【作者】 羊燕群；

【导师】 陈蔚文；

【作者基本信息】 广州中医药大学 ， 中西医结合临床， 2009， 博士

【摘要】 目的:观察理中汤对脾阳虚证模型大鼠吸收功能的影响,探讨其调节小肠上皮细胞刷状缘膜囊(BBMV)上肽转运载体PepT1转运功能、影响PepT1的mRNA和蛋白表达的作用机制;并进一步寻找其中可能的调节途径及因素。方法:1.建立脾阳虚大鼠模型造模方法:采用SD雄性大鼠,灌胃4℃的10g/kg番泻叶液每日一次;皮下注射利血平0.5mg/kg隔日一次,游泳10min(水温38℃)隔日一次,利血平注射与游泳交替进行,以上造模因素持续3周后,通过一般症状观察、尿D-木糖排泄率检测、采集小肠组织,HE染色病理学检查等指标的观测评价,建立利血平复合因素脾阳虚证大鼠造模方法。2.理中汤疗效观察:雄性SD大鼠分为模型组和正常对照组。模型组大鼠用利血平复合因素造模法造模,正常对照组隔日皮下注射等体积生理盐水,每日灌胃蒸馏水但不游泳,第3周末通过一般症状观察、尿D-木糖排泄率检测判断模型成立。模型大鼠分为理中汤给药组和未治疗模型组,分别给予理中汤灌胃和水灌胃处理4周,通过一般症状观察、尿D-木糖排泄率检测、采集小肠组织,HE染色病理学检查和透射电镜下观察小肠黏膜细胞超微结构等指标的观测评价理中汤的治疗效果。3.雄性SD大鼠分为模型组和正常对照组。模型组大鼠用利血平复合因素造模法造模,正常对照组隔日皮下注射等体积生理盐水,每日灌胃蒸馏水但不游泳,第3周末通过一般症状观察、尿D-木糖排泄率检测判断模型是否成立。随机选取部分模型大鼠和对照组大鼠,采集血清,用ELISA法测定小肠黏膜上皮细胞内cAMP水平及血清中相关影响因素Leptin及细胞因子--TNF-α、IFN-γ水平;通过给大鼠十二指肠内注射肽类似药物β-内酰氨类抗生素头孢氨苄(cefalexin,作为小肠内PepT1转运标志物),采集给药后2h血清,用反相高效液相法测定其体内血药浓度,观察PepT1转运的状态;采集大鼠小肠黏膜,用RT-PCR法测定小肠粘膜PepT1mRNA含量;采集小肠组织,免疫组化法半定量检测PepT1蛋白在小肠黏膜上皮细胞刷状缘膜囊(BBMV)的表达;剩余大鼠停止造模因素分为治疗组和非治疗模型组:治疗组每日给理中汤水煎液灌胃,非治疗模型组每日给水灌胃,第4周末即实验第7周末处死大鼠,同前法采集标本测定同前法采集标本。结果:1.利血平复合因素造模大鼠至第3周,出现饮食减少、消瘦、皮毛杂乱无光、倦怠懒动、泻下烂便、体温降低、喜扎堆;尿D-木糖排泄率明显低于正常大鼠(P＜0.05);透射电镜下观察,小肠黏膜柱状上皮细胞间隙变宽,粗面内质网间隙增宽,线粒体肿胀,大小不一,嵴断裂、减少或结构模糊,甚至有空泡样改变,与正常组结构有明显变化。2.脾阳虚模型组大鼠造模3周后,与正常对照组大鼠比较,cefalexin血药浓度显著升高(P＜0.01),小肠黏膜上皮细胞刷状缘膜囊(BBMV)上PepT1蛋白表达明显升高(P＜0.01),小肠黏膜PepT1 mRNA水平与正常组比较无显著性差异(P＞0.05),小肠黏膜cAMP与正常组比较,显著升高(P＜0.05)。3.停止造模4周后,理中汤治疗组大鼠活动度增加,大便软硬适中,皮毛恢复光泽,尿D-木糖排泄率与正常对照组比较无显著性差异(P＞0.05),大鼠小肠上皮细胞线粒体大小与正常对照组相似,结构清晰,大多数嵴较清楚,偶见嵴断裂现象;非治疗模型组大鼠活动度增加,大便软硬适中,皮毛恢复光泽,尿D-木糖排泄率与正常对照组比较显著性降低(P＜0.05),大鼠小肠上皮细胞线粒体大小不均匀,仍有嵴断裂、残留、空泡样改变。4.停止造模后4周,脾阳虚大鼠模型理中汤治疗组与正常组相比,cefalexin血药浓度、大鼠小肠黏膜BBMV上PepT1蛋白表达下降至正常水平(P＜0.05);小肠黏膜cAMP与非治疗组比较,显著升高(P＜0.05);非治疗模型组cefalexin下降至正常对照组水平,小肠黏膜上皮细胞BBMV上PepT1蛋白表达与正常组比较仍处高水平(P＜0.01);小肠黏膜PepT1 mRNA水平在3组动物中无明显差异。结论1.复合因素合利血平造模法建立脾阳虚证大鼠模型,经一般症状观察,尿D-木糖排泄率检测,细胞内超微结构观察,认为符合脾阳虚证模型要求,适用于理中汤小肠吸收药理学研究。2.温阳健脾方药理中汤对脾阳虚证大鼠的小肠吸收功能有改善作用,体现于改善症状,提高尿D-木糖排泄率,改善小肠黏膜柱状上皮细胞内超微结构。3.理中汤可使脾阳虚大鼠BBMV异常升高的PepT1蛋白恢复正常水平,提高cefalexin血药浓度和cAMP水平,提示理中汤治疗脾阳虚吸收功能不良的作用可能与cAMP-PepT1调节机制相关。更多还原

【Abstract】 Object:To explore the effects of Li-zhong decoction on small intestinal peptide transporter(PepT1) in Spleen-Yang deficiency rats,and regulate factors connecting them.Methods:1) Set up the spleen-yang deficiency rat model:Using the composite factors method to set up the spleen-yang deficiency rat model for 3 weeks on male SD rats,which includes intragastric administration of cold senna water at 10g/kg everyday,hypodermic injection apoplon at 0.5mg/kg every other day,and swimming for lOmins every the other day. Verifying models based on manifestation,urine D-xylose excretory rate, andultramicrostructureunder electron microscope.2) Observe the curative function of Li-zhong decotction:Male SD rats were deivded into 2 groups: model group and blank group.The model group rats were bearing the composite factors method to set up the spleen-yang deficiency rat model,and the blank group rats were giving subcutaneous injection and intragastric administration with saline but without swimming for 3 weeks.Stopping all of the factors and giving part of the model rats Li-zhong decoction and the rest water as well as the blank group rats for 4 weeks.Evaluating function of Li-zhong decoction based on manifestation,urine D-xylose excretory rate,and ultramicrostructure under electron microscope.3) Set up spleen yang deficiency rat models with the composite factors method and select parts of normal rats and model rats to observe translating activity of PepT1 by RP-HPLC detecting the blood concentration of cefalexin,which has the similar chemical construction with peptide,after being administrated in duodenum with pylorus ligated.Detect PepT1 protein in small intestine mucosa by way of Western Blot.Detect PepT1 mRNA by way of RT-PCR.Detect influential factors of PepT1 such as cAMP,TNF-α,IFN-γand leptin by way of ELISA.Give model rats with or without Li-Zhong decoction for 4 weeks and observe the same projects.According to different experiment purposes,serum and mucosa of small intestine were collected.Results:1) Spleen-yang deficiency rats modeled with the composite factors method for 3weeks were thin and lassitude with poor appetite,dirty fur,loose stool,low temperature,decreased urinary D-xylose excretory rate,and construction changes of chondrosome with swell,collapse or disappear of cristae.2) The blood concentration of cefalexin was higher of spleen-yang deficiency group than that of normals(P＜0.01),which had no difference with normal group and groups treated with or without Li-Zhong decoction(P＞0.05).3) Protein of PepT1 in spleen-yang deficiency group higher than the normal group(P＜0.01),and that of the group treated with Li-Zhong decoction recovered to have no difference with the normal(P＞0.05), which still remain higher than the normal that of the group treated without Li-Zhong decoction(P＜0.05).4) There was no difference of mRNA between groups normal and spleen-yang deficiency,as well as between groups treated with or without Li-Zhong decoction(P＞0.05).5) cAMP in intestine mucosa of spleen-yang deficiency was higher than that of normal(P＜0.05),which was much lower in rats treated without Li-Zhong decoction than that of rats treated with decoction(P＜0.05).Conclutions:1) The composite factors method modeling spleen yang deficiency rats is suitable for pharmacology study of Li-zhong decoction evaluated by manifestation,urinary D-xylose excretory rate, ultramicrostructure of cellula columnoepithelialis.2) Li-zhong decoction has the function of improving absorption of small intestine which was verified by relieving manifestation of spleen yang deficiency,increasing urinary D-xylose excretory rate,ameliorating ultramicrostructure of cellula columnoepithelialis in small intestine.3) Li-zhong decoction treating malfunction of absorption of small intestine in spleen yang deficiency may through way of cAMP-PepT1 regulatory road.更多还原