重组腺病毒Ad5-KAI1对骨髓瘤细胞的功能调节作用

Regulation of Ad5-KAI1 on multiple myeloma cell functions

目的研究抗癌一号KAI1(又称CD82)基因对骨髓瘤细胞生物学特性的影响及机制。方法将携带KAI1基因克隆至重组腺病毒,制备Ad5-KAI1重组腺病毒,并感染骨髓瘤细胞SKO-007。Ad5-GFP作为阴性对照,未感染细胞为空白对照,利用CCK-8和AnnexinⅤ-PI等方法分别检测不同时间点(24 h,48 h)的细胞活率和凋亡水平。另外,利用W estern印迹方法检测不同组别细胞表达ERK及其磷酸化水平。结果 Ad5-KAI1组细胞活率抑制水平和凋亡水平明显高于对照组,且剪切型胱天蛋白酶(caspase)3、ERK磷酸化水平在实验组高表达,而抑制ERK磷酸化则导致凋亡进一步增加。结论 KAI1显著抑制骨髓瘤细胞增殖,并诱导其caspase依赖的凋亡;KAI1在促进凋亡的同时,其应激性诱导了ERK磷酸化,后者又对细胞具有保护作用。

Objective To investigate the function and potential mechanism of anticancer gene 1 KAI1（or CD82） on multiple myeloma cells.Methods The full-length KAI1 gene was cloned to recombinant adenovirus（Ad5） before Ad5-KAI1 was prepared.SKO-007 cells were infected with Ad5-KAI1 and Ad5-GFP（as the negative control group）.Uninfected cells were described as a control group.The cell viability and apoptosis of infected cells were detected using CCK-8 and Annexin Ⅴ-PI double staining at different times（24 h and 48 h）,respectively.Results In Ad5-KAI1 group,the inhibition of cell proliferation and levels of apoptosis were significantly higher than in Ad5-GFP and control groups.Moreover,the expression of cleared caspase 3 and ERK phosphorylation was upregulated in Ad5-KAI1 group.To block ERK phosphorylation by specific inhibitors,levels of apoptotic cells were amplified in Ad5-KAI1 group.Conclusion KAI1 inhibits SKO-007 cells proliferation and caspase-dependent apoptosis.Meanwhile,ERK phosphorylation is activated by KAI1stress,which protects cells against apoptosis by KAI1 in turn.