沙利度胺对TNBS诱导的SD幼鼠结肠炎的治疗作用和机理研究

发布时间：2018-03-18 09:32

本文选题：TNBS　切入点：结肠炎　出处：《复旦大学》2011年硕士论文　论文类型：学位论文

【摘要】：第一部分 TNBS诱导SD幼鼠结肠炎模型的建立背景：炎症性肠病(Inflammatory Bowel Disease, IBD)是一类病因不明的慢性肠道炎症性疾病,主要包括溃疡性结肠炎(Ulcerative Colitis, UC)和克罗恩病(Crohn's Disease, CD)。其发病率在欧洲和北美最高,在亚洲等地区也呈逐年上升的态势。IBD确切的病因和发病机制尚不清楚,目前缺乏有效的治疗手段,因此利用适当的结肠炎动物模型来开发具有治疗作用的临床新药和研究其作用机制,对提高人类炎症性肠病的治疗效果以及对炎症性肠病致病机理进行研究提供了极大的帮助。目的：建立TNBS诱导的SD幼鼠结肠炎模型,通过观察幼鼠进食、毛色、活动情况和体质量增长情况；大便性状；结肠大体标本；肠粘膜损伤与修复等情况明确炎症性肠病对幼鼠生长发育的影响,验证造模是否成功,为后续研究奠定实验基础。方法：将25只4-5周龄清洁级SD幼鼠,随机分为对照组(n=12)和TNBS模型组(n=13)。对照组胃针自肛门插入,灌入生理盐水；TNBS模型组灌入造模药物150mg/kg TNBS。每天记录幼鼠进食、毛色以及活动情况,称量和记录幼鼠体质量,记录大便性状及隐血情况。于第4,7,14天分别处死4只幼鼠,进行结肠大体形态观察,按照Wallace和Keenan评分标准进行结肠黏膜大体损伤评分,根据Cooper组织学评分法观察组织病理改变。结果：对照组幼鼠毛发有光泽,饮食、活动、精神状态均正常,腹泻和血便恢复较快,分别持续2.08±0.67天和1.83±0.72天,无幼鼠死亡。TNBS模型组幼鼠,毛发色泽灰暗,精神萎靡,食量下降,造模后立即出现腹胀、腹泻和血便等情况,腹泻和血便分别持续6.00±1.71天和6.08±1.68天,与对照组相比有显著性差异(P0.01)。TNBS模型组幼鼠死亡率7.69%。前4天体质量不增加反而下降,在第7天时恢复到原有水平并逐渐增长,但仍明显落后于对照组(P0.01)。模型组幼鼠造模后结肠明显扩张,粘膜充血水肿,肠壁变薄,肠道粘膜可见较大的溃疡病灶,表面糜烂成糊状,腹腔可见积液。至第14天,肠道炎性症状有所改善,部分溃疡处疤痕形成,局部肠壁明显增厚,多数结肠与相邻脏器粘连,大体标本形态学第4、7、14天评分分别为4.25±0.50,4.25±0.96,2.75±0.50；对照组评分0,两组存在显著性差异(P0.01)。模型组幼鼠光镜下结肠粘膜不完整可见较多溃疡,杯状细胞减少,隐窝和大部分腺体被破坏,部分病灶表面上皮仅少许残留或完全被破坏。腺体正常结构丧失,排列紊乱,腺腔消失,可见大量炎性细胞浸润,累及全层。至第14天,小溃疡局限,边缘见隐窝和杯状细胞再生、修复以及腺体再生,仍可见较多炎性细胞浸润,主要累及黏膜和黏膜下层,偶可至肌层,可见增生的成纤维细胞,第4、7、14天光镜下组织学评分分别为3.75±0.50,3.75±0.50,2.50±0.58；对照组评分0,两组存在显著性差异(P0.01)。结论：TNBS诱导的幼鼠结肠炎模型,具有腹泻血便时间长、体重增加缓慢等临床特点,其肠道粘膜充血水肿、溃疡、肠壁增厚等大体改变,粘膜大量炎性细胞浸润、隐窝腺体破坏等组织病理改变,可在一定程度上反映炎症性肠病的疾病特点。幼鼠结肠炎模型可能更适合儿童炎症性肠病的相关研究。第二部分沙利度胺对TNBS诱导的SD幼鼠结肠炎的治疗作用和机理研究背景：国外以及本课题组前期的临床研究发现应用沙利度胺(thalidomide)治疗难治性炎症性肠病,常可使患者的临床症状缓解乃至消失,瘘管愈合。沙利度胺对炎症性肠病的治疗作用引起了人们的关注。目的：通过TNBS诱导的幼鼠结肠炎动物模型,观察沙利度胺对炎症性肠病的治疗作用,并初步探讨其可能的作用机制,为推广沙利度胺在儿童炎症性肠病中的临床应用提供实验依据。方法：将82只4-5周龄清洁级SD幼鼠,随机分为三组,正常对照组(n=25),TNBS模型组(n=29)和沙利度胺干预组(n=28)。正常对照组,胃针自肛门插入,灌入生理盐水;TNBS模型组,灌入造模药物150mg/kg TNBS；干预组,灌入造模药物150mg/kg TNBS,并经口喂服沙利度胺150mg/kg/d.每天记录幼鼠进食、毛色和活动情况,称量和记录幼鼠体质量,记录大便性状及隐血情况。于第4,7,14天分别处死至少8只幼鼠,用ELISA法检测血清中TNF-α和白细胞介素(IL-1β,IL-6,IL-10和IL-12)水平的变化。按照Wallace和Keenan评分标准进行结肠黏膜的大体损伤评分,Cooper组织学评分标准评价组织病理学变化。用免疫组化的方法检测结肠粘膜核因子(NF)-kB的表达。结果：对照组幼鼠腹泻和血便恢复很快(2.04±0.68天和1.96±0.84天),无幼鼠死亡。TNBS模型组腹泻和血便持续时间较长(5.77±1.70天和5.92±1.74天),与对照组相比有显著性差异(P0.01),幼鼠死亡率10.34%。观察期间模型组幼鼠体质量增长均显著落后于对照组。沙利度胺干预组,幼鼠腹泻和血便分别持续3.78±0.58天和3.89±1.15天,明显快于模型组(P0.01)。干预组幼鼠第7天起体重增长速度明显快于模型组,但仍低于对照组,幼鼠死亡率3.57%。模型组幼鼠第4、7、14天大体标本形态学评分分别为4.30±0.48,4.25±0.71和2.88±0.64;光镜下组织学评分分别为3.80±0.42,3.88±0.35和2.38±0.52;和对照组相比,存在显著性差异(P0.01)。沙利度胺干预组幼鼠第4天结肠大体形态学评分(4.00±0.71)和组织学评分(3.78±0.44),与模型组无显著性差异(P0.05)；第7天结肠扩张好转,与周围组织有轻微粘连,溃疡缩小,光镜下溃疡局限,边缘见隐窝和杯状细胞再生、修复以及腺体再生,大体形态学评分(3.55±0.73)和组织学评分(3.00±0.50)与第4天相比明显好转(P0.01)。第14天,肠道结肠无明显扩张,溃疡处疤痕形成,光镜下组织结构较清晰,黏膜趋完整,炎性细胞明显减少。大体形态学评分(1.11±0.60)和组织学评分(0.89±0.60),较第7天显著降低(P0.01),并且显著低于模型组(P0.01),提示干预组幼鼠结肠病理改变恢复较模型组好。对照组肠道粘膜较少表达NF-κB,主要分布在细胞胞浆中；TNBS模型组和沙利度胺干预组第4天均可见大量的核阳性染色及细胞浆染色,以核染色为主,TNBS模型组(59.80%±9.30%)和沙利度胺干预组(56.78%±6.72%)明显高于对照组(6.75%±1.39%,P0.01)。第7和14天时,模型组和干预组核阳性细胞虽逐渐下降,但仍高于对照组。沙利度胺干预组第7,14天NF-κB核阳性细胞百分比(36.00%±9.97%,13.22%±2.91%)均明显低于模型组(52.63%±5.13%,31.13%±6.66%,P0.01)。对照组血清中炎性细胞因子(TNF-α,IL-1β,IL-6,IL-10和IL-12)均处在低表达水平,模型组和干预组血清中上述炎性细胞因子在第4天时已经明显上升(P对照-模型,P对照-干预0.01)。第7天,模型组和干预组炎性细胞因子较第4天有所下降,但仍明显高于对照组(P对照-模型,P对照-干预0.01)。第14天,干预组炎性细胞因子水平明显低于模型组(P模型-干预0.05)。造模后第4天,干预组IL-10水平明显升高(44.90±21.66pg/ml),与对照组(12.41±4.03pg/ml)和模型组(18.81±7.07pg/ml)相比,差异有显著性(P对照-干预,P模型-干预0.01),之后虽逐渐下降,但仍保持较高水平,第7、14天IL-10水平分别为32.11±10.10pg/ml和31.14±7.46pg/ml。模型组IL-10的表达水平高峰出现较干预组晚,第4天和对照组相比无显著性差异,第7、14天表达水平(25.84±10.07pg/ml;24.91±8.44pg/ml)与对照组(11.98±4.09pg/ml; 12.28±3.10pg/ml)相比有显著性差异(P对照-模型0.01),但低于干预组。结论：沙利度胺对TNBS诱导的幼鼠结肠炎治疗作用肯定,明显缩短腹泻和血便的天数,加速肠道粘膜病理损伤的修复,调节肠道炎症反应。沙利度胺在治疗TNBS诱导的幼鼠结肠炎模型中表现出良好的抗炎作用和免疫调节的双重作用,可能与抑制和下调核因子NF-κB及其下游炎性细胞因子(TNF-α、IL-1β、IL-6和IL-12)的表达,以及上调抗炎细胞因子(IL-10)的表达有关。
[Abstract]:Part one
Establishment of TNBS induced colitis model in young SD rats
Background: inflammatory bowel disease (Inflammatory Bowel, Disease, IBD) is a kind of chronic inflammatory bowel disease of unknown etiology, including ulcerative colitis (Ulcerative Colitis, UC) and Crohn's disease (Crohn's Disease, CD). The incidence rate in Europe and North America, the most high in Asia also showed etiology and pathogenesis the mechanism of the upward trend of.IBD was still unclear, the current lack of effective treatment, the mechanism of the colitis appropriate animal model to develop therapeutic drugs and clinical research, to improve human inflammatory bowel disease treatment effect and provides a great help to study the pathogenic mechanism of inflammatory bowel disease.
Objective: to establish a SD rat model of TNBS induced colitis in rats by observing, eating, hair color, activity and the increase of body mass; stool specimens; colon; intestinal mucosal injury and repair of inflammatory bowel disease clearly influence on the growth and development of young rats, to verify the success of modeling, lay the foundation for the following study.
Methods: 25 clean SD rats aged 4-5 weeks were randomly divided into control group, model group (n=12) and TNBS (n=13). The control group gastric needle from the anal insertion, filled with saline; TNBS model group into the model drug 150mg/kg TNBS. rats were recorded every day to eat, hair color and activity, weighing and record in body weight, stool occult blood and record. In the 4,7,14 rats were sacrificed at day 4, colon gross observation, Wallace and Keenan of colonic mucosa in accordance with the standard for evaluation of macroscopic damage score, according to the score of the observation group woven Cooper pathology organization.
Results: the rats in the control group hair glossy, diet, activity, mental state were normal, diarrhea and bloody stool and fast recovery, respectively for 2.08 days and 1.83 + 0.67 + 0.72 days, no death in.TNBS model rats, gray hair color, listlessness, decreased appetite, abdominal distension immediately after modeling, diarrhea and bloody stool so, diarrhea and bloody stool respectively for 6 days and 6.08 + 1.71 + 1.68 days, compared with the control group there were significant differences (P0.01).TNBS model group rats 7.69%. mortality before 4 body weights did not increase but decrease, restored to the original level in seventh days and gradually increase, but still lags behind the control group (P0.01).
Rats in the model group after the model of colonic mucosal hyperemia and edema, dilatation, intestinal wall thinning, intestinal mucosa visible large ulcer lesions, surface erosion and paste into the peritoneal effusion. Visible to fourteenth days, inflammatory bowel symptoms improved, part of the ulcer scar formation, local bowel wall thickening, most of the colon and adjacent organs the gross morphology of adhesion, day 4,7,14 score was 4.25 + 0.50,4.25 + 0.96,2.75 + 0.50; the control group was 0, two groups had significant difference (P0.01).
The model group rats under light microscope are seen in the colonic mucosa ulcer, goblet cells decreased, and the majority of crypt glands were destroyed, only a little part of the surface epithelium or residual lesion was completely destroyed. The normal structure of the gland loss, disordered glandular cavity disappeared, showing a large number of inflammatory cell infiltration, full-thickness. To fourteenth days. Small ulcer limitation, edge crypt and goblet cells regeneration, repair and regeneration of the gland, still visible more infiltration of inflammatory cells, mainly involving the mucosa and submucosa, even to the muscle layer, fibroblast hyperplasia could be seen, the organization 4,7,14 sky mirror school scores were 3.75 + 0.50,3.75 + 0.50,2.50 + 0.58; control the score of group 0, two groups had significant difference (P0.01).
Conclusion: the rat model of TNBS induced colitis with diarrhea, bloody long, slow weight gain and other clinical features, the intestinal mucosal hyperemia and edema, ulcer, intestinal wall thickening by change, mucosal inflammatory cells, destruction of glands of crypt pathological changes, can reflect the disease characteristics of inflammatory bowel disease in a certain extent the related research is more suitable for children. Inflammatory bowel disease may in colitis model.
The second part
The therapeutic effect and mechanism of thalidomide on TNBS induced SD colitis in young rats
Background: our previous studies and clinical application of thalidomide (thalidomide) found that the treatment of refractory inflammatory bowel disease, often can make the patient's clinical symptoms relieved and disappeared fistulas. Therapeutic effect of thalidomide on inflammatory bowel disease has aroused people's attention.
Objective: To observe the therapeutic effect of thalidomide on inflammatory bowel disease and to explore its possible mechanism through the animal model of colitis induced by TNBS, so as to provide experimental evidence for promoting the clinical application of thalidomide in children with inflammatory bowel disease.
Methods: 82 clean SD rats aged 4-5 weeks were randomly divided into three groups, normal control group (n=25), TNBS model group (n=29) and thalidomide group (n=28). The normal control group, gastric needle from the anus inserted into the saline irrigation; TNBS model group, poured into the modeling of drug 150mg /kg TNBS; the intervention group, poured into the modeling of drug 150mg/kg TNBS, and oral feeding of thalidomide 150mg/kg/d. mice are recorded everyday eating, hair color and activity, weighing and recording in body weight, stool occult blood and record. In the first 4,7,14 days were killed at least 8 young rats, serum alpha TNF- and interleukin ELISA (IL-1 beta, IL-6, IL-10 and IL-12) levels. In injury of colonic mucosa score according to Wallace and Keenan standard for evaluation, Cooper evaluation standard for evaluation of histological pathological changes. Immunohistochemical method was used in detection of colonic mucosa nuclear factor (NF) expression of -kB.
Results: in the control group were diarrhea and bloody stool recovered quickly (2.04 + 0.68 and 1.96 + 0.84 days), no death in.TNBS model group, diarrhea and bloody stool continues for a long time (5.77 + 1.70 and 5.92 + 1.74 days), compared with the control group there were significant differences (P0.01), 10.34%. rats mortality observation period the increase of body mass in model group rats were significantly behind the control group. Thalidomide intervention group, diarrhea and bloody stool were respectively for 3.78 days + 0.58 and 3.89 + 1.15 days, significantly faster than the model group (P0.01). The intervention group in the seventh day weight growth rate was faster than the model group, but still lower than the control group, rats the mortality rate of 3.57%.
The model group rats 4,7,14 days gross morphological scores were 4.30 + 0.48,4.25 + 0.71 and 2.88 + 0.64; under the histological scores were 3.80 + 0.42,3.88 + 0.35 and 2.38 + 0.52 light microscope; compared with the control group, there was significant difference (P0.01). Rats in group fourth days of thalidomide (colonic morphology score 4 + 0.71) scores and organization (3.78 + 0.44), with no significant difference between the model group (P0.05); seventh days colectasia improved, there is a slight adhesion with the surrounding tissue, ulcers, ulcer under limited light, see the crypt and goblet cell edge regeneration, repair and regeneration of the gland, the gross morphological score (3.55 + 0.73) scores and organization (3 + 0.50) compared with fourth days significantly improved (P0.01). Fourteenth days, no obvious expansion of colon intestinal ulcer, scar formation, under the light microscope, organizational structure is more clear, more complete mucosal inflammatory cells, generally decreased significantly. The morphological score (1.11 + 0.60) and histological score (0.89 + 0.60) were significantly lower than those on the seventh day (P0.01), and significantly lower than that in the model group (P0.01), suggesting that the pathological changes in the colon of the young rats in the intervention group were better than those in the model group.
The control group of intestinal mucosa less expression of NF- kappa B, mainly distributed in the cytoplasm; the TNBS model group and thalidomide group in the fourth day showed positive nuclear staining and cytoplasmic staining with large, nuclear staining, TNBS model group (59.80% + 9.30%) and thalidomide group (56.78% + 6.72%) was significantly higher than that of control group (6.75% + 1.39%, P0.01). The seventh and fourteenth day, the model group and the intervention group although nuclear positive cells gradually decreased, but still higher than the control group. The intervention group of thalidomide day 7,14 NF- kappa B nuclear positive cells percentage (36% + 9.97%, 13.22% + 2.91%) was significantly lower than the model group (52.63% + 5.13%, 31.13% + 6.66%, P0.01).
The control of inflammatory cytokines in serum (TNF- alpha, IL-1 beta, IL-6, IL-10 and IL-12) were in the low expression level of model group and intervention group in the serum inflammatory cytokines has been significantly increased on day fourth (P - P control model, the control intervention 0.01). The seventh day, the model group the intervention group and inflammatory cytokines than fourth days decreased, but still significantly higher than the control group (P control model, P control intervention 0.01). For fourteenth days, the intervention group of inflammatory cytokine levels were significantly lower than the model group (P model intervention 0.05). Fourth days after modeling, the intervention group IL-10 level increased (44.90 + 21.66pg/ml), and control group (12.41 + 4.03pg/ml) and model group (18.81 + 7.07pg/ml) compared with significant difference (P - P model - control intervention, intervention after 0.01), although gradually decreased, but remained at a high level, the 7,14 levels of IL-10 were 32.11 + 10.10pg/ml 31.14 + 7.46pg/ml. and IL- in the model group 10 the expression peak of a late intervention group, there was no significant difference between the fourth day and the control group, the expression level of 7,14 days (25.84 + 10.07pg/ml; 24.91 + 8.44pg/ml) and control group (11.98 + 12.28 + 4.09pg/ml; 3.10pg/ml) compared with significant difference (P control - model 0.01), but lower than the intervention group.
Conclusion: the therapeutic effect of thalidomide on rat colitis induced by TNBS, diarrhea and bloody stool days shortened obviously, accelerate the repair of injury of intestinal mucosa pathology, regulation of intestinal inflammation. Play a double role in anti inflammation and immune regulation in good colitis model induced by thalidomide in the treatment of TNBS, may be related to inhibition and down-regulation of nuclear factor the expression of NF- B and its downstream inflammatory cytokines (TNF- alpha, IL-1 beta, IL-6 and IL-12) expression and up regulation of anti-inflammatory cytokines (IL-10) expression.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R-332;R574.62

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