摘要
目的采用一段人工合成的互补于多药耐药基因（ｍｄｒ１基因）的反义硫代磷酸寡核苷酸（ＡＳＳＯＤＮ，反义核酸）转染白血病多药耐药细胞株Ｋ５６２／ＡＤＭ（耐阿霉素的Ｋ５６２细胞株），来逆转白血病细胞的多药耐药。方法四唑蓝快速比色法（ＭＴＴ法）检测Ｋ５６２／ＡＤＭ细胞对阿霉素的半数抑制量（ＩＣ５０），流式细胞仪分析细胞内的柔红霉素含量，免疫细胞化学染色方法确定Ｐ１７０的表达水平，逆转录聚合酶链反应（ＲＴＰＣＲ）检测ｍｄｒ１ｍＲＮＡ的相对水平（ｍｄｒ１与β２微球蛋白之比，ｍｄｒ１／β２ＭＧ）。结果浓度为２μｍｏｌ／Ｌ的反义核酸使Ｋ５６２／ＡＤＭ细胞对阿霉素的ＩＣ５０从处理前的２５７２ｍｇ／Ｌ降至处理后的６９７ｍｇ／Ｌ，相对逆转效率为７３０１％。反义核酸亦使Ｋ５６２／ＡＤＭ细胞内的柔红霉素含量增加。Ｐ１７０表达阳性率从处理前的（９８２６±１３５）％降至处理后的（１８３±６５）％，ｍｄｒ１／β２ＭＧ亦由处理前的２０７±０１６降至１５７±０１４，ｍｄｒ１ｍＲＮＡ相对水平有所下降。结论反义核酸通过下调ｍｄｒ１基因，阻断Ｐ１７０的表达，从而降低Ｋ５６２／ＡＤＭ的耐药性。
Objective To
investigate the effectiveness of 14 mer antisense phosphorothiaoate oligonucleotide (ASSODN)
complementary to the published multidrug resistance gene (mdr1 gene) sequence to reverse
multidrug resistance of leukemia cell line. Methods 50% inhibitory concentration (IC50) of K562
cell line resistant to adriamycin (K562/ADM cell line) was determined by the tetrazolium dye,
3(4,5dimethylthiazol2yl)2,5diphenyltetrazolium bromide (MTT) method, the concentration of
daunorubicin (DNR) in the cell by flow cytometry, the level of P170 by immunohistochemical
staining technique and the level of mdr1mRNA by halfquantitative RTPCR (the ratio of
mdr1/2MG). Results Treatment of K562/ADM cell line with 2 mol/L ASSODN reduced the level of
both the mdr1mRNA and P170. The ratio of mdr1/2MG reduced from (2.070.16) to (1.570.14), and
positive rate of P170 expression decreased from (98.261.35)% to (18.306.50)%. Accordingly, the
concentration of DNR in cell increased. With these findings, IC50 to ADM reduced from 25.72
mg/L to 6.97 mg/L and relative effciency of modulation was 73.01%. Conclusion Antisense
oligonucleotide targeting mdr1 gene can reduce mdr1mRNA, block P170 expression so as to
increase the sensitivity of K562/ADM cell line to chemotherapy drugs.
出处
《中华内科杂志》
CAS
CSCD
北大核心
1999年第6期373-376,共4页
Chinese Journal of Internal Medicine
关键词
反义寡核苷酸类
多药抗药性
白血病
Oligonucleotides, antisenseDrug resistance, multipleLeukemia
