针刺和辛伐他汀治疗小鼠高胆固醇血症的基因表达研究

发布时间：2018-03-17 18:10

本文选题：小鼠高胆固醇血症　切入点：肝脏　出处：《四川大学》2006年博士论文　论文类型：学位论文

【摘要】： 针刺在临床降低高胆固醇血症中已具有较肯定的疗效。本文以高胆固醇血症小鼠为模型，对针刺和辛伐他汀降低高胆固醇血症后肝脏组织基因表达变化进行比较研究，为针刺和辛伐他汀的降脂机制、以及辛伐他汀对机体和细胞的可能副作用提供分子水平的实验证据。在本实验室的前期工作中，共获得6组不同处理的小鼠肝脏样品，分别是正常组(NM)、高脂组(HC)、针治组(EA)、他汀组(ST)、针预组(EP)、针非组(EN)，对各组小鼠血液多项生化指标的分析结果表明，针刺丰隆穴和他汀药物治疗可以有效降低高胆固醇小鼠血浆总胆固醇、低密度脂蛋白胆固醇、高密度脂蛋白胆固醇水平。在本实验中，我们选取正常组、高脂组、针治组、他汀组等4组小鼠肝脏组织，采用抑制消减杂交(Suppression Subtractive Hybridization，SSH)技术，在Mirror orientation selection(MOS)方法去除部分假阳性的基础上，成功构建了5个抑制消减cDNA文库，它们分别是：针刺治疗高胆固醇血症双向差异表达基因文库(分别称之为ssh-EH和ssh-HE)；辛伐他汀治疗高胆固醇血症双向差异表达基因文库(分别称之为ssh-SH和ssh-HS)；以及辛伐他汀治疗高胆固醇血症与正常小鼠肝脏单向差异表达文库(ssh-SN)。在SSH富集差异基因的基础上，经Targeted Display(TD)方法对5个文库进行了差异基因的筛选，共获得142个差异片段(针刺后86个，他汀治疗后56个)。经序列测定和分析，这些片段共代表了116个差异表达基因，分别为针刺作用后差异表达的已知功能基因52个，未知功能基因25个，其中有40个基因上调表达，37个基因下调表达。辛伐他汀作用后差异表达的已知功能基因26个，未知功能基因13个，其中有16个基因上调表达，23个基因下调表达。根据已有功能进行分类，结果表明，针刺作用后差异表达的基因主要归为以下几类：代谢相关基因(其中脂类代谢基因占多数)、基因表达调控相关基因、免疫反应相关基因等。辛伐他汀作用引起的表达变化基因主要有代谢过程相关基因、转录调控基因、免疫过程相关基因等。比较针刺和辛伐他汀作用后二者差异表达基因的分类和数目发现，针刺作用调控了脂类代谢过程中关键酶acyl-Coenzyme A oxidase(Acox)和stearoyl-Coenzyme A desaturase 1(Scd 1)基因的表达；且针刺后上、下调表达基因数目接近，说明电针的刺激对机体产生的是一种整体调节作用，使失衡的机体代谢恢复正常。辛伐他汀作用后下调表达的基因多于上调表达基因，核因子1(nuclear factorⅠ)基因的下调，一定程度上可能会解释这一现象。对脂联素受体2(Adiponectin recptor 2，AdipoR2)、赖氨酸tRNA合成酶(lysyl-tRNA synthetase，Krs)、肿瘤坏死因子α(tumor necrosis factorα，TNF-α)、Dystrophin相关蛋白(Utrophin，Utr)等4个基因进行定量PCR检测，在不同处理组小鼠肝脏中的表达情况有所不同。在正常组、高脂组、辛伐他汀组、针治组、针预组、针非组6个组中，AdipoR2和Krs在针治组表达量最低，TNF-α在针非组中最低，Utr在他汀组中最低，与差异基因的筛选结果一致；同时也说明AdipoR2、Krs、TNF-α在针刺组中下调表达可能与其降脂作用有关；而Utr基因在他汀组中表达减少，可能与该药对肝细胞的损伤作用有关。进一步采用C2C12细胞研究不同浓度的辛伐他汀对细胞生长和基因表达的影响，结果表明，在0、1、3、10、30到100μmol／L浓度的药物处理后，随着浓度的增加和作用时间的延长，C2C12细胞活力降低，细胞死亡数增加。对Adiponectin recptor 2、TNF-α、Lysyl-tRNA synthetase、Utrophin 4个基因进行的定量RT-PCR检测结果显示，药物处理24h的不同浓度组与对照组相比，，这4个基因的表达均发生上调变化，因此，辛伐他汀对肝脏组织和体外培养的肌细胞基因表达的影响是不同的。本论文首次采用SSH结合TD方法研究小鼠肝脏组织中基因表达变化，结果表明，电针刺激可能通过影响肌体脂质代谢过程关键酶的基因表达，发挥降低血脂的作用；辛伐他汀通过影响转录因子的表达来调控组织和细胞的基因表达。这些结果对于从基因表达水平上阐明针刺降脂的机理，以及寻找辛伐他汀治疗作用的多效性机制有着积极意义。
[Abstract]:Acupuncture in the clinical curative effect is reduced certainly has high cholesterol in hypercholesterolemic mice. Based on the model of acupuncture and simvastatin reduced hypercholesterolemia after liver tissue gene expression changes compare for lipid-lowering mechanism of acupuncture and simvastatin, and simvastatin on the body and cells may provide molecular level side effects the experimental evidence.
In our previous work, a total of 6 different groups of liver samples were normal group (NM), high fat group (HC), acupuncture treatment group (EA), atorvastatin group (ST), pre group (EP), needle needle group (EN), the results of each group mouse blood biochemical index showed that acupuncture Fenglong and statin therapy can effectively reduce cholesterol in plasma total cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol levels. In this experiment, we selected normal group, high fat group, acupuncture treatment group, atorvastatin group and other 4 groups of mice liver tissue by using suppression subtractive hybridization (Suppression, Subtractive Hybridization, SSH Mirror orientation) technology in selection (MOS) removal method based on some false positives, successfully constructed 5 suppression subtractive cDNA library, they are: the gene expression of acupuncture in the treatment of hypercholesterolemia two-way difference Libraries (called ssh-EH and ssh-HE respectively); simvastatin in the treatment of hypercholesterolemia bidirectional differentially expressed gene library (respectively called ssh-SH and ssh-HS), and simvastatin treatment of hypercholesterolemia and normal mice liver one-way differential expression library (ssh-SN).
Based on the differences of gene enrichment in SSH, by Targeted Display (TD) of 5 libraries were screened differentially expressed genes, we obtained 142 fragments (86 after acupuncture, statin therapy after 56). By sequence analysis, these fragments were representative of 116 differentially expressed genes. Respectively, known functional genes differentially expressed after the acupuncture effect 52, 25 unknown genes, including 40 genes up-regulated and 37 genes were downregulated. Known functional genes differentially expressed after simvastatin treatment 26, 13 unknown genes, 16 genes up-regulated in the expression of 23. A gene classification, according to the existing function. The results show that the differential expression of acupuncture effect after gene is mainly classified into the following categories: metabolism related genes (including lipid metabolism genes in the majority), regulation of gene expression related genes, immune response related genes The expression change genes caused by the action of simvastatin mainly include metabolic process related genes, transcriptional regulation genes, immune process related genes and so on.
Comparison of acupuncture and simvastatin after two differential expression classification and number of gene discovery, the effect of acupuncture regulating lipid metabolism in the process of the key enzyme acyl-Coenzyme A oxidase (Acox) and stearoyl-Coenzyme A desaturase 1 (Scd 1) gene expression; and after acupuncture, down regulate the expression of gene number close, indicating that Electroacupuncture stimulation of the body is a kind of overall regulation, so that the metabolism imbalance recovered. After simvastatin treatment downregulated the expression of genes than up-regulated gene expression of nuclear factor 1 (nuclear factor 1) gene under the regulation, may to some extent can explain this phenomenon.
The adiponectin receptor 2 (Adiponectin recptor 2, AdipoR2), tRNA lysine synthetase (lysyl-tRNA synthetase, Krs), tumor necrosis factor alpha (tumor necrosis factor TNF-, Dystrophin alpha, alpha) related proteins (Utrophin, Utr) by quantitative PCR detection of 4 genes expression in different treatment groups in the liver of mice different. In normal group, high fat group, simvastatin group, acupuncture treatment group, acupuncture pretreatment group, needle group of 6 groups, AdipoR2 and Krs in the acupuncture treatment group the lowest expression level of TNF- alpha in needles was the lowest, Utr in statin group was lowest, and differences in gene screening results; at the same time that the AdipoR2, Krs, down-regulation of TNF- expression may be related to lipid lowering effect of alpha in the acupuncture group; and Utr gene in the statin group reduced expression, may be associated with the damage effect of the drug on liver cells.
The effects of different concentrations of simvastatin on C2C12 cells on cell growth and gene expression results showed that the drug in 0,1,3,10,30 to 100 mol / L concentration after treatment increased with the increase of concentration and time, C2C12 decreased the cell viability, cell death. The increase in the number of Adiponectin recptor 2, TNF- alpha. Lysyl-tRNA synthetase RT-PCR, quantitative Utrophin test results of 4 genes showed that 24h treatment with different doses of drug group compared with the control group, the expression of these 4 genes were up-regulated, so the effects of simvastatin on the expression of genes in cultured muscle cells and liver tissue in vitro is different.
This paper firstly adopts the changes of SSH gene expression with TD method of mouse liver tissue showed that electroacupuncture may affect the body lipid metabolism key enzyme gene expression, play the effect of lowering blood lipids; simvastatin by affecting the expression regulation of tissue and cell transcription factor gene expression. These results from the gene expression level to clarify the mechanism of acupuncture lipid-lowering, has a positive meaning and find the pleiotropic action of simvastatin treatment mechanism.

【学位授予单位】：四川大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R245;R96

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