目的:探讨不同细胞生长因子和小分子组分对小鼠乳腺上皮类器官生长可能存在的影响,构建TP53(tumor protein 53)缺失型(knockout,KO)小鼠乳腺上皮类器官模型以研究疾病的发生.方法:配置含有不同生长因子成分的类器官培养基(B1～B12),测试它们对原代乳腺上皮细胞形成类器官影响的差异.使用光学显微镜观察和记录乳腺上皮类器官的数目和大小,用CCK-8试验检测乳腺上皮类器官的增殖能力.基于TP53条件性基因敲除转基因小鼠构建TP53缺失型乳腺上皮类器官,分析TP53野生型(wild-type,WT)和TP53 KO乳腺上皮类器官生长情况的差异.结果:头蛋白(noggin)介导的Wnt/β-连环蛋白(β-catenin)信号通路和骨形态发生蛋白质(bone morphogenetic protein,BMP)信号通路是乳腺上皮类器官形成的必要条件.完全培养基(B11)中形成的类器官数目优于其他培养基(B1～B6),并能长期稳定地维持乳腺上皮异质性特征.B12培养基中的小鼠乳腺类器官大多分化为基底型乳腺上皮细胞,这可能与Wnt-3a的添加有关.B11培养基培养下的TP53 KO乳腺上皮类器官较TP53 WT乳腺上皮类器官增殖速度更快,类器官的直径更大.结论:B11培养基能用于构建稳定、可持续传代的体外乳腺上皮类器官模型;Wnt-3a可能是基底型乳腺上皮类器官形成的关键因子.
Establishment of a model of TP53 knockout mouse mammary epithelial organoids
Objective:To delve into the impact of various growth factors and small molecule components on the growth of mouse mammary epithelial organoids,and to establish a tumor protein 53(TP53)knockout(KO)mouse mammary epithelial organoid model for disease research.Methods:The effect of various culture media(B1-B12),each containing distinct growth factors and small molecule components,on the growth of organoids derived from primary mammary epithelial cells were prepared.The number and size of mammary epithelial organoids were observed and documented through optical microscopy,while their proliferative capacity was assessed using the CCK-8 assay.Subsequently,a TP53 KO mammary epithelial organoid model was established using TP53 conditional KO mice.The difference in the growth and phenotypic characteristics of TP53 wild-type(WT)and TP53 KO organoids were recorded and compared.Results:Noggin-mediated Wnt/β-catenin signaling pathway and bone morphogenetic protein(BMP)signaling pathway were identified crucial for the formation of mammary epithelial organoids.Compared with other media(B1-B6),the complete medium B11 demonstrated better capability in facilitating organoid formation and maintaining the heterogeneity of mammary epithelial cells consistently.In contrast,mammary gland organoids cultured in B12 medium predominantly differentiated into basal-type mammary epithelial cells,possibly due to the addition of Wnt-3a.TP53 KO organoids cultured in B11 medium exhibited a faster growth rate and larger size compared to TP53 WT organoids.Conclusion:The B11 medium can reliably establish an in vitro organoid model of mammary epithelium with the capability of serial passage.Wnt-3a appears to play a crucial role in the formation of basal-type mammary epithelial organoids.
万方数据
维普
