慢病毒载体介导SPROUTY2基因过表达对多发性骨髓瘤RPMI8226细胞增殖作用的影响

The effect of lentiviral vector mediated SPROUTY2 over-expression on proliferation of multiple myeloma RPMI8226 cells

目的 探讨慢病毒表达载体介导的人SPROUTY2基因过表达对多发性骨髓瘤RPMI8226细胞增殖、克隆性生长和细胞外调节蛋白激酶（ERK）抑制剂敏感性等生物学特性的影响.方法 克隆人SPROUTY2基因,构建SPROUTY2基因与绿色荧光蛋白（GFP）的重组慢病毒表达载体LV-S-GFP及对照载体LV-GFP.脂质体法包装病毒;优化慢病毒感染RPMI8226细胞的条件;荧光显微镜观察GFP表达;反转录聚合酶链反应（PCR）、蛋白质印迹法（Western blot）方法分别检测目的基因及其蛋白的表达,CCK-8法检测RPMI8226细胞增殖及其对ERK 1 /2抑制剂AS703026敏感性的影响,软琼脂克隆形成实验检测细胞的克隆性生长能力.结果 成功地构建了共表达SPROUTY2基因和报告GFP的高滴度慢病毒颗粒.LV-S-GFP实验组RPMI8226细胞中SPROUTY2的表达明显高于LV-GFP对照组,灰度值分别为（230.85±32.12）％和（140.35±36.62）％（P＜ 0.05）.过表达SPROUTY2基因对骨髓瘤细胞增殖具有抑制作用,并可以增强ERK1/2抑制剂AS703026对RPMI8226细胞的杀伤作用.过表达SPROUTY2基因可明显抑制RPMI8226细胞的克隆性生长.结论 慢病毒载体系统可高效介导SPROUTY2基因在RPMI8226细胞中的表达,抑制骨髓瘤细胞增殖及克隆性生长,并能够提高其对ERK抑制剂的敏感性.

Objective To establish a myeloma cell line with SPROUTY2 over-expression using lentiviral vector,and preliminary evaluate its proliferation,clone growth and ERK inhibitors sensitivity and other biological effect.Methods The human SPROUTY2 gene was cloned and the recombinant lentiviral vector including SPROUTY2 and green fluorescent protein （GFP） name as LV-S-GFP and the control vector as LV-GFP were constructed.Both plasmids were co-transfected into 293FT packaging cell line with packaging plasmid pSPAX2 and enveloping pMD.2G using Lipofectamine 2000 to produce lentiviral virus,respectively.The recombinant virus was harvested and the virus titer was determined by limiting dilution.Cells with high level of SPROUTY2 were selected through optimizing the condition of lentiviral infection.The florescence microscope was used to observe the express level of GFP.The level of SPROUTY2 was detected by reverse transcription polymerase chain reaction （RT-PCR） and Western blot.CCK-8 assay was used to detect the effect of SPROUTY2 on cell proliferation and the sensitivity of RPMI8226 cells to ERK1/2 inhibitors AS703026.The colony grow ability of the gene-transfected cell was measured by soft agar colony formation assay.Results The SPROUTY2 fragment was amplified by RT-PCR and verified by DNA sequencing.The restriction enzyme digestion experiment demonstrated that the recombinant lentiviral vector LV-S-GFP and the control vector LV-GFP were successfully constructed.The result of RT-PCR and Western blot showed that the level of SPROUTY2 significantly increased in LV-S-GFP group cells compared with that in the control cells （（230.85± 32.12） % vs （140.35±36.62） %） （P 〈 0.05）.The result of CCK-8 assay showed that SPROUTY2 inhibited cell proliferation in RPMI8226 cells,and enhanced the inhibitory effect of AS703026 on RPMI8226 cells.The result of colony formation assay demonstrated that SPROUTY2 could inhibit the grow ability as single clone of RPMI8226 cells.Conclusion The lentiviral vector with SPROUTY2 was successfully constructed.The lentiviral vector can efficiently transferred SPROUTY2 into RPMI8226 cells and SPROUTY2 inhibited myeloma cell proliferation and clone formation,improved the sensitivity of ERK inhibitors.