本文選題：膿毒癥　切入點(diǎn)：腸屏障　出處：《皖南醫(yī)學(xué)院》2015年碩士論文

【摘要】：目的通過(guò)在體實(shí)驗(yàn)和離體實(shí)驗(yàn),探討PLK1和NF-?B信號(hào)通路在膿毒癥腸屏障功能障礙中的作用。方法在體實(shí)驗(yàn):取健康清潔成年雄性C57BL小鼠40只,采用隨機(jī)數(shù)字法分為2組(n=20):正常對(duì)照組(C組)和膿毒癥組(LPS組),采用腹腔內(nèi)注射脂多糖(LPS)40mg/kg制備膿毒癥小鼠模型,對(duì)照組腹腔注射等量生理鹽水。造模12h后,取小鼠靜脈血,測(cè)定血清TNF-?及二胺氧化酶(DAO)水平,然后用頸椎脫臼法處死,取回盲部小腸5cm.,4%多聚甲醛固定,常規(guī)脫水石蠟包埋,切片,HE染色,觀察小腸絨毛與黏膜炎癥等病理改變。采用TUNEL法進(jìn)行原位細(xì)胞凋亡檢測(cè)。小鼠小腸PLK1表達(dá)水平檢測(cè)采用Western blotting及免疫組織化學(xué)的方法。離體實(shí)驗(yàn):在HT29細(xì)胞培養(yǎng)基中加入不同濃度(0,10,20,30?g/ml)的LPS處理24h,收集細(xì)胞行Annexin V/Propidium Iodide染色后用流式細(xì)胞分析儀檢測(cè)細(xì)胞凋亡情況,同時(shí)提取蛋白行Western blotting檢測(cè)PARP1、Caspase3、PLK1、I?B-?等蛋白表達(dá)水平,并采用細(xì)胞免疫熒光法檢測(cè)NF-?B蛋白定位;在HT29細(xì)胞培養(yǎng)基中加入不同濃度(0,2,10,20?ng/ml)的NF-?B通路抑制劑PDTC預(yù)處理4h,再加入LPS(30?g/ml)處理24h,收集細(xì)胞行Annexin V/Propidium Iodide染色后用流式細(xì)胞分析儀檢測(cè)細(xì)胞凋亡情況,同時(shí)提取蛋白行Western blotting檢測(cè)Caspase3、I?B-?等蛋白表達(dá)水平;在HT29細(xì)胞培養(yǎng)基中加入PLK1抑制劑BI2536(50nM)處理24h,收集細(xì)胞提取蛋白行Western blotting檢測(cè)PARP1、PLK1、I?B-?等蛋白表達(dá)水平,并采用細(xì)胞免疫熒光法檢測(cè)NF-?B蛋白定位;在HT29細(xì)胞中正義轉(zhuǎn)染pCDNA3.1-PLK1-myc質(zhì)粒,再加入LPS(30?g/ml)處理24h,收集細(xì)胞用Annexin V/Propidium Iodide染色后行流式細(xì)胞分析儀檢測(cè)細(xì)胞凋亡情況,同時(shí)提取蛋白行Western blotting檢測(cè)PARP1、Caspase3等蛋白表達(dá)水平。結(jié)果在體實(shí)驗(yàn):(1)腹腔注射LPS(40mg/kg)后,與C組相比,LPS組小鼠精神萎靡、動(dòng)作遲緩,腹腔內(nèi)有大量膿性積液,腸管充血水腫,血清TNF-?濃度顯著增加(P0.001)。(2)與C組相比,LPS組小鼠腸黏膜明顯萎縮,小腸絨毛脫落,腺體結(jié)構(gòu)喪失,血清DAO濃度顯著增加(P0.001)。(3)與C組相比,LPS組小鼠腸黏膜上皮細(xì)胞發(fā)生凋亡,小腸組織中PARP1,Caspase3蛋白表達(dá)水平明顯下調(diào)。(4)與C組相比,LPS組小鼠小腸組織中PLK1表達(dá)減少,I?B-?蛋白的表達(dá)水平下調(diào)。離體實(shí)驗(yàn):(1)不同濃度LPS處理HT29細(xì)胞24h,可誘導(dǎo)細(xì)胞發(fā)生凋亡,且凋亡細(xì)胞比例隨LPS濃度的增加而增大。(2)不同濃度LPS處理HT29細(xì)胞24h,與對(duì)照組相比,細(xì)胞核中NF-?B蛋白表達(dá)增加,同時(shí)I?B-?蛋白的表達(dá)水平下調(diào)。(3)用PDTC(10 ng/ml,20?ng/ml)預(yù)處理HT29細(xì)胞4h,與對(duì)照組相比,可顯著減少LPS引起的HT29細(xì)胞凋亡比例(P0.01或P0.001)。(4)不同濃度LPS處理HT29細(xì)胞24h,與對(duì)照組相比,PLK1蛋白的表達(dá)水平下調(diào)。(5)BI2536(50nM)處理HT29細(xì)胞24h,與對(duì)照組相比,細(xì)胞核中NF-?B蛋白表達(dá)增加,同時(shí)I?B-?、PARP1蛋白的表達(dá)水平下調(diào)。(6)在HT29細(xì)胞中正義轉(zhuǎn)染pCDNA3.1-PLK1-myc質(zhì)粒,與未正義轉(zhuǎn)染組細(xì)胞相比,LPS(30?g/ml)處理誘導(dǎo)細(xì)胞產(chǎn)生的凋亡比例顯著減少(P0.01)。結(jié)論P(yáng)LK1蛋白表達(dá)下調(diào)和NF-?B通路激活在膿毒癥腸黏膜屏障障礙機(jī)制中發(fā)揮重要作用;膿毒癥時(shí),PLK1通過(guò)負(fù)向調(diào)節(jié)NF-?B通路,加重腸上皮細(xì)胞凋亡,引起腸黏膜通透性增加,導(dǎo)致腸屏障功能障礙。
[Abstract]:Objective by in vivo and in vitro experiments, to investigate the PLK1 and NF-? B signal pathway in sepsis with intestinal barrier dysfunction in in vivo. Methods: healthy clean adult male C57BL 40 mice were randomly divided into 2 groups (n=20): normal control group (C group) and sepsis group (LPS group), by intraperitoneal injection of lipopolysaccharide (LPS) sepsis mice model of 40mg/kg prepared by the control group, intraperitoneal injection of saline. 12h after modeling, the mice blood serum TNF-? Two and monoamine oxidase (DAO), and then were executed by cervical dislocation. Ileocecal intestinal 5cm., 4% paraformaldehyde, conventional dehydration, paraffin embedding, slice, HE staining, and the intestinal villi mucosa inflammation and other pathological changes were observed by TUNEL assay. Cell apoptosis in mouse intestinal. The expression level of PLK1 detected by Western blotting and immunohistochemistry. In vitro experiments: in the medium added with the different concentrations of HT29 cell culture (0,10,20,30? G/ml) LPS 24h, Annexin V/Propidium cells were collected after Iodide staining by flow cytometry and cell apoptosis, simultaneous extraction of protein by Western blotting detection of PARP1, Caspase3, PLK1, I? B-? Protein expression level, and by immunofluorescence detection of NF-? B protein; in the medium added with the different concentrations of HT29 cell culture (0,2,10,20? Ng/ml) NF-? B pathway inhibitor PDTC pretreated 4h, adding LPS (30? G/ml) 24h, Annexin V/Propidium cells were collected after Iodide staining by flow cytometry and cell apoptosis at the same time, Western protein extraction blotting detection Caspase3, I? B-? Protein expression level; in addition of the PLK1 inhibitor BI2536 based in HT29 cell culture (50nM) at 24h, Western blotting cells were collected to extract protein The detection of PARP1, PLK1, I? B-? Protein expression level, and the immunofluorescence detection of NF-? B protein in HT29 cells; justice transfected with pCDNA3.1-PLK1-myc plasmid, then add LPS (30? G/ml) 24h, flow cytometry and apoptosis cells were collected with Annexin V/Propidium staining for Iodide at the same time, Western blotting detection of PARP1 protein extraction, the expression level of Caspase3 protein. Results: (1) intraperitoneal injection of LPS (40mg/kg), compared with the C group, LPS group, apathetic, slow, there is a lot of purulent fluid of abdominal cavity, bowel edema, serum TNF- concentration increased significantly? (P0.001). (2) compared with C group, LPS group of mice intestinal mucosal atrophy, intestinal villus shedding, glandular structure loss, the concentration of serum DAO increased significantly (P0.001). (3) compared with the C group, LPS group of mice intestinal mucosal epithelial cell apoptosis of intestinal tissue 涓璓ARP1,Caspase3铔嬬櫧琛ㄨ揪姘村鉤鏄庢樉涓嬭皟.(4)涓嶤緇勭浉姣，

本文編號(hào)：1723531