小鼠Ⅱ型肺泡上皮细胞类器官三维培养体系的建立

Establishment of a three-dimensional organoid culture system for mouse type 2 alveolar epithelial cells

本研究旨在建立小鼠Ⅱ型肺泡上皮细胞(type 2 alveolar epithelial cells,AT2)类器官三维(three-dimensional,3D)培养体系的方法。采用酶消化与磁珠分选分离和纯化ICR小鼠肺AT2细胞,proSPC免疫荧光染色鉴定AT2细胞纯度;2维(two-dimensional,2D)培养8 d,5-乙炔基-2’脱氧尿嘧啶核苷(5-ethynyl-2’-deoxyuridine,EdU)掺入和荧光染色法观察AT2细胞的增殖与分化;将AT2细胞与小鼠肺成纤维细胞(mouse lung fibroblasts,Mlg)3D共培养,光学显微镜下观察类器官生长情况,收集生长13d的类器官,2%多聚甲醛固定后行HE染色,荧光染色观察类器官内部形态结构。结果显示,提取AT2细胞纯度超过95%;在体外培养1~8 d期间,AT2细胞EdU荧光染色阴性,未见增殖;AT2细胞形状逐渐趋向扁平鳞状、细胞表面积显著增大;在体外培养3 d后,有部分AT2细胞特异性标志物proSPC阳性细胞开始出现I型肺泡上皮细胞(type 1 alveolar epithelial cells,AT1)标志物T1α和HopX表达,培养5 d和8 d后T1α和HopX表达量进一步增加,proSPC的表达量和proSPC阳性细胞比例逐渐减小。AT2细胞与Mlg 3D共培养4 d时形成圆球样细胞团块,生长至13 d时形成肺泡类器官。组织学分析显示:肺泡类器官呈一空心小体;表达proSPC的AT2细胞主要分布于类器官的外侧;表达HopX、由AT2细胞转分化形成的AT1细胞则主要位于类器官小体的内部。proSPC/EdU双荧光染色显示:位于肺泡类器官外侧圈层的proSPC阳性AT2细胞中,有部分细胞表现为proSPC/EdU双阳性,表明类器官内的AT2细胞是具有增殖能力的细胞。以上结果显示,本研究成功建立了小鼠AT2类器官3D培养体系。

The purpose of this study was to establish a three-dimensional(3D)organoid culture system for type 2 alveolar epithelial(AT2)cells in mice.AT2 cells were isolated from ICR mouse lung and purified by enzymatic digestion and MicroBeads sorting.The purity of AT2 cells was determined by immunofluorescence(IF)staining using an antibody against proSPC.The AT2 differentiation was examined by IF staining with proSPC/HopX and proSPC/T1αantibodies,and proliferation of AT2 cells was assessed by EdU incorporation assays after two-dimensional(2D)culture for 8 days.In addition,AT2 cells were co-cultured with mouse lung fibroblasts(Mlg)in three-dimensional(3D)culture system.After 13 days of co-culture,the organoids were fixed in 2%paraformaldehyde for histological analysis and IF staining.The results showed that the purity of the AT2 cells was over 95%,as assessed by proSPC staining.2D cultured AT2 cells were negative for EdU staining,which indicates that no proliferation occurs.proSPC expression was gradually disappeared,whereas T1αand HopX expression was gradually increased after 3,5 and 8 days of culture.In 3D culture system,the alveolar organoids were formed after co-culturing AT2 cells with Mlg for 4 days.Histological analysis showed that alveolar organoids displayed a hollow morphology.proSPC was highly expressed in the peripheral cells,whereas type 1 alveolar epithelial(AT1)cells transdifferentiated from AT2 cells expressing HopX were mainly located in the interior of organoid bodies after 13 days.Some of the proSPC-positive AT2 cells located in the outer circle of alveolar organoids were stained positive for both proSPC and EdU,indicating that the AT2 cells in the alveolar organoids were proliferative.These results showed that the 3D organoid culture system of mouse AT2 cells was successfully established.