本文選題：肝臟 + 酒精��； 參考：《南方醫(yī)科大學(xué)》2014年碩士論文

【摘要】：研究背景 隨著物質(zhì)生活水平的提高,各種酒精飲料越加廣泛地成為人們生活的一部分。隨之,酒精相關(guān)的疾病也在增加。例如,大量飲酒可導(dǎo)致肝臟脂肪性變,形成酒精性肝病,進(jìn)一步可發(fā)展為酒精性肝硬化,甚至發(fā)展到酒精性肝癌。肝臟脂肪性病變后可導(dǎo)致多種病理結(jié)果,其中一個(gè)重要方面則是導(dǎo)致胰島素抵抗,即機(jī)體對(duì)胰島素生理作用的反應(yīng)性降低或敏感性降低。而胰島素抵抗與其它多種疾病(如糖尿病、肥胖、脂代謝紊亂和高血壓等)的發(fā)生和發(fā)展密切關(guān)聯(lián)[1-2]。 PI3K/AKT和MAPK/ERK是胰島素調(diào)節(jié)細(xì)胞代謝或生長的兩條重要通路。正常情況下,胰島素與胞膜上的受體結(jié)合后,引起胰島素受體自身磷酸化,從而激活酪氨酸激酶和胰島素受體底物(Insulin Receptor Substrate, IRS)。在PI3K/AKT信號(hào)通路中,活化的IRS進(jìn)而激活下游的3-磷酸肌醇激酶(phosphoinositide-3-kinase, PI3K)的活性。P13K的激活可使磷脂酰肌醇4,5二磷酸(phosphatidylinositol-4,5-bisphosphate, PI(4,5)P2)轉(zhuǎn)化為磷脂酰肌醇3,4,5三磷酸(phosphatidylinositol-4,5-triphosphate, PI(3,4,5)P3),后者可進(jìn)一步激活蛋白絲氨酸-蘇氨酸蛋白激酶B(serine-threonine protein kinase B, AKT),進(jìn)而引起葡萄糖轉(zhuǎn)運(yùn)體易位[3-4]。在MAPK/ERK通路中,Ras和Raf結(jié)合,將Raf從細(xì)胞漿轉(zhuǎn)移到細(xì)胞膜,在細(xì)胞膜上Raf絲／蘇氨酸發(fā)生磷酸化而被激活。激活后的Raf以其C端催化區(qū)與MEK結(jié)合,并使其催化區(qū)Ser磷酸化,從而使MEK激活�；罨腗EK進(jìn)一步激活下游的ERK。抑癌基因第10號(hào)染色體同源性磷酸酶-張力蛋白基因(phosphatase and tensin homologue, PTEN)是Insulin/PI3K/AKT信號(hào)通路的主要負(fù)調(diào)控分子,其行為的變化直接影響胰島素信號(hào)通路的穩(wěn)態(tài)[5-6]。 因?yàn)镮nsulin信號(hào)通路接受許多分子的調(diào)控,并進(jìn)而介導(dǎo)許多的下游分子事件,因而,該通路的調(diào)節(jié)異常也與細(xì)胞的許多病變相聯(lián)系。例如癌癥、糖尿病、胰島素抵抗。慢性酒精刺激可以導(dǎo)致胰島素抵抗。然而,酒精刺激導(dǎo)致胰島素抵抗的分子機(jī)制不甚清楚,有待深入的研究。本課題是國家基金慢性酒精對(duì)肝臟胰島素抵抗形成的影響的一部分。該基金旨在探討抑癌基因PTEN在慢性酒精刺激損害Insulin/PI3K/AKT信號(hào)通路的分子機(jī)制。而本課題,主要是該基金的前期工作,即研究酒精小鼠的模型建立。我們使用液體喂養(yǎng)模型,采用Lieber-DeCarli液體飼料。本課題開展了體外細(xì)胞的培養(yǎng),以印證體內(nèi)試驗(yàn)的結(jié)果。 目的 慢性酒精刺激對(duì)肝臟胰島素信號(hào)通路的研究影響及其機(jī)制。 方法 一、體外實(shí)驗(yàn)部分 1、本基金用肝癌細(xì)胞系HepG2細(xì)胞為研究對(duì)象。細(xì)胞常規(guī)培養(yǎng)(10%胎牛血清的DMEM培養(yǎng)液培養(yǎng),溫度37℃、5%二氧化碳恒溫箱)。 1) HepG2細(xì)胞胰島素刺激下PI3K/AKT信號(hào)通路的激活。 細(xì)胞分為對(duì)照組和胰島素刺激組。無血清饑餓12小時(shí)后,胰島素刺激10min,對(duì)照組用PBS刺激。裂解細(xì)胞,提取蛋白。測(cè)量蛋白濃度后,經(jīng)過western blot(制膠、電泳、電轉(zhuǎn)移、封閉、一抗孵育、二抗孵育、漂洗PVDF膜、膠片曝光)蛋白分析法檢測(cè)對(duì)照組和胰島素刺激組P-AKT蛋白表達(dá)水平。 2)慢性酒精效應(yīng) 細(xì)胞分為酒精刺激組和對(duì)照組。無血清饑餓12小時(shí)后,無水乙醇刺激(100mM/L)72小時(shí),對(duì)照組為無血清DMEM培養(yǎng)液。血清饑餓12小時(shí)后,胰島素刺激10min,裂解細(xì)胞,提取蛋白。測(cè)量蛋白濃度后,P-AKT、P-ERK蛋白表達(dá)水平的檢測(cè)見上節(jié)1。 2、凝膠圖像分析。將膠片進(jìn)行掃描,利用Image J軟件分析信號(hào)灰度值,以GAPDH作為內(nèi)參,P-AKT以總AKT作為參照,P-ERK以總ERK作為參照,以相應(yīng)蛋白條帶與GAPDH或AKT或ERK的比值計(jì)算目的蛋白的相對(duì)表達(dá)量。 二、體內(nèi)實(shí)驗(yàn)部分 1、實(shí)驗(yàn)設(shè)計(jì)：SPF級(jí)Balb/c小鼠購自南方醫(yī)科大學(xué)動(dòng)物中心,南方醫(yī)院動(dòng)物房按清潔級(jí)標(biāo)準(zhǔn)飼養(yǎng)。周齡為6至8周,雄性小鼠,14只,隨機(jī)分為2組：對(duì)照組和慢性酒精組。根據(jù)Lieber-DeCarli液體飼料配方配制含酒精和不含酒精而熱卡相同的液體飼料。予小鼠每日定量喂養(yǎng)：對(duì)照組喂對(duì)照液體飼料(熱量比例：脂肪35%,碳水化合物47%,蛋白質(zhì)18%,不含酒精),慢性酒精組喂酒精液體飼料(與對(duì)照液體飼料相同,但用酒精代替其中的一部分碳水化合物,酒精占36%,酒精體積分?jǐn)?shù)為5%),兩組小鼠最終熱卡攝入量基本一致。整個(gè)造模過程分成過渡期和造模期,小鼠共飼養(yǎng)56天。在整個(gè)造模過程中,每7天給小鼠測(cè)一次空腹體重。處死當(dāng)天,再次測(cè)量小鼠空腹體重。56天后,予腹腔注射戊巴比妥鈉溶液,待小鼠麻醉充分后打開腹腔,采集肝臟組織,取一小部分肝臟組織用4%甲醛溶液固定,剩余部分立即放于液氮速凍,再移至-80℃冰箱長期保存。 2、小鼠肝臟組織的制片與觀察。肝臟組織在4%甲醛溶液中固定。然后組織塊經(jīng)過脫水、透明、浸蠟、包埋、切片、烤片、脫蠟、復(fù)水、染色、脫水、透明、封片,制成病理切片,顯微鏡觀察對(duì)照組和慢性酒精組兩組肝臟組織的結(jié)構(gòu)變化。 3、Western blot檢測(cè)對(duì)照組和慢性酒精組肝臟組織中P-AKT、P-ERK和PTEN的表達(dá)量。-80℃碎冰上剪取肝臟組織,并于普通冰上裂解研磨肝臟組織(RIPA buffer液),提取蛋白,蛋白樣品用western blot(制膠、電泳、電轉(zhuǎn)移、封閉、一抗孵育、二抗孵育、漂洗PVDF膜、膠片曝光)檢測(cè)P-AKT、P-ERK和PTEN表達(dá)量。 4、凝膠圖像分析。將膠片進(jìn)行掃描,利用Image J軟件分析信號(hào)灰度值,以GAPDH作為內(nèi)參,P-AKT以總AKT作為參照,P-ERK以總ERK作為參照,以相應(yīng)蛋白條帶與GAPDH或AKT或ERK的比值計(jì)算目的蛋白的相對(duì)表達(dá)量。 三、統(tǒng)計(jì)分析：所有結(jié)果經(jīng)Exce12007與SPSS13.0軟件統(tǒng)計(jì)分析,結(jié)果以“算術(shù)平均值±標(biāo)準(zhǔn)差(arithmetic mean±standard deviation,M±SD)"表示。兩組間比較采用兩獨(dú)立樣本t檢驗(yàn)(Independent-Samples T Test)或wilcoxon秩和檢驗(yàn)(Mann-Whitney U檢驗(yàn)),P值0.05定義為有顯著性差異。 實(shí)驗(yàn)結(jié)果 一、體外實(shí)驗(yàn)結(jié)果 1、Western blot檢測(cè)結(jié)果顯示,相比對(duì)照組,胰島素刺激組在胰島素刺激10min后,P-AKT水平顯著提高(t=-12.758,P=0.009),兩組灰度值比分別為0.313±0.156及2.765±0.222。 2、我們的檢測(cè)結(jié)果中,酒精刺激組較對(duì)照組P-AKT蛋白表達(dá)水平下降(t=2.645,P=0.048),兩組灰度值比分別為0.454±0.085及0.681±±0.150,AKT蛋白無顯著差異,兩組灰度值比值為0.588±±0.213及0.705±±0.052(t=-1.063,P=0.329)。 3、酒精刺激組較對(duì)照組P-ERK蛋白表達(dá)水平下降(t=2.911,P=0.027),兩組灰度值比分別為0.058±0.031及0.125±±0.034,ERK蛋白無顯著差異,兩組灰度值比值為1.725±±0.539及1.727±0.196(t=0.008,P=0.994)。 二、體內(nèi)實(shí)驗(yàn)結(jié)果 1、酒精喂養(yǎng)前,各組小鼠精神狀態(tài)好,食欲佳,毛發(fā)色澤光亮。實(shí)驗(yàn)過程中,對(duì)照組小鼠與之前對(duì)比,變化不大；慢性酒精組小鼠與之前對(duì)比,精神差,食欲差,毛發(fā)色澤較晦暗,且有2只小鼠死亡。 2、體重經(jīng)過56天慢性酒精刺激后,對(duì)照組實(shí)驗(yàn)結(jié)束時(shí)平均體重(31.943±±1.381)較實(shí)驗(yàn)前重(18.300±1.253),差異有統(tǒng)計(jì)學(xué)意義(P0.01)。慢性酒精組實(shí)驗(yàn)結(jié)束時(shí)平均體重(19.160±1.539)與實(shí)驗(yàn)前(18.900±0.860)對(duì)比,變化不大,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。對(duì)照組體重的增加(13.643±2.343)顯著多于酒精組小鼠的體重增加(0.420±0.550),差異有統(tǒng)計(jì)學(xué)意義(P0.01)。 3、HE染色結(jié)果顯示,對(duì)照組的肝臟病理切片基本正常,肝小葉結(jié)構(gòu)較清晰,肝細(xì)胞呈多角形,由中央靜脈向四周呈放射性排列。慢性酒精組肝小葉結(jié)構(gòu)輪廓基本消失,肝細(xì)胞排列不規(guī)則,細(xì)胞形態(tài)不規(guī)則,胞質(zhì)中可見大小不等的空泡,為脂肪滴,部分細(xì)胞呈印戒狀,即以酒精性脂肪肝病理表現(xiàn)為主。 4、Western blot檢測(cè)結(jié)果 1)慢性酒精刺激降低小鼠肝臟內(nèi)P-AKT的表達(dá)水平,慢性酒精組P-AKT水平(0.268±0.208)顯著低于對(duì)照組(0.646±0.309)(t=3.216,P=0.005),但肝臟內(nèi)總AKT蛋白水平無明顯差異,灰度值比值為慢性酒精組(1.735±1.003)及對(duì)照組(1.644±0.618)(t=-0.255,P=0.801),western blot以GAPDH作為內(nèi)參。 2)慢性酒精刺激降低小鼠肝臟內(nèi)P-ERK的表達(dá)水平,慢性酒精組P-ERK水平(0.072±0.028)顯著低于對(duì)照組(0.178±0.019)(t=6.322,P=0.001),但肝臟內(nèi)總ERK蛋白水平無明顯差異,灰度值比值為慢性酒精組(1.938±0.320)及對(duì)照組(1.582±0.218)(t=-1.842,P=0.115),western blot以GAPDH作為內(nèi)參。 3)慢性酒精刺激增加小鼠肝臟內(nèi)PTEN的表達(dá)水平,慢性酒精組PTEN水平(1.173±0.536)顯著高于對(duì)照組(0.725±0.304)(t=-2.516,P=0.020)。結(jié)論 1、慢性酒精組小鼠體重實(shí)驗(yàn)前后沒有增長,對(duì)比對(duì)照組有明顯差異,這可能表明工業(yè)酒精對(duì)小鼠有一定的毒性,或小鼠對(duì)工業(yè)酒精飼料有一定抵制。從而,實(shí)驗(yàn)表明,目前廣泛應(yīng)用的工業(yè)酒精可能不適宜小鼠此類試驗(yàn)的研究。從而提示,下一步的研究,將選擇不同的食用酒精。 2、通過顯微鏡觀察對(duì)照組和慢性酒精組的病理切片,慢性酒精刺激可導(dǎo)致正常小鼠肝臟脂肪性變,形成酒精性肝病。 3、體外實(shí)驗(yàn)中,胰島素刺激激活PI3K/AKT信號(hào)通路,上調(diào)P-AKT的表達(dá)水平。 4、體外體內(nèi)實(shí)驗(yàn)western blot檢測(cè),慢性酒精組P-AK1、P-ERK的表達(dá)水平較對(duì)照組低,慢性酒精刺激降低小鼠肝臟內(nèi)P-AKT、P-ERK的表達(dá)水平,形成肝內(nèi)胰島素抵抗。 5、體內(nèi)western blot檢測(cè),慢性酒精組PTEN的表達(dá)水平較對(duì)照組高,慢性酒精刺激增加小鼠肝臟內(nèi)PTEN的表達(dá)水平。從而可知,在健康小鼠中,慢性酒精刺激可能通過上調(diào)PTEN的表達(dá)量進(jìn)而誘導(dǎo)其P-AKT的表達(dá)水平下降,形成肝內(nèi)胰島素抵抗。然而,PTEN表達(dá)水平的提高,是因?yàn)榭赡艿墓I(yè)酒精的潛在毒性,還是因?yàn)榫凭旧硇?yīng)的因素,有待于進(jìn)一步研究。該問題的理清對(duì)酒精肝胰島素抵抗的機(jī)制具有重要的作用。
[Abstract]:Research background
With the improvement of the living standard of material, alcoholic beverages have become more and more widely used as part of people's life. Accordingly, alcohol related diseases are also increasing. For example, a large amount of alcohol can lead to fatty changes in the liver, form alcoholic liver disease, further develop into alcoholic liver harden, and even develop to alcoholic liver cancer. Fatty disease of the liver. A variety of pathological results can lead to a variety of pathological results, one of which is the cause of insulin resistance, that is, the body's response to insulin physiology is reduced or reduced, and insulin resistance is closely associated with the development and development of many other diseases, such as diabetes, obesity, lipid metabolic disorders, and hypertension, [1-2].
PI3K/AKT and MAPK/ERK are two important pathways for insulin to regulate cell metabolism or growth. Under normal conditions, insulin receptor binding to the receptor on the membrane causes the phosphorylation of the insulin receptor itself, which activates the tyrosine kinase and the insulin receptor substrate (Insulin Receptor Substrate, IRS). In the PI3K/AKT signaling pathway, the activated IRS Activation of the active.P13K in the downstream 3- phosphoric acid inositol kinase (phosphoinositide-3-kinase, PI3K) can convert phosphatidylinositol 4,5 two phosphoric acid (phosphatidylinositol-4,5-bisphosphate, PI (4,5) P2) into phosphatidyl inositol 3,4,5 three phosphoric acid (phosphatidylinositol-4,5-triphosphate, PI), which can further activate the eggs. B (serine-threonine protein kinase B, AKT), resulting in a glucose transporter translocation [3-4]. in the MAPK/ERK pathway, Ras and Raf binding, and Raf from cytoplasm to cell membrane, activated by phosphorylation of Raf filament / threonine on the cell membrane. The active MEK further activates the ERK. suppressor gene tenth homologous phosphatase - tension protein gene (phosphatase and tensin homologue, PTEN), the main negative regulator of the Insulin/PI3K/AKT signaling pathway, which directly affects the insulin signal. The steady-state [5-6]. of the path
Because the Insulin signaling pathway is regulated by many molecules and thus mediates many downstream molecular events, the regulation of the pathway is associated with many lesions of the cell, such as cancer, diabetes, insulin resistance. Chronic alcohol stimulation can lead to insulin resistance. However, alcohol stimulates the molecules that cause insulin resistance. The study is part of the impact of the national fund chronic alcohol on the formation of insulin resistance in the liver. The fund aims to explore the molecular mechanism of the tumor suppressor gene PTEN in the chronic alcohol irritation damage Insulin/PI3K/AKT signaling pathway. This subject is mainly the early work of the fund, that is, to study alcohol The mice model was established. We used the liquid feeding model and used the Lieber-DeCarli liquid feed. The culture of the cells in vitro was carried out in this subject to verify the results of the test in vivo.
objective
Effects of chronic ethanol stimulation on hepatic insulin signaling pathway and its mechanism.
Method
First, in vitro experimental section
1, the fund used the hepatoma cell line HepG2 cells as the research object. Cell culture (the DMEM culture medium of 10% fetal bovine serum, temperature 37, 5% carbon dioxide thermostat).
1) activation of PI3K/AKT signaling pathway stimulated by insulin in HepG2 cells.
The cells were divided into the control group and the insulin stimulation group. After 12 hours of serum free starvation, insulin stimulated 10min, and the control group was stimulated by PBS. The protein concentration was measured. After the measurement of protein concentration, the control group was detected by Western blot (glue, electrophoresis, electrical transfer, closure, incubation, two anti incubation, rinsing PVDF membrane, film exposure) protein analysis. The level of P-AKT protein expression in the insulin stimulation group.
2) chronic alcohol effect
The cells were divided into the alcohol stimulation group and the control group. After 12 hours without serum starvation, anhydrous ethanol stimulation (100mM/L) was 72 hours, and the control group was serum-free DMEM culture. After 12 hours of serum starvation, insulin stimulated 10min, lysis cells and protein extraction. After measuring protein concentration, the level of P-AKT and P-ERK protein expression was detected in the previous section 1..
2, gel image analysis. The film is scanned and the Image J software is used to analyze the gray value of the signal. GAPDH is used as the internal reference. P-AKT is used as the reference for the total AKT. P-ERK is used as the reference of the total ERK. The relative expression of the target protein is calculated by the ratio of the corresponding protein strip to GAPDH or AKT or ERK.
Two, the experimental part of the body
1, experimental design: SPF class Balb/c mice were bought from the animal center of Southern Medical University, and the animal room of the southern hospital was kept in accordance with the standard of clean grade. The week age was 6 to 8 weeks, male mice and 14 rats were randomly divided into 2 groups: the control group and the chronic alcohol group. The same liquid diet containing alcohol and non alcohol was made according to the formula of Lieber-DeCarli liquid feed. Feed. Give the mice daily quantitative feeding: the control group was fed the control liquid feed (the proportion of heat: fat 35%, carbohydrate 47%, protein 18%, no alcohol), and the chronic alcohol group was fed with alcohol liquid feed (the same as the control liquid feed, but alcohol replaced part of the carbohydrate, alcohol accounted for 36%, the alcohol volume fraction was 5%), two groups. The final intake of heat card was basically the same. The whole process was divided into the transition period and the mold making period. The mice were fed for 56 days. During the whole process, the mice were given an empty abdominal body weight every 7 days. The day of death, the abdominal body weight of.56 days after the abdominal injection of pentobarbital sodium was given, and the abdominal cavity was opened after the mice were fully anesthetized. Liver tissue was collected and a small amount of liver tissue was fixed with 4% Formaldehyde Solution. The rest was immediately frozen in liquid nitrogen and then moved to -80 C refrigerator for long-term preservation.
2, the production and observation of liver tissue in mice. The liver tissue was fixed in 4% Formaldehyde Solution. Then the tissue block was dehydrated, transparent, impregnated, embedded, sliced, roasted, dewaxed, rehydrated, dyed, dehydrated, transparent, and made into pathological sections, and the structural changes of two groups of liver tissues in the control group and the chronic alcohol group were observed by microscope.
3, Western blot detected the expression of P-AKT, P-ERK and PTEN in the liver tissues of the control group and the chronic alcohol group at.-80 centigrade ice to cut the liver tissue, and lysed and lapping the liver tissue (RIPA buffer liquid) on the ordinary ice. The protein samples were extracted with Western blot (glue, electrostroke, electrotransfer, sealing, one anti incubation, two anti incubation, and rinse PVDF membrane, " The expression of P-AKT, P-ERK and PTEN was detected by film exposure.
4, gel image analysis. The film is scanned and the Image J software is used to analyze the gray value of the signal. GAPDH is used as the internal reference. P-AKT is used as the reference for the total AKT. P-ERK is used as the reference of the total ERK. The relative expression of the target protein is calculated by the ratio of the corresponding protein strip to GAPDH or AKT or ERK.
Three, statistical analysis: all results were analyzed by Exce12007 and SPSS13.0 software, and the results were "arithmetic mean + standard deviation (M + SD)". The two groups were compared with two independent sample t test (Independent-Samples T Test) or rank sum test (0.05). Significant differences.
experimental result
First, in vitro experimental results
1, the results of Western blot detection showed that compared with the control group, the insulin stimulation group increased the level of P-AKT significantly (t=-12.758, P=0.009) after insulin stimulation of 10min, and the ratio of the two groups was 0.313 + 0.156 and 2.765 + 0.222., respectively.
2, in our test results, the expression level of P-AKT protein in the alcohol stimulation group was lower than that in the control group (t=2.645, P=0.048). The ratio of gray value of the two groups was 0.454 + 0.085 and 0.681 + 0.150 respectively, and the AKT protein was not significantly different. The ratio of the gray value of the two groups was 0.588 + + + + 0.213 and 0.705 + 0.052 (t=-1.063, P=0.329).
3, the expression level of P-ERK protein in the alcohol stimulation group was lower than that in the control group (t=2.911, P=0.027). The ratio of gray value of the two groups was 0.058 + 0.031 and 0.125 + 0.034 respectively. There was no significant difference in the ERK protein. The ratio of the gray value of the two groups was 1.725 + + + 0.539 and 1.727 + 0.196 (t=0.008, P=0.994).
Two, experimental results in vivo
1, before the alcohol was fed, the mice in each group had good mental state, good appetite, and bright hair color. In the experiment, the control group had little change, and the mice in the chronic alcohol group were compared with the previous, poor spirit, poor appetite, dark hair and dark color, and 2 mice died.
2, after 56 days of chronic alcohol stimulation, the average weight of the control group was 31.943 + 1.381 (18.300 + 1.253) at the end of the experiment (18.300 + 1.253), and the difference was statistically significant (P0.01). The average weight (19.160 + 1.539) at the end of the chronic alcohol group was compared with the pre experiment (18.900 + 0.860), and the difference was not significant (P0.05). Body weight gain (13.643 + 2.343) was significantly higher than that of alcohol group (0.420 + 0.550), and the difference was statistically significant (P0.01).
3, the results of HE staining showed that the pathological sections of the liver of the control group were basically normal, the structure of hepatic lobule was clearer, the liver cells were polygonal, and the central vein was arranged radially from the central vein to the surrounding area. The structure of the hepatic lobule in the chronic alcohol group disappeared basically, the liver cells were irregular, the cell shape was irregular, and the vacuoles of different sizes were visible in the cytoplasm, and the fat was fat. Some of the cells showed a signet ring, which was mainly alcoholic fatty liver.
4, Western blot detection results
1) chronic alcohol stimulation reduced the expression level of P-AKT in the liver of mice. The P-AKT level of chronic alcohol group (0.268 + 0.208) was significantly lower than that of the control group (0.646 + 0.309) (t=3.216, P=0.005), but there was no significant difference in the total AKT protein level in the liver, the ratio of gray value was (1.735 + 1.003) and the control group (1.644 + 0.618) (t=-0.255, P=0.801), Wester N blot takes GAPDH as the internal reference.
2) chronic alcohol stimulation reduced the expression level of P-ERK in the liver of mice. The P-ERK level of chronic alcohol group (0.072 + 0.028) was significantly lower than that of the control group (0.178 + 0.019) (t=6.322, P=0.001), but there was no significant difference in the total ERK protein level in the liver, the ratio of gray value was (1.938 + 0.320) and the control group (1.582 + 0.218) (t=-1.842, P=0.115), Wester N blot takes GAPDH as the internal reference.
3) chronic alcohol stimulation increased the expression level of PTEN in the liver of mice, and the level of PTEN in chronic alcohol group (1.173 + 0.536) was significantly higher than that of the control group (0.725 + 0.304) (t=-2.516, P=0.020).
1, the mice in the chronic alcohol group did not increase before and after the weight test, and there was a significant difference between the control group and the control group. This may indicate that the industrial alcohol has some toxicity to the mice, or the mice have a certain resistance to the industrial alcohol feed. One step of the study will choose different edible alcohol.
2, the pathological sections of the control group and chronic alcohol group were observed by microscope. Chronic alcohol stimulation could cause fatty liver in normal mice to form alcoholic liver disease.
3, in vitro, insulin stimulates the activation of PI3K/AKT signaling pathway and upregulates the expression level of P-AKT.
4, in vitro test of Western blot, the expression level of P-AK1 and P-ERK in chronic alcohol group was lower than that of the control group. Chronic alcohol stimulation reduced the expression level of P-AKT and P-ERK in the liver of mice, and formed the insulin resistance in the liver.
5, in vivo Western blot detection, the expression level of PTEN in chronic alcohol group is higher than that of the control group. Chronic alcohol stimulation increases the expression level of PTEN in the liver of mice. Thus, in healthy mice, chronic alcohol stimulation may induce the decrease of the expression level of P-AKT by up regulating the expression of PTEN and forming the insulin resistance in the liver. However, PT The increase in the expression level of EN is due to the potential toxicity of industrial alcohol, or the factors of alcohol itself, which need to be further studied. It is important to clarify the mechanism of insulin resistance to alcohol and liver.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R575.5

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