【作者】 康耀霞；

【导师】 张义正；

【作者基本信息】 四川大学 ， 遗传学， 2006， 博士

【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。更多还原

【Abstract】 The efficiency of electroacupuncture(EA) has been concerned in treating clinical hypercholesterolemia. In order to provide with molecular evidence to understand the mechanism of acupuncture and the side function of simvastain in treating hypercholesterolemia, identification of differentially expressed genes responding to the hypercholesterolemia have been carried out in hypercholesterolcmic mice liver after acupuncture and simvastatin treatment.In previous study, the mice model of hypercholesterolemia group(HC), normal group(NM), EA treatment group(EA), simvastatin treatment group(ST), EA preventive group(EP), EA non-acupoint group(EN) had been established, and the results of blood biochemical indicators manifested that the treatment of electroacupuncture on fenglong acupoint or simvastatin could markedly reduce the levels of total cholesterol, high density cholesterol and low density cholesterol of plasma. In this study, four groups of NM, HC, EA, and ST mice was chosen for constructing Suppression Subtractive Hybridization(SSH) libraries. Mirror orientation selection(MOS) method was applied to subtracted cDNA populations to reduce the level of false positives before the ligation between vector and cDNA. A total of five independent SSH libraries were constructed and named as ssh-EH and ssh-HE, ssh-SH and ssh-HS, ssh-SN from reciprocal substractive hybridization between EA and HC, EA and ST, NM and ST.Targeted Display(TD), a novel procedure of differentially displaying method was used to select for and amplify truc differentially expressed cDNAs. Total 142 differential displayed fragments were cloned and sequenced. The corresponding genes of 116 were identified by BLAST with the sequenced fragments against GenBank and the mice genome sequence. Forty and thirty-seven genes were up-and down-regulated, respectively, in response to electroacupuncture. However, functions of 52 genes are known and 25 unknown among them. After simvastatin treatment, 16 genes were up-regulated and 23 down-regulated, while among them the functions of 23 genes are known and the others unknown. The differentially expressioned genes were found to be involved in a lot of physiological processes in EA treated mice. The genes related to metabolism(major of lipid metabolism), gene expressed regulation and immunity were differentially expressed after EA stimulation. Simvastatin treatment was mainly relateted to those genes involved in metabolism, signal transduction, immune response, and so on.The number of down-regulated genes after simvastatin treatment was exceeded that of up-regulated. It was suggested that this might be caused by the down-expressed nuclear factor I. At the same time, the number of differentially expressed genes in up-and down-regulation was almost equal after EA treatment, and the genes encoding key enzyme of acyl-Coenzyme A oxidase(Acox) and stearoyl-Coenzyme A desaturase 1(Scd 1) in lipid metabolism were down-regulated, implying that the modulated action to balance body metabolism was contributed to electroacupuncture treatment.The expression of genes encoding adiponectin recptor 2(Adipo R2), lysyl-tRNA synthetase(Krs), tumor necrosis factorα(TNF-α), Utrophin(Utr) was analyzed by real-time RT-PCR in the livers of groups NM, HC, EA, ST, EP and EN. Adipo R2 and Krs genes were lowest expressed in EA group. The genes of TNF-αand Utr were lowest expressed in the groups of EN and ST, respectively. This result was coincidence with that of SSH. It means that decreased expression of Adipo R2, Krs, TNF-αmight be relateted to the action of lipid metabolism by EA treatment, and the down-regulation of Utr gene expression was might be involved in cell damage after simvastatin treatment.To confirm the effect of simvastatin on the changes of gene expression and the state of cell growth, C2C12 was treated in 0(control group), 1, 3, 10, 30 and 100μmol/L of simvastatin. The depressant effect was found dose and time dependent as increase in concentration increased the inhibition. The RNA level of Utr and other three genes compared with control cells were all increased after 24h treatment with different doses of drug. It was suggested that simvastatin coule be able to result in different changes of genes expression between liver and C2C12 cells.This study was the first time using SSH accompanyed by TD to study gene expression of liver in hypercholesterolemic mice. The results demonstrated that EA could modulate gene expression of key enzymes in lipid oxidation, and simvastatin reduced the expression of transductional factor gene to accommodate the gene expression in tissues and C2C12 striated muscle cells.The consequence obtained from EA stimulation and simvastatin treatment might be able to arouse our interest to further study the mechanism of acupuncture on gene level. At the same time, it also help us understand the pleiotropic action of simvastatin.更多还原