本文選題：S型雌馬酚 + 胰島素分泌��； 參考：《第三軍醫(yī)大學(xué)》2015年碩士論文

【摘要】：糖尿病(diabetic mellitus,DM)是最常見的慢性代謝性疾病之一,嚴(yán)重危害著人類身體健康,給個(gè)人及家庭帶來(lái)極重的精神和經(jīng)濟(jì)負(fù)擔(dān)。并且DM發(fā)病機(jī)制極為復(fù)雜,至今仍未完全闡明。目前認(rèn)為胰島素抵抗和胰島素分泌功能缺陷是DM發(fā)病的兩大主要因素,已成為糖尿病防治的重要靶點(diǎn)。然而,臨床上針對(duì)其使用的藥物大都存在諸多不良反應(yīng)。因此,不斷尋找和開辟糖尿病防治的新途徑已成為當(dāng)今醫(yī)學(xué)研究的焦點(diǎn),尤其是經(jīng)過(guò)膳食途徑改善糖尿病及其并發(fā)癥具有非常重要的意義。雌馬酚(equol,Eq)是由大豆生長(zhǎng)過(guò)程中形成的次生代謝物大豆異黃酮(soy isoflavones,SI)經(jīng)微生物代謝形成的重要產(chǎn)物,包括S、R兩種異構(gòu)體,而在動(dòng)物體內(nèi)均為S型[1]。大量研究表明,SI干預(yù)可增加胰島素分泌,促進(jìn)葡萄糖攝取和利用,從而有效控制血糖濃度,改善糖尿病癥狀。而進(jìn)一步研究發(fā)現(xiàn),SI的生物學(xué)活性主要由Eq來(lái)實(shí)現(xiàn)[2,3]。Eq具有比SI更高的生物活性和生物利用度,能夠有效的發(fā)揮雌激素樣、抗炎和抗氧化等作用,不僅在骨質(zhì)疏松癥、更年期綜合癥以及心血管疾病等方面表現(xiàn)出較好的防治效果,而且對(duì)乳腺癌及前列腺癌也具有很好的預(yù)防作用[4]。已有研究表明,Eq可顯著增加葡萄糖耐量[5],促進(jìn)葡萄糖攝取和利用,提高胰島素敏感性[6],但其具體的作用機(jī)制有待進(jìn)一步研究。最新研究發(fā)現(xiàn),硫氧還蛋白相互作用蛋白(thioredoxin-interacting protein,Txnip)在調(diào)控胰島素分泌中發(fā)揮著關(guān)鍵作用。糖尿病發(fā)病過(guò)程中,胰島素敏感性改變、高血糖、糖皮質(zhì)激素變化和β細(xì)胞凋亡均可誘導(dǎo)Txnip表達(dá),抑制葡萄糖攝取與利用。Txnip的表達(dá)受多條細(xì)胞外葡萄糖調(diào)控路徑影響,其中碳水化合物反應(yīng)元件結(jié)合蛋白(carbohydrate response element binding protein,ChREBP)和Max樣蛋白(Max-like protein X,MLx)是調(diào)節(jié)Txnip表達(dá)的最主要轉(zhuǎn)錄因子。高糖可促進(jìn)ChREBP去磷酸化,形成活化的ChREBP。當(dāng)活化ChREBP進(jìn)入細(xì)胞核,與異質(zhì)二聚體伴侶MLX,Txnip募集的輔助劑和組蛋白乙�；疨300結(jié)合,導(dǎo)致組蛋白H4乙�；�,從而進(jìn)行染色質(zhì)修飾,同時(shí)促使RNA聚合酶Ⅱ(PolⅡ)轉(zhuǎn)移到啟動(dòng)子區(qū),致使Txnip基因開始轉(zhuǎn)錄[7]。那么,S-Eq是否能夠通過(guò)ChREBP-Txnip信號(hào)通路調(diào)節(jié)胰島細(xì)胞分泌功能,從而實(shí)現(xiàn)其抗DM的作用,相關(guān)研究未見報(bào)道。本課題采用體外培養(yǎng)大鼠胰島素瘤(INS-1)細(xì)胞株,利用高糖刺激建立體外胰島細(xì)胞損傷模型,采用CCK-8法檢測(cè)細(xì)胞活力,ELISA法測(cè)定葡萄糖刺激胰島素分泌(glucose-stimulated insulin secretion,GSIS)功能,TUNEL法聯(lián)合Annexin V-FITC/PI流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡,Realtime PCR檢測(cè)前胰島素原(preproinsulin,PPI)、葡萄糖轉(zhuǎn)運(yùn)蛋白2(glucose transporter 2,Glut2)、線粒體陰離子載體解偶聯(lián)蛋白2(uncoupling protein2,UCP2)mRNA表達(dá),Western blot檢測(cè)Glut2及UCP2蛋白表達(dá),以探討S-Eq對(duì)INS-1細(xì)胞胰島素分泌功能的影響。并在此基礎(chǔ)上,采用PKA及PP2A試劑盒分別檢測(cè)PKA及PP2A活性,同時(shí)通過(guò)轉(zhuǎn)染si RNA和Western blot檢測(cè)ChREBP、Txnip蛋白表達(dá),結(jié)合Chip及雙熒光素酶報(bào)告基因等技術(shù)深入研究ChREBP-Txnip信號(hào)通路在S-Eq調(diào)控胰島細(xì)胞分泌功能中的作用。本實(shí)驗(yàn)的主要實(shí)驗(yàn)結(jié)果和結(jié)論如下:(1)S-Eq可顯著增加高糖培養(yǎng)條件下INS-1細(xì)胞活力,減少細(xì)胞凋亡(P0.05)。(2)S-Eq可顯著上調(diào)高糖培養(yǎng)條件下INS-1細(xì)胞PPI mRNA表達(dá)水平,改善高糖培養(yǎng)條件下INS-1細(xì)胞GSIS功能。同時(shí)可顯著增加高糖處理后細(xì)胞Glut2的轉(zhuǎn)錄表達(dá)而降低UCP2的轉(zhuǎn)錄表達(dá)水平(P0.05)。(3)S-Eq可顯著降低高糖培養(yǎng)INS-1細(xì)胞內(nèi)PP2A活性,升高PKA活性,抑制INS-1細(xì)胞內(nèi)ChREBP蛋白表達(dá)(P0.05)。(4)S-Eq可顯著減少ChREBP在ChoRE順式作用元件上募集,下調(diào)ChREBP(wt)轉(zhuǎn)染后INS-1細(xì)胞內(nèi)Txnip啟動(dòng)子區(qū)轉(zhuǎn)錄活性,進(jìn)而抑制Txnip表達(dá)。而在轉(zhuǎn)染ChREBP(dm)質(zhì)粒后,各組INS-1細(xì)胞內(nèi)Txnip啟動(dòng)子區(qū)轉(zhuǎn)錄活性均顯著降低。進(jìn)一步采用siRNA沉默ChREBP基因表達(dá)后,ChREBP和Txnip表達(dá)均顯著降低,同時(shí)發(fā)現(xiàn)S-Eq對(duì)高糖誘導(dǎo)Txnip的表達(dá)有顯著抑制作用(P0.05)。全文結(jié)論:S-Eq可能通過(guò)調(diào)節(jié)ChREBP活性進(jìn)而抑制Txnip信號(hào)通路,從而減少高糖培養(yǎng)條件下INS-1細(xì)胞凋亡、增強(qiáng)Glut2表達(dá)和降低UCP2的轉(zhuǎn)錄表達(dá),進(jìn)而改善INS-1細(xì)胞功能,增加胰島素分泌。
[Abstract]:Diabetic mellitus (DM) is one of the most common chronic metabolic diseases. It seriously endangers the health of human beings and brings serious mental and economic burden to individuals and families. And the pathogenesis of DM is very complex and is still not fully elucidated. At present, insulin resistance and insulin secretion deficiency are the two major causes of DM. The main factors have become an important target for the prevention and control of diabetes. However, most of the drugs used in clinical use have many adverse reactions. Therefore, it has become the focus of modern medical research to find and open up new ways to prevent and cure diabetes. Especially, it is very important to improve diabetes and its complications through dietary ways. Equol (Eq) is an important product formed by the metabolism of soy isoflavones (SI), the secondary metabolite of Soybean (soy isoflavones, SI), formed in the process of soybean growth, including S, R two isomers, and in the animal body, a large amount of S type [1]. studies show that SI intervention can increase insulin secretion, promote glucose uptake and utilization, so as to promote the uptake and utilization of glucose. Further studies have found that the biological activity of SI is mainly based on Eq to achieve higher bioactivity and bioavailability of [2,3].Eq than SI, and can effectively play the role of estrogen like, anti-inflammatory and antioxidant activities, not only in osteoporosis, menopause syndrome, and cardiovascular disease, and so on. [4]. has shown good preventive effects and has a good preventive effect on breast and prostate cancer. Studies have shown that Eq can significantly increase glucose tolerance [5], promote glucose uptake and utilization, improve insulin sensitivity [6], but its specific mechanism remains to be further studied. The latest research has found that sulphur and oxygen are still eggs. Thioredoxin-interacting protein (Txnip) plays a key role in the regulation of insulin secretion. In the course of diabetes, insulin sensitivity changes, hyperglycemia, glucocorticoid changes and beta cell apoptosis can induce Txnip expression, and the inhibition of glucose uptake and use of.Txnip is affected by multiple extracellular Portuguese. The effects of glucose regulation pathway, including carbohydrate response element binding protein (ChREBP) and Max like protein (Max-like protein X, MLx), are the most important transcription factors regulating Txnip expression. Hetero two polymer chaperone MLX, Txnip raised auxiliary and histone acetylation P300 binding, causing histone H4 acetylation, making chromatin modification, and promoting the transfer of RNA polymerase II (Pol II) to the promoter region, causing the Txnip gene to begin to transcribe [7]., then whether S-Eq can regulate islet cell scores through the ChREBP-Txnip signaling pathway. The role of secreting function to achieve its anti DM effect was not reported. This topic used in vitro culture of rat insulinoma (INS-1) cell lines, using high glucose to build a three-dimensional external islet cell damage model, using the CCK-8 method to detect cell viability, ELISA assay of glucose stimulated insulin secretion (glucose-stimulated insulin secretion, GS). IS) function, TUNEL method combined with Annexin V-FITC/PI flow cytometry to detect cell apoptosis. Realtime PCR was used to detect proinsulin (preproinsulin, PPI), glucose transporter 2 (glucose transporter 2, Glut2), mitochondrial anion carrier uncoupling protein 2 In order to investigate the effect of S-Eq on the insulin secretion of INS-1 cells, the activity of PKA and PP2A was detected by PKA and PP2A kits respectively. Meanwhile, the expression of Txnip protein was detected by the transfection of Si RNA and Western blot, and the expression of Txnip protein, combined with the double luciferase reporter gene and other techniques, was deeply studied. The main experimental results and conclusions of this experiment are as follows: (1) S-Eq can significantly increase the activity of INS-1 cells and decrease apoptosis (P0.05). (2) S-Eq can significantly increase the level of PPI mRNA in INS-1 cells under high glucose culture, and improve the GSIS function of INS-1 cells under high glucose conditions. At the same time, the transcriptional expression of Glut2 was significantly increased after high glucose treatment, and the transcriptional expression level of UCP2 (P0.05) was reduced. (3) S-Eq significantly reduced the PP2A activity in the INS-1 cells of high glucose, increased the PKA activity and inhibited the ChREBP protein expression in INS-1 cells (P0.05). (4) S-Eq can significantly reduce the recruitment of ChREBP in the cis acting element. The transcriptional activity of Txnip promoter in INS-1 cells after transfection of BP (WT), and then the expression of Txnip was inhibited. The transcriptional activity of Txnip promoter in INS-1 cells was significantly reduced after transfection of ChREBP (DM) plasmid. The ChREBP and expression were significantly reduced after the siRNA silent ChREBP gene was expressed. The expression of P has significant inhibitory effect (P0.05). Conclusion: the full text conclusion: S-Eq may inhibit the Txnip signaling pathway by regulating the activity of ChREBP, thus reducing the apoptosis of INS-1 cells under high glucose conditions, enhancing the expression of Glut2 and reducing the transcriptional expression of UCP2, and then improving the function of INS-1 cells and increasing the secretion of insulin.

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R587.1

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相關(guān)期刊論文 前2條

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本文編號(hào)：1891787