类固醇激素分泌功能的肾上腺三维培养体系的建立

Establishment of three-dimensional culture system of adrenal gland with steroid hormone secretion function

目的:探索类固醇分泌功能的肾上腺皮质三维培养体系。方法:选取2019—2021年于广西医科大学第二附属医院的流产胚胎肾上腺组织,消化成细胞悬液,基质胶包裹三维培养。培养基中添加FGF2生长因子,Wnt信号通路激活因子(CHIR99021、R-spondin1),通过病理组织染色、实时荧光定量逆转录聚合酶链反应(RT-qPCR)、免疫荧光和透射电镜等方法检测相关基因及蛋白表达水平,并分析其对于类固醇分泌功能培养体系建成的影响。结果:(1)添加FGF2生长因子后,3D培养球体直径明显增加。(2)添加CHIR99021、R-spondin1后,球状体具有显著外层致密内层疏松的肾上腺特殊结构,更有利3D培养肾上腺立体结构的形成;(3)RT-qPCR显示3D培养形成的球状类器官结构表达SF-1、CYP17A1、CYP11A1、CYP11B1、HSD3B2类固醇合成关键酶表达,免疫荧光染色证实了CYP17A1蛋白在类器官球状结构的表达。(4)球体结构的类器官表达黑皮质素2受体(MC2R)受体,在受促肾上腺皮质激素(ACTH)刺激下,皮质醇分泌增加。结论:添加FGF2、Wnt信号通路激活因子CHIR99021、R-spondin1,可建立一种具有类固醇激素分泌功能且受促肾上腺皮质激素调控的肾上腺皮质三维培养体系。

Objective:To investigate the preparation method and system of adrenal cortex three-dimensional culture with steroid hormone secretion function.Methods:The human fetal adrenal tissues were obtained from aborted embryos at the Second Affiliated Hospital of Guangxi Medical University from 2019 to 2021.They were digested into cell suspension and wrapped with matrigel for three-dimensional culture.FGF2 growth factor,and Wnt signaling pathway activator(CHIR99021,R-spondin1) were added to the culture medium.The expression levels of related genes and proteins were detected by pathological tissue staining,fluorescence real-time quantitative reverse transcription polymerase chain reaction(RT-qPCR),immunofluorescence,and transmission electron microscopy to analyze the influence of different conditioned medium on the establishment of culture system with steroid secretion function.Results:(1) Adding FGF2 growth factor significantly increased the sphere diameter in 3 D culture compared with that of the group without adding FGF2 growth factor.(2)After adding Wnt signaling pathway activators CHIR99021 and R-spondin1,the globules had the special structure of dense outer layer and porous inner layer,which was more favorable for the formation of 3 D culture adrenal stereo structure;(3) RT-qPCR indicated that the spherical organoid structure formed by 3 D culture expressed SF-1,CYP17 A1,CYP11 A1,CYP11 B1,and HSD3 B2 enzymes of steroid synthesis,and immunofluorescence staining confirmed the expression of CYP17 A1 marker genes in the spherical organoid structure.(4)Globoid organoids expressed melanocortin 2 receptor(MC2 R),and cortisol secretion increased under adrenocorticotroph hormone(ACTH) stimulation.Conclusion:By adding FGF2,Wnt signaling pathway activator CHIR99021 and R-spondin1,a three-dimensional culture system of adrenal cortex with steroid secretion function can be established which regulated by ACTH.