摘要
目的 研究转染了逆转录病毒载体G1CEACDNa致SW 480肠癌细胞的死亡机制。方法 脂质体法转染G 1CEACDNa至SW 480细胞。行RT PCR间接验证CD基因的表达情况。细胞计数测量生长曲线 ;用含 1mmol/L 5 -氟胞嘧啶 ( 5 FC)的 164 0培养基培养细胞 ,MTT法测生长抑制率和旁观者效应 ;透射电镜观察细胞的超微结构 ;流式细胞仪annexinV和PI双重染色行细胞凋亡检查。结果 RT PCR产物电泳显示在 1.5kb处可见相应的显影条带。MTT比色实验示 1mmol/L 5 FC作用下CD+SW 480细胞在 48h开始生长抑制 ,72 ,96,12 0h抑制率分别约为 3 0 .0 % ,5 0 .0 % ,80 .0 %。电镜下可见凋亡的早、中、晚期细胞及凋亡小体 ,同时可见部分细胞呈坏死改变。流式细胞仪显示 48h开始有少量细胞出现凋亡 ,72 ,96h细胞群凋亡和坏死的比率分别为 2 0 .2 % ,3 0 .7%和 19.6% ,2 1.1%。结论 转染G 1CEACDNa的SW 480细胞给予 5 FC处理后 ,其细胞死亡机制为坏死和凋亡并存 ,细胞凋亡过程的诱导可能是该治疗过程中的旁观者效应。
Objective To study the mechanism of SW480 cell line death caused by transfection of retroviral vector-G1CEACDNa.Methods Plasmid G1CEACDNa was transferred into the SW480 cell line using liposomes method. RT-PCR was performed to examin the expression of CD gene indrectly.The cell growth curve was measured by means of cell counting. When the CD+SW480 cells were exposed to 5-FC (1mmol/L), the growth inhibition rate and the 'bystander effect' were detected by MTT method.The ultrastructure was observed by electron microscope.Apoptosis was verified by flow cytometer .Results The product of RT-PCR showed a band at 1.5kb on the photo of electrophoresis. The growth of CD+ SW480 cells was inhibited 24h after administrating 5-FC,and the inhibition rates at 72h,96h,120h were 30.0%,50.0% and 80.0%,respectively.Apoptosis cells in different phases and apoptotic bodies in the field of electron microscope were observed. Meanwhile ,a few cells showed necrosis.Flow cytometer verified that a few cells appearred apoptosis 48h after exposed to 5-FC (1mmol/L), the apoptosis rate and the necrosis rate at 72h,96h were 20.2%,30.7% and 19.6%,21.1% respectively.Conclusions The death mechanism of SW480 cells transfected with G1CEACDNa followed by 5-FC treatment includes both necrosis and apoptosis.Apoptosis is possibly the bystander effect.
出处
《中国普通外科杂志》
CAS
CSCD
2003年第11期831-835,共5页
China Journal of General Surgery
