發(fā)布時(shí)間：2018-04-30 06:24

  本文選題：氧化應(yīng)激 + 胰島素抵抗�。� 參考：《吉林大學(xué)》2017年碩士論文

【摘要】：研究背景:目前糖尿病是全世界嚴(yán)重的公共衛(wèi)生問題,患病率呈現(xiàn)世界性上升趨勢(shì)。胰島素抵抗是2型糖尿病的重要發(fā)病機(jī)制,氧化應(yīng)激是骨骼肌發(fā)生胰島素抵抗的一個(gè)重要病因,ROS會(huì)激活細(xì)胞應(yīng)激敏感性通路炎癥信號(hào)通路NF-κB,進(jìn)而影響胰島素信號(hào)通路的傳導(dǎo),引起胰島素抵抗。NF-κB是氧化應(yīng)激重要的一個(gè)靶點(diǎn),了解及明確NF-κB在氧化應(yīng)激引起的胰島素抵抗過程中的具體機(jī)制尤其重要。氧化應(yīng)激可被內(nèi)在和外在的因素可控制,通過降低氧化應(yīng)激預(yù)防和治療胰島素抵抗是目前的一個(gè)新的研究方向。本研究通過一定濃度過氧化氫刺激大鼠骨骼肌細(xì)胞(L6細(xì)胞)建立氧化應(yīng)激模型,檢測(cè)葡萄糖攝取能力及胰島素抵抗情況,并應(yīng)用NF-κB抑制劑PDTC預(yù)處理,觀察胰島素抵抗水平情況,探討其具體機(jī)制;在了解過氧化氫誘導(dǎo)大鼠骨骼肌胰島素抵抗的具體機(jī)制之后,我們欲探討天然中藥銀杏葉提取物能否改善L6細(xì)胞的胰島素抵抗?fàn)顟B(tài),并探討具體機(jī)制。本研究分為以下兩個(gè)部分。第一部分:過氧化氫誘導(dǎo)的胰島素抵抗及機(jī)制研究目的:研究過氧化氫對(duì)L6細(xì)胞胰島素抵抗的影響,并探討具體機(jī)制。方法:首先體外培養(yǎng)L6細(xì)胞并誘導(dǎo)分化為成熟的骨骼肌細(xì)胞,MTT法測(cè)定細(xì)胞存活率,確定H_2O_2和PDTC的作用濃度和作用時(shí)間。將細(xì)胞分為4組:正常組、H_2O_2組、H_2O_2+PDTC組、PDTC組。應(yīng)用DCFH-DA探針檢測(cè)細(xì)胞內(nèi)ROS的生成,DAPI觀察細(xì)胞凋亡情況,2-NBDG法檢測(cè)細(xì)胞攝取葡萄糖能力,ELISA法檢測(cè)細(xì)胞上清中炎性因子TNF-α、IL-1β、IL-6的分泌水平,Western Blot檢測(cè)細(xì)胞中Bax、Bcl2、Caspase-8、IκB-a、P-IκB-a、P38-MAPK、P-P38-MAPK、NF-κB、P-NF-κB、IRS1、P-IRS1 Tyr612、PI3K、GLUT4等蛋白的表達(dá)。結(jié)果:1.L6細(xì)胞的誘導(dǎo)分化:誘導(dǎo)分化后第7d細(xì)胞融合成明顯的肌管。2.氧化應(yīng)激模型建立:H_2O_2濃度1.0mmol/L時(shí),細(xì)胞存活率70%左右,確定H_2O_2的刺激濃度為1.0mmol/L。當(dāng)PDTC濃度為100μmol/L時(shí),細(xì)胞存活率為80%左右,高于其他實(shí)驗(yàn)濃度組。孵育4h時(shí),PDTC對(duì)細(xì)胞存活率抑制作用相對(duì)較小,確定PDTC的孵育濃度和時(shí)間為100μmol/L,4h。DCFH-DA結(jié)果顯示,1.0mmol/LH_2O_2處理L6細(xì)胞4h后,細(xì)胞內(nèi)ROS生成顯著升高,確定氧化應(yīng)激模型建立。3.細(xì)胞凋亡情況:正常組細(xì)胞核完整,邊緣清晰,染色質(zhì)均一;H_2O_2組細(xì)胞出現(xiàn)典型凋亡特征,如細(xì)胞核皺縮,細(xì)胞核破碎,凋亡小體形成;而H_2O_2+PDTC組除個(gè)別細(xì)胞出現(xiàn)凋亡,大多數(shù)均正常。與正常組相比較,H_2O_2組Bax相對(duì)表達(dá)量、Bax/Bcl2比值、Caspase-8相對(duì)表達(dá)量顯著升高,Bcl2相對(duì)表達(dá)量顯著降低,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。與H_2O_2組相比較,H_2O_2+PDTC組Bax相對(duì)表達(dá)量無(wú)變化,差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05);Bcl2相對(duì)表達(dá)量顯著升高,Bax/Bcl2比值、Caspase-8相對(duì)表達(dá)量均顯著降低,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。4.NF-κB活化及胰島素信號(hào)傳導(dǎo)改變:2-NBDG攝取試驗(yàn)結(jié)果顯示,與正常組相比較,H_2O_2組2-NBDG攝取能力顯著下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05);與H_2O_2組相比較,H_2O_2+PDTC組2-NBDG攝取能力顯著升高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。ELISA結(jié)果顯示,與正常組相比較,H_2O_2組炎癥因子TNF-α、IL-1β、IL-6產(chǎn)生顯著增加,差異均有統(tǒng)計(jì)學(xué)意義(P0.05);與H_2O_2組相比較,H_2O_2+PDTC組TNF-α、IL-1β、IL-6產(chǎn)生顯著降低,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。與正常組相比較,H_2O_2組IκB-a、P38-MAPK、NF-κB相對(duì)表達(dá)量均顯著升高,P-IκB-a/IκB-a、P-P38/P38、P-NF-κB/NF-κB相對(duì)水平均顯著升高,IRS1、PI3K、GLUT4相對(duì)表達(dá)量及P-IRS1 Tyr612/IRS1相對(duì)水平均顯著降低,差異均有統(tǒng)計(jì)學(xué)意義(P0.05);與H_2O_2組相比較,H_2O_2+PDTC組IκB-a、P38-MAPK、NF-κB相對(duì)表達(dá)量均顯著降低,P-IκB-a/IκB-a、P-P38/P38、P-NF-κB/NF-κB相對(duì)水平均顯著降低,H_2O_2+PDTC組IRS1、PI3K、GLUT4相對(duì)表達(dá)量,P-IRS1 Tyr612/IRS1相對(duì)水平均顯著升高,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:1.H_2O_2能夠?qū)е翷6細(xì)胞出現(xiàn)氧化應(yīng)激和凋亡。2.H_2O_2能夠?qū)е录?xì)胞炎性因子TNF-α、IL-1β、IL-6產(chǎn)生增加。3.H_2O_2能夠?qū)е录?xì)胞攝取葡萄糖能力下降,引起胰島素抵抗,具體機(jī)制與NF-κB通路激活有關(guān)。4.應(yīng)用PDTC可以緩解細(xì)胞凋亡、減少ROS和炎性因子的產(chǎn)生、提高攝取葡萄糖能力、抑制NF-κB通路的激活,進(jìn)而改善胰島素抵抗。第二部分銀杏葉提取物對(duì)過氧化氫誘導(dǎo)的胰島素抵抗的改善作用目的:研究EGB對(duì)過氧化氫誘導(dǎo)的胰島素抵抗的改善作用及具體機(jī)制。方法:首先體外培養(yǎng)L6細(xì)胞并誘導(dǎo)分化為成熟的骨骼肌細(xì)胞,MTT法測(cè)定細(xì)胞存活率,確定EGB的作用濃度。將細(xì)胞分為7組:正常組、H_2O_2組、H_2O_2+EGB組、H_2O_2+PDTC組、H_2O_2+EGB+PDTC組、PDTC組、EGB組。應(yīng)用DCFH-DA探針檢測(cè)細(xì)胞內(nèi)ROS的生成,2-NBDG攝取試驗(yàn)檢測(cè)細(xì)胞攝取葡萄糖能力,ELISA法檢測(cè)細(xì)胞上清中炎性因子TNF-α、IL-1β、IL-6的分泌水平,Western Blot檢測(cè)L6細(xì)胞中IκB-a、P-IκB-a、NF-κB、P-NF-κB、IRS1、P-IRS1 Tyr612、GLUT4等蛋白的表達(dá)。結(jié)果:1.EGB對(duì)氧化應(yīng)激的改善:MTT結(jié)果顯示,隨著EGB濃度的增加,細(xì)胞的存活率呈先升高后穩(wěn)定,反而稍微下降的趨勢(shì)。在EGB濃度為200μg/m L時(shí),細(xì)胞存活率達(dá)到最大值108%,確定EGB的預(yù)處理濃度為200μg/m L。DCFH-DA流式結(jié)果顯示,與正常組相比較,H_2O_2組細(xì)胞內(nèi)ROS顯著升高,差異有統(tǒng)計(jì)學(xué)意義(P0.05),確定氧化應(yīng)激模型建立;與H_2O_2組相比較,H_2O_2+EGB、H_2O_2+PDTC和H_2O_2+EGB+PDTC組細(xì)胞內(nèi)ROS顯著降低,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。且H_2O_2+EGB+PDTC組細(xì)胞內(nèi)ROS均低于H_2O_2+EGB組和H_2O_2+PDTC組,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。2.EGB對(duì)NF-κB活化及胰島素信號(hào)傳導(dǎo)的影響:2-NBDG攝取試驗(yàn)結(jié)果顯示,與正常組相比較,H_2O_2組2-NBDG攝取能力顯著下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05);與H_2O_2組相比較,H_2O_2+EGB、H_2O_2+PDTC和H_2O_2+EGB+PDTC組2-NBDG攝取能力均顯著升高,差異均具有統(tǒng)計(jì)學(xué)意義(P0.05)。并且H_2O_2+EGB+PDTC組細(xì)胞2-NBDG攝取能力顯著高于H_2O_2+PDTC組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。ELISA結(jié)果顯示,與正常組相比較,H_2O_2組炎癥因子TNF-α、IL-1β、IL-6產(chǎn)生顯著增加,差異均有統(tǒng)計(jì)學(xué)意義(P0.05);與H_2O_2組相比較,H_2O_2+EGB、H_2O_2+PDTC和H_2O_2+EGB+PDTC組TNF-α、IL-1β、IL-6產(chǎn)生均顯著降低,差異均有統(tǒng)計(jì)學(xué)意義(P0.05);且H_2O_2+EGB+PDTC組TNF-α產(chǎn)生低于H_2O_2+PDTC組,差異有統(tǒng)計(jì)學(xué)意義(P0.05);Western blot結(jié)果顯示,與正常組相比較,H_2O_2組IκB-a、NF-κB、P-IκB-a/IκB-a、P-NF-κB/NF-κB相對(duì)表達(dá)量均顯著升高,IRS1、GLUT4、P-IRS1 Tyr612/IRS1相對(duì)表達(dá)量均顯著降低,差異均有統(tǒng)計(jì)學(xué)意義(P0.05);與H_2O_2組相比較,H_2O_2+EGB、H_2O_2+PDTC和H_2O_2+EGB+PDTC組IκB-a、NF-κB、P-IκB-a/IκB-a、P-NF-κB/NF-κB相對(duì)表達(dá)量均顯著降低,IRS1、GLUT4、P-IRS1 Tyr612/IRS1相對(duì)表達(dá)量均顯著升高,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:1.EGB能夠提高L6細(xì)胞存活率,降低氧化應(yīng)激程度,且聯(lián)合應(yīng)用EGB和PDTC在降低細(xì)胞氧化應(yīng)激程度上優(yōu)于單獨(dú)應(yīng)用EGB和單獨(dú)應(yīng)用PDTC。2.EGB能夠減少L6細(xì)胞炎性因子TNF-α、IL-1β、IL-6產(chǎn)生,且聯(lián)合應(yīng)用EGB和PDTC在減少TNF-α產(chǎn)生上優(yōu)于單獨(dú)應(yīng)用PDTC。3.EGB能夠提高細(xì)胞攝取葡萄糖能力,改善胰島素抵抗,具體機(jī)制與抑制NF-κB通路的激活有關(guān),且聯(lián)合應(yīng)用EGB和PDTC在提高細(xì)胞攝取葡萄糖能力上優(yōu)于單獨(dú)應(yīng)用PDTC。
[Abstract]:Background: at present, diabetes is a serious public health problem in the world. The incidence of disease is rising worldwide. Insulin resistance is an important pathogenesis of type 2 diabetes. Oxidative stress is an important cause of insulin resistance in skeletal muscle. ROS activates the inflammatory signaling pathway NF- kappa B of the cell stress sensitivity pathway and further shadows The transmission of the insulin signaling pathway causes insulin resistance to.NF- kappa B as an important target for oxidative stress. It is particularly important to understand and clarify the specific mechanism of NF- kappa B in the process of insulin resistance induced by oxidative stress. Oxidative stress can be controlled by internal and external factors, and the prevention and treatment of insulin against oxidative stress can be used to prevent and treat insulin. Resistance is a new research direction at present. In this study, the oxidative stress model of rat skeletal muscle cells (L6 cells) was stimulated by a certain concentration of hydrogen peroxide, and the ability of glucose uptake and insulin resistance was detected. The condition of islet resistance was observed by NF- kappa B inhibitor PDTC, and the specific mechanism was discussed. After the specific mechanism of insulin resistance induced by hydrogen peroxide in rat skeletal muscle, we would like to explore whether the extract of Ginkgo biloba leaves can improve the insulin resistance of L6 cells and discuss the specific mechanism. This study is divided into two parts. The first part: the purpose of the study of hydrogen peroxide induced insulin resistance and its mechanism: the study of peroxidation The effect of hydrogen on insulin resistance in L6 cells was discussed. Methods: first, L6 cells were cultured in vitro and induced into mature skeletal muscle cells. The survival rate of cells was determined by MTT method. The action concentration and time of H_2O_2 and PDTC were determined. The cells were divided into 4 groups: normal group, H_2O_2 group, H_2O_2+PDTC group, PDTC group. Detection of ROS production in cells, DAPI observation of cell apoptosis, 2-NBDG method to detect cell uptake of glucose, ELISA method to detect the secretion of TNF- alpha, IL-1 beta, IL-6 in cell supernatant, Western Blot detection cells Bax, Bcl2, Caspase-8. The expression of UT4 and other proteins. Results: induced differentiation of 1.L6 cells: the differentiation of 7D cells after induction of differentiation into a distinct.2. oxidative stress model of myotubes: the survival rate of cells was about 70% when H_2O_2 concentration was 1.0mmol/L, and the survival rate of H_2O_2 was about 80% when PDTC concentration was 100 micron mol/L, which was higher than that of other experiments. Concentration group. When incubated for 4h, the inhibitory effect of PDTC on cell survival was relatively small, and the incubation concentration and time of PDTC were 100 u mol/L. The results of 4h.DCFH-DA showed that after 1.0mmol/LH_2O_2 treated L6 cell 4h, the formation of ROS in the cells increased significantly, and the oxidative stress model established.3. fine cell apoptosis: normal group nuclei were complete, edge clear, dyed. The cells of group H_2O_2 showed typical characteristics of apoptosis, such as nuclear crinkle, nucleus fragmentation, and apoptotic body formation, while group H_2O_2+PDTC except some cells appeared apoptosis, most of them were normal. Compared with normal group, the relative expression of Bax in group H_2O_2, Bax/Bcl2 ratio, relative expression of Caspase-8 increased significantly, and the relative expression of Bcl2 was significant. The difference was statistically significant (P0.05). Compared with the H_2O_2 group, the relative expression of Bax in group H_2O_2+PDTC had no significant difference (P0.05), the relative expression of Bcl2 increased significantly, the ratio of Bax/Bcl2 and the relative expression of Caspase-8 decreased significantly, and the difference was statistically significant (P0.05).4.NF- kappa B activation and insulin signal transduction Changes: 2-NBDG uptake test results showed that compared with the normal group, the 2-NBDG uptake ability of H_2O_2 group decreased significantly (P0.05). Compared with the H_2O_2 group, the 2-NBDG uptake ability of H_2O_2+PDTC group increased significantly, and the difference was statistically significant (P0.05).ELISA results, compared with the normal group, the H_2O_2 group inflammatory factor TNF- alpha, IL-1 beta, IL-6 produced a significant increase, the difference was statistically significant (P0.05). Compared with the H_2O_2 group, TNF- alpha, IL-1 beta and IL-6 produced a significant decrease in H_2O_2+PDTC group, and the difference was statistically significant (P0.05). The relative levels of IRS1, PI3K, GLUT4 and the relative level of P-IRS1 Tyr612/IRS1 were significantly decreased, and the difference was statistically significant (P0.05). Compared with the H_2O_2 group, the relative expression of I kappa B-a, P38-MAPK, NF- kappa, H_2O_2+PDTC group was significantly reduced, and the relative level of kappa kappa was significantly reduced. The relative expression of IRS1, PI3K, GLUT4 in group H_2O_2+PDTC, P-IRS1 Tyr612/IRS1 relative levels were significantly increased, and the difference was statistically significant (P0.05). Conclusion: 1.H_2O_2 can lead to oxidative stress and apoptosis of L6 cells, and.2.H_2O_2 can lead to the inflammatory factor TNF- alpha, IL-1 beta, which can lead to the uptake of grapes. The decrease of sugar capacity causes insulin resistance. The specific mechanism related to the activation of NF- kappa B pathway related to the activation of.4. application PDTC can alleviate apoptosis, reduce the production of ROS and inflammatory factors, increase the uptake of glucose, inhibit the activation of NF- kappa B pathway, and then improve insulin resistance. Second parts of Ginkgo biloba extract against hydrogen peroxide induced insulin against the insulin resistance. Objective: To study the effect of EGB on the improvement of insulin resistance induced by hydrogen peroxide and the specific mechanism. Methods: first, L6 cells were cultured in vitro and induced into mature skeletal muscle cells. MTT method was used to determine the cell survival rate and determine the concentration of EGB. The cells were divided into 7 groups: normal group, H_2O_2 group, H_2O_2+EGB group, H_2O_2+PDTC Group H_2O_2+EGB+PDTC, group PDTC, group EGB. Using DCFH-DA probe to detect the formation of ROS in cells, 2-NBDG uptake test to detect the ability of cell uptake of glucose, ELISA method to detect the inflammatory factor TNF- a, IL-1 beta, IL-6 secreted in cell supernatant. Results: the results of the expression of equal protein. Results: the improvement of oxidative stress by 1.EGB: MTT results showed that with the increase of EGB concentration, the survival rate of cells increased first and then decreased slightly. The cell survival rate reached the maximum of 108% when the concentration of EGB was 200 mu g/m L, and the preconditioning concentration of EGB was shown to be 200 u g/m L.DCFH-DA flow results. Compared with the normal group, the intracellular ROS in the H_2O_2 group increased significantly, and the difference was statistically significant (P0.05), and the oxidative stress model was established. Compared with the H_2O_2 group, the intracellular ROS in H_2O_2+EGB, H_2O_2+PDTC and H_2O_2+EGB+PDTC groups decreased significantly (P0.05), and the ROS in the H_2O_2+EGB+PDTC group was lower than that in the H_2O_2+EGB group and in the H_2O_2+EGB+PDTC group. H_2O_2+PDTC group, the difference was statistically significant (P0.05) the effect of.2.EGB on the activation of NF- kappa B and insulin signal transduction. 2-NBDG uptake test results showed that the 2-NBDG uptake ability of H_2O_2 group decreased significantly compared with the normal group, and the difference was statistically significant (P0.05); H_2O_2+EGB, H_2O_2+PDTC, and groups were compared with the H_2O_2 group. The ability of G uptake increased significantly (P0.05), and the ability of 2-NBDG uptake in H_2O_2+EGB+PDTC group was significantly higher than that in H_2O_2+PDTC group, and the difference was statistically significant (P0.05).ELISA results showed that the inflammatory factors TNF- alpha, IL-1 beta and IL-6 were significantly increased in H_2O_2 group, and the difference was statistically significant. (P0.05); compared with group H_2O_2, the production of TNF- alpha, IL-1 beta and IL-6 in H_2O_2+EGB, H_2O_2+PDTC and H_2O_2+EGB+PDTC groups decreased significantly, and the difference was statistically significant (P0.05), and TNF- alpha production in H_2O_2+EGB+PDTC group was lower than that of H_2O_2+PDTC group. - kappa B, P-I kappa B-a/I kappa B-a, P-NF- kappa B/NF- kappa B significantly increased, IRS1, GLUT4, P-IRS1 Tyr612/IRS1 relative expression decreased significantly, the difference was statistically significant (P0.05). Low, IRS1, GLUT4, P-IRS1 Tyr612/IRS1 relative expression increased significantly, the difference was statistically significant (P0.05). Conclusion: 1.EGB can improve the survival rate of L6 cells and reduce oxidative stress, and the combination of EGB and PDTC in reducing cell oxidative stress is better than single application EGB and single application PDTC.2.EGB can reduce L6 cell inflammation Factor TNF- alpha, IL-1 beta, IL-6 production, and combined use of EGB and PDTC in reducing TNF- alpha production is superior to single application PDTC.3.EGB can improve cell uptake of glucose ability and improve insulin resistance. The specific mechanism is related to the inhibition of NF- kappa B pathway activation, and the combined application of EGB and PDTC is superior to the ability to increase cell uptake of glucose. Using PDTC.

【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)】：R587.1

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