"两步法"建立裸鼠肾癌活体生物发光肿瘤模型

Establishing bioluminescent nude mouse renal cancer model using a two-step method

目的:建立人肾癌细胞裸鼠活体生物发光成像肿瘤模型。方法:将可表达荧光素酶基因的pcDNA3.1-Luc2质粒转染至肾癌细胞系A498细胞中,利用G418筛选法构建能够表达荧光素酶的稳定转染细胞系,命名为"A498-Luc2"细胞,并进行体外细胞生物发光成像检测。将A498-Luc2细胞通过细胞悬液注射法建立重度联合免疫缺陷(SCID)鼠皮下荷瘤模型,并应用小动物活体成像系统观察肿瘤发光情况,SCID鼠左侧肿瘤组织的发光强度为9.758×105 p/s,右侧的发光强度为7.100×105 p/s。待皮下肿瘤生长至一定体积后,处死SCID小鼠获取肾癌组织,并通过组织块接种法接种至BALB/c裸鼠皮下,即通过两步法建立肾癌裸鼠皮下荷瘤模型。成瘤后,定期观察肿瘤生长情况,并应用小动物活体成像系统观察肿瘤组织发光情况。各组数据均满足正态性且两组方差相等,采用t检验进行组间比较,不满足则考虑非参数秩和检验。结果:通过G418筛选法成功建立稳定表达荧光素酶的稳定转染细胞系"A498-Luc2"细胞。生物发光成像结果显示,A498-Luc2细胞发光强度与细胞数目呈正相关。当细胞数量达到1.000×106个时,细胞发光强度为1.776×108 p/s。组织块接种法接种BALB/c裸鼠皮下约14 d后长出肿瘤,成瘤率为95%(P<0.01),差异有统计学意义。并随天数的增加体积逐渐增大、肿瘤发光信号逐渐增强。在第32天时,裸鼠肿瘤组织的发光强度达到最强为2.374×108 p/s,差异有统计学意义。结论:本研究构建稳定表达荧光素酶的人肾癌细胞系,并通过两步法成功建立裸鼠肾癌生物发光肿瘤模型,为后续研究奠定基础。

Objective To establish a bioluminescent nude mouse renal cancer model using a two-step method,and provide ideal experimental tools for screening anti-renal cancer drug and investigating the molecular mechanism of renal cancer.Methods The pcDNA3.1-Luc2 plasmid was transfected into renal carcinoma cell line A498.The A498 cell line with stable expression of luciferase was constructed by G418 resistance screening and named"A498-Luc2"cell line.The A498-luc2 cells were injected subcutaneously into severe combined immune-deficiency(SCID)mice to establish a subcutaneous xenotransplantation tumor model,the luminescence intensity of left tumor tissue in SCID mice was 9.758×105 p/s and that of right tumor tissue was 7.100×105 p/s.The SCID mice were killed and the tumor tissues were excised and then inoculated subcutaneously to establish the subcutaneous nude mice xenotransplantation model,and tumor tissue luminescence was observed with small animal imaging system.All data were first tested for normality,and if all groups satisfied normality and the two groups had equal variances,t-test,otherwise the non-parametric rank-sum test was considered.Results The A498 cell line with stable expression of luciferase was constructed,and bioluminescence imaging showed that the luminescence intensity of A498-Luc2 was positively correlated with the number of cells.When the number of cells reached 1.000×106,the cell luminescence intensity was 1.776×108 p/s.A bioluminescent nude mouse renal cancer model was successfully established using a two-step method,and the development of renal cancer was effectively monitored.On the 32nd day,the luminescence intensity of tumor tissue of nude mice reached the highest value of 2.374×108 p/s.Histological examination revealed that the tumor cells were of different shapes and sizes,and pathological nuclear division was observed.Conclusion In this study,the A498 cell line with stable expression of luciferase was constructed,and a subcutaneous bioluminescent xenotransplantation tumor model of nude mice was successfully established by a two-step method,laying a foundation for subsequent research.