应用AFLP法检测小鼠黑色素瘤B16基因组的变异

Detection of genomic variation of melanoma cell strain B16 with amplified fragment length polymorphism in C57 mice

目的 应用AFLP技术检测小鼠黑色素瘤B1 6的遗传改变。方法 肿瘤及正常组织基因组DNA经限制性内切酶酶切 ,连接特异性接头 ,采用 2 0条与接头相识别的引物进行扩增 ,扩增产物经含溴化乙锭的 1 .5%琼脂糖凝胶电泳 ,紫外透视仪分析、照相。结果 2 0条引物均扩增出清晰条带 ,2 0条引物扩增结果显示B1 6瘤细胞株与移植瘤基因组间无差异 ,1 7条引物扩增结果显示肿瘤与正常组织间无差异 ,3条引物扩增结果显示肿瘤与正常组织间有差异 ,表现为扩增带的丢失或增加。结论 黑色素瘤的发生可能与基因组不稳定性有关。

Objective To detect the genetic variant of melanoma cell B16 in C57 mice with amplified fragment length polymorphism (AFLP). Methods Melanoma B16 cells were inoculated into C57 mice after being cultured for 3 to 4 passages. Genomic DNA from melanoma samples and corresponding normal tissue were digested with restriction endonuclease, ligated with specific adapter, and amplified with 20 primers. PCR products were electrophoretically separated in agarose gel and banding profiles were visualized with ethidium bromide staining. Results Clear banding profiles were obtained through the 20 primers and the amplified products showed no difference between those from B16 cell line and from implanted tumor. The amplified products produced by 17 primers were monomorphic, while those by the other 3 primers revealed changes when the products from B16 melanoma AFLP were compared with those from normal tissue DNA. The alterations in tumor DNA included the loss of a normal band and appearance of a new band. Conclusion The occurrence of melanoma might be associated with genetic genomic instability.