本文選題：DEHP + 糖代謝��； 參考：《吉林大學(xué)》2017年碩士論文

【摘要】：隨著社會(huì)經(jīng)濟(jì)的發(fā)展和人民生活水平的提高,糖尿病患病率逐年呈上升趨勢(shì),已成為影響人類生命健康的主要慢性非傳染性疾病之一。其中兒童青少年患病比例也在逐步擴(kuò)大,特別是2型糖尿病。而2型糖尿病的發(fā)生又與代謝綜合征聯(lián)系緊密,已有研究提示環(huán)境中存在的內(nèi)分泌干擾物等外源性化學(xué)物質(zhì)可以干擾機(jī)體的正常代謝,特別是以DEHP為主要代表的鄰苯二甲酸酯類物質(zhì),由于DEHP作為增塑劑使用廣泛,而其又容易從材料中揮發(fā)和溶解出來(lái),因此不但對(duì)生態(tài)環(huán)境造成污染,還可通過(guò)空氣、水、食物等多種媒介進(jìn)入機(jī)體,進(jìn)而危害機(jī)體健康。目的:觀察DEHP暴露對(duì)大鼠糖代謝的影響,了解大鼠肝臟和胰腺組織胰島素受體、瘦素受體以及JAK2-STAT3-SOCS3通路相關(guān)蛋白的表達(dá)情況,探討DEHP致大鼠糖代謝紊亂的可能機(jī)制,為深入了解環(huán)境內(nèi)分泌干擾物DEHP的毒性,保護(hù)人群健康提供科學(xué)依據(jù),為Met S和T2D等代謝性疾病的研究以及預(yù)防提供新思路。方法:選擇健康清潔級(jí)Wistar大鼠80只,雌雄各半,21日齡,重量為(50±5)g,將80只青春期大鼠按照性別分別隨機(jī)分為四組:(1)對(duì)照組(玉米油);(2)低劑量組(5mg/kg/d DEHP,1/6000 LD50);(3)中劑量組(50 mg/kg/d DEHP,1/600 LD50);(4)高劑量組(500 mg/kg/d DEHP,1/60 LD50)。適應(yīng)性飼養(yǎng)一周后,用玉米油配制相應(yīng)濃度的DEHP后,每天上午9:00進(jìn)行灌胃染毒,稱量飲水量、攝食量和體重,并對(duì)實(shí)驗(yàn)動(dòng)物進(jìn)行大體觀察,持續(xù)染毒4周。染毒結(jié)束后,禁食12h,將大鼠麻醉,心臟取血,離心后收集血清,并剪取肝臟和胰腺;用生化分析儀檢測(cè)空腹血糖,用ELISA法測(cè)定血清胰島素和瘦素水平,并計(jì)算各組青春期大鼠的HOMA-IR值;計(jì)算肝臟和胰腺的臟器系數(shù);HE染色法觀察青春期大鼠肝臟和胰腺的病理組織變化;實(shí)時(shí)熒光定量PCR法測(cè)定肝臟和胰腺中JAK2、STAT3、SOCS3和Ob-R基因m RNA的表達(dá)情況;免疫組織化學(xué)法對(duì)JAK2、STAT3、SOCS3和Ob-R蛋白在肝臟和胰腺中進(jìn)行定位,并應(yīng)用Image-Pro Plus 6.0軟件根據(jù)不同組間的IOD值進(jìn)行半定量蛋白分析;Western Blot法測(cè)定肝臟和胰腺中JAK2、STAT3、SOCS3、Ob-R和IR蛋白表達(dá)水平,并進(jìn)行分析。結(jié)果:1.DEHP對(duì)青春期大鼠的一般毒性(1)50mg/kg/d和500mg/kg/d DEHP暴露組的青春期大鼠出現(xiàn)行動(dòng)遲緩、精神萎靡不振、被毛疏松無(wú)光澤并且伴有脫毛現(xiàn)象,而對(duì)照組和5mg/kg/d DEHP暴露組的青春期大鼠精神狀態(tài)良好、活動(dòng)正常、皮毛光亮。(2)50mg/kg/d和500mg/kg/d DEHP暴露組的青春期大鼠攝食、飲水和體重均顯著低于5mg/kg/d DEHP暴露組(P0.05),而5mg/kg/d DEHP暴露組青春期大鼠攝食量、飲水量和體重顯著高于對(duì)照組(P0.05)。(3)500mg/kg/d DEHP暴露組的青春期大鼠肝臟臟器系數(shù)顯著高于其他各組(P0.05),而各組大鼠胰腺臟器系數(shù)均無(wú)顯著差異(P0.05)。2.DEHP對(duì)青春期大鼠的血糖穩(wěn)態(tài)和相關(guān)激素的影響50mg/kg/d和500mg/kg/d DEHP暴露組的青春期大鼠空腹血糖、血清胰島素,血清瘦素和HOMA-IR值均顯著高于對(duì)照組(P0.05),并且500mg/kg/d DEHP暴露組顯著高于5mg/kg/d DEHP暴露組(P0.05)。3.DEHP對(duì)青春期大鼠肝臟和胰腺組織形態(tài)的影響(1)肝臟:50mg/kg/d和500mg/kg/d DEHP暴露組青春期大鼠肝細(xì)胞染色較淺、排列疏松、部分水腫、胞漿比較清亮,有炎性細(xì)胞浸潤(rùn)和空泡樣變,并且500mg/kg/d染毒組青春期大鼠肝細(xì)胞出現(xiàn)輕度壞死現(xiàn)象。(2)胰腺:50mg/kg/d和500mg/kg/d染毒組青春期大鼠胰島數(shù)量相對(duì)減少,分布零散,邊界不清,并且細(xì)胞體積稍有增大4.DEHP對(duì)青春期大鼠肝臟和胰腺相關(guān)基因m RNA表達(dá)的影響(1)肝臟:500mg/kg/d DEHP暴露組大鼠肝臟Ob-R基因m RNA表達(dá)顯著低于5mg/kg/d DEHP暴露組(P0.05);500mg/kg/d DEHP暴露組大鼠肝臟SOCS3 m RNA表達(dá)顯著高于對(duì)照組和5mg/kg/d DEHP暴露組(P0.05);JAK2和STAT3 m RNA表達(dá)各組間均無(wú)顯著差異(P0.05)。(2)胰腺:500mg/kg/d DEHP暴露組大鼠胰腺Ob-R基因m RNA表達(dá)顯著低于其他各組(P0.05);500mg/kg/d DEHP暴露組大鼠胰腺SOCS3基因m RNA表達(dá)顯著高于對(duì)照組(P0.05);50mg/kg/d和500mg/kg/d DEHP暴露組大鼠胰腺STAT3基因m RNA表達(dá)均顯著高于對(duì)照組(P0.05);JAK2 m RNA表達(dá)各組均無(wú)顯著差異(P0.05)。5.DEHP對(duì)青春期大鼠肝臟和胰腺相關(guān)蛋白表達(dá)的影響(1)肝臟:免疫組織結(jié)果顯示,500mg/kg/d DEHP暴露組大鼠肝臟Ob-R蛋白表達(dá)顯著低于對(duì)照組(P0.05);50mg/kg/d和500mg/kg/d DEHP暴露組大鼠肝臟SOCS3蛋白表達(dá)均顯著高于對(duì)照組(P0.05);500mg/kg/d DEHP暴露組大鼠肝臟JAK2和STAT3蛋白表達(dá)均顯著高于對(duì)照組(P0.05)。Western Blot結(jié)果顯示,500mg/kg/d DEHP暴露組大鼠肝臟IR蛋白表達(dá)顯著低于對(duì)照組(P0.05);5mg/kg/d、50mg/kg/d和500mg/kg/d DEHP暴露組大鼠肝臟Ob-R蛋白表達(dá)均顯著低于對(duì)照組(P0.05);50mg/kg/d和500mg/kg/d DEHP暴露組大鼠肝臟SOCS3蛋白表達(dá)均顯著高于對(duì)照組和5mg/kg/d DEHP暴露組(P0.05);50mg/kg/d和500mg/kg/d DEHP暴露組大鼠肝臟JAK2和STAT3蛋白表達(dá)均顯著高于對(duì)照組(P0.05)。(2)胰腺:免疫組織結(jié)果顯示,500mg/kg/d DEHP暴露組大鼠胰腺Ob-R蛋白表達(dá)顯著低于其他各組(P0.05);500mg/kg/d和50mg/kg/d DEHP暴露組大鼠胰腺SOCS3蛋白表達(dá)均顯著高于對(duì)照組(P0.05);JAK2和STAT3蛋白表達(dá)各組間均無(wú)顯著差異(P0.05)。Western Blot結(jié)果顯示,500mg/kg/d DEHP暴露組大鼠胰腺IR蛋白表達(dá)顯著低于對(duì)照組和5mg/kg/d DEHP暴露組(P0.05);50mg/kg/d和500mg/kg/d DEHP暴露組大鼠胰腺Ob-R蛋白表達(dá)均顯著低于對(duì)照組(P0.05);500mg/kg/d DEHP暴露組大鼠胰腺SOCS3蛋白表達(dá)顯著高于其他各組(P0.05);500mg/kg/d DEHP暴露組大鼠胰腺JAK2和STAT3蛋白表達(dá)均顯著高于對(duì)照組(P0.05)。結(jié)論:(1)低劑量DEHP暴露可能會(huì)促進(jìn)青春期大鼠生長(zhǎng)發(fā)育,體重增速加快,而高劑量的DEHP暴露可能會(huì)減緩青春期大鼠體重增加;高劑量DEHP暴露可使青春期大鼠肝臟的臟器系數(shù)增加。(2)高劑量DEHP暴露可能會(huì)影響青春期大鼠正常糖代謝,擾亂血糖穩(wěn)態(tài),引起胰島素和瘦素水平升高,出現(xiàn)以胰島素抵抗為典型癥狀的代謝綜合征。(3)高劑量DEHP暴露可降低青春期大鼠肝臟和胰腺胰島素受體和瘦素受體的表達(dá),受體信號(hào)傳導(dǎo)受阻可能是高劑量DEHP暴露所導(dǎo)致青春期大鼠瘦素水平升高的機(jī)制之一(4)高劑量DEHP暴露可能會(huì)增加肝臟和胰腺SOCS3 m RNA表達(dá),并且使青春期大鼠肝臟和胰腺JAK2、STAT3和SOCS3蛋白表達(dá)增加。由于SOCS3的高表達(dá)而引起的受體后信號(hào)轉(zhuǎn)導(dǎo)抑制可能是青春期大鼠產(chǎn)生胰島素抵抗和瘦素水平升高的機(jī)制之一。
[Abstract]:With the development of social economy and the improvement of people's living standard, the prevalence of diabetes has been increasing year by year, and has become one of the main chronic non infectious diseases affecting human life and health. Among them, the proportion of children and adolescents is gradually expanding, especially type 2 diabetes. And the incidence of type 2 diabetes is associated with metabolic syndrome. Closely, it has been suggested that exogenous chemicals such as endocrine disruptors, such as endocrine disruptors, can interfere the normal metabolism of the body, especially the DEHP as the main representative of phthalic acid esters. Because DEHP is widely used as plasticizer, it is easily volatilized and dissolved from the material. Therefore, it is not only made to the ecological environment. In order to investigate the effect of DEHP exposure on the metabolism of sugar in rats, the effects of exposure on glucose metabolism in rats, the expression of insulin receptor, leptin receptor and JAK2-STAT3-SOCS3 pathway related egg white in rat liver and pancreas were observed, and the disorder of glucose metabolism caused by DEHP in rats was investigated. The possible mechanism provides a scientific basis for understanding the toxicity of environmental endocrine disrupting interferon DEHP, protecting the health of the population, and providing new ideas for the study and prevention of metabolic diseases such as Met S and T2D. Methods: 80 healthy and clean Wistar rats were selected, the male and the male were half, 21 days old, and the weight was (50 + 5) g, and 80 puberty rats were divided by sex respectively. Randomly divided into four groups: (1) the control group (corn oil); (2) low dose group (5mg/kg/d DEHP, 1/6000 LD50); (3) medium dose group (50 mg/kg/d DEHP, 1/600 LD50); (4) high dose group (500 mg/kg/d DEHP, 1/60 LD50). After adaptation to a week, using jade rice oil to prepare the corresponding concentration, at 9:00 every morning, gavage, weighing water quantity, feeding, feeding The quantity and weight were observed, and the experimental animals were observed for 4 weeks. After the end of the infection, the rats were fasted 12h, the rats were anaesthetized, the heart was collected, the serum was collected, the liver and the pancreas were collected, the liver and the pancreas were cut, the fasting blood glucose was detected by the biochemical analyzer, the serum islet and leptin levels were measured by the ELISA method, and the HOMA-IR value of the rats in each group was calculated. The organ coefficients of the liver and pancreas were calculated. The pathological changes of the liver and pancreas in the puberty rats were observed by HE staining. The expression of M RNA, JAK2, STAT3, SOCS3 and Ob-R in the liver and pancreas was measured by real time fluorescence quantitative PCR, and the immunohistochemical method was used to locate JAK2, STAT3, SOCS3 and Ob-R protein in the liver and pancreas, and should be located in the liver and pancreas. Image-Pro Plus 6 software was used for semi quantitative protein analysis based on IOD values between different groups. Western Blot was used to determine the protein expression levels of JAK2, STAT3, SOCS3, Ob-R and IR in the liver and pancreas. Results: 1.DEHP toxicity of 1.DEHP on adolescent rats (1) action in adolescent rats Retardation, depressed spirit, glossy hair loosening and hair removal, while the adolescent rats in the control group and the 5mg/kg/d DEHP exposed group had good mental state, normal activity and bright fur. (2) the feeding of puberty rats in 50mg/kg/d and 500mg/kg/d DEHP exposure groups were significantly lower than those of 5mg/kg/d DEHP exposure group (P0.05). In 5mg/kg/d DEHP exposure group, the intake of puberty rats was significantly higher than that of the control group (P0.05). (3) the liver organ coefficient of the puberty rats in the 500mg/kg/d DEHP exposure group was significantly higher than that of the other groups (P0.05), but the pancreas organ coefficient of the rats in each group had no significant difference (P0.05).2.DEHP to the blood glucose homeostasis and related excitation in the puberty rats. The effect of 50mg/kg/d and 500mg/kg/d DEHP exposure group on fasting blood glucose, serum insulin, serum leptin and HOMA-IR were significantly higher than that of the control group (P0.05), and the 500mg/kg/d DEHP exposure group was significantly higher than 5mg/kg/d DEHP exposure group (P0.05).3.DEHP on the liver and pancreatic tissue morphology of the puberty rats (1) livers: 50mg/ The liver cells of kg/d and 500mg/kg/d DEHP exposed rats were dyed shallow, loosely arranged, partial edema, cytoplasm relatively clear, inflammatory cell infiltration and vacuolating, and there was a slight necrosis in the liver cells of the puberty rats of 500mg/kg/d. (2) pancreas: the number of pancreatic islets in 50mg/ kg/d and 500mg/kg/d exposed rats was relative to the number of islets of the rats. The effect of 4.DEHP on the expression of M RNA in liver and pancreas related genes in puberty rats (1) liver: Ob-R gene m RNA expression in liver of 500mg/kg/d DEHP exposure group was significantly lower than 5mg/kg/d DEHP exposure group (P0.05). There was no significant difference between the control group and the 5mg/kg/d DEHP exposure group (P0.05), and there was no significant difference in the expression of JAK2 and STAT3 m RNA (P0.05). (2) the pancreas Ob-R gene was significantly lower in the pancreas of the 500mg/kg/d DEHP exposure group than in the other groups. The expression of STAT3 gene m RNA in the pancreas of g/kg/d and 500mg/kg/d DEHP exposed rats was significantly higher than that in the control group (P0.05), and there was no significant difference in the expression of JAK2 m RNA in each group (P0.05).5.DEHP on the expression of liver and pancreas related proteins in puberty rats (1) liver: immune tissue results showed the liver protein table of rats exposed group The expression of SOCS3 protein in liver of 50mg/kg/d and 500mg/kg/d DEHP exposed rats was significantly higher than that of control group (P0.05), and the expression of JAK2 and STAT3 protein in liver of 500mg/kg/d DEHP exposed rats was significantly higher than that of control group (P0.05) (P0.05). The expression of Ob-R protein in liver of rats exposed to 5mg/kg/d, 50mg/kg/d and 500mg/kg/d DEHP was significantly lower than that of the control group (P0.05), and the expression of SOCS3 protein in the liver of 50mg/kg/d and 500mg/kg/d DEHP exposed rats was significantly higher than that of the control group and the 5mg/kg/d exposed group. The expression of JAK2 and STAT3 protein was significantly higher than that of the control group (P0.05). (2) pancreas: the results of immuno tissue showed that the expression of Ob-R protein in the pancreas of 500mg/kg/d DEHP exposed rats was significantly lower than that of the other groups (P0.05), and the expression of SOCS3 protein in the pancreas of 500mg/kg/d and 50mg/kg/d DEHP exposed rats was significantly higher than that of the control group (P0.05). There was no significant difference between each group (P0.05).Western Blot results showed that the expression of IR protein in the pancreas of 500mg/kg/d DEHP exposed rats was significantly lower than that of the control group and 5mg/kg/d DEHP exposure group (P0.05), and the expression of pancreatic protein in the pancreas of 50mg/kg/d and 500mg/kg/d DEHP exposed rats was significantly lower than that of the control group. The expression of S3 protein was significantly higher than that in other groups (P0.05), and the expression of JAK2 and STAT3 protein in the pancreas of 500mg/kg/d DEHP exposed rats was significantly higher than that of the control group (P0.05). Conclusion: (1) low dose DEHP exposure may promote the growth and development of puberty rats and accelerate the growth of body weight, and high dose of DEHP exposure may slow the weight gain of adolescent rats. High dose DEHP exposure can increase the organ coefficient of liver in puberty rats. (2) high dose DEHP exposure may affect normal glucose metabolism in puberty rats, disturb blood glucose homeostasis, increase insulin and leptin levels, and develop metabolic syndrome with insulin resistance as typical symptoms. (3) high dose DEHP exposure can reduce the liver of puberty rats The expression of insulin receptor and leptin receptor in the pancreas and pancreas, and the obstruction of receptor signal conduction may be one of the mechanisms of high levels of leptin in puberty rats induced by high dose DEHP exposure (4) high dose DEHP exposure may increase the expression of SOCS3 m RNA in the liver and pancreas, and make the JAK2, STAT3 and SOCS3 protein tables of the liver and pancreas of the puberty rats The inhibition of post receptor signal transduction caused by the high expression of SOCS3 may be one of the mechanisms of insulin resistance and leptin levels in puberty rats.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R114

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