本文選題：Fox + O1　； 參考：《第四軍醫(yī)大學(xué)》2015年碩士論文

【摘要】：研究背景:Fox O1是胰島β細(xì)胞的轉(zhuǎn)錄因子之一,在細(xì)胞內(nèi)參與多種信號(hào)轉(zhuǎn)導(dǎo)通路,能夠調(diào)節(jié)細(xì)胞分化、增殖和凋亡,對(duì)胰島β細(xì)胞的功能具有重要影響。Fox O1-EGFP實(shí)時(shí)成像研究顯示,在小鼠胰島β細(xì)胞或MIN6細(xì)胞,用3mmol/L葡萄糖孵育時(shí),Fox O1一直在細(xì)胞核顯著分布。當(dāng)分別以16.7mmol/L、30mmol/L葡萄糖或116.16ng/m L、1161.6ng/m L胰島素孵育細(xì)胞10-20min時(shí),可檢測(cè)到胞質(zhì)熒光增多而細(xì)胞核熒光減少。另有研究表明,用25mmol/L葡萄糖刺激MIN6細(xì)胞和小鼠胰島β細(xì)胞30min時(shí)Fox O1發(fā)生急劇磷酸化并且由細(xì)胞核轉(zhuǎn)位至細(xì)胞質(zhì),這一效應(yīng)具有時(shí)間(0.5-2h)和劑量(2.5-20mmol/L)依賴性。而分別以0.58ng/m L、5.80ng/m L、580ng/m L和1161.6ng/m L胰島素刺激MIN6細(xì)胞和小鼠胰島β細(xì)胞同樣能夠觀察到Fox O1發(fā)生磷酸化改變,但卻未對(duì)Fox O1的胞質(zhì)-胞核轉(zhuǎn)位進(jìn)行觀察。為探討胰島素對(duì)Fox O1表達(dá)部位的影響,本實(shí)驗(yàn)擬通過(guò)給予不同濃度胰島素孵育小鼠MIN6細(xì)胞,觀察不同濃度胰島素在不同時(shí)間對(duì)β細(xì)胞Fox O1細(xì)胞核-細(xì)胞質(zhì)穿梭定位的影響。目的:采用激光掃描共聚焦顯微鏡對(duì)MIN6細(xì)胞胰島素作用后免疫化學(xué)染色觀察Fox O1在不同濃度刺激不同時(shí)間后表達(dá)位置的變化。方法:1.小鼠胰島素瘤MIN6細(xì)胞用DMEM(high glucose)培養(yǎng)基(含有25mmol/L葡萄糖、15%FBS、100U/m L青霉素、100μg/m L鏈霉素)于25ml培養(yǎng)瓶放置在37℃、5%CO2濃度的細(xì)胞孵箱中培養(yǎng)。2.分別以含25mmol/L和5.5mmol/L葡萄糖培養(yǎng)基孵育12小時(shí)并檢測(cè)培養(yǎng)上清液胰島素濃度。3.在含5.5mmol/L葡萄糖培養(yǎng)基基礎(chǔ)上給予50μIU/m L(1.75ng/m L)、100μIU/m L(3.5ng/m L)和150μIU/m L(5.25ng/m L)濃度胰島素分別作用12、24和48小時(shí)。4.細(xì)胞免疫化學(xué)染色結(jié)合激光掃描共聚焦顯微鏡觀察Fox O1的細(xì)胞內(nèi)定位。結(jié)果:1.25mmol/L葡萄糖培養(yǎng)MIN6細(xì)胞12小時(shí)胰島素分泌濃度為74.37ng/m L,Fox O1主要在細(xì)胞質(zhì)表達(dá);5.5mmol/L葡萄糖培養(yǎng)MIN6細(xì)胞12小時(shí)胰島素分泌濃度為37.46ng/m L,Fox O1主要在細(xì)胞核表達(dá)。2.與對(duì)照組相比,胰島素濃度50μIU/m L組分別在刺激12、24、48小時(shí)后Fox O1熒光強(qiáng)度的胞質(zhì)/胞核比增加,12小時(shí)組差異不具有統(tǒng)計(jì)學(xué)意義(P=0.084),24小時(shí)和48小時(shí)組存在統(tǒng)計(jì)學(xué)差異(P0.05);胰島素濃度100μIU/m L組分別在刺激12、24和48小時(shí)后Fox O1熒光強(qiáng)度胞質(zhì)/胞核比增加,差異具有統(tǒng)計(jì)學(xué)意義(P0.05);胰島素濃度150μIU/m L組分別在刺激12、24和48小時(shí)后Fox O1熒光強(qiáng)度胞質(zhì)/胞核比增加,均存在統(tǒng)計(jì)學(xué)差異(P0.05)。50μIU/m L胰島素組、100μIU/m L胰島素組和150μIU/m L胰島素組分別在不同時(shí)間的組內(nèi)比較存在統(tǒng)計(jì)學(xué)差異(P0.05)。結(jié)論:1.25mmol/L葡萄糖水平下Fox O1主要表達(dá)于細(xì)胞質(zhì);5.5mmol/L葡萄糖水平下Fox O1主要表達(dá)于細(xì)胞核。2.外源性胰島素作用使Fox O1出細(xì)胞核轉(zhuǎn)位至細(xì)胞質(zhì),且與胰島素濃度和刺激時(shí)間的遞增成正比。
[Abstract]:Background: 1: Fox O1 is one of the transcription factors of islet 尾 cells. It participates in many signal transduction pathways in cells and can regulate cell differentiation, proliferation and apoptosis, which has an important effect on the function of islet 尾 cells.In mouse islet 尾 cells or MIN6 cells, 3mmol/L glucose was used to incubate 3mmol/L O1 in the nucleus.When 10-20min was incubated with 16.7 mmol / L 30 mmol / L glucose or 116.16ng/m L 1161.6 ng / mL insulin, the cytoplasmic fluorescence increased and the nuclear fluorescence decreased.Other studies showed that Fox O1 was phosphorylated sharply and translocated from nucleus to cytoplasm when 25mmol/L glucose was used to stimulate 30min in MIN6 cells and mouse islet 尾 cells. This effect was time-dependent and dose-dependent (2.5-20 mmol / L).However, the phosphorylation of Fox O 1 was also observed in MIN6 cells and 尾 cells of mouse islets stimulated with 0.58ng/m L 5.80 ng / m L and 1161.6ng/m L insulin 580ng / mL, respectively, but the cytoplasmic nuclear translocation of Fox O 1 was not observed.In order to investigate the effect of insulin on the expression site of Fox O1, the effect of different concentrations of insulin on the localization of Fox O1 nucleus-cytoplasmic shuttle in 尾 cells was observed by incubating MIN6 cells with different concentrations of insulin at different times.Aim: to observe the expression of Fox O1 in MIN6 cells after insulin treatment with laser scanning confocal microscope (LSCM).Method 1: 1.Mouse insulinoma MIN6 cells were cultured on DMEM(high glucose medium (containing 25mmol/L glucose, 100 渭 g / mL penicillin 100 渭 g / m L streptomycin) in 25ml culture flask and cultured in a cell incubator with CO 2 concentration at 37 鈩，

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