CN113462635A - 一种绵羊卵巢颗粒细胞分离、培养及鉴定方法 - Google Patents
一种绵羊卵巢颗粒细胞分离、培养及鉴定方法 Download PDFInfo
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Abstract
本发明公开了一种绵羊卵巢颗粒细胞分离、培养及鉴定方法,属于动物细胞培养技术领域。本申请通过特殊的收集、消毒、切割等处理方式获得卵巢颗粒细胞。本申请的方法具有简便、省力、污染率低、卵泡液获得率高等优点,为卵巢颗粒细胞、卵母细胞及卵泡发育的研究打下基础。
Description
技术领域
本发明属于动物细胞培养技术领域,具体涉及一种绵羊卵巢颗粒细胞分离、培养及鉴定方法。
背景技术
绵羊卵泡发育是由内分泌调控和外部调控相结合的一个十分复杂有序过程,颗粒细胞和卵母细胞作为卵泡的主要成分,在卵泡募集、选择、优势化、成熟、排卵及黄体形成等各个阶段都发挥着重要作用。了解绵羊卵泡发育过程及调控机制,可以有效提高绵羊排卵率,进一步提高绵羊繁殖效率。
颗粒细胞(granulosa cells,GCs)作为哺乳动物卵巢的主要功能细胞,在卵泡发育过程中发挥至关重要的作用。卵巢颗粒细胞表面存在卵泡刺激素(folliclestimulating hormone,FSH)和黄体生成素(luteinizing hormone,LH)受体,能在促性腺激素的作用下促使颗粒细胞发生增殖并分泌卵泡液,促使卵泡逐渐成熟。颗粒细胞同时能通过间隙连接为卵母细胞提供所需营养调节代谢,对于维持卵母细胞的正常功能起着重要的作用。此外,雄激素、雌激素、促性腺激素、生长激素等激素、EGF、IGF2I、TNF2ɑ等细胞因子及p53、Bcl-2、Fas等基因直接或间接参与或调控颗粒细胞凋亡,从而启动卵泡闭锁,影响卵泡发育。
因此,为了研究卵巢颗粒细胞增殖和凋亡对绵羊卵泡发育的调控机制,颗粒细胞的分离、培养及鉴定就成了首要任务。
目前,绵羊卵巢颗粒细胞分离多采用针头刺破卵泡或注射器吸取卵泡液的方法,虽然操作简便,但仍存在吸液费力、卵泡液浪费等现象;且绵羊卵巢颗粒细胞的分离或培养操作过程中存在细胞污染、细胞传代死亡等现象,鉴定过程存在染色不均、掉片、荧光过重等问题。鉴于此,如何提供一种高收率、低污染、高存活的绵羊卵巢颗粒细胞分离、培养及鉴定方法是本领域亟待解决的问题。
发明内容
本发明公开了一种绵羊卵巢颗粒细胞分离、培养及鉴定方法。
为了实现上述目的,本发明采用如下技术方案:
一种卵巢颗粒细胞分离方法,步骤如下:
(1)收集新鲜卵巢,消毒后,置于预热生理盐水中,浸泡2-4h;
(2)取出浸泡卵巢,消毒后,用预热生理盐水清洗;
(3)将步骤(2)处理后的卵巢剔除脂肪及系膜,之后用预热生理盐水清洗;
(4)将步骤(3)处理后的卵巢转移至培养基中,将刀片与水平呈30°-60°切割卵巢,释放卵泡液,操作在液面下完成,得离体卵泡液;
优选的,步骤(1)、(2)和(3)中,所述的预热生理盐水为37℃;
优选的,步骤(2)和(3)中,所述清洗的次数为3次;
优选的,步骤(4)中,所述新鲜卵巢为含直径3-7mm卵泡的卵巢;
优选的,步骤(4)中,所述培养基为DMEM/F12培养基;
一种卵巢颗粒细胞的培养方法,步骤如下:
(1)将权利要求1得到的离体卵泡液,离心去上清后,按0.4-1.5×105个/mL细胞密度加入完全培养基,混匀后,置于37℃、5%CO2条件下培养;
(2)24h换液后继续贴壁培养,每2天更换一次培养基;
(3)当细胞汇合度达70%-90%时进行传代;
优选的,步骤(1)中,所述离心参数为1500r/min 10min;
优选的,步骤(1)和(2)中,所述完全培养基为由以下成分配制而成:
DMEM/F1245mL+FBS 5mL+双抗500μL+50μg/mL丙酮酸钠1μL;
所述双抗包括100U/mL青霉素和100μg/mL链霉素;
一种卵巢颗粒细胞的鉴定方法,步骤如下:
以0.4-1.5×105个/mL的密度接种细胞,置于37℃、5%CO2培养箱中,当细胞汇合度达70%-90%,进行爬片,通过HE染色和FSHR免疫荧光结合鉴定。
综上所述,本发明公开了一种绵羊卵巢颗粒细胞分离、培养及鉴定方法。本申请通过特殊的收集、消毒、切割等处理方式获得卵巢颗粒细胞。本申请的方法具有简便、省力、污染率低、卵泡液获得率高等优点,为卵巢颗粒细胞、卵母细胞及卵泡发育的研究打下基础。
附图说明
图1消毒、清洗后的绵羊卵巢;A(含直径3-7mm卵泡的绵羊卵巢),B(含直径大于7mm卵泡的绵羊卵巢),C(含直径3-7mm卵泡及红体的绵羊卵巢),D(超排后含红体和黄体的绵羊卵巢),E(含白体的绵羊卵巢);
图2分离时卵巢颗粒细胞(40×);分离培养24h换液时观察到的卵巢颗粒细胞。
图3传代时卵巢颗粒细胞(40×);当细胞汇合度达70%-90%时,进行传代,原代细胞4-5天。
图4被污染的卵巢颗粒细胞(40×);分离培养换液后第3d观察到的卵巢颗粒细胞。
图5绵羊卵巢颗粒细胞形态学鉴定(100×);
图6绵羊卵巢颗粒细胞FSHR免疫荧光鉴定(200×)。
具体实施方式
下面对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
仪器
试剂
HE染色:
当细胞汇合度达80%时,使用4%多聚甲醛固定15min,PBS洗3次,每次5min;晾干后,苏木素染色15min,自来水洗涤3min;盐酸酒精分色5s,自来水浸洗3min,流水冲洗3min;浸入伊红染液染色3min,自来水洗涤,梯度酒精脱色,二甲苯透明,晾干后中性树胶封片,显微镜下观察细胞形态。
FSHR免疫荧光:
当盖玻片细胞汇合度达80%时,使用4%多聚甲醛固定20min,PBS洗3次,每次3min;0.2%Triton X-100通透10min,PBS洗3次,每次3min;BSA封闭30min,PBS清洗后,滴加FSHR一抗,4℃湿盒内过夜;PBS清洗后,滴加二抗,37℃孵育90min。然后进行DAPI染色,利用荧光显微镜观察细胞并拍照;最后用抗荧光淬灭封片液封片。
实施例1
(1)将成年经产的绵羊进行颈部放血处死,收集卵巢,酒精喷洗消毒,置于(含1%双抗,100U/mL青霉素和100μg/mL链霉素)预热生理盐水(37℃)中2h-4h内转移至无菌室。
(2)将步骤(1)收集的卵巢经75%酒精浸泡10s,37℃生理盐水清洗3遍后,除去卵巢上多余的脂肪和系膜,再用37℃生理盐水清洗3遍后,转置超净台内备好的10mL DMEM/F12完全培养基[DMEM/F12(45mL)+FBS(5mL)+双抗(500μL,所述双抗包括100U/mL青霉素和100μg/mL链霉素)+50μg/mL丙酮酸钠(1μL)]中,用刀片切割3-7mm卵泡(如图1所示),释放卵泡液,全程液面下操作。
(3)将细胞培养基混悬液置于15mL离心管,1500r/min离心10min,弃上清;PBS冲洗沉淀,1500r/min离心10min,弃上清,该过程重复3次。
(4)将上述所得沉淀加入适量DMEM/F12完全培养基[DMEM/F12(45mL)+FBS(5mL)+双抗(500μL,所述双抗包括100U/mL青霉素和100μg/mL链霉素)+50μg/mL丙酮酸钠(1μL)],吹打混匀后,接种于细胞瓶,置于37℃、5%CO2培养箱中培养。24h换液,倒置显微镜观察细胞,结果如图2所示,之后继续培养。
实施例2
(1)将成年经产的绵羊进行颈部放血处死,收集卵巢,酒精喷洗消毒,置于(含1%双抗,100U/mL青霉素和100μg/mL链霉素)预热生理盐水(37℃)中4h内转移至无菌室。
(2)将步骤(1)收集的卵巢经75%酒精浸泡10s,37℃生理盐水清洗3遍后,除去卵巢上多余的脂肪和系膜,再用37℃生理盐水清洗5遍后,转置超净台内备好的10mL DMEM/F12完全培养基[DMEM/F12(45mL)+FBS(5mL)+双抗(500μL,所述双抗包括100U/mL青霉素和100μg/mL链霉素)+50μg/mL丙酮酸钠(1μL)]中,用刀片切割3-7mm卵泡,释放卵泡液。
(3)将细胞培养基混悬液置于15mL离心管,1500r/min离心10min,弃上清;PBS冲洗沉淀,1500r/min离心10min,弃上清。该过程重复3次。
(4)将上述所得沉淀加入适量DMEM/F12完全培养基[DMEM/F12(45mL)+FBS(5mL)+双抗(500μL,所述双抗包括100U/mL青霉素和100μg/mL链霉素)+50μg/mL丙酮酸钠(1μL)],吹打混匀后,接种于细胞瓶,置于37℃、5%CO2培养箱中培养。24h换液后继续培养,每2d换次液。第4d进行传代(细胞汇合度达80%),倒置显微镜观察细胞,结果如图3所示。
实施例3
(1)将成年经产的绵羊进行颈部放血处死,收集卵巢,置于(含1%双抗,100U/mL青霉素和100μg/mL链霉素)预热生理盐水(37℃)中4h内转移至无菌室。
(2)将步骤(1)收集的卵巢经75%酒精浸泡10s,37℃生理盐水清洗3遍后,除去卵巢上多余的脂肪和系膜,再用37℃生理盐水清洗5遍后,转置超净台内备好的10mL DMEM/F12完全培养基[DMEM/F12(45mL)+FBS(5mL)+双抗(500μL,所述双抗包括100U/mL青霉素和100μg/mL链霉素)+50μg/mL丙酮酸钠(1μL)]中,用刀片切割3-7mm卵泡,释放卵泡液。
(3)将细胞培养基混悬液置于15mL离心管,1500r/min离心10min,弃上清;PBS冲洗沉淀,1500r/min离心10min,弃上清。该过程重复3次。
(4)将上述所得沉淀加入适量DMEM/F12完全培养基[DMEM/F12(45mL)+FBS(5mL)+双抗(500μL,所述双抗包括100U/mL青霉素和100μg/mL链霉素)+50μg/mL丙酮酸钠(1μL)],吹打混匀后,接种于细胞瓶,置于培养箱中培养。24h换液后继续培养,每2d换次液。
实施例4
(1)将成年经产的绵羊进行颈部放血处死,收集卵巢,酒精喷洗消毒置于(含1%双抗,100U/mL青霉素和100μg/mL链霉素)预热生理盐水(37℃)中4h内转移至无菌室。
(2)将步骤(1)收集的卵巢经37℃生理盐水清洗3遍后,除去卵巢上多余的脂肪和系膜,再用37℃生理盐水清洗5遍后,转置超净台内备好的10mL DMEM/F12完全培养基[DMEM/F12(45mL)+FBS(5mL)+双抗(500μL,所述双抗包括100U/mL青霉素和100μg/mL链霉素)+50μg/mL丙酮酸钠(1μL)]中,用刀片切割3-7mm卵泡,释放卵泡液。
(3)将细胞培养基混悬液置于15mL离心管,1500r/min离心10min,弃上清;PBS冲洗沉淀,1500r/min离心10min,弃上清。该过程重复3次。
(4)将上述所得沉淀加入适量DMEM/F12完全培养基[DMEM/F12(45mL)+FBS(5mL)+双抗(500μL,所述双抗包括100U/mL青霉素和100μg/mL链霉素)+50μg/mL丙酮酸钠(1μL)],吹打混匀后,接种于细胞瓶,置于培养箱中培养。24h换液后继续培养,每2d换次液。
实施例5
该绵羊卵巢颗粒细胞分离及培养方法,包括以下步骤:
(1)将成年经产的绵羊进行颈部放血处死,收集卵巢,酒精喷洗消毒,置于(含1%双抗,100U/mL青霉素和100μg/mL链霉素)预热生理盐水(37℃)中4h内转移至无菌室。
(2)将步骤(1)收集的卵巢经75%酒精浸泡10s,37℃生理盐水清洗3遍后,除去卵巢上多余的脂肪和系膜,再用37℃生理盐水清洗3遍后。
(3)将步骤2所得卵巢转置超净台内备好的10mL DMEM/F12完全培养基[DMEM/F12(45mL)+FBS(5mL)+双抗(500μL,所述双抗包括100U/mL青霉素和100μg/mL链霉素)+50μg/mL丙酮酸钠(1μL)]中,用刀片切割3-7mm卵泡,释放卵泡液。
(4)将细胞培养基混悬液置于15mL离心管,1500r/min离心10min,弃上清;PBS冲洗沉淀,1500r/min离心10min,弃上清。该过程重复3次。
(5)将上述所得沉淀加入适量DMEM/F12完全培养基[DMEM/F12(45mL)+FBS(5mL)+双抗(500μL,所述双抗包括100U/mL青霉素和100μg/mL链霉素)+50μg/mL丙酮酸钠(1μL)],吹打混匀后,接种于细胞瓶,置于37℃、5%CO2培养箱中培养。24h换液后继续培养。
(6)将上述分离所得细胞制备细胞爬片,用4%多聚甲醛固定,一方面用于HE染色进行细胞形态学观察,另一方面用于FSHR免疫荧光鉴定进行细胞纯度检测,结果如图5和6所示。
对分离的绵羊卵巢颗粒细胞进行鉴定,在倒置显微镜下可以观察到HE染色的绵羊卵巢颗粒细胞形态清晰完整,呈梭形、三角形或不规则的多边形,状态良好。免疫荧光结果显示FSHR绿色荧光蛋白在颗粒细胞的细胞质和细胞核均有表达,利用DAPI染核呈蓝色,经Merge后可见细胞核和细胞质相对应,FSHR阳性表达率高达99%,表明分离培养的原代细胞为绵羊卵巢颗粒细胞。
结果分析
实施例3和实施例4细胞分离过程分别缺少步骤(1)中酒精喷洗消毒和步骤(2)中75%酒精浸泡10s,易造成细胞污染,如图4。
实施例2基本按照该发明绵羊卵巢颗粒细胞分离、培养过程,与实例1一致,得到清晰、单一的细胞,如图2和图3。
实施例5添加了颗粒细胞鉴定过程,鉴定方法和步骤完全参照该发明,观察细胞形态和FSHR免疫荧光表达,从而判定该细胞是否为颗粒细胞,如图5和图6。
本说明书中各个实施例采用递进的方式描述,每个实施例重点说明的都是与其他实施例的不同之处,各个实施例之间相同相似部分互相参见即可。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对上述实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (8)
1.一种卵巢颗粒细胞分离方法,其特征在于,步骤如下:
(1)收集新鲜卵巢,消毒后,置于预热生理盐水中,浸泡2-4h;
(2)取出浸泡卵巢,消毒后,用预热生理盐水清洗;
(3)将步骤(2)处理后的卵巢剔除脂肪及系膜,之后用预热生理盐水清洗;
(4)将步骤(3)处理后的卵巢转移至培养基中,将刀片与水平呈30°-60°切割卵巢,释放卵泡液,操作在液面下完成,得离体卵泡液。
2.如权利要求1所述的卵巢颗粒细胞分离方法,其特征在于,步骤(1)、(2)和(3)中,所述的预热生理盐水为37℃。
3.如权利要求1所述的卵巢颗粒细胞分离方法,其特征在于,步骤(1)中,所述新鲜卵巢为含直径3-7mm卵泡的卵巢。
4.如权利要求1所述的卵巢颗粒细胞分离方法,其特征在于,步骤(4)中,所述培养基为DMEM/F12培养基。
5.一种卵巢颗粒细胞的培养方法,其特征在于,步骤如下:
(1)将权利要求1得到的离体卵泡液,离心去上清后,按0.4-1.5×105个/mL细胞密度加入完全培养基,混匀后,置于37℃、5%CO2条件下培养;
(2)24h后更换完全培养基,继续贴壁培养,每2天更换一次培养基;
(3)当细胞汇合度达70%-90%时进行传代。
6.如权利要求5所述的卵巢颗粒细胞的培养方法,其特征在于,步骤(1)中,所述离心参数为1500r/min 10min。
7.如权利要求5所述的卵巢颗粒细胞的培养方法,其特征在于,步骤(1)和(2)中,所述完全培养基为由以下成分配制而成:
DMEM/F1245mL+FBS 5mL+双抗500μL+50μg/mL丙酮酸钠1μL;
所述双抗包括100U/mL青霉素和100μg/mL链霉素。
8.一种卵巢颗粒细胞的鉴定方法,其特征在于,步骤如下:
以0.4-1.5×105个/mL的密度接种细胞,置于37℃、5%CO2培养箱中,当细胞汇合度达70%-90%进行爬片,通过HE染色和FSHR免疫荧光结合鉴定。
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