巨噬细胞活化对NEC肠道ICCs表型改变的影响及其机理研究

发布时间：2018-02-23 20:49

本文关键词： NEC 巨噬细胞 TNF-α ICCs NEC 巨噬细胞氯膦酸二钠脂质体 TNF-α ICCs TNF-α miR-222 c-kit ICCs　出处：《华中科技大学》2016年博士论文　论文类型：学位论文

【摘要】：第一部分NEC患儿肠道巨噬细胞的活化、TNF-α的表达及ICCs的表型改变目的：研究NEC患儿肠道巨噬细胞的活化、TNF-a的表达及ICCs的表型改变情况,探究其在NEC发病中的作用。方法：采用HE染色、免疫荧光双染、免疫组化、Westen blot 及 RT-PCR等技术对正常小肠组织和NEC小肠组织进行对比研究。结果：HE染色—正常小肠绒毛排列整齐,肠壁无水肿及充血,而NEC患儿小肠绒毛坏死,肠壁充血,肠壁有大量炎性细胞浸润。免疫荧光双染—正常小肠有少量CD68阳性的巨噬细胞表达,未见巨噬细胞活化,而NEC肠道有大量M1型巨噬细胞浸润,未见M2型巨噬细胞表达。免疫组化和western blot-NEC组肠道TNF-a蛋白表达明显高于正常组,且TNF-a阳性表达区域主要分布于粘膜层以及环行肌和纵行肌之间。c-kit的免疫组化染色可见正常小肠ICCs主要分布于环行肌和纵行肌之间、环行肌内和纵行肌内,NEC组小肠未见c-kit阳性表达。RT-PCR结果与蛋白表达结果一致,可见NEC组小肠CD68、iNOS 及 TNF-α mRNA的表达明显高于正常组,而c-kit mRNA的表达明显低于正常组。结论：NEC肠道有大量M1型巨噬细胞浸润,TNF-α表达显著升高。在NEC发病过程中,肠道ICCs发生表型改变,这可能与肠道运动功能紊乱有关。第二部分巨噬细胞对NEC小鼠肠道ICCs表型改变的影响目的：进一步在动物模型中研究NEC肠道巨噬细胞活化、TNF-α表达及ICCs的表型改变情况,研究巨噬细胞对NEC肠道TNFF-α表达及ICCs表型改变的影响。方法：新生鼠随机分为4组：正常组,未进行缺氧、注射用药及冷刺激；NEC组,采用缺氧冷刺激的方式制作NEC动物模型：NEC+NS组,在NEC模型制作前24小时腹腔注射生理盐水60μl；NEC+CL组,在NEC模型制作前24小时腹腔注射氯膦酸二钠脂质体溶液60g1。分别于实验第2天(NEC早期)、第4天(NEC进展期)、第8天(NEC恢复期)及第15天(肠道恢复)提取小鼠小肠组织,行HE染色对肠道进行病理评分,观察各组NEC发生率,采用免疫荧光双染、免疫组化、Westen blot及RT-PCR等技术检测各组肠道巨噬细胞的活化、TNF-a的表达及ICCs的表型改变情况。结果：氯膦酸二钠脂质体在NEC早期和NEC恢复期能明显减少M1型巨噬细胞的表达,但在NEC进展期这种抑制作用减弱。在NEC早期M1型巨噬细胞低表达能明显减轻NEC肠道的损伤,降低NEC发生率和TNF-a的表达,并能减轻肠道ICCs的损伤。在NEC恢复期M1型巨噬细胞低表达能降低TNF-a的表达,促进肠道组织修复及ICCs表型的恢复。结论：M1型巨噬细胞在NEC肠道的炎性损伤中具有重要作用,抑制M1型巨噬细胞不仅能降低TNF-a的表达,减轻肠道的炎性损伤和ICCs的损伤,还能在恢复期促进肠道组织的修复及ICCs表型的恢复。第三部分TNF-α对肠道ICCs表型改变的影响及其机理研究目的：研究TNF-a抑制c-kit的表达,导致ICCs发生表型改变的机制。方法：出生24小时内将B6小鼠随机分为4组：正常对照组,对肠管进行组织培养,培养液中未加TNF-α;空白对照组,未培养的肠管；INF-α组,对肠管进行组织培养,培养液中加入不同浓度的TNF-α;miR-222拮抗组,新生小鼠腹腔注射miR-222拮抗剂24小时后对肠管进行组织培养,培养液中加入不同浓度的TNF-α。小肠组织培养48小时后采用RT-PCR及Western blot技术检测肠组织中miRNA-222、c-kit mRNA及c-kit蛋白的表达。双荧光素酶实验鉴定c-kit是否为miRNA-222的靶基因。结果：当培养液中’NF-a浓度在100-500ng/ ml时,miRNA-222的表达上调并抑制c-kitmRNA和蛋白的表达。使用miR-222拮抗剂之后,在培养液TNF-α浓度在100-200ng/ml时,可逆转这种抑制作用,但是当培养液中TNF-a浓度高于200ng/ml时,c-kit mRNA和蛋白的表达仍然被抑制。双荧光素酶实验证实c-kit是miR-222的靶基因,miR-222可通过与c-kit mRNA结合抑制c-kit的表达。结论：TNF-a可通过上调miR-222抑制c-kit的表达,miR-222拮抗剂在一定范围内可逆转这种抑制作用。
[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.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R722.1

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