本文關(guān)鍵詞： NEC 巨噬細(xì)胞 TNF-α ICCs NEC 巨噬細(xì)胞 氯膦酸二鈉脂質(zhì)體 TNF-α ICCs TNF-α miR-222 c-kit ICCs　出處：《華中科技大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分NEC患兒腸道巨噬細(xì)胞的活化、TNF-α的表達(dá)及ICCs的表型改變目的：研究NEC患兒腸道巨噬細(xì)胞的活化、TNF-a的表達(dá)及ICCs的表型改變情況,探究其在NEC發(fā)病中的作用。方法：采用HE染色、免疫熒光雙染、免疫組化、Westen blot 及 RT-PCR等技術(shù)對(duì)正常小腸組織和NEC小腸組織進(jìn)行對(duì)比研究。結(jié)果：HE染色—正常小腸絨毛排列整齊,腸壁無(wú)水腫及充血,而NEC患兒小腸絨毛壞死,腸壁充血,腸壁有大量炎性細(xì)胞浸潤(rùn)。免疫熒光雙染—正常小腸有少量CD68陽(yáng)性的巨噬細(xì)胞表達(dá),未見(jiàn)巨噬細(xì)胞活化,而NEC腸道有大量M1型巨噬細(xì)胞浸潤(rùn),未見(jiàn)M2型巨噬細(xì)胞表達(dá)。免疫組化和western blot-NEC組腸道TNF-a蛋白表達(dá)明顯高于正常組,且TNF-a陽(yáng)性表達(dá)區(qū)域主要分布于粘膜層以及環(huán)行肌和縱行肌之間。c-kit的免疫組化染色可見(jiàn)正常小腸ICCs主要分布于環(huán)行肌和縱行肌之間、環(huán)行肌內(nèi)和縱行肌內(nèi),NEC組小腸未見(jiàn)c-kit陽(yáng)性表達(dá)。RT-PCR結(jié)果與蛋白表達(dá)結(jié)果一致,可見(jiàn)NEC組小腸CD68、iNOS 及 TNF-α mRNA的表達(dá)明顯高于正常組,而c-kit mRNA的表達(dá)明顯低于正常組。結(jié)論：NEC腸道有大量M1型巨噬細(xì)胞浸潤(rùn),TNF-α表達(dá)顯著升高。在NEC發(fā)病過(guò)程中,腸道ICCs發(fā)生表型改變,這可能與腸道運(yùn)動(dòng)功能紊亂有關(guān)。第二部分巨噬細(xì)胞對(duì)NEC小鼠腸道ICCs表型改變的影響目的：進(jìn)一步在動(dòng)物模型中研究NEC腸道巨噬細(xì)胞活化、TNF-α表達(dá)及ICCs的表型改變情況,研究巨噬細(xì)胞對(duì)NEC腸道TNFF-α表達(dá)及ICCs表型改變的影響。方法：新生鼠隨機(jī)分為4組：正常組,未進(jìn)行缺氧、注射用藥及冷刺激；NEC組,采用缺氧冷刺激的方式制作NEC動(dòng)物模型：NEC+NS組,在NEC模型制作前24小時(shí)腹腔注射生理鹽水60μl；NEC+CL組,在NEC模型制作前24小時(shí)腹腔注射氯膦酸二鈉脂質(zhì)體溶液60g1。分別于實(shí)驗(yàn)第2天(NEC早期)、第4天(NEC進(jìn)展期)、第8天(NEC恢復(fù)期)及第15天(腸道恢復(fù))提取小鼠小腸組織,行HE染色對(duì)腸道進(jìn)行病理評(píng)分,觀察各組NEC發(fā)生率,采用免疫熒光雙染、免疫組化、Westen blot及RT-PCR等技術(shù)檢測(cè)各組腸道巨噬細(xì)胞的活化、TNF-a的表達(dá)及ICCs的表型改變情況。結(jié)果：氯膦酸二鈉脂質(zhì)體在NEC早期和NEC恢復(fù)期能明顯減少M(fèi)1型巨噬細(xì)胞的表達(dá),但在NEC進(jìn)展期這種抑制作用減弱。在NEC早期M1型巨噬細(xì)胞低表達(dá)能明顯減輕NEC腸道的損傷,降低NEC發(fā)生率和TNF-a的表達(dá),并能減輕腸道ICCs的損傷。在NEC恢復(fù)期M1型巨噬細(xì)胞低表達(dá)能降低TNF-a的表達(dá),促進(jìn)腸道組織修復(fù)及ICCs表型的恢復(fù)。結(jié)論：M1型巨噬細(xì)胞在NEC腸道的炎性損傷中具有重要作用,抑制M1型巨噬細(xì)胞不僅能降低TNF-a的表達(dá),減輕腸道的炎性損傷和ICCs的損傷,還能在恢復(fù)期促進(jìn)腸道組織的修復(fù)及ICCs表型的恢復(fù)。第三部分TNF-α對(duì)腸道ICCs表型改變的影響及其機(jī)理研究目的：研究TNF-a抑制c-kit的表達(dá),導(dǎo)致ICCs發(fā)生表型改變的機(jī)制。方法：出生24小時(shí)內(nèi)將B6小鼠隨機(jī)分為4組：正常對(duì)照組,對(duì)腸管進(jìn)行組織培養(yǎng),培養(yǎng)液中未加TNF-α;空白對(duì)照組,未培養(yǎng)的腸管；INF-α組,對(duì)腸管進(jìn)行組織培養(yǎng),培養(yǎng)液中加入不同濃度的TNF-α;miR-222拮抗組,新生小鼠腹腔注射miR-222拮抗劑24小時(shí)后對(duì)腸管進(jìn)行組織培養(yǎng),培養(yǎng)液中加入不同濃度的TNF-α。小腸組織培養(yǎng)48小時(shí)后采用RT-PCR及Western blot技術(shù)檢測(cè)腸組織中miRNA-222、c-kit mRNA及c-kit蛋白的表達(dá)。雙熒光素酶實(shí)驗(yàn)鑒定c-kit是否為miRNA-222的靶基因。結(jié)果：當(dāng)培養(yǎng)液中’NF-a濃度在100-500ng/ ml時(shí),miRNA-222的表達(dá)上調(diào)并抑制c-kitmRNA和蛋白的表達(dá)。使用miR-222拮抗劑之后,在培養(yǎng)液TNF-α濃度在100-200ng/ml時(shí),可逆轉(zhuǎn)這種抑制作用,但是當(dāng)培養(yǎng)液中TNF-a濃度高于200ng/ml時(shí),c-kit mRNA和蛋白的表達(dá)仍然被抑制。雙熒光素酶實(shí)驗(yàn)證實(shí)c-kit是miR-222的靶基因,miR-222可通過(guò)與c-kit mRNA結(jié)合抑制c-kit的表達(dá)。結(jié)論：TNF-a可通過(guò)上調(diào)miR-222抑制c-kit的表達(dá),miR-222拮抗劑在一定范圍內(nèi)可逆轉(zhuǎn)這種抑制作用。
[Abstract]:The first part of NEC with intestinal macrophage activation, phenotype and the expression of ICCs TNF- alpha changes Objective: activation of NEC with intestinal macrophage phenotype, expression of ICCs and TNF-a changes, to explore its role in the pathogenesis of NEC. Methods: HE staining, immunofluorescence staining, immunohistochemistry, Westen blot and the technique of RT-PCR, the comparative study of normal intestinal tissue and NEC in intestinal tissue. Results: HE staining: normal intestinal villi arranged neatly, no intestinal wall edema and hyperemia, and NEC children with intestinal villi necrosis, intestinal hyperemia, intestinal inflammatory cell infiltration. Immunofluorescence double staining, normal bowel little expression no CD68 positive macrophages, macrophage activation, and intestinal NEC infiltrated M1 macrophages, there was no expression of M2 macrophages. Immunohistochemistry and western group blot-NEC, TNF-a egg white form Damien Significantly higher than the normal group, TNF-a group and the immune positive expression area mainly distributed between.C-kit and mucosal layer of circular muscle and longitudinal muscle of staining of normal small intestine ICCs mainly distributed in the circular muscle and longitudinal muscle, circular muscle and longitudinal muscle in the small intestine of the NEC group no c-kit positive expression of.RT-PCR and protein expression of the results consistent, visible small intestine of the NEC group CD68, the expression of iNOS and TNF- alpha mRNA was significantly higher than the normal group, the expression of c-kit and mRNA were significantly lower than that of normal group. Conclusion: NEC has a large number of intestinal infiltration of M1 macrophages, TNF- expression increased significantly. In the pathogenesis of NEC, intestinal ICCs phenotype change, which may be related to intestinal movement the second part macrophage dysfunction. The influences of the NEC phenotype of ICCs mice Objective: to further study NEC intestinal macrophage activation in animal models, TNF- expression and ICCs expression Type changes, effects of macrophages on the change of NEC TNFF- expression and intestinal alpha ICCs phenotype. Methods: neonatal rats were randomly divided into 4 groups: normal group, without hypoxia, injection and cold stimulation; NEC group, NEC animal model making by hypoxia and cold stimulation methods: NEC+NS group, NEC in model preparation 24 hours of intraperitoneal injection of saline 60 l; group NEC+CL, NEC in model 24 hours before the intraperitoneal injection of two sodium clodronate liposome solution 60g1. respectively on the second day of the experiment (early NEC), fourth days (eighth days, NEC advanced) (NEC recovery) Ji Di 15 days (bowel recovery) extraction small intestine of mice by HE staining on the intestinal pathology score, incidence of the observation group NEC, double immunofluorescence staining, immunohistochemistry, activation of Westen blot and RT-PCR technology to detect the intestinal macrophage phenotype, expression of ICCs and TNF-a. Results: the change of chlorine Phosphonic acid two sodium liposome recovery can significantly reduce the expression of M1 type macrophages at the early stage of NEC and NEC, but in NEC during the progression of this inhibition decreased. Expression can significantly reduce the NEC of intestinal injury in the early stage of NEC M1 macrophages is low, the incidence of NEC and decrease the expression of TNF-a, and can reduce the intestinal ICCs injury in the recovery phase of NEC low expression of M1 macrophages could reduce TNF-a expression and promote intestinal tissue repair and restore the ICCs phenotype. Conclusion: M1 macrophages play an important role in inflammatory injury of intestinal NEC in inhibition of M1 macrophages can not only reduce the expression of TNF-a, reduce the intestinal inflammatory injury and ICCs injury. Also, during the recovery period to promote intestinal tissue repair and restore the ICCs phenotype. The third part of the TNF- alpha objective to study the effect and mechanism of the change of intestinal phenotype of ICCs: expression of TNF-a inhibits c-kit, lead to ICCs Mechanism of phenotypic changes within 24 hours of birth. Methods: B6 mice were randomly divided into 4 groups: normal control group, the intestinal tissue culture, culture medium without TNF- alpha; blank control group, uncultured bowel; INF- Alpha Group on intestinal tissue culture, different concentrations was added into the medium TNF- alpha; miR-222 antagonist group, neonatal mice by intraperitoneal injection of miR-222 antagonist after 24 hours of bowel tissue culture, cultured with medium containing different concentrations of TNF- alpha. Intestinal tissue after 48 hours of incubation was detected by miRNA-222 RT-PCR and Western blot in intestinal tissue, the expression of c-kit mRNA and c-kit protein. Dual luciferase assay whether c-kit is the target gene of miRNA-222. Results: when cultured in NF-a concentration in 100-500ng/ ml, up regulate the expression of miRNA-222 and inhibit the expression of c-kitmRNA and protein. After the use of miR-222 antagonists in medium TN The serum level of F- in 100-200ng/ml, reversed this inhibition, but when the concentration of TNF-a in culture solution was higher than 200ng/ml, the expression of c-kit mRNA and protein was still inhibited. Dual luciferase experiments confirmed that c-kit is the target gene of miR-222, miR-222 and c-kit combined with mRNA can inhibit the expression of c-kit. Conclusion: TNF-a can inhibit the expression of c-kit through the up regulation of miR-222 and miR-222 antagonists in a certain range can reverse the inhibitory effect.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R722.1

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