以慢病毒构建的GFP阳性卵巢肿瘤细胞中侧群细胞的分离和动物活体内示踪

Isolation and tracing of side population from lentivirus-transduced GFP-positive ovarian cancer cells

目的通过慢病毒将绿色荧光蛋白(green fluorescent protein,GFP)基因转入卵巢癌细胞中,研究慢病毒感染对细胞泵出Hoechst 33342能力的影响,追踪GFP(+)侧群(side population,SP)细胞在裸鼠体内的荧光表达和肿瘤成像。方法选用人卵巢癌细胞株HO-8910PM,以慢病毒感染GFP基因至人卵巢癌细胞株HO-8910PM,摸索以Ubiquitin启动子构建的慢病毒颗粒对该细胞的最佳感染复数(multiplicity of infection,MOI)。以流式细胞仪分选表达GFP的卵巢癌细胞株,获得高纯度的GFP(+)HO-8910PM,检测GFP表达对Hoechst 33342染色的影响。再以流式细胞仪分选GFP(+)SP细胞,将分选的GFP(+)SP细胞注射入裸鼠皮下,观察GFP(+)SP细胞的致瘤能力,并在体内观测GFP(+)SP来源肿瘤的荧光成像。结果 Ubiquitin启动子构建的慢病毒感染不影响卵巢癌细胞拒染Hoechst 33342的能力,分选的GFP(+)SP细胞仍能在裸鼠体内形成表达GFP的肿瘤,成瘤能力和未感染细胞相比无显著变化,GFP阳性肿瘤在成像系统中可见荧光表达。结论慢病毒感染不影响卵巢癌细胞拒染Hoechst 33342的能力,慢病毒感染后分选的GFP(+)SP细胞仍能在裸鼠体内形成表达GFP的肿瘤,成瘤能力和未感染细胞相比无显著变化,GFP标记可以作为研究卵巢癌细胞在动物活体内生物学行为的示踪标记。

Objective To testify whether lentivirus transduction has influence on the capability of ovarian cancer cells to pump out Hoechst 33342 and whether GFP can be used as a marker to trace cancer cell mobility in vivo.Methods HO-8910PM cells were transduced by lentivirus at various multiplicity of infection（MOI）.Expression of transgene was clarified by FACs analysis.The best MOI under the control of Ubiquitin promoter was determined referring to GFP expression.GFP（＋）HO-8910PM were collected by FACsorting and passaged under regular culture condition.GFP（＋）SP cells were sorted after stained with Hoechst 33342 and injected subcutaneously into nude mice.The local tumorigenecity of GFP（＋）SP cells at injection site was compared to that of non-transduced cells.The fluorescence images of tumor-bearing mice were taken under a small animal fluorescence imaging system. Results Lentiviral transduction under the control of Ubiquitin promoter had no effect on the capability of HO-8910PM cell to pump Hoechst 33342 out.GFP（＋）SP cells from HO-8910PM could form GFP（＋） tumor in nude mice.There was no difference in tumorigenecity between GFP（＋）SP cells and non-transduced SP cells.Fluorescence in tumor cells at subcutaneous injection site could be detected under a fluorescence imaging system. Conclusions Lentiviral transduction has no affect on the capability of ovarian cancer SP cells-derived tumor formation.Lentiviral transduction can be a useful tool in the study of tumorigenesis and development of ovarian cancer.GFP can be a marker to trace cancer cells in vivo.