长春新碱诱导的白血病多药耐药细胞株CEM/VCR的建立及其生物学特性  

Establishment of leukemia multidrug resistant cell line CEM/VCR induced by VCR and its biological features

作　　者：朱大诚[1] 陈秀珍[2] 何莲花[1] 

机构地区：[1]江西中医药大学,江西南昌330004 [2]江西省肿瘤医院,江西南昌330029

出　　处：《中国现代医学杂志》2014年第20期10-15,共6页China Journal of Modern Medicine

基　　金：国家自然科学基金(No:81260580);江西省科技支撑计划资助(No:2008SBA03300);江西省自然科学基金(No:2007GZY0915)

摘　　要：目的建立白血病多药耐药细胞株CEM/VCR,探讨其生物学特性。方法以白血病CEM细胞为亲本细胞,通过逐步增加长春新碱的浓度联合大剂量间断冲击诱导方法建立对长春新碱耐药的白血病CEM/VCR细胞株。MTT实验检测耐药细胞株对常用化疗药物阿霉素(ADM)、阿糖胞苷(Ara-C)、表柔比星(EPI)、长春新碱(VCR)、柔红霉素(DNR)及门冬酰胺酶(L-ASP)的耐药性,计算出CEM和CEM/VCR细胞的半数抑制浓度(IC50)和耐药倍数(RI);光镜下观察其细胞形态学变化,绘制细胞生长曲线,并计算细胞倍增时间;流式细胞仪检测细胞周期分布;RT-PCR半定量检测MDR1 mRNA的表达。结果历时14个月,传代110代,成功建立可操作的、重复性好的CEM/VCR耐药细胞株,该细胞株能够在含0.01μg/mL VCR条件下长期生存。ADM、Ara-C、EPI、VCR、DNR及L-ASP作用CEM细胞72 h的IC50分别为(0.009 9±0.001 8)、(0.0097±0.001 7)、(0.011 8±0.005 1)、(0.002 7±0.000 3)和(0.0 085±0.000 9)μg/mL及(7.679 4±0.058 3)u/mL,而作用CEM/VCR细胞株72 h的IC50分别为(5.597 6±0.113 8)、(0.105 5±0.004 1)、(0.897 9±0.069 0)、(2.5259±0.098 2)和(1.356 3±0.033 5)μg/mL及(3 354.796 5±9.012 8)u/mL,其RI分别为563.71、10.79、75.55、905.34、158.08和436.85倍,差异有统计学意义(P<0.05),说明耐药细胞株CEM/VCR不仅对VCR耐药,还存在交叉耐药;光镜下,CEM/VCR细胞与CEM细胞相比,其体积较小;CEM细胞、CEM/VCR细胞的倍增时间分别为(25.09±0.78)h和(24.27±0.79)h,差异无统计学意义(P>0.05);两种细胞在G0/G1期、S期的分布差异无统计学意义(P>0.05),在G2/M期的分布差异有统计学意义(P<0.05);耐药细胞株CEM/VCR的MDR1mRNA表达水平高于亲本细胞CEM,分别为(1.012±0.031和0.187±0.006)(P<0.01)。结论成功建立了一株白血病多药耐药细胞株CEM/VCR。该细胞株可以成为研究长春新碱获得性耐药机制及开展多药耐药逆转剂筛选较好的体外模型。[Objective] To establish leukemia muhidrug resistant cell line, CEM/VCR, and discuss its bi- ological characteristics. [Methods] Use CEM cell as parent cell and establish CEM/VCR cell line which is resistant to VCR by gradually increasing the VCR concentration and high-dose intermittent inducing. Use MTF test to detect the resistance of cell line to commonly used chemotherapeutics ADM, Ara-C, EPI, VCR, DNR and L-ASP, and figure out the IC50 and RI of CEM and CEM/VCR cells. Observe the cell morphology in light microscope, and draw the cell growth curve, calculate the cell doubling time. Use FCM to detect the cell cycle distribution. Use RT-PCR to make semi-quantitative detection on description of MDR1 mRNA. [Results] Successfully established the operable and well repeated CEM/VCR resistant cell line after 110 passages last 14 months, which can be long term survival in the concentration of 0.01μg/mL VCR. The separate IC50 of CEM cell affected by ADM, Ara-C, EPI, VCR, DNR and L-ASP after 72 h is （0.009 9 ±0.001 8）, （0.009 7 ±0.001 7）, （0.011 8 ± 0.005 1）, （0.002 7 ± 0.000 3）, （0.008 5 ±0.000 9） （1μg/mL） and （7.679 4 ± 0.058 3） （u/mL）. The separate IC50 of CEM/VCR cell affected by ADM, Ara-C, EPI, VCR, DNR and L-ASP after 72 h is （5.597 6 ± 0.113 8）, （0.105 5 ±0.004 1）, （0.897 9 ±0.069 0）, （2.525 9 ±0.098 2）, （1.356 3 ± 0,033 5） （μg/mL） and （3 354.796 5 ± 9.012 8） （u/mL）, and the RI is 563.71, 10.79, 75.55, 905.34, 158.08 and 436.85. It appears that the drug-resistance cell line CEM/VCR is not only resistant to VCR, but also has eross resistance. The CEM/VCR cell is smaller than CEM cell. The doubling time of CEM and CEM/VCR cell is （25.09 ＋ 0.78） h and （24.27 ＋ 0.79）h, not significant difference （P〉0.05）. The 2 kinds of cell distributions differences are not obvious in G0/G1 and S period （P〉0.05）, and have significant differenees in G2/M period （P 〈0.05）. The drug resistant cell strain CEM/VCR is much higher

关 键 词：白血病 多药耐药 长春新碱 CEM/VCR细胞 CEM细胞 

分 类 号：R733.7[医药卫生—肿瘤]