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目的:通过浸泡加震荡法制备猪部分卵巢组织去细胞生物支架,并对其进行鉴定。方法:健康未成年猪卵巢30个,横切成厚为6 mm的切片,室温条件下,1%SDS溶液浸泡,同时平板震荡器震荡,最后用去离子水清洗其内残留SDS及DNA碎片。获得去细胞卵巢生物支架后,对其进行组织化学染色、DNA残留量定量测定、扫描电镜检查及免疫荧光染色,观察细胞去除情况、支架的三维结构以及细胞外基质中胶原与蛋白质的保留情况。结果:DNA定量分析得出仅15.4%的DNA残留;HE染色、扫描电镜及免疫荧光染色提示支架内未见细胞核残留,并且支架的结构、胶原成分保留较完整。结论:运用浸泡加震荡法能够有效地去除卵巢组织中的细胞成分,并且较完整地保持细胞外基质的支架结构,不同程度地保留一些胶原成分及蛋白质。
OBJECTIVE: To prepare some ovarian tissue acellular scaffolds by immersion plus shaking method and identify them. METHODS: Thirty healthy ovariectomized ovariectomized rabbits were cut transversely into 6 mm thick sections and immersed in 1% SDS solution at room temperature. Simultaneously the plate shaker was shaken. Finally, residual SDS and DNA fragments were washed with deionized water. After obtaining acellular scaffold of ovary, histochemical staining, quantitative determination of DNA residues, scanning electron microscopy and immunofluorescence staining were performed to observe the cell removal, the three-dimensional structure of the scaffold and the retention of collagen and protein in the extracellular matrix. Results: Quantitative DNA analysis showed that only 15.4% of DNA residues were found. HE staining, scanning electron microscopy and immunofluorescence staining showed no nuclear residues in the scaffolds, and the structure and collagen components of scaffolds remained intact. Conclusion: Soaking and adding shock method can effectively remove the cellular components in ovarian tissue, and maintain the integrity of the extracellular matrix scaffold structure, to varying degrees, retain some collagen components and proteins.