左归丸联合人脐带间充质干细胞移植防治大鼠宫腔粘连的实验研究

发布时间：2018-01-31 21:34

  本文关键词： 宫腔粘连 脐带间充质干细胞 炎症反应 免疫调节　出处：《湖北中医药大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的观察轻、中度宫腔粘连患者血清中TNF-α、IL-1及IL-6等促炎因子的表达,探讨炎症反应在宫腔粘连(intrauterine adhesions,IUA)发病机制中作用;在成功建立大鼠IUA模型的基础上,观察左归丸及人脐带间充质干细胞移植对IUA模型大鼠的宫腔纤维化是否具有改善作用,并从免疫调节方面初步探讨其可能机制。方法第一部分临床研究,选取已确诊轻、中度宫腔粘连患者30例及同期因继发不孕行宫腔镜检查且子宫内膜正常的患者30例,确诊后无菌采血约3ml,离心后取上清。酶联免疫吸附法(ELISA)测定两组患者血清中TNF-α、IL-1、IL-6的表达水平。第二部分动物造模,48只SD大鼠随机分为4组,每组12只。A假手术组:仅开腹穿刺左侧子宫,无热水损伤;B热损伤10s组:导管穿刺左侧宫腔,连接100℃沸腾热水损伤10s;C热损伤15s组;D热损伤20s组。分别于造模后第3d、7d、14d、21d处死各组大鼠3只,收集双侧子宫。HE染色观察各组大鼠子宫内膜形态学变化及子宫内膜腺体数目改变;Masson染色观察各组大鼠子宫内膜间质纤维化情况,并利用IPP图像分析软件分析各组子宫内膜纤维化面积比。第三部分实验研究,30只SD大鼠随机分为5组,每组6只。A假手术组:仅穿刺左侧中段子宫,不予热损伤,不予任何干预;B模型组:造模后不予其他干预;C药物组:造模后给予左归丸灌胃;D人脐带间充质干细胞(human umbilical cord mesenchymal stem cell,h UC-MSC)移植组:造模后尾静脉注射P3代h UC-MSCs;E联合组:造模后给与左归丸+h UC-MSC移植。治疗28d后处死各组大鼠,取双侧子宫,同时采集心脏血3ml。HE染色和Masson染色观察各组大鼠子宫内膜形态学变化、内膜腺体数目及子宫内膜纤维化面积;ELISA检测血清中TNF-α、IL-1、IL-6的表达水平;免疫荧光双重染色法观察子宫内膜中CD29及P38MAPK的定位及表达;Western blot检测子宫内膜组织中TGF-β1、P38MAPK、P65NF-k B蛋白的表达水平。结果第一部分临床研究,与正常对照组相比,IUA患者血清中TNF-α、IL-1、IL-6呈高表达,差异具有统计学意义(P0.05)。第二部分动物造模,形态学观察结果,A组宫腔呈星型,子宫内膜完整,腺体丰富,结构规则;B、C、D三组子宫内膜呈不同程度损伤,其中B组在损伤后14d及21d宫腔形态已基本恢复,可见少量新生腺体,无明显纤维化改变;C组在损伤后14d及21d宫腔狭小,肌层完整,间质纤维化,大量胶原纤维聚集,呈纤维样改变,可见少量腺体结构;D组子宫内膜过度损伤,肌层紊乱结构模糊,在损伤后7d、14d及21d宫腔完全封闭,瘢痕修复。综合各组结果显示,根据建模后14d子宫内膜的形态及纤维化情况可判断及鉴定是否成功建立IUA模型。第三部分实验研究,(1)形态学观察,与B组相比,3个治疗组宫腔形态均不同程度恢复,腺体数目增加,纤维化面积减少,其中D、E两组的腺体数目增加最明显,纤维化面积减少最明显(P0.05),但对比A组,宫腔形态仍未完全恢复(P0.05);(2)血清炎性因子表达,与B模型组相比,3个治疗组大鼠血清中TNF-α、IL-1、IL-6的表达均显著减少(P0.05);其中D、E两组减少最明显(P0.05),但仍高于A组(P0.05);(3)免疫荧光双重染色,CD29阳性细胞显色为红色荧光,P38MAPK阳性显色为绿色荧光,A、B、C三组CD29的光密度相互比较无统计学差异(P0.05),D、E两组的CD29表达明显高于A、B、C三组(P0.05),其中D h UC-MSC移植组CD29阳性表达最高(P0.05);P38MAPK的表达在B模型组最高,在3个治疗组中均不同程度降低(P0.05),其中D、E两组P38MAPK表达降低最明显(P0.05),但仍高于A组(P0.05),D与E比较,P38MAPK表达无统计学差异(P0.05);(4)Western blot检测,B模型组TGF-β1、P38MAPK、P65NF-k B蛋白的表达最高(P0.05),3个治疗组均不同程度降低(P0.05),其中D、E两组降低最明显(P0.05),但仍高于A组(P0.05),D组TGF-β1的表达低于E组(P0.05),高于A组(P0.05),D、E两组中P38MAPK、P65NF-k B蛋白的表达无统计学差异(P0.05)。结论⑴宫腔粘连患者血清中炎性因子TNF-α、IL-1及IL-6呈高表达;炎性因子TNF-α、IL-1及IL-6的高表达可能参与了宫腔粘连的发病机制。⑵以100℃的热水流动导管损伤大鼠宫腔15s,可造成子宫内膜基底层损伤,14d后形成纤维化粘连带,从而成功建立稳定的大鼠IUA模型。⑶左归丸联合人脐带间充质干细胞移植能有效改善大鼠宫腔粘连的程度,其作用机制可能是通过抑制P38MAPK/NF-k B炎症信号通路,下调TNF-α、TGF-β1、IL-1及IL-6等促炎及致纤维化因子的表达而实现的。
[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, 绾ょ淮鍖栭潰绉�鍑忓皯鏈�鏄庢樉(P0.05),浣嗗�规瘮A缁，

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