利用Cre-loxP系统研究细胞间mRNA的传递  

Intercellular mRNA transfer investigated by a Cre-loxP system

作　　者：王梦雪 韩小松 倪超 王小宁 WANG Mengxue;HAN Xiaosong;NI Chao;WANG Xiaoning(Institute of Psychiatry and Neurology,Xinxiang Medical University,Xinxiang 453000;State Key Laboratory of Genetic Engineering,School of Life Sciences,Fudan University,Shanghai 200438;Municipal Key Laboratory of Geriatric,Chinese PLA General Hospital,Beijing 100853,China)

机构地区：[1]新乡医学院精神神经医学研究院,河南新乡453000 [2]复旦大学生命科学学院遗传工程国家重点实验室,上海200438 [3]中国人民解放军总医院老年医学研究所,北京100853

出　　处：《细胞与分子免疫学杂志》2020年第9期769-775,共7页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(31770975);中国博士后科学基金(2018M640335)。

摘　　要：目的利用Cre-loxP重组酶系统研究mRNA在细胞间传递过程及意义。方法构建和筛选稳定表达Cre重组酶的供体细胞及稳定表达loxP-DsRed-loxP-eGFP的受体细胞。将供、受体细胞共培养或在Transwell^TM中共培养,设置单独受体细胞组为对照,72 h后观察统计各组受体细胞eGFP阳性率,利用细胞免疫荧光细胞染色检测eGFP阳性细胞中Cre表达情况,并探讨供体细胞死亡、细胞膜纳米管、Rho相关激酶(ROCK)信号通路对Cre mRNA传递的影响。结果将供、受体细胞共培养72 h后发现,共培养组eGFP为阳性,单独受体细胞组、Transwell^TM共培养组eGFP均为阴性,表明细胞接触可以介导Cre mRNA传递,免疫荧光染色结果显示Cre蛋白可在受体细胞中翻译和入核。此外,Cre mRNA的传递主要通过活细胞,而膜纳米管及ROCK信号通路抑制剂可部分阻滞该过程。结论mRNA可以通过细胞间接触在活细胞中传递,进入受体细胞后还可翻译成蛋白质发挥生物学功能。Objective To study the process and significance of intercellular mRNA transfer by using a Cre-loxP system.Methods Cre-expressing donor cells and loxP-DsRed-loxP-eGFP-expressing recipient cells were established.Co-culture and Transwell^TM co-culture of both donor and recipient cells were analyzed with a separate culture of recipient cells only as controls.After 72 hours,the eGFP+recipient cells in each group were observed and analyzed.Immunofluorescence staining was used to detect Cre expression in eGFP+cells.The effects of donor cell death,cell membrane nanotubes,and ROCK signaling pathway on Cre mRNA transfer were explored.Results After co-culture for 72 hours,the eGFP+cells were only observed in co-culture group,but not in the recipient cells alone group and the Transwell^TM co-culture group,indicating that cell-cell contact could mediate Cre mRNA transfer.Immunofluorescence staining showed that Cre mRNA could be translated into proteins and transported to the nucleus in eGFP+recipient cells.Moreover,the transport of Cre mRNA between living cells could be partially blocked by membrane nanotubes or ROCK signaling pathway inhibitors.Conclusion The mRNA can be exchanged among living cells through intercellular contact,thus leading to the changes of biological functions in the recipient cells.

关 键 词：细胞接触 mRNA传递 Cre-loxP重组酶系统 膜纳米管 

分 类 号：Q813.11[生物学—生物工程] R392.12[医药卫生—免疫学]