缝隙连接通讯抑制剂对Cx43cDNA转染后化疗旁观者效应的影响

The Effect of Cx43cDNA Transfection on the Bystander Effect of Glioma Chemotherapy

背景与目的:有研究通过转染Cx43cDNA观察到C6细胞化疗过程中存在旁观者效应,并推测缝隙连接细胞间通讯可能为化疗旁观者效应机制之一,本研究旨在探讨脑胶质瘤化疗旁观者效应与缝隙连接细胞间通讯功能的关系。方法:(1)质粒扩增,提取纯化及酶切鉴定:氯化钙法将质粒转入JM109菌株扩增,提取纯化后酶切鉴定;(2)基因转染:LipofectAMINE2000介导C6细胞质粒DNA转染,G418筛选,半定量RT-PCR及划痕荷载染料传输实验(SLDT)鉴定。(3)将转染后细胞分为实验组和对照组:将VM-26处理细胞与VM-26未处理细胞按不同比例在无VM-26环境混合培养,培养液中加入缝隙连接细胞间通讯功能抑制剂18α-次甘草酸(AGA)为实验组,不加AGA为对照组,以MTT法及流式细胞仪技术观察AGA对Cx43cDNA转染后化疗旁观者效应的影响。结果:(1)获得转染了相应质粒的细胞株C6-Cx43及C6-Non;(2)C6-Cx43细胞Cx43mRNA表达显著增加,染料传输能力较C6-Non明显增强;(3)C6胶质细胞缝隙连接细胞间通讯功能低下;(4)当转染Cx43cDNA,明显上调C6细胞缝隙连接细胞间通讯功能后,化疗药物的细胞毒性作用得到明显放大;AGA可显著抑制化疗旁观者效应。结论:(1)单纯C6细胞化疗旁观者效应不明显;(2)转染Cx43cDNA上调缝隙连接细胞间通讯功能后,C6细胞化疗表现出显著的旁观者效应;缝隙连接细胞间通讯功能抑制剂AGA能显著抑制化疗旁观者效应,表明化疗旁观者效应与缝隙连接细胞间通讯功能有关。

BACKGROUND & OBJECTIVE: To investigate the relationship between the gap junctional intercellular communication(GJIC) and the bystander effect of glioma chemotherapy. METHODS:(1)The plasmids were transformed into JM109 bacterium through calcium chlorid method, purified and identified with endonuclease. (2)Gene transfection:C6 cells were transfected with plasmid DNA through LipofectAMINE2000.The transfected cells were obtained by using G418.Then transfected cells were identified by RT-PCR and scrape loading d...