CN112646772A - 一种山羊精原干细胞体外诱导分化培养方法 - Google Patents
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Abstract
本发明公开了一种山羊精原干细胞体外诱导分化培养方法。所公开的方法包括采用同时添加有RA和BMP4的细胞培养液对山羊精原干细胞进行体外诱导分化培养。本发明通过外源添加RA和BMP4构建山羊SSCs体外诱导分化体系，有效提高了SSCs分化基因Stra8、c‑KIT的表达，为SSCs应用于转基因动物制备、生殖医学和家畜资源保护等提供有效途径。
Description
技术领域
本发明属于生物技术领域，具体通过外源添加RA和BMP4构建一种山羊精原干细胞体外诱导分化体系。
背景技术
精原干细胞(SSCs)在精子发生中发挥举足轻重的作用。SSCs是精子发生的基础，既确保了源源不断的产生精子，又保证了雄性动物生命过程中的生殖基础，对于繁衍后代具有非常重要的作用。SSCs的分化和自我更新能够保证SSCs数量处于动态平衡，如果在精子发生过程中出现分化受阻，将导致精原细胞的积累，从而影响精子的持续发生。SSCs的分化受到内在和外在因素的严格控调，睾丸内的细胞和细胞因子对于SSCs的分化具有重要的调控作用。
精原干细胞体外培养的建立首先是啮齿动物，为家畜SSCs体外培养体系的建立提供了基础。除目前公认的GDNF外，通过添加其他生长因子和激素也可以实现SSCs的长期培养。目前，在兔子、日本鹌鹑、树鼩中成功建立了有效的长期培养体系，但关于大家畜尤其是山羊的SSCs研究进展十分缓慢，目前还没有成功建立大家畜SSCs体外培养体系。
发明内容
针对现有技术的缺陷或不足，本发明提供了一种山羊精原干细胞体外诱导分化培养方法。
为此，本发明提供的山羊精原干细胞体外诱导分化培养方法包括：采用同时添加有RA和BMP4的细胞培养液对山羊精原干细胞进行体外诱导分化培养。
进一步，采用细胞培养液对山羊精原干细胞进行传代培养，之后采用同时添加有RA和BMP4的细胞培养液对经传达培养1～3次后的精原干细胞进行体外诱导分化培养。
进一步，本发明的方法包括：
(1)分离纯化山羊精原干细胞；
(2)采用细胞培养液对分离纯化的山羊精原干细胞进行传代培养，之后采用同时添加有RA和BMP4的细胞培养液对经传达培养1～3次后的精原干细胞进行体外诱导分化培养。
优选的，所述RA和BMP4的添加量分别为10±0.1nM和50±0.1ng/mL。
可选的，所述细胞培养液为含有FBS的DMEM培养基。
本发明通过外源添加RA和BMP4构建山羊SSCs体外诱导分化体系，有效提高了SSCs分化基因Stra8、c-KIT的表达，为SSCs应用于转基因动物制备、生殖医学和家畜资源保护等提供有效途径。
附图说明
图1为分离纯化前后的山羊精原干细胞电镜图，A为差贴前，B为差贴后；
图2为山羊精原干细胞传代培养3天后的克隆团电镜图，A为传代培养前，B为经3传代培养后；
图3为传代长期培养的山羊精原干细胞通过CD90/PLZF免疫荧光双标记染色法鉴定及双标记结果Merge融合图；
图4为实施例培养液中添加RA和BMP4培养24h后山羊精原干细胞分化相关基因表达图。
具体实施方式
除非另有说明，本文中的术语根据相关领域普通技术人员的常规认识理解。
本发明是用同时添加有RA和BMP4的基础细胞培养液对山羊精原干细胞进行体外诱导分化培养，其中基础细胞培养液为适合山羊精原干细胞体外分化生长的培养液，包括但不限于现有技术已经公开的培养液，也可以是已与培养液自身的作用改进后的细胞培养液。
本发明所述山羊精原干细胞可为市售产品，也可存在用现有相关技术进行分离纯化后获得，所得山羊精原干细胞经传代培养合理代数后，采用本发明的方法进行体外诱导分化。所述的分离纯化方法可采用现有技术中公开的相关方法或为获得山羊精原干细胞所改进的分离纯化方法，一种山羊精原干细胞分离纯化方法示例如下(本文示例中所用试剂如无特殊说明均购自Sigma公司)：
(1)分离纯化山羊精原干细胞
(1.1)睾丸的处理与单细胞悬液的制备
活体阉割法取下1～2月龄山羊睾丸，首先用75％酒精进行初步杀菌，然后转移至含有5％双抗的PBS溶液中；
在培养皿中加入PBS，用眼科剪刀将睾丸剪开去除白膜，同时去除位于睾丸组织中间的结缔组织，然后将睾丸组织剪碎，加入PBS清洗3次后，加入V胶原酶和透明质酸酶消化25min，4℃低温离心后弃上清，得到睾丸组织碎片；
之后加入重悬后静置于离心管中使其自然沉降，弃上清得到曲细精管的碎片，随后首先加入细胞裂解液离心，弃上清，然后加入0.25％胰酶使细胞分散均匀后加入含有10％FBS的DMEM溶液终止消化，4℃低温离心，弃上清；最后加入5％FBS的DMEM溶液重悬，400目的细胞筛过筛，收集细胞悬液，调整细胞密度为1×107个/mL左右，得含有支持细胞和SSCs的细胞悬液；
(1.2)精原干细胞的纯化
采用差速贴壁法对细胞悬液进行纯化：接种的细胞在37℃、CO2含量为5％的细胞培养箱中培养，将培养皿中没有贴壁的细胞转移至新的提前用明胶包被的培养皿中继续培养；细胞培养时分别进行3次差贴(约24h)，之后收集细胞悬液，离心后加入2mL含2％FBS的DMEM进行SSCs的培养；其中在第一次差贴的贴壁细胞的密度达到70％～80％时，吸出培养基，用PBS清洗3遍后加入10μg/mL丝裂霉素C，处理3h后吸出丝裂霉素C，用PBS清洗细胞3遍，再加入2％FBS的DMEM培养2h后即可接入SSCs，电镜图如图1所示。
本发明的山羊精原干细胞传代培养可采用现有技术或为获得传代培养后的细胞所改进的相应技术。一种示例如下：
继上述分离纯化示例之后，在SSCs的培养过程中，每24h更换一次培养液，并观察细胞状态，当细胞密度达到70％～80％时即可进行细胞传代培养，传代比例是1:3，传代培养后细胞如图2所示。
为验证传代所得细胞的纯度，发明人上述示例传代3次后的SSCs利用荧光免疫鉴定已知抗原(标记分子CD90和PLZF)的表达：
将细胞接种于24孔板中，每孔1×104个细胞，待细胞融合度为60％～70％进行染色；
用新配置的4％多聚甲醛固定细胞30min，然后用2％多聚甲醛固定15～30min。PBS清洗后加入0.5％的Triton X-100增加细胞的通透性；
PBS稀释血清，室温封闭60～90min后将封闭液吸出，直接加入一抗稀释液(一般抗体稀释比例为1:100～200)，室温封闭2h或4℃封闭过夜。PBS清洗后加入生物素标记的二抗(稀释比例1:100～200)，室温孵育30min。PBS清洗后加入SABC-Cy3(稀释比例1:100～200)，室温孵育30min，PBS清洗3次。最后进行核染DAPI，PBS清洗3次后荧光显微镜下观察拍照。
结果显示：免疫荧光染色的具体结果如图3所示，纯化后的山羊SSCs表达生殖干细胞的标记分子CD90和PLZF，图中DAPI为可以穿透细胞膜的蓝色荧光染料，用于活细胞的染色；Merge表明所染色的细胞是活细胞且具有精原干细胞的标记分子。
实施例：
将传代3次后的山羊精原干细胞在细胞培养液中添加RA或/和BMP4联合进行诱导分化：
示例1：RA和BMP4在细胞培养液中的终浓度分别为10nM RA和50ng/mL BMP4；
示例2：细胞培养基+10nM RA；
示例3：细胞培养基+50ng/mL BMP4；
示例4：细胞培养基+10nM RA+2ng/ml Noggin；
上述细胞培养液为含有2％FBS的美国Gibco公司生产的DMEM培养基；Noggin购自Sigama公司。
连续培养24h测定SSCs分化相关基因的表达情况，测定方法如下：
(1)总RNA提取
采用Trizol试剂盒(Takara)提取样品的总RNA；将样品从-80℃冰箱中取出，置于冰上解冻后，4℃离心5min；吸取上清液至新的1.5mL的试管中，加入1/5RNAiso Plus体积量的氯仿，振荡至溶液充分乳化；4℃离心后小心吸取上清至新的离心管中，加入等体积的异丙醇，用手上下摇晃使之充分混匀，静置10min；离心弃上清，加入1mL 75％乙醇，重复2次。再次离心后干燥沉淀，加入DEPC处理水，吹打溶解。
(2)总RNA完整性及浓度鉴定
1)总RNA电泳检测：Total RNA与6×Loading Buffer按照5:1混合后加样，调整电压、电流及电泳时间进行跑胶；RNA经电泳检测可见清晰的18S和28S两个条带，以证明RNA的完整性良好；
2)RNA浓度的测定：利用核酸蛋白测定仪检测总RNA的浓度和纯度。其中OD 260/280比值在1.8～2.2间可用，说明RNA纯度较高；OD 260/280<1.8，表明RNA中污染了蛋白质或有机物，OD 260/280>2.2，表明RNA已经降解。
先将反转录试剂及模版RNA解冻，然后配制gDNA去除反应体系。按照表1去除基因组DNA的混合液，完全混匀后离心，并在42℃条件下反应，然后在冰面上进行冷却。配制反转录体系，并加入gDNA去除反应液，在42℃条件下反应5min，然后置于95℃条件下反应3min，再置于冰面上冷却，最后获取cDNA；
表1 gDNA去除反应体系
3)Real-time PCR反应体系建立：将2×SuperReal Color PreMix、cDNA模板、上下游引物(序列图表3所示)、40×Dilution Buffer和Rnase-Free ddH2O等物质置于室温下解冻，震荡混匀；在冰面上配制Real-time PCR反应体系，具体见表2。
表2实时荧光定量PCR反应体系
表3相关引物序列
4)Real-time PCR具体反应步骤：混匀反应物，离心1min；向PCR仪加入反应体系，进行反应；反应程序见表4：
表4实时荧光定量PCR反应程序
结果显示：如图4所示，SSCs分化基因Stra8、c-KIT的表达在RA+BMP4组分化基因的表达量最高，说明在山羊SSCs基础培养液中，联合添加RA和BMP4的诱导分化效果优于单独添加RA和BMP4。
上述实施例只是为清楚地说明发明内容所列举的例子，而并非对具体实施方式和技术做出的限定。相关领域的技术人员可以在上述说明的基础上，根据所学或从业的经验做出合理改动，以符合当时或当地的具体情况，而由此发明所引伸出的任何改动应属于本发明的范围。
Claims (5)
1.一种山羊精原干细胞体外诱导分化培养方法，其特征在于，方法包括：采用同时添加有RA和BMP4的细胞培养液对山羊精原干细胞进行体外诱导分化培养。
2.如权利要求1所述的山羊精原干细胞体外诱导分化培养方法，其特征在于，采用细胞培养液对山羊精原干细胞进行传代培养，之后采用同时添加有RA和BMP4的细胞培养液对经传达培养1～3次后的精原干细胞进行体外诱导分化培养。
3.如权利要求2所述的山羊精原干细胞体外诱导分化培养方法，其特征在于：方法包括：
(1)分离纯化山羊精原干细胞；
(2)采用细胞培养液对分离纯化的山羊精原干细胞进行传代培养，之后采用同时添加有RA和BMP4的细胞培养液对经传达培养1～3次后的精原干细胞进行体外诱导分化培养。
4.如权利要求1-3任一权利要求所述的山羊精原干细胞体外诱导分化培养方法，其特征在于，所述RA和BMP4的添加量分别为10±0.1nM和50±0.1ng/mL。
5.如权利要求1-3任一权利要求所述的山羊精原干细胞体外诱导分化培养方法，其特征在于，所述细胞培养液为含有FBS的DMEM培养基。
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