【作者】 李国慧；

【导师】 颉玉欣；

【作者基本信息】 河北医科大学 ， 临床检验诊断学， 2008， 硕士

【摘要】 目的:当前恶性肿瘤的发病率逐年增加,已经成为严重威胁人类健康的一大杀手,对恶性肿瘤的防治成为医务工作者面临的重要课题。越来越多的研究表明肿瘤发生不仅是由于瘤细胞的过度增生,还因为瘤细胞死亡过少,凋亡机制障碍。细胞凋亡参与肿瘤起始过程,并对肿瘤发生发展起负调控作用。本研究运用多种实验技术,探讨苦参碱(matrine)对人卵巢癌细胞A21-2和人骨肉瘤细胞MG63的增殖抑制和诱导凋亡效应,并对促凋亡因子caspase-3进行进行检测和分析,以期进一步阐明苦参碱抗癌的作用机制,为苦参碱的抗癌治疗提供实验基础。方法:将A21-2细胞和MG63细胞置于37℃,饱和湿度5%的CO2培养箱常规条件下培养,待细胞进入对数生长期后用于实验。1应用倒置相差显微镜观察细胞形态学变化:将不同浓度的苦参碱作用于A21-2细胞和MG63细胞,在倒置相差显微镜下定时观察细胞的生长情况并照相。2透射电子显微镜观察:0.6mg/ml的苦参碱作用A21-2细胞和MG63细胞72h后收集细胞,体积分数为2.5%戊二醛4℃固定,用10g/L锇酸后固定,丙酮系列脱水,最后用Epon812环氧树脂包埋,超薄切片机切片,醋酸铀与柠檬酸铅双重染色,电镜下观察并照相。3采用四甲基偶氮唑蓝(MTT)法检测药物对细胞的生长抑制作用。4流式细胞术(FCM)检测细胞凋亡、细胞周期分布:收集经苦参碱作用72h的A21-2细胞和MG63细胞及对照组细胞,冷PBS漂洗,预冷70%乙醇,4℃固定,上机检测前离心去固定液,0.5%胃蛋白酶消化,碘化丙锭(Propidium Iodide,PI:50mg/L Trinton X-100 1.0%)室温避光染色。流式细胞仪上机检测,应用软件进行分析。5 FCM检测Caspase-3蛋白的表达:收集经苦参碱作用72h的A21-2细胞和MG63细胞及对照组细胞,按常规方法进行荧光标记,流式细胞仪检测荧光强度,以荧光指数(FI)表示蛋白的表达情况。6反转录聚合酶链反应(RT-PCR)检测Caspase-3mRNA的表达:利用TRIZOL试剂盒提取经苦参碱作用72h后的A21-2细胞、MG63细胞及对照组细胞RNA,反转录、扩增后得到的PCR产物经8%聚丙烯酰氨凝胶电泳,溴化乙锭(EB)显色,Bio-profif凝胶图像分析系统进行摄像分析。结果:(1)经0.2、0.4、0.6mg/ml苦参碱处理A21-2细胞和MG63细胞72h后,倒置相差显微镜下观察,与对照组相比,用药组的细胞生长明显被抑制,胞浆内颗粒增多增粗,部分细胞变圆而悬浮在培养基内。透射电镜下观察到染色体凝集边聚,粗面内质网肿胀,细胞表面出现空泡。(2)分别用苦参碱(0.2、0.4、0.6、0.8、1.0mg/ml)作用于A21-2细胞和MG63细胞48、72h,MTT结果显示苦参碱能明显抑制A21-2细胞和MG63细胞的增殖,并呈剂量和时间依赖性。作用48h和72h后,A21-2细胞半数抑制率(inhibitory concentration giving 50% inhibition, IC50)分别为1.017mg/ml、0.751 mg/ml,MG63细胞半数抑制率(inhibitory concentration giving 50% inhibition, IC50)分别为1.019mg/ml与0.701mg/ml。1.0mg/ml苦参碱作用A21-2细胞和MG63细胞72h后抑制率可分别达到71.89%、71.16%(p<0.01)。(3)FCM分析发现,0.2、0.4、0.6 mg/ml的苦参碱处理细胞72h后,G0/G1期前面出现凋亡峰。A21-2细胞凋亡率分别为13.54%±0.39%、17.93%±0.19%、26.35%±0.11%,细胞G0/G1期比例由对照组54.12%±1.23%升至59.35%±1.31%,67.52%±0.42%,77.53%±0.19%, S期、G2/M期细胞比例减少,MG63细胞凋亡率分别为11.37%±0.53%、14.28%±0.16%、24.13%±0.23%(p<0.01),处理后的细胞G0/G1期比例增高(由对照组63.21%±1.52%升至69.14%±1.12%,75.31%±0.33%,82.64%±0.16%)。caspase-3蛋白表达升高, A21-2细胞的荧光指数分别为1.60±0.03、1.78±0.02、2.08±0.05,MG63细胞的荧光指数分别为1.64±0.01、1.84±0.03、2.08±0.02。(4)RT-PCR结果表明:经0.2、0.4、0.6 mg/ml的苦参碱处理A21-2细胞和MG63细胞72h后,随着药物浓度的增加,Caspase-3mRNA表达均逐渐升高。结论:1苦参碱能明显抑制肿瘤细胞MG63和A21-2增殖,使细胞周期受到阻滞,并进一步诱导其凋亡。2苦参碱抗肿瘤的作用机制可能与促进Caspase-3的表达有关。其它机制有待于进一步研究。更多还原

【Abstract】 Objective: At present, the attack rate of malignant tumor increase year by year, malignant tumor has become a major cause which threatens people’s health severely, the prevention and cure of malignant tumor become the important topic for medical workers. More and more researches indicate that tumor is a kind of disease with cell apoptosis disturbance.,The cell apoptosis gene get involve in the happen of malignant tumor, and regulate it’s development. In this investigation, different methods were employed to study the effects of apoptosis-inducing and growth-inhibition on the human osteosarcoma cells line MG63 and ovarian cancer cells line A21-2 by matrine, at the same time we detect and analyse the apoptosis activator caspase-3. The purpose of this study is to clarify the anti-tumor mechanism of matrine, it may provide experimental foundation for matrine on anti-tumor medicine.Methods: A21-2cells and MG63 cells were cultured in the environment of 37℃, 5 % CO2 with Dulbecco Eagle’s minimum essential medium (DMEM) and RPMI1640. The logarithmically growing A21-2 cells and MG63 cells were used.1 Using light microscope observe the change of morphology: A21-2 cells and MG63 cells were treated with different concentrations of matrine, the change of morphology was observed by inverted phase contrast microscope in different time and photographed.2 Morphology analysis by transmission electron microscopy: A21-2 cells and MG63 cells treated with 0.6mg/ml matrine for 72 hour were harvested , and pre-fixed with 2.5% glutaraldehyde at 4℃, The cells were postfixed for 1 hour with 1% osmium tetroxide, then dehydrated through agraded ethanol series, and embedded in Epon 812. The ultrastructure of cells was analyzed in ultrathin sections in a transmission electron microscope after the sections were stained with uranylacetate and lead citrate.3 The suppressive effects of matrine on the proliferaition of A21-2 cells and MG63 cells were evaluated in vitro by MTT colrimetric assay.4 Flow cytometric analysis: A21-2 cells and MG63 cells were treated with matrine for 72 hour. Experimental group and control group cells were harvested and washed twice with PBS. Cells were fixed with ice cold 70% ethanol at 4℃. Precipitates were digested with 0.5% pepsin after centrifugation. And then cells were resuspended in 0.5ml propideium iodide (PI)/RNAase A solution. Cells were incubated in the dark at room temperature for 15 min. The fluorescence emission of stained cells was measured with a flow cytometer. Data were analyzed with Multipcycle software. 5 Analysis of caspase-3 protein by flow cytometry: A21-2 cells and MG63 cells were treated with matrine for 72 hour.. At the end of the treatment, adherent and floating cells were combined and centrifuged, cells were stained by indirect immunofluorescence labing method. A histogram plot of FITC-fluorescence intensity (in logarithmic fluorescence intensity) (χ-axis) versus counts (у-axis) has been shown by flow cytometry. Fluorescence index was used to analysis the expression of caspase-3 proteins.6 Reverse transcription polymerase chain reaction (RT-PCR) were used to observed caspase-3mRNA expression. After A21-2 cells and MG63 cells were treated with matrine for 72 hour, total cellular RNA was extracted with TRIZOL reagent according to the manufacturer’s instructions. Reverse transcription reaction and amplification were carried out. PCR products were electropheresed on 8% polyacrylamide gels(1×TBE running buffer ) and visualized by gels imaging system (BIO-PROFIL, VL company, France) stained by ethidium bromide. The fluorescence intensity ofβ-actin fragments served as the criterion for the fragments.Results: 1 A21-2 cells and MG63 cells were treated with 0.2mg/ml、0.4mg/ml、0.6mg/ml matrine for 72 hour. Inverted phase contrast microscope observed, comparing with control group cells, the growth of drug-treated group cells were inhibited, cell granulations were increased and thicken, some cells became round and suspension in the medium. Under transmission electron microscope, the nuclear chromatin of drug-treated group cells became condensed, marginated and segregated and the cytopolasm was vacuolated. The membrane of the cell, nucleus and cell organ were all complete.2 A21-2 cells and MG63 cells were treated with matrine at various concentrations for 48h and 72h respectively, the growth of cells was inhibited significantly in a time- and dose- dependent fashion. The IC50 of A21-2 cells of 48h and 72h were 1.017mg/ml、0.751mg/ml, The IC50 of MG63 cells of 48h and 72h were 1.019mg/ml、0.701mg/ml, respectively. The biggest inhibitory rate of matrine for A21-2 cells and MG63 cells were 71.89% and 71.16% at the concentration of 1.0 mg/ml for 72h(p<0.01)3 The analysis of cellular DNA content by FCM showed that there was a sub-G0/G1 peak in the graph of drug-treated groups. That was a typical apoptotic peak, which was not shown in the graph of control groups. After A21-2 cells were exposed to matrine of 0.2 mg/ml、0.4mg/ml、0.6mg/ml for 72h, the apoptosis rates were 13.54%±0.39%、17.93%±0.19%、26.35%±0.11%,respectively. while the apoptosis rates of control cell is 5.34%±0.41%. the quantity of treated cells in G0/G1 phase were 59.35 %±1.31% , 67.52 %±0.42% , 77.53 %±0.19%,respectively, while the quantity of control cells in G0/G1 phase were 54.12%±1.23%. After MG63 cells were exposed to matrine (0.2、0.4mg/ml、0.6mg/ml) for 72h, the apoptosis rates were 11.37%±0.53%、14.28%±0.16%、24.13% ±0.23% respectively, which were significantly higher than control (3.27%±0.11)(p<0.01).The quantity of treated cells in G0/G1 phase increased but that in S、G2/M phase decreased. Most MG63 cells were blocked at G0/G1stage (from 63.21%±1.52% to 69.14%±1.12%, 75.31%±0.33%,82.64%±0.16%, respectively). Expressions of caspase-3 protein was increased (Fluorescence index of A21-2 were 1.60±0.03、1.78±0.02、2.08±0.05, respectively. Fluorescence index of MG63 were 1.64±0.01、1.84±0.03、2.08±0.02, respectively.).4 The result of RT-PCR showed that expression of caspase-3mRNA was up-regulated .Conclusions: Mtrine can inhibit proliferation and induce apoptosis of A21-2 cells and MG63 cells.Its possible molecular mechanisms might be related to modulation the expression of caspase-3, Other mechanisms should be investigated further.更多还原