本文關(guān)鍵詞：ABL-N對(duì)L1210細(xì)胞增殖和凋亡的影響及其作用機(jī)制　出處：《河北醫(yī)科大學(xué)》2011年碩士論文　論文類(lèi)型：學(xué)位論文

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【摘要】：1-O-乙�；蠡ㄐ不▋�(nèi)酯(ABL)是歐亞旋覆花中含量較高的活性成分之一,為倍半萜內(nèi)酯類(lèi)化合物,具有抗腫瘤活性,但其穩(wěn)定性和溶解性都不太好,本實(shí)驗(yàn)所用的藥物是ABL的衍生物,簡(jiǎn)稱(chēng)ABL-N。目的:觀(guān)察ABL-N對(duì)小鼠白血病細(xì)胞L1210增殖和凋亡的影響,并初步探討其作用的機(jī)制。 方法: (1) MTT法測(cè)定ABL-N對(duì)小鼠白血病細(xì)胞L1210增殖的影響:取細(xì)胞培養(yǎng)于96孔培養(yǎng)板內(nèi),培養(yǎng)24 h后,給藥組加入不同濃度的ABL-N溶液,對(duì)照組加與最高濃度組等量的DMSO。分別培養(yǎng)24 h、48 h、72 h后用Fluo-star在檢測(cè)波長(zhǎng)570 nm條件下測(cè)定光密度值(OD value),計(jì)算各濃度的ABL-N對(duì)L1210細(xì)胞的抑制率。(2)臺(tái)盼藍(lán)拒染法檢測(cè)ABL-N對(duì)L1210存活細(xì)胞數(shù)的影響。取對(duì)數(shù)生長(zhǎng)期的細(xì)胞,給藥組給予不同濃度的ABL-N溶液,對(duì)照組加與最高濃度組等量的DMSO。培養(yǎng)后的分別于第24 h、48 h、72 h取樣,臺(tái)盼藍(lán)染色,計(jì)數(shù)活細(xì)胞,用活細(xì)胞數(shù)對(duì)時(shí)間繪制生長(zhǎng)曲線(xiàn)。(3)通過(guò)瑞氏-吉姆薩染色和Hochest 33258染色觀(guān)察ABL-N作用48 h后L1210細(xì)胞的形態(tài)變化。收集ABL-N(20 mg/L)處理后的細(xì)胞,進(jìn)行染色,觀(guān)察細(xì)胞形態(tài)學(xué)變化。(4)用流式細(xì)胞儀測(cè)定ABL-N作用后L1210細(xì)胞的凋亡率和周期分布。取對(duì)數(shù)生長(zhǎng)期的細(xì)胞,給予ABL-N(10、20 mg/L)作用48 h后,PI染色,在流式細(xì)胞儀上測(cè)定各組細(xì)胞的凋亡率和周期分布。(5)Western-blot法檢測(cè)ABL-N作用后L1210細(xì)胞Bax和Bcl-2蛋白表達(dá)的變化。(6)免疫熒光法檢測(cè)ABL-N作用后L1210細(xì)胞p53蛋白表達(dá)的變化。 結(jié)果: (1)用不同濃度ABL-N(5、10、20 mg/L)分別處理L1210細(xì)胞24 h后抑制率分別為14.6%、38.5%、47.4%。作用48 h時(shí)抑制率分別為28.8%、63.2%、74.8%。72 h后抑制率分別為35.7%、81.5%、91.6%。表明ABL-N可明顯抑制L1210細(xì)胞的增殖,并呈現(xiàn)出濃度和時(shí)間依賴(lài)性。(2)用不同濃度ABL-N(5、10、20 mg/L)作用于L1210后,從生長(zhǎng)曲線(xiàn)可以看出明顯的濃度效應(yīng)關(guān)系,且高濃度組加藥1 d后及中濃度組加藥2 d后,活細(xì)胞數(shù)量減少,說(shuō)明ABL-N對(duì)體外培養(yǎng)的小鼠白血病L1210細(xì)胞有較強(qiáng)的抑制作用及殺滅作用。(3)本研究采用Giemsa染色及Hoechst 33258熒光染色方法分別觀(guān)察了ABL-N作用于L1210細(xì)胞48 h后的形態(tài)變化,2種染色方法均顯示給藥組細(xì)胞呈現(xiàn)核聚集和碎裂等凋亡特征性形態(tài)學(xué)變化。(4)不同濃度的ABL-N(10、20 mg/L)作用48 h后凋亡率分別為13.6%、29.9%。溶劑對(duì)照組的凋亡率為3.57%�？梢�(jiàn)ABL-N作用后凋亡率明顯的增加。(5)ABL-N(10、20 mg/L)作用48 h后G1期細(xì)胞數(shù)量由26.8%增加到30.9%、51.4%,S期細(xì)胞數(shù)量由73.2%下降到69.1%、48.6%。(6)不同濃度的ABL-N(5、10、20 mg/L)作用48 h后,Bax/β-actin比例由0.49分別增加到0.58、0.71(P0.01)、0.82(P0.01)。Bcl-2/β-actin比例由0.66分別下降到0.61、0.57(P0.05)、0.28(P0.01)。Bcl-2/Bax比值由1.36分別下降到1.17、0.80(P0.01)、0.34(P0.01)。(7)對(duì)照組p53蛋白表達(dá)的熒光強(qiáng)度為63.5。不同濃度的ABL-N(5、10、20 mg/L)作用48 h后的熒光強(qiáng)度分別為83.3(P0.05)、188.0(P0.01)、242.5(P0.01),有了明顯的增強(qiáng)。 結(jié)論: (1)ABL-N能明顯抑制小鼠白血病細(xì)胞L1210的增殖。(2)ABL-N對(duì)L1210細(xì)胞有明顯的細(xì)胞毒性。(3)ABL-N影響細(xì)胞周期動(dòng)力學(xué),使細(xì)胞停滯在G1期。(4)ABL-N抑制L1210細(xì)胞增殖作用可能與影響細(xì)胞周期動(dòng)力學(xué)有關(guān)。使細(xì)胞停滯在G1期,影響了DNA的合成。(5)ABL-N能誘導(dǎo)L1210細(xì)胞凋亡,這可能與上調(diào)p53和Bax,下調(diào)Bcl-2蛋白的表達(dá)有關(guān)。其上調(diào)p53蛋白表達(dá)可能與使細(xì)胞停滯在G1期有關(guān)。
[Abstract]:1-O- acetyl flower 1-o-acetylbritannilactone (ABL) is one of the active ingredients of Inula britannica content high, is a sesquiterpene lactone compound, has antitumor activity, but its stability and solubility are not too good, drugs used in this experiment is a derivative of ABL ABL-N. Objective: the effect of ABL-N on proliferation and apoptosis of L1210 mouse leukemia cells, and to explore its mechanism.
Method:
(1) the effect of ABL-N on the proliferation of leukemia cells in L1210 mice was determined by MTT assay: cells cultured in 96 well plates, cultured 24 h, the drug group added different concentrations of ABL-N solution, the control group was treated with the highest concentration of equal amounts of DMSO. were cultured for 24 h, 48 h, determination of optical density values in the the detection wavelength of 570 nm under the condition of 72 h Fluo-star (OD value), to calculate the concentration of ABL-N the inhibition rate of L1210 cells. (2) effect of trypan blue dye exclusion assay for the detection of ABL-N L1210. The number of viable cells in logarithmic growth phase cells, ABL-N solution treatment groups were given different concentrations. The control group was treated with the highest concentration of equal amounts of DMSO. cultured in twenty-fourth h, 48 h, 72 h sampling, trypan blue staining, counting live cells, draw the growth curve of time with the number of living cells. (3) by Wright Giemsa staining and Hochest staining was observed in 33258 ABL-N 48 h L1210 cell morphology Change. Collect ABL-N (20 mg/L) treated cells, staining, morphological changes were observed. (4) with the rate of apoptosis and L1210 cell cycle distribution was determined by ABL-N flow cytometry. After logarithmic growth phase cells treated with ABL-N (10,20 mg/L) 48 h, PI staining, apoptosis the rate and cycle distribution of cells was measured by flow cytometer. (5) the expression changes of L1210 cell Bax detection ABL-N Western-blot method and Bcl-2 protein. (6) the expression changes of L1210 cells p53 ABL-N protein was detected by immunofluorescence assay after effect.
Result:
(1) with different concentrations of ABL-N (5,10,20 mg/L) respectively. The treatment rate was 14.6%, suppressed after 24 h L1210 cells 38.5%, 47.4%. 48 h when the inhibition rates were 28.8%, 63.2%, 35.7% respectively, the inhibition of H 74.8%.72 81.5%, 91.6%. showed that ABL-N can inhibit the proliferation of L1210 cells, and showed a time and concentration dependent. (2) with different concentrations of ABL-N (5,10,20 mg/L) in L1210, see the obvious concentration effect relationship from the growth curve, and the high concentration group after 1 D and the concentration of drug dosing group after 2 D, the number of living cells decreased, indicating that ABL-N has strong inhibition and killing effect the in vitro murine leukemia L1210 cells. (3) the study of morphological changes by Giemsa staining and Hoechst were used to observe the effect of ABL-N on L1210 cells after 48 h 33258 fluorescence staining method, 2 kinds of staining methods showed that drug treated cells showed nuclear accumulation and broken 瑁傜瓑鍑嬩骸鐗瑰緛鎬у艦鎬佸鍙樺寲.(4)涓嶅悓嫻撳害鐨凙BL-N(10,20 mg/L)浣滅敤48 h鍚庡噵浜＄巼鍒嗗埆涓，

本文編號(hào)：1389494