论文部分内容阅读
目的 研究转染逆转录病毒载体 (G1CEACDNa)的肠癌细胞LoVo株的死亡机制。方法 脂质体法转染G1CEACDNa至LoVo细胞。TRIzol试剂提取总RNA ,行RT -PCR间接验证CD基因的表达情况。用含 1mmol·L-15 -FC的 164 0培养基培养细胞 ,MTT法测细胞生长曲线和旁观者效应 ;透射电镜观察细胞的超微结构 ;流式细胞仪annexinⅤ和PI双重染色行凋亡相关检查。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-15 -FC作用下CD+ LoVo细胞 48h开始生长抑制 ,72h、96h、12 0h抑制率分别约为 3 0 %、5 0 %、80 %。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 ,72h、96h细胞群凋亡和坏死的比例分别为 2 3 8%、3 5 1%和 2 0 4%、2 6 0 %。结论 转染G1CEACDNa的LoVo细胞给予 5 -FC治疗 ,其细胞死亡机制为坏死和凋亡并存 ,凋亡相关过程的诱导可能是该治疗过程中旁观者效应的重要机制。
Objective To study the mechanism of death of LoVo strain of colorectal cancer cells transfected with retroviral vector (G1CEACDNa). Methods Liposome method was used to transfect G1CEACDNa into LoVo cells. Total RNA was extracted with TRIzol reagent, and CD gene expression was indirectly verified by RT-PCR. The cells were cultured with 164 0 medium containing 1 mmol·L-15-FC. The cell growth curve and bystander effect were measured by MTT assay. The ultrastructure of the cells was observed by transmission electron microscopy. Flow cytometry annexinⅤ and PI double staining were used to detect apoptosis an examination. Results Electrophoresis of RT-PCR products showed that the corresponding development bands were visible at 15kb. MTT colorimetric assay showed that CD + LoVo cells started to grow 48h after treated with 1mmol·L-15-FC, and the inhibitory rates were 30%, 50% and 80% at 72h, 96h and 120h respectively. Under the electron microscope, early, middle and late apoptotic cells and apoptotic bodies were observed. At the same time, some of the cells showed necrotic changes. Flow cytometry showed that a small amount of cells began to show apoptosis at 48h, and the rates of apoptosis and necrosis at 72h and 96h were 23.8%, 35.1% and 20.4%, 26.0% respectively. CONCLUSION: LoVo cells transfected with G1CEACDNa are treated with 5-FC. The mechanism of cell death is necrosis and apoptosis co-exist. The induction of apoptosis-related process may be an important mechanism of bystander effect in this process.