人脂肪来源干细胞与膀胱脱细胞基质-丝素蛋白双层支架的生物相容性研究

摘要

Objective To observe the growth of human adipose-derived stem cells (hASCs) in bladder acellular matrix graft-silk fibroin (BAMG-SF) bilayer scaffold and to analyze the biological compatibility of BAMG-SF with hASCs. Methods hASCs were isolated from human subcutaneous adipose tissue after collagenase digesting, filtrating and centrifuging, then cultured in the leaching solution of BAMG-SF. The cytotoxicity of scaffold was evaluated by CCK-8 cell viability assay, and the growth curves were also observed. Surface morphology on BAMG-SF was observed by scanning electron microscopy (SEM). The hASCs of passage 3 were seeded onto the BAMG-SF bilayer scaffolds for 1 week, then the BAMG-SF bilayer scaffolds seeded with hASCs were transplanted into nude mouse for 1 week or 2 weeks. The growth of cells in BAMG-SF biomaterials was observed by HE staining. The species origin of these cells in the BAMG-SF scaffolds cultured in vivo was detected by Immunofluorescence. Results hASCs maintained high proliferation rate in the leaching solution of BAMG-SF and the BAMG-SF scaffolds were nontoxic absolutely. According to the growth curves of hASCs cultured in the leaching solution of the BAMG-SF and DMEM, BAMG-SF scaffolds were conducive to the growth of hASCs. The histological study found that hASCs could grow into the space of the BAMG-SF scaffolds after cultured in vitro and in vivo. There were more cells in the scaffolds cultured in vivo than in vitro. Immuno-fluorescence suggested that some of the cells inside the scaffolds were hASCs. Conclusion BAMG-SF bilayer scaffolds are nontoxic and have a good biocompatibility with hASCs, which can be used as a vehicle for hASCs in bladder defect reconstruction.%目的：观察人脂肪来源干细胞（Human adipose derived stem cells，hASCs）在膀胱黏膜下脱细胞基质-丝素蛋白（Bladder acellular matrix graft-silk fibroin，BAMG-SF）双层支架材料中的生长情况，分析其生物相容性。方法取hASCs，置于BAMG-SF浸提液中培养，CCK-8法检测其细胞活力，评价BAMG-SF支架的细胞毒性并绘制生长曲线。扫描电镜观察BAMG-SF双层支架材料的表面形貌。将hASCs传代扩增后接种到BAMG-SF双层支架材料上，体外培养1周后，转至裸鼠皮下培养1周、2周，HE染色观察细胞在支架上的生长情况。HLA免疫荧光鉴定裸鼠皮下双层支架上细胞的种属来源。结果 hASCs在BAMG-SF双层支架浸提液中可保持较高的增殖率，根据细胞相对增殖率与细胞毒性分级关系证实BAMG-SF双层支架浸提液无细胞毒性。由hASCs在BAMG-SF浸提液和DMEM培养基中的生长曲线可知，BAMG-SF有利于hASCs的生长。将hASCs接种到BAMG-SF双层支架材料上，经过体外、内培养，hASCs均能长入支架的空隙内，且体内培养比体外培养有更多的hASCs细胞长入支架。 HLA检测显示支架内细胞部分为hASCs。结论新型BAMG-SF双层支架材料安全无毒，与hASCs生物相容性好，可作为细胞载体应用于组织工程膀胱的研究。