【摘要】 目的建立早产大鼠肺泡Ⅱ型上皮细胞(AECⅡ)与肺成纤维细胞(LF)共培养模型,为研究肺发育和肺损伤时AECⅡ和LF的相互作用提供实验基础。方法分离、纯化胎鼠AECⅡ和LF,通过插入式培养皿和培养板来构建AECⅡ-LF共培养模型。AECⅡ以5×105／mL的密度接种到6孔板中,LF以1×106／mL的密度接种到PCF插入式Millicell培养皿中,以6孔板中不放入套皿单独培养的AECⅡ为对照。用倒置显微镜观察AECⅡ形态和基本生长情况,台盼蓝染色了解细胞成活情况,免疫细胞化学染色法检测AECⅡ Ki67表达。结果早产大鼠AECⅡ和LF共培养模型成功构建。共培养2 d与单独培养2 d相比,AECⅡ的形态、活力和增殖无明显差异。共培养4 d,AECⅡ仍可保持其细胞形态,细胞数目、细胞活力;AECⅡ Ki67表达较单独培养4d 明显增加。结论体外构建的早产大鼠AECⅡ与LF共培养模型,部分模拟了体内微环境。AECⅡ和LF共培养体系有利于保持 AECⅡ的增殖和分化功能,可用于体外研究肺发育和肺损伤时AECⅡ和LF的相互作用。更多还原

【Abstract】 Objective To set up a coculture model of typeⅡ alveolar epithelial cells (AECⅡ) and lung fibroblasts (LF) from fetal rat lung so as to study the interaction between AECⅡ and LF in lung development and injury in vitro. Methods AECⅡ and LF was isolated and purified. AECⅡ was plated into the wells of six - well cluster dishes at a density of 5×105/mL. LF was plated into the Millicell culture plate inserts at a density of 1×106 /mL. The morphological change and growth of cells were observed under inverted phase contrast microscope. Cell viability was determined by Trypan blue dye exclusion. The expression of Ki67 was detected by immunocytochemical staining. Results There were no notable difference between coculture and control culture on 2 days in morphology, viability and proliferation of AECII. In cocluture on 4 days, AECⅡ also kept its morphology. Cell viability and proliferation of AECⅡ were increased significantly than those of control culture on 4 days. Conclusions The coculture model constructes in vitro mimics microenvironment in vivo. AEC Ⅱ differentiation and proliferation can be maintained in this coculture model, this can be used to study the the interaction between AEC Ⅱ and LF in lung development and injury in vitro.更多还原