本文選題：TLR2 + 脂肪酸�。� 參考：《河北醫(yī)科大學(xué)》2017年碩士論文

【摘要】：近年來對(duì)胰島素抵抗(insulin resistance,IR)的病因?qū)W研究成為一個(gè)熱點(diǎn),因?yàn)镮R貫穿于高脂血癥、糖尿病、高血壓、痛風(fēng)等多種代謝相關(guān)疾病,是這些疾病的共同土壤。眾多研究資料表明炎癥反應(yīng)是IR發(fā)生的重要機(jī)制。從發(fā)現(xiàn)第一個(gè)與胰島素抵抗相關(guān)的炎性細(xì)胞因子腫瘤壞死因子α(TNFα)以來,炎癥機(jī)制已成為肥胖相關(guān)胰島素抵抗的研究熱點(diǎn)。肥胖狀態(tài)下的炎癥是一種低度的慢性炎癥反應(yīng)。TOLL樣受體(Toll-like receptors,TLRs)作為模式識(shí)別受體不僅在感染免疫和損傷誘導(dǎo)的炎癥反應(yīng)中發(fā)揮重要作用,還參與代謝相關(guān)疾病的發(fā)生發(fā)展。TOLL樣受體2(TLR2)是已克隆出的TOLL樣受體家族中表達(dá)范圍最廣,識(shí)別病原微生物種類最多的成員。TLR2主要在哺乳動(dòng)物的肺臟、心臟、大腦和肌肉組織中表達(dá),其最突出的生物學(xué)功能就是直接或間接促進(jìn)炎癥因子的合成與釋放。人類及嚙齒類動(dòng)物血漿葡萄糖60%~70%在骨骼肌代謝,骨骼肌是胰島素刺激葡萄糖攝取的主要部位。脂代謝在骨骼肌胰島素抵抗形成中扮演重要角色,線粒體是脂肪酸氧化的部位,表明線粒體功能在骨骼肌胰島素抵抗形成中的重要地位。因此研究TLR2對(duì)骨骼肌線粒體生物發(fā)生、胰島素抵抗的影響對(duì)代謝性疾病的防治具有重要的意義,為尋求糖尿病治療新靶點(diǎn)提供科學(xué)依據(jù)。棕櫚酸(Palmitate acid,PA)是游離脂肪酸中最主要的飽和脂肪酸,用其孵育可致細(xì)胞產(chǎn)生胰島素抵抗,是建立體外細(xì)胞胰島素抵抗模型的常用方法之一。本課題采用PA孵育L6成肌細(xì)胞構(gòu)建胰島素抵抗模型,并通過質(zhì)粒上調(diào)和siRNA下調(diào)肌細(xì)胞中TLR2的表達(dá),探究飽和脂肪酸是否是通過TLR2的表達(dá)影響骨骼肌線粒體生物發(fā)生,進(jìn)而導(dǎo)致胰島素抵抗。目的:研究TLR2對(duì)棕櫚酸所致骨骼肌胰島素抵抗的作用及其機(jī)制。方法:培養(yǎng)原始L6成肌細(xì)胞,使其分化成骨骼肌細(xì)胞,并用PA孵育,建立胰島素抵抗模型。隨后進(jìn)行如下分組:對(duì)照組(control),棕櫚酸組(PA),棕櫚酸siRNA陰性對(duì)照組(PA+NC-siRNA),棕櫚酸TLR2敲低組(PA+si-TLR2),棕櫚酸空質(zhì)粒組(PA+pcDNA),棕櫚酸TLR2過表達(dá)組(pa+pcdna/tlr2)。通過rt-pcr及westernblot方法檢測(cè)tlr2mrna及蛋白表達(dá)水平,確定轉(zhuǎn)染成功后,用葡萄糖氧化酶法測(cè)定各組細(xì)胞培養(yǎng)基中葡萄糖濃度,評(píng)價(jià)胰島素的敏感性。采用elisa方法測(cè)定六組培養(yǎng)基中炎癥因子tnfα及il-1β的濃度。采用rt-pcr和westernblotting方法檢測(cè)六組樣本骨骼肌細(xì)胞中tlr2蛋白的表達(dá)水平,采用westernblotting方法檢測(cè)六組樣本骨骼肌細(xì)胞中線粒體生物發(fā)生相關(guān)蛋白pgc1α、mfn2、nrf-1以及胰島素信號(hào)通路pi3k、p-akt、glut4蛋白的表達(dá)水平。多樣本比較采用單因素方差分析,兩樣本間比較采用t檢驗(yàn)。結(jié)果:1成功分化骨骼肌細(xì)胞分化組標(biāo)志性分化基因desmin和myogenin的mrna表達(dá)較未分化組明顯增加,差異具有統(tǒng)計(jì)學(xué)意義(p0.05);2胰島素抵抗模型的建立成功分化的骨骼肌細(xì)胞,分為正常對(duì)照組和棕櫚酸干預(yù)組。分別在12h、16h、20h、24h測(cè)定兩組培養(yǎng)基葡萄糖濃度,在24h時(shí),棕櫚酸干預(yù)組葡萄糖濃度明顯高于對(duì)照組,差異具有統(tǒng)計(jì)學(xué)意義(p0.05);3轉(zhuǎn)染成功后六組樣本葡萄糖濃度及炎癥因子tnfα和il-1β的變化pa組培養(yǎng)基中葡萄糖濃度及炎癥因子tnfα和il-1β較control組明顯升高,差異具有統(tǒng)計(jì)學(xué)意義(p0.05);pa+nc-sirna組和pa+pcdna組較pa組葡萄糖濃度及炎癥因子tnfα和il-1β無明顯變化,差異無統(tǒng)計(jì)學(xué)意義(p0.05);pa+si-tlr2組較pa+nc-sirna組葡萄糖濃度及炎癥因子tnfα和il-1β明顯下降,差異具有統(tǒng)計(jì)學(xué)意義(p0.05);pa+pcdna/tlr2組較pa+pcdna組葡萄糖濃度及炎癥因子tnfα和il-1β明顯升高,差異具有統(tǒng)計(jì)學(xué)意義(p0.05);4轉(zhuǎn)染成功后六組樣本tlr2的表達(dá)與control組相比,pa組tlr2的表達(dá)明顯升高,差異具有統(tǒng)計(jì)學(xué)意義(p0.05);pa+nc-sirna組和pa+pcdna組較pa組tlr2的表達(dá)無明顯變化,差異無統(tǒng)計(jì)學(xué)意義(p0.05);PA+si-TLR2組較PA+NC-siRNA組TLR2的表達(dá)明顯下降,差異具有統(tǒng)計(jì)學(xué)意義(P0.05),表明si-TLR2轉(zhuǎn)染成功;PA+pcDNA/TLR2組較PA+pcDNA組TLR2的表達(dá)明顯升高,差異具有統(tǒng)計(jì)學(xué)意義(P0.05)表明TLR2質(zhì)粒轉(zhuǎn)染成功;5轉(zhuǎn)染成功后六組樣本線粒體生物發(fā)生相關(guān)蛋白指標(biāo)及胰島素信號(hào)通路指標(biāo)的變化與control組相比,PA組線粒體生物發(fā)生相關(guān)蛋白指標(biāo)PGC1α、MFN2、NRF-1以及胰島素信號(hào)通路PI3K、P-AKT、GLUT4的蛋白表達(dá)水平下降,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);PA+NC-siRNA組和PA+pcDNA組較PA組線粒體生物發(fā)生相關(guān)蛋白指標(biāo)PGC1α、MFN2、NRF-1以及胰島素信號(hào)通路PI3K、P-AKT、GLUT4的蛋白表達(dá)水平無明顯變化,差異無統(tǒng)計(jì)學(xué)意義(P0.05);PA+si-TLR2組較PA+NC-si RNA組線粒體生物發(fā)生相關(guān)蛋白指標(biāo)PGC1α、MFN2、NRF-1以及胰島素信號(hào)通路PI3K、P-AKT、GLUT4的蛋白表達(dá)水平升高,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);PA+pcDNA/TLR2組較PA+pcDNA組線粒體生物發(fā)生相關(guān)蛋白指標(biāo)PGC1α、MFN2、NRF-1以及胰島素信號(hào)通路PI3K、P-AKT、GLUT4的蛋白表達(dá)水平下降,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);結(jié)論:1高脂孵育可以誘導(dǎo)骨骼肌細(xì)胞胰島素抵抗,表現(xiàn)為葡萄糖攝取減少,同時(shí)可使TLR2增加,炎癥因子TNFα和IL-1β釋放增加。2抑制或過表達(dá)TLR2可影響葡萄糖攝取,炎癥因子分泌,線粒體生物發(fā)生相關(guān)蛋白PGC1α、MFN2、NRF-1及胰島素信號(hào)通路PI3K、P-AKT、GLUT4蛋白的表達(dá),說明TLR2表達(dá)增加損傷了線粒體生物發(fā)生相關(guān)蛋白,進(jìn)而引起胰島素抵抗。
[Abstract]:The etiological study of insulin resistance (insulin resistance, IR) has become a hot spot in recent years, because IR runs through many metabolic related diseases such as hyperlipidemia, diabetes, hypertension and gout, and is the common soil of these diseases. Many research data show that the inflammatory response is an important mechanism for the occurrence of IR. Since the anti related inflammatory cytokine tumor necrosis factor alpha (TNF alpha), the inflammatory mechanism has become a hot spot in obesity related insulin resistance. Inflammation in the state of obesity is a low degree chronic inflammatory response, the.TOLL like receptor (Toll-like receptors, TLRs) as a pattern recognition receptor not only in the infection of immune and injury induced inflammation. .TOLL like receptor 2 (TLR2), a member of the cloned TOLL like receptor family, is the most widely expressed, and the most widely recognized member of the pathogenic microorganism,.TLR2, is mainly expressed in the lungs, heart, brain and muscle tissue of mammalian, and its most prominent biological function. It is a direct or indirect promotion of the synthesis and release of inflammatory factors. Human and rodent plasma glucose 60%~70% is in skeletal muscle metabolism, the skeletal muscle is the main part of insulin stimulation of glucose uptake. Lipid metabolism plays an important role in the formation of insulin resistance in skeletal muscle. Mitochondria are the part of fatty acid oxidation, indicating mitochondrial work. It is important in the formation of insulin resistance in skeletal muscle. Therefore, the study of the mitochondrial biogenesis of skeletal muscle, the effect of insulin resistance on the prevention and control of metabolic diseases is of great significance, and provides a scientific basis for the search for new targets for the treatment of diabetes. Palmitic acid (Palmitate acid, PA) is the most important saturation in free fatty acids. Fatty acids, which can induce insulin resistance in cells, are one of the commonly used methods to establish insulin resistance models in vitro. This topic uses PA to incubate L6 myoblasts to construct insulin resistance model, and to explore whether saturated fatty acids are expressed through TLR2 expression through up-regulation and siRNA down regulation of TLR2 expression in myocytes. Mitochondrial biogenesis of skeletal muscle and insulin resistance. Objective: To study the effect and mechanism of TLR2 on the insulin resistance of skeletal muscle induced by palmitic acid. Methods: the primary L6 myoblasts were cultured to differentiate into skeletal muscle cells, and the insulin resistance model was incubated with PA. The following groups were grouped as follows: the control group (control), brown. Acid group (PA), palmitic acid siRNA negative control group (PA+NC-siRNA), palmitic acid TLR2 knockout group (PA+si-TLR2), palmitic acid empty plasmid group (PA+pcDNA), palmitic acid TLR2 overexpression group (pa+pcdna/tlr2). The expression of tlr2mrna and protein was detected by RT-PCR and Westernblot methods, and the cells of each group were determined by glucose oxidase method after the transfection was successful. The concentration of glucose in the medium was evaluated and the sensitivity of insulin was evaluated. The concentrations of inflammatory factors TNF a and IL-1 beta in the six groups were measured by ELISA method. The expression of TLR2 protein in the skeletal muscle cells of the six groups was detected by RT-PCR and westernblotting, and the midline of six groups of skeletal muscle cells was detected by westernblotting method. The expression level of PGC1 alpha, Mfn2, NRF-1, and insulin signaling pathway PI3K, p-Akt, GLUT4 protein in granular biogenetic protein. The diversity was compared with single factor analysis of variance, and t test was used for comparison between two samples. Results: 1 the expression of desmin and myogenin in the differentiation group of skeletal muscle cell differentiation was more than that of the undifferentiated group. The difference was statistically significant (P0.05); 2 insulin resistance model established the differentiated skeletal muscle cells, divided into the normal control group and the palmitic acid intervention group. The glucose concentration in two groups was measured in the 12h, 16h, 20h, 24h respectively. The glucose concentration in the palmitic acid intervention group was significantly higher than the control group at 24h, and the difference was statistically significant. Study significance (P0.05); 3 the glucose concentration and inflammatory factors TNF a and IL-1 beta in the six groups after the transfection, the glucose concentration and the inflammatory factor TNF alpha and IL-1 beta in the group PA were significantly higher than those in the control group, the difference was statistically significant (P0.05), and the concentration of glucose and the inflammatory factors in the pa+nc-sirna group and pa+ pcDNA group were compared with those of the PA group. There was no significant difference in beta (P0.05), but in group pa+si-tlr2, glucose concentration and inflammatory factors TNF a and IL-1 beta were significantly decreased, and the difference was statistically significant (P0.05); the glucose concentration and inflammatory factors in the pa+pcdna/tlr2 group were significantly higher than those in the pa+pcdna group, and the difference was statistically significant (P0.05); the 4 turn was 4. Compared with the control group, the expression of TLR2 was significantly higher in the group PA than that in the control group. The difference was statistically significant (P0.05), and there was no significant change in the expression of TLR2 in the pa+nc-sirna and pa+pcdna groups. The difference was statistically significant (P0.05), and the difference was statistically significant compared with that of the PA+NC-siRNA group. Significance (P0.05) showed that the transfection of si-TLR2 was successful, and the expression of TLR2 in group PA+pcDNA/TLR2 was significantly higher than that in group PA+pcDNA, and the difference was statistically significant (P0.05) indicated that the transfection of TLR2 plasmid was successful; 5 after the successful transfection, the changes of mitochondrial biogenesis related protein index and insulin signaling pathway index of 6 groups were compared with that of the control group, and the PA group mitochondria The protein expression levels of PGC1 alpha, MFN2, NRF-1 and insulin signaling pathway PI3K, P-AKT, GLUT4 were decreased, and the difference was statistically significant (P0.05); PA+NC-siRNA and PA+pcDNA groups were compared to PGC1 alpha, MFN2, and insulin signaling pathway. There was no significant difference in expression level and no significant difference (P0.05), and in group PA+si-TLR2, the mitochondrial biogenesis associated protein index of PA+NC-si RNA group, PGC1 alpha, MFN2, NRF-1, and insulin signaling pathway PI3K, P-AKT, GLUT4 protein expression level increased, and the difference was statistically significant (P0.05); PA+pcDNA/TLR2 group was more than the mitochondrial organisms. The protein expression levels of PGC1 alpha, MFN2, NRF-1 and insulin signaling pathway PI3K, P-AKT, GLUT4 were decreased, and the difference was statistically significant (P0.05). Conclusion: 1 high fat incubation can induce insulin resistance in skeletal muscle cells, showing a decrease in glucose uptake, increasing TLR2, and increasing the release of inflammatory factors TNF A and IL-1 beta. .2 inhibition or overexpression of TLR2 can affect glucose uptake, inflammatory factor secretion, mitochondrial biogenetic protein PGC1 alpha, MFN2, NRF-1 and insulin signaling pathway PI3K, P-AKT, GLUT4 protein expression, indicating that the increase of TLR2 expression damage the mitochondrial biogenetic protein, and then cause insulin resistance.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R58

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 ;Curcumin,a Potential Inhibitor of Up-regulation of TNF-alpha and IL-6 Induced by Palmitate in 3T3-L1 Adipocytes through NF-kappaB and JNK Pathway[J];Biomedical and Environmental Sciences;2009年01期

2 崔麗娟;都健;;脂聯(lián)素與炎癥及胰島素抵抗關(guān)系的研究進(jìn)展[J];國(guó)際內(nèi)分泌代謝雜志;2006年S1期

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本文編號(hào)：1804633