应用Lv-pNanog-GFP慢病毒载体探讨Nanog在调控结直肠癌细胞的自我更新与转移能力中的作用  

Role of Nanog on self-renewal and metastasis in colorectal cancer cells by Lv-pNanog-GFP lentiviral vector

作　　者：朱传琳[1] 姚超[2] 钱程[2] 徐先林[1] 欧刚卫[1] 

机构地区：[1]遵义医学院生物化学与分子生物学教研室,贵州遵义310018 [2]第三军医大学西南医院全军病理学研究所,重庆400038

出　　处：《第三军医大学学报》2014年第1期29-32,共4页Journal of Third Military Medical University

基　　金：国家重点基础研究发展计划("973"计划;2004CB518804)~~

摘　　要：目的探讨干细胞标记物Nanog对结直肠癌细胞的自我更新以及转移能力的影响。方法将本实验室已构建的Nanog启动子携带绿色荧光蛋白的慢病毒载体Lv-pNanog-GFP感染人结直肠癌细胞系HCT116,并经流式分选得到GFP表达阳性和阴性的细胞,通过Western blot检测病毒的有效性,克隆形成实验和成球实验检测肿瘤细胞的自我更新能力,Transwell实验检测肿瘤细胞的转移能力。结果细胞系在感染病毒12 h后,倒置荧光显微镜下可观察到GFP的荧光表达,说明病毒Lv-pNanog-GFP可稳定感染人结直肠癌细胞HCT116,且GFP的阳性率为5.9%。Western blot结果显示,根据GFP所分选出的细胞能准确的对应Nanog的表达量。Nanog阳性细胞的克隆形成率[(56.3±2.87)%]显著大于Nanog阴性细胞的克隆形成率[(19±2.16)%](P<0.05)。Nanog阳性细胞所形成的干细胞球体积明显增大且成球率[(34±4)%]显著大于Nanog阴性细胞[(10.67±6.43)%](P<0.05),说明Nanog阳性细胞具有更强的自我更新能力。Nanog阳性细胞穿过Transwell小室的细胞数(175.8±12.89)比Nanog阴性细胞(74.6±16.55)显著增多(P<0.05),说明Nanog阳性细胞具有更强的转移能力。结论在结直肠癌细胞中Nanog调控肿瘤细胞的自我更新以及转移能力。Objective To determine the role of the stem cell marker,Nanog,in the self-renewal and metastasis of colorectal cancer cells. Methods The constructed Nanog lentiviral vector Lv-pNanog-GFP that carrying green fluorescent protein（ GFP） was used to infect human colorectal cancer cell line HCT116,and the cells with positive and negative expression of GFP were sorted by flow cytometry. Western blotting was employed to detect the effectiveness of the virus infection. Clone formation and sphere formation were applied to determine self-renewal capacity. Metastatic ability of tumor cells were detected by Transwell assay. Results After 12 h of the cells infected with the virus,inverted fluorescence microscopy indicated that the expression of GFP fluorescence,suggesting that the virus Lv-pNanog-GFP stably infected HCT116 cells,with a GFP positive rate of 5. 9%. Western blotting results showed that GFP sorting the cells expressed exactly the amount of corresponding Nanog. Clone formation rate of Nanog positive cells [（ 56. 3 ± 2. 87） %]was significantly higher than that of Nanog negative cells [（ 19 ± 2. 16） %,P〈0. 05]. Sphere volume of Nanog positive cells was increased significantly and the sphere formation rate [（ 34 ± 4） %]was significantly greater than Nanog negative cells [（ 10. 67 ± 6. 43） %,P〈0. 05]. We demonstrated that Nanog positive cells exhibited enhanced ability of self-renewal. The number of Nanog positive cells through the Transwell cells（ 175. 8 ± 12. 89） was significantly larger than that of Nanog negative cells（ 74. 6 ± 16. 55,P〈0. 05）. Nanog positive cells exhibited enhanced ability of metastasis. Conclusion Nanog regulates self-renewal and metastasis in colorectal cancer cells.

关 键 词：结直肠癌 NANOG 肿瘤干细胞 

分 类 号：R394.3[医药卫生—医学遗传学] R73-37[医药卫生—基础医学]