卵巢去细胞生物支架的制备及鉴定

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摘要目的：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。

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	兰莉
	王严
	付养华
	刘帅良
	黎婷
	王志斌
	习海涛
	杨运俊
	梅劲

Abstract：Objective: To prepare decullularized biologic scaffolds of part of porcine ovarian tissue by soaking with rotating, and then indentify their characteristics. Methods: Thirty ovaries were excised from healthy immature pigs, and then cut transversely into pieces at the thickness of 6 mm uniformly. At room temperature, they were soaked into 1% SDS solutions and rotated on a tablet oscillator at the speed of 120 r/min for 36 h. At last they were washed with deionized water to get rid of residual SDS and DNA fragments. After decellularization, histochemical staining was done, the residual DNA was quantitative determined, examined under scanning electron microscope examination and did immunofluorescence to observe the removal of cells, the three-dimensional structure of the scaffolds, and the reservation of collagens and proteins in the extracellular matrix. Results: Quantitative analysis showed that only 15.4% DNA remained in the decellularized scaffolds compared to the normal control tissues; HE straining, scanning electron microscope and immunofluorescence showed there were no cells left in the scaffolds, at the same time, the three-dimensional structure and the collagens in the extracellular matrix kept intact. Conclusion: The method of soaking with rotating can remove all cells from the ovarian tissue, maintain the supporting structure of extracellular matrix relatively completely, and reserve collagens and proteins at a certain degree.

Key words： decellularization ovary tissue scaffolds extracellular matrix ovarian follicle

收稿日期: 2016-11-01

基金资助:浙江省自然科学基金资助项目（LQ16H040002）。

通讯作者: 梅劲，副教授，硕士生导师，Email：tibetcn@email.com。

作者简介: 兰莉（1975-），女，湖北武汉人，主治医师，硕士。

链接本文:

https://xb.wmu.edu.cn/CN/ 或 https://xb.wmu.edu.cn/CN/Y2017/V47/I3/182

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