细胞间隧道纳米管介导的线粒体转运诱导急性单核细胞白血病细胞抵抗凋亡的研究  

Intercellular tunneling nanotube-mediated mitochondrial transport induces apoptosis resistance in acute monocytic leukemia cells

作　　者：徐前飞 刘亚蒙[1] 周震[2] 田志祥 王丽红[1] 胡艳[1] 李静[1] 刘晓颖 葛健[1] Xu Qianfei;Liu Yameng;Zhou Zhen(Dept of Hematology, The First Affiliated Hospital of Anhui Medical University, Hefei 230022;Health Management Center, The Affiliated Hospital of Qingdao University, Qingdao 266000)

机构地区：[1]安徽医科大学第一附属医院血液内科,合肥230022 [2]青岛大学附属医院健康管理中心,青岛266000 [3]安徽医科大学生命科学学院生物学教研室,合肥230032

出　　处：《安徽医科大学学报》2019年第9期1377-1382,共6页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81200371);高等学校博士学科点专项科研基金联合资助课题(编号:20123420120011);安徽省自然科学基金(编号:1208085QH154、1708085MH224);安徽省教育厅高校优秀拔尖人才培育资助项目(编号:gxyqZD2017029)

摘　　要：目的观察人类急性单核细胞白血病(AML-M5)细胞系THP-1与骨髓间充质干细胞(BM-MSCs)之间隧道纳米管(TNTs)结构的形成;探讨TNTs中线粒体转运对THP-1细胞凋亡的影响及线粒体在TNTs中转运的分子机制。方法使用特异性荧光染料分别对线粒体、细胞骨架蛋白F-Actin和细胞核进行荧光标记,共聚焦显微镜观察BM-MSCs与THP-1细胞间TNTs的形成及TNTs中线粒体的传递。BM-MSCs与THP-1细胞共培养5 d后运用流式细胞术检测THP-1细胞凋亡;共培养体系分为4组:直接共培养(SC组)、Transwell透膜共培养(TC组)、直接共培养体系中加入细胞松弛素(TNTs抑制剂)Latrunculin B(LC组)、无BM-MSCs而仅有培养液(MC组);Western blot检测细胞器转运相关马达蛋白(KIF5B)表达水平。结果 BM-MSCs与THP-1细胞间可观察到TNTs形成,BM-MSCs中线粒体通过TNTs单向传递到THP-1细胞,并未观察到线粒体逆向传递。Annexin V-FITC/PI检测THP-1细胞凋亡结果显示,SC组跟LC组凋亡率较MC组明显减低( P <0.01),LC组高于SC组( P <0.01),TC组较SC组和LC组明显升高( P <0.01),TC组与MC组凋亡率无明显差异( P >0.05)。BM-MSCs中 KIF5B表达水平明显高于THP-1细胞( P <0.001),Rotenone处理BM-MSCs后显著抑制KIF5B的表达( P <0.01)。结论 BM-MSCs线粒体通过TNTs向THP-1细胞单向传递,THP-1细胞摄取BM-MSCs线粒体后可能诱导凋亡抵抗;细胞间线粒体转运可能与马达蛋白KIF5B相关。Objective To observe the formation of tunneling nanotubes(TNTs) between human acute monocytic leukemia(AML-M5) cell line THP-1 and bone marrow mesenchymal stem cells(BM-MSCs);and investigate the effect of mitochondrial transport on apoptosis of THP-1 cells in TNTs, then to explore its molecular mechanism. Methods Specific fluorescent dyes were used to label mitochondria, cytoskeleton protein F-Actin and nucleus respectively. The formation of TNTs between BM-MSCs and THP-1 cells and the transmission of mitochondria in TNTs were observed by confocal microscope. After 5 days of co-culture of BM-MSCs and THP-1 cells, apoptosis of THP-1 cells was detected by flow cytometry. The co-culture system was divided into four groups: Direct co-culture(SC), Transwell transmembrane co-culture(TC), direct co-culture system with cytochalasin(TNTs inhibitor) Latrunculin B(LC), no BM-MSCs but only culture medium(MC);The expression of organelle transporter associated motor protein(KIF5B) was detected by Western blot. Results TNTs formation was observed between BM-MSCs and THP-1 cells, mitochondria in BM-MSCs cells were unidirectionally transmitted to THP-1 cells via TNTs, but no mitochondrial reverse transmission was observed. Annexin V-FITC/PI cell apoptosis assay results showed that the apoptosis rate of THP-1 cells in SC,LC groups were significantly lower compared with MC group( P <0.01), The apoptosis rate of group LC was higher than that of group SC( P <0.01), and that of group TC was significantly higher than that of group SC and group LC( P <0.01),there was no significant difference in apoptosis rate between group TC and group MC( P >0.05). The expression level of KIF5B in BM-MSCs was higher than that in THP-1 cells( P <0.001). Rotenone significantly inhibited the expression of KIF5B in BM-MSCs( P <0.01). Conclusion Mitochondria of BM-MSCs are unidirectionally transmitted to THP-1 cells via TNTs, which may induces apoptosis resistance in THP-1 cells;intercellular mitochondrial transport may be associated with motor protein KIF

关 键 词：急性单核细胞白血病 人骨髓间充质干细胞 THP-1细胞 凋亡 

分 类 号：R733.713[医药卫生—肿瘤]