慢病毒介导shRNA干扰ADAM10基因对多发性骨髓瘤MM.1S细胞增殖作用的研究  

Effect of Interfering the ADAM10 by Lentivirul Vector-Mediated shRNA on Multiple Myeloma MM.1S Cell Proliferation

作　　者：许利凡[1] 罗建萍[1] 李护君[1] 陆倩[1] 张倩楠[1] 姚瑶[1] 姚若斯 徐开林[1] 李振宇[1] 

机构地区：[1]徐州医学院附属医院血液科,江苏徐州221002

出　　处：《中国实验血液学杂志》2017年第4期1074-1079,共6页Journal of Experimental Hematology

基　　金：国家自然科学基金资助项目(81570183)

摘　　要：目的:探讨干扰ADAM10基因对多发性骨髓瘤MM.1S细胞增殖与凋亡的影响及其作用机制。方法:设计4对针对ADAM10 mRNA不同位点的shRNA编码序列,分别连接到慢病毒载体质粒p LVshRNA-EGFP(2A)Puro中去,构建sh/ADAM10-1,sh/ADAM10-2,sh/ADAM10-3,sh/ADAM10-4和sh/Con;将重组质粒与慢病毒包装质粒及包膜质粒共转染293FT细胞,收获病毒颗粒,浓缩后测定病毒滴度,感染MM.1S细胞,流式细胞术分选GFP+细胞。利用实时定量PCR及Westem blot检测慢病毒载体介导的shRNA干扰ADAM10基因的作用。选取ADAM10下调最显著的细胞株,CCK-8法检测干扰ADAM10后细胞的增殖,Annexin V/7-AAD双染流式细胞术检测细胞存活与凋亡,定量PCR检测促凋亡基因BAD、BAK、BIK抑凋亡基因BCL-2、c-Myc以及Notch1及其下游靶基因Hes-1的转录变化。结果:成功构建了特异性干扰ADAM10表达的慢病毒载体,干扰ADAM10基因对MM.1S细胞的增殖具有抑制作用,并能诱导MM.1S细胞的凋亡,且促凋亡基因BAD、BAK表达升高,抑凋亡基因BCL-2、c-Myc表达降低。Q-PCR结果显示,干扰ADAM10后Notch1水平升高,Hes-1水平降低。结论:干扰ADAM10基因可显著抑制MM.1S细胞的增殖,促进其凋亡,其机制与Notch1信号通路有关。Objective： To explore the effect of interfering ADAM10 on proliferation and apoptosis of multiple myeloma MM. 1S cells,and its possible mechanism. Methods： Four pairs of shRNA-coding sequences directed against different sites of ADAM10 mRNA were designed and inserted into lentiviral vector plasimd pLVshRNA-EGFP（ 2A） Puro for constructing the sh/ADAM10-1,sh/ADAM10-2,sh/ADAM10-3,sh/ADAM10-4 and sh/Con. These plasmids and lentiviral packaging plasmids were co-transfected into the packaging cells 293 FT,then the virus particles were collected and the viral titer was assayed after concentration,and these viral particles were transfected to MM. 1S cells. The flow cytometry was used to sort GFP ＋ cells. Real-time quantitative PCR,and Western blot were used to detect the effect of interfering the ADAM10 gene by lentiviral vector mediated shRNA. The proliferation-inhibition curve was plotted by CCK-8 method,the cell viability and apoptosis were detected by flow cytometry with Annexin V and 7-AAD staining,the transcripts of pro-apoptosis gene BAD,BAK,BIK,anti-apoptotic genes BCL-2,c-Myc and Notch1 target gene Hes-1 were detected by real-time PCR. Results： Lentivirus vector was successfully constructed,that could specifically interfere ADAM10 expression. Interfering ADAM10 gene could inhibit the MM. 1S cell proliferation and induce apoptosis. After the interferencing ADAM10 gene the mRNA levels of pro-apoptosis gene BAD,BAK and BIK were increased,and the mRNA levels of anti-apoptotic genes BCL-2 and c-Myc were reduced. Q-PCR results showed that the mRNA level of Notch1 were increased,but that of Hes-1 were reduced. Conclusion： Down-regulated ADAM10 expression can significantly inhibit multiple myeloma MM. 1S cell proliferation and promote the apotosis. Its mechanism may be related to Notch1 signaling pathways.

关 键 词：ADAM10基因 Notch1信号通路 慢病毒载体 多发性骨髓瘤 MM.1S细胞 

分 类 号：R733.3[医药卫生—肿瘤]