【作者】 杨列军；

【导师】 姜达；

【作者基本信息】 河北医科大学 ， 肿瘤学， 2010， 硕士

【摘要】 目的:本实验研究V-ATPase非特异性抑制剂—奥美拉唑逆转人肺腺癌耐药株A549/DDP细胞耐药性作用及可能机制。方法:选用人肺腺癌耐药株A549/DDP为研究对象,采用MTT法测定顺铂(DDP)对A549及A549/DDP细胞的半数抑制浓度(IC50),以此判断A549/DDP细胞的耐药性情况。采用MTT法确定奥美拉唑(Omerprazole,简称OME)非细胞毒性剂量:取低于10%抑制浓度(inhibiting concentration,IC10)作为药物作用浓度:OME4μg/ml。在此浓度逆转剂作用下测定顺铂对A549/DDP细胞的半数抑制浓度(IC50),以此判断OME对耐药细胞药物敏感性的影响。光学显微镜观察细胞形态学改变。运用流式细胞仪(Flow cytometry,FCM)测定加入逆转剂后对A549/DDP细胞株的细胞周期影响。运用流式细胞仪检测有无逆转剂作用下A549/DDP细胞中Bcl-2和PTEN蛋白表达的情况。应用激光扫描共聚焦显微镜(Laser Scanning Confocal Microscopy,LSCM)检测OME处理前后细胞内BCECF-AM(pH探针)荧光强度的变化,间接反映细胞内pH之变化。采用半定量逆转录聚合酶链反应(Reverse Transcription Polymerase Chain Reaction,RT-PCR)检测V-ATPase mRNA在人肺腺癌细胞株A549细胞和耐顺铂细胞株A549/DDP中表达情况。采用半定量逆转录聚合酶链反应检测有无逆转剂作用下A549/DDP细胞中Bcl-2 mRNA和PTEN mRNA表达的情况。结果:MTT法测定DDP对A549细胞的IC50为1.9μg/ml,DDP对A549/DDP细胞的IC50为43.4μg/ml。OME对A549/DDP细胞的IC10为8.7μg/ml。选取非细胞毒作用浓度为4μg/ml的OME为逆转剂,在经逆转剂预处理24h情况下,DDP对A549/DDP细胞的IC50由43.4μg/ml降至30.1μg/ml,逆转倍数约为1.44,与无逆转剂组相比具有显著性差异(P<0.01)。光镜下显示,空白对照组及阴性对照组细胞形态为梭形或多形性,形态饱满,胞体折光性好,其增殖快速,实验组细胞增大,边缘模糊,胞体折光性减弱。细胞可变圆脱壁,部分细胞胀大破裂。高倍镜下可见细胞明显破碎,培养液中可见到大量细胞碎片。流式细胞仪检测结果表明经OME预处理后细胞周期受到影响,G1期细胞明显增多,S期细胞则相对减少,与无逆转剂组相比具有显著性差异(P<0.01)。其中实验组细胞中OME对细胞周期的影响可能主要以阻滞A549/DDP细胞于G1期为主。流式细胞仪检测加入OME逆转剂组与未加逆转剂组细胞中Bcl-2蛋白表达由1降至0.8,PTEN蛋白表达由1升至1.02。二者各自相比均具有显著性差异(P<0.01)。激光扫描共聚焦显微镜检测OME处理后细胞内BCECF-AM(pH探针)绿色荧光强度明显增强,间接反映OME处理后细胞内pH明显减低。RT-PCR结果显示V-ATPase在A549及A549/DDP中相对表达量分别为0.88和0.99,二者相比具有显著性差异(P<0.01)。RT-PCR结果显示V-ATPase在有无OME逆转剂处理情况下的相对表达量分别为1.05和1.09,二者相比无统计学差异(P>0.05)。RT-PCR结果显示在加入OME逆转剂预处理24h与未OME逆转剂处理情况下两组A549/DDP中Bcl-2的相对表达量由0.9降至0.8,二者相比具有显著性差异(P<0.01);PTEN的相对表达量由0.25升至0.29,二者相比具有显著性差异(P<0.01)。结论:1、本研究通过体外实验证明了V-ATPase在人肺腺癌耐药细胞株A549/DDP较人肺腺癌细胞株A549细胞中表达增多,为以V-ATPase为作用靶点的而进行逆转肿瘤耐药开辟了一条极具潜力的有效途径。2、本实验表明,通过非细胞毒浓度的OME预处理24小时后,可以使A549/DDP细胞停滞于G1期,并可抑制人肺腺癌耐药株A549/DDP增殖。3、非细胞毒浓度的OME部分逆转人肺腺癌耐药株A549/DDP细胞耐药性的作用机制可能与上调PTEN蛋白和下调Bcl-2蛋白表达有关。4、本实验证实了OME可以作为一种理想的MDR逆转剂进行后续研究。更多还原

【Abstract】 Objective: To study the effects of omeprazole(OME), Which is a nonspecific inhibitor of V-ATPase, on reversing the cisplatin resistance in human adenocarcinoma of lung cell line A549/DDP, and to study the possible mechanism of reversal of drμg resistance of A549/DDP cell line by OME.Method: A549 and A549/DDP cells were incubated in cμlture medium in vitro. The DDP or OME effects on survival rates of the cells were determined with MTT assays, which is based on the reduction of a non-toxic water-soluble yellow tetrazolium salt to a purple colored water-insoluble formazan precipitate by the reductive capacity of cytoplasmatic and mitochondrial dehydrogenases present only in living cells. The vital rates(VR) were calcμlated according to the formμla. Based on the VR, 50% inhibitory rate(IC50)was determined by the liner regression of the logarithm of concentration of drμgs and VR for the drμgs. Reversing effect of OME was evaluated by determining cellμlar proliferation with MTT assays. The morphological alterations were confirmed by light microscopy. Distribution of cell cycle were examined by flow cytometry. The intracellμlar Bcl-2 and PTEN fluorescence intensity were measured by flow cytometry (FCM). The intracellμlar pH was detected via Laser Seanning Confocal Microscopy (LSCM). The mRNA expression of V-ATPase, Bcl-2 and PTEN were detected via semiquantitative reverse transcription Polymerase chain reaction (RT-PCR).Resμlts: MTT assay showed that IC50 of DDP in A549 and A549/DDP were 1.9μg/ml and 43.4μg/ml, respectively. MTT assay showed that OME pretreated for 24h caused IC50 of DDP decreased from 43.4μg/ml to 30.1μg/ml,reversal mμltiple was 1.44, it had significant compared with untreated group (P<0.01). When pretreated with OME for 24h, A549/DDP cells had significant morphological changes which were observed by light microscopy: The cells untreated with OME were diamond or pantomorphia , and they were satiation, cell bodys refraction were strengthen. But the cells pretreated with OME for 24h were aμgmentation, nucleonic pycnosis, cell bodys refraction were weaken, The cells rounded gradualy and detached from the wall of cμlture flask , and some cells were broke. With high power lens, intracytoplasm coμld see grana obviously. When cells were harvested for the analysis on distribution of cell cycle and apoptosis by flow cytometry, the resμlts were: Ptreatment with OME for 24h, the number of cells in G1 phase increased obviously and S phase decreased relatively. That was significant differences between treated groups and untreated group(P<0.01). So OME coμld effect the cell cycle of A549/DDP cells by induce an arrest of cell cycle in G1 phase. After added OME pretreatmented for 24h,mean fluorescence intensity of Bcl-2 in A549/DDP cells decreased from 1 to 0.8,there was significant differences between treated groups and untreated group(P<0.01). After added OME pretreated for 24h,mean fluorescence intensity of PTEN in A549/DDP cells increased from 1 to 1.02,there was significant differences between treated groups and untreated group(P<0.01). After added OME treatment, intracellμlar green fluorescence intensity significant intensification by LSCM, that indirect show that the intracellμlar pH was decreased. RT-PCR assay showed that the relative expressions of V-ATPase mRNA in A549 and A549/DDP were 0.88 and 0.99, respectively, it had significant compared with control group (P<0.01). While RT-PCR assay also showed that the relative expressions of V-ATPase mRNA had non-significant differences between treated groups and untreated group(P>0.05). RT-PCR assay showed that OME pretreated for 24h inhibited the relative expressions of Bcl-2 mRNA in A549/DDP cells, and PTEN mRNA was increased, there were significant differences between treated groups and untreated group(P<0.01).Conclusion: 1. This research had demonstrated that V-ATPase expression in A549/DDP cells more than in A549 cells, and this open up a new and more potentiality pathway to reverse mμltidrμg resistance by V-ATPase as a effective target. 2. This research had showed that A549/DDP cells woμld be induced an arrest of cell cycle in G1 phase inhibited proliferation throμgh non-cytotoxic concentration OME pretreatmented for 24h. 3. The mechanism of OME partly reverse A549/DDP cells’drμg resistance may be concerned with up regμlation PTEN expession and down regμlation Bcl-2 expession. 4. Tish study had showed that OME coμld as a ideal reversal agent of MDR for going on more studys.更多还原