【作者】 张发艳；

【导师】 王世军；

【作者基本信息】 山东中医药大学 ， 中西医结合基础， 2012， 博士

【摘要】 目的：利用基因芯片技术检测实热证模型大鼠肝全基因表达谱的改变，经典寒性中药大黄、黄连及其提取物对实热证模型大鼠肝全基因表达谱的影响，从功能基因组角度探讨寒性中药的物质基础及其性效发生机制，探讨寒性中药属性界定的可能依据和中药药性理论研究的思路和方法。方法：SPF级Wistar大鼠96只。实验分2批完成，每批48只。第一批动物分为空白对照组，模型对照组，大黄水煎液、大黄乙酸乙酯萃取物，大黄正丁醇萃取物、大黄水萃取物组。其中大黄水煎液治疗组、各萃取物组按照10ml/Kg灌胃相应液体，空白对照组给予等量蒸馏水。第二批动物分为空白对照组，模型对照组，黄连水煎液、黄连乙酸乙酯萃取物，黄连正丁醇萃取物、黄连水萃取物组。其中黄连水煎液治疗组、各萃取物组按照10ml/Kg灌胃相应液体，空白对照组给予等量蒸馏水。大鼠背部皮下注射2,4-二硝基苯酚（DNP）生理盐水溶液复制实热证模型，造模后0.5h给药治疗，2h、4h、6h测肛温。之后提取肝组织总RNA，应用基因芯片检测各组大鼠肝脏基因表达，筛选差异表达基因，进行基因聚类分析和功能分类注释。选择部分差异基因进行荧光实时定量PCR实验验证芯片结果的准确性。结果：实热模型对照组与空白对照组比较有167条差异表达基因；大黄乙酸乙酯组、大黄正丁醇组、大黄水提物组和大黄水煎液组与实热模型对照组比较分别有177、139、287和177条差异表达基因；黄连乙酸乙酯组、黄连正丁醇组、黄连水提物组和黄连水煎液组与实热模型对照组比较有336、362、417和218条差异表达基因；对差异基因进行基因功能分类注释，实热模型对照组与空白对照组相比，查询到15项显著性基因功能.。大黄乙酸乙酯组、大黄正丁醇组、大黄水提物组、大黄水煎液组与实热模型对照组相比分别查询到34、31、53和29项显著性基因功能，主要为代谢过程基因功能。黄连乙酸乙酯组、黄连正丁醇组、黄连水提物组、黄连水煎液组与实热模型对照组相比分别查询到59、78、72、32项显著性基因功能，主要为代谢过程功能，催化活性功能项。结论：实热证大鼠主要通过调节代谢过程、钙离子平衡和异生物质刺激应答相关基因实现对机体活动的调节。大黄水煎液主要通过调节代谢过程功能基因，尤其是糖代谢相关基因表达，降低机体过高的能量代谢过程。黄连水煎液主要通过调节代谢过程功能基因，尤其是氨基酸代谢相关基因表达，发挥其对机体的调节作用。寒性中药大黄、黄连及其提取物均能通过上调Gclc、Adh1、Rpl6、Nqo1、RGD1562920_predicted基因表达，下调Ubd、Hamp、Sds、LOC683385基因表达发挥作用。以上基因改变可能是寒性中药发挥其清热、泻火、解毒作用的分子机制之一。更多还原

【Abstract】 Objective：To detect the change of hepatic gene expression profile in rats with excessheat syndrome using gene chip technology, to study the effects of herbs with cold propertysuch as rhubarb, Rhizoma coptidis and their extracts on the change of hepatic geneexpression profiling, to discover the material foundation of cold property of Chinesemedicine and to discuss the mechanism of it from the aspect of functional genomicsperspective.Methods：96SPF Wistar rats were divided into two batches to complete theexperiment. The first batch animals were divided into the blank control group, excess heatsyndrome group, Rhubarb decoction group, Rhubarb ethyl acetate extract group, Rhubarbn-butanol extract group, Rhubarb water extracts group. The Rhubarb decoction group andthe extracts of Rhubarb groups were given the corresponding liquid in accordance with the10ml/Kg, the excess heat syndrome group and the blank control group were given10ml/Kgdistilled water. The second batch animals were also divided into6groups. The Rhizomacoptidis decoction group and the extracts of Rhizoma coptidis groups were given thecorresponding liquid in accordance with the10ml/Kg, the excess heat syndrome group andthe blank control group were given10ml/Kg distilled water.The rats were given subcutaneous injection of2,4-dinitrophenol saline solution toreplicate the excess heat syndrome model,0.5h later giving medication treatment,2h,4h,6h point later, the rectal temperature of rats was measured. After the extraction of totalRNA from liver tissue, the gene clustering analysis and functional classification annotationwere carried on. We selected the differential expression genes, analyzed the genes bycluster and conducted the significant analysis on the genetic function of differential genes. A part of genes was selected to test the accuracy of results by RT-PCR.Results：There were167strips of differential expression genes in excess heatsyndrome group as compared to blank control group. There were177、139、287and177strips of differential expressions in Rhubarb ethyl acetate extract group, Rhubarbn-butanol extract group, Rhubarb water extracts group and Rhubarb decoction group,respectively compared with heat syndrome rat group.336、362、417and218strips ofdifferential expressions were found in Rhizoma coptidis ethyl acetate extract group,Rhizoma coptidis n-butanol extract group, Rhizoma coptidisis water extracts group andRhizoma coptidis decoction group compared with excess heat syndrome group.As compared to the blank control group, there were15items of significant genefunction in the excess heat syndrome group. There were34,31,53and29items ofsignificant gene function in Rhubarb ethyl acetate extract group, Rhubarb n-butanol extractgroup, Rhubarb water extracts group and Rhubarb decoction group respectively comparedwith excess heat syndrome group. There were59,78,72,32items of significant genefunction in Rhizoma coptidis ethyl acetate extract group, Rhizoma coptidis n-butanolextract group, Rhizoma coptidis water extracts group and Rhizoma coptidis decoctiongroup respectively compared with the excess heat syndrome group.Conclusion：Excess heat syndrome rats regulate body function mainly throughchanging the expression of genes related to metabolic processes, calcium ion homeostasisand xenobiotic stimuli response. Rhubarb decoction reduces the excessive energymetabolism of the body mainly through modulating the expression of genes involved inmetabolic processes, particularly glucose metabolism. Rhizoma coptidis Decoction plays aregulatory role in the rats with excess heat syndrome mainly through modulating theexpression of genes involved in metabolic processes, especially amino acid metabolism.The herbs with cold property such as rhubarb, Rhizoma coptidis and their extractsmay realize their antibacterial anti-inflammatory, antiviral, anti infection effect byincreasing the gene expression of Gclc, Adh1, Rpl6, Nqo1, RGD1562920_predicted anddecreasing the gene expression of Ubd, Hamp, Sds, LOC683385. The above genes may beone of the main molecular mechanisms of clearing away heat, purging fire and eliminatingtoxic materials of the Chinese medicinal herbs with cold nature.更多还原