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

发布时间：2018-05-13 23:21

本文选题：ADAM基因 + Notch信号通路　；参考：《中国实验血液学杂志》2017年04期

【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。
[Abstract]:Aim: to investigate the effect and mechanism of interfering ADAM10 gene on proliferation and apoptosis of multiple myeloma MM.1S cells. Methods: four pairs of shRNA coding sequences aimed at different sites of ADAM10 mRNA were designed and ligated into lentivirus vector p LVshRNA-EGFP(2A)Puro, respectively, to construct sh/ ADAM10-1 / ADAM10-2 / ADAM10-3shr / ADAM10-4 and shrCon.Recombinant plasmids were co-transfected with lentivirus packaging plasmids and encapsulated plasmids into 293FT cells to harvest viral particles. After concentration, virus titers were detected, MM.1S cells were infected, and GFP cells were sorted by flow cytometry. The effect of shRNA mediated by lentivirus vector on ADAM10 gene was detected by real time quantitative PCR and Westem blot. ADAM10 down-regulated cell line (CCK-8) was selected to detect cell proliferation after interfering with ADAM10 by Annexin V/7-AAD double staining flow cytometry to detect cell survival and apoptosis. Quantitative PCR was used to detect the transcription changes of BCL-2BIK, BCL-2c-Myc, Notch1 and its downstream target gene Hes-1. Results: the lentivirus vector which specifically interfered with the expression of ADAM10 was successfully constructed. Interfering ADAM10 gene could inhibit the proliferation of MM.1S cells and induce the apoptosis of MM.1S cells. The results of Q-PCR showed that the level of Notch1 increased after interfering with ADAM10 and decreased the level of Hes-1. Conclusion: interfering with ADAM10 gene can significantly inhibit the proliferation and promote the apoptosis of MM.1S cells, and its mechanism is related to the Notch1 signaling pathway.
【作者单位】：徐州医学院附属医院血液科;
【基金】：国家自然科学基金资助项目(81570183)
【分类号】：R733.3

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