Q61R和V112A突变的HRAS基因在NIH小鼠体内诱发肿瘤的实验研究

doi:10.3969/j.issn.1004-616x.2019.01.001

癌变·畸变·突变 ›› 2019, Vol. 31 ›› Issue (1): 1-8.doi: 10.3969/j.issn.1004-616x.2019.01.001

• 论著 • 下一篇

Q61R和V112A突变的HRAS基因在NIH小鼠体内诱发肿瘤的实验研究

张峰¹, 赵龙², 樊金萍¹, 吴雪伶¹, 孟淑芳¹

1. 中国食品药品检定研究院, 北京 100050;
2. 包头市肿瘤医院, 内蒙古包头 014030

收稿日期:2017-11-27 修回日期:2018-11-18 出版日期:2019-01-31 发布日期:2019-01-31
通讯作者: 孟淑芳,E-mail:mengsf@263.net E-mail:mengsf@263.net
作者简介:张峰,E-mail:zhangfeng5122@163.com;赵龙,E-mail:Zhaolo123@163.com。
基金资助:
国家863计划（2012AA020902）

Induction of tumor in NIH mice by the plasmid containing the V112A and Q61R mutated HRAS

ZHANG Feng¹, ZHAO Long², FAN Jinping¹, WU Xueling¹, MENG Shufang¹

1. National Institutes for Food and Drug Control, Beijing 100050;
2. Baotou Cancer Hospital, Baotou 014030, Inner Mongolia, China

Received:2017-11-27 Revised:2018-11-18 Online:2019-01-31 Published:2019-01-31

摘要/Abstract

摘要： 目的：分析含有HRAS（V112A）基因及其G12C、G12C、Q61R和G12C/G12C/Q61R突变体的质粒DNA在NIH小鼠体内的致癌性。方法：从人结肠癌DiFi细胞中扩增含有V112A突变的HRAS（V112A）基因，采用PCR点突变法在HRAS（V112A）中分别引入G12C[HRAS（V112A/G12C）]、G12C[HRAS（V112A/G12C）]、Q61R[HRAS（V112A/Q61R）]或3位点联合突变[HRAS（V112A/G12C/G12C/Q61R）]，将HRAS（V112A）及上述突变基因克隆入真核表达载体pCDNA3.1（+），Western blot确证HRAS（V112A）及各突变体可体外表达后，每种质粒按每只100 μg剂量皮内注射6~8周龄雌性NIH小鼠，阴性对照组注射PBS，每组8只小鼠，持续观察小鼠致瘤情况。注射后4个月，取小鼠瘤样组织分别采用PCR和Western blot方法检测病变组织中的HRAS基因及其表达产物。结果：从DiFi细胞中成功扩增出含有V112A突变的HRAS（V112A）基因，并构建4个突变体，体外转染结果表明各HRAS均可在L929细胞中表达。重组质粒注射小鼠后，HRAS（V112A）组有4只小鼠注射后4个月于注射部位出现增生样改变，该病变在1个月后自愈；HRAS（V112A/Q61R）组有1只小鼠于注射后4个月出现明显的瘤样增生。至观察期末，HRAS（V112A/G12C）、HRAS（V112A/G12C）和HRAS（V112A/G12C/G12C/Q61R）组小鼠均未出现可见增生样结节。病变组织的PCR和Western blot检测结果表明HRAS（V112A）组小鼠增生样组织和HRAS（V112A/Q61R）组小鼠肿瘤组织中均有对应的HRAS基因存在和表达。结论：HRAS（V112A）可在NIH小鼠体内导致注射部位皮肤的增生样改变，含有Q61R突变的HRAS（V112A）可在NIH小鼠体内诱导肿瘤形成，含有G12C、G12C及G12C/G12C/Q61R突变的HRAS（V112A）在小鼠体内不会引起明显病变。

关键词: HRAS, 点突变, 致瘤性, 小鼠成瘤实验

Abstract: OBJECTIVE:To evaluate oncogenicity of HRAS (V112A) and its G12C, G12C,Q61R,G12C/G12/Q61R mutants in NIH mice. METHODS:HRAS (V112A) containing the V112A mutation was amplified from the DiFi human colon cancer cells. G12C[HRAS(V112A/G12C)],G12C[HRAS(V112A/G12C)],Q61R[HRAS(V112A/Q61R)] and 3 sites joint[HRAS(V112A/G12C/G12C/Q61R)] mutants were generated with site directed mutation PCR. After HRAS (V112A) and the above 4 mutants were inserted into pCDNA3.1(+) and the in vitro expression was verified with Western blot,each plasmind was injected into 8 6-8 weeks aged female NIH mice intracutaneously with a dosage of 100 μg per mouse to observe the expression of tumorgenicity. Pathological tissues was assayed with PCR and Western blot was used to detect the presence of the HRAS gene and protein. RESULTS:HRAS(V112A) and the 4 designed mutants were amplified successfully. After transfection of L929,HRAS expression was detected. Four months after the injection,4 mice from the HRAS (V112A) injected group developed hyperplastic lesions and 1 mouse from the HRAS(V112A/Q61R) group developed tumor. No lesions were observed in the HRAS (V112A/G12C),HRAS(V112A/G12C) and HRAS(V112A/G12C/G12C/Q61R) groups. PCR and Western blot results show the existence and expression of the corresponding HRAS genes. CONCLUSION:HRAS containing the V112A mutation could induce hyperplastic lesions in mice,while HRAS containing V112A and Q61R mutations could induce tumors in NIH mice. The HRAS(V112A) containing G12C,G12C and G12C/G12C/Q61R did not induce observable lesions.

Key words: HRAS, point mutation, oncogenicity, mouse tumor formation experiment

中图分类号:

R73-35+4

张峰, 赵龙, 樊金萍, 吴雪伶, 孟淑芳. Q61R和V112A突变的HRAS基因在NIH小鼠体内诱发肿瘤的实验研究[J]. 癌变·畸变·突变, 2019, 31(1): 1-8.

ZHANG Feng, ZHAO Long, FAN Jinping, WU Xueling, MENG Shufang. Induction of tumor in NIH mice by the plasmid containing the V112A and Q61R mutated HRAS[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2019, 31(1): 1-8.

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Q61R和V112A突变的HRAS基因在NIH小鼠体内诱发肿瘤的实验研究

Induction of tumor in NIH mice by the plasmid containing the V112A and Q61R mutated HRAS

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