本文選題：白藜蘆醇　切入點(diǎn)：糖尿病視網(wǎng)膜病變　出處：《武漢大學(xué)》2016年博士論文

【摘要】：第一部分白藜蘆醇對(duì)STZ誘導(dǎo)的糖尿病大鼠視網(wǎng)膜病變的影響目的：觀察白藜蘆醇(Resveratrol,Res)對(duì)鏈脲佐菌素(Streptozotocin,STZ)誘導(dǎo)的大鼠糖尿病視網(wǎng)膜病變的防治作用,探討其可能的作用機(jī)制。方法隨機(jī)將35只SD大鼠中的10只分為正常對(duì)照組(Control),剩余的25只腹腔注射STZ建立糖尿病模型,將造模成功的20只大鼠隨機(jī)均分為糖尿病模型組(DM)和白藜蘆醇治療組(DM+Res)。確定造模成功次日即開(kāi)始灌胃給藥,分別于灌胃后的第0、4、8、12周時(shí)測(cè)量大鼠的體重和血糖,12周時(shí)處死大鼠取眼球,然后測(cè)定各組大鼠視網(wǎng)膜丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性；采用蘇木素-伊紅(HE)染色觀察大鼠視網(wǎng)膜組織的形態(tài)學(xué)改變并測(cè)量視網(wǎng)膜厚度；脫氧核糖核苷酸末端轉(zhuǎn)移酶介導(dǎo)的缺口末端標(biāo)記法(TUNEL)檢測(cè)大鼠視網(wǎng)膜神經(jīng)細(xì)胞的凋亡：透射電鏡下觀察大鼠視網(wǎng)膜神經(jīng)節(jié)細(xì)胞和毛細(xì)血管管壁超微結(jié)構(gòu)的變化；免疫組織化學(xué)染色法檢測(cè)大鼠視網(wǎng)膜Bax和Bcl-2蛋白的表達(dá)。結(jié)果：DM+Res組大鼠4、8、12周時(shí)體重均較DM組重,但低于Control組；其血糖在上述3個(gè)時(shí)間點(diǎn)均低于DM組。與DM組相比,DM+Res組大鼠視網(wǎng)膜MDA含量明顯降低(P0.01),SOD活性明顯升高(P0.01),視網(wǎng)膜總厚度及視網(wǎng)膜內(nèi)、外核層厚度均顯著增加(p0.01),神經(jīng)節(jié)細(xì)胞層和內(nèi)核層凋亡指數(shù)均降低(P0.01)；電鏡下,DM組可見(jiàn)神經(jīng)節(jié)細(xì)胞核染色質(zhì)凝聚,線粒體腫脹,胞質(zhì)空泡化；DM+Res組神經(jīng)節(jié)細(xì)胞未見(jiàn)明顯的核染色質(zhì)凝聚、邊集,線粒體腫脹、嵴丟失及胞質(zhì)空泡化；免疫組織化學(xué)法顯示與DM組比較,DM+Res組Bcl-2表達(dá)增強(qiáng),Bax表達(dá)強(qiáng)度減弱,Bcl-2/Bax比值顯著升高。結(jié)論： Res可改善糖尿病大鼠體重,降低其血糖,增強(qiáng)其視網(wǎng)膜的抗氧化能力,從而減輕視網(wǎng)膜的氧化損傷,改善糖尿病所致的視網(wǎng)膜的病理改變,并可通過(guò)上調(diào)Bcl-2和下調(diào)Bax的表達(dá),抑制視網(wǎng)膜神經(jīng)節(jié)細(xì)胞凋亡,發(fā)揮對(duì)糖尿病視網(wǎng)膜病變的防治作用。第二部分 白藜蘆醇對(duì)高糖狀態(tài)ARPE-19細(xì)胞的影響目的：研究高糖條件下不同時(shí)間及不同濃度Res對(duì)ARPE-19細(xì)胞活力的影響,以探尋最適宜ARPE-19細(xì)胞高糖損傷模型,篩選適宜干預(yù)的Res濃度,并探討Res對(duì)高糖狀態(tài)ARPE-19細(xì)胞的影響方法：將 ARPE-19細(xì)胞株分別置于正常糖狀態(tài)(5.5mmol/L葡萄糖)、高糖狀態(tài)(30mmol/L葡萄糖)和高滲狀態(tài)(5.5mmol/L葡萄糖+24.5mmol/L甘露醇)培養(yǎng)24h、48h、72h,CCK-8法檢測(cè)各個(gè)時(shí)間點(diǎn)細(xì)胞活性并進(jìn)行對(duì)比,選擇最佳作用時(shí)間點(diǎn)；再用不同濃度白藜蘆醇(Oμmol/L、5μmol/L、10μmol/L、25μmol/L、 50μmol/L)處理高糖狀態(tài)(30mmol/L葡萄糖)ARPE-19細(xì)胞株48h,通過(guò)CCK-8法檢測(cè)各組細(xì)胞活性,選擇最佳濃度；將ARPE-19細(xì)胞株置于含30mmol/L葡萄糖和10 μmol/L Res的DMEM培養(yǎng)基培養(yǎng)48h(同時(shí)設(shè)置正常、高糖對(duì)照和高滲對(duì)照)后用倒置顯微鏡觀察各組細(xì)胞生長(zhǎng)情況和形態(tài)變化。結(jié)果：CCK-8法檢測(cè)顯示,與正常對(duì)照組相比,高糖組ARPE-19細(xì)胞,24h時(shí)細(xì)胞活性降低但無(wú)統(tǒng)計(jì)學(xué)差異,48h時(shí)出現(xiàn)統(tǒng)計(jì)學(xué)差異,72h時(shí)細(xì)胞活性降低但與48h時(shí)相比無(wú)統(tǒng)計(jì)學(xué)差異。故后續(xù)實(shí)驗(yàn)取48h為最佳作用時(shí)間。而高滲對(duì)照組在24h、48h、72h細(xì)胞活性與正常對(duì)照組無(wú)明顯差異。高糖狀態(tài)(30mmol/L葡萄糖)白藜蘆醇濃度為10 μmol/L時(shí),培養(yǎng)48h后細(xì)胞活性最高；高糖能引起ARPE-19細(xì)胞形態(tài)的異常改變,Res可緩解高糖所致的細(xì)胞形態(tài)的異常改變。結(jié)論：30mM的高糖作用體外培養(yǎng)的ARPE-19細(xì)胞48h可抑制其活性；白藜蘆醇作用于高糖狀態(tài)ARPE-19細(xì)胞48h時(shí)能增加其活性,濃度為10μM時(shí)效果最佳;Res可緩解高糖對(duì)ARPE-19細(xì)胞的損傷。第三部分 白藜蘆醇對(duì)高糖狀態(tài)下ARPE-19田胞的保護(hù)作用及機(jī)制研究目的：探討白藜蘆醇對(duì)高糖狀態(tài)下ARPE-19細(xì)胞的保護(hù)作用及其機(jī)制方法：ARPE-19細(xì)胞株分為3組：正常對(duì)照組(Control):5.5mmol/L葡萄糖；高糖組(HG):30mmol/L葡萄糖；Res干預(yù)組(HG+Res):30mmol/L葡萄糖+10μmol/LRes,三組細(xì)胞條件培養(yǎng)48h,流式細(xì)胞儀檢測(cè)細(xì)胞凋亡率,免疫熒光法和Western-Blot檢測(cè)各組中bax、bcl-2的表達(dá),流式細(xì)胞儀和免疫熒光法檢測(cè)ROS的產(chǎn)生,RT-PCR法檢測(cè)IL-1βmRNA、TNF-αmRNA的表達(dá)結(jié)果：與HG組相比,HG+Res干預(yù)組細(xì)胞的凋亡率降低,表達(dá)bcl-2增加、bax減少,bcl-2/bax值升高；且ROS的產(chǎn)生減少、IL-1βmRNA、TNF-αmRNA的表達(dá)降低。結(jié)論：Res能降低細(xì)胞的凋亡率,可能是通過(guò)增加bcl-2、減少bax、升高bc1-2/bax值,減少ROS的產(chǎn)生和減少炎癥相關(guān)細(xì)胞因子的釋放實(shí)現(xiàn)的
[Abstract]:Part 1 Effects of resveratrol on STZ induced diabetic retinopathy in rats Objective: To observe the effect of resveratrol (Resveratrol, Res) on streptozotocin (Streptozotocin, STZ) prevention and treatment induced diabetic retinopathy in rats, and to explore its possible mechanism. Methods 10 only 35 SD the rats were divided into normal control group (Control), 25 with intraperitoneal injection of STZ remaining to establish diabetes model. Then 20 rats were randomly divided into model for diabetic model group (DM) and resveratrol treatment group (DM+Res). The next day to determine the successful model intragastrically. Measurement of body weight and blood glucose of rats after intragastric administration respectively at 0,4,8,12 weeks, 12 weeks, the rats were sacrificed, the eye, and then determined the Rats Retinal malondialdehyde (MDA) content and superoxide dismutase (SOD) activity; using hematoxylin eosin (HE) staining of rat optic Omental tissue morphological changes and measurement of retinal thickness; terminal deoxynucleotidyl transfer enzyme mediated nick end labeling (TUNEL) detection of apoptosis of retinal nerve cells in rats: Observation of rat retinal ganglion cells and capillary wall micro structure changes under electron microscope; immunohistochemical staining was used to detect the expression of Bax in rat retina and the Bcl-2 protein. Results: DM+Res rats, 4,8,12 weeks of weight when compared with the DM group, but lower than that of Control group; the blood glucose levels were lower than that of group DM at the 3 time points. Compared with the DM group, retinal MDA in DM+Res rats were significantly decreased (P0.01), the activity of SOD was significantly increased (P0.01) the total retinal thickness and retinal, and the thickness of the outer nuclear layer were significantly increased (P0.01), ganglion cell layer and inner nuclear layer decreased the apoptosis index (P0.01); under electron microscope, DM group showed ganglion cell nuclear chromatin Condensation, mitochondrial swelling, cytoplasm vacuolation of ganglion cells; DM+Res group had no obvious nuclear chromatin condensation, margination, mitochondrial swelling, cristae loss and cytoplasmic vacuolization; immunohistochemical method showed that compared with DM group, DM+Res group enhanced the expression of Bcl-2, reduce the strength weak expression of Bax, Bcl-2/Bax ratio was significantly increased. Conclusion: Res can improve the body weight of diabetic rats, lower blood sugar, enhance the antioxidative ability of retina, thereby reducing oxidative damage in the retina, improve the pathological changes in diabetic retina, and through the upregulation of the expression of Bcl-2 and Bax under the regulation, inhibit the apoptosis of retinal ganglion cells, play a role in the prevention and treatment of diabetic retinopathy. The second part of resveratrol on high glucose ARPE-19 cells Objective: different time and different concentration of Res on high glucose conditions on ARPE-19 cell activity influence, to explore The most suitable ARPE-19 cells in high glucose injury model, screening the appropriate concentration of Res intervention, and to explore the effect of Res on high glucose ARPE-19 cells methods: ARPE-19 cells were cultured in normal glucose (5.5mmol/L glucose), high glucose (30mmol/L glucose) and hyperosmolar state (5.5mmol/L glucose +24.5mmol/L mannitol) cultured in 24h, 48h, 72h. Comparison of CCK-8 assay at various time points and cell activity, choose the best time point; with different concentrations of resveratrol (O mol/L, 5 mol/L, 10 mol/L, 25 mol/L, 50 mol/L) and high glucose (30mmol/L glucose) ARPE-19 cell line 48h cells were activated through CCK-8 method detection, choose the best concentration; the ARPE-19 cell line was placed in the containing 30mmol/L glucose and 10 mol/L Res DMEM 48h (medium and normal, high glucose control and hypertonic control) with inverted microscope The growth and morphological changes of cells in each group. Results: CCK-8 assay showed that compared with normal control group, high glucose group ARPE-19 cells, 24h cell activity decreased but the difference was not statistically significant, statistically significant differences between the 48h, 72h cell activity decreased when compared with 48h but no statistically significant difference. So the follow-up experiment 48h is the best the role of time. While hypertonic group in 24h, 48h, 72h cell activity had no significant difference with normal control group. High glucose (30mmol/L glucose) resveratrol concentration is 10 mol/L, the highest 48h activity of cultured cells; high glucose can cause abnormal changes of the morphology of ARPE-19 cells, Res can alleviate the abnormal changes induced by high glucose cell morphology. Conclusion: ARPE-19 48h cells were cultured with high glucose in vitro effect of 30mM can inhibit the activity of resveratrol in high glucose; ARPE-19 cells can increase the activity of 48h, the concentration of 10 M aging The best fruit; Res can alleviate the damage of high glucose on ARPE-19 cells. The third part of resveratrol on the objective to study the protective effect and mechanism of ARPE-19 field cells under high glucose condition: To investigate the protective effect and mechanism of resveratrol on ARPE-19 cells under high glucose condition: ARPE-19 cells were divided into 3 groups: normal control group (Control) 5.5mmol/L: glucose; high glucose group (HG): 30mmol/L glucose; Res intervention group (HG+Res): 30mmol/L glucose +10 mol/LRes, 48h three groups of cell culture conditions, cell apoptosis was detected by flow cytometry, immunofluorescence and Western-Blot were detected in Bax, the expression of Bcl-2, flow cytometry and immunofluorescence method the detection of ROS, detection of IL-1 beta mRNA RT-PCR method, the expression of TNF- alpha mRNA: compared with HG group, the apoptosis of HG+Res cells decreased the rate of the intervention group, the expression of Bcl-2 increased, Bax decreased, bcl-2/bax increased and ROS; The expression of IL-1 beta mRNA and TNF- alpha mRNA decreased. Conclusion: Res can reduce the apoptosis rate of cells, which may be achieved by increasing Bcl-2, decreasing Bax, increasing bc1-2/bax value, reducing ROS production and reducing inflammatory related cytokines release.

【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R587.2;R774.1

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