【作者】 周毅；

【导师】 邓虹珠；

【作者基本信息】 南方医科大学 ， 中药学， 2007， 硕士

【摘要】 目的:探讨苦豆子总碱(TASA)治疗大鼠溃疡性结肠炎的作用机制。方法:1.观察TASA对溃疡性结肠炎(UC)大鼠模型病理改变的影响,大鼠UC模型由三硝基苯磺酸(TNBS)/乙醇灌肠制成,评价UC大鼠在急慢性期(第7、21天)的症状和观察结肠组织学变化;2.通过流式细胞术观察TASA对UC大鼠外周血(PB)和结肠组织(COLON)中调节性T细胞(regulatory T cells,Tr)CD4~+CD25~+,CD8~+CD28~-分别在急慢性期中表达的影响;3.通过酶联免疫吸附法(ELISA)和逆转录酶PCR(RT-PCR)观察TASA对UC大鼠细胞因子IL-10表达的影响;4.探讨COLON炎症慢性期的疾病活动指数评分(DAI)与CD8~+CD28~-Tr之间的相关性,COLON炎症慢性期的组织学损伤与CD8~+CD28~-Tr之间的相关性;PB CD4~+CD25~+Tr和细胞因子IL-10表达的相关性;COLON CD4~+CD25~+Tr和细胞因子IL-10表达的相关性。结果:1.在急慢性期中,模型组大鼠DAI和组织学损伤评分显著高于正常组(P＜0.05),TASA三个剂量组(60、30、15mg/kg)在慢性期中均低于模型组(P＜0.05)。2.①建模后第7天:与正常对照组相比,模型组COLON内T细胞亚群CD4~+CD25~+,CD4~+CD25~+/CD25~-显著降低,CD8~+CD28~-及CD8~+CD28~-/CD28~+显著升高(P＜0.05),TASA三个剂量组虽有调节作用,但统计学差异不显著(P＞0.05)。②建模后第21天:与正常对照组相比,模型组COLON中的CD4~+CD25~+、CD4~+CD25~+/CD25~-表达显著降低,CD8~+D28~-、CD8~+CD28~-/CD28~+的表达显著升高(P＜0.01),TASA三个剂量组能有效的调节其表达(P＜0.05);模型组大鼠PB中的CD8~+CD28~-和CD8~+CD28~-/CD28~+的表达高于正常对照组,TASA三个剂量组能有效的下调其高表达(P＜0.05)。③除正常组外,模型组、5-ASA组、苦豆子三个剂量组的COLONCD8~+CD28~-表达的增长幅度与PB的相比,分别在第7天和第21天中都有显著性意义(P＜0.05)。3.在ELISA检测中,模型组COLON和PB中IL-10的表达较对照组均显著降低(P＜0.01),其中以COLON中的IL-10降低的更加明显(P＜0.01);且TASA高、中、低三个剂量组都能显著性上调COLON和PB中的IL-10的表达(P＜0.05),但COLON的治疗变化与PB的相比差异不明显。4.COLON炎症慢性期的DAI与CD8~+CD28~-Tr之间存在的正相关性(Pearson r=0.828,p＜0.01),COLON炎症慢性期的组织学损伤与CD8~+CD28~-Tr之间存在正相关性(Pearson r=0.819,P＜0.01);COLON炎症慢性期的CD4~+CD25~+Tr与细胞因子IL-10表达成正相关(Pearson r=0.789,P＜0.01);PB炎症慢性期的CD4~+CD25~+Tr与细胞因子IL-10表达无相关性(Pearson r=-0.191,P＞0.05),每组检测都为双侧,样本容量N=42。结论:1.苦豆子总碱能改善UC大鼠结肠的一般病理变化:各给药组可见黏膜脱落或坏死减轻,黏膜及黏膜下层炎细胞(淋巴细胞,中性粒细胞,嗜酸性粒细胞)浸润减少,纤维组织只有少量增生,偶见小溃疡,症状改善明显;2.TASA通过有效的调节CD4~+D25~+Tr、CD8~+CD28~-Tr,缓解炎症反应;3.TASA能升高UC大鼠血清细胞因子IL-10的表达,改善炎症状态;4.COLON炎症慢性期的DAI与CD8~+CD28~-Tr之间存在的正相关性,COLON炎症慢性期的组织学损伤与CD8~+CD28~-Tr之间存在正相关性;COLON炎症慢性期的CD4~+CD25~+Tr与细胞因子IL-10表达成正相关;PB炎症慢性期的CD4~+CD25~+Tr与细胞因子IL-10表达无相关性。更多还原

【Abstract】 Obeject:To study the mechanism of action about treatment of total alkaloid of Sophora alopecuroides(TASA)on rats with experimental colitis.Methods:1.To observe the treated effects of TASA on pathological change of rats with experimental colitis In the 7th day and the 21th day,the experimental colitis model of rats was induced by administrating 2,4,6-trinitrobenzesulphonic acid (TNBS)/ethanol rectally,the symptoms and colonic histology were evaluated.2.To observe the effects of TASA on the expression of CD4~C+D25~+Tr、CD8~+CD28~- Tr of rats with experimental colitis by FCM.3.To observe the effects of TASA on IL-10 cytokine of rats with experimental colitis by ELISA and RT-PCR.4.To respectively study the correlation between the expression of CD8~+CD28~-Tr and DAI,between the expression of CD8~+CD28~-Tr and histological grading of colitis in the colon of rats and between the expression of CD4~+CD25~+Tr and IL-10 cytokine in the colon and peripheral blood(PB)of rats.Results:1.A significant decrease was found in DAI and Histological grading of colitis of normal group in the 7th day and the 21th day and all TASA groups, compared with model group(P＜0.01),could alleviate the symptoms and histological damages of model rats in the 21th day(P＜0.05).2.①In the 7th day after modelling: The proportion of CD8~+CD28~-Tr and CD8~+CD28~-/CD28~+ in the colon of colitis rats of model group was higher than that in normal control group,but the proportion of CD4~+CD25~+Tr,CD4~+CD25~+/CD25~- is lower than that in normal control group (P＜0.01).Compared all TASA groups with model group,the statistical differences were not significant(P＞0.05).②In the 21th day after modelling:The expressions of CD8~+CD28~-Tr and CD8~+CD28~-/CD28~+ Tr in the PB and colon of rats of model group were obviously higher than those in normal control group and all TASA groups(P＜0.05),and the expressions of CD4~+CD25~+Tr and CD4~+CD25~+/CD25~- in the colon tissues were lower than those in normal control group and all TASA groups(P＜0.01).③Growth rates of colon’s CD8~+CD28~-Tr expression of model,5-ASA and all TASA groups obviously are higher in the colon of colitis rats than that of PB(P＜0.05).3①The expression oflL-10 cytokine in the colon and the PB of colitis rats was lower than that of normal control group(P＜0.01),moreover, the expression in the colon tissue was the lowest of them.The expression of IL-10 cytokine in the rats with TASA was almost as well as that of normal control group.4 The positive correlations were demonstrated between the expression of CD8~+CD28Tr and DAI(Pearson r=0.828,P＜0.01),between the expression of CD8~+CD28~-Tr and histological grading of colitis(Pearson r=0.819,P＜0.01),also existed CD4~+CD25~+Tr and IL-10 cytokine in the colon of rats(Pearson r=0.789, P＜0.01),whereas,no correlation was found between CD4~+CD25~+Tr and IL-10 cytokine in the PB of rats(r=-0.191,P＞0.05). Conclusions:1.TASA could significantly alleviate the pathological changes of rats with experimental colitis,which can obviously reduce exfoliation or necrosis of mucosa and decreased inflammatory cell in the mucosa and mucosa underlayer on the rats with colitis.The colon only contain a few small fibroplasias or ulcers.2.TASA could upgrade the expression of CD4~+CD25~+Tr and degrade the expression of CD8~+CD28~- Tr of rats with experimental colitis,relieve the inflammatory reaction.3. TASA could significantly regulate the expression of IL-10 cytokine of UC rats, improve the symptoms.4 The positive correlations were demonstrated between the expression of CD8~+CD28~-Tr and DAI,between the expression of CD8~+CD28~-Tr and histological grading of colitis,also existed CD4~+CD25~+Tr and IL-10 cytokine in the colon fo rats,whereas,no correlation was found between CD4~+CD25~+Tr and IL-10 cytokine in the PB of rats更多还原