氨甲喋呤对映体诱导白血病BALL-1、CCRF-CEM细胞系耐药后EGFR信号通路相关基因表达分析  

Differentially expression of EGFR signaling pathway-related genes in BALL-1 and CCRF-CEM cell lines after drug-resistance induced by methotrexate enantiomers

作　　者：索美芳[1] 何晓东[1] 张培[1] 陈洋[1] 沈佐君[1] 

机构地区：[1]安徽省立医院临床检验中心,合肥230001

出　　处：《临床检验杂志》2014年第4期262-266,共5页Chinese Journal of Clinical Laboratory Science

基　　金：国家自然科学基金项目(30672011)

摘　　要：目的建立耐氨甲蝶呤(MTX)对映体的人白血病BALL-1和CCRF-CEM细胞,观察其生物学特性,分析不同MTX对映体耐药细胞表皮生长因子受体(EGFR)信号通路相关基因的表达。方法用浓度递增结合低剂量持续诱导的方法建立L-(+)-MTX、D-(-)-MTX耐药细胞并绘制细胞生长曲线,用流式细胞仪检测细胞周期,用RT-PCR法检测各细胞EGFR、KRAS、TGF、PI3K mRNA的表达情况。结果 L-(+)-MTX、D-(-)-MTX耐药细胞自第4日增殖速度明显较亲本细胞慢,而L-(-)-MTX耐药细胞增殖速度明显较D-(+)-MTX耐药细胞慢。与亲本细胞BALL-1、CCRF-CEM相比,两种耐药细胞周期分布中G0/G1期所占比例增加,S期细胞所占比例减少。L-(+)-MTX/BALL-1、D-(-)-MTX/BALL-1间EGFR、K-Ras mRNA差异有统计学意义(P均<0.05)。L-(+)-MTX/CCRF-CEM、D-(-)-MTX/CCRF-CEM组间EGFR、K-Ras mRNA差异有统计学意义(P均<0.05)。BALL-1细胞组、CCRF-CEM细胞TGF mRNA表达水平差异均无统计学意义(P均>0.05),而两种细胞均无PI3K mRNA表达。结论耐D-(-)-MTX细胞增殖能力大于L-(-)-MTX细胞;耐L-(-)-MTX、D-(-)-MTX细胞EGFR、K-Ras mRNA表达存在差异。Objective To estabhsh methotrexate （MTX） enantiomers-resistant human leukemia BALL-1 and CCRF-CEM cell lines, observe their biological characters and analyze the expressions of relevant genes of epidermal growth factor receptor （EGFR） signal pathway in different enantiomers-resistant cell lines. Methods The different MTX enantiomer-resistant cell lines were established by using the method of increasing concentrations combined with continuous low-dose induction. The cell growth curve was plotted by cell counting, the cell cycle distribution was analyzed by flow cytometry assay, and the mRNA expressions of KRAS, TGF and PI3K genes were detected by RT-PCR. Results The proliferation rates of L-（ ＋ ）-MTX and D-（ - ）-MTX resistant cell lines were significantly slower than that of parental ceils since the fourth day, and the proliferation rate of D-（ ＋ ）-MTX resistant cell line was faster than that of L-（ - ） -MTX resistant cell lines. The cell cycle distribution showed that the S phase of resistant cell lines （ L-type and D-type） re- duced （P 〈 0.05 ） while G0/G, phase increased compared with parental cell lines （BALL-1 and CCRF-CEM） （P 〈 0.05）. The def- ferences of mRNA of EGFR and K-Ras between L-（ ＋ ）-MTX/CCRF-CEM and D-（ - ）-MTX/ CCRF-CEM showed statistical signifi- cance （P 〈 0.05）, while No significant differences of TGF mRNA in BALL-1 cell and CCRF-CEM cell group were observed （ P 〉 0.05）. No expression of mRNA of PI3K gene was detectable. Conclusion The proliferation rate of D-（ - ）-MTX resistant cell line was higher than that of L-（ ＋ ） -MTX resistant cell line. The mRNA expressions of EGFR and K-Ras presented significant differences between L-（ ＋ ）-MTX and D-（ - ）-MTX resistant cell lines.

关 键 词：白血病 氨甲喋呤 对映体 聚合酶链反应 

分 类 号：R4[医药卫生—临床医学]