摘要
目的:探讨5-氟胞嘧啶(5-fluorocytosine,5-FC)热化疗对转染组织特异性胞嘧啶脱氨基酶(cytosinedeaminase,CD)基因的大肠癌细胞SW480的作用及机制.方法:脂质体法将CEA基因顺式转录调控序列(TRS)驱动CD基因的组织特异性逆转录病毒载体G1CEACDNa转导入大肠癌细胞SW480,以G418筛选阳性克隆扩增后,采用水浴加温法,43℃作用30min共3次,同时给予前药5-FC进行敏感试验;RT-PCR法检测目的基因的表达,MTT法检测细胞存活率,电镜检测细胞的形态学变化,流式细胞术检测细胞周期变化及凋亡.结果:SW480-CEACD细胞在43℃作用30min共3次条件下,CD基因能稳定表达;热疗本身对SW480细胞有一定的杀伤作用(P<0.05),转CD基因后,SW480细胞对5-FC的敏感性明显提高(P<0.01),热疗与前药5-FC合用,对SW480-CEACD的杀伤作用显著大于对未转基因细胞的杀伤作用(P<0.01,t=4.356,n=9),亦大于单独应用5-FC时对SW480-CEACD细胞的杀伤作用(P<0.05,t=2.376,n=9),增加了SW480-CEACD细胞对5-FC的敏感性,电镜及流式细胞术检测显示,细胞死亡以细胞凋亡为主,前药热化疗导致结肠癌细胞SW480产生G1期阻滞,凋亡细胞比例增加.结论:热疗与前药5-FC联合应用,导致结肠癌细胞SW480产生G1期阻滞,细胞凋亡比例增加,提高了CEA组织特异性CD/5-FC系统的靶向性杀伤作用.
AIM: To investigate the mechanism of thermochemotherapy with 5-fluorocytosine (5-FC) on human colon cancer cell line SW480 transfected carcinoembryonic antigen (CEA) tissue-specific cytosine deaminase (CD) in vitro. METHODS: Recombinant retroviral vector GlCEACDNa, in which the CD gene was controlled under the CEA promoter, was introduced through liposome technique to human colorectal carcinoma cell line SW480, and then the cells were selectively cultured in G418. The proliferated colonies were treated with the combined therapy of 5-FC and hyper thermia at a temperature of 43 ℃ for 30 min, 3 times. RT PCR was performed to detect the expression of CD gene in target cells after being heated. The cell survival rate was detected by MTT method. The ultrastructures of cells were observed by electron microscopy and apoptosis was verified by flow cytometry. RESULTS: The expression of CD genes in target cells was detected after being heated. After transfertion, SW480 CEACD cells were more sensitive than their parental cells (P<0.01, t=5.620, n =9) to 5-FC, the killing effect of hyper thermia on SW480 cells was observed (P <0.05, t =2.999, n =9). Furthermore, after being treated with thermochemotherapy of 5-FC at a temperature of 43 ℃ for 30 min, the killing effect on SW480-CEACD cells was more significant than that on SW480 cells (P<0.01, t =4.356, n =9). Treatment with the combination of 5-FC and hyperthermia displayed a higher anti-tumor effect than that with 5-FC alone on SW480-CEACD cells in vitro (P<0.05,t=2.376, n =9). Apoptotic bodies in the field of electron microscope were observed. G1 blockage was confirmed and the increased rate of apoptosis cells was verified after hyper thermia with 5-FC by flow cytometry. CONCLUSION: The combination of 5-FC and hyperther mia will result in Gl blockage of human colorectal carcinoma cell lines SW480 transfected with the CEA tissue specific CD genes, which will improve the outcome of the anti-tumor effect on that cell line.
出处
《世界华人消化杂志》
CAS
2004年第10期2307-2311,共5页
World Chinese Journal of Digestology
