野生型-p53与JunB融合基因对裸鼠肝细胞肝癌移植瘤生长抑制作用的实验研究

Suppression of wild-type p53 and JunB fusion gene to the growth of transplanted tumor of hepatocellular carcinoma in nude mice

目的探讨野生型(wt)-p53与JunB融合基因对裸鼠肝细胞肝癌(肝癌)移植瘤的抑制作用。方法分别应用携带绿色荧光蛋白(GFP)的wt-p53与JunB融合基因质粒和脂质体Lipofectmine 2000(融合基因载体)、携带GFP的空质粒和Lipofectmine 2000(空载体)及单纯Lipofectmine 2000转染HepG2肝癌细胞。应用G418筛选抗生素培养基筛选阳性克隆,应用逆转录聚合酶链反应进行阳性克隆鉴定。将18只BALB/c裸鼠按随机数字表法随机分为实验组、阴性对照组和空白对照组,每组6只,分别取融合基因载体、空载体转染的HepG2肝癌细胞及单纯的HepG2肝癌细胞接种裸鼠,将2×107个肿瘤细胞悬液皮下注射至裸鼠右侧背部,于注射后的28 d处死荷瘤裸鼠,测量肿瘤体积和重量,计算肿瘤抑制率。3组裸鼠的肿瘤体积和肿瘤重量比较采用单因素方差分析,两两比较采用LSD-t检验。结果融合基因载体和空载体转染HepG2肝癌细胞24 h后,转染效率均达到40%。G418筛选抗生素培养基筛选10 d,融合基因载体和空载体转染的HepG2肝癌细胞出现较大的单细胞阳性克隆,扩大培养后获得稳定表达融合基因的HepG2细胞。凝胶电泳显示融合基因载体转染的HepG2肝癌细胞的p53和JunB基因表达量较空载体转染的肝癌细胞增高。融合基因载体转染的HepG2肝癌细胞的p53基因相对含量为6.09,JunB为0.13;空载体转染的HepG2肝癌细胞的p53基因相对含量为0.42,JunB为0.04。3组裸鼠移植瘤位于背侧皮下,实验组裸鼠移植瘤较小,直径为数毫米,阴性对照组与空白对照组裸鼠移植瘤均明显隆起于裸鼠背部皮下,直径介于十数毫米与数十毫米之间,移植瘤均呈菜花样突出,表面光滑无包膜,与周围组织分界明显,触之质硬,与深部组织无明显粘连,腹腔各脏器表面光滑,未见有明显的转移灶。实验组与阴性对照组移植瘤的体积分别为(47±21)mm3和(384±166)mm3,两组差异有统计学意义(LSD-t=4.638,P<0.05);两者重量分别为(2.6±0.5)g和(10.1±0.6)g,差异亦有统计学意义(LSD-t=19.560,P<0.05)。实验组与空白对照组移植瘤的体积(292±103)mm3比较,差异有统计学意义(LSD-t=5.740,P<0.05),实验组与空白对照组移植瘤的重量(9.6±0.7)g比较,差异亦有统计学意义(LSD-t=27.225,P<0.05)。实验组平均肿瘤抑制率为84%。结论 wt-p53和JunB融合基因可明显抑制裸鼠肝癌移植瘤的生长。

Objective To explore the suppressive effects of wt-p53 and JunB fusion gene to transplanted tumor of hepatocellular carcinoma in nude mice. Methods Hepatocellular carcinoma HepG2 cells were transfected by wt-p53/junB fusion gene plasmid and lipidosome Lipofectmine 2000 with green fluorescent protein （GFP） （fusion gene vector）, empty plasmid and Lipofectmine 2000 with GFP （empty vector） and pure Lipofectmine 2000. The positive clone was selected by using G418 antibiotics selection culture medium and RT-PCR was performed to identify the positive clone. Afterwards, 18 BALB/c nude mice were randomly divided into experimental group, negative control group and blank control group with 6 mice in each group using computer random number table method. The nude mice in 3 groups were inoculated with HepG2 cells transfected by fusion gene vector, empty vector and pure HepG2 ceils respectively and 2×10^7 tumor cell suspension was injected subcutaneously on the right side of nude mice＇s back. The nude mice were killed 28 days after injection and the gross tumor volume, tumor weight and tumor control rate were calculated. The gross tumor volume and tumor weight were compared using one-way analysis of variance and pairwise comparison was performed using LSD-t test. Results The transfecting rate of HepG2 cells was 40% 24 hours after transfected by fusion gene vector and empty vector. After 10 days selection by using G418 antibiotics selection culture medium, HepG2 cells transfected by fusion gene vector and empty vector acquired large single cell positive clone and fusion gene stably-expressed HepG2 cells was gained after the cultivation was amplified. HepG2 cells transfected by fusion gene vector expressed higher level of p53 and JunB gene compared with the HepG2 cells transfected by empty vector through gel electrophoresis. The gene relative amount of p53 in the experimental group was 6.09 and JunB was 0.13, while in the negative control group were 0.42 and 0.04. The transplanted tumors were located subcutaneously on the backside of nude mice of 3 groups. The tumors of mice in the experimental group were smaller with a few millimeters in diameter. The tumors of mice in the negative control group and blank control group obviously uplifted on the mice back with the diameter between ten to dozens millimeters. The transplanted tumors were cauliflower-like, had smooth surface without coating and obvious interface with surrounding tissue. The tumors were hard-texture without adhesion with deep tissue. The surfaces of abdominal organs were smooth and no obvious metastasis was observed. The gross tumor volume of mice in the experimental group and negative control group were （47±21）mm3, （384±166）mm3 respectively and there was significant difference between two groups （LSD-t=4.638,P〈0.05）. And the tumor weight in two groups were（2.6±0.5）g and （10.1±0.6）g respectively and there was also significant difference （LSD-t=19.560, P〈0.05）. There was significant difference in the gross tumor volume and tumor weight between the experimental group and blank control group [（292±103）mm3, （9.6±0.7）g] （LSD-t=5.740,27.225; P〈0.05）. The tumor control rate of experimental group was 84%. Conclusion The growth of transplanted tumor of hepatoeellular carcinoma in nude mice can be obviously inhibited by fusion gene of wt-p53 and JunB.