人卵巢癌顺铂耐药细胞株OVCAR-3/DDP的建立及相关基因表达水平的研究
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摘要
目的建立人卵巢癌顺铂耐药细胞株OVCAR-3/DDP,初步探讨其耐药机制。方法采用顺铂(DDP)浓度梯度递增诱导法,建立人卵巢癌OVCAR-3细胞株的顺铂耐药细胞株OVCAR-3/DDP;通过细胞计数和MTT法评估细胞增殖情况,流式细胞仪检测细胞周期情况,以评价OVCAR-3/DDP的生物学特性;采用RT-PCR法检测Mfn2、EGFR及MMP2 mRNA水平。结果成功建立了OVCAR-3/DDP顺铂耐药细胞株,其对DDP耐药指数是1.92;与亲本细胞株OVCAR-3相比,OVCAR-3/DDP生长速度减慢,倍增期是OVCAR-3细胞的1.36倍(P<0.05);流式细胞术检测结果显示细胞主要停滞于G2/M期(P<0.05);与OVCAR-3细胞相比,OVCAR-3/DDP细胞中Mfn2、EGFR及MMP2 mRNA水平均明显升高(P<0.05)。结论建立的OVCAR-3/DDP细胞株对顺铂耐药性稳定;OVCAR-3/DDP细胞对顺铂耐药可能与Mfn2、EGFR、MMP2基因转录水平上调有关。
Objectives To establish cisplatin-resistant human ovarian cancer cell line OVCAR-3/DDP,and discuss its resistance mechanism.Methods Stepwise selection in increasing concentration of cisplatin was adopted to establish cisplatin-resistant OVCAR-3/DDP cell line.Cell count and MTT method were used to evaluate cell proliferation;Flow cytometry was used to detect the cell cycle;RT-PCR was used to analyze the mRNA levels of Mfn2,EGFR and MMP2.Results Cisplatin-resistance cell line OVCAR-3/DDP was successfully established,DDP resistance index of which was 1.92;Cell growth of OVCAR-3/DDP cells was slow,its doubling time was 1.36-fold longer than that of OVCAR-3 cells(P<0.05);Flow cytometry results show that the cells were main stagnation in G2/M phase(P<0.05);The mRNA levels of Mfn2,EGFR and MMP2 were increased significantly in OVCAR-3/DDP compared with those in OVCAR-3 cells(P<0.05).Conclusion The cisplatin resistance of OVCAR-3/DDP cell line is stable,which probably is related to the increased expression of Mfn2,EGFR and MMP2 genes.
Objectives To establish cisplatin-resistant human ovarian cancer cell line OVCAR-3/DDP,and discuss its resistance mechanism.Methods Stepwise selection in increasing concentration of cisplatin was adopted to establish cisplatin-resistant OVCAR-3/DDP cell line.Cell count and MTT method were used to evaluate cell proliferation;Flow cytometry was used to detect the cell cycle;RT-PCR was used to analyze the mRNA levels of Mfn2,EGFR and MMP2.Results Cisplatin-resistance cell line OVCAR-3/DDP was successfully established,DDP resistance index of which was 1.92;Cell growth of OVCAR-3/DDP cells was slow,its doubling time was 1.36-fold longer than that of OVCAR-3 cells(P<0.05);Flow cytometry results show that the cells were main stagnation in G2/M phase(P<0.05);The mRNA levels of Mfn2,EGFR and MMP2 were increased significantly in OVCAR-3/DDP compared with those in OVCAR-3 cells(P<0.05).Conclusion The cisplatin resistance of OVCAR-3/DDP cell line is stable,which probably is related to the increased expression of Mfn2,EGFR and MMP2 genes.
引文
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[32]Fishman DA,Liu Y,Ellerbroek SM,et al.Lysophosphatidic acid promotes matrix metallo proteinase(MMP)activation and MMP dependent invasion in ovarian cancer cells[J].Cancer Res,2001,61(4):3194-3199
[33]Kamel H,Abdelazim I,Habib SM,et al.Immunoexpression of matrix metallo proteinase-2(MMP-2)in malignant ovarian epithelial tumours[J].J Obstet Gynaecol Can,2010,32(6):580-586
[2]周莹莹,王敏,吴建磊,等.耐药及敏感卵巢癌组织与细胞系中microRNA的表达及意义[J].肿瘤学杂志,2014,3(20):197-202
[3]Chen G,Liu N,Zhou A,et al.The role of hypertension-related gene in aortic vascular smooth muscle cells from mice and rats[J].Chin Med J,2001,114(8):833-836
[4]Santel A,Fuller MT.Control of mitochondrial morphology by a human mitofusin[J].Cell Sci,2001,114(pt5):867-874
[5]Dorn GW,Song M,Walsh K.Functional implications of mitofusin 2-mediated mitochondrial-SR tethering[J].J Mol Cell Cardiol,2014,pii:S0022-2828(14)00296-X
[6]Nasrallah CM,Horvath TL.Mitochondrial dynamics in the central regulation of metabolism[J].Nat Rev Endocrinol,2014,doi:10.1038/nrendo.2014.160
[7]白云,熊艳杰,刘晓光.MFN2、Survivin和nm23在卵巢癌中的表达及其意义[J].中国妇幼保健,2014,13(29):2079-2081
[8]Chen KH,Dasgupta A,Ding J,et al.Role of mitofusin 2(Mfn2)in controlling cellular proliferation[J].FASEB J,2014,28(1):382-394
[9]姚玉霜,张婷婷,戚玉言,等.卵巢癌组织EGFR及下游信号分子的表达及其临床意义[J].中华肿瘤防治杂志,2011,18(3):212-215
[10]Glaysher S,Bolton LM,Johnson P,et al.Targeting EGFR and PI3K pathways in ovarian cancer[J].Br J Cancer.2013,109(7):1786-1794
[11]Bull Phelps SL,Schorge JO,Peyton MJ,et al.Implications of EGFR inhibition in ovarian cancer cell proliferation[J].Gynecol Oncol.2008,109(3):411-417
[12]Zhang J,Chen AP,Wang B,et al.Correlations of EGFR and LRP to chemotherapy resistance and prognosis of ovarian cancer[J].Ai Zheng,2008,27(12):1331-1336
[13]Xiao yue Tan,Ying jian Li,You hua Liu.Paricalcitol attenuates renal interstitial fibrosis in obstructive nephropathy[J].J Am Soc Nephrol,2006,17(12):3382-3393
[14]栾英姿,李力,黎丹戎,等.五种卵巢癌耐药细胞系的建立及其部分耐药相关基因的表达[J].中华妇产科杂志,2004,6(39):403-405
[15]高瑞萍,葛秀君,蒋立艳,等.人卵巢腺癌多药耐药细胞株OVCAR/DDP的建立及生物学特性评价[J].新医学,2012,1(43):22-25
[16]Sorlano FX,Liesa M,Bach D,et al.Evidence for a mitochondrial regulatory pathway defined by peroxisome proliferator activated receptor-gamma,coactivator-1 alpha,estrogen-related receptor-alpha and mitofusin2[J].Diabetes,2006,55(6):1783-1791
[17]Guo X,Chen KH,Guo Y,et al.Mitofusin 2 triggers vascular smooth muscle cell apoptosis via mitochondrial death pathway[J].Circ Res,2007,101(11):1113-1122
[18]白云,姜广建,李卫红,等.HSG/Mfn2和nm23在卵巢癌中的表达及其意义[J].中国妇幼保健,2009,24(15):2132-2134
[19]Xia Y,Wu Y,He X,et al.Effects of mitofusin-2gene on cell proliferation and chemotherapy sensitivity of MCF-7[J].Hua Zhong Univ Sci Technolog Med Sci,2008,28(2):185-189
[20]Yun Xia,Yaqun Wu,Xiaojun He,et al.Effects of Mitofusin-2 Gene on Cell Proliferation and Chemotherapy Sensitivity of MCF-7[J].Journal of Huazhong University of Science and Technology(Medical Sciences),2008,28(2):185-189
[21]王尧,陈艳红.线粒体融合素基因2对肝癌细胞的增殖及对化疗敏感性的影响[J].中国普通外科杂志,2009,18(7):708-712
[22]Wu L,Li Z,Zhang P,et al.Adenovirus-expressed human hyperplasia suppressor gene induces apoptosis in cancer cells[J].Mol Cancer Ther,2008,7(1):222-232
[23]章骏,赵怡,王群,等.乳腺癌组织中HSG和EGFR基因mRNA表达与其临床病理因素的相关性研究[J].实用癌症杂志,2014,29(6):629-632
[24]MimoriK,Nagahara H,Sudo T,et al.The epidermal growth factor receptor gene sequence is highly conserved in primary gastric cancers[J].J Surg Onco,2006,93(1):44-46
[25]Wu X,Deng Y,Wang G.Combining siRNAs at two different sites in the EGFR to suppress its expression,induce apoptosis,and enhance 5-fluorouracil sensitivity of colon cancer cells[J].J Surg Res,2007,138(1):56-63
[26]Nielsen JS,Jakobsen E,Holund B,et al.Prognostic significance of p53,Her-2,and EGFR overexpression in borderline and epithelial ovarian cancer[J].Int J Gynecol Cancer,2004,14(6):1086-1096
[27]PsyrriA,Kassar M,Yu Z,et al.Effect of epidermal growth factor receptor expression level on survival in patients with epithelial ovarian cancer[J].Clin Cancer Res,2005,11(12):8637-8643
[28]Park SJ,Armstrong S,Kim CH,et al.Lack of EGF receptor contributes to drug sensitivity of human germline cell[J].Br J Cancer,2005,92(2):334-345
[29]陈爱平,张晶,刘辉,等.EGFR、MVD和LRP在上皮性卵巢癌中的表达及其临床意义[J].中国肿瘤生物治疗杂志,2008,15(3):248-253
[30]Kim TJ,Rho SB,Choi YL,et al.High expression of tissue inhibitor of metallo proteinase-2 in serous ovarian carcinomas and the role of this expression in ovarian tumorigenesis[J].Human Pathology,2006,37(7):906-913
[31]黄晓智,熊伟,张晓斌,等.MMPRIN通过调控MMP-2的表达参与乳腺癌恶性侵袭进程[J].中国煤炭工业医学杂志,2014,17(8):1205-1208
[32]Fishman DA,Liu Y,Ellerbroek SM,et al.Lysophosphatidic acid promotes matrix metallo proteinase(MMP)activation and MMP dependent invasion in ovarian cancer cells[J].Cancer Res,2001,61(4):3194-3199
[33]Kamel H,Abdelazim I,Habib SM,et al.Immunoexpression of matrix metallo proteinase-2(MMP-2)in malignant ovarian epithelial tumours[J].J Obstet Gynaecol Can,2010,32(6):580-586