组成活化型Rac1 GTP酶通过上调促血小板血成素受体的表达促进白血病细胞的静息  

Constitutively active Rac1 GTPase confers leukemia cells quiescence through upregulating myeloproliferative leukemia

作　　者：李欢[1] 王继英[1] 于沛[1] 陈树英[1] 邢海燕[1] 田征[1] 唐克晶[1] 王敏[1] 饶青[1] 

机构地区：[1]中国医学科学院北京协和医学院血液学研究所血液病医院实验血液学国家重点实验室,天津300020

出　　处：《中国肿瘤生物治疗杂志》2017年第8期828-832,共5页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金资助项目(No.81370599)~~

摘　　要：目的:研究Rac1 GTP酶的活化对白血病细胞静息的影响,并探索其机制。方法:构建Rac1组成活化型慢病毒载体,以此感染急性髓系白血病KG-1a细胞,比较感染了组成活化型Rac1的KG-1a(Rac1-V12-KG-1a)细胞和感染空载体的KG-1a(pCDH-KG-1a)细胞在G0期的比例差异。以Rac1特异性抑制剂NSC23766处理感染后KG-1a细胞,4 d后检测两组细胞G0期比例的变化。实时定量PCR检测两组细胞中与细胞周期和细胞静息相关分子的表达。流式细胞术检测小鼠Rac1GTP酶高度活化的AMLI-ETO9a白血病细胞模型中促血小板生成素受体(myeloproliferative leukemia MPL)-和MPL+细胞群在G0期的比例。结果:感染了组成活化型Rac1的Rac1-V12-KG-1a细胞的G0期细胞比例显著高于感染空载体的p CDH-KG-1a细胞的比例[(15.30±0.60)%vs(11.50±0.17)%,P<0.05];抑制剂处理KG-1a细胞后,随着抑制剂浓度的增加G0期细胞比例显著下降(P<0.05)。实时定量PCR检测结果显示,周期抑制因子P21、P27、P57的mRNA表达水平在Rac1-V12-KG-1a细胞表达均有不同程度的上调,静息相关调节分子N-Cadherin和MPL m RNA表达水平均显著升高(P<0.05);流式检测结果显示,MPL+白血病细胞群的比例在G0期显著高于MPL-组[(40.3±3.5)%vs(19.05±7.65)%,P<0.05]。结论:白血病细胞中Rac1 GTP酶的活化通过上调MPL等细胞外在调节因子的表达增加休眠期细胞的比例,从而促进白血病细胞维持静息状态。Objective： To determine the role of Rac1 GTPase activation in the regulation of leukemia cell quiescence and investigate the possible mechanism. Methods： Constitutively active Rac1 lenti-virus vector was constructed to transfect KG-1a leukemia cells（Rac1-V12-KG-1a）. The G0 phase cell ratio in sorted Rac1-V12-KG-1a and KG-1a cells transfected with empty vector（p CDH-KG-1a） was compared. Furthermore, after 4 days treatment with NSC23766, the Rac1-specific inhibitor, the G0 phase cell ratio in KG-1a cells was also detected. The expression levels of cell quiescence and cell cycle associated moleculars were then determined by RT-PCR. The G0 phase cell ratio of MPL-and MPL＋leukemia cells in AML1-ETO9 a leukemia mouse was detected by flow cytometry. Results：The results showed that the G0 phase cell ratio in Rac1-V12-KG-1a cells was significantly higher than that in p CDH-KG-1a cells（[15.30±0.60]% vs [11.50 ± 0.17]%, P〈0.05）. After the treatment of Rac1-specific inhibitor, the G0 phase cell ratio of the KG-1a cells was decreased significantly（P〈0.05）, and the effect was concentration dependent. RT-PCR showed that m RNA transcription levels of cell cycle related factors（P21,P27,P57） were up-regulated in Rac1-V12-KG-1a cells at different level; moreover, the expressions of N-Cadherin and MPL were also significantly higher in Rac1-V12-KG-1a cells（P〈0.05）. Flow cytometry analysis showed that the percentage of G0 phase MPL＋leukemia cells was significantly higher than that of MPL-leukemia cells（[40.3±3.5]）% vs [19.05±7.65]%, P〈0.05）. Conclusion： Activation of Rac1 GTPase could increase the ratio of leukemia cells at G0 phase, and promote leukemia cells quiescence by up-regulating MPL.

关 键 词：RAC1 GTP酶 白血病细胞 静息 促血小板生成素受体 微环境 

分 类 号：R733.7[医药卫生—肿瘤] R730.2[医药卫生—临床医学]