本文關(guān)鍵詞： HSP90 17-DMAG 人肝星狀細(xì)胞LX2 細(xì)胞凋亡 α-SMA　出處：《青島大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：目的研究HSP90抑制劑17-DMAG在體外對人LX2肝星狀細(xì)胞的生長增殖及凋亡機(jī)制。方法體外培養(yǎng)人LX2肝星狀細(xì)胞作為受試細(xì)胞,實驗設(shè)計為對照組和17-DMAG處理組,以24h為作用時間點,采用細(xì)胞毒試驗(MTT法)觀察并計算不同濃度(100.250.500.1000nmol/L)和作用時間(24h、48h、72h)的17-DMAG對LX2細(xì)胞的生長抑制率并計算IC50值；應(yīng)用流式細(xì)胞儀Annexin V-FITC／PI試劑雙染法檢測藥物作用48h、72h作用后LX2肝星狀細(xì)胞的凋亡率；Western blot檢測藥物作用48h、72h作用后α-SMA的表達(dá),分析灰度值并與對照組進(jìn)行對比。結(jié)果LX2肝星狀細(xì)胞在DMEM培養(yǎng)液中生長良好,并且當(dāng)細(xì)胞濃度5.0×104m1時,培養(yǎng)4-5天后,細(xì)胞進(jìn)入平臺期；17-DMAG臺抑制人LX2肝星狀細(xì)胞生長,且在100-1000nmol/L濃度范圍內(nèi)及1-5天的時間范圍內(nèi)呈濃度和時間依賴性(p0.01)并且能夠抑制細(xì)胞增殖,隨藥物濃度(100-1000nmol/L)加大和作用時間(24h、48h、72h)延長,存活細(xì)胞數(shù)量逐漸減少,并計算在24h、48h、72h的IC50值為：757.380.12lnmol/L:流式細(xì)胞儀PI雙染法證實400nmol/L的17-DMAG可誘導(dǎo)LX2細(xì)胞凋亡,與24h、48h對照組(3.07±0.72、7.50±0.95)比較,17-DMAG處理組細(xì)胞凋亡率分別為(46.68±2.46、62.84±2.73)明顯增加(p0.01); Westernblot研究結(jié)果表明經(jīng)過17-DMAG處理作用48h、72h,可使LX2肝星狀細(xì)胞α-SMA表達(dá)減少,與對照組比較差異有統(tǒng)計學(xué)意義(p0.05)。結(jié)論17-DMAG在100-1000nmol/L濃度范圍內(nèi)及1-5天的時間范圍內(nèi)具有量效和時效關(guān)系；17-DMAG能夠抑制LX2細(xì)胞增殖,并且流式細(xì)胞儀PI雙染法證實400nmol/L的17-DMAG可以誘導(dǎo)該細(xì)胞凋亡,這與降低α-SMA表達(dá)有關(guān)。
[Abstract]:Objective to study the growth, proliferation and apoptosis mechanism of HSP90 inhibitor 17-DMAG on human LX2 hepatic stellate cells in vitro. Methods Human LX2 hepatic stellate cells were cultured in vitro. MTT assay was used to observe and calculate the growth inhibition rate of 17-DMAG (100.250.500.1000nmol / L) and the time of treatment (24h ~ 48h ~ 72h) on LX2 cells, and to calculate the IC50 value. The apoptosis rate of hepatic stellate cells of LX2 was detected by flow cytometry with Annexin V-FITC / Pi reagent double staining method. The expression of 偽 -SMA in hepatic stellate cells of LX2 was detected by Western blot after 48 h and 72 h treatment. Results LX2 hepatic stellate cells grew well in DMEM medium, and when the cell concentration was 5.0 脳 10 ~ (4) m ~ (1), after 4-5 days of culture, the growth of human LX2 hepatic stellate cells was inhibited by entering the platform-phase 17-DMAG platform. In the range of 100-1000 nmol / L and 1-5 days, the cell proliferation was inhibited in a concentration and time-dependent manner (p0.01). With the increase of drug concentration of 100-1000 nmol / L and the prolongation of the time of treatment, the number of viable cells gradually decreased. The IC50 value was calculated to be: 1 757.380.12 lnmol / L at 24 h after 48 h. Flow cytometry Pi double staining showed that 400nmol / L 17-DMAG could induce apoptosis of LX2 cells. The apoptotic rate of 17-DMAG group was 46.68 鹵2.46 鹵62.84 鹵2.73, respectively, and the expression of 偽 -SMA in LX2 hepatic stellate cells was decreased after treatment with 17-DMAG for 48 h and 72 h after treatment with 17-DMAG for 48 h and 72 h, respectively, and the apoptotic rate was 46.68 鹵2.46 and 62.84 鹵2.73, respectively. Conclusion 17-DMAG can inhibit the proliferation of LX2 cells in the range of 100-1000 nmol / L and in the time range of 1-5 days. Flow cytometry Pi double staining showed that 400nmol / L 17-DMAG could induce apoptosis of the cell, which was related to the decrease of 偽 -SMA expression.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R96

【共引文獻(xiàn)】

中國博士學(xué)位論文全文數(shù)據(jù)庫 前1條

1 徐磊;非酒精性脂肪性肝病與內(nèi)分泌代謝紊亂的關(guān)系的研究[D];浙江大學(xué);2014年

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本文編號：1505941