干细胞因子逆转录病毒表达载体的构建及体外性质研究

目的通过逆转录病毒介导两种类型人干细胞因子在NIH3T3细胞中稳定表达，并研究它们对白血病细胞的作用。方法用DNA重组技术构建并鉴定可溶型及膜结合型干细胞因子的重组逆转录病毒表达载体MSCV—PGK—GFP—sSCF、MSCV—PGK—GFP—mSCF，与空载体对照MSCV—PGK—GFP分别转染Phoenix细胞包装病毒，并感染NIH3T3细胞，流式分选术获得3种阳性细胞，CCK8法分别检测与其共培养的K562细胞的增殖情况。结果成功构建了sSCF、mSCF逆转录病毒表达载体；经Phoenix包装的重组及对照逆转录病毒成功感染NIH3T3细胞，获得了稳定表达细胞株NIH3T3-S、NIH3T3-M和对照细胞株NIH3T3-V。共培养中NIH3T3-S、NIH3T3-M均可促进K562细胞的增殖，且在低血清条件下，NIH3T3-M的作用高于NIH3T3-S。结论可溶性及膜结合SCF分别通过旁分泌和并置性作用促进白血病细胞的增殖。

Construction of Retroviral Expression Vector Containing Human Stem Cell Factor and Study on Its Property in Vitro

Objective To express human stem cell factor （SCF） in NIH3T3 cells by retrovirus-mediated method and study SCF effects on the proliferation of leukemia cells. Methods The retroviral vectors MSCV-PGK-GFP-sSCF and MSCV-PGK-GFP-mSCF were constructed by DNA recom- binant techniques. Recombinant and empty vectors were packed in Phoenix cells to produce retroviral particules. NIH3T3 cells were infected by the viral supernatants. After sorted by flow cytometry, three positive cell lines were obtained. The proliferation of K562 cells in co-cuhure system was detected by means of CCK8 methods. Results The recombinant retroviral vectors expressing sSCF and mSCF were successfully constructed. High titer recombinant retroviruses were obtained in packaging Phoenix cells. NIH3T3 cells were successfully infected by retroviruses. Stable expressing cell lines, NIH3T3-S and NIH3T3-M, as well as control cell line NIH3T3-V, were established. Both NIH3T3-S and NIH3T3-M in co-culture systems displayed growth promotion effects on K562 cells. Furthermore, under low serum culture conditions, NIH3T3-M showed higher effects than NIH3T3- S. Conclusions Soluble and membrane form SCF could promote the proliferation of leukemia cells through paracrine or juxtacrine mechanisms.