人乳腺癌MCF-7耐紫杉醇细胞株的建立及耐药性形成与Twist基因表达相关性分析
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摘要
目的：本实验采用大剂量紫杉醇(Taxol)冲击诱导方法建立紫杉醇耐药的人乳腺癌细胞株MCF-7/Taxol,建立实时荧光定量PCR(Real-time PCR)方法分析Twist基因表达,并采用该方法动态定量检测紫杉醇耐药性形成过程中MCF-7细胞株Twist基因表达量的改变,为研究乳腺癌获得性紫杉醇耐药与Twist基因之间的关系提供更加确凿的证据,从而进一步探讨乳腺癌耐药机制,为临床上乳腺癌治疗和耐药逆转提供实验模型。方法：(1)采用Taxol大剂量冲击方法处理MCF-7,历时4个月,经历4个加药筛选阶段,直至细胞能在15μg/ml紫杉醇中正常生长增殖。用四甲基偶氮唑蓝(MTT)法检测细胞耐药性以及紫杉醇对耐药诱导过程中各阶段细胞的抑制率,观察细胞形态学变化,同时检测MCF-7/Taxol对长春新碱的交叉耐药性。(2)建立Twist基因的Real-time PCR检测方法,并用该法检测MCF-7耐药性诱导形成过程中Twist基因表达量的变化情况。结果：(1)MTT法检测结果显示,亲代MCF-7细胞及4个加药阶段细胞的紫杉醇半数抑制浓度(50% inhibiting concentration, IC50)分别是3.827±0.04μg/ml, 3.76±0.07μg/ml,10.41±0.27μg/ml (P<0.05),24.82±0.32μg/ml(P<0.01),107.05±1.79μg/ml(P<0.01)；同时这4组细胞对紫杉醇表现出逐渐升高的耐受能力：与亲代MCF-7相比,对紫杉醇的耐药指数(resistance index, RI)分别为0.94±0.04、2.36±0.33(P<0.05)、7.51±1.23(P<0.01)、28.83±1.05(P<0.01)。随着耐药性的稳定形成,细胞在形态上发生了较为明显的改变：亲代MCF-7细胞大小均一,核分裂像少,细胞边缘光滑,折光性良好,细胞成团生长；MCF-7/Taxol细胞则明显变圆回缩、大小不一,细胞间相互连接疏松、细胞间距离明显增宽,细胞边缘不规则,胞质颜色加深颗粒明显、’核仁松散变大、核分裂像增多,少数细胞形大,部分细胞幼稚、胞体明显增大,折光性差；耐药指数最高的MCF-7/TaxolⅣ细胞(RI=28.83±1.05)与亲代MCF-7相比,细胞形态从原来规则圆梭状明显转变为不规整圆形,细胞间排列呈铺路石样改变。(2)MTT法检测结果显示,亲代MCF-7细胞与MCF-7/TaxolⅣ细胞的长春新碱半数抑制浓度IC50分别是12.86±0.34μg/ml、22.06±1.12μg/ml(P<0.05)。(3) Real time-PCR标准曲线的制作：回收RT-PCR扩增的目的基因Twist及内参GAPDH,转化到大肠杆菌并成功构建分别含有目的基因及内参的PMD18T质粒,利用构建的质粒制作的标准曲线循环阈值与模板浓度具有良好的线性关系,内参GAPDH和目的基因Twist标准曲线相关系数分别为0.99897、0.99927,其最低检测拷贝数为1,Twist熔解曲线呈单个特异峰。(4)Realtime-PCR检测结果显示：在加药筛选获得MCF-7/Taxol细胞株的过程中,Twist基因的表达量出现逐渐增高的趋势：四个筛选阶段细胞中Twist的表达情况依次是0.84±0.05(P>0.05)、1.63±0.30(P<0.05)、3.38±0.14(P<0.01)、65.28±0.26(P<0.01)。结论：(1)通过大剂量冲击方法成功地建立了获得性紫杉醇耐药的人乳腺癌细胞株MCF-7/Taxol,最高耐药指数达到28.83±1.05。该细胞和亲代MCF-7细胞相比,不仅在生物形态上发生了改变,同时对紫杉醇的敏感性也显著减低,其耐药倍数随着耐药性的获得而升高；(2)MCF-7/Taxol对长春新碱的耐药性也显著升高,MCF-7/TaxolIV细胞耐药指数达到1.71±0.13(P<0.05),表明耐紫杉醇的MCF-7同时对长春新碱也产生了交叉耐药；(3)成功构建了Twist的原核表达载体,建立的SYBR GREEN I荧光染料实时定量PCR法特异性和稳定性良好,可以用于对MCF-7及各耐药阶段细胞中Twist表达水平的动态定量检测；(4)随着MCF-7细胞耐药指数的升高,Twist的表达水平也明显上升,Twist的表达水平与细胞耐药指数之间的相关系数达到0.999,P<0.05,表明两者之间的相关性成立。提示：Twist基因的高表达在MCF-7细胞紫杉醇获得性耐药的形成中具有重要作用。
Abstract:objective:To establish taxol-resistant human mammary adenocarcinoma cell subline(MCF-7/Taxol) and investigate the relationship between twist gene expression and taxol resistance dynamically. Methods:(1)A taxol-resistant human breast cancer cell subline (MCF-7/Taxol) was established by repeatedly exposing human mammary adenocarcinoma cell subline (MCF-7) to high concentrations of taxol (up to 15μg/ml). Inhibiting growth curve of MCF-7 and MCF-7/Taxol,50% inhibiting concentration (IC50) and taxol resistance index(RI) were got based on MTT assay. The morphological featuers of MCF-7 and MCF-7/Taxol were observed under light microscopy.The dynamic analysis for 4 different stages of taxol resistance MCF-7(named MCF-7/Taxol I,MCF-7/Taxol II,MCF-7/TaxolⅢ,MCF-7/TaxolIV respectively) were carried out. (2)Real-time PCR for twist gene was established as following steps:twist gene was amplified by RT-PCR and subcloned into PMD18T vector which was transformed into E.coil, then the positive recombinant plasmid was used as quantitative template to generate standard curve. GAPDH as the internal standard and Rotor-Gene 3000 series sofeware was performed to detect the level of Twist in the MCF-7. Results:(1) Taxol IC50 of MCF-7、MCF-7/TaxolⅠ、 MCF-7/TaxolⅡ、MCF-7/TaxolⅢand MCF-7/TaxolIV were 3.83±0.04μg/ml,3.76±0.07μg/ml (P>0.05),10.41±0.27μg/ml (P<0.05),24.82±0.32μg/ml (P<0.01),107.05±1.79μg/ml (P<0.01) respectively. Index number of drug resistance (RI) to taxol were 0.94±0.04 (MCF-7/TaxolⅠ)、2.36±0.33 (MCF-7/TaxolⅡ,P<0.05)、7.51±1.23 (MCF-7/TaxolⅢ,P<0.01)、28.83±1.05 ((MCF-7/TaxolⅣ,P<0.01), indicating MCF-7/TaxolⅡ、MCF-7/TaxolⅢand MCF-7/TaxolⅣshowed stable resistance to taxol. (2)The vinblastine IC50 of MCF-7 and MCF-7/TaxolⅣwere 12.86±0.34μg/ml、22.06±1.12μg/ml(P<0.05) respectively,suggesting MCF-7/TaxolIV also showed tolenrance to vinblastine. (3)The standard curve of Real-time PCR for twist indicated the linear relationship between CT(cycle threshold) and template concentration. The lowest value of copy for SYBR GREEN I for Twist was 1.The melting cure showed a peak of Twist, and the coefficient correlation of GAPDH and Twist were 0.99897and 0.99927 respectively.(4)Quantitative analysis of twist gene by Real-time PCR showed that Twist expression were 0.84±0.05 (MCF-7/TaxolⅠ, P>0.05)、1.63±0.30 (MCF-7/TaxolⅡ, P<0.05)、3.38±0.14 (MCF-7/TaxolⅢ，P<0.01)、65.28±0.26 (MCF-7/TaxolⅣ, P<0.01) respectively. The correlation coefficient of RI and Twist expression was 0.999,P<0.05.Conclusions:(1)MCF-7/Taxol cell sublines was established successfully by 4-step taxol shock method lasting 4 months.It is an ideal model for screening drug-resistant gene and drug-reversal agent. MCF-7/Taxol was tolerant to vinblastine while showing stable resistance to taxol suggesting it had the characteristic of multidrug resistance.The expression of Twist in MCF-7 was getting higher following the tendency of acquired taxol resistance. There was significant correlation between drug resistant and Twist expression during the establishment of MCF-7/Taxol, indicating high expression of Twist is of significance for taxol resistance in MCF-7.
Abstract:objective:To establish taxol-resistant human mammary adenocarcinoma cell subline(MCF-7/Taxol) and investigate the relationship between twist gene expression and taxol resistance dynamically. Methods:(1)A taxol-resistant human breast cancer cell subline (MCF-7/Taxol) was established by repeatedly exposing human mammary adenocarcinoma cell subline (MCF-7) to high concentrations of taxol (up to 15μg/ml). Inhibiting growth curve of MCF-7 and MCF-7/Taxol,50% inhibiting concentration (IC50) and taxol resistance index(RI) were got based on MTT assay. The morphological featuers of MCF-7 and MCF-7/Taxol were observed under light microscopy.The dynamic analysis for 4 different stages of taxol resistance MCF-7(named MCF-7/Taxol I,MCF-7/Taxol II,MCF-7/TaxolⅢ,MCF-7/TaxolIV respectively) were carried out. (2)Real-time PCR for twist gene was established as following steps:twist gene was amplified by RT-PCR and subcloned into PMD18T vector which was transformed into E.coil, then the positive recombinant plasmid was used as quantitative template to generate standard curve. GAPDH as the internal standard and Rotor-Gene 3000 series sofeware was performed to detect the level of Twist in the MCF-7. Results:(1) Taxol IC50 of MCF-7、MCF-7/TaxolⅠ、 MCF-7/TaxolⅡ、MCF-7/TaxolⅢand MCF-7/TaxolIV were 3.83±0.04μg/ml,3.76±0.07μg/ml (P>0.05),10.41±0.27μg/ml (P<0.05),24.82±0.32μg/ml (P<0.01),107.05±1.79μg/ml (P<0.01) respectively. Index number of drug resistance (RI) to taxol were 0.94±0.04 (MCF-7/TaxolⅠ)、2.36±0.33 (MCF-7/TaxolⅡ,P<0.05)、7.51±1.23 (MCF-7/TaxolⅢ,P<0.01)、28.83±1.05 ((MCF-7/TaxolⅣ,P<0.01), indicating MCF-7/TaxolⅡ、MCF-7/TaxolⅢand MCF-7/TaxolⅣshowed stable resistance to taxol. (2)The vinblastine IC50 of MCF-7 and MCF-7/TaxolⅣwere 12.86±0.34μg/ml、22.06±1.12μg/ml(P<0.05) respectively,suggesting MCF-7/TaxolIV also showed tolenrance to vinblastine. (3)The standard curve of Real-time PCR for twist indicated the linear relationship between CT(cycle threshold) and template concentration. The lowest value of copy for SYBR GREEN I for Twist was 1.The melting cure showed a peak of Twist, and the coefficient correlation of GAPDH and Twist were 0.99897and 0.99927 respectively.(4)Quantitative analysis of twist gene by Real-time PCR showed that Twist expression were 0.84±0.05 (MCF-7/TaxolⅠ, P>0.05)、1.63±0.30 (MCF-7/TaxolⅡ, P<0.05)、3.38±0.14 (MCF-7/TaxolⅢ，P<0.01)、65.28±0.26 (MCF-7/TaxolⅣ, P<0.01) respectively. The correlation coefficient of RI and Twist expression was 0.999,P<0.05.Conclusions:(1)MCF-7/Taxol cell sublines was established successfully by 4-step taxol shock method lasting 4 months.It is an ideal model for screening drug-resistant gene and drug-reversal agent. MCF-7/Taxol was tolerant to vinblastine while showing stable resistance to taxol suggesting it had the characteristic of multidrug resistance.The expression of Twist in MCF-7 was getting higher following the tendency of acquired taxol resistance. There was significant correlation between drug resistant and Twist expression during the establishment of MCF-7/Taxol, indicating high expression of Twist is of significance for taxol resistance in MCF-7.
引文
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[3]李坤,陈建明,蒋耀光,等.Twist在食管鳞癌中的表达及其对预后的影响.重庆医学[J],2008,37(8)：837-839.
[4]曹笠.胃癌组织Twist基因的荧光实时定量PCR检测.中国现代医生[J],2008,46(33)：20-21.
[5]宫奇林,林梅青,时昌文,等.Twist基因检测对大肠癌临床病理诊断价值的探讨.中华肿瘤防治杂志[J],2008,15(16)：1259-1260.
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[8]Zhang X, Wang Q, Ling MT,et al. Anti-apoptotic role of TWIST and its association with Akt pathway in mediating taxol resistance in nasopharyngeal carcinoma cells.Int J Cancer,2007,120(9):1891-1898.
[9]Cheng GZ, Chan J,Wang Q, et al.Twist transcriptionally up-regulates AKT2 in breast cancer cells leading to increased migration, invasion, and resistance to paclitaxel. Cancer Res [J],2007,67(5):1979-1987.
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[11]Kasper S, Cookson MS. Mechanisms Leading to the Development of Hormone-Resistant Prostate Cancer. Urol Clin North Am [J],2006,33(2): 201-210
[12]Mani SA, Yang J, Brooks M, et al. Mesenchyme Forkhead 1 (FOXC2) plays a key role in metastasis and is associated with aggressive basal-like breast cancers. Proc Natl Acad Sci U S A[J],2007,104(24):10069-10074.
[13]刘芳,江泽飞,宋三泰,等.单药紫杉醇治疗晚期乳腺癌剂量强度与疗效和毒性的关系.中华肿瘤杂志[J],2005,27(1)：56-58.
[14]黄在菊,杨守华,李敏,等。不同方法建立卵巢癌紫杉醇耐药细胞株对临床用药方案的启发.中国药师[J],2008,11(10)：1135-1137.
[15]陶永辉,张熔熔,金坚,等.两株耐紫杉醇人乳腺癌细胞MCF-7的比较.细胞生物学杂志[J],2008,30：515-521
[16]杨晓华,沙慧芳,冯久贤,等.人肺腺癌紫杉醇耐药细胞株的建立及其 特性研究.现代医学[J],2009,37(1)：34-37.
[17]邵泽勇,沈鼎明,伍峰,等.两种方法建立的人肝癌细胞多药耐药模型的比较.肿瘤[J],2005,25(1)：51-54.
[18]Florian K, David A. Twist induce an epithelial-mesenchymal transition to facilitate tumor metatasis[J]. Cancer Biology&Therapy,2004,3(11): 1058-1059.
[1]杨静,顾玉超,于文功,等.Twist对小鼠乳腺癌细胞基因表达谱的调控研究.中国生物工程杂志[J],2008,28(4)：1-6.
[2]Cheng GZ, Chan J, Wang Q,et al.Twist transcriptionally up-regulates AKT2 in breast cancer cells leading to increased migration, invasion, and resistance to paclitaxel. Cancer Res[J],2007,67(5):1979-1987.
[3]李坤,陈建明,蒋耀光,等.Twist在食管鳞癌中的表达及其对预后的影响.重庆医学[J],2008,37(8)：837-839.
[4]曹笠.胃癌组织Twist基因的荧光实时定量PCR检测.中国现代医生[J],2008,46(33)：20-21.
[5]宫奇林,林梅青,时昌文,等.Twist基因检测对大肠癌临床病理诊断价值的探讨.中华肿瘤防治杂志[J],2008,15(16)：1259-1260.
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[8]张飞,史玉荣,张霖,等.乳腺癌多药耐药细胞MCF-7/ADR中Twist的表达与EMT现象的实验研究.中国肿瘤临床[J]2007,34(7)：361-365.
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[2]Schrder R. Vasa mRNA accumulates at the posterior pole during blastoderm formation in the flour beetle Tribolium castaneum.Dev Genes Evol[J],2006,216(5):277-283.
[3]李坤,陈建明,蒋耀光,等.Twist在食管鳞癌中的表达及其对预后的影响.重庆医学[J],2008,37(8)：837-839.
[4]曹笠.胃癌组织Twist基因的荧光实时定量PCR检测.中国现代医生[J],2008,46(33)：20-21.
[5]宫奇林,林梅青,时昌文,等.Twist基因检测对大肠癌临床病理诊断价值的探讨.中华肿瘤防治杂志[J],2008,15(16)：1259-1260.
[6]Wang X, Ling MT, Guan XY,et al.Identification of a novel function of TWIST, a bHLH protein, in the development of acquired taxol resistance in human cancer cells. Oncogene[J],2004,23(2):474-482.
[7]张飞,史玉荣,张霖,等.乳腺癌多药耐药细胞MCF-7/ADR中Twist的表达与EMT现象的实验研究.中国肿瘤临床[J],2007,34(7)：361-365.
[8]Zhang X, Wang Q, Ling MT,et al. Anti-apoptotic role of TWIST and its association with Akt pathway in mediating taxol resistance in nasopharyngeal carcinoma cells.Int J Cancer,2007,120(9):1891-1898.
[9]Cheng GZ, Chan J,Wang Q, et al.Twist transcriptionally up-regulates AKT2 in breast cancer cells leading to increased migration, invasion, and resistance to paclitaxel. Cancer Res [J],2007,67(5):1979-1987.
[10]Zhang X, Wang Q, Ling MT, et al. Anti-apoptotic role of TWIST and its association with Akt pathway in mediating taxol resistance in nasopharyngeal carcinoma cells.Int J Cancer [J],2007,120(9):1891-1898.
[11]Kasper S, Cookson MS. Mechanisms Leading to the Development of Hormone-Resistant Prostate Cancer. Urol Clin North Am [J],2006,33(2): 201-210
[12]Mani SA, Yang J, Brooks M, et al. Mesenchyme Forkhead 1 (FOXC2) plays a key role in metastasis and is associated with aggressive basal-like breast cancers. Proc Natl Acad Sci U S A[J],2007,104(24):10069-10074.
[13]刘芳,江泽飞,宋三泰,等.单药紫杉醇治疗晚期乳腺癌剂量强度与疗效和毒性的关系.中华肿瘤杂志[J],2005,27(1)：56-58.
[14]黄在菊,杨守华,李敏,等。不同方法建立卵巢癌紫杉醇耐药细胞株对临床用药方案的启发.中国药师[J],2008,11(10)：1135-1137.
[15]陶永辉,张熔熔,金坚,等.两株耐紫杉醇人乳腺癌细胞MCF-7的比较.细胞生物学杂志[J],2008,30：515-521
[16]杨晓华,沙慧芳,冯久贤,等.人肺腺癌紫杉醇耐药细胞株的建立及其 特性研究.现代医学[J],2009,37(1)：34-37.
[17]邵泽勇,沈鼎明,伍峰,等.两种方法建立的人肝癌细胞多药耐药模型的比较.肿瘤[J],2005,25(1)：51-54.
[18]Florian K, David A. Twist induce an epithelial-mesenchymal transition to facilitate tumor metatasis[J]. Cancer Biology&Therapy,2004,3(11): 1058-1059.
[1]杨静,顾玉超,于文功,等.Twist对小鼠乳腺癌细胞基因表达谱的调控研究.中国生物工程杂志[J],2008,28(4)：1-6.
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