本文選題：壞死性小腸結(jié)腸炎 + 人β-防御素3��； 參考：《上海交通大學(xué)》2014年博士論文

【摘要】：目的評(píng)價(jià)重組人β-防御素3（rhBD3）對(duì)新生大鼠壞死性小腸結(jié)腸炎（NEC）模型的干預(yù)作用，并探討Rho-ROCK信號(hào)通路和細(xì)胞內(nèi)鈣離子在rhBD3干預(yù)NEC中可能的分子機(jī)制。 方法體外實(shí)驗(yàn)以人和大鼠腸上皮細(xì)胞Caco-2和IEC-6為研究對(duì)象，觀察rhBD3對(duì)腸上皮細(xì)胞的毒性作用、誘導(dǎo)腸上皮細(xì)胞遷移和增生的作用（CCK-8、BrdU摻入實(shí)驗(yàn)、細(xì)胞周期檢測(cè)、劃痕實(shí)驗(yàn)和RTCA）。檢測(cè)趨化因子受體CCR6在大鼠小腸和結(jié)腸中的表達(dá)情況。通過(guò)中和抗體、抑制劑和siRNA預(yù)處理觀察CCR6介導(dǎo)rhBD3誘導(dǎo)腸上皮細(xì)胞遷移中的作用。并通過(guò)沉淀實(shí)驗(yàn)、細(xì)胞免疫熒光染色、Western Blot等方法檢測(cè)rhBD3處理后細(xì)胞內(nèi)鈣離子濃度、RhoA的活性水平、MLC2磷酸化、以及F-actin的表達(dá)。然后聯(lián)合人工喂養(yǎng)+缺氧+冷刺激作用于新生SD大鼠建立NEC模型，并通過(guò)腸道組織病理檢查證實(shí)模型的可靠性和成功率。體內(nèi)實(shí)驗(yàn)以新生大鼠NEC模型為研究對(duì)象，實(shí)驗(yàn)分組：A組為正常組（經(jīng)胃管注入生理鹽水），B組為正常rhBD3干預(yù)組（經(jīng)胃管注入rhBD3），，C組為NEC模型組（經(jīng)胃管注入生理鹽水），D組為NEC模型rhBD3干預(yù)組（經(jīng)胃管注入rhBD3），收集腸管和血液標(biāo)本。利用real-time PCR、ELISA和免疫組織化學(xué)染色等方法檢測(cè)腸壁組織和血清中炎癥因子的表達(dá)、腸壁粘膜屏障的完整性、以及腸上皮細(xì)胞遷移和增生的情況。 結(jié)果rhBD3在一定的pH范圍內(nèi)（pH5.8-pH8.5）仍保持抗菌活性，對(duì)腸上皮細(xì)胞無(wú)明顯毒性作用，在體內(nèi)和體外均能誘導(dǎo)腸上皮細(xì)胞的遷移，但是對(duì)增生無(wú)明顯促進(jìn)作用。趨化因子受體CCR6在新生大鼠和成年大鼠的小腸和結(jié)腸中穩(wěn)定表達(dá)。rhBD3通過(guò)細(xì)胞跨膜受體CCR6，激活細(xì)胞內(nèi)小分子GTP結(jié)合蛋白R(shí)ho（由Rho-GDP轉(zhuǎn)變?yōu)镽ho-GTP），通過(guò)下游效應(yīng)分子Rho激酶磷酸化肌球蛋白輕鏈MLC2（Ser19），增加腸上皮細(xì)胞中纖維狀肌動(dòng)蛋白F-actin的表達(dá)，同時(shí)通過(guò)動(dòng)員細(xì)胞內(nèi)鈣離子，調(diào)節(jié)細(xì)胞骨架的重組和細(xì)胞的遷移。在成功建立新生SD大鼠NEC模型的基礎(chǔ)上，發(fā)現(xiàn)rhBD3對(duì)正常新生大鼠無(wú)明顯影響，但能夠維持NEC模型大鼠的體重、降低NEC發(fā)病率（P=0.039）、提高生存率（P0.001）、改善NEC疾病的嚴(yán)重程度（P=0.01）。其次，rhBD3可以降低腸壁組織和血清中炎癥因子的表達(dá)、增加血清中DAO的水平、并增加緊密連接蛋白ZO-1的表達(dá)。 結(jié)論小分子抗菌肽rhBD3能夠通過(guò)趨化因子受體CCR6動(dòng)員細(xì)胞內(nèi)鈣離子和激活Rho-ROCK信號(hào)通路，誘導(dǎo)腸上皮細(xì)胞的遷移。經(jīng)胃管給予rhBD3對(duì)新生大鼠NEC模型具有一定的保護(hù)作用，其機(jī)制可能與降低炎癥介質(zhì)的表達(dá)、促進(jìn)腸道粘膜屏障的修復(fù)以及保持腸道粘膜屏障的完整性有關(guān)。
[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.
【學(xué)位授予單位】：上海交通大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R722.1

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