代謝性內(nèi)毒素血癥在肝胰島素抵抗中對(duì)肝細(xì)胞線粒體功能的影響
發(fā)布時(shí)間：2018-09-16 20:19
【摘要】：目的:1、通過(guò)建立大鼠代謝性內(nèi)毒素血癥及肝胰島素抵抗模型,探討代謝性內(nèi)毒素血癥對(duì)肝線粒體結(jié)構(gòu)與能量代謝的影響;2、通過(guò)體外實(shí)驗(yàn),探討內(nèi)毒素(LPS)對(duì)大鼠正常肝細(xì)胞線粒體結(jié)構(gòu)及能量代謝的直接影響。方法:在體實(shí)驗(yàn):30只雄性SD大鼠隨機(jī)分三組(每組10只):正常對(duì)照組(NC組)、高果糖組(HFD組,10%果糖水喂養(yǎng))、LPS組(皮下注射LPS 300μg·kg-1·d-1)。每周檢測(cè)并記錄大鼠體重,8周末,禁食12 h以上,腹腔注射糖耐量實(shí)驗(yàn)結(jié)束后,經(jīng)乙醚麻醉腹主動(dòng)脈采血,4℃3500 r/min離心10 min,取血漿分裝后置于-40℃保存。分離肝臟組織,部分置于4%多聚甲醛溶液中固定12~24 h,沖洗修整,經(jīng)常規(guī)石蠟包埋切片,行蘇木素伊紅染色(HE染色);剩余樣本置液氮速凍后轉(zhuǎn)-80℃保存。1、腹腔注射糖耐量實(shí)驗(yàn)(IPGTT)每周檢測(cè)大鼠體重變化,8周末,空腹腹腔注射50%葡萄糖溶液(2 g/kg)。尾靜脈采血,采用快速血糖儀(羅氏活力型)分別檢測(cè)注射前(0 min)與注射后(15min、30 min、120 min)血糖水平。2、血漿肝酶、LPS檢測(cè)及肝胰島素抵抗評(píng)估采用酶法測(cè)定血糖、血漿AST及ALT;鱟試劑法檢測(cè)血漿LPS水平;ELISA法檢測(cè)胰島素變化,計(jì)算胰島素抵抗指數(shù)(Homeostasis model assessment-insulin resistance,HOMA-IR),公式為:HOMA-IR=空腹血糖(FPG,mmol/L)×空腹胰島素(FINS,EU/ml)/22.5。3、氧化損傷產(chǎn)物及能量代謝指標(biāo)檢測(cè)采用酶法檢測(cè)并計(jì)算血漿中GSH-PX的水平;ELISA法檢測(cè)血漿中氧化損傷產(chǎn)物(8-Ohd G、MDA、4-HNE)及能量代謝指標(biāo)(ADP、ATP)的表達(dá)。4、肝臟組織病理學(xué)檢測(cè)于4%多聚甲醛固定液中取出肝臟組織,沖洗修整后,梯度酒精脫水,二甲苯透明,常規(guī)石蠟包埋切片,厚度5μm,行HE染色,光鏡下觀察肝組織病理學(xué)變化。5、Western blot分析采用Western blot檢測(cè)肝組織胰島素信號(hào)轉(zhuǎn)導(dǎo)關(guān)鍵蛋白(p-IRS1Tyr632、IRS1、p-PI3KTyr458、PI3K)及線粒體內(nèi)膜受體蛋白(UCP2)表達(dá)。離體實(shí)驗(yàn):采用改良膠原酶二步灌流法,體外分離培養(yǎng)大鼠肝細(xì)胞并隨機(jī)分為四組:正常對(duì)照組(NC組,DMEM培養(yǎng)液培養(yǎng))、高果糖組(HFD組,培養(yǎng)液+4.5 g/L果糖水)、LPS組(培養(yǎng)液+10 mg/L LPS)、果糖并LPS干預(yù)組(H+L組,培養(yǎng)液+4.5g/L果糖水+10 mg/L LPS)。20 h后,吸取并分裝細(xì)胞上清液,胰酶(含EDTA)消化,2000 r/min離心3 min后棄上清,收取肝細(xì)胞于-40℃冷凍保存。1、氧化損傷產(chǎn)物及能量代謝指標(biāo)檢測(cè)采用ELISA法檢測(cè)細(xì)胞上清液中氧化損傷產(chǎn)物(8-Ohd G、MDA、4-HNE)及能量代謝指標(biāo)(ADP、ATP)的表達(dá)。2、Western blot分析采用Western blot檢測(cè)肝細(xì)胞中胰島素信號(hào)轉(zhuǎn)導(dǎo)關(guān)鍵蛋白(p-IRS1Tyr632、IRS1、p-PI3KTyr458、PI3K)、線粒體內(nèi)膜受體蛋白(UCP2)表達(dá)。結(jié)果:在體實(shí)驗(yàn):1、體重變化及糖耐量結(jié)果實(shí)驗(yàn)期間,各組大鼠總體狀態(tài)良好。與NC組相比,HFD組與LPS組大鼠體重在2~8周顯著增高(P0.01,P0.05)。HFD組與LPS組各時(shí)間點(diǎn)血糖均顯著高于NC組(P0.01,P0.05),表明HFD組與LPS組大鼠發(fā)生糖耐量異常。2、血漿肝酶、LPS檢測(cè)及肝胰島素抵抗評(píng)估結(jié)果HFD組與LPS組的肝酶、FINS、FPG、LPS水平及HOMA-IR顯著高于NC組(P0.01),HFD組與LPS組無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。3、氧化損傷產(chǎn)物及能量代謝指標(biāo)變化HFD組與LPS組8-OhdG、MDA、4-HNE及GSH-PX顯著高于NC組(P0.01),HFD組與LPS組差異無(wú)統(tǒng)計(jì)學(xué)意義。能量代謝指標(biāo),HFD組與LPS組ADP、ATP與NC組相比顯著降低(P0.01),HFD組與LPS組差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。4、肝組織病理學(xué)變化NC組肝臟呈暗紅色,無(wú)油膩感,HE染色后光鏡下可見(jiàn)肝索呈放射狀排列。HFD組與LPS組肝臟有明顯油膩感;鏡下可見(jiàn)肝細(xì)胞包漿內(nèi)出現(xiàn)脂滴空泡并伴有氣球樣變。5、Western blot結(jié)果與NC組相比,HFD組與LPS組肝組織IRS1、PI3K表達(dá)、p-IRS1Tyr632/IRS1和p-PI3KTyr458/PI3K比值則顯著降低(P0.01),UCP2表達(dá)顯著升高(P0.01),HFD組與LPS組相比無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。離體實(shí)驗(yàn):1、氧化損傷產(chǎn)物及能量代謝指標(biāo)變化HFD組、LPS組及H+L組8-Ohd G、MDA及4-HNE顯著高于NC組(P0.01,P0.05),能量代謝指標(biāo)與NC組比較,HFD組、LPS組及H+L組ADP、ATP顯著降低(P0.05)。HFD組、LPS組及HFD+LPS組差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。2、Western blot結(jié)果與NC組相比,HFD組、LPS組及H+L組肝細(xì)胞IRS1、PI3K表達(dá)、p-IRS1Tyr632/IRS1和p-PI3KTyr458/PI3K比值則顯著降低(P0.01,P0.05),UCP2表達(dá)顯著升高(P0.01),HFD組、LPS組及H+L組相比無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:1、高果糖飲食及皮下注射LPS可誘發(fā)大鼠胰島素抵抗和代謝性內(nèi)毒素血癥,并伴有氧化應(yīng)激和肝細(xì)胞線粒體功能障礙。2、LPS可誘導(dǎo)氧化中間產(chǎn)物生成增多,清除減少,致使肝臟處于氧化應(yīng)激狀態(tài),進(jìn)而破壞肝細(xì)胞線粒體結(jié)構(gòu),影響其能量代謝,加速胰島素抵抗及代謝性疾病的發(fā)生發(fā)展。
[Abstract]:Objective: 1. To investigate the effects of metabolic endotoxemia on hepatic mitochondrial structure and energy metabolism by establishing rat models of metabolic endotoxemia and hepatic insulin resistance; 2. To investigate the direct effects of lipopolysaccharide (LPS) on mitochondrial structure and energy metabolism of rat normal hepatocytes in vitro. Rats were randomly divided into three groups (10 rats in each group): normal control group (NC group), high fructose group (HFD group, fed with 10% fructose water) and LPS group (subcutaneous injection of LPS 300 UG kg 1 D 1). Liver tissues were separated and fixed in 4% paraformaldehyde solution for 12-24 hours, washed and trimmed, paraffin embedded sections were stained with hematoxylin-eosin (HE) staining, and the remaining samples were frozen and stored at - 80 C. 1. Intraperitoneal glucose tolerance test (IPGTT) was performed weekly for 8 weeks. At the end of the study, 50% glucose solution (2 g / kg) was injected intraperitoneally on an empty stomach. Blood samples were collected from the tail vein. Blood glucose levels were measured before injection (0 min) and after injection (15 min, 30 min, 120 min) by rapid glucose meter (Roche's activity type). Blood glucose levels were measured by enzymatic method, plasma AST and ALT by LPS detection and liver insulin resistance assessment. The levels of insulin were measured by ELISA, and the Homeostasis model assessment-insulin resistance (HOMA-IR) was calculated. The formula was: HOMA-IR = fasting blood glucose (FPG, mmol/L) * fasting insulin (FINS, EU/ml) / 22.5.3. The levels of GSH-PX in plasma were measured by enzyme method. The expression of oxidative damage products (8-Ohd G, MDA, 4-HNE) and energy metabolism index (ADP, ATP) in plasma were detected by LISA. The liver tissues were removed from 4% paraformaldehyde fixed solution by histopathological examination. After washing and dressing, the liver tissues were dehydrated by graded alcohol, dimethylbenzene was transparent, paraffin embedded and sliced, and the thickness was 5 microns. The liver tissues were observed by HE staining and light microscopy. Pathological changes. 5. Western blot analysis was used to detect the expression of key insulin signal transduction proteins (p-IRS1Tyr632, IRS1, p-PI3KTyr458, PI3K) and mitochondrial endometrial receptor protein (UCP2) in liver tissue. In vitro experiment: rat hepatocytes were isolated and cultured by modified two-step perfusion of collagenase and randomly divided into four groups: normal control group Group C (NC group, DMEM culture medium), high fructose group (HFD group, culture medium + 4.5 g/L fructose water), LPS group (culture medium + 10 mg/L LPS), fructose and LPS intervention group (H + L group, culture medium + 4.5 g/L fructose water + 10 mg/L LPS). 20 hours later, the supernatant of cells was absorbed and packed, digested with trypsin (containing EDTA), and centrifuged for 3 minutes after 2000 r/min, the supernatant was discarded and collected. The expression of oxidative damage products (8-Ohd G, MDA, 4-HNE) and energy metabolism index (ADP, ATP) in cell supernatant were detected by ELISA. 2. The key proteins of insulin signal transduction (p-IRS1Tyr632, IRS1, p-PI3KTyr458, PI3K) were detected by Western blot. Mitochondrial endometrial receptor protein (UCP2) expression. Results: In vivo experiment: 1. Body weight changes and glucose tolerance results during the experiment, the overall state of rats in each group was good. Compared with NC group, the weight of HFD group and LPS group increased significantly in 2-8 weeks (P 0.01, P 0.05). The levels of liver enzymes, FINS, FPG, LPS and HOMA-IR in HFD group and LPS group were significantly higher than those in NC group (P 0.01). There was no significant difference between HFD group and LPS group (P 0.05). The changes of oxidative damage products and energy metabolism indexes in HFD group and LPS group were 8-OhdG, MDA, 4-HNE and GSH-PX. The ADP and ATP of HFD group and LPS group were significantly lower than those of NC group (P Compared with NC group, the expression of IRS1 and PI3K, the ratio of p-IRS1Tyr632/IRS1 and p-PI3KTyr458/PI3K in HFD group and LPS group were significantly decreased (P 0.01), and the expression of UCP2 was significantly increased (P 0.01) in HFD group and LPS group. In vitro experiment: 1. The changes of oxidative damage products and energy metabolism indexes in HFD group, LPS group and H + L group were significantly higher than those in NC group (P 0.01, P 0.05). Compared with NC group, ADP and ATP in HFD group, LPS group and H + L group were significantly lower (P 0.05). There was no significant difference between HFD group, LPS group and HFD + LPS group (P 0.05). (2) Compared with NC group, the expression of IRS1, PI3K, p-IRS1Tyr632/IRS1 and p-PI3KTyr458/PI3K in hepatocytes of HFD group, LPS group and H+L group were significantly decreased (P 0.01, P 0.05), and the expression of UCP2 was significantly increased (P 0.01). There was no significant difference between HFD group, LPS group and H+L group (P 0.05). Insulin resistance and metabolic endotoxemia, accompanied by oxidative stress and hepatocyte mitochondrial dysfunction. 2, LPS can induce the production of oxidative intermediates increased, clearance decreased, resulting in oxidative stress in the liver, thereby destroying the structure of hepatocyte mitochondria, affecting its energy metabolism, accelerating insulin resistance and metabolic diseases. The development of life.
【學(xué)位授予單位】：山西醫(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
本文編號(hào)：2244679
[Abstract]:Objective: 1. To investigate the effects of metabolic endotoxemia on hepatic mitochondrial structure and energy metabolism by establishing rat models of metabolic endotoxemia and hepatic insulin resistance; 2. To investigate the direct effects of lipopolysaccharide (LPS) on mitochondrial structure and energy metabolism of rat normal hepatocytes in vitro. Rats were randomly divided into three groups (10 rats in each group): normal control group (NC group), high fructose group (HFD group, fed with 10% fructose water) and LPS group (subcutaneous injection of LPS 300 UG kg 1 D 1). Liver tissues were separated and fixed in 4% paraformaldehyde solution for 12-24 hours, washed and trimmed, paraffin embedded sections were stained with hematoxylin-eosin (HE) staining, and the remaining samples were frozen and stored at - 80 C. 1. Intraperitoneal glucose tolerance test (IPGTT) was performed weekly for 8 weeks. At the end of the study, 50% glucose solution (2 g / kg) was injected intraperitoneally on an empty stomach. Blood samples were collected from the tail vein. Blood glucose levels were measured before injection (0 min) and after injection (15 min, 30 min, 120 min) by rapid glucose meter (Roche's activity type). Blood glucose levels were measured by enzymatic method, plasma AST and ALT by LPS detection and liver insulin resistance assessment. The levels of insulin were measured by ELISA, and the Homeostasis model assessment-insulin resistance (HOMA-IR) was calculated. The formula was: HOMA-IR = fasting blood glucose (FPG, mmol/L) * fasting insulin (FINS, EU/ml) / 22.5.3. The levels of GSH-PX in plasma were measured by enzyme method. The expression of oxidative damage products (8-Ohd G, MDA, 4-HNE) and energy metabolism index (ADP, ATP) in plasma were detected by LISA. The liver tissues were removed from 4% paraformaldehyde fixed solution by histopathological examination. After washing and dressing, the liver tissues were dehydrated by graded alcohol, dimethylbenzene was transparent, paraffin embedded and sliced, and the thickness was 5 microns. The liver tissues were observed by HE staining and light microscopy. Pathological changes. 5. Western blot analysis was used to detect the expression of key insulin signal transduction proteins (p-IRS1Tyr632, IRS1, p-PI3KTyr458, PI3K) and mitochondrial endometrial receptor protein (UCP2) in liver tissue. In vitro experiment: rat hepatocytes were isolated and cultured by modified two-step perfusion of collagenase and randomly divided into four groups: normal control group Group C (NC group, DMEM culture medium), high fructose group (HFD group, culture medium + 4.5 g/L fructose water), LPS group (culture medium + 10 mg/L LPS), fructose and LPS intervention group (H + L group, culture medium + 4.5 g/L fructose water + 10 mg/L LPS). 20 hours later, the supernatant of cells was absorbed and packed, digested with trypsin (containing EDTA), and centrifuged for 3 minutes after 2000 r/min, the supernatant was discarded and collected. The expression of oxidative damage products (8-Ohd G, MDA, 4-HNE) and energy metabolism index (ADP, ATP) in cell supernatant were detected by ELISA. 2. The key proteins of insulin signal transduction (p-IRS1Tyr632, IRS1, p-PI3KTyr458, PI3K) were detected by Western blot. Mitochondrial endometrial receptor protein (UCP2) expression. Results: In vivo experiment: 1. Body weight changes and glucose tolerance results during the experiment, the overall state of rats in each group was good. Compared with NC group, the weight of HFD group and LPS group increased significantly in 2-8 weeks (P 0.01, P 0.05). The levels of liver enzymes, FINS, FPG, LPS and HOMA-IR in HFD group and LPS group were significantly higher than those in NC group (P 0.01). There was no significant difference between HFD group and LPS group (P 0.05). The changes of oxidative damage products and energy metabolism indexes in HFD group and LPS group were 8-OhdG, MDA, 4-HNE and GSH-PX. The ADP and ATP of HFD group and LPS group were significantly lower than those of NC group (P Compared with NC group, the expression of IRS1 and PI3K, the ratio of p-IRS1Tyr632/IRS1 and p-PI3KTyr458/PI3K in HFD group and LPS group were significantly decreased (P 0.01), and the expression of UCP2 was significantly increased (P 0.01) in HFD group and LPS group. In vitro experiment: 1. The changes of oxidative damage products and energy metabolism indexes in HFD group, LPS group and H + L group were significantly higher than those in NC group (P 0.01, P 0.05). Compared with NC group, ADP and ATP in HFD group, LPS group and H + L group were significantly lower (P 0.05). There was no significant difference between HFD group, LPS group and HFD + LPS group (P 0.05). (2) Compared with NC group, the expression of IRS1, PI3K, p-IRS1Tyr632/IRS1 and p-PI3KTyr458/PI3K in hepatocytes of HFD group, LPS group and H+L group were significantly decreased (P 0.01, P 0.05), and the expression of UCP2 was significantly increased (P 0.01). There was no significant difference between HFD group, LPS group and H+L group (P 0.05). Insulin resistance and metabolic endotoxemia, accompanied by oxidative stress and hepatocyte mitochondrial dysfunction. 2, LPS can induce the production of oxidative intermediates increased, clearance decreased, resulting in oxidative stress in the liver, thereby destroying the structure of hepatocyte mitochondria, affecting its energy metabolism, accelerating insulin resistance and metabolic diseases. The development of life.
【學(xué)位授予單位】：山西醫(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10條
1 秦苗;王文遞;王明亮;宋彬妤;吳惠文;;代謝性內(nèi)毒素血癥在果糖誘發(fā)的NAFLD等代謝疾病中的作用研究[J];現(xiàn)代生物醫(yī)學(xué)進(jìn)展;2016年33期
2 顏斌;李婷;彭正良;;維格列汀對(duì)非酒精性脂肪肝病大鼠脂肪酸w氧化關(guān)鍵酶CYP1A1及CYP2E1表達(dá)的影響[J];廣東醫(yī)學(xué);2016年05期
3 楊友生;瞿祥春;胡松;;三種大鼠肝細(xì)胞分離及原代培養(yǎng)方法的比較[J];中華實(shí)驗(yàn)外科雜志;2016年03期
4 Licia Polimeni;Maria Del Ben;Francesco Baratta;Ludovica Perri;Fabiana Albanese;Daniele Pastori;Francesco Violi;Francesco Angelico;;Oxidative stress:New insights on the association of nonalcoholic fatty liver disease and atherosclerosis[J];World Journal of Hepatology;2015年10期
5 劉建華;朱芬芳;阮國(guó)永;;羅格列酮聯(lián)合維生素E治療非酒精性脂肪性肝病的臨床研究[J];中國(guó)醫(yī)學(xué)創(chuàng)新;2014年07期
6 葉娟;王群;付溪;鄭銳丹;應(yīng)艷琴;羅小平;;原代大鼠肝細(xì)胞分離及培養(yǎng)鑒定[J];實(shí)用兒科臨床雜志;2012年07期
7 安紅敏;;維生素與微量元素抗氧化作用的研究進(jìn)展[J];天津科技;2008年05期
8 R Scott Rector;John P Thyfault;Jamal A Ibdah;;Nonalcoholic fatty liver disease and mitochondrial dysfunction[J];World Journal of Gastroenterology;2008年02期
9 趙文霞;劉曉彥;段榮章;董靖;聶山文;石艷芬;;消脂護(hù)肝膠囊對(duì)非酒精性脂肪肝大鼠病理及CYP2E1基因的影響[J];中華中醫(yī)藥雜志;2007年06期
10 黃進(jìn),楊國(guó)宇,李宏基,熊程輝,李留安,吳玉臣;抗氧化劑作用機(jī)制研究進(jìn)展[J];自然雜志;2004年02期
，本文編號(hào)：2244679
本文鏈接：http://sikaile.net/yixuelunwen/nfm/2244679.html
最近更新
教材專著
