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

doi:10.3969/j.issn.2095-4344.2017.33.006

Abstract

Abstract:

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 at 21 days 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 plus 6.0 software.
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. After 21-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 (21 days) co-culture.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Adipose Tissue, Mesenchymal Stem Cells, Pulmonary Alveoli, Epithelial Cells, Coculture Techniques, Cell Differentiation, Tissue Engineering

CLC Number:

R394.2

Chen Shang-ya, Cui Guan-qun, Bo Cun-xiang, Zhang Yu, Zhang En-guo, Yang Ye, Du Zhong-jun, Shao Hua. In vitro differentiation of rat adipose-derived mesenchymal stem cells induced by rat lung epithelial-T-antigen negative cell line[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(33): 5280-5286.

Figures/Tables 1

2.1 脂肪间充质干细胞的分离、培养、鉴定结果原代细胞提取后，接种至培养瓶中，贴壁细胞主要为梭形、纺锤形，部分为多角形或卵圆形；第2天换液后，大部分贴壁细胞为长梭形、成纤维细胞样；至第4，5天细胞密度达到80%-90%，进行胰酶消化、传代。传至第3代时，细胞呈长梭形，分布均匀，密度达到100%时呈漩涡样、菊花样聚集，见图1。随传代次数升高，杂细胞数量明显降低，脂肪间充质干细胞增殖速度逐渐下降。流式细胞术检测结果表明，第4代脂肪间充质干细胞CD90、CD73表达阳性(阳性率分别为99.6%和99.2%)，CD45、CD11b表达阴性(阳性率分别为0.3%和3.6%)，见图2；脂肪间充质干细胞成脂、成骨、成软骨分化成功，见图3，证实为间充质干细胞。 2.2 RLE-6TN的培养、鉴定结果 RLE-6TN是肺泡Ⅱ型上皮细胞，培养液选用与脂肪间充质干细胞一致的完全培养基，约48 h可铺满瓶底。培养至第3代时，倒置显微镜下观察，细胞呈卵圆形、多角形(图4A)；免疫荧光染色后，荧光显微镜下观察，细胞核呈蓝色荧光表达，细胞呈SP-C蛋白红色荧光表达(图4B)。 2.3 脂肪间充质干细胞共培养后形态变化共培养48 h后，各小室细胞密度达到80%以上。21 d后，对照组脂肪间充质干细胞无形态改变，仍为长梭形、成纤维细胞样生长，实验组部分脂肪间充质干细胞形态开始向不规则细胞转化，部分变圆、多角形，见图5。 2.4 荧光显微镜观察脂肪间充质干细胞荧光表达共培养21d后，将各组脂肪间充质干细胞进行DAPI细胞核染色和SP-C蛋白的免疫荧光染色，荧光显微镜下观察。实验组可见红色荧光的SP-C蛋白表达，对照组均不见红色荧光表达，见图6。采用Image pro plus 6.0软件，对每张图片进行吸光度矫正后，分析各图红色荧光区域的积分吸光度值。统计结果显示，实验组的积分吸光度值明显高于对照组，差异有显著性意义(P < 0.01)，见表1。"

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