突变的人DHFR肿瘤耐药基因转导脐血干细胞的体外实验研究

In vitro study on the transduction of mutant human dihydrofolate reductase cDNA into human umbilical cord blood CD 34 + cells

目的探讨突变的人二氢叶酸还原酶（ｍＤＨＦＲ）耐药基因在人造血干细胞中抗甲氨蝶呤（ＭＴＸ）的效应。方法应用免疫磁珠分选系统（ＭＡＣＳ）分离纯化脐血ＣＤ３４＋细胞后，用含ｍＤＨＦＲ的逆转录病毒上清转染脐血ＣＤ３４＋细胞，采用造血干细胞集落形成实验进行转导后细胞抗ＭＴＸ分析。结果应用ＭＡＣＳ能高度纯化人ＣＤ３４＋细胞，使分选后的脐血ＣＤ３４＋细胞的纯度平均达９０％，回收率为７１．１％。在ＭＴＸ浓度为２０ｎｍｏｌ／Ｌ的条件下培养１４天，转导ｍＤＨＦＲ耐药基因的脐血干细胞集落形成数明显高于对照组（Ｐ＜０．０１），同时对ＭＴＸ的抗性提高了约２倍。结论突变的人ＤＨＦＲ耐药基因能提高人的造血干细胞对化疗药物ＭＴＸ的抗性。

Objective To explore whether human umbilical cord blood hematopoietic progenitor cells transduced with mutant dihydrofolate reductase(mDHFR) gene increase resistance to metrotrexate(MTX). Methods Cord blood CD 34 + cells were enriched with a high gradient magnetic cell sorting system(MACS), and then transfected with supernatant of retrovirus containing human mDHFR cDNA. Hematopoietic progenitor clonogenic assay was used to evaluate mDHFR conferred resistance to MTX. Results The purity of cord blood CD 34 + cells was approximately 90% and recovery rate was 71.1%.The transduced and mock transduced CD 34 + cells were cultured in mediums containing 20nmol/L MTX for 14 days. The percentages of progenitor colonies of transduced and mock transduced CD 34 + cells were higher than that of non transduced (P<0.01), obviously. The resistance of the former to MTX is nearly twofold of the latter. Conclusion Transduction of mDHFR gene could confer the resistance of human hematopoietic progenitor cells to MTX toxicity.