缝隙连接通讯抑制剂对Cx43cDNA转染后化疗旁观者效应的影响

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...

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

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 dye transfer test (SLDT). (3)Transfected cells were divided into experimental and control group : VM-26-treated and VM-26-untreated were co-cultured with different ratio mixed in non-VM-26 environment. GJIC inhibitor,AGA was added ,to culture medium of the experimental group. While there was no AGA in control group. The influence of AGA on bystander effect in Cx43cDNA-transfected cells was investigated with MTT and flow cytometry. RESULT:(1)According to the different transfected plasmids,C6-Cx43 and C6-Non,relevant to different plasmids ,were havested. (2)The level of Cx43mRNA was obviously elevated in C6-Cx43.And the dye transfer ability of C6-Cx43 was stronger than that of C6-Non. (3)C6 glioma cells have poor GJIC. (4)Amplified cytotoxicity of VM-26 detected after GJIC of C6-Cx43 was upregulated by Cx43-cDNA transfected. (5)AGA can obviously inhibit bystander effect in chemotherapy. CONCLUSIONS: (1)Weak or no bystander effect can be detected in chemotherapy on C6; (2)The bystander effect of glioma chemotherapy can be detected while GJIC being upregulated in Cx43-cDNA-transfected cells. (3)GJIC inhibitor AGA can inhibit bystander effect in chemotherapy, which indicates that bystander effect of glioma chemotherapy is related to GJIC.