摘要
目的探讨OPCML在体外、体内对卵巢癌细胞的影响。方法将OPCML用重组慢病毒转导入人卵巢癌细胞A2780、OCC1和正常小鼠卵巢上皮细胞CD1。通过细胞增殖试验、细胞聚合力试验、细胞周期的分析和体内成瘤试验等来研究OPCML在卵巢癌细胞中的功能。结果(1)OPCML能被重组慢病毒高效地转导入靶细胞,转导效率几乎达100%,同时达到稳定的表达,Westernblot能检测到OPCML(60kDa)和GFP(27kDa)基因蛋白在靶细胞中的表达;(2)在A2780细胞系,转导入OPCML后的细胞(A2780-OPCML)的增殖明显的比未转基因(A2780)或仅转空载体(A2780-pWPI)者慢(P<0.01),但在OCC1和CD1细胞系,OPCML对细胞的增殖无明显的影响(P>0.05);(3)用流式细胞仪法对细胞周期分析显示,OPCML对A2780的细胞周期有明显的滞留作用(P<0.05),但对OCC1、CD1细胞则无明显滞留作用;(4)细胞聚合力试验提示OPCML能明显增强细胞的粘附力;(5)表达OPCML的A2780细胞在裸鼠皮下仅有一个(1/4)有肿瘤生长,体积显著小于A2780及A2780-pWPI组(4/4)(P<0.001),免疫组织化学染色法能够检测到OPCML蛋白在肿瘤组织中的表达。结论慢病毒载体作为转基因的工具,具有高效转导率和稳定表达的特点;OPCML能够增加细胞的粘附能力,抑制A2780的增殖和体内成瘤,提示OPCML可能是一个新的抑癌基因。
Objective To study the effects of the OPCML in ovarian cancer cell lines. Methods The OPCML was transferred to the human ovarian cancer cell lines A2780 and OCC1 in addition to normal CD1 mouse ovarian surface epithelial cells by recombinant lentiviruses which carrying OPCML, the infected cells were then characterized by cell proliferation assays, cell aggregation assays, cell cycle analysis by flow cytometry（FCM） and tumorigenicity assays following injection into nude mice. Results （1） The efficiency of infection of the cell lines using the lentiviral vectors was almost 100% allowing the stable expression of OPCML in nearly all cells. Stable expression of OPCML （60kDa） and GFP（27kDa） proteins was confirmed by Western blot analysis. （2） A2780 cells expressing OPCML grew slowly compared to A2780 parental or control virus infected cells （P〈. 01）, but the expression of OPCML had no effect on the proliferation rates of OCC1 and the normal CD1 cells when compared to their respective parental or controls （P〉0. 05）. （3） Flow cytometry based cell cycle assays showed that the expression of OPCML can arrest A2780 cells （P〈0. 05） ; but not in OCC1, CD1 cells. （4） Cell aggregation assays indicated the OPCML can increase cell surface adhesion mediated. （5） A2780 cells expressing OPCML only formed a single tumor in mice and which was significantly smaller than controls（4/4）（P〈0. 001 ）. Expression of OPCML in tumor was confirmed by immunohistochemisty. Conclusion The use of lentiviral vectors allowed the efficient expression of OPCML in nearly 100% of target cells. Expression of the OPCML resuited in an increase of cell adhesion in all cell lines tested, and decreased the proliferation and tumorigenicity of the A2780 ovarian cancer cell line. It suggests that the OPCML maybe a new tumor suppressor gene.
出处
《肿瘤防治研究》
CAS
CSCD
北大核心
2007年第2期121-124,164,共5页
Cancer Research on Prevention and Treatment
基金
广西回国基金资助项目(桂科回0575020)
