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目的:细胞凋亡的调节紊乱与肿瘤的发生密切相关,已发现维甲类化合物全反式维甲酸ATRA在一些实体瘤细胞系中具有诱导凋亡作用。但在大肠癌中无报道,我们就此作一研究。材料和方法:结肠癌细胞株Lo-Vo在1640培养液中常规培养,取对数生长期细胞进行实验,ATPA溶解在无水乙醇中,实验和对照组乙醇终浓度均为0.05%。①MTT法反映活细胞数量,用MTT法测细胞生长抑制率;②流式细胞仪(FCM)分析细胞周期。FCM测定采用单染色单激光法。标本为10~(-5)molATRA作用后2、4、6d和传2、4、6代的LoVo细胞株;③IXl0~(-5)mol、IXl0~(-6)molATRA作用6d后在电镜下观察凋亡细胞;④用DNA断端标记(TDT)法测定凋亡细胞:凋亡细胞DNA断裂,末端被FITC标记,在荧光显微镜下计算凋亡细胞;⑤核酸电泳:按照Wessenlborg方法进行。结果:①ATRA浓度在10~(-5)~10~(-7)mol对LoVo细胞均有生长抑制作用(P<0．01),是有效药物浓度,生长抑制显示时间剂量依赖性;②流式细胞仪分析细胞周期,结果ATRA能改变LoVo细胞的细胞周期,使G_0/G_1期比例增高,S期下降,细胞被阻滞于G_1:期。10(-5)mol组第6天及传代2、4、6代后G_0/G_1期细胞比例下降,S期逐渐上升并出现亚“G_0/G_1”凋亡峰;③透射电镜下见特征性凋亡细胞,其特点是核膜完整染色质固缩,形成新月形,而对照组
OBJECTIVE: Apoptosis regulation is closely related to tumorigenesis. Retinoid ATRA has been shown to induce apoptosis in some solid tumor cell lines. However, there is no report on colorectal cancer. We have made a study on this. Materials and Methods: Colon cancer cell line Lo-Vo was routinely cultured in 1640 culture medium. Logarithmic growth phase cells were used for experiments. ATPA was dissolved in anhydrous ethanol. The final concentration of ethanol was 0.05% in the experimental and control groups. 1MTT method reflects the number of living cells, MTT method was used to measure the cell growth inhibition rate; 2 The flow cytometry (FCM) was used to analyze the cell cycle. The FCM assay uses a single staining single laser method. The specimens were treated with 10~(-5)mol ATRA for 2, 4 and 6 days, and passaged for 2, 4 and 6 generations of LoVo cell line; 3IXlO~(-5)mol, IXl0~(6)molATRA for 6 days after electron microscopy. Observe the apoptotic cells; 4 Apoptotic cells were determined by DNA fragmentation (TDT) method: Apoptotic cells were broken by DNA, the ends were labeled with FITC, and apoptotic cells were counted under a fluorescence microscope; 5 Nucleic Acid Electrophoresis: Performed according to the Wessenlborg method. RESULTS: 1ATRA concentration in the range of 10~(-5)~10~(-7)mol had inhibitory effect on LoVo cells (P<0.01). It was an effective drug concentration, and growth inhibition showed time-and dose-dependent; Cytometry analysis of the cell cycle revealed that ATRA could alter the cell cycle of LoVo cells, increasing the ratio of G_0/G1 phase, decreasing S phase, and arresting cells at the G_1 phase. In the 10(-5)mol group on the 6th day and on the 2nd, 4th, and 6th passages, the proportion of cells in the G_0/G_1 phase decreased, S phase gradually increased and sub-“G_0/G_1” apoptotic peaks appeared; 3 the characteristics were observed under the transmission electron microscope. Apoptotic cells characterized by nuclear chromatin condensation and crescent formation, whereas the control group