CN110938589A - 一种许氏平鲉精原干细胞的分离和移植方法 - Google Patents
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Abstract
本发明涉及卵胎生鱼类精原干细胞分离及移植方法,具体的说是一种许氏平鲉精原干细胞的分离和移植方法。将处于5月龄‑1龄或性腺发育初期的雄性许氏平鲉的完整性腺经消化酶消化离心，获得精原干细胞，然后通过显微注射将精原干细胞移植至仔鱼腹腔。本发明提供了一种卵胎生鱼类精原干细胞分离和移植方法，该方法分离并移植后的精原干细胞经荧光标记后可在受体鱼内观测到供体生殖细胞的迁移和增殖过程。该方法的为海水鱼类种质资源的保存、保护以及新品种的培育提供了新方法。
Description
技术领域
本发明涉及海水鱼类精原干细胞的分离,具体的说是一种卵胎生鱼许氏平鲉类精原干细胞的分离和移植方法。
背景技术
许氏平鲉是我国北方经济价值很高的海水养殖鱼类，其肉质细嫩，营养丰富，深受消费者欢迎。许氏平鲉属鮋科、平鮋属，是卵胎生鱼类，由于在国内长累代的养殖，近亲交配较为严重，导致病害频发和产量下降。精巢中精原干细胞属于生殖干细胞,既可自我更新,又具有分化的全能型，可以分化形成各阶段的生殖细胞直至成熟配子。日本学者已经成功的在虹鳟鱼实现精原干细胞的移植，并成功获得后代。
但对于卵胎生鱼类并未相关记载，本发明建立了海水卵胎生鱼类精原干细胞的分离和移植的方法对于海洋种植资源的保存、保护以及海水养殖鱼类的可持续发展具有重要意义。
发明内容
本发明目的在于提供一种卵胎生鱼许氏平鲉类精原干细胞的分离和移植方法。
为实现上述目的，本发明采用技术方案为：
一种许氏平鲉精原干细胞的分离，将处于5月龄-1龄或性腺发育初期的雄性许氏平鲉的完整性腺经消化酶消化离心，分离获得精原干细胞。
将许氏平鲉的精巢(性腺)剪碎后加入至5-10倍体积的L-15培养基中混合，加入混合物质量0.004％的胶原酶和混合物质量0.03％的中性酶消化，消化过程中反复进行吹打混匀，使精原干细胞充分解离3-4h。
将消化分离后细胞分解液过滤得解离的细胞团，而后向其中加入L-15培养基，离心收集沉淀，沉淀在经L-15培养基洗涤，洗涤后经L-15培养基重悬，并通过胎盘蓝染色确定精原干细胞的比率以及活性；即获得结构清晰、无杂质、高成活率的精原干细胞。
一种许氏平鲉精原干细胞的移植方法，将权利要求1所述精原干细胞悬液经终浓度为0.2％荧光染料PKH26进行染色，并用L-15培养基重悬细胞，洗涤染料，收集细胞悬液。
采集初孵的许氏平鲉仔鱼并用玻璃针将细胞悬液注入7-10日龄仔鱼腹腔脊椎骨鱼肠中间部位，每尾仔鱼注射约105-106个精原干细胞，即实现精原干细胞的移植。
采集初孵的许氏平鲉仔鱼采用玻璃针，将细胞悬液注入仔鱼腹腔脊椎骨鱼肠中间部位，每尾仔鱼注射约105-106个精原干细胞，即实现精原干细胞的移植。
所述移植于4℃下24-30h，而后将移植后仔鱼置于1-5％双抗(青霉素/链霉素)灭菌海水中静水孵化，微充气，即得嵌合体仔鱼。
本发明所具有的优点：
本发明卵胎生鱼类精原干细胞分离和移植方法，该方法分离并移植后的精原干细胞经荧光标记后可在受体鱼内观测到供体生殖细胞的迁移和增殖过程。该方法的为海水鱼类种质资源的保存、保护以及新品种的培育提供了新方法；
采用本发明方法获得适宜的发育期雄鱼性腺可获得高纯度的精原干细胞；筛选出适宜日龄的仔鱼进行移植，外源精原干细胞整合到供体性腺的比例高；本发明分离精原干细胞的方法简便、快速、重复性好；且，移植方法简单、对仔鱼损伤小，移植后仔鱼成活率高，嵌合体比率为100％。
附图说明
图1为本发明实施例提供采用本发明方法分离后的精原干细胞效果图；其中，A为分离获得精原干细胞悬液；B为荧光染色后的精原干细胞。
图2为本发明实施例提供采用本发明方法移植后60天幼鱼体内的外源精原干细胞效果图(外源精原干细胞移植入受体仔鱼后在性腺中的分布)；其中，箭头所示为红色荧光标记的精原干细胞。
具体实施方式
以下结合附图和实例对本发明的具体实施方式做进一步说明，应当指出的是，此处所描述的具体实施方式只是为了说明和解释本发明，并不局限于本发明。
实施例
1.分离精原干细胞过程：
1)取5月龄-1龄300-400g许氏平鮋雄鱼性腺，放入离心管，并用剪碎呈浆状；
2)配置500mL消化液，25mLFBS、0.02g胶原酶H、0.0165中性酶Ⅱ、475mL L-15。向上述剪碎呈浆状性腺中加入250mL消化液，混合后于25℃温育2.5-3h，温育过程中用移液管轻柔吹打细胞分散；
3)采用42um孔径尼龙筛对上述获得细胞分散液过滤，收集滤液，向滤液中加1mLL-15培养基洗涤，200g离心5min，弃上清；
4)向上述离心后沉淀中加入200uL L-15培养基将细胞重悬，而后经台盼蓝染色，观察细胞状态(参见图1)，由图1观察可见细胞结构清晰，无杂质，经统计，A型精原细胞比率60-80％；
2.移植精原干细胞过程：
1)将上述分离所得1mL2×悬液快速加入1mL2×染料溶液中，立即吹打混匀，每管相当于500uL悬液加500uL染料，孵育5min，向孵化液中加等体积的1％FBS(FBS用培养基稀释后得1％FBS)，孵育1min；
2×染料溶液为PKH26。
2)将孵育后细胞经200g离心5min，小心去除上清，加1mL L-15培养基获重悬细胞，而后再次离心再经重悬，进而除去未结合的染料，得细胞浓度为107cells/mL的重悬细胞液；
3)采集初孵的许氏平鲉仔鱼并用玻璃针将细胞悬液注入仔鱼腹腔脊椎骨鱼肠中间部位，移植于4℃下每尾仔鱼注射约105-106个精原干细胞，24h移植完成，移植后的2天内在1-5％双抗(青霉素/链霉素)灭菌自然海水中培育，投喂轮虫，两天后加入微藻环境，约10天后开始喂卤虫，逐渐转向配合饲料(参见图2)。
由图2可见移植后在受体的性腺内观察到供体的精原干细胞。
Claims (6)
1.一种许氏平鲉精原干细胞的分离，其特征在于：将处于5月龄-1龄或性腺发育初期的雄性许氏平鲉的完整性腺经消化酶消化离心，分离获得精原干细胞。
2.按权利要求1所述的许氏平鲉精原干细胞的分离，其特征在于：将许氏平鲉的精巢(性腺)剪碎后加入至5-10倍体积的L-15培养基液中混合，加入混合物质量0.004％的胶原酶和混合物质量0.03％的中性酶消化，消化过程中反复进行吹打混匀，使精原干细胞充分解离3-4h。
3.按权利要求1或2所述的许氏平鲉精原干细胞的分离方法，其特征在于：将消化分离后细胞分解液过滤得解离的细胞团，而后向其中加入L-15培养基离心收集沉淀，沉淀在经L-15培养基础洗涤，洗涤后经L-15培养基重悬，并通过胎盘蓝染色确定精原干细胞的比率以及活性；即获得结构清晰、无杂质、高成活率的精原干细胞。
4.一种许氏平鲉精原干细胞的移植方法，其特征在于：将权利要求1所述精原干细胞悬液经终浓度为0.2％荧光染料PKH26进行染色，并用L-15培养基重悬细胞，洗涤染料，收集细胞悬液。
5.按权利要求4所述的许氏平鲉精原干细胞的移植方法，其特征在于：采集许氏平鲉7-10日龄仔鱼并用玻璃针将细胞悬液注入仔鱼腹腔脊椎骨与肠中间部位，每尾仔鱼注射约105-106个精原干细胞，即实现精原干细胞的移植。
6.按权利要求5所述的许氏平鲉精原干细胞的移植方法，其特征在于：所述移植于4℃下24-30h，而后将移植后仔鱼置于1-5％双抗(青霉素/链霉素)灭菌自然海水中静水孵化，微充气，即得嵌合体仔鱼。
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