结直肠癌缺失基因对卵巢癌细胞系SKOV-3体内外增殖的影响。

Influence of deleted in colorectal carcinoma gene on proliferation of ovarian cancer cell line SKOV-3 in vivo and in vitro.

作者信息

Cai Yan, Hu Chun-jie, Wang Jia, Wang Ze-hua

机构信息

Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.

出版信息

Chin Med Sci J. 2011 Sep;26(3):175-81. doi: 10.1016/s1001-9294(11)60045-x.

DOI:10.1016/s1001-9294(11)60045-x

PMID:22207928

Abstract

OBJECTIVE

To elucidate the effects of the deleted in colorectal carcinoma (DCC) gene on proliferation of ovarian cancer cell line SKOV-3.

METHOD

An exogenous recombinant eukaryotic expression vector pcDNA3.1(+)-DCC, containing human DCC cDNA coding sequences, was constructed and transfected into SKOV-3 cells (SKOV-3/DCC). The pcDNA3.1 (+) transfected cells (SKOV-3/Neo) and SKOV-3 cells were used as the positive and negative controls, respectively. Expressions of DCC mRNA and protein were analyzed by RT-PCR and immunocytochemical analysis, respectively. Cell growth was detected by soft agar colony formation assay and MTT assay. Flow cytometry and transmission electron microscopy were used to assess the effects of DCC on cell cycle distribution and ultrastructure, respectively. BALB/c mice were used to evaluate the effects of DCC on tumorigenicity in vivo.

RESULTS

RT-PCR and immunocytochemical analysis revealed the exogenous DCC gene was successfully transfected into SKOV-3 cell lines and obtained permanent expression. The half maximal inhibitory concentration (IC50) of SKOV-3/DCC cells was significantly lower than that of SKOV-3 or SKOV-3/Neo cells (all P<0.05). DCC expression caused SKOV-3 cells to be arrested in G1 phase (78.0%), and electron microscopic analysis showed SKOV-3/DCC cells displayed typical morphological changes of apoptosis. Two mice xenografted with SKOV-3/DCC cells showed no tumor tumorigenecity. The tumor volume of BALB/c mice bearing SKOV-3/DCC cells (3.403 mm(3)) was smaller than that of SKOV-3 cells (9.206 mm(3)).

CONCLUSION

DCC gene may play an important role in suppressing the growth of SKOV-3 cell line and inducing apoptosis.

摘要

目的

阐明结直肠癌缺失基因（DCC）对卵巢癌细胞系SKOV-3增殖的影响。

方法

构建含人DCC cDNA编码序列的外源性重组真核表达载体pcDNA3.1(+)-DCC，并转染至SKOV-3细胞（SKOV-3/DCC）。分别将转染pcDNA3.1(+)的细胞（SKOV-3/Neo）和SKOV-3细胞作为阳性和阴性对照。分别通过逆转录聚合酶链反应（RT-PCR）和免疫细胞化学分析检测DCC mRNA和蛋白的表达。采用软琼脂集落形成试验和MTT试验检测细胞生长情况。分别用流式细胞术和透射电子显微镜评估DCC对细胞周期分布和超微结构的影响。采用BALB/c小鼠评估DCC对体内致瘤性的影响。

结果

RT-PCR和免疫细胞化学分析显示外源性DCC基因成功转染至SKOV-3细胞系并获得稳定表达。SKOV-3/DCC细胞的半数最大抑制浓度（IC50）显著低于SKOV-3或SKOV-3/Neo细胞（均P<0.05）。DCC表达导致SKOV-3细胞停滞于G1期（78.0%），电子显微镜分析显示SKOV-3/DCC细胞呈现典型的凋亡形态学改变。两只接种SKOV-3/DCC细胞的小鼠未出现肿瘤致瘤性。接种SKOV-3/DCC细胞的BALB/c小鼠肿瘤体积（3.403 mm³）小于接种SKOV-3细胞的小鼠（9.206 mm³）。