DCC基因对裸鼠皮下结直肠肿瘤的生长和淋巴管生成的影响

Effects of DCC gene on growth and lymphangiogenesis of subcutaneous colorectal tumors in nude mice

目的研究DCC(Deleted in Colorectal Carcinoma)基因对裸鼠皮下结直肠肿瘤的生长和淋巴管的生成及抑制机制。方法获取基因片段,将目的基因插入到表达载体,将人结直肠癌SW1116细胞悬液注射到裸鼠皮下,使裸鼠成瘤。建模第15天,观察裸鼠成瘤情况,测量瘤大小。将裸鼠随机分为3组(15只体重、年龄相等的雄性裸鼠),第1组为生理盐水组(n=5,腹腔注射生理盐水);第2组为DCC干预治疗组(n=5,瘤体内多点注射DCC质粒,注射50μg,质粒浓度908ng/μl);第3组为5-FU治疗组(20mg/kg体重,腹腔注射,连续15d)。每周测量瘤体大小,称量瘤重,采用流式细胞仪检测各组裸鼠的细胞凋亡率;采用Western blotting检测各组中DCC蛋白水平的表达量;采用苏木精-伊红(HE)染色对淋巴管进行计数。结果建模第30天,观察裸鼠成瘤情况,测量肿瘤大小。生理盐水组、DCC干预治疗组、5-FU治疗组裸鼠的肿瘤重量分别为(0.353±0.119)g、(0.123±0.015)g、(0.147±0.041)g,差异具有统计学意义(F=8.903,P=0.016);与生理盐水组相比,DCC干预治疗组、5-FU治疗组裸鼠肿瘤重量均有所减轻,差异均具有统计学意义(P<0.05)。细胞凋亡实验结果显示,生理盐水组、DCC干预治疗组和5-FU治疗组的总凋亡率分别为23%、54%、59%,差异有统计学意义(χ~2=21.362,P=0.000)。Western blotting检测DCC蛋白的变化,结果显示重复3次,曝光900秒未检测到条带(DCC蛋白)。生理盐水组、DCC干预治疗组、5-FU治疗组的DCC蛋白表达分别为1.556±0.088、2.061±0.075和2.052±0.114,差异有统计学意义(F=28.427,P=0.001)。淋巴管计数结果显示,生理盐水组、DCC干预治疗组、5-FU治疗组的淋巴管计数情况分别是(14.67±0.626)个、(8.43±1.002)个、(7.49±0.514)个,差异有统计学意义(F=6.489,P=0.039)。结论 DCC基因能够抑制裸鼠结直肠肿瘤的生长和淋巴管的生成,且抑癌机制与其能够诱导细胞凋亡有关。

Objective To discuss whether DCC(Deleted in Colorectal Carcinoma)gene therapy can inhibit subcutaneous tumor growth and lymph vessels in nude mice or not.Methods We got the gene fragment and inserted the target gene into the expression vector.The suspension of SW1116 cells of human colorectal cancer was injected subcutaneously to form tumors in nude mice.On the 15 th day of modeling,tumor formation in nude mice was observed and tumor size was measured.The nude mice were randomly divided into three groups(the weight and age of 15 nude mice were similar.select 15 two-digit random number table and sort them from small to large.The first five numbers are the first group,the middle five numbers are the second group,and the last five numbers are the third group).and the following intervention experiments were conducted on nude mice.Group 1:model group(n=5,intraperitoneal injection of normal saline).Group 2:the model+DCC intervention treatment group(n=5,multipoint DCC plasmid injection in the tumor,injection of 50 plus carmine g,plasmid concentration of 908 ng/displacement l).Group 3:5-Fu positive control group(20 mg/kg body weight,intraperitoneal injection,15 days in a row).The tumor size was measured weekly and the tumor weight was weighed.Apoptosis rate of each group of nude mice was measured by flow cytometry.The expression level of DCC protein in each group was detected by Western blotting.Hematoxylin-Eosin(HE)staining was used to count the lymphatic vessels.Results The tumor formation in nude mice was observed and the tumor size was measured on the thirtieth day of model construction.The tumor weights of nude mice in saline group,DCC intervention treatment group and the 5-FU treatment group were(0.353±0.119)g,(0.123±0.015)g,and(0.147±0.041)g respectively,and the difference was statistically significant(F=8.903,P=0.016).Compared with the saline group,the tumor weights of nude mice in the DCC intervention group and the 5-FU treatment group were reduced,and the differences were statistically significant(P<0.05).The results of the cell apoptosis experiment showed that the total apoptosis rates in the saline group,DCC intervention group and 5-FU treatment group were 23%,54%and 59%respectively,and the difference was statistically significant(χ2=21.362,P=0.000).The change of DCC protein was detected by Western blotting,and the result showed that the strip(DCC protein)was not detected after repeated 3 times and exposure for 900 seconds.The expressions of DCC protein in saline group,DCC intervention group and 5-FU treatment group were 1.556±0.088,2.061±0.075 and 2.052±0.114 respectively,and the difference was statistically significant(F=28.427,P=0.001).The results of lymphatic counting showed that the majority of lymphatic vessels were in saline group,DCC group and 5-FU group were 8.43±1.002,7.49±0.514 and 14.67±0.626 respectively,and the difference was statistically significant(F=6.489,P=0.039).Conclusion DCC gene can inhibit the growth of colorectal tumors and lymph angiogenesis.The cancer inhibition mechanism of DCC gene is related to its ability to induce cell apoptosis.