發(fā)布時間：2018-01-31 21:34

  本文關(guān)鍵詞： 宮腔粘連 臍帶間充質(zhì)干細胞 炎癥反應(yīng) 免疫調(diào)節(jié)　出處：《湖北中醫(yī)藥大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的觀察輕、中度宮腔粘連患者血清中TNF-α、IL-1及IL-6等促炎因子的表達,探討炎癥反應(yīng)在宮腔粘連(intrauterine adhesions,IUA)發(fā)病機制中作用;在成功建立大鼠IUA模型的基礎(chǔ)上,觀察左歸丸及人臍帶間充質(zhì)干細胞移植對IUA模型大鼠的宮腔纖維化是否具有改善作用,并從免疫調(diào)節(jié)方面初步探討其可能機制。方法第一部分臨床研究,選取已確診輕、中度宮腔粘連患者30例及同期因繼發(fā)不孕行宮腔鏡檢查且子宮內(nèi)膜正常的患者30例,確診后無菌采血約3ml,離心后取上清。酶聯(lián)免疫吸附法(ELISA)測定兩組患者血清中TNF-α、IL-1、IL-6的表達水平。第二部分動物造模,48只SD大鼠隨機分為4組,每組12只。A假手術(shù)組:僅開腹穿刺左側(cè)子宮,無熱水損傷;B熱損傷10s組:導(dǎo)管穿刺左側(cè)宮腔,連接100℃沸騰熱水損傷10s;C熱損傷15s組;D熱損傷20s組。分別于造模后第3d、7d、14d、21d處死各組大鼠3只,收集雙側(cè)子宮。HE染色觀察各組大鼠子宮內(nèi)膜形態(tài)學(xué)變化及子宮內(nèi)膜腺體數(shù)目改變;Masson染色觀察各組大鼠子宮內(nèi)膜間質(zhì)纖維化情況,并利用IPP圖像分析軟件分析各組子宮內(nèi)膜纖維化面積比。第三部分實驗研究,30只SD大鼠隨機分為5組,每組6只。A假手術(shù)組:僅穿刺左側(cè)中段子宮,不予熱損傷,不予任何干預(yù);B模型組:造模后不予其他干預(yù);C藥物組:造模后給予左歸丸灌胃;D人臍帶間充質(zhì)干細胞(human umbilical cord mesenchymal stem cell,h UC-MSC)移植組:造模后尾靜脈注射P3代h UC-MSCs;E聯(lián)合組:造模后給與左歸丸+h UC-MSC移植。治療28d后處死各組大鼠,取雙側(cè)子宮,同時采集心臟血3ml。HE染色和Masson染色觀察各組大鼠子宮內(nèi)膜形態(tài)學(xué)變化、內(nèi)膜腺體數(shù)目及子宮內(nèi)膜纖維化面積;ELISA檢測血清中TNF-α、IL-1、IL-6的表達水平;免疫熒光雙重染色法觀察子宮內(nèi)膜中CD29及P38MAPK的定位及表達;Western blot檢測子宮內(nèi)膜組織中TGF-β1、P38MAPK、P65NF-k B蛋白的表達水平。結(jié)果第一部分臨床研究,與正常對照組相比,IUA患者血清中TNF-α、IL-1、IL-6呈高表達,差異具有統(tǒng)計學(xué)意義(P0.05)。第二部分動物造模,形態(tài)學(xué)觀察結(jié)果,A組宮腔呈星型,子宮內(nèi)膜完整,腺體豐富,結(jié)構(gòu)規(guī)則;B、C、D三組子宮內(nèi)膜呈不同程度損傷,其中B組在損傷后14d及21d宮腔形態(tài)已基本恢復(fù),可見少量新生腺體,無明顯纖維化改變;C組在損傷后14d及21d宮腔狹小,肌層完整,間質(zhì)纖維化,大量膠原纖維聚集,呈纖維樣改變,可見少量腺體結(jié)構(gòu);D組子宮內(nèi)膜過度損傷,肌層紊亂結(jié)構(gòu)模糊,在損傷后7d、14d及21d宮腔完全封閉,瘢痕修復(fù)。綜合各組結(jié)果顯示,根據(jù)建模后14d子宮內(nèi)膜的形態(tài)及纖維化情況可判斷及鑒定是否成功建立IUA模型。第三部分實驗研究,(1)形態(tài)學(xué)觀察,與B組相比,3個治療組宮腔形態(tài)均不同程度恢復(fù),腺體數(shù)目增加,纖維化面積減少,其中D、E兩組的腺體數(shù)目增加最明顯,纖維化面積減少最明顯(P0.05),但對比A組,宮腔形態(tài)仍未完全恢復(fù)(P0.05);(2)血清炎性因子表達,與B模型組相比,3個治療組大鼠血清中TNF-α、IL-1、IL-6的表達均顯著減少(P0.05);其中D、E兩組減少最明顯(P0.05),但仍高于A組(P0.05);(3)免疫熒光雙重染色,CD29陽性細胞顯色為紅色熒光,P38MAPK陽性顯色為綠色熒光,A、B、C三組CD29的光密度相互比較無統(tǒng)計學(xué)差異(P0.05),D、E兩組的CD29表達明顯高于A、B、C三組(P0.05),其中D h UC-MSC移植組CD29陽性表達最高(P0.05);P38MAPK的表達在B模型組最高,在3個治療組中均不同程度降低(P0.05),其中D、E兩組P38MAPK表達降低最明顯(P0.05),但仍高于A組(P0.05),D與E比較,P38MAPK表達無統(tǒng)計學(xué)差異(P0.05);(4)Western blot檢測,B模型組TGF-β1、P38MAPK、P65NF-k B蛋白的表達最高(P0.05),3個治療組均不同程度降低(P0.05),其中D、E兩組降低最明顯(P0.05),但仍高于A組(P0.05),D組TGF-β1的表達低于E組(P0.05),高于A組(P0.05),D、E兩組中P38MAPK、P65NF-k B蛋白的表達無統(tǒng)計學(xué)差異(P0.05)。結(jié)論⑴宮腔粘連患者血清中炎性因子TNF-α、IL-1及IL-6呈高表達;炎性因子TNF-α、IL-1及IL-6的高表達可能參與了宮腔粘連的發(fā)病機制。⑵以100℃的熱水流動導(dǎo)管損傷大鼠宮腔15s,可造成子宮內(nèi)膜基底層損傷,14d后形成纖維化粘連帶,從而成功建立穩(wěn)定的大鼠IUA模型。⑶左歸丸聯(lián)合人臍帶間充質(zhì)干細胞移植能有效改善大鼠宮腔粘連的程度,其作用機制可能是通過抑制P38MAPK/NF-k B炎癥信號通路,下調(diào)TNF-α、TGF-β1、IL-1及IL-6等促炎及致纖維化因子的表達而實現(xiàn)的。
[Abstract]:Objective To observe the light, TNF- alpha in serum of patients with moderate intrauterine adhesion, the expression of IL-1 and IL-6 of pro-inflammatory factor, to investigate the inflammatory reaction in the intrauterine adhesion (intrauterine adhesions IUA) mechanism; based on the successfully established IUA rat model, observe the effect of Zuogui pill and human umbilical cord mesenchymal stem is fibrosis of uterine cavity cell transplantation on rat model of IUA with improved function, from the aspects of immune regulation and explore its possible mechanism. Methods: the first part of clinical research, we selected 30 cases of diagnosed light, and the same moderate intrauterine adhesions in patients with secondary infertility by hysteroscopy and endometrial normal in 30 cases, after the diagnosis of aseptic the blood is about 3ml, the supernatant after centrifugation. Enzyme linked immunosorbent assay (ELISA) determination of TNF- alpha, two groups of patients with serum IL-1, the expression level of IL-6. The second part of the animal model, 48 SD rats were randomly divided into 4 groups, 12 rats in each group of.A prosthetic hand Operation group: open the left uterine puncture, no hot water damage; B thermal injury group 10s: catheter puncture left uterine cavity, connecting 100 degrees of boiling water damage 10s C; thermal damage in 15s group; D group 20s respectively. The thermal damage after modeling in 3D, 7d, 14d, 21d rats were killed only 3 collect, bilateral uterus of rats changed.HE staining to observe the morphological changes of endometrium and endometrial gland number; observe endometrial interstitial fibrosis rats Masson staining and image analysis were analyzed by IPP software. The endometrial fibrosis research area than the third part of the experiment, 30 SD rats were randomly divided into 5 group.A, 6 rats in each group: sham operation group only puncture of the left middle uterus, no thermal damage, without any intervention; B model group: no other intervention after modeling; C group: after modeling given Zuoguiwan intragastric D; human umbilical cord mesenchymal stem cells (human umbilical cord mesenchymal st EM cell, H UC-MSC) transplantation group: after modeling the tail vein injection of P3 generation h UC-MSCs; E group: after modeling +h UC-MSC to Zuoguiwan transplantation. After the treatment of 28d rats, bilateral uterus, and uterine morphological changes of membrane staining of rats were collected in heart blood stained with 3ml.HE Masson, the number of endometrial glands and endometrial fibrosis area; TNF- alpha, ELISA detection of serum IL-1, the expression level of IL-6; double immunofluorescence staining was used to observe the localization of CD29 and P38MAPK in endometrium and endometrial tissue expression; detection Western blot TGF- beta 1, P38MAPK, P65NF-k expression level of B protein. The results of the first part of clinical research, compared with the normal control group, TNF- alpha, IUA in blood serum of patients with IL-1, IL-6 showed high expression, the difference was statistically significant (P0.05). The second part of the animal model, the results of morphological observation, A group of uterine cavity was star, endometrial integrity The gland, rich, structure of B, C, D rules; three groups of endometrium showed different degrees of injury in the B group at 14d after injury and 21d uterine cavity have been restored, a small amount of new glands, no obvious fibrosis; group C after injury, 14d and 21d in uterine cavity narrow, intact muscular layer, between a large number of interstitial fibrosis, collagen fiber aggregation, a fiber like change, a small amount of glandular structure; D endometrium excessive injury, muscle disorder fuzzy structure after injury 7d, 14d and 21d in uterine cavity completely closed, scar repair. All results show that according to the modeling form and fibrosis of 14d in endometrium can the judgment and identification of whether the IUA model was successfully established. The third part of the experimental study, (1) morphological observation, compared with the B group, 3 treatment groups of uterine cavity were different degree of recovery, the number of glands increased, fibrosis area decreased, the number of D, E two groups of glands increased obviously, 綰ょ淮鍖栭潰縐噺灝戞渶鏄庢樉(P0.05),浣嗗姣擜緇，

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