鹅原始生殖细胞体外分离与冷冻保存方法的比较研究

发布时间：2018-10-15 13:22

【摘要】：为了建立针对鹅原始生殖细胞(primordial germ cells,PGCs)的分离纯化与冷冻保存方法,对血液Ficoll密度梯度离心法、血液ACK裂解法、生殖腺胰酶-EDTA消化法与生殖腺消化结合体外短时间培养法4种PGCs分离方法以及STEM-CELLBANKER、CELLBANKER 2与10%DMSO加90%血清等3种冻存液进行了比较研究。通过形态学观察、糖原染色与PGCs特异免疫荧光染色鉴定PGCs,同时利用台盼蓝染色检测PGCs活力等方法对其分离和保存效果进行评价。结果表明,从鹅胚血液与生殖腺中均已经成功分离到鹅PGCs;生殖腺消化结合体外短期培养法与传统的血液分离法相比,获得的鹅PGCs数量、纯度与活力最高,每枚胚胎获得的鹅PGCs数量达到115个、细胞纯度达80.8%、细胞活率达88.3%;而商业化的干细胞冷冻保护液STEM-CELLBANKER同一般的体细胞冻存液相比更适合鹅PGCs的冷冻保存,冷冻解冻后的细胞存活率达90%左右。试验可以得出结论:生殖腺消化结合体外短期培养法成功分离到数量与纯度都可观的鹅PGCs,STEM-CELLBANKER冻存液能够用于鹅PGCs的长期冷冻保存。
[Abstract]:In order to establish a method for isolation, purification and cryopreservation of goose primordial germ cell (primordial germ cells,PGCs), blood Ficoll density gradient centrifugation and blood ACK lysis were studied. Four methods of isolation of PGCs from gonadal trypsin-EDTA digestion and gonadal digestion combined with short time culture in vitro were compared. Three kinds of cryopreservation solutions including STEM-CELLBANKER,CELLBANKER 2 and 10%DMSO plus 90% serum were compared. Morphological observation, glycogen staining and PGCs specific immunofluorescence staining were used to identify PGCs, and Trypan blue staining was used to detect the activity of PGCs. The results showed that goose PGCs; was successfully digested from goose embryo blood and gonads, and compared with the traditional blood separation method, goose PGCs was obtained with the highest quantity, purity and activity. The number of goose PGCs per embryo reached 115, the cell purity reached 80.8 and the cell viability rate reached 88.3.The commercial STEM-CELLBANKER was more suitable for the freezing preservation of goose PGCs than that of normal somatic cell cryopreservation. The survival rate of frozen and thawed cells was about 90%. The results showed that gonadal digestion combined with short-term culture in vitro successfully isolated goose PGCs,STEM-CELLBANKER cryopreservation solution with considerable quantity and purity, which could be used for long-term freezing preservation of goose PGCs.
【作者单位】：江苏省农业科学院畜牧研究所/动物品种改良和繁育重点实验室;
【基金】：江苏省农业科技自主创新资金项目[CX(14)2068] 江苏省自然科学基金(BK20130718)
【分类号】：S835

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