毛囊干细胞与异种膀胱脱细胞基质生物相容性的研究

Biocompatibflity of hair follicle stem cells and heterogeneous bladder acellular matrixat

目的研究毛囊干细胞（hair follicle stem cells，HFSCs）与异种膀胱脱细胞基质（blad—der acellular matrix，BAM）的体内外生物相容性，探讨HFSCs和异种BAM构建组织工程膀胱的可行性。方法2011年6月至2012年7月，我们运用二步酶法及差速贴壁法获取第3代大鼠HFSCs，流式细胞术检测FITC标记的β1整合素对HFSCs进行鉴定；显微分离及化学洗涤法制备新西兰兔BAM，Masson染色和扫描电镜检测材料脱细胞效果。采用二次沉淀法将HFSCs静态接种于BAM表面，体外复合培养1周后组织学检测和扫描电镜观察细胞在材料表面的生长状况。将体外培养的细胞材料复合物植入大鼠皮下，分别在1、2、4周取材，大体观察、组织学检测观察细胞在支架上的生长情况。结果制备的BAM为白色半透明膜状，扫描电镜下观察为纤维网状结构，未见细胞残留。Masson染色提示BAM为胶原结构，无明显细胞残留。细胞材料体外复合培养48h后倒置显微镜下观察BAM周围HFSCs生长状态良好，1周后扫描电镜下观察HFSCs伸展、黏附于支架表面。细胞材料复合物大鼠皮下植入后无明显的炎症反应，1周后行HE染色可见单层细胞结构，4周后行HE染色可见多层细胞结构。结论HFSCs与异种BAM有良好的生物相容性，为HFSCs修复膀胱缺损疾病的研究奠定了基础。

Objective To study the compatibility and feasibility of construct tissue engineer bladder through bioeompatibility of hair follicle stem cells （HFSCs） and heterogeneous bladder acellular matrix （BAM） in vitro and vivo. Methods The third-generation of rat HFSCs were cultured with the two-step enzymatic and different adhesion time method. The cells were identified by flow cytometry through detection expression of β1 integrin FITC-Conjugated. The New Zealand rabbit BAM were deeellularized by the method of microdissection and chemical washing, and then examined by Masson staing and electron microscope to confirm no cell elements remained. The HFSCs of the rats were implanted in BAM and cultured for about 24 hours. Then the cells growth conditions on the material surface were examined by histology and scanning electron microscopy. The cells-scaffold composites were implanted in rats subcutaneously, samples and his- tological examination were harvested at 1, 2 and 4 weeks after implantation. Results The BAM was white and translucent membranous. There were fiber network structures without cell elements remained under the examination of electron microscope. And the BAM prompted for collagen tissue composition under Masson staining, without significant residual cells. The growth condition of HFSCs beside the BAM was well that ob- served by inverted microscope at 48 h of co-culture. After 1 week the HFSCs extended and adhered to the matrix surface observed under the scanning electron microscope. No significant inflammatory response in ratsubcutaneous implantation experiments, the single-layer cell structure in histological examination could be seen after 1 week, and multi-layer cell structure in histological examination could be seen after 4 weeks of implantation. Conclusion The biocompatibility of HFSCs and heterogeneous BAM is good, which pro- vides a good experiment support for HFSCs to repair the bladder defects disease.