【摘要】 目的:观察Galectin-3表达与肿瘤细胞增殖和凋亡的关系,探讨利用RNA干扰技术作为肿瘤基因治疗的方法。方法:实验于2005-06/2006-12在南方医院消化研究所完成。从PubMed的序列号NM-002306中按照引物设计软件设计并化学合成4条Galectin-3小干扰片断,从中选出有效的干扰片断,并顺势转染大肠癌细胞系LOVO(为Galectin-3siRNA组),用未转染细胞做为正常对照,以转染阴性干扰片断作为空白对照,免疫印记法验证干扰结果,MTT方法观察干扰后24,48和72h各组细胞增殖吸光度(A值),流式细胞仪检测各组细胞凋亡情况。结果:①用顺势转染的方法成功转染了Gal-3siRNA片断,转染后72h收集细胞并提取细胞浆蛋白,免疫印记法验证结果显示Galectin-3siRNA组灰度值(55.790)与正常对照(97.234)及空白对照的灰度值(90.459)相比,galectin-3表达明显减弱(P<0.05)。②细胞增殖情况:干扰后24,48和72hGalectin-3siRNA组细胞继续生长,但细胞生长减慢,A值低于正常对照和空白对照(24h:1.018±0.002,1.478±0.185,1.169±0.005;48h:2.049±0.008,2.635±0.003,2.532±0.009;72h:2.512±0.506,3.213±0.006,3.060±0.002;P均=0.000)。③细胞凋亡率:各组中凋亡中晚期均多于早期,Galectin-3siRNA组高于正常对照和空白对照[早期:(15.900±2.508)%,(3.813±1.305)%,(5.780±1.56)%;中晚期:(19.750±0.780)%,(5.050±0.120)%,(7.667±0.145)%;P均=0.000]。结论:成功转染并抑制了galectin-3在大肠癌细胞系lovo中的表达,干扰后细胞增殖减慢,凋亡增加。更多还原

【Abstract】 AIM:To observe the relationship of expression of Galectin-3 with the proliferation and apoptosis of tumor cells and explore the RNA interference technique for anticancer gene therapy.METHODS:The experiment was performed at the Institute of Gastroenterology,Nanfang Hospital from June 2005 to December 2006.Four galectin-3 small interfering RNA(siRNA)was constructed by chemistry synthesis according to primer design software(accession No.NM-002306 in PubMed).Effective siRNA was collected,and then transfected into colorectal cancer LOVO(as Galectin-3siRNA group).Non-transfected cells were used as normal control,and negative RNA as blank control.Western-blot was used to verify the interference result.Cell proliferation(absorbance value)was observed by MTT after interference for 24,48 and 72 hours.Cell apoptosis was measured with flow cytometer.RESULTS:①Gal-3siRNA was successfully transfected by direct transfer.After 72 hours,the cytoplasm protein was extracted after collecting cells.Western-blot showed that the expression of Galectin-3 was markedly decreased in Galectin-3siRNA group in gray value(55.790)than normal control(97.234)and blank control group(90.459)(P < 0.05).②Cell proliferation:Cells proliferated still in the hGalectin-3siRNA group after 24,48 and 72 hours,but very slow,and the A value was lower than the normal control group and blank control group(24 hours:1.018±0.002,1.478±0.185,1.169±0.005;48 hours:2.049±0.008,2.635±0.003,2.532±0.009;72 hours:2.512±0.506,3.213±0.006,3.060±0.002;P all =0.000).③Cell apoptosis rate:The apoptosis mostly appeared in the middle and advanced stages.It was higher in the Galectin-3siRNA group than the normal control group and blank control group [early stage:(15.900±2.508)%,(3.813±1.305)%,(5.780±1.56)%;middle and advanced stages:(19.750±0.780)%,(5.050±0.120)%,(7.667±0.145)%;P all =0.000].CONCLUSION:The galectin-3siRNA is successfully transfected in lovo and the expression is significantly decreased.The interference of galectin-3siRNA inhibits the proliferation of the tumor cells,but promotes the apoptosis of tumor cells.更多还原