發(fā)布時(shí)間：2018-06-05 18:58

  本文選題：炎癥性腸病 + 獨(dú)一味�。� 參考：《重慶醫(yī)科大學(xué)》2015年碩士論文

【摘要】：實(shí)驗(yàn)?zāi)康模貉芯开?dú)一味環(huán)烯醚萜苷(IGLR)對(duì)大鼠急性IBD模型的治療作用；為將IGLR開(kāi)發(fā)成為治療IBD的有效新藥提供實(shí)驗(yàn)依據(jù)和理論基礎(chǔ)。實(shí)驗(yàn)方法：1分組：將90只Wistar大鼠隨機(jī)分成9組：正常對(duì)照組(C)、TNBS模型對(duì)照組(M1)、TNBS模型的IGLR高、中、低(300mg/kg、 150mg/kg、75mg/kg)三種劑量治療組(T1h、T1m、T1l)、柳氮磺吡啶(SASP)陽(yáng)性對(duì)照組(S1), DSS模型對(duì)照組(M2)、DSS模型的IGLR 300mg/kg治療組(T2)、SASP陽(yáng)性對(duì)照組(S2)。2大鼠IBD模型制作：用TNBS-無(wú)水乙醇(1：1)溶液一次性灌腸誘導(dǎo)大鼠TNBS性IBD模型；給予6% DSS溶液自由飲用8天誘導(dǎo)大鼠DSS性IBD模型；C組以等量蒸餾水灌腸或飲用。3給藥和結(jié)果觀察：造模完成后次日開(kāi)始灌胃給藥,各對(duì)照組給等容積蒸餾水,期間觀察大鼠活動(dòng)、大便性狀及隱血檢測(cè)；連續(xù)處理7天后處死大鼠,取結(jié)腸組織標(biāo)本,觀察記錄各組大鼠結(jié)腸病變；HE染色光鏡下觀察各組組織學(xué)改變并進(jìn)行組織學(xué)評(píng)分；取結(jié)腸標(biāo)本做分子生物學(xué)機(jī)制研究。4分子生物學(xué)相關(guān)指標(biāo)檢測(cè)：采用IHC法檢測(cè)結(jié)腸組織MPO及EPO的表達(dá)；采用ELISA法檢測(cè)血清標(biāo)本中INF-γ、IL-4、IL-10及IL-17的表達(dá)；采用Western Blot法及RT-PCR法檢測(cè)結(jié)腸組織中GATA-3、T-bet、Foxp3、ROR-yt蛋白及其mRNA的表達(dá)。實(shí)驗(yàn)結(jié)果：1一般狀況：M1,T11組大鼠實(shí)驗(yàn)期間期間毛發(fā)豎立,進(jìn)食、飲水及活動(dòng)顯著減少,體重明顯下降,腹脹,無(wú)排便,DAI呈較恒定高水平,與M1相比,S1、T1m及T1h大鼠在給藥第2-4天起開(kāi)始排便,體重回升,一般狀況明顯好轉(zhuǎn),給藥兩天后起,DAI呈明顯下降趨勢(shì)；M2組大鼠在實(shí)驗(yàn)期間一般狀況差,出現(xiàn)大量稀便及膿血便,體重明顯降低,DAI呈持續(xù)高水平,無(wú)明顯下降,與M2相比S2、T2組于治療開(kāi)始后2-3天基本無(wú)肉眼血便,6-7天時(shí)大便呈梭形固體顆粒,隱血(-),體重逐漸恢復(fù),但未達(dá)C組水平,給藥后3天起DAI下降趨勢(shì)明顯。2結(jié)腸組織大體形態(tài)及組織學(xué)改變：結(jié)腸組織肉眼觀察表明,M1組存在大片腸壁潰瘍,部分節(jié)段腸壁僵硬,完全失去活性,組織學(xué)評(píng)分與C組差異顯著(p0.05),T11組有一定緩解,但不明顯,S1、T1h及Tim潰瘍面明顯縮小,仍存在部分小片及點(diǎn)狀潰瘍,評(píng)分與M1存在顯著差異(p0.05)；M2組大鼠結(jié)腸壁潰瘍、水腫、充血嚴(yán)重,有明顯出血灶及血絲,評(píng)分與C組相較差異顯著(p0.05),S2及T2組結(jié)腸壁潰瘍、水腫、充血、出血明顯減少,存在少量出血點(diǎn),與M2相比評(píng)分下降具有統(tǒng)計(jì)學(xué)意義(p0.05)。HE染色后光鏡下可見(jiàn)M1組腸壁組織破壞深達(dá)肌層,大量組織壞死及炎癥細(xì)胞浸潤(rùn)；與M1組相較,S1、Ti1、T1h及Tim組有不同程度緩解,呈劑量依賴性；M2組可見(jiàn)腸黏膜細(xì)胞腫脹壞死,但損傷未達(dá)肌層,S2及T2可見(jiàn)炎癥修復(fù)和及再生的腺體及杯狀細(xì)胞。與C組相比,M1、M2組織學(xué)評(píng)分顯著升高(p0.05),而各治療組均呈顯著下降(p0.05)。3 HIC檢測(cè)MPO及EPO：與C組對(duì)照,M1及M2組大鼠結(jié)腸潰瘍組織處MPO及EPO均表達(dá)增強(qiáng),呈棕黃色深染,其主要分布于胞漿內(nèi),炎癥致細(xì)胞破壞后,MPO及EPO大量溢出,檢測(cè)評(píng)分結(jié)果升高顯著(p0.05)。經(jīng)IGLR和SASP處理7天后,與M1或M2組相比,各給藥組大鼠結(jié)腸病變區(qū)MPO及EPO均表達(dá)均呈現(xiàn)不同程度的降低(p0.05)。4 ELISA檢測(cè)血清蛋白：檢測(cè)結(jié)果顯示,與C組相比,INF-γ及IL-17在M1、M2組中均有顯著升高,IL-10則顯著下降,IL-4僅在M2中出現(xiàn)明顯下降(p0.05)；治療后,各治療組INF-γ均出現(xiàn)顯著下降(p0.05), IL-10均出現(xiàn)顯著上升(p0.05), IL-17在S1、T1m、T1h、S2及T2組中出現(xiàn)明顯下降(p0.05),在TNBS誘導(dǎo)的炎癥性腸病模型中IL-4無(wú)顯著變化,而在DSS模型的治療組S2、T2中有明顯回升(p0.05)。5結(jié)腸潰瘍組織蛋白質(zhì)及mRNA表達(dá)：檢測(cè)結(jié)果提示,與C組對(duì)照,Ml及M2組在潰瘍組織處T-bet和ROR-yt的蛋白及mRNA表達(dá)均顯著提升(p0.05),經(jīng)過(guò)治療,T2、S2、Tim及T1h均出現(xiàn)顯著下降(p0.05); GATA-3蛋白及mRNA在M1組中表達(dá)量改變不顯著,而在M2組中明顯上升(p0.05),治療后T2、S2及T1h出現(xiàn)表達(dá)量下降,其該變量具有統(tǒng)計(jì)學(xué)意義(p0.05); Foxp3的表達(dá)在M1及M2組中均顯著降低(p0.05),而與之相比,所有治療組表達(dá)均出現(xiàn)明顯升高(p0.05)。實(shí)驗(yàn)結(jié)論：1 TNBS誘導(dǎo)的大鼠IBD模型主要表現(xiàn)為腹脹、便秘及體重急劇下降,嚴(yán)重的結(jié)腸炎性反應(yīng),大片結(jié)腸壁潰瘍,部分節(jié)段腸壁僵硬,潰瘍深達(dá)肌層,其免疫病理學(xué)改變以Thl及Th17細(xì)胞因子作用為主導(dǎo)；DSS誘導(dǎo)的大鼠IBD模型以腹瀉、膿血便及體重急劇下降為主要表現(xiàn),結(jié)腸炎性反應(yīng)嚴(yán)重,潰瘍主要損傷結(jié)腸黏膜層,以水腫、出血為主,鏡下可見(jiàn)腺窩缺損和杯狀細(xì)胞破壞,其免疫病理學(xué)改變可能包括Th1、Th2及Th17細(xì)胞因子等的共同作用。2 IGLR對(duì)TNBS及DSS誘導(dǎo)的兩類大鼠IBD模型均有顯著療效,可有效抑制IBD的炎癥反應(yīng)和結(jié)腸損傷。3 IGLR對(duì)大鼠1BD治療作用的機(jī)制可能是通過(guò)調(diào)節(jié)Th1、Th2及Th17細(xì)胞因子及其轉(zhuǎn)錄因子T-bet、GATA-3及RORyt的表達(dá),抑制其炎癥反應(yīng)及結(jié)腸損傷而產(chǎn)生。4 IGLR還通過(guò)促進(jìn)Treg細(xì)胞因子及其轉(zhuǎn)錄因子Foxp3的表達(dá),產(chǎn)生其抗炎及治療IBD的作用。
[Abstract]:Objective: To study the therapeutic effect of cyclo iridoid glycoside (IGLR) on acute IBD model in rats; to provide experimental basis and theoretical basis for developing IGLR as an effective new drug for the treatment of IBD. Experimental methods: 1 groups: 90 Wistar rats were randomly divided into 9 groups: normal control group (C), TNBS model control group (M1), TNBS model IGLR Three doses of 300mg/kg, 150mg/kg, 75mg/kg, T1h, T1m, T1l, SASP positive control group (S1), DSS model control group (M2), DSS model IGLR treatment group. The model of DSS induced rat IBD was induced free drinking of 6% DSS solution for 8 days. Group C was treated with equal amount of distilled water enema or drinking.3, and the results were observed: after the model was completed the next day, the rats were given the medicine, and the control groups were given equal volume distilled water. During the period, the rats' activity, stool character and occult blood test were observed, and the rats were killed after 7 days of treatment. Colonic tissue specimens were taken to observe the colonic lesions of the rats in each group. The histological changes were observed and the histological scores were observed under the HE staining light microscope. The molecular biological mechanism of the colon specimens was examined for the detection of the.4 molecular biology related indexes: the expression of MPO and EPO in the colon tissue was detected by the IHC method; the serum specimens were detected by ELISA method. The expression of INF- gamma, IL-4, IL-10 and IL-17, Western Blot and RT-PCR methods were used to detect the expression of GATA-3, T-bet, Foxp3, ROR-yt protein and mRNA in colon tissue. Experimental results: 1 general conditions: during the period of experiment, the hair was erected, eating, drinking and activity decreased significantly, weight decreased obviously, abdominal distention and defecation were presented. Compared with the constant high level, compared with M1, S1, T1m and T1h rats began to defecate on the 2-4 day of administration, the body weight recovered and the general condition obviously improved. After two days of administration, DAI showed a significant decline trend. In the M2 group, the general condition of the rats was poor during the experiment, a large number of dilute stool and pus and blood were found, the body weight was significantly reduced, the DAI showed a sustained high level, and no significant decline. Compared with M2, S2, group T2 had no naked eye blood stool at 2-3 days after treatment, and the stool was spindle solid particles, occult blood (-) and body weight gradually recovered on the 6-7 day, but not in group C. The trend of DAI descending after 3 days was obviously.2 colonic tissue general morphology and histological changes: intestinal tissue naked eye observation showed that there were large bowel wall ulcers in group M1. The intestinal wall of the segment was stiff and completely lost the activity. The histological score was significantly different from the C group (P0.05), and the T11 group had some relief, but the S1, T1h and Tim ulcer surfaces were obviously narrowed, there were still some small fragments and punctate ulcers, and there were significant differences between the scores and M1 (P0.05), and the colon wall ulcers, edema, severe congestion, obvious hemorrhagic foci and blood in the group M2 rats. The score was significantly different from that of the C group (P0.05). The colonic wall ulcers in S2 and T2 groups were sored, edema, hyperemia, bleeding obviously decreased, and there was a small number of bleeding points. Compared with M2, the score decreased significantly (P0.05).HE staining showed that the intestinal wall tissue of M1 group destroyed the deep myometrium, a large number of tissue necrosis and inflammatory cell infiltration, compared with the M1 group, S1, T. The I1, T1h and Tim groups were in a dose-dependent manner. In the M2 group, the intestinal mucosa cells were swollen and necrotic, but the damage did not reach the myometrium, and the S2 and T2 showed the glandular and goblet cells of the inflammatory repair and regeneration. Compared with the C group, the histological score of M1 and M2 increased significantly (P0.05). Compared with group C, the expression of MPO and EPO in the tissues of colon ulcers in M1 and M2 rats was enhanced and stained with brown yellow, which was mainly distributed in the cytoplasm. After the destruction of the cells, a large number of MPO and EPO spillovers, and the results of the detection score increased significantly (P0.05). After 7 days of IGLR and SASP treatment, the colon lesion areas of the rats were compared with M1 or M2 groups. All the expression showed different degrees of decrease (P0.05).4 ELISA detection of serum protein: the results showed that compared with the C group, INF- and IL-17 were significantly increased in M1, M2 group, IL-10 decreased significantly, IL-4 only decreased in M2 (P0.05). The increase (P0.05), IL-17 in the S1, T1m, T1h, S2 and T2 groups decreased significantly (P0.05), and there was no significant change in the TNBS induced inflammatory bowel disease model. The protein and mRNA expression of ET and ROR-yt increased significantly (P0.05). After treatment, T2, S2, Tim and T1h decreased significantly (P0.05), and the expression of GATA-3 protein and mRNA in the M1 group was not significant. The expression in M1 and M2 groups was significantly decreased (P0.05), and the expression of all the treatment groups increased significantly (P0.05). Experimental conclusion: 1 TNBS induced rat IBD models were mainly abdominal distention, constipation and weight decline, severe colitis, large colonic wall ulcers, partial segmental stiffness of the bowel wall, ulcers reaching the myometrium, The immunhistopathological changes were dominated by the action of Thl and Th17 cytokine, and DSS induced rat IBD model was characterized by diarrhea, pus and blood stool and body weight drastic decline. The colitis was seriously damaged, the ulcers mainly damaged the colonic mucosa layer, with edema and hemorrhage, and the pathological changes of the gland fossa and goblet cells could be seen under the microscope. The interaction of Th1, Th2 and Th17 cytokines, such as.2 IGLR, has a significant effect on TNBS and DSS induced IBD models of two types of rats. It can effectively inhibit the inflammatory reaction of IBD and the mechanism of colon injury.3 IGLR on rat 1BD treatment. The expression of YT, the inhibition of its inflammatory response and colon injury, and the production of.4 IGLR by promoting the expression of Treg cytokine and its transcription factor Foxp3, and producing its anti-inflammatory and therapeutic effect on IBD.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R574.62

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