【作者】 冯俊；

【导师】 夏先明；

【作者基本信息】 泸州医学院 ， 外科学， 2011， 硕士

【摘要】 目的:本研究通过建立高脂血症大鼠模型,以肠道Na~+/胆汁酸转运体(ileal bile acid transporter,IBAT)为研究对象,探讨肠道Na~+/胆汁酸转运体与胆汁酸代谢及高脂血症形成的相关性,为防治高脂血症提供新的依据和分子理论基础,有望为治疗胆汁酸代谢紊乱及相关性疾病提供一种新的思路。方法:取Wistar雄性大鼠(体重145±8.5g)60只,随机分为2组,每组30只:普通饮食对照组(简称对照组)、高脂饮食实验组(简称实验组)。对照组予以普通饮食,实验组予以高脂饮食(配方为:在基础饲料中加入质量分数为0.04胆固醇、0.1猪油、0.05蔗糖、0.005胆酸钠、0.002丙基硫氧嘧啶),喂食90天,建立高脂血症模型,定期取血用自动生化仪检测胆固醇含量,4-6周监测血胆固醇水平,观察建模是否达到要求。两组入选大鼠均需排除已饮用含其它类药物饮食,高脂血症﹑糖尿病﹑畸形等疾病存在。建模成功后,所用实验方法分为以下两个环节:1.分别提取对照组和实验组的空肠末端、回肠末端、盲肠及结肠近端组织的RNA,应用逆转录-聚合酶链反应(RT-polymerase chain reaction; RT-PCR)技术检测两组模型肠组织的IBAT基因表达的强度;2.取对照组和实验组的空肠末端、回肠末端、盲肠及结肠近端组织,石蜡切片后用免疫组化SP(streptavidin-perosidase)法检测两组模型肠组织的IBAT表达的强度。统计数据运用SPSS14.0 for windows统计软件包作统计学处理,P <0.05显示有统计学意义。结果: 1.电泳结果显示:实验中空肠末端、回肠末端、盲肠扩增产物都出现了内参β-actin基因的220bp扩增带和IBAT基因的355bp扩增带,且实验组IBAT基因扩增带亮度明显强于对照组。结肠近端中未见IBAT基因扩增带。半定量结果分析:利用Quantity One软件计算比较每一泳道中PCR产物条带的光密度值,计算IBAT基因与β-actin条带的光密度的相对比值,通过比较各样本IBAT基因的相对值,最终得出IBAT基因在空肠、回肠末端、盲肠组织中mRNA表达的相对含量。半定量结果的相对光密度值需每组每一实验对象同等处理条件下均重复测定3次,取其最终平均值作为统计用数据。最后结果显示:结肠近端在实验组和对照组均无表达,其余在对照组IBAT基因mRNA表达较低,空肠末端(0.88427±0.041664)、回肠末端(0.92013±0.049935)、盲肠(0.86777±0.0336157),实验组IBAT基因mRNA表达较高,空肠末端(1.12817±0.050841)、回肠末端(1.14613±0.061868)、盲肠(1.14320±0.048016)表达差异性显著,空肠近端(t=-25.6988 P<0.05)、回肠末端(t=-15.446 P<0.05)、盲肠(t=-24.007 P<0.05),均有统计学意义。2.免疫组化SP(streptavidin-perosidase)法结果显示:在高倍显微镜镜下肠道组织中出现黄色染色颗粒为阳性细胞。每张切片随机选取5个视野,每个视野内观察100个细胞,阳性细胞≥10%为阳性,否则为阴性,利用四格表的Fisher确切概率法检验。结果显示:实验组空肠末端、回肠末端、盲肠表达阳性率分别为66.3%、78.5%、55.3%;对照组空肠末端、回肠末端、盲肠表达阳性率为10.3%、12.5%、8.95%。它们之间的差异有显著性,空肠末端IBAT基因(X2=9.631 P<0.05),回肠末端IBAT基因(X2=9.873 P<0.05),盲肠IBAT基因(X2=9.209 P<0.05)。结论:1.实验组大鼠的IBAT基因mRNA的表达较对照组明显增加。2.实验组和对照组大鼠的结肠近端中未见IBAT基因的表达。3.实验组空肠末端,回肠末端及盲肠IBAT的表达明显高于对照组,故在高脂血症动物回肠中胆汁酸的主动转运及空肠中胆汁酸的被动转运过程增强,及盲肠参与胆汁酸转运增强,进而胆汁酸摄取率增加,增加体内胆酸池大小和肝内胆固醇含量,从而引起血清胆固醇增加,加速高脂血症的形成。。更多还原

【Abstract】 Objective: In this study, through the establishment of hyperlipidemia rats to ileum Na~+ / bile acid transporter as the research object of ileal Na~+ / bile acid transporter and bile acid metabolism and hyperlipidemia in the formation of the correlation, for the prevention and control high fat hyperlipidemia disease providing a new way of thinking.Method: Take Wistar rats ( male, weight 145±8.5g) 60 rats were randomly divided into 2 groups: normal diet control group, high fat diet experimental group. Be compared to normal diet control group, the experimental group to high fat diet . (Formula: in the basal feed Concentration 0.04 cholesterol, 0.1 lard, 0.05 sucrose, 0.005 sodium cholate, 0.002 propylthiouracil), through 90 days of feeding, the establishment of hyperlipidemia model, using of automatic biochemical analyzer blood cholesterol testing regularly, monit oring the blood cholesterol levels four to six weeks, observe whether the requirements modeling. Two groups which selected required to exclude non-Winstar type rats, whether eating food with other drugs, hyperlipidemia, diabetes, deformities or other diseases . After the success of mode ling, experimental methods used were divided into the following two aspects:1. Extraction of the control group and experimental group ileal intestinal tissue and colon tissue of RNA, reverse transcription polymerase chain reaction detection of two model ileal intestinal tissue of IBAT gene expre- ssion intensity. 2. Extraction of the control group and experimental group ileal intestinal tissue, paraffin sections by indirect immunofluorescence after staining were detected ileal intestinal tissue model of the IBAT expression intensity. Statistical data used SPSS14.0 for windows statis- tical package X2,Fish paired t test and exact test for statistical analysis, P <0.05 indicate statistical significance. Result: 1. Electrophoresis results showed that: the experiment Ileum PCR products have emerged internal referenceβ-actin gene of 355bp amplified 220bp band and IBAT gene was amplified with the experimental group, with a brightness of IBAT gene amplification was significantly stronger than the control group. Semiquantitative results analysis: calculated using quantity One software more lanes in each PCR product band of optical density, calculated IBAT gene andβ-actin band of the optical density of the relative ratio of IBAT gene by comparing the relative value of the sample, eventually come to IBAT gene expression in ileal tissue mRNA relative content. Semiquan- titative results of the relative optical density of each of the subjects in each group required the same processing conditions are determined to repeat 3 times, whichever is the final average of data for statistical use. The final results: the experimental group gene mRNA expression in rat IBAT higher Jejunum end(1.12817±0.050841) ,Ileum end(1.14613±0.061868)、caecum(1.14320±0.048016),control rat IBAT gene mRNA expression was lower Jejunum end (0.88427±0.041664)、Ileum end(0.92013±0.049935)、caecum(0.86777±0.0336157),the expression of significant differences was statistically significant (t=-25.6988, t=-15.446 t=-24.007 P <0.05).2 Indirect immunoflu- orescence staining showed that: in the high-powered microscope, fluore- scence microscope ileal tissue particles appear yellow green staining positive cells. 5 randomly selected for each slice view, each view to obse- rve the 100 cells,≥20% positive. Use tables from the Fisher exact prob- ability test results show: the experimental group IBAT expression was 66.3%,78.5%,55.3%; the control group IBAT expression was 10.3%,12.5%,8.95% . The difference between them was significant, there was significant (X2=9.631 ,X2=9.873,X2=9.209 P <0. 05).Conclusion: 1. Experimental group of IBAT mRNA gene expression more than control group increased. 2. Experimental group and control group in the rat colon no IBAT gene expressi on. 3.Ileal experim- ental group was significantly higher expression of IBAT, so ileum bile acid active trans port process of enhanced bile acid uptake was increased, theincreaseingin cholesterol and lipid absorption, accelerate the formation of hyperli pidemia更多还原