肌肉注射sRAGE基因?qū)Ω咧痛笫笾|(zhì)代謝、慢性炎癥狀態(tài)及重要器官的作用
發(fā)布時(shí)間：2018-08-26 07:19
【摘要】： 背景及目的:高脂餐飲食促使動(dòng)脈粥樣硬化、血脂異常、胰島素抵抗等發(fā)病,現(xiàn)代生活習(xí)慣使之呈流行趨勢(shì),近年研究表明慢性炎癥和胰島素抵抗相輔相成�，F(xiàn)代飲食以高脂、高熱量為特征,高溫烹制的食物富含晚期糖基化終末產(chǎn)物(AGE),AGE與與其受體(RAGE)結(jié)合,通過(guò)NADPH及NF-κB通路,激活巨噬細(xì)胞,可導(dǎo)致氧化應(yīng)激,引起多種促炎因子產(chǎn)生,一次攝入雖可使循環(huán)中AGE升高,使機(jī)體短時(shí)間內(nèi)產(chǎn)生氧化應(yīng)激狀態(tài),炎癥因子水平升高,但停止攝入后機(jī)體AGE水平逐漸下降,氧化應(yīng)激逐漸消退,但如果長(zhǎng)期大劑量攝入,則會(huì)超出機(jī)體的代償能力,表現(xiàn)為慢性炎癥狀態(tài)。減少食物中的AGE、阻斷AGE與RAGE結(jié)合可降低機(jī)體慢性炎癥狀態(tài),使db/db大鼠胰島素抵抗減輕,NOD小鼠和高脂餐C57/B6小鼠糖尿病發(fā)生減少,糖尿病慢性并發(fā)癥(動(dòng)脈粥樣硬化、微血管并發(fā)癥)減輕。新近研究發(fā)現(xiàn)體內(nèi)存在一種缺失細(xì)胞跨膜段及細(xì)胞內(nèi)段的RAGE(又稱為sRAGE)具有與AGE結(jié)合的能力,限制了AGE與細(xì)胞膜上完整的RAGE結(jié)合的能力,阻斷AGE所導(dǎo)致的細(xì)胞氧化應(yīng)激,減輕機(jī)體慢性免疫反應(yīng)狀態(tài)。本研究的目的是通過(guò)肌肉注射sRAGE基因,觀察sRAGE對(duì)高脂餐大鼠機(jī)體氧化應(yīng)激狀態(tài)、脂質(zhì)代謝、胰島素抵抗及主動(dòng)脈內(nèi)膜、肝臟、腎臟的改變的影響。探討肌肉注射sRAGE基因防治高脂餐大鼠代謝紊亂及重要器官損傷的作用。 方法: 1.動(dòng)物實(shí)驗(yàn):取SD大鼠28只(體重150～160g,6周齡),隨機(jī)分為3組:高脂餐治療組、高脂餐對(duì)照組和普通餐對(duì)照組。高脂餐大鼠先以高脂餐喂養(yǎng)8周,第9周10只在雙側(cè)后腿肌內(nèi)各注射pLNCX_2-sRAGE 300μg(高脂餐治療組),10只注射pLNCX_2質(zhì)粒300μg/側(cè)(高脂餐對(duì)照組):分別于第12周、第15周重復(fù)注射一次,第17周處死。8只同期普通飼料喂養(yǎng)的大鼠(普通餐對(duì)照組)在第17周處死。骨骼肌注射pLNCX_2-sRAGE和pLNCX_2質(zhì)粒方法:4%水合氯醛腹腔麻醉大鼠,暴露雙側(cè)后腿肌肉,各注射質(zhì)粒300μg,以電極相距20mm給予方波電脈沖(電壓200V/cm,波寬40CM,脈沖次數(shù)9,頻率1HZ)刺激,以增加基因轉(zhuǎn)移效率。 2.三組大鼠分別于每次注射后的1、2、3周斷尾取血測(cè)定SOD、MDA值。第17周處死前測(cè)空腹血糖和體重,處死后立即取心臟血、主動(dòng)脈、肝臟和腎臟,進(jìn)行血清胰島素、甘油三脂、總膽固醇、TNF-α、超敏CRP、SOD、MDA測(cè)定,腎臟和主動(dòng)脈病理HE染色,應(yīng)用免疫組織化學(xué)染色觀察經(jīng)不同措施處理的大鼠的腎臟TGF-β1、肝臟NF-κB的表達(dá)。 結(jié)果: 1.高脂餐對(duì)照組體重、血糖、血清胰島素、甘油三脂、總膽固醇、超敏CRP、TNF-α較普通餐對(duì)照組明顯升高,與高脂餐對(duì)照組相比高脂餐治療組除體重和超敏CRP輕度下降無(wú)統(tǒng)計(jì)學(xué)差異、甘油三脂顯著升高外,其余各指標(biāo)下降有統(tǒng)計(jì)學(xué)差異。 2.高脂餐治療組大鼠注射基因后SOD水平逐漸上升,到第1次注射后3周與高脂餐對(duì)照組呈現(xiàn)統(tǒng)計(jì)學(xué)差異(P0.05),高脂餐治療組大鼠注射基因后血MDA水平開始下降,1次注射后2周與高脂餐對(duì)照組有統(tǒng)計(jì)學(xué)差異(P0.05),在1次注射后3周有所升高;重復(fù)注射SOD繼續(xù)升高,至接近普通餐對(duì)照組不再升高,MDA再次下降。 3. sRAGE對(duì)重要器官作用情況:普通餐對(duì)照組和高脂餐治療組均未見(jiàn)動(dòng)脈粥樣硬化,但高脂餐對(duì)照組動(dòng)脈內(nèi)膜不完整,內(nèi)皮壞死脫落,局部?jī)?nèi)膜略增厚,有動(dòng)脈粥樣硬化早期損傷的表現(xiàn);高脂餐對(duì)照組腎小球系膜細(xì)胞和細(xì)胞外基質(zhì)增多,TGF-β1呈現(xiàn)高表達(dá),而高脂餐治療組以上變化則較輕微,普通餐對(duì)照組皮質(zhì)部腎小體和腎小管結(jié)構(gòu)完好,TGF-β1低水平表達(dá),TGF-β1三組間差異顯著(P0.01);高脂餐對(duì)照組肝臟NF-κB表達(dá)增加,普通餐對(duì)照組呈低水平表達(dá),高脂餐治療組介于兩者之間,三組間差異顯著(P0.01)。 結(jié)論: 1.高脂飲食使大鼠體內(nèi)氧化應(yīng)激水平升高,抗氧化能力減弱,骨骼肌注射sRAGE基因可以降低高脂餐大鼠的氧化應(yīng)激水平,抗氧化能力增強(qiáng)。 2.高脂餐可引起胰島素抵抗,膽固醇水平升高;出現(xiàn)動(dòng)脈粥樣硬化、腎小球硬化的早期表現(xiàn),骨骼肌注射sRAGE可一定程度地減輕此種改變。
[Abstract]:BACKGROUND & OBJECTIVE: High-fat diet promotes atherosclerosis, dyslipidemia, insulin resistance and other diseases. Modern living habits make it popular. Recent studies have shown that chronic inflammation and insulin resistance complement each other. Modern diet is characterized by high fat and high calorie content. High-temperature cooked food is rich in advanced glycation end products (AGE), AGE. Combining with its receptor (RAGE) and activating macrophages through NADPH and NF-kappa B pathway can lead to oxidative stress and induce the production of many pro-inflammatory factors. One intake can elevate the circulating AGE, which results in oxidative stress in a short period of time. The level of inflammatory factors increases, but the level of AGE decreases gradually after stopping intake and oxidative stress drives. Reducing AGE in food and blocking the combination of AGE and RAGE can reduce the chronic inflammatory state of the body, reduce insulin resistance in dB / DB rats, decrease the incidence of diabetes in NOD mice and C57 / B6 mice with high fat diet, and reduce the chronic complications of diabetes mellitus. Recent studies have found that a kind of RAGE (also known as sRAGE) lacking cell transmembrane and intracellular segments in vivo has the ability to bind to AGE, which limits the ability of AGE to bind to the complete RAGE on the cell membrane, blocks the oxidative stress induced by AGE and alleviates the chronic immune response symptoms. The aim of this study was to observe the effects of sRAGE on oxidative stress, lipid metabolism, insulin resistance and changes of aortic intima, liver and kidney in rats fed high-fat diet by intramuscular injection of sRAGE gene.
Method:
1. Animal experiment: 28 SD rats (weighing 150-160g, 6 weeks old) were randomly divided into three groups: high-fat meal treatment group, high-fat meal control group and normal meal control group. Eight rats fed with normal diet were sacrificed at the seventeenth week. The skeletal muscles were injected with pLNCX_2-sRAGE and pLNCX_2 plasmids. The rats were anesthetized with 4% chloral hydrate intraperitoneally. The hind leg muscles were exposed. The plasmids were injected at 300 UG and the distance between the electrodes was 20 mm. The gene transfer efficiency was increased by stimulating with square wave electric pulse (voltage 200V/cm, wave width 40CM, pulse number 9, frequency 1HZ).
2. Blood samples were taken at the end of 1, 2, and 3 weeks after each injection to determine SOD and MDA. Fasting blood glucose and body weight were measured at the seventeenth week before execution. Cardiac blood, aorta, liver and kidney were taken immediately after execution. Serum insulin, triglyceride, total cholesterol, TNF-a, hypersensitive CRP, SOD, MDA were measured. Pathological HE staining of kidney and aorta was used. Histochemical staining was used to observe the expression of TGF- NF- 1 and liver NF- kappa B in rats treated with different measures.
Result:
1. The body weight, blood glucose, serum insulin, triglyceride, total cholesterol, hypersensitive CRP and TNF-alpha in the high-fat meal control group were significantly higher than those in the normal meal control group. Compared with the high-fat meal control group, there was no significant difference in weight and hypersensitive CRP, except triglyceride.
2. The level of SOD in the high-fat diet group increased gradually after gene injection, and there was significant difference between the high-fat diet group and the control group at 3 weeks after the first injection (P 0.05). Repeated injection of SOD continued to rise to nearly normal meal. The control group no longer increased, and MDA decreased again.
3. Effect of sRAGE on important organs: There was no atherosclerosis in the control group and the high-fat diet group, but in the high-fat diet control group, the intima was incomplete, endothelial necrosis and exfoliation, local intima was slightly thickened, showing early atherosclerotic injury; in the high-fat diet control group, mesangial cells and extracellular matrix increased, TGF-ECM increased. The expression of TGF-beta 1 was significantly higher in the control group than that in the control group (P There was a significant difference between the three groups (P0.01).
Conclusion:
1. High-fat diet can elevate the level of oxidative stress and weaken the antioxidant capacity of rats. Injecting sRAGE gene into skeletal muscle can reduce the level of oxidative stress and enhance the antioxidant capacity of rats with high-fat diet.
2. High-fat diet can induce insulin resistance and elevated cholesterol levels, and early manifestations of atherosclerosis and glomerulosclerosis can be alleviated by intramuscular injection of sRAGE.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R363
本文編號(hào)：2204134
[Abstract]:BACKGROUND & OBJECTIVE: High-fat diet promotes atherosclerosis, dyslipidemia, insulin resistance and other diseases. Modern living habits make it popular. Recent studies have shown that chronic inflammation and insulin resistance complement each other. Modern diet is characterized by high fat and high calorie content. High-temperature cooked food is rich in advanced glycation end products (AGE), AGE. Combining with its receptor (RAGE) and activating macrophages through NADPH and NF-kappa B pathway can lead to oxidative stress and induce the production of many pro-inflammatory factors. One intake can elevate the circulating AGE, which results in oxidative stress in a short period of time. The level of inflammatory factors increases, but the level of AGE decreases gradually after stopping intake and oxidative stress drives. Reducing AGE in food and blocking the combination of AGE and RAGE can reduce the chronic inflammatory state of the body, reduce insulin resistance in dB / DB rats, decrease the incidence of diabetes in NOD mice and C57 / B6 mice with high fat diet, and reduce the chronic complications of diabetes mellitus. Recent studies have found that a kind of RAGE (also known as sRAGE) lacking cell transmembrane and intracellular segments in vivo has the ability to bind to AGE, which limits the ability of AGE to bind to the complete RAGE on the cell membrane, blocks the oxidative stress induced by AGE and alleviates the chronic immune response symptoms. The aim of this study was to observe the effects of sRAGE on oxidative stress, lipid metabolism, insulin resistance and changes of aortic intima, liver and kidney in rats fed high-fat diet by intramuscular injection of sRAGE gene.
Method:
1. Animal experiment: 28 SD rats (weighing 150-160g, 6 weeks old) were randomly divided into three groups: high-fat meal treatment group, high-fat meal control group and normal meal control group. Eight rats fed with normal diet were sacrificed at the seventeenth week. The skeletal muscles were injected with pLNCX_2-sRAGE and pLNCX_2 plasmids. The rats were anesthetized with 4% chloral hydrate intraperitoneally. The hind leg muscles were exposed. The plasmids were injected at 300 UG and the distance between the electrodes was 20 mm. The gene transfer efficiency was increased by stimulating with square wave electric pulse (voltage 200V/cm, wave width 40CM, pulse number 9, frequency 1HZ).
2. Blood samples were taken at the end of 1, 2, and 3 weeks after each injection to determine SOD and MDA. Fasting blood glucose and body weight were measured at the seventeenth week before execution. Cardiac blood, aorta, liver and kidney were taken immediately after execution. Serum insulin, triglyceride, total cholesterol, TNF-a, hypersensitive CRP, SOD, MDA were measured. Pathological HE staining of kidney and aorta was used. Histochemical staining was used to observe the expression of TGF- NF- 1 and liver NF- kappa B in rats treated with different measures.
Result:
1. The body weight, blood glucose, serum insulin, triglyceride, total cholesterol, hypersensitive CRP and TNF-alpha in the high-fat meal control group were significantly higher than those in the normal meal control group. Compared with the high-fat meal control group, there was no significant difference in weight and hypersensitive CRP, except triglyceride.
2. The level of SOD in the high-fat diet group increased gradually after gene injection, and there was significant difference between the high-fat diet group and the control group at 3 weeks after the first injection (P 0.05). Repeated injection of SOD continued to rise to nearly normal meal. The control group no longer increased, and MDA decreased again.
3. Effect of sRAGE on important organs: There was no atherosclerosis in the control group and the high-fat diet group, but in the high-fat diet control group, the intima was incomplete, endothelial necrosis and exfoliation, local intima was slightly thickened, showing early atherosclerotic injury; in the high-fat diet control group, mesangial cells and extracellular matrix increased, TGF-ECM increased. The expression of TGF-beta 1 was significantly higher in the control group than that in the control group (P There was a significant difference between the three groups (P0.01).
Conclusion:
1. High-fat diet can elevate the level of oxidative stress and weaken the antioxidant capacity of rats. Injecting sRAGE gene into skeletal muscle can reduce the level of oxidative stress and enhance the antioxidant capacity of rats with high-fat diet.
2. High-fat diet can induce insulin resistance and elevated cholesterol levels, and early manifestations of atherosclerosis and glomerulosclerosis can be alleviated by intramuscular injection of sRAGE.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R363
【參考文獻(xiàn)】
相關(guān)期刊論文 前3條
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2 蔣家華，，熊愛(ài)華，鐘玲;糖尿病大鼠心肌MDA含量、SOD和Na￣+-K￣+-ATP酶活性的變化[J];中國(guó)病理生理雜志;1997年01期
3 徐成,王莉莉,曹穎林,李松;PPARs:脂代謝調(diào)節(jié)與胰島素增敏治療藥物的作用靶標(biāo)[J];中國(guó)藥理學(xué)通報(bào);2004年03期
本文編號(hào)：2204134
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