本文選題：TNBS　切入點(diǎn)：結(jié)腸炎　出處：《復(fù)旦大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：第一部分 TNBS誘導(dǎo)SD幼鼠結(jié)腸炎模型的建立 背景：炎癥性腸病(Inflammatory Bowel Disease, IBD)是一類病因不明的慢性腸道炎癥性疾病,主要包括潰瘍性結(jié)腸炎(Ulcerative Colitis, UC)和克羅恩病(Crohn's Disease, CD)。其發(fā)病率在歐洲和北美最高,在亞洲等地區(qū)也呈逐年上升的態(tài)勢。IBD確切的病因和發(fā)病機(jī)制尚不清楚,目前缺乏有效的治療手段,因此利用適當(dāng)?shù)慕Y(jié)腸炎動物模型來開發(fā)具有治療作用的臨床新藥和研究其作用機(jī)制,對提高人類炎癥性腸病的治療效果以及對炎癥性腸病致病機(jī)理進(jìn)行研究提供了極大的幫助。 目的：建立TNBS誘導(dǎo)的SD幼鼠結(jié)腸炎模型,通過觀察幼鼠進(jìn)食、毛色、活動情況和體質(zhì)量增長情況；大便性狀；結(jié)腸大體標(biāo)本；腸粘膜損傷與修復(fù)等情況明確炎癥性腸病對幼鼠生長發(fā)育的影響,驗證造模是否成功,為后續(xù)研究奠定實驗基礎(chǔ)。 方法：將25只4-5周齡清潔級SD幼鼠,隨機(jī)分為對照組(n=12)和TNBS模型組(n=13)。對照組胃針自肛門插入,灌入生理鹽水；TNBS模型組灌入造模藥物150mg/kg TNBS。每天記錄幼鼠進(jìn)食、毛色以及活動情況,稱量和記錄幼鼠體質(zhì)量,記錄大便性狀及隱血情況。于第4,7,14天分別處死4只幼鼠,進(jìn)行結(jié)腸大體形態(tài)觀察,按照Wallace和Keenan評分標(biāo)準(zhǔn)進(jìn)行結(jié)腸黏膜大體損傷評分,根據(jù)Cooper組織學(xué)評分法觀察組織病理改變。 結(jié)果：對照組幼鼠毛發(fā)有光澤,飲食、活動、精神狀態(tài)均正常,腹瀉和血便恢復(fù)較快,分別持續(xù)2.08±0.67天和1.83±0.72天,無幼鼠死亡。TNBS模型組幼鼠,毛發(fā)色澤灰暗,精神萎靡,食量下降,造模后立即出現(xiàn)腹脹、腹瀉和血便等情況,腹瀉和血便分別持續(xù)6.00±1.71天和6.08±1.68天,與對照組相比有顯著性差異(P0.01)。TNBS模型組幼鼠死亡率7.69%。前4天體質(zhì)量不增加反而下降,在第7天時恢復(fù)到原有水平并逐漸增長,但仍明顯落后于對照組(P0.01)。 模型組幼鼠造模后結(jié)腸明顯擴(kuò)張,粘膜充血水腫,腸壁變薄,腸道粘膜可見較大的潰瘍病灶,表面糜爛成糊狀,腹腔可見積液。至第14天,腸道炎性癥狀有所改善,部分潰瘍處疤痕形成,局部腸壁明顯增厚,多數(shù)結(jié)腸與相鄰臟器粘連,大體標(biāo)本形態(tài)學(xué)第4、7、14天評分分別為4.25±0.50,4.25±0.96,2.75±0.50；對照組評分0,兩組存在顯著性差異(P0.01)。 模型組幼鼠光鏡下結(jié)腸粘膜不完整可見較多潰瘍,杯狀細(xì)胞減少,隱窩和大部分腺體被破壞,部分病灶表面上皮僅少許殘留或完全被破壞。腺體正常結(jié)構(gòu)喪失,排列紊亂,腺腔消失,可見大量炎性細(xì)胞浸潤,累及全層。至第14天,小潰瘍局限,邊緣見隱窩和杯狀細(xì)胞再生、修復(fù)以及腺體再生,仍可見較多炎性細(xì)胞浸潤,主要累及黏膜和黏膜下層,偶可至肌層,可見增生的成纖維細(xì)胞,第4、7、14天光鏡下組織學(xué)評分分別為3.75±0.50,3.75±0.50,2.50±0.58；對照組評分0,兩組存在顯著性差異(P0.01)。 結(jié)論：TNBS誘導(dǎo)的幼鼠結(jié)腸炎模型,具有腹瀉血便時間長、體重增加緩慢等臨床特點(diǎn),其腸道粘膜充血水腫、潰瘍、腸壁增厚等大體改變,粘膜大量炎性細(xì)胞浸潤、隱窩腺體破壞等組織病理改變,可在一定程度上反映炎癥性腸病的疾病特點(diǎn)。幼鼠結(jié)腸炎模型可能更適合兒童炎癥性腸病的相關(guān)研究。 第二部分 沙利度胺對TNBS誘導(dǎo)的SD幼鼠結(jié)腸炎的治療作用和機(jī)理研究 背景：國外以及本課題組前期的臨床研究發(fā)現(xiàn)應(yīng)用沙利度胺(thalidomide)治療難治性炎癥性腸病,�？墒够颊叩呐R床癥狀緩解乃至消失,瘺管愈合。沙利度胺對炎癥性腸病的治療作用引起了人們的關(guān)注。 目的：通過TNBS誘導(dǎo)的幼鼠結(jié)腸炎動物模型,觀察沙利度胺對炎癥性腸病的治療作用,并初步探討其可能的作用機(jī)制,為推廣沙利度胺在兒童炎癥性腸病中的臨床應(yīng)用提供實驗依據(jù)。 方法：將82只4-5周齡清潔級SD幼鼠,隨機(jī)分為三組,正常對照組(n=25),TNBS模型組(n=29)和沙利度胺干預(yù)組(n=28)。正常對照組,胃針自肛門插入,灌入生理鹽水;TNBS模型組,灌入造模藥物150mg/kg TNBS；干預(yù)組,灌入造模藥物150mg/kg TNBS,并經(jīng)口喂服沙利度胺150mg/kg/d.每天記錄幼鼠進(jìn)食、毛色和活動情況,稱量和記錄幼鼠體質(zhì)量,記錄大便性狀及隱血情況。于第4,7,14天分別處死至少8只幼鼠,用ELISA法檢測血清中TNF-α和白細(xì)胞介素(IL-1β,IL-6,IL-10和IL-12)水平的變化。按照Wallace和Keenan評分標(biāo)準(zhǔn)進(jìn)行結(jié)腸黏膜的大體損傷評分,Cooper組織學(xué)評分標(biāo)準(zhǔn)評價組織病理學(xué)變化。用免疫組化的方法檢測結(jié)腸粘膜核因子(NF)-kB的表達(dá)。 結(jié)果：對照組幼鼠腹瀉和血便恢復(fù)很快(2.04±0.68天和1.96±0.84天),無幼鼠死亡。TNBS模型組腹瀉和血便持續(xù)時間較長(5.77±1.70天和5.92±1.74天),與對照組相比有顯著性差異(P0.01),幼鼠死亡率10.34%。觀察期間模型組幼鼠體質(zhì)量增長均顯著落后于對照組。沙利度胺干預(yù)組,幼鼠腹瀉和血便分別持續(xù)3.78±0.58天和3.89±1.15天,明顯快于模型組(P0.01)。干預(yù)組幼鼠第7天起體重增長速度明顯快于模型組,但仍低于對照組,幼鼠死亡率3.57%。 模型組幼鼠第4、7、14天大體標(biāo)本形態(tài)學(xué)評分分別為4.30±0.48,4.25±0.71和2.88±0.64;光鏡下組織學(xué)評分分別為3.80±0.42,3.88±0.35和2.38±0.52;和對照組相比,存在顯著性差異(P0.01)。沙利度胺干預(yù)組幼鼠第4天結(jié)腸大體形態(tài)學(xué)評分(4.00±0.71)和組織學(xué)評分(3.78±0.44),與模型組無顯著性差異(P0.05)；第7天結(jié)腸擴(kuò)張好轉(zhuǎn),與周圍組織有輕微粘連,潰瘍縮小,光鏡下潰瘍局限,邊緣見隱窩和杯狀細(xì)胞再生、修復(fù)以及腺體再生,大體形態(tài)學(xué)評分(3.55±0.73)和組織學(xué)評分(3.00±0.50)與第4天相比明顯好轉(zhuǎn)(P0.01)。第14天,腸道結(jié)腸無明顯擴(kuò)張,潰瘍處疤痕形成,光鏡下組織結(jié)構(gòu)較清晰,黏膜趨完整,炎性細(xì)胞明顯減少。大體形態(tài)學(xué)評分(1.11±0.60)和組織學(xué)評分(0.89±0.60),較第7天顯著降低(P0.01),并且顯著低于模型組(P0.01),提示干預(yù)組幼鼠結(jié)腸病理改變恢復(fù)較模型組好。 對照組腸道粘膜較少表達(dá)NF-κB,主要分布在細(xì)胞胞漿中；TNBS模型組和沙利度胺干預(yù)組第4天均可見大量的核陽性染色及細(xì)胞漿染色,以核染色為主,TNBS模型組(59.80%±9.30%)和沙利度胺干預(yù)組(56.78%±6.72%)明顯高于對照組(6.75%±1.39%,P0.01)。第7和14天時,模型組和干預(yù)組核陽性細(xì)胞雖逐漸下降,但仍高于對照組。沙利度胺干預(yù)組第7,14天NF-κB核陽性細(xì)胞百分比(36.00%±9.97%,13.22%±2.91%)均明顯低于模型組(52.63%±5.13%,31.13%±6.66%,P0.01)。 對照組血清中炎性細(xì)胞因子(TNF-α,IL-1β,IL-6,IL-10和IL-12)均處在低表達(dá)水平,模型組和干預(yù)組血清中上述炎性細(xì)胞因子在第4天時已經(jīng)明顯上升(P對照-模型,P對照-干預(yù)0.01)。第7天,模型組和干預(yù)組炎性細(xì)胞因子較第4天有所下降,但仍明顯高于對照組(P對照-模型,P對照-干預(yù)0.01)。第14天,干預(yù)組炎性細(xì)胞因子水平明顯低于模型組(P模型-干預(yù)0.05)。造模后第4天,干預(yù)組IL-10水平明顯升高(44.90±21.66pg/ml),與對照組(12.41±4.03pg/ml)和模型組(18.81±7.07pg/ml)相比,差異有顯著性(P對照-干預(yù),P模型-干預(yù)0.01),之后雖逐漸下降,但仍保持較高水平,第7、14天IL-10水平分別為32.11±10.10pg/ml和31.14±7.46pg/ml。模型組IL-10的表達(dá)水平高峰出現(xiàn)較干預(yù)組晚,第4天和對照組相比無顯著性差異,第7、14天表達(dá)水平(25.84±10.07pg/ml;24.91±8.44pg/ml)與對照組(11.98±4.09pg/ml; 12.28±3.10pg/ml)相比有顯著性差異(P對照-模型0.01),但低于干預(yù)組。 結(jié)論：沙利度胺對TNBS誘導(dǎo)的幼鼠結(jié)腸炎治療作用肯定,明顯縮短腹瀉和血便的天數(shù),加速腸道粘膜病理損傷的修復(fù),調(diào)節(jié)腸道炎癥反應(yīng)。沙利度胺在治療TNBS誘導(dǎo)的幼鼠結(jié)腸炎模型中表現(xiàn)出良好的抗炎作用和免疫調(diào)節(jié)的雙重作用,可能與抑制和下調(diào)核因子NF-κB及其下游炎性細(xì)胞因子(TNF-α、IL-1β、IL-6和IL-12)的表達(dá),以及上調(diào)抗炎細(xì)胞因子(IL-10)的表達(dá)有關(guān)。
[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.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R-332;R574.62

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