【作者】 苏全武；

【导师】 邱明义；

【作者基本信息】 湖北中医学院 ， 中医临床基础， 2009， 博士

【摘要】 目的：研究加味四逆散对肝硬化大鼠胃肠动力障碍防治作用，并对其机制进行初步探讨。通过加味四逆散对肝硬化大鼠一般情况、肝功能、肝脏形态的疗效观察，对肝硬化大鼠胃电图异常的防治作用，从胃肠肽角度探讨实验性肝硬化大鼠胃肠动力紊乱的机制以及加味四逆散防治作用。以ICC（Interstitial cells of Cajal，Cajal间质细胞）表达c-kit（Ⅲ型跨膜蛋白酪氨酸激酶生长因子受体蛋白）为切入点，研究肝硬化大鼠的c-kit表达量变化，以及加味四逆散对其的影响，以GASR（胃泌素受体）、VIPR2（VIP-2 receptor，血管活性肠肽Ⅱ型受体）为代表，在胃肠组织对胃肠肽受体水平测定探讨肝硬化大鼠胃肠动力信号接收系统变化，探讨加味四逆散治疗机理。方法：Wister雄性大鼠48只被随机分为4组，每组12只，除空白组外，其他三组均给予大鼠皮下注射40％CCL₄花生油溶液2．5ml／kg，每周2次造模，连续造模11周。第八周造模同时中药组用“加味四逆散”煎剂灌胃治疗，西药组以西沙比利灌胃治疗，空白、模型组则给予等体积生理盐水灌胃。于11周末检测大鼠胃电图、肝功能、肝组织病理；放免法测定血清、胃、小肠的MTL（Motilin，胃动素）、GAS（gastrin，胃泌素）、VIP（Vosoactiveintestinepoly Peptide，血管活性肠肽）、SS（somatostatins，生长抑素）含量变化。免疫组化法测定胃组织GAS、SS，小肠组织MTL、VIP含量变化；免疫组化法测定胃组织c-kit阳性表达量变化；RT—PCR（Rever se Transcription Polymerase Chain Reaction，逆转录聚合酶联反应）法测定胃组织GASR、空肠组织VIPR2两种受体mRNA的表达及定量分析，检测完毕后对空白组、模型组、西药组及中药组相应数据分别进行统计学分析。结果：（1）模型组一般情况、肝功能、肝脏形态与空白组相比均有显著性差异（P＜0．01或P＜0．05），加味四逆散组与模型组比较肝功能TP（total protein，总蛋白）、ALB（albumin，白蛋白）含量明显升高，ALT（alaninetransamina se，谷丙转氨酶）、AST（aspartate transaminase天冬氨酸转氨酶）含量明显下降，均有显著性差异（P＜0．01或P＜0．05）；肝组织病理改变明显减轻；“加味四逆散”组与西药组比较：肝功能TP、ALB含量明显升高，ALT、AST含量明显下降，均有显著性差异（P＜0．01、P＜0．01或P＜0．05），肝组织病理改变减轻。（2）模型组慢波节律、主功率、主频率、快波频率明显低于正常组（P＜0．05、P＜0．01），胃电慢波振幅无明显改变；中药组慢波节律、主频率、主功率、快波频率与模型组比较明显增强（P＜0．05、P＜0．01），胃电慢波振幅无明显改变，胃电波接近正常对照水平，与西药组比较慢波节律及快波频率均有明显差异（P＜0．05、P＜0．01）；西药组主频率、快波频率与模型组比较明显增强（P＜0．05），主功率、慢波节律与模型组比较无明显差异（P＞0．05）。（3）放免法测定，模型组与空白对照组比较，模型组大鼠血浆及胃肠组织中GAS、SS、MTL、VIP的含量均明显增加（P＜0．01或P＜0．05）；中药组与模型组比较，中药组大鼠血浆及胃组织中GAS、SS、MTL、VIP的含量明显降低（P＜0．01或P＜0．05），肠组织中MTL、SS、VIP的含量明显降低（P＜0．01或P＜0．05），肠组织中GAS的含量无明显降低（P＞0．05）。中药组与西药组比较血浆MTL升高明显（P＜0．05），VIP明显降低（P＜0．01），GAS、SS无明显差异（P＞0．05），胃组织中GAS含量明显升高（P＜0．01），VIP的含量明显降低（P＜0．05），MTL、SS的含量无明显差距（P＞0．05），肠组织中GAS的含量明显升高（P＜0．05），SS的含量明显降低（P＜0．05）MTL、VIP的含量无明显差异（P＞0．05）但有不同程度升高或降低；西药组与模型组比较大鼠血浆MTL、VIP、SS的含量明显降低（P＜0．01或P＜0．05），GAS接近（P＞0．05），胃组织中MTL、GAS、SS、VIP明显降低（P＜0．05），肠组织中MTL、GAS、VIP明显降低（P＜0．05），SS接近（P＞0．05）。免疫组化法测定，模型组与空白组比较大鼠胃肠组织中GAS、SS、MTL、VIP的含量明显升高（P＜0．05）。中药组与模型组比较大鼠胃肠组织中GAS、SS、MTL、VIP的含量明显降低（P＜0．05），与西药组比较胃肠组织中GAS、SS、MTL、VIP的含量接近，无明显差异（P＜0．05）；西药组与模型组比较大鼠胃肠组织中GAS、MTL、VIP的含量明显降低（P＜0．05），SS接近。（4）各组胃组织c-kit均有阳性表达，肝硬化组与对照组相比c-kit量明显降低（P＜0．05）。中药组的阳性表达量与肝硬化组比较明显升高（P＜0．05），接近空白组，与西药组比较明显升高（P＜0．05）。西药组与模型组比较阳性表达量无显著差异（P＞0．05）。（5）各组在胃组织中GAS受体mRNA均有表达，肝硬化组在胃组织GASRmRNA表达与空白组相比含量明星降低（P＜0．01）；中药组与肝硬化组比较含量明显升高（P＜0．01）与西药组比较明显升高（P＜0．05），。在空肠各组VIPR2受体mRNA均有表达，肝硬化组与空白组相比VIPR2mRNA含量升高（P＜0．01），中药组与组肝硬化VIPR2mRNA含量明显降低（P＜0．01），与西药组比较明显降低（P＜0．05）。结论：本实验得出以下结论，（1）加味四逆散：显著提高TP、ALB含量，降低ALT、AST含量。因此，加味四逆散能有效地保护肝细胞，恢复肝功能，具有较好的防治实验大鼠肝硬化病理改变的作用。（2）加味四逆散能够明显改善实验性肝硬化大鼠的胃电波异常，从而有明显促胃动力作用。（3）肝硬化大鼠血浆、胃、小肠不同部位的GAS、SS、MTL、VIP的变化方向基本一致，但程度不一。可能与肝硬化时肝脏灭活胃肠激素功能减退有关。加味四逆散对实验性肝硬化大鼠胃肠肽分泌异常有一定调节作用。（4）肝硬化大鼠胃c-kit含量明显降低、加味四逆散具有增加c-kit阳性ICC作用。（5）GAS、VIP2受体在胃肠道分布差异可能与肝硬化所致胃肠道动力紊乱表现有一定关系，加味四逆散对肝硬化大鼠胃肠肽受体水平的异常有调节作用。更多还原

【Abstract】 Object:to study how Supplemented Sini powdercan cure rats withliver cirrhosis with gastric motility dysfunction and probe itsmechanism preliminarily by liver function、liver tissuepathology.research of Jiawei Sinisans’s prophylaxis the big rats electrogastrograms with liver cirrhosis and conduct a preliminary probe.toprobe into the signal conduction dysfunction of experimental ratscaused by liver cirrhosis with gastric motility dysfunction fromgastrointestinal peptide signal cell level and Supplemented Sinipowdertherapymechanism.touseICC to express c-kit and set it asthe point of entry to study the c-kit change of rats with livercirrhosis and the influence of Jiawei Sinisan,and expecting toexplain partly mechanism how the liver cirrhosis gastric motilitydysfunction happens,using GAS、VIP2receptorsasrepresentatives,to detect the signal receiving system change in gastrointestinalpeptide smooth muscle cells receptor level,and probe into therapycure mechanism of the Jiawei Sinisan.Method:We divide 48 Wister male big rats into 4 groups randomlyand each group has 12 rats,then inject the other 3 groups with40%C C 1₄peanut oil solution with concentration of 2.5ml/kg except blank group,and construct model twice every week and continuouslyfor 11 weeks.After the construction of 4 weeks,using SupplementedSini powderto do gastric perfusion therapy to cure the traditionalChinese medicine group,western group with cisapride gastricperfusion therapy,bank group and model group with physiologicalsaline of the same volume.After 11 weeks,testing all the rats’s electrogastrograms、liver function、liver tissue pathology.detect the content changes of MTL、GAS、VIP、SS in serum、gastric、small intestines by radiation immunological.Detect gastrictissue’ GAS、SS、c-kit and small intestines’ MTL、VIP contentchanges by immunohistochemical technique.detecting the gastricGASR and the jejunum these two receptors’ distribution andquantitative analysis by RT-PCR technique.After the test,doingstatistics comparison to the data of traditional Chinese medicinegroup、bank group、modeling group and western medicine group.Results:（1）Rats in normal situation、liver function、liver histolomorphof liver cirrhosis group all had evident differents compared to theblank group（P＜0.01 or P＜0.05）.comparison of Supplemented Sinipowdergrouptomodelinggroup:liver function’ s TP and ALB contentsincreased obviously,while ALT and AST contents decreased evidentlyand had obvious discrepancies（P＜0.01 or P＜0.05）;Liver tissuepathology change alleviated apparently.Comparison of SupplementedSini powdergroup to western medicine group:liver function’ s TPand ALB increasedobviously,while ALT、ASTdecreasedevidentlyandhad obvious discrepancies（P＜0.01 or P＜0.05）;Liver tissuepathology change alleviated;Supplemented Sini powdergroup’ selectrogastrograms was close to normal while western group’ s electrogastrograms the amplitude of vibration had slight recover.（2）Modelinggroup’ s slow wave rhythm、main frequency、mainpower and fast wave is lower than normal group obviously（P＜0.05orP＜0.01）,and have no obvious change of electrogastrogramamplitude of vibration at gastric antrum（P＞0.05）;The traditionalChinese medicine group’ s slow wave rhythm、main frequency、mainpower and fast wave are bigger than the modeling group（P＜0.05 orP＜0.01）,and close to the normal comparison level（P＞0.05）,andamplitude of vibration has no evident change than the modeling group（P＞0.05）,slow wave rhythm and fast wave are bigger than thewestern medicine group’（P＜0.05orP＜0.01）;the’smainfrequencyand fast wave are bigger than the modeling group（P＜0.05）,westernmedicine group main power and slow wave rhythm has no evident changethan the modeling group（P＞0.05）（3）To test MTL、GAS、VIP、SS by radiation immunological,theGAS、SS、MTL、VIPcontent inblood、gastric and intestines of livercirrhosis group all had evident increases compared to the bankgroup（P＜0.01orP＜0.05）,theGAS、SS、MTL、VIPcontent in blood andgastric of Chinese traditional medicine group all had evidentdecreases compared to the liver cirrhosis group（P＜0.01orP＜0.05）,MTL、SS、VIP content in intestines had evident decreases（P＜0.01orP＜0.05）,GAS content in intestines had no evidentdecreases（P＞0.05）,MTL content in blood had evident increasescompared to the modeling group（P＜0.05）,VIPhadevident decreases（P＜0.01）,GAS、SS had no evident different（P＞0.05）,but haddifferent level of increase or decrease.GAS content in gastric hadevident increases（P＜0.01orP＜0.05）,VIP had evident decreases,MTL、SS had no evident different（P＞0.05）.GAS in intestines had evident increases（P＜0.05）,SShadevidentdecreases（P＜0.05）,MTL、VIP hadno evident different（P＞0.05）.the GAS、MTL、VIPcontent in bloodand gastric of Chinese traditional medicine group all had evidentdecreases compared to the liver cirrhosis group（P＜0.01orP＜0.05）,SShad no evident different（P＞0.05）.the GAS、SS、MTL、VIP contentin gastric and intestines of the liver cirrhosis detected byimmunohistochemical technique had obvious increases compared tothe comparison group（P＜0.05）,The GAS、SS、MTL、VIP content ingastric and intestines of the Chinese traditional medicine groupall had evident decreases compared to the liver cirrhosis group（P＜0.05）,And theMTL、GAS、SS、VIPwas close to the the westernmedicine group,TheGAS、MTL、VIPcontent in gastric and intestinesof the western medicine group all had evident decreases comparedto the liver cirrhosis group（P＜0.05）,SS、had no evident different（P＞0.05）.（4）In different levels of gastric,c-kit Mean optical density（MOD）and positive area percentage of liver cirrhosis group all hadobvious decreases compared to the comparison group（P＜0.05）.Meanoptical density and positive area percentage of the Chinesetraditional medicine group all increased evidently（P＜0.05）,andhad no obvious discrepancies to the comparison group.Mean opticaldensity and positive area percentage of the western medicine groupall decreased evidently（P＜0.05）,and had no obvious discrepanciesto the modeling group（P＞0.05）.（5）All groups had GAS receptor mRNA expression in gastricantrum,and liver cirrhosis group had obvious decreases of GASRmRNAexpression compared to the normal group（P＜0.01）;the Chinesetraditional medicine group had evidently increase of GASRmRNA expression compared to the liver cirrhosis group（P＜0.05）,hadevidently increase of GASRmRNA expression compared to the westernmedicine group（P＜0.05）,and had no obvious discrepancies comparedto the normal group（P＞0.05）.All groups hadVIPR2 receptor mRNAexpression in the jejunum,and liver cirrhosis had a higherVIPR2mRNA content than the comparison group（P＜0.01）,and theChinese traditional medicine group obviously had a lower VIPR2mRNAcontent than the liver cirrhosis group（P＜0.01）.had a lowerVIPR2mRNA content than the western medicine group（P＜0.05）.Conclusion:（1）Supplemented Sini powderevident increases of TP and ALBcontents and decreased of ALT and AST content.So,Supplemented Sinipowdercan effectively protect liver cells,recover liver function,and has a good cure of experiment rats’ liver cirrhosis andameliorates Gastric motility function（2）Jiawei Sinisans can better experimental rats’ electrogastrogram wave dysfunction evidently and promote gastric motility（3）In gastrointestinal peptide signal cell level,GAS、SS、MTL、VIP in blood、gastric and intestines of rats with livercirrhosis had nearly the same change direction,but different inlevel.It maybe relate to liver cirrhosis gastric motilitydysfunction、malabsorption、ascites,etc.Supplemented Sinipowderhad some therapy cure to the experimental liver cirrhosisrats with gastrointestinal peptide parasecretion.（4）Gastric c-kit content in rats liver cirrhosis had evidentdecreased,and Supplemented Sini powdermay have the function ofincreasing c-kit positive ICC.（5）GAS and VIP receptors having different distributions in gastrointestinal tract may have some relationship to the differentgastric motility dysfunction expression caused by liver cirrhosis.Supplemented Sini powderhad a therapy cure to disnormalgastrointestinal peptide receptor level of rats with livercirrhosis.更多还原