摘要
[目的]建立斑马鱼卵巢和精巢组织的原代细胞培养技术，获得原代和传代培养细胞单层。[方法]采用组织块贴壁法进行斑马鱼卵巢和精巢组织的原代细胞培养，采用0．25％胰蛋白酶消化法进行传代培养。[结果]斑马鱼性腺组织的原代细胞培养条件为：最适培养基为DMEM／F12（pH 7．0～7．2），培养温度为24℃，在添加20 ng／mL 表皮生长因子（EGF）、20 ngm／L 碱性成纤维细胞生长因子（bFGF）、0．5 mmol／L β－巯基乙醇和15％胎牛血清时更有利于细胞的存活。其中，卵巢的组织块接种3～4 d后，上皮样细胞首先开始迁出，迁出的上皮样细胞生长5 d后，逐渐凋亡；此后，成纤维样细胞和另一种上皮样细胞开始大量迁出，10 d后可生长汇合成80％的原代细胞单层；可成功传代培养1次，但传代后的上皮样细胞不贴壁，很快死亡，而传代后的成纤维样细胞可贴壁生长，但基本不分裂，健康存活7 d后开始逐渐凋亡。精巢组织接种5 d后，上皮样细胞和成纤维样细胞同时开始迁出，培养16 d后逐渐凋亡，仅得到60％原代培养细胞单层。[结论]初步建立了斑马鱼性腺组织的原代细胞培养条件，并将卵巢组织块迁出的成纤维样细胞成功传代1次。
Abstract
Objective] To develop a primary cell culture system for the cells from ovary and testis tissues of zebrafish and to obtain the corre -sponding cell monolayer of both primary culture and subculture.[Method]Primary cell cultures were derived using the explants culture method. The primary cells were sub-cultured by 0.25% trypsin digestion.[Result] The zebrafish gonad-derived cells were cultured in DMEM/F12 (pH 7.0-7.2) at an optimal temperature of 24 ℃.The cells had better growth and survivor when the medium mentioned above was supplemented with 20 ng/mL epidermal growth factor (EGF),20 ng/mL basic fibroblast factor (bFGF),0.5 mmol/Lβ-mercaptoethanol and 15%fetal bovine serum (FBS).For ovary tissues,epithelium-like cells first migrated from the explants at 3~4 days after seeding.These epithelium-like cells could only survive for about 5 days in vitro,and then tended to apoptosis.However, active migration of fibroblast-like cells and another kind of epitheli-um-like cells were then observed and a primary cell monolayer with 80%confluence could be formed within10 days.Then the primary cell mono-layer could be successfully sub-cultured once.The epithelium-like cells can’t re-attach and die soon after subculture.However,the re-attached fi-broblast-like cells could healthily survive for another 7 days and then tend to apoptosis.No obvious cell division was observed in both the primary and subculture cells.For testis tissues,both epithelium-like cells and fibroblast-like cells migrated from the explants at 5 days after seeding.At 16 days after seeding,a primary cell monolayer with 60%confluence was obtained and then tended to apoptosis.[Conclusion] This study has estab-lished a primary cell culture system suitable for the gonad tissues of zebrafish ,and the fibroblast-like cell monolayer derived from the ovary tissues has been successfully sub-cultured for one time.
基金项目
国家自然科学基金(31472274)
国家自然科学基金(31172391)
海洋经济创新发展区域示范项目(12 PYY001 SF08)
中国海洋大学本科生SRDP项目()
国家科技期刊平台
NSTL
维普
万方数据