自杀基因TK联合细胞因子FL真核表达载体的构建及其相关特性的研究

Construction and characterization of TK/FL recombinant protein

目的 探讨自杀基因TK(thymidinekinase ,胸苷激酶 )与细胞因子FL(Flt 3ligand ,Flt 3配体 )在同一细胞克隆中表达的特性 ,为肿瘤的基因治疗提供实验基础。方法 构建以IRES(内部核糖体进入位点 )相连的TK与FL的真核表达质粒 ,脂质体法转入人乳腺癌细胞株MCF 7中。MTT法检测GCV(Ganciclovir,更昔洛韦 )对MCF 7/TK FL细胞的杀伤活性 ,电镜、光镜及FACS法检测凋亡情况。ELISA法及Westernblot对该细胞克隆分泌的FL进行定量、定性分析并检测其对CD34+细胞的促增殖活性。结果 构建的pIRES TK FL真核表达质粒以脂质体法可成功转入MCF 7细胞中 ,MCF 7/TK FL细胞可被GCV有效杀伤并存在凋亡情况 ,IC50 值为 0 5 μg/ml。该细胞分泌的FL有蛋白水平的表达 ,ELISA法测定FL分泌量为每 4天 10 5个细胞中 15 7ng/ml,它具有刺激CD34+细胞增殖的活性。结论MCF 7/TK FL细胞可同时表达TK与FL基因 ,在自杀基因瘤苗的基础上 ,其分泌的细胞因子FL具有生物活性 。

Objective To determine the in vitro behavior of simultaneously expressed TK and FL in cells. Methods A recombinant vector carrying herpes simplex virus thymidine kinase gene (TK), internal ribosome entry site (IRES) and Flt 3 ligand gene (FL) was constructed, and transfected into the MCF 7 tumor cells (MCF 7/TK FL). FL in the supernatant was detected by ELISA. MTT was adopted to determine the sensitivity of MCF 7/TK FL cells to GCV (ganciclovir). The apoptosis of cells was monitored using electro microscope and flow cytometry. Results MCF 7/TK FL cells expressed both TK and FL genes simultaneously. Dose dependent cell killing by a transduction of the HSV TK gene followed by GCV treatment was observed. Apoptosis was also obvious in the MCF 7/TK FL cells GCV system. The amount of FL secreted in culture supernatant of MCF 7/TK FL cells was 15.7ng/ml from 10 5 cells during 4 days, as determined by ELISA. The supernatant was able to stimulate proliferation of CD34 + cells from human cord blood. Conclusion MCF 7/TK FL cells may be used as a experimental vaccine for tumor therapy, they are also valuable in producing large quantity of FL.