温度敏感性材料培养的脂肪源性干细胞作为眼表重建种子细胞的可行性研究

Feasibility of adipose tissue-derived stem cell sheet fabricated ex vivo on temperature-responsive scaffolds as seed cells for ocular surface reconstruction

背景 组织工程角膜学的发展为角膜盲的治疗提供了新的选择,但目前暂未发现培养方法简单、可行性好的角膜上皮种子细胞和理想的支架材料.研究表明,脂肪源性干细胞（ADSCs）具有自我更新能力同时也具有类上皮的特质,而温度敏感性材料（TRSs）作为支架进行干细胞培养也为细胞层片技术的进步提供了技术支撑. 目的 研究兔ADSCs在TRSs上的培养特性,并与经典的口腔黏膜上皮细胞（OMECs）特性进行比较,探讨ADSCs作为眼表重建种子细胞的可行性.方法 异丙基丙烯酰胺溶于二丙醇后均匀涂于直径35 mm的聚苯乙烯培养皿表面,利用电子束照射制备TRSs,兔颈背部皮下脂肪组织2～3g经消化培养后获得ADSCs,同时取出兔口腔黏膜组织进行消化培养,获得OMECs.将2种干细胞均接种于TRSs上继续培养,比较2种细胞形态、生长速度、脱附时间和成活细胞的总计数,将ADSCs层片和OMECs层片行组织病理学检查,观察2种细胞的形态特征;采用免疫组织化学法检测2种细胞中干细胞标志物和上皮细胞标志物的表达;应用扫描电子显微镜检查2种细胞层片的表面超微结构.结果 自制TRSs透明性和光滑度与普通培养皿接近,任意观察的5个样品中有4个水接触角＞10°,成功率为80％.TRSs上培养的ADSCs呈长梭形,OMECs呈不规则圆形.ADSCs生长周期在TRSs上为12～ 14 d,脱附时间为（46.0±9.6） min,细胞总计数为（7.9±1.1）×105/片,而TRSs培养的OMECs生长周期为14～16 d,脱附时间为（91.9±10.9） min,细胞计数为（45.8±26.5） ×105/片,2种细胞间层片脱附时间和细胞计数的差异均有统计学意义（P=0.002、0.028）.组织病理学检查显示,TRSs上的ADSCs呈1～3层排列,而OMECs呈4～5层覆层结构.免疫组织化学染色显示,ADSCs和OMECs细胞层片细胞角蛋白12（CK12）及干细胞标志物p63、ATP结合转运蛋白G超家族成员2（ABCG2）均呈阳性表达.扫描电子显微镜下观察可见ADSCs和OMECs表面均有致密的上皮微绒毛结构,细胞间连接紧密.结论 自制的TRSs可作为脂肪干细胞的培养支架,ADSCs取材广泛,TRSs上培养的ADSCs层片细胞活力好,可操作性强,可作为眼表重建新的种子来源。

Background Development of corneal tissue engineering creates a new therapeutic method for severe corneal diseases.However,ideal seed cells and scaffold for corneal surface reconstruction have not yet been investigated well.Adipose-derived stem cells （ADSCs） are varified to have a self-renewal ability and epithelioid features,and temperature-responsive scaffolds （TRSs） can offer technical support for stem cell sheet.Objective This study was to investigate the characteristics of ADSCs cultured on TRSs and compare these features to typical oral mucosal epithelial cells （OMECs）,and therefore to explore the feasibility of reconstruction of ocular surface with ADSCs as seed cells.Methods Self-made TRSs were prepared by adding isopropyl alcohol dissolved poly-Nisopropylacrylamide （PNIPAAm） to each polystyrene tissue culture dish and then irradiating using an election beam.Subcutaneious fatty tissue of rabbit neck was obtained to culture ADSCs,and 4 pieces of oral cavity mucosal tissue were digested and cultured to obtain OMECs.Then the ADSCs and OMECs were incubated on TRSs,and cell morphology,growth rate,detached duration and survival counts were compared between ADSCs and OMECs.The ADSCs sheet and OMECs sheet were stained with hematoxylin and eosin for morphological examination.Immunochemistry was used to observe the expressions of stem-cell biomakers and epithelioid-cell biomakers in the cells.The ultrastructure of cell surface was observed under the scanning electron microscope.Results Self-made TRSs were similar to ordinary culture dish in the transparancy and smoothness.The water contact angle of 4 in 5 samples were 〉10° with the effective rate upto 80%.A DSCs showed the elongated fusiform in shape,while OMECs showed a cobblestone appearance.The growth cycle,detached duration and cell number of ADSCs were 12-14 days,（46.0 ±9.6） minutes and （7.9 ±1.1）×105/sheet,and those of OMECs were 14-16 days,（91.9 ±10.9） minutes and （45.8 ±26.5）×105/sheet,respectively,showing statistically significant differences in the detached duration and cell counts between ADSCs and OMECs （P=0.002,0.028）.Hematoxylin and eosin staining showed that ADSCs sheet comprised only 1-3 layer cells,while OMECs showed 4-5 layer cells.ATP-binding cassette superfamily G member 2 （ABCG2）,p63 and cytokeratin 12 （CK12） were positively expressed in both ADSCs sheet and OMECs sheet.Closely packed cells and typical eithelial microvilli in the cell surface were exhibited in both ADSCs sheet and OMECs sheet under the scanning electron microscope.Conclusions Self-made TRSs can be used as scaffold of ADSCs.The ADSCs sheet on the TRSs appears to have a good cell vitality and therefore is a new seed source of ocular surface reconstruction.