鸡胚胎原始生殖细胞的冷冻保存和体外培养的研究
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摘要
本研究采用Fieoll密度梯度离心提取发育至第19期和第28期鸡胚胎性腺中的原始生殖细胞(Primordial Germ Cells，PGCs)，在不同的冷冻保护液、同一种冷冻保护液的不同浓度、不同的冷冻保护程序下进行超低温冷冻保存，1周后复苏，并进行体外培养、分化试验，结果如下：
1．当各种冷冻保护剂的浓度为10％时，同一种冷冻保护液。冷冻程序Ⅰ和冷冻程序Ⅱ之间，PGCs的存活率存在显著(P＜0.05)或极显著(P＜0.01)差异。而同一种冷冻程序。冷冻保护液1、冷冻保护液Ⅱ、冷冻保护液Ⅲ、冷冻保护液Ⅳ、冷冻保护液Ⅴ、冷冻保护液Ⅵ之间存在显著(P＜0.05)或极显著(P＜0.01)差异，比较其结果显示冷冻保护液Ⅲ的保护效果最好。而在同一冷冻保存条件下，第19期和第28期性腺中的PGCs存活率差异不显著(P＞0.05)。
2．当冷冻保护剂的浓度为10％时，在其他冷冻保存条件相同的情况下，分离后直接进行冷冻保存的PGCs，复苏后的存活率极显著(P＜0.01)高于经体外培养24 h后再进行冷冻保存的PGCs。
3．同一种冷冻保存条件下，冷冻保护液不同的浓度之间表现为：10％浓度与15％浓度相比较，细胞的存活率除冷冻保护液Ⅳ外，其余差异不显著(P＞0.05)：20％浓度下，细胞的存活率最低，且与10％及15％浓度下的存活率相比存在显著(P＜0.05)或极显著(P＜0.01)差异。
4．PGCs复苏后，当把其培养在以DMEM为基础液，添加10％的胎牛血清、2％的鸡血清、2mmol／L谷氨酰胺、1mmol／L丙酮酸钠、5.5×10~(-5)mol／L β-巯基乙醇、100U／mL硫酸庆大霉素、以鸡胚成纤维细胞作饲养层的培养体系中时，PGCs
可贴壁，并可聚集成细胞团，但不能克隆、增殖，继续培养下去，PGCs最终死亡。
5.PGCs复苏后，当把其培养在添加10%的胎牛血清、2%的鸡血清、2幻。roo比
谷氨酞胺、1 mmo比丙酮酸钠、5，sxlo一smo比p一疏基乙醇、sng/inLhseF、一oul/mL
毗正、long/mL bFGF、0.04ng/mL hIL一11、lon创mL IGF、100U加LL硫酸庆大霉
素、以鸡胚成纤维细胞作饲养层的培养体系中时，PGCs可贴壁，并可聚集增殖。
传代培养后，PGCs仍具有聚集增殖能力，并能进一步分化为其他类型的细胞。
This paper was focus on isolating primordial germ cells (PGCs) from gonads at stage 19 and stage 28 by Ficoll density-gradient centrifugation. Cryopreserved the PGCs at different freezing media, cryopretectant concentration and crypreservation procedure, then thawed after 7 days. PGCs was cultured in vitro in DMEM medium, let the PGCs differentiated spontaneously. The results showed:
1. Under the condition of 10% of the cryopretectant concentration. At the same freezing media, the viability of the frozen-thawed PGCs showed significant difference (P<0.05 )or very significant difference(P<0.01) between the different cryopreservation procedures. At the same cryopreservation procedure, the viability of the frozen-thawed PGCs showed significant difference (P<0.05) or very significant difference(P<0.01) among freezing media I , freezing media II, freezing media III, freezing media IV, freezing media V, freezing media VI. At the same cryopreservation procedure and freezing media, the viability of the frozen-thawed PGCs showed no significant difference between stage 19 and stage28.
Under the condition of 10% of the cryopretectant concentration. At the same cryopreservative condition, cryopreserved the PGCs after 24h cultured in vitro, the viability of the frozen-thawed PGCs showed very significant(P<0.01) lower than that of fresh PGCs cryoprederved .
3. Among the cryoprotectant concentration, the results were: between the concentration of 10% and 15%, the viability of the frozen-thawed PGCs showed no significant difference (P>0.05) except freezing media IV. When the cryoprotectant concentration was 20%, the viability of the frozen-thawed PGCs was the lowest, and showed significant difference (P<0.05 ) or very significant difference(P<0.01) compared to other concentration.
4. The thawed PGCs were cultured in DMEM media, supplemented with 10% fetal bovine serum, 2% chicken serum, 2 mmol glutamine, 1mmol sodium pyruvate, 5.5 x 10-5mol -mercaptoethanol, 100U/mL gentamycin sulfate. The thawed PGCs can adhere to the cell layer and form cell mass, but the PGCs can not proliferation. The PGCs gradually died when cultured continue.
5. The thawed PGCs were cultured in DMEM media, supplemented with 10% fetal bovine serum, 2% chicken serum, 2 mmol glutamine, Immol sodium pyruvate, 5.5 x 10"5mol p-mercaptoethanol, 5ng/mL hSCF, 10UI/mL mLIF, 10ng/mL bFGF, 0.04ng/mL hIL-11, 10ng/mL IGF, 100u/mL gentamycin sulfate. The PGCs can adhere to the cell layer, form cell mass and the PGCs can proliferation. When subcultured, PGCs still could form cell mass and proliferation, it also could differentiated into other cells.
1．当各种冷冻保护剂的浓度为10％时，同一种冷冻保护液。冷冻程序Ⅰ和冷冻程序Ⅱ之间，PGCs的存活率存在显著(P＜0.05)或极显著(P＜0.01)差异。而同一种冷冻程序。冷冻保护液1、冷冻保护液Ⅱ、冷冻保护液Ⅲ、冷冻保护液Ⅳ、冷冻保护液Ⅴ、冷冻保护液Ⅵ之间存在显著(P＜0.05)或极显著(P＜0.01)差异，比较其结果显示冷冻保护液Ⅲ的保护效果最好。而在同一冷冻保存条件下，第19期和第28期性腺中的PGCs存活率差异不显著(P＞0.05)。
2．当冷冻保护剂的浓度为10％时，在其他冷冻保存条件相同的情况下，分离后直接进行冷冻保存的PGCs，复苏后的存活率极显著(P＜0.01)高于经体外培养24 h后再进行冷冻保存的PGCs。
3．同一种冷冻保存条件下，冷冻保护液不同的浓度之间表现为：10％浓度与15％浓度相比较，细胞的存活率除冷冻保护液Ⅳ外，其余差异不显著(P＞0.05)：20％浓度下，细胞的存活率最低，且与10％及15％浓度下的存活率相比存在显著(P＜0.05)或极显著(P＜0.01)差异。
4．PGCs复苏后，当把其培养在以DMEM为基础液，添加10％的胎牛血清、2％的鸡血清、2mmol／L谷氨酰胺、1mmol／L丙酮酸钠、5.5×10~(-5)mol／L β-巯基乙醇、100U／mL硫酸庆大霉素、以鸡胚成纤维细胞作饲养层的培养体系中时，PGCs
可贴壁，并可聚集成细胞团，但不能克隆、增殖，继续培养下去，PGCs最终死亡。
5.PGCs复苏后，当把其培养在添加10%的胎牛血清、2%的鸡血清、2幻。roo比
谷氨酞胺、1 mmo比丙酮酸钠、5，sxlo一smo比p一疏基乙醇、sng/inLhseF、一oul/mL
毗正、long/mL bFGF、0.04ng/mL hIL一11、lon创mL IGF、100U加LL硫酸庆大霉
素、以鸡胚成纤维细胞作饲养层的培养体系中时，PGCs可贴壁，并可聚集增殖。
传代培养后，PGCs仍具有聚集增殖能力，并能进一步分化为其他类型的细胞。
This paper was focus on isolating primordial germ cells (PGCs) from gonads at stage 19 and stage 28 by Ficoll density-gradient centrifugation. Cryopreserved the PGCs at different freezing media, cryopretectant concentration and crypreservation procedure, then thawed after 7 days. PGCs was cultured in vitro in DMEM medium, let the PGCs differentiated spontaneously. The results showed:
1. Under the condition of 10% of the cryopretectant concentration. At the same freezing media, the viability of the frozen-thawed PGCs showed significant difference (P<0.05 )or very significant difference(P<0.01) between the different cryopreservation procedures. At the same cryopreservation procedure, the viability of the frozen-thawed PGCs showed significant difference (P<0.05) or very significant difference(P<0.01) among freezing media I , freezing media II, freezing media III, freezing media IV, freezing media V, freezing media VI. At the same cryopreservation procedure and freezing media, the viability of the frozen-thawed PGCs showed no significant difference between stage 19 and stage28.
Under the condition of 10% of the cryopretectant concentration. At the same cryopreservative condition, cryopreserved the PGCs after 24h cultured in vitro, the viability of the frozen-thawed PGCs showed very significant(P<0.01) lower than that of fresh PGCs cryoprederved .
3. Among the cryoprotectant concentration, the results were: between the concentration of 10% and 15%, the viability of the frozen-thawed PGCs showed no significant difference (P>0.05) except freezing media IV. When the cryoprotectant concentration was 20%, the viability of the frozen-thawed PGCs was the lowest, and showed significant difference (P<0.05 ) or very significant difference(P<0.01) compared to other concentration.
4. The thawed PGCs were cultured in DMEM media, supplemented with 10% fetal bovine serum, 2% chicken serum, 2 mmol glutamine, 1mmol sodium pyruvate, 5.5 x 10-5mol -mercaptoethanol, 100U/mL gentamycin sulfate. The thawed PGCs can adhere to the cell layer and form cell mass, but the PGCs can not proliferation. The PGCs gradually died when cultured continue.
5. The thawed PGCs were cultured in DMEM media, supplemented with 10% fetal bovine serum, 2% chicken serum, 2 mmol glutamine, Immol sodium pyruvate, 5.5 x 10"5mol p-mercaptoethanol, 5ng/mL hSCF, 10UI/mL mLIF, 10ng/mL bFGF, 0.04ng/mL hIL-11, 10ng/mL IGF, 100u/mL gentamycin sulfate. The PGCs can adhere to the cell layer, form cell mass and the PGCs can proliferation. When subcultured, PGCs still could form cell mass and proliferation, it also could differentiated into other cells.
引文
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