【作者】 宋士一；

【导师】 王德山；

【作者基本信息】 辽宁中医药大学 ， 中西医结合基础， 2010， 博士

【摘要】 目的：本研究以分子生物学、形态学等检测技术,检测眼针治疗对IBS模型大鼠结肠和血清5-HT及5-HIAA水平,结肠EC细胞表达,结肠TPH1、SERT、5-HT3R和5-HT4R蛋白和mRNA表达水平的影响,旨在从分子水平探讨眼针治疗IBS的5-HT机制,为眼针治疗IBS提供实验依据。材料与方法：1.动物造模及眼针治疗健康SPF级Wistar雄性大鼠30只,分成对照组、模型组、眼针组,采用束缚刺激与应激刺激相结合的方法建立IBS动物模型。模型组、眼针组每日放入自制束缚性鼠笼1h进行束缚刺激,并每日依次交替进行一项下列应激刺激：①禁水24h②置于温箱40℃,5min③电击刺激30V,电击5s间歇5s共120s④冷水游泳14℃,5min⑤禁食24h⑥夹尾180s；共循环3次计18日造模结束。对照组无处理。眼针组在IBS大鼠造模成功后第二天开始,取下焦区、大肠区、肝区、脾区针刺,定位参照人体取穴方法；用31号13mm毫针,于大鼠眶周2mm处平刺,进针3mm,留针20min,留针期间捻转行针3次,每次1min,每12h针刺1次,针刺7天。2.指标检测采用免疫组化方法检测结肠EC细胞并进行分析；ELISA法检测血清和结肠5-HT和5-HIAA水平；Western-Blot法、RT-PCR法检测结肠TPH1、SERT、5-HT3R、5-HT4R的蛋白和mRNA表达。结果：1.眼针对IBS模型大鼠一般状态影响实验过程中动物无死亡。与对照组比较,IBS模型大鼠出现易怒好斗、毛色晦暗、食欲下降等表现；稀便状态持续存在,慢性腹泻形成；观察结肠病理学、结肠转运功能和肠道敏感性等的变化对模型进行评价,依据成模标准表明造模成功。经过眼针治疗后大鼠一般状态明显改善,便质及转运功能逐渐恢复正常。2.眼针对IBS模型大鼠结肠5-HT和5-HIAA水平的影响与对照组比较,模型组5-HT和5-HIAA水平均升高(P<0.05),5-HT转化百分率也升高(P<0.05)；而与模型组比较,眼针组5-HT和5-HIAA水平下降(P<0.05),眼针组5-HT转化百分率降低(P<0.05)。3.眼针对IBS模型大鼠结肠EC细胞表达的影响与对照组比较,模型组5-HT免疫阳性细胞数量增多(P<0.05),免疫组化染色强度也增强(P<0.05)；与模型组比较,眼针组5-HT免疫阳性细胞数量、免疫组化染色强度显著降低(P<0.05)。4.眼针对IBS模型大鼠结肠TPH1和SERT表达水平的影响与对照组比较,模型组TPH1的蛋白和mRNA表达均上调(P<0.05)；与模型组比较,眼针组TPHl表达下调(P<0.05)。与对照组比较,模型组SERT的蛋白和mRNA表达均下调(P<0.05)；而与模型组比较,眼针组SERT表达上调(P<0.05)5.眼针对IBS模型大鼠结肠5-HT3R和5-HT4R表达水平的影响与对照组比较,模型组5-HT3R的蛋白和mRNA表达均上调(P<0.05)；而与模型组比较,眼针组5-HT3R表达下调(P<0.05)。与对照组比较,模型组5-HT4R的蛋白和mRNA表达均下调(P<0.05)；而与模型组比较,眼针组5-HT4R表达上调(P<0.05)。眼针治疗通过下调结肠EC细胞及TPH1表达而降低了5-HT合成代谢水平,同时通过上调SERT的表达而提高5-HT分解代谢水平,使5-HT的合成与分解得以相匹配而协调,5-HT恢复到正常水平,进而IBS大鼠结肠功能得以恢复正常。眼针治疗下调5-HT3R表达,上调5-HT4R表达,使两者恢复至正常水平,这可能是眼针治疗IBS的机制之一。结论：1.IBS模型大鼠结肠和血清5-HT和5-HIAA水平升高,5-HT转化百分率也升高；眼针治疗可降低5-HT、5-HIAA水平和5-HT转化百分率,恢复到对照水平。2.IBS模型大鼠结肠EC细胞及TPH1表达上调,SERT的表达下调；眼针治疗能够使EC及TPH1表达下调,SERT的表达上调。3.IBS模型大鼠结肠5-HT3R表达上调,5-HT4R表达下调；眼针治疗使5-HT3R表达下调上调,5-HT4R表达上调。更多还原

【Abstract】 Purpose:To detect colonic EC cells by morphology, serum and colon 5-HT,5-HIAA by ELISA, colon TPH1, SERT,5-HT3R,5-HT4R mRNA and protein expression by molecular biology, and to explore the 5-HT molecular mechanism of OAT on IBS and to provide experimental evidence for OAT on IBS.Material and method:1. Setting up animal model and given OAT30 SPF Wistar male rats were randomly divided into control, IBS model and OAT groups. IBS model was established by method of restraint stress and combining bound. IBS model and OAT groups were put into selfmade cages 1 h and given a stimulus alternately daily①water deprivation,24h②40℃,5min③electric shock,30V,5s aff and on 5s, totally120s④swimming 14℃,5min⑤fasting,24h⑥tail clipped 180s; a total of 18 days 3 cycles. Control group had no treatment.OAT group was given OAT from 2nd day after model set endding. With No.31, 13mm needles, OAT was given 3mm deep acupuncture in parallel for 20min, during retention the needles were moved softly 3 times×1min,2 times everyday for 7 days.2. Indexes were detected Immunohistochemistry was used to detect and analyze colonic EC cells. ELISA was used to detect serum and colon 5-HT and 5-HIAA. Western-Blot and RT-PCR were used to detect and analysis colon TPH1, SERT,5-HT 3 R,5-HT 4 R mRNA and protein.Results:1. The general state of ratsNo animals died during the experiment. Compared with control group, irritable aggressive IBS model rats appeared dull coat, loss of appetite, watery feces and chronic diarrhea. Based on the model standard to observe colonic pathology, transit function and sensitivity, we confirmed that IBS model was successful. OAT significantly improved IBS model rats general state.2. Effects of OAT on colonic 5-HT and 5-HIAA levels in IBS modelCompared with control group, the levels of 5-HT and 5-HIAA in IBS model increased remarkably (P<0.05).Percent conversion of 5-HT in IBS model increased remarkably too. Compared with those in IBS model, the levels of 5-HT and 5-HIAA and percent conversion of 5-HT in OAT decreased remarkably (P<0.05).3. Effects of OAT on colonic EC cells in IBS modelCompared with control group, the stained intensity and the number of EC cells in IBS model increased remarkably (P<0.05). The stained intensity and the number of EC cells in OAT decreased remarkably (P<0.05)compared with those in IBS model.4. Effects of OAT on colonic TPH1 and SERT expression in IBS modelCompared with control group, IBS model TPH1 expression increased (P<0.05) and SERT protein and mRNA expression reduced(P<0.05); OAT TPH1 expression reduced (P<0.05) and SERT protein and mRNA expression upregulated(P<0.05) compared with IBS model group. 5. Effects of OAT on colonic 5-HT 3 R and 5-HT 4 R expression in IBS modelCompared with control group, IBS model 5-HT 3 R expression increased (P<0.05) and 5-HT 4 R protein and mRNA expression reduced (P<0.05); OAT 5-HT 3 R expression reduced (P<0.05) and 5-HT4 R protein and mRNA expression upregulated(P<0.05) compared with IBS model group.OAT decreased 5-HT synthesis by downregulated EC cells and TPH1 expression, while the expression of SERT was upregulated to improve the level of 5-HT catabolism, so that the 5-HT synthesis and catabolism can be matched and coordinated,5-HT levels returned to normal, then IBS rat colon to restore normal function. OAT downregulated the expression of 5-HT3R, upregulated 5-HT4R expression, so that the two returned to normal levels, which may be the mechanism of OAT for IBS.Conclusion:1. The levels of colon and serum 5-HT,5-HIAA, and Percent conversion of 5-HT increase in IBS model rats. OAT can downregulate those.2. IBS model rats have increase in EC number and stained intensity, increase TPH1 expression and reduce SERT expression. OAT can reduce EC number and stained intensity, TPH1 expression and increase expression of SERT.3. IBS model rats increase 5-HT 3 R expression and reduce expression of 5-HT 4 R. OAT can reduce 5-HT 3 R expression and increase expression of 5-HT 4 R.更多还原