人胆囊类器官培养体系的建立与鉴定

Establishment and identification of human gallbladder organoid culture system

目的构建人胆囊类器官的培养体系,实现人胆囊类器官体外稳定快速扩增,并鉴定其特性。方法分离人来源胆囊组织上皮细胞,运用三维培养体系将细胞嵌入基质胶中进行3D培养。观察胆囊类器官生长过程中球囊样结构的面积、周长与形态,计算形状因子。利用细胞免疫荧光染色技术检测胆囊类器官的干性标志物CD133、富含亮氨酸重复序列的G蛋白偶联受体5(LGR5)以及胆管上皮细胞标志物肝细胞核因子1β(HNF1β)、上皮细胞黏附分子(Ep CAM)、细胞角蛋白7、细胞角蛋白19、性别决定区Y框9(SOX9)的表达情况。使用5-溴脱氧尿嘧啶核苷(Brd U)摄入实验检测小分子CHIR-99021和blebbistatin对类器官增殖特性的影响。结果胆囊类器官在体外培养过程中,类器官形成的球囊面积逐渐增大,第7天的类器官面积与第1天比较差异有统计学意义(P<0.01),第7天时其形状因子从第1天的0.80(0.75,0.84)增高至0.83(0.81,0.85)(P<0.01),表明类器官在体外稳定生长且形状逐渐趋于一个完美的圆。免疫荧光染色可见类器官表达胆管上皮细胞标志物。在培养基中加入小分子CHIR-99021和blebbistatin后,Brd U检测显示类器官的增殖能力增强(P<0.01)。结论在体外成功培养获得了具有增殖能力的人胆囊类器官,其具有胆管上皮细胞的特性,可用于胆管疾病的研究与建模。

Objective To construct a culture system of human gallbladder organoids,successfully culture them in vitro and identify their characteristics.Methods Human gallbladder epithelial cells were isolated and embedded in matrix glue using a 3-dimensional culture system for 3-dimensional culture.The area,perimeter and morphology were observed during the growth of gallbladder organoids and the form factor was calculated.The expression of the stemness markers(CD133 and leucine rich repeat containing G protein coupled receptor 5[LGR5])and the bile duct epithelial cell markers(hepatocyte nuclear factor 1β[HNF1β],epithelial cell adhesion molecule[EpCAM],cytokeratin 7,cytokeratin 19,sex determining region Y box 9[SOX9])of gallbladder organoids was detected by immunofluorescence staining.After adding small molecules CHIR-99021 and blebbistatin into the growth medium,5-bromodeoxyuridinc(BrdU)incorporation assay was used to detect the proliferation characteristics of organoids.Results During the in vitro culture process of gallbladder organoids,the cell area was gradually increased,and there was a significant difference between the area on day 7 and day 1(P<0.01).The form factor was increased from 0.80(0.75,0.84)on day 1 to 0.83(0.81,0.85)on day 7(P<0.01),indicating that the organoids grew stably in vitro and tended to form a perfect circle.Immunofluorescence staining showed that the bile duct epithelial cell markers were expressed in organoids.After adding small molecules CHIR-99021 and blebbistatin into the medium,BrdU detection showed that the proliferation of the organoids was increased(P<0.01).Conclusion Human gallbladder organoids with proliferative ability can be successfully cultured in vitro,and it has the characteristics of bile duct epithelial cells and can be used for the research and modeling of biliary diseases.