發(fā)布時間：2018-06-01 07:20

  本文選題：痛瀉二草方 + 潰瘍性結(jié)腸炎��； 參考：《甘肅中醫(yī)學(xué)院》2014年碩士論文

【摘要】：目的：本課題采用束縛刺激加三硝基苯磺酸（TNBS）和乙醇混合灌腸的病證結(jié)合方式建立肝郁脾虛型潰瘍性結(jié)腸炎（Ulcerative colitis，UC）大鼠模型，觀察痛瀉二草方（TXECF）對肝郁脾虛型UC大鼠結(jié)腸組織病理學(xué)的影響及NF-κB信號通路相關(guān)基因和蛋白的影響，從而闡明TXECF治療肝郁脾虛型UC的作用機理和治療靶點。 方法：將90只SPF級Wistar大鼠，根據(jù)體重及雌雄隨機分為空白組、模型組、TXECF高劑量組、TXECF中劑量組、TXECF低劑量組、陽性組（SASP組），每組15只�？瞻捉M給予蒸餾水等體積灌腸，其余各組懫用束縛刺激加TNBS和乙醇混合灌腸的病證結(jié)合法建立肝郁脾虛型UC大鼠模型，動物模型制備成功后，中藥各治療組給予相應(yīng)濃度的TXECF灌胃，陽性組給予柳氮磺胺吡啶（SASP）灌胃，空白組及模型組給予等體積的蒸餾水灌胃，每日1次，連續(xù)21d。各組大鼠末次灌胃結(jié)束后禁食不禁水24h，水合氯醛麻醉，剖開整個結(jié)腸組織，先用肉眼仔細(xì)觀察各組大鼠結(jié)腸黏膜的充血、水腫、潰瘍和糜爛數(shù)目及面積病理改變情況，然后取少量結(jié)腸組織石蠟包埋，進(jìn)行HE染色，光鏡下觀察各組結(jié)腸組織病理形態(tài)變化。剩余的結(jié)腸組織標(biāo)本用于PCR-Array及RT-PCR和蛋白免疫印跡(Western blot)等技術(shù)檢測。 結(jié)果：1.與空白組比較，模型組及各治療組的結(jié)腸黏膜在肉眼觀察下存在一定程度的充血、水腫、潰瘍及糜爛；光鏡下觀察也存在不同程度的潰瘍、炎癥及并發(fā)的肉芽和纖維化增生。給予痛瀉二草方治療后與模型組比較，各組大鼠的結(jié)腸黏膜損傷評分和病理切片評分均降低，差異顯著（P0.01或P0.05）。 2.與空白組比較，模型組結(jié)腸組織中Nfkb1、Nfrkb、Ikbkb基因表達(dá)量均升高，差異有統(tǒng)計學(xué)意義（P0.01）。給予痛瀉二草方治療后Nfkb1、Nfrkb、Ikbkb基因表達(dá)量與模型組比較均有所降低（P0.01或P0.05），其中高劑量組各基因的表達(dá)量差異顯著（P0.01）。痛瀉二草方高劑量組與SASP治療組比較，Nfkb1、Nfrkb表達(dá)差異無統(tǒng)計學(xué)意義，Ikbkb表達(dá)差異有統(tǒng)計學(xué)意義（P0.05）。 3.與空白組比較，模型組結(jié)腸組織中NF-κB-P65、IκB-α、IκB-β蛋白相對表達(dá)水平增高，差異顯著（P0.01）。給予痛瀉二草方治療后NF-κB-P65、IκB-α、IκB-β蛋白表達(dá)水平降低（P0.01或P0.05），且高劑量組各蛋白表達(dá)量降低差異顯著（P0.01）。痛瀉二草方高劑量組與SASP治療組比較各蛋白表達(dá)差異均有統(tǒng)計學(xué)意義（P0.01或P0.05）。 結(jié)論：1.痛瀉二草方可以抑制肝郁脾虛型UC病程中炎癥細(xì)胞浸潤，減輕炎癥反應(yīng)，對腸黏膜的損傷修復(fù)有促進(jìn)作用，，能夠使腸黏膜分泌功能恢復(fù)正常。說明本研究肝郁脾虛型UC模型復(fù)建成功，且痛瀉二草方可以通過減輕炎癥反應(yīng)，修復(fù)腸黏膜，進(jìn)而起到治療肝郁脾虛型UC的作用。 2.肝郁脾虛型UC結(jié)腸黏膜Nfkb1、Nfrkb、Ikbkb的表達(dá)水平能夠反映結(jié)腸黏膜的損傷程度，對病情的評估及治療效果的判斷可能具有潛在指導(dǎo)意義。 3.痛瀉二草方的治療作用可能與下調(diào)Nfkb1、Nfrkb、Ikbkb基因的表達(dá)水平，進(jìn)而減輕黏膜炎癥反應(yīng)，起到修復(fù)黏膜的作用有關(guān)。提示Nfkb1、Nfrkb、Ikbkb基因位點可能是痛瀉二草方治療肝郁脾虛型UC的關(guān)鍵靶位。 4.痛瀉二草方對肝郁脾虛型UC大鼠結(jié)腸黏膜NF-κB-P65、IκB-α、IκB-β蛋白的表達(dá)具有下調(diào)作用，進(jìn)而可以抑制炎癥的發(fā)生發(fā)展，促進(jìn)修復(fù)損傷的黏膜。提示痛瀉二草方對肝郁脾虛型UC的治療作用與下調(diào)NF-κB-P65、IκB-α及IκB-β蛋白表達(dá)水平有關(guān)。
[Abstract]:Objective: to establish a rat model of Ulcerative colitis (UC) with liver depression and spleen deficiency type ulcerative colitis (UC) by combination of binding stimulation plus three nitrobenzene sulfonic acid (TNBS) and ethanol mixed enema, and observe the effect of TXECF on the histopathology of intestinal histology in UC rats with liver depression and spleen deficiency and the genes related to NF- kappa B signaling pathway Thus, we can elucidate the mechanism and therapeutic targets of TXECF in treating UC of liver depression and spleen deficiency.
Methods: 90 SPF grade Wistar rats were divided into blank group, model group, TXECF high dose group, middle dose group, TXECF low dose group, TXECF low dose group, positive group (SASP group), 15 rats in each group. The blank group was treated with distilled water and equal volume enema. The rest groups were combined with the combination of TNBS and ethanol mixed enema with the combination of TNBS and ethanol enema. After the success of the model of UC rats with spleen deficiency and spleen deficiency, the treatment group was given the corresponding concentration of TXECF. The positive group was given the gavage of sulfasalazine (SASP). The blank group and the model group were given equal volume of distilled water to the stomach, 1 times a day. After the last time of the end of the last perfusion of the 21d. rats, the fasting was 24h, and chloral hydrate anaesthetized with hydrated. The whole colonic tissue was opened, and the colonic mucosa in each group was carefully observed with the naked eye. The number and area of the colonic mucosa were carefully observed. Then a small amount of colonic tissue was embedded in paraffin and stained with HE. The pathological changes of the colon tissue were observed under light microscope. The remaining colonic tissue specimens were used for PCR-Array and RT-PCR. And protein immunoblot (Western blot) technology detection.
Results: 1. compared with the blank group, there was a certain degree of congestion, edema, ulcers and erosions in the colonic mucosa of the model group and the treatment groups. Under the light microscope, there were different degrees of ulcers, inflammation and complicated granuloma and fibrotic hyperplasia. After the treatment of the two herbs of pain and diarrhea, the colon was compared with the model group, and the colon of the rats in each group Mucosal injury score and pathological section score decreased significantly (P0.01 or P0.05).
2. compared with the blank group, the expression of Nfkb1, Nfrkb and Ikbkb in the colon tissue of the model group increased, and the difference was statistically significant (P0.01). The expression of Nfkb1, Nfrkb, Ikbkb in the group of Nfkb1, Nfrkb, Ikbkb decreased (P0.01 or P0.05), and the expression of each gene in the middle high dose group was significantly different (P0.01). Two of the pain diarrhea. There was no significant difference in the expression of Nfkb1 and Nfrkb between the high dose group and the SASP group. The difference of Ikbkb expression was statistically significant (P0.05).
3. compared with the blank group, the relative expression level of NF- kappa B-P65, I kappa B- alpha and I kappa B- beta protein in the colon tissue of the model group was increased significantly (P0.01). The expression level of NF- kappa B-P65, I kappa B- alpha, I kappa protein expression level decreased after the treatment of pain diarrhea two grass prescription, and the expression of protein expression in the high dose group decreased significantly. Compared with the SASP treatment group, the difference of protein expression was statistically significant (P0.01 or P0.05).
Conclusion: 1. pain and diarrhea two grass can inhibit the infiltration of inflammatory cells in the course of liver depression and spleen deficiency of UC, alleviate the inflammatory reaction, promote the repair of intestinal mucosa damage and restore the secretion function of the intestinal mucosa to normal. It shows that the liver depression and spleen deficiency type UC model is successfully rebuilt, and the pain and diarrhea two grass can repair the intestine by alleviating the inflammatory reaction and repairing intestines. Mucosa plays a role in the treatment of UC of liver depression and spleen deficiency.
2. the expression level of Nfkb1, Nfrkb, Ikbkb in the UC colon mucosa of the liver depression and spleen deficiency can reflect the damage degree of the colonic mucosa, and the evaluation of the disease and the judgment of the therapeutic effect may be of potential guiding significance.
3. the therapeutic effect of Tong Xie two grass recipe may be related to the reduction of the expression level of Nfkb1, Nfrkb, Ikbkb gene and the mucosal inflammation, which may play a role in the repair of mucous membrane. It is suggested that the Nfkb1, Nfrkb, and Ikbkb gene loci may be the key target for the treatment of the liver depression and spleen deficiency type UC.
4. pain and diarrhea two grass recipe has a down-regulation effect on the expression of NF- kappa B-P65, I kappa B- alpha and I kappa B- beta protein in the colon mucosa of UC rats with liver depression and spleen deficiency, which can inhibit the occurrence and development of inflammation and promote the repair of damaged mucosa. It suggests that the expression level of NF- kappa B-P65, I kappa B- alpha and kappa beta protein in the treatment of the liver depression and spleen deficiency type UC by the pain diarrhea two herb prescription. Close.
【學(xué)位授予單位】：甘肅中醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R259;R285.5

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