轻链MLC2（Ser19），增加肠上皮细胞中纤维状肌动蛋白F-actin的表达，同时通过动员细胞内钙离子，调节细胞骨架的重组和细胞的迁移。在成功建立新生SD大鼠NEC模型的基础上，发现rhBD3对正常新生大鼠无明显影响，但能够维持NEC模型大鼠的体重、降低NEC发病率（P=0.039）、提高生存率（P0.001）、改善NEC疾病的严重程度（P=0.01）。其次，rhBD3可以降低肠壁组织和血清中炎症因子的表达、增加血清中DAO的水平、并增加紧密连接蛋白ZO-1的表达。结论小分子抗菌肽rhBD3能够通过趋化因子受体CCR6动员细胞内钙离子和激活Rho-ROCK信号通路，诱导肠上皮细胞的迁移。经胃管给予rhBD3对新生大鼠NEC模型具有一定的保护作用，其机制可能与降低炎症介质的表达、促进肠道粘膜屏障的修复以及保持肠道粘膜屏障的完整性有关。
[Abstract]:Objective to evaluate the effect of recombinant human 尾 -defensin 3 (rhBD3) on necrotizing enterocolitis (NEC) model in neonatal rats, and to explore the possible molecular mechanism of Rho-ROCK signaling pathway and intracellular calcium in rhBD3 intervention of NEC. Methods the Caco-2 and IEC-6 of human and rat intestinal epithelial cells were studied in vitro. The toxic effects of rhBD3 on intestinal epithelial cells were observed, and the effects of rhBD3 on the migration and proliferation of intestinal epithelial cells were observed. The expression of chemokine receptor CCR6 in small intestine and colon of rats was detected. The role of CCR6 in inducing intestinal epithelial cell migration induced by rhBD3 was observed by neutralizing antibody, inhibitor and siRNA pretreatment. The phosphorylation of MLC2 and the expression of F-actin in the cells treated with rhBD3 were detected by precipitation assay and immunofluorescence staining with Western Blot. Then the NEC model of neonatal SD rats was established by artificial feeding combined with hypoxia and cold stimulation, and the reliability and success rate of the model were confirmed by pathological examination of intestinal tissue. In vivo, the NEC model of newborn rats was used as the research object. The experimental group was divided into two groups: normal group (group B: normal rhBD3 intervention group, group C: NEC model group, group D: NEC model group, group D: NEC model rhBD3 intervention group, group B: injection of rhBD3N via gastric tube; Collect intestinal duct and blood samples. The expression of inflammatory factors in intestinal wall tissue and serum, the integrity of mucosal barrier of intestinal wall, and the migration and proliferation of intestinal epithelial cells were detected by real-time PCR Elisa and immunohistochemical staining. Results rhBD3 maintained antibacterial activity in a certain pH range (pH 5.8-pH 8.5), and had no obvious toxicity to intestinal epithelial cells. It could induce the migration of intestinal epithelial cells in vivo and in vitro, but had no obvious effect on proliferation. Stable expression of chemokine receptor CCR6 in the small intestine and colon of neonatal and adult rats. RhBD3 activates the intracellular small molecule GTP binding protein (from Rho-GDP to Rho-GTPN, via downstream effector molecule Rho kinase phosphorous) through the cellular transmembrane receptor CCR6. Acidified myosin light chain (MLC2) Ser19C increased the expression of fibronectin F-actin in intestinal epithelial cells. At the same time, the recombination of cytoskeleton and cell migration were regulated by mobilization of intracellular calcium ions. On the basis of successful establishment of NEC model of neonatal SD rats, it was found that rhBD3 had no obvious effect on normal neonatal rats, but it could maintain the weight of NEC model rats, reduce the incidence of NEC, increase the survival rate and improve the severity of NEC disease. Secondly, rhBD3 could decrease the expression of inflammatory factors in intestinal wall and serum, increase the level of DAO in serum, and increase the expression of tight junction protein ZO-1. Conclusion small molecule antimicrobial peptide rhBD3 can mobilize intracellular Ca ~ (2 +) and activate Rho-ROCK signaling pathway through chemokine receptor (CCR6) and induce the migration of intestinal epithelial cells. Gastric tube administration of rhBD3 has a protective effect on neonatal rat NEC model. The mechanism may be related to reducing the expression of inflammatory mediators, promoting the repair of intestinal mucosal barrier and maintaining the integrity of intestinal mucosal barrier.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R722.1

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