大鼠肺泡Ⅱ型上皮细胞系RLE-6TN诱导脂肪间充质干细胞体外分化  

In vitro differentiation of rat adipose-derived mesenchymal stem cells induced by rat lung epithelial-T-antigen negative cell line

作　　者：陈尚雅 崔冠群 薄存香[2] 张玉[2] 张恩国 杨叶 杜忠君[1,2] 邵华[1,2] Chen Shang-ya;Cui Guan-qun;Bo Cun-xiang;Zhang Yu;Zhang En-guo;Yang Ye;Du Zhong-jun;Shao Hua(Shandong University of Traditional Chinese Medicine, Jinan 250355, Shandong Province, China;Shandong Academy of Occupational Health and Occupational Medicine, Jinan 250062, Shandong Province, China;Department of Respiration, Qilu Children's Hospital of Shandong University, Jinan 250022, Shandong Province, China;School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Science, Jinan 250062, Shandong Province, China)

机构地区：[1]山东中医药大学,山东省济南市250355 [2]山东省职业卫生与职业病防治研究院,山东省济南市250062 [3]山东大学齐鲁儿童医院呼吸内科,山东省济南市250022 [4]济南大学山东省医学科学院医学与生命科学学院,山东省济南市250062

出　　处：《中国组织工程研究》2017年第33期5280-5286,共7页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(81470145,81602893);山东省自然科学基金(ZR2015YL049);山东省医药卫生科技发展计划(2016WS0540);山东省医学科学院创新工程~~

摘　　要：背景:研究表明,骨髓间充质干细胞具有在体外分化为肺泡上皮细胞的可能性,但至今未见脂肪间充质干细胞与肺泡上皮细胞通过长时间Transwell共培养,诱导脂肪间充质干细胞分化的相关报道。目的:观察大鼠肺泡Ⅱ型上皮细胞系RLE-6TN诱导大鼠脂肪间充质干细胞分化为肺泡Ⅱ型上皮细胞的可行性。方法:取3只SPF级健康雌性SD大鼠作为供体,分离、提取、培养、鉴定脂肪间充质干细胞。实验分2组:实验组(脂肪间充质干细胞与大鼠肺泡Ⅱ型上皮细胞系RLE-6TN进行Transwell共培养)和对照组(脂肪间充质干细胞单独培养)。共培养第21天,倒置显微镜观察各组脂肪间充质干细胞的形态变化,对脂肪间充质干细胞进行SP-C免疫荧光染色,荧光显微镜下观察蛋白表达并拍照,采用Image pro plus 6.0软件分析荧光表达的积分吸光度值。结果与结论:(1)共培养21 d,实验组的脂肪间充质干细胞形态由长梭形、成纤维细胞样,逐渐转化为卵圆形、多边形,对照组脂肪间充质干细胞形态保持成纤维细胞样;(2)荧光显微镜下观察,实验组脂肪间充质干细胞可见红色荧光表达,对照组无红色荧光表达,实验组的荧光表达较对照组增强(P<0.05);(3)结果表明,大鼠肺泡Ⅱ型上皮细胞系RLE-6TN能够在长时间(21 d)共培养条件下,诱导脂肪间充质干细胞分化为肺泡上皮细胞。BACKGROUND:Studies have shown that bone marrow mesenchymal stem cells have the potential of differentiation into alveolar epithelial cells in vitro,but so far no study has indicated that adipose-derived mesenchymal stem cells(ADSCs)can be differentiated into alveolar epithelial cells through long-term Transwell co-culture.OBJECTIVE:To observe whether rat lung epithelial-T-antigen negative cell lines(RLE-6TN)can induce rat ADSCs to differentiate into type II alveolar epithelial cells by long-term Transwell co-culture.METHODS:Three SPF health female Sprague-Dawley rats were used as donors to separate,extract,culture and identity ADSCs.The experimental group was subjected to the Transwell co-culture of ADSCs and RLE-6TN,while the control group was subjected to the culture of ADSCs alone.The morphological changes of ADSCs were observed by the inverted phase contrast microscope at21days after co-culture.Immunofluorescence staining using surfactant protein C(SP-C)was performed on the co-cultured ADSCs.The fluorescence staining was observed using the inverted fluorescence microscope.Integral optical density(IOD)analysis was conducted by Image pro plus6.0software.RESULTS AND CONCLUSION:RLE-6TN cells were identified by fluorescence staining with stable expression of SP-C protein(red fluorescence)in the experimental group,and there was no red fluorescence in the control group.After21-day co-culture,the cell shape in the experimental group was transformed from the long spindle shape into oval or polygon shape gradually,while the cell shape in the control group remained fibroblast-like.These results show that RLE-6TN can induce ADSCs to differentiate into type II alveolar epithelial cells after a long-term(21days)co-culture.

关 键 词：干细胞 脂肪肝细胞 脂肪间充质干细胞 肺泡上皮细胞 RLE-6TN 共培养 TRANSWELL 细胞分化 大鼠 国家自然科学基金 

分 类 号：R394.2[医药卫生—医学遗传学]