人卵巢颗粒细胞分离培养方法的改进

发布时间：2018-02-14 09:43

  本文关键词： 卵巢 细胞 培养的 受精 体外 组织构建 组织工程 卵巢颗粒细胞 裂解法 沉淀法 密度梯度离心法 Ⅰ型胶原酶 透明质酸酶 自体卵泡液　出处：《中国组织工程研究》2015年46期 　论文类型：期刊论文

【摘要】：背景:建立分离培养颗粒细胞快速有效的方法也是提高体外受精-胚胎移植成功率关键的一步。虽然目前文献中有较多关于人卵巢颗粒细胞分离方法的报道,但在细胞数量、纯度及后续生长等方面不尽人意。目的:建立有效的分离提纯、体外培养人卵巢黄素化颗粒细胞的方法。方法:收集体外受精-胚胎移植取卵时的卵泡液,用裂解法、沉淀法、密度梯度离心法分离,Ⅰ型胶原酶或透明质酸酶消化颗粒细胞黏液团并接种在培养皿中进行培养,培养液中加入或不加自体卵泡液。结果与结论:用裂解法得到的颗粒细胞数较沉淀法和密度梯度离心法得到的细胞数多(P0.05,P0.05);3种方法提取的颗粒细胞活性比较无明显差异;培养24 h后沉淀法贴壁细胞数最多(P0.05),而密度梯度离心法贴壁细胞数最少(P0.05);透明质酸酶消化颗粒细胞较Ⅰ型胶原酶耗时少且消化彻底;加入自体卵泡液能够促进颗粒细胞生长和存活。结果证实,沉淀法提取颗粒细胞虽然耗时长,但培养的细胞存活率高、培养后收获的细胞数多;透明质酸酶较Ⅰ型胶原酶更适合消化颗粒细胞黏液团;在培养液中加入自体卵泡液更有益于颗粒细胞生长。
[Abstract]:Background: to establish a rapid and effective method for isolation and culture of granulosa cells is also a key step to improve the success rate of in vitro fertilization and embryo transfer. Objective: to establish an effective method for isolation and purification of human ovarian luteinized granulosa cells in vitro. By density gradient centrifugation, granulosa cell mucus was digested by type I collagenase or hyaluronidase and cultured in culture dish. Results and conclusion: the number of granulosa cells obtained by cleavage method was more than that by precipitation method and density gradient centrifugation method. After 24 h culture, the number of adherent cells by precipitation method was the highest, and the number of adherent cells by density gradient centrifugation was the least, and hyaluronidase digestion of granulosa cells took less time and digested thoroughly than that of type 鈪，

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