發(fā)布時(shí)間：2018-01-25 09:10

  本文關(guān)鍵詞： 低氧 低氧誘導(dǎo)因子1 T98G細(xì)胞株 RNA干擾 增殖 凋亡　出處：《昆明醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：[目的]：本研究在于模擬腦膠質(zhì)瘤所生存的低氧微環(huán)境,通過(guò)倒置顯微鏡觀察膠質(zhì)瘤細(xì)胞在低氧下的形態(tài),檢測(cè)1%氧濃度對(duì)腦膠質(zhì)瘤細(xì)胞凋亡的影響,并應(yīng)用免疫熒光及Western Blot技術(shù)對(duì)HIF-1a進(jìn)行定量檢測(cè),檢查低氧下不同時(shí)間梯度及細(xì)胞密度對(duì)HIF-1α表達(dá)的影響,并在體外實(shí)驗(yàn)中運(yùn)用RNA干擾技術(shù),沉默低氧誘導(dǎo)因子-1,觀察其對(duì)低氧下膠質(zhì)瘤的增殖、凋亡的影響。為進(jìn)一步研究以HIF-1為靶點(diǎn)治療膠質(zhì)瘤提供實(shí)驗(yàn)和理論依據(jù)。 [方法]：1、通過(guò)低氧培養(yǎng)箱模擬低氧微環(huán)境,通過(guò)倒置顯微鏡下觀察和Hoechest染色、MTT檢測(cè)低氧對(duì)膠質(zhì)瘤細(xì)胞增殖凋亡的影響；在低氧(1%02)培養(yǎng)箱中培養(yǎng)4h-72h。通過(guò)western blot檢查T98G細(xì)胞在低氧條件下4h-72h低氧誘導(dǎo)因子-1a表達(dá)變化.2、通過(guò)平時(shí)的T98G細(xì)胞培養(yǎng)、計(jì)數(shù),設(shè)計(jì)一組細(xì)胞密度梯度并于低氧(1%02濃度下)處理24h,通過(guò)Western blot定量分析HIF-1a表達(dá)變化。3、采用脂質(zhì)體轉(zhuǎn)染法進(jìn)行HIF-1a SiRNA質(zhì)粒轉(zhuǎn)染膠質(zhì)瘤細(xì)胞系T98G細(xì)胞,運(yùn)用Western blot技術(shù)在蛋白水平檢測(cè)感染后穩(wěn)定細(xì)胞系干擾的有效性。4、選取有效的SiRNA質(zhì)粒轉(zhuǎn)染T98G細(xì)胞,分別設(shè)為干擾組和空白對(duì)照,于1%02和21%氧濃度下培養(yǎng)24h、48h、72h、96h、120h,MTT檢測(cè)各組細(xì)胞存活及增殖情況；流式細(xì)胞儀檢測(cè)細(xì)胞凋亡情況。 [結(jié)果]1、正常腦膠質(zhì)瘤細(xì)胞T98G于1%氧濃度下培養(yǎng)與21%氧濃度下培養(yǎng),倒置顯微鏡下觀察和Hoechest染色未見(jiàn)明顯細(xì)胞凋亡,MTT檢測(cè)低氧組與常氧組均呈增殖狀態(tài)。1%氧濃度培養(yǎng)T98G細(xì)胞株4h、6h、12h、24h(24h),HIF-1α蛋白表達(dá)快速升高,24h、48h、72h(24h),HIF-1a蛋白表達(dá)是逐漸下降的。2、在不同細(xì)胞密度對(duì)T98G細(xì)胞HIF-1a表達(dá)的影響中,其中8.5萬(wàn)個(gè)細(xì)胞/Cm2密度的T98G細(xì)胞HIF-1a表達(dá)最高,細(xì)胞密度過(guò)高或過(guò)低都會(huì)導(dǎo)致HIF-1α表達(dá)降低。3、脂質(zhì)體法包裹HIF-1α-SHRNA轉(zhuǎn)染T98G細(xì)胞,經(jīng)過(guò)G418篩選后,HIF-1α蛋白表達(dá)抑制率較高,可達(dá)至92.15%。4、Hoechest染色、流式細(xì)胞儀檢測(cè)1%氧濃度下下干擾組和空白對(duì)照組細(xì)胞均無(wú)明顯凋亡,MTT檢測(cè)發(fā)現(xiàn),低氧干擾組較低氧對(duì)照組增殖緩慢(P0.05),常氧干擾組細(xì)胞較低氧干擾組生長(zhǎng)好(P0.05)。 [結(jié)論]1%低氧微環(huán)境不能誘導(dǎo)腦膠質(zhì)瘤細(xì)胞發(fā)生凋亡；在1%02濃度低氧微環(huán)境中,T98G細(xì)胞低氧處理在24小時(shí)以內(nèi),其HIF-1α蛋白的表達(dá)是快速升高,T98G細(xì)胞低氧處理24h、48h、72h,其HIF-1α的表達(dá)是逐漸降低的。在低氧下其匯合度至95%左右HIF-1α的表達(dá)最高,細(xì)胞密度過(guò)高或過(guò)低均會(huì)降低HIF-1α的表達(dá)。干擾沉默T98G細(xì)胞HIF-1α并不能誘導(dǎo)T98G細(xì)胞凋亡,卻能降低T98G細(xì)胞的增殖速度。
[Abstract]:[Objective: the aim of this study was to simulate the hypoxic microenvironment of glioma, observe the morphology of glioma cells under hypoxia by inverted microscope, and detect the effect of 1% oxygen concentration on apoptosis of glioma cells. Immunofluorescence and Western Blot techniques were used to detect the effect of different time gradient and cell density on the expression of HIF-1 偽. In vitro, RNA interference technique was used to silence hypoxia inducible factor-1 (HIF-1) to observe the proliferation of hypoxic glioma. The effect of apoptosis provides experimental and theoretical basis for further study on the treatment of glioma with HIF-1 as the target. [Methods: the hypoxic microenvironment was simulated by hypoxia incubator. The effect of hypoxia on the proliferation and apoptosis of glioma cells was detected by inverted microscope and Hoechest staining. T98G cells were cultured in hypoxia incubator for 4h-72 h. The expression of hypoxia inducible factor-1a (HIF-1 a) in T98G cells was detected by western blot at 4h-72 h after hypoxia. T98G cells were cultured and counted. A group of cell density gradient was designed and treated with hypoxia for 24 hours. The changes of HIF-1a expression were quantitatively analyzed by Western blot. The HIF-1a SiRNA plasmid was transfected into glioma cell line T98G by liposome transfection. Western blot technique was used to detect the interference effectiveness of stable cell lines after infection at the protein level. The effective SiRNA plasmid was selected to transfect T98G cells. The cells were cultured for 24 h, 48 h, 72 h, 96 h and 120 h, respectively as interference group and blank control group. The survival and proliferation of the cells in each group were detected by MTT. Apoptosis was detected by flow cytometry. [Results: 1. Normal glioma cells T98G were cultured at 1% oxygen concentration and 21% oxygen concentration respectively. No apoptosis was observed under inverted microscope and Hoechest staining. The expression of HIF-1 偽 protein in T98G cells cultured with 1% oxygen concentration in hypoxia group and normoxic group was rapidly increased at 4 h, 12h, 12h, 12h, 24h and 24h, respectively. The expression of HIF-1a protein decreased gradually in T98G cells with different cell densities. The expression of HIF-1a was the highest in T98G cells with 85,000 cell / / cm ~ 2 density. Too high or too low cell density could lead to the decrease of HIF-1 偽 expression in T98G cells. HIF-1 偽 -SHRNA was transfected into T98G cells by liposome method, and the inhibition rate of HIF-1 偽 protein expression reached 92.15.4 after G418 screening. Hoechest staining, flow cytometry detection of 1% oxygen concentration under the interference group and blank control cells were not significantly apoptotic. The proliferation of cells in hypoxic interference group was slower than that in hypoxic control group, while that in normoxic interference group was better than that in hypoxic interference group. [Conclusion: 1% hypoxia microenvironment can not induce apoptosis of glioma cells. The expression of HIF-1 偽 protein in T98G cells treated with hypoxia for 24 hours was increased rapidly after hypoxia for 24 h or 72 h. The expression of HIF-1 偽 decreased gradually, and its confluence degree reached about 95% under hypoxia. The expression of HIF-1 偽 was the highest. Too high or too low cell density could decrease the expression of HIF-1 偽, and interfering with silencing HIF-1 偽 of T98G cells could not induce the apoptosis of T98G cells, but could decrease the proliferation rate of T98G cells.
【學(xué)位授予單位】：昆明醫(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)】：R739.41

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