摘要
本实验利用小鼠β-珠蛋白基因探针和7种癌基因探针,对细胞的基因表达状态进行探测。含有基因片段的探针经过扩增、DNA碱抽提以及鉴定回收,然后经缺口翻译法制备高比活性的^(32)P标记的cDNA探针,通过原位杂交方法在细胞水平检测基因转录物。结果发现:1.小鼠浆细胞瘤(SP2/0)细胞始终未有小鼠β-珠蛋白基因的转录活动,在它与大鼠中幼、晚幼红细胞杂交后72小时的标本中,也未测到相应的转录产物;但自第4代杂交细胞起,直至第26代杂交细胞,均可测到信号强弱不同的小鼠β-珠蛋白基因产物。2.对SP2/0细胞进行7种癌基因的检测,发现除SiS表达较微弱外,其余6种癌基因均有较强的表达。对杂交细胞各代进行同样的原位杂交检测,可见癌基因的表达均有不同程度的减弱,其中尤以C-myc和Ki-ras癌基因受到抑制的现象最为明显。在较长时间传代(26代)之后,有些杂交细胞几乎又可测到类似SP2/0细胞的癌基因表达程度,但C-myc和Ki-ras的表达水平仍较SP2/0细胞弱。上述结果表明,多阶段肿瘤发生过程涉及多癌基因的活化和表达;而肿瘤细胞去恶性化,可能与相应癌基因表达受抑制以及分化基因的激活和持续表达有关。
The gene expression of both the mouse plasmocytoma (SP2/0) and the hybrid cells crossed with rat nucleated erythroblasts were detected by in situ hybridization technique using the probes of mouse β-globin gene and 7 oncogenes (v-Ki-ras, v-H-ras, v-sis, erb-B, v-abl, v-fos, c-myc). After plasmic amplification, DNA was isolated by alkali lysis, purified and recovered, the DNA containing gene fragments were labelled with ^(32)P to become high activity ^(32)P-cDNA probes through nick translation, and the labelled probes were used to detect the gene transcripts in cellular level. The results indicated that: (1) no mouse β-globin gene transcripts could be detected in the cytoplasm of SP2/0 cells, as well as in hybrid cells within 72 hours after cell fusion, but transcript signals could be observed in hybrid cells from 4th to 26th passages. (2) Active expression of multioncogenes in SP2/0 cells was demonstrated, all the 7 oneogenes tested, except v-sis, were expressed more strongly. On the other hand, the expression of oncogenes in hybrid cells was found to be dramatically decreased, among them, the oncogenes of c-myc and Ki-ras been suppressed completely. After long term of passages (26th subcultures), the expression of c-myc and Ki-ras was still lower than that of SP2/o ceils although in some cases other oncogenes increased in their expression levels. These results confirmed that the multistep carcinogenesis involved multi-oncogenes expression and that the decancerization of tumor cells may be due to the suppression of multi-oncogenes activity as well as to activate the expression of differentiation genes.
出处
《解剖学报》
CAS
CSCD
北大核心
1990年第2期178-183,共6页
Acta Anatomica Sinica
关键词
细胞杂交
基因表达
核酸杂交
肿瘤
In situ hybridization
Oneogenes and β-globin gene
Cell differentiation
Cellular hybridization
Rat erythroblast
Plasmocytoma SP2/0 cell
