本文選題：小鼠高膽固醇血癥　切入點(diǎn)：肝臟　出處：《四川大學(xué)》2006年博士論文　論文類型：學(xué)位論文

【摘要】： 針刺在臨床降低高膽固醇血癥中已具有較肯定的療效。本文以高膽固醇血癥小鼠為模型，對(duì)針刺和辛伐他汀降低高膽固醇血癥后肝臟組織基因表達(dá)變化進(jìn)行比較研究，為針刺和辛伐他汀的降脂機(jī)制、以及辛伐他汀對(duì)機(jī)體和細(xì)胞的可能副作用提供分子水平的實(shí)驗(yàn)證據(jù)。 在本實(shí)驗(yàn)室的前期工作中，共獲得6組不同處理的小鼠肝臟樣品，分別是正常組(NM)、高脂組(HC)、針治組(EA)、他汀組(ST)、針預(yù)組(EP)、針非組(EN)，對(duì)各組小鼠血液多項(xiàng)生化指標(biāo)的分析結(jié)果表明，針刺豐隆穴和他汀藥物治療可以有效降低高膽固醇小鼠血漿總膽固醇、低密度脂蛋白膽固醇、高密度脂蛋白膽固醇水平。在本實(shí)驗(yàn)中，我們選取正常組、高脂組、針治組、他汀組等4組小鼠肝臟組織，采用抑制消減雜交(Suppression Subtractive Hybridization，SSH)技術(shù)，在Mirror orientation selection(MOS)方法去除部分假陽性的基礎(chǔ)上，成功構(gòu)建了5個(gè)抑制消減cDNA文庫，它們分別是：針刺治療高膽固醇血癥雙向差異表達(dá)基因文庫(分別稱之為ssh-EH和ssh-HE)；辛伐他汀治療高膽固醇血癥雙向差異表達(dá)基因文庫(分別稱之為ssh-SH和ssh-HS)；以及辛伐他汀治療高膽固醇血癥與正常小鼠肝臟單向差異表達(dá)文庫(ssh-SN)。 在SSH富集差異基因的基礎(chǔ)上，經(jīng)Targeted Display(TD)方法對(duì)5個(gè)文庫進(jìn)行了差異基因的篩選，共獲得142個(gè)差異片段(針刺后86個(gè)，他汀治療后56個(gè))。經(jīng)序列測(cè)定和分析，這些片段共代表了116個(gè)差異表達(dá)基因，分別為針刺作用后差異表達(dá)的已知功能基因52個(gè)，未知功能基因25個(gè)，其中有40個(gè)基因上調(diào)表達(dá)，37個(gè)基因下調(diào)表達(dá)。辛伐他汀作用后差異表達(dá)的已知功能基因26個(gè)，未知功能基因13個(gè)，其中有16個(gè)基因上調(diào)表達(dá)，23個(gè)基因下調(diào)表達(dá)。根據(jù)已有功能進(jìn)行分類，結(jié)果表明，針刺作用后差異表達(dá)的基因主要?dú)w為以下幾類：代謝相關(guān)基因(其中脂類代謝基因占多數(shù))、基因表達(dá)調(diào)控相關(guān)基因、免疫反應(yīng)相關(guān)基因等。辛伐他汀作用引起的表達(dá)變化基因主要有代謝過程相關(guān)基因、轉(zhuǎn)錄調(diào)控基因、免疫過程相關(guān)基因等。 比較針刺和辛伐他汀作用后二者差異表達(dá)基因的分類和數(shù)目發(fā)現(xiàn)，針刺作用調(diào)控了脂類代謝過程中關(guān)鍵酶acyl-Coenzyme A oxidase(Acox)和stearoyl-Coenzyme A desaturase 1(Scd 1)基因的表達(dá)；且針刺后上、下調(diào)表達(dá)基因數(shù)目接近，說明電針的刺激對(duì)機(jī)體產(chǎn)生的是一種整體調(diào)節(jié)作用，使失衡的機(jī)體代謝恢復(fù)正常。辛伐他汀作用后下調(diào)表達(dá)的基因多于上調(diào)表達(dá)基因，核因子1(nuclear factorⅠ)基因的下調(diào)，一定程度上可能會(huì)解釋這一現(xiàn)象。 對(duì)脂聯(lián)素受體2(Adiponectin recptor 2，AdipoR2)、賴氨酸t(yī)RNA合成酶(lysyl-tRNA synthetase，Krs)、腫瘤壞死因子α(tumor necrosis factorα，TNF-α)、Dystrophin相關(guān)蛋白(Utrophin，Utr)等4個(gè)基因進(jìn)行定量PCR檢測(cè)，在不同處理組小鼠肝臟中的表達(dá)情況有所不同。在正常組、高脂組、辛伐他汀組、針治組、針預(yù)組、針非組6個(gè)組中，AdipoR2和Krs在針治組表達(dá)量最低，TNF-α在針非組中最低，Utr在他汀組中最低，與差異基因的篩選結(jié)果一致；同時(shí)也說明AdipoR2、Krs、TNF-α在針刺組中下調(diào)表達(dá)可能與其降脂作用有關(guān)；而Utr基因在他汀組中表達(dá)減少，可能與該藥對(duì)肝細(xì)胞的損傷作用有關(guān)。 進(jìn)一步采用C2C12細(xì)胞研究不同濃度的辛伐他汀對(duì)細(xì)胞生長(zhǎng)和基因表達(dá)的影響，結(jié)果表明，在0、1、3、10、30到100μmol／L濃度的藥物處理后，隨著濃度的增加和作用時(shí)間的延長(zhǎng)，C2C12細(xì)胞活力降低，細(xì)胞死亡數(shù)增加。對(duì)Adiponectin recptor 2、TNF-α、Lysyl-tRNA synthetase、Utrophin 4個(gè)基因進(jìn)行的定量RT-PCR檢測(cè)結(jié)果顯示，藥物處理24h的不同濃度組與對(duì)照組相比，，這4個(gè)基因的表達(dá)均發(fā)生上調(diào)變化，因此，辛伐他汀對(duì)肝臟組織和體外培養(yǎng)的肌細(xì)胞基因表達(dá)的影響是不同的。 本論文首次采用SSH結(jié)合TD方法研究小鼠肝臟組織中基因表達(dá)變化，結(jié)果表明，電針刺激可能通過影響肌體脂質(zhì)代謝過程關(guān)鍵酶的基因表達(dá)，發(fā)揮降低血脂的作用；辛伐他汀通過影響轉(zhuǎn)錄因子的表達(dá)來調(diào)控組織和細(xì)胞的基因表達(dá)。這些結(jié)果對(duì)于從基因表達(dá)水平上闡明針刺降脂的機(jī)理，以及尋找辛伐他汀治療作用的多效性機(jī)制有著積極意義。
[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.

【學(xué)位授予單位】：四川大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R245;R96

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