CN112458041A - 一种用于绵羊卵巢皮质组织体外培养的无血清培养液 - Google Patents
一种用于绵羊卵巢皮质组织体外培养的无血清培养液 Download PDFInfo
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Abstract
本发明公开了一种用于绵羊卵巢皮质组织体外培养的无血清培养液,所述培养液包括基础培养液和添加物,所述基础培养基为α‑MEM和DMEM的混合液;所述添加物包括以下含量的组分:3~10mg/mL牛血清白蛋白、0.10~0.50mM丙酮酸钠、1~5 mM谷氨酰胺、1~5mM次黄嘌呤、3~10μg/mL胰岛素、1~5μg/mL转铁蛋白、2~8ng/mL亚硒酸钠、10~200μg/mL VC、50~200IU/mL青霉素和50~200IU/mL链霉素,pH 6.9~7.5。本发明通过各组分间的协同作用,既抑制间质细胞的增殖速度,又促进颗粒细胞增殖,解决了卵巢皮质组织在培养过程中间质细胞过度增殖导致组织塌陷无法维持原始卵泡发育的立体环境的缺陷,有效提高了卵巢皮质组织中原始卵泡在体外的生长发育情况和存活时间,同时避免了血清中未知成分对研究的影响,具有良好的应用前景。
Description
技术领域
本发明细胞生物学技术领域,特别是涉及一种用于绵羊卵巢皮质组织体外培养的无血清培养液。
背景技术
卵巢作为雌性生育的繁殖基础,含有大量的处于不同发育阶段的卵泡,是雌性繁殖力的储备库。在生理状态下,90%以上的卵泡以休眠状态的原始卵泡形式存在于卵巢皮质中,并在生殖相关激素激素、生长因子作用下一批批被募集激活进入发育卵泡阶段。遗憾的是随着个体的发育进行,大部分卵泡都由于退化而损失,只有不到1%卵泡会发育至成熟卵子并最终受精产生后代,这是对雌性动物繁殖力的极大浪费。目前对雌性绵羊卵巢内卵泡的利用主要集中在次级卵泡以后的卵泡尤其是有腔卵泡,这部分卵泡对数以万计的卵巢卵泡而言,只是其中很少的一部分。随着克隆和转基因技术的发展,将来对卵母细胞的需求越来越大,光靠回收窦腔卵泡中的卵子已不能满足需求。通过体外培养卵巢组织或卵泡,复苏卵巢中占主导地位的原始卵泡的生长和发育,可以加深我们对卵泡发育,排卵和闭锁的生理调节机制的了解,为辅助生育技术及其他生物工程技术提供潜在的额外的同种质卵母细胞来源,对于提高高遗传和经济价值的家畜以及在科研上具有重要价值的动物的繁殖潜力,珍稀或濒危动物的保护以及人类生育能力的保存和辅助受孕具有重大意义。
目前对卵巢组织或分离卵泡的体外培养已经开展了许多研究,方法实例包括分离卵泡的三维培养、卵巢皮质和髓质共培养、整个卵巢的器官培养、卵巢皮质组织的培养等。在体内环境下,原始卵泡的激活和生长发育的发生受到年龄、营养水平、激素环境以及多种生长因子等诸多因素的综合影响,且原始卵泡与卵巢间质细胞、卵巢髓质细胞、颗粒细胞以及其他发育阶段卵母细胞等的细胞间交流通讯也会对其激活过程产生影响,所以这一过程在体内环境下存在许多的不可控因素或相关抑制干扰。而在体外培养模式下,可以人为的去除大部分干扰因素,为研究原始卵泡的激活和生长发育过程提供了一种条件限定的可控研究环境,这种可控研究环境可以帮助我们鉴定调节卵巢功能的相关因子对卵泡发育的影响,这些因子的鉴定在体内条件下可能不易完成或很难确定。通过限定条件的体外培养体系的研究我们可以对各种因子,激素或相关生理信号途径的促进剂或抑制剂,以及某些对生殖有毒有害的化学物质进行系统的鉴定。而如何开发能有效支持卵巢原始卵泡长期存活、成分已知且更接近于生理状态营养需求的培养液是进行原始卵泡体外激活研究的基础。
近年来,研究者针对卵泡发育也不断开发了一系列不同的培养液配方,如发明专利CN102250831A提供制备促进离体绵羊腔前卵泡体外发育的培养基的方法,将胰岛素、转铁蛋白、亚硒酸钠、丙酮酸钠、谷氨酰胺、次黄嘌呤、血清、促卵泡素、促黄体素、雌激素、PTEN抑制剂和 α-MEM 培养基混合,得到培养基。但该培养基中含有血清,由于血清成分复杂,批次之间波动较大,使用有血清系统不利于后续对影响卵泡发育的各种细胞因子和激素的检测和分析,更重要的是该方法应用于初级卵泡阶段以后的分离腔前卵泡培养有较好的效果,目前国内可查阅的文献与专利,腔前卵泡一般指的是初级卵泡阶段以后的卵泡,此阶段的卵泡已经处于生长阶段,而原始卵泡是处于休眠状态中的卵泡,还需要有激活的过程,受到更加复杂的调控,目前对于其激活机制研究尚不明确,在体外培养中需要激活其发育进程,同时抑制其凋亡。因此原始卵泡阶段的生长发育营养需求与其他发育阶段卵泡发育需求也有较大差异。且绵羊原始卵泡在体外的生长需要较长的时间,但在体外长时间培养过程中常常会产生间质细胞过度增殖而导致组织塌陷无法维持卵泡发育的立体环境的缺陷,至今专门针对绵羊组织尤其是卵巢皮质组织中原始卵泡的专用培养基还未有相关报道。
无血清培养基由于其各种添加成份明确,实验条件易于控制的优点,已成为发展的趋势。因此开发一种成本较低、能支撑原始卵泡生长和一定的组织环境以维持其立体结构的新型无血清培养对于研究卵巢原始卵泡发育机制至关重要。
发明内容
针对现有存在的上述不足,本发明的目的在于提供一种用于绵羊卵巢皮质组织体外培养的无血清培养液,解决卵巢皮质组织中原始卵泡不能进行长时间的体外培养,大大限制了其产业发展和教学科研的问题。
为实现上述目标,本发明的主要技术方案如下:一种用于绵羊卵巢皮质组织体外培养的无血清培养液,所述培养液包括基础培养液和添加物,所述基础培养基为α-MEM和DMEM的混合液;所述添加物包括以下含量的组分:3~10 mg/mL牛血清白蛋白、0.10~0.50 mM丙酮酸钠、1~5 mM谷氨酰胺、1~5 mM次黄嘌呤、3~10 μg/mL胰岛素、1~5 μg/mL转铁蛋白、2~8ng/mL亚硒酸钠、10~200 μg/mL VC、50~200 IU/mL 青霉素和50~200 IU/mL 链霉素,pH 6.9~7.5。
这样,采用α-MEM和DMEM培养基作为基本培养基,其中营养元素的种类选择更能保证卵巢皮质组织中原始卵泡的基本生长需求,牛血清白蛋白可以很好的替代血清中所含血清蛋白的基本功能,作为一种功能营养分子,更加适用于多细胞状态的组织长期体外培养,具有抗氧化、调节体外生理环境,维持体外培养组织的渗透压和pH值,有利于组织细胞中营养分子的运输等优点。本发明中牛血清白蛋白浓度低于1 mg/mL时,不利于细胞生长增殖,高于30 mg/mL时颗粒细胞增殖受到抑制,不利于卵泡发育与存活;然后通过额外添加营养因子谷氨酰胺和丙酮酸钠弥补α-MEM培养基能量水平相对较低的缺陷;添加胰岛素-转铁蛋白-硒系统可以有效防止卵泡闭锁,并维持颗粒细胞增殖与存活,同时结合VC的添加可以有效清除培养基中的游离自由基,降低细胞凋亡的发生,而且在卵泡细胞增殖和发育过程中需要大量的胶原来维持基底膜完整,VC可以促进胶原的合成,对于维持组织正常生长环境有利;通过无血清培养液中各营养因子之间的协同配伍作用,既能抑制间质细胞的增殖速度,又能促进颗粒细胞增殖,有效解决卵巢皮质组织在培养过程中因间质细胞过度增殖导致组织塌陷而无法维持卵泡发育所需的立体环境的缺陷,从而有效提高卵巢皮质组织中原始卵泡在体外存活时间,实现卵巢皮质组织中原始卵泡在体外长时间的培养。
使用前,每10 mL培养液中添加20~100 μL HEPES溶液(100 mmol/L),较佳的为100μL HEPES溶液(100 mmol/L);和/或培养前培养液用1 mol/L NaOH溶液和1 mol/L HCl溶液调整pH至6.9~7.5。
作为优选的,所述基础培养液中α-MEM和DMEM的体积比为6~10: 1~4;进一步,所述基础培养液中α-MEM和DMEM的体积比为4: 1。
作为优选的,所述添加物包括以下含量的组分:0.2 mM丙酮酸钠、2 mM谷氨酰胺、2mM次黄嘌呤、8 μg/mL胰岛素、5 μg/mL转铁蛋白、6.0 ng/mL亚硒酸钠、50 μg/mL VC、100IU/mL青霉素和100 IU/mL链霉素。
本发明的另一个目的在于,还提供了上述无血清培养液在用于绵羊卵巢皮质组织体外培养方面的应用。
本发明的另一个目的在于,还提供了一种体外培养绵羊卵巢皮质组织的方法,具体包括以下步骤:获取绵羊的卵巢皮质组织并切割成碎块,然后将得到的皮质碎块置于嵌入式细胞培养小室表面,再加入上述无血清培养液,使无血清培养液的液面没过皮质碎块的下表面但不高于所述皮质碎块的上表面,再将其置于5% CO2,35~38℃的培养箱中进行气液相界面培养。
进一步,所述皮质碎块大小为0.5~4 mm3,优选为1 mm3。
进一步,所述培养小室表面的皮质碎块的放置密度为6~20块/小室,优选为12块/小室。
进一步,所述绵羊选自新疆军垦型细毛羊,亦可是其他品种绵羊或山羊。
进一步,所述培养时间为6~10天。
相比现有技术,本发明具有如下有益效果:
1、本发明采用的培养液是以α-MEM培养基和DMEM培养基的混合液作为基本培养基,能够满足卵巢皮质组织的基本生长需求,再通过额外添加营养因子的协同作用,即抑制间质细胞的增殖速度,又维持颗粒细胞正常增殖和原始卵泡的正常激活以及存活,有效解决了卵巢皮质组织在培养过程中因间质细胞过度增殖导致组织塌陷而无法维持卵泡发育所需的立体环境的缺陷。经试验证明,本发明构建的培养体系能有效支持卵巢皮质组织体外存活10天以上,仍可观察到正常增殖的颗粒细胞和卵母细胞,且组织形态完整没有发生组织塌陷的现象;最长培养42天仍可观察到正常的卵泡结构和存活的间质细胞,可见,本发明有效提高了卵巢皮质组织中原始卵泡在体外的生长发育情况和存活时间。
2、本发明提供的培养液的配料均为常见化合物,廉价易得,成本低,制备方法简便,体系成分清晰含量明确,提高了培养过程的可控性,便于有效分析添加各类激素和生长因子对卵泡体外发育的影响,为研究卵泡发育生物学机制提供了一种有效的体外培养环境,无需使用血清,最大程度上解决了由于血清成分复杂及批次波动对研究各类因子和激素对卵泡发育作用的影响。
3、本发明构建的卵巢皮质组织体外培养体系,具有易操作,培养程序简单等优点,该培养体系既能维持组织存活和原始卵泡的正常发育,又创造一个已知成分且环境稳定的实验条件,不仅可用于任何对卵泡发育起作用的激素和生长因子的直接或间接作用的研究,还可以用于卵巢发育和卵泡发育中各类活性物质或毒性物质的安全性和功效性检测(毒理学研究),拓宽了对体系的应用范围,尤其是对原始卵泡体外发育规律的研究是一种理想模式,为卵泡发育研究提供了理论基础,具有良好的应用前景。
说明书附图
图1为培养前后的卵巢碎块形态图;a为培养前,b为基础培养液培养4天,c为无血清培养液培养10天。
图2为培养前后卵巢皮质组织切片HE染色图;a为培养前,b为基础培养液培养4天,c为无血清培养液培养10天。
图3为培养前后卵泡超微结构图;a为培养前,b为基础培养液培养4天,c为无血清培养液培养10天。
图4为培养前后组织PCNA检测图;a为培养前,b为基础培养液培养4天,c为无血清培养液培养10天。
图5为培养前后组织TUNEL检测图;a为培养前,b为基础培养液培养4天,c为无血清培养液培养10天。
具体实施方式
以下结合附图和具体实施方式对本发明作进一步说明。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。以下实施例采用的卵巢皮质组织取自新疆军垦型细毛羊为例说明,但该方法也适用于其它品种绵羊或山羊,在此未一一列举。
实施例
1、配置培养基
基础培养液:将α-MEM和DMEM按照体积比4:1添加。
无血清培养液:在上述基础培养液中依次添加BSA 5 μg/mL,丙酮酸钠0.2 mM,谷氨酰胺3 mM,次黄嘌呤3 mM,胰岛素10 μg/mL,转铁蛋白6.25 μg/mL,亚硒酸钠6.25 ng/mL,VC 50 μg/mL,青霉素100 IU/mL,链霉素100 IU/mL。
2、获取卵巢皮质组织及碎块
使用手术法采集新疆军垦型细毛羊羔羊卵巢组织样本,保存于37.5℃添加有200 IU/mL青霉素和200 IU/mL链霉素的0.9%生理盐水中1h内送回实验室,依次经添加有100 IU/mL青霉素和100 IU/mL链霉素的生理盐水中漂洗2次、酒精漂洗1次和生理盐水漂洗2次,然后在体式显微镜下去除卵巢表面筋膜及附属物。
再在体式显微镜下,用手术刀片在卵巢幽门部十字剖分,生理盐水漂洗,切取四分之一卵巢,在体式显微镜下小心去除卵巢髓质,保留皮质于MEM液中,将皮质分割成0.5-1mm3大小卵巢皮质碎块依次经过生理盐水—生理盐水-磷酸缓冲液-α-MEM漂洗(每步5s),然后置于预热α-MEM培养液中备用,即得到边沿齐整的卵巢皮质碎块。
3、皮质碎块的体外培养
在体式显微镜下用移液器小心吸取得到的卵巢皮质碎块并均匀置于预涂层处理的嵌入式细胞培养小室表面,每个小室放置6-9块碎块,将放有碎块的小室随机分为对照组和试验组,向对照组中每个培养孔内添加2 mL基础培养液,向试验组的每个培养孔内添加2 mL无血清培养液,使得基础培养液或无血清培养液的液面浸润皮质碎块下表面但未没过碎块上表面,分别在对照组和试验组的每个碎块上表面滴加100 μL基础培养液或无血清培养液。将上述培养小室置于37℃,5% CO2培养箱中进行培养,每两天换液一次,在培养的0、2、4、6、8、10天每组分别取样,进行组织学和免疫组化分析。
4、培养结果分析
(1)分别在培养的0、2、4、6、8、10天取对照组和试验组的卵巢碎块,在倒置显微镜下直接观察培养状态,结果如图1所示。
从图1中可以看出,用基础培养液培养的卵巢碎块在培养第4天,组织形态已出现塌陷呈弥散状,而用本发明无血清培养液培养的卵巢碎块在培养10天时虽然边沿有间质细胞扩散生长,但组织形态仍保持基本正常,说明本发明培养体系能有效支持卵巢组织体外存活。
(2)将对照组和试验组培养后的卵巢碎块用4% PBS-多聚甲醛溶液在4℃固定24h,按照常规石蜡包埋、切片和苏木精-伊红染色方法,经梯度酒精脱水,二甲苯透明,石蜡包埋后,7μm连续切片,每10张取一张,每组不同时间段,一个块取三张切片做染色,每组取3个块,重复实验3次,HE染色后光镜下观察,计数,进行组织学观察分析,结果如图2所示。卵泡分类标准参考文献分类标准,分为原始卵泡和发育卵泡。形态正常卵泡具有完整的卵母细胞,颗粒细胞排列规则,无固缩细胞核,异常卵泡卵母细胞皱褶或颗粒细胞分布杂乱与基底膜分离,核固缩。
从图2中可以看出,试验组在培养10天的卵巢碎块中仍然可以观察到形态正常的卵泡,且正常卵泡比例明显高于对照组培养4天的卵巢碎块,说明应用本发明中所述培养体系可维持体外培养绵羊卵巢皮质碎块中卵泡结构和形态。
(3)将试验组和对照组培养后的卵巢组织碎块在2%多聚甲醛+2.5%戊二醛的0.1MPBS缓冲液中固定3-4h,经PBS洗后,在1%四氧化锇溶液中固定1h,PBS漂洗3次,梯度丙酮脱水后,用环氧树脂浸透包埋,3μm切片,甲苯胺蓝染色光镜下定位形态正常的原始卵泡,初级卵泡,次级卵泡以及闭锁卵泡,经柠檬酸三钴和醋酸双氧铀电子染色后在透射电镜下观察卵泡超微结构变化,如胞质电子密度和完整性、颗粒细胞细胞器、空泡化、基底膜和核膜的完整性、胞质内细胞器的构成和分布、有无透明带等,进行卵泡超微结构观察,结果如图3所示。
从图3中可以看出,试验组培养10天的卵巢碎块中卵母细胞中虽然已出现空泡化现象,但仍可观察到较为完整的超微结构,而对照组卵巢碎块在培养第4天就已经无法找到完整卵泡结构,且空泡化和固缩现象明显。实验结果表明应用本发明所述培养体系对体外培养卵巢皮质中卵泡超微结构在10天内不会造成较大的损伤。
(4)将试验组和对照组培养后的卵巢组织碎块,经石蜡切片(7μm连续切片,每10张取一张,每组不同时间段,一个块取三张切片做染色,每组取3个块,重复实验3次)和常规脱蜡复水后,用0.1%柠檬酸抗体修复液中进行抗体热修复,PBS洗后,按照PCNA免疫组化染色试剂盒说明用3%双氧水室温孵育8 min,消除内源性过氧化物酶活性;正常羊血清封闭10min,1: 50稀释的鼠抗PCNA标记,4℃过夜;样本常温复温后,用PBS漂洗3次,滴加生物素标记羊抗小鼠IgG,室温孵育12 min后用PBS洗3遍,每次三分钟;然后再加入辣根酶标记链酶卵白素室温孵育15 min,PBS洗后DAB染色5-7 min(光镜下观察细胞核呈棕黄终止),自来水冲洗,苏木素复染,进行细胞核增殖抗原检测分析(PCNA),若颗粒细胞棕黄则判定为PCNA阳性卵泡,结果如图4所示,并统计阳性卵泡数和总共观察到卵泡数及二者比例,如表1所示。
表1 不同培养液对培养卵巢皮质中卵泡颗粒细胞增殖的影响
培养液 | 未培养组织(阳性卵泡率) | 培养2天(阳性卵泡率) | 培养4天(阳性卵泡率) | 培养10天(阳性卵泡率) |
对照组 | 16.95% (10/59) | 15.56 (7/45) | 0 (0/16) | — |
试验组 | 16.95% (10/59) | 49.12 (28/57)* | 46.51 (20/43)* | 30.76 (12/39)* |
注:“—”表示未检测到卵泡,“*”表示差异显著。
从表1结果可以看出,未培养组织PCNA检测阳性卵泡比例为16.95%(10/59),试验组在培养2天时,卵巢碎块中PCNA阳性卵泡比例约达到50%,然后随着培养时间的增长,卵巢碎块中PCNA阳性卵泡比例呈缓慢下降趋势,培养10天后在卵巢碎块中仍可检测到30%的PCNA阳性卵泡存在。而对照组在培养过程中卵巢碎块中PCNA阳性比例显著低于试验组,其中,培养4天后未观察到PCNA阳性卵泡的存在。说明应用本发明所述培养体系,能够有效支持卵泡的存活和激活发育。
(5)将试验组和对照组培养后的卵巢组织碎块,经石蜡切片(7μm连续切片,每10张取一张,每组不同时间段,一个块取三张切片做染色,每组取3个块,重复实验3次)和常规脱蜡入水后,按照TUNEL检测试剂盒说明,室温滴加3%双氧水处理8 min,PBS洗涤5min×3次;滴加用TBS 1: 200稀释的蛋白酶K,37℃消化7 min,0.01M TBS洗涤5min×3次;滴加标记缓冲液20 μL/张,保持切片湿润。按每张取TdT和DIG-d-UTP各1μL,加入18μL标记缓冲液中,混匀;甩去切片上多余液体后,加入标记液20 μL/张,置于湿盒中37℃标记2h;经0.01M TBS洗涤5 min×3次后,加入封闭液50 μL/张,室温处理30 min,甩掉封闭液,不洗;用抗体稀释液1: 100稀释生物素抗地高辛抗体,混匀后按50 μL/张滴加在样本上,置于湿盒中37℃反应30 min;0.01M TBS洗涤5 min×3次后,用抗体稀释液1:100稀释SABC,混匀后湿盒中37℃反应30 min;0.01M TBS洗涤5 min×3次后DAB染色10-15 min,水洗后苏木素轻度复染,水洗后常规脱水,进行组织细胞凋亡TUNEL检测,透明封片显微镜下观察细胞核中出现棕黄色颗粒者为阳性细胞,即凋亡的细胞,结果如图5所示,并统计凋亡卵泡数和总共观察到卵泡数及二者比例,如表2所示。
表2 不同培养液对培养卵巢皮质中卵泡凋亡的影响
培养液 | 未培养组织(卵泡凋亡率) | 培养2天(卵泡凋亡率) | 培养4天(卵泡凋亡率) | 培养10天(卵泡凋亡率) |
对照组 | 9.43%(5/53) | 15.56% (7/45) | 100% (12/12) | — |
试验组 | 9.43%(5/53) | 25.49% (13/51)* | 23.91 % (11/46)* | 13.89% (5/36)* |
注:“—”表示未检测到卵泡,“*”表示差异显著。
从表2结果可以看出,未培养组织TUNEL检测阳性卵泡比例9.43% (5/53),试验组的卵巢碎块在培养过程中,卵巢碎块中卵泡凋亡比例前期呈缓慢上升趋势,后期变化平缓,至培养10天凋亡阳性率为13.89%,即约86%的卵泡未发生凋亡;而对照组在培养第4天开始已检测不到TUNEL阴性细胞的存在,即卵泡均已凋亡。说明应用本发明所述培养体系,能够有效支持卵泡的存活和激活发育。
综上,采用本发明的无血清培养体系既能较好的长时间维持卵泡存活,又能维持组织正常形态和卵泡正常结构,说明本发明能够较大程度模拟卵巢内维持原始卵泡存活和生长所需的基础营养条件,进而为研究卵巢内原始卵泡在体外的发育机制提供较好的支撑,可用于后续鉴定和分析各类促性腺激素、生长因子等对原始卵泡发育机制的研究。
以上所述仅为本发明的较佳实施例而已,并不以本发明为限制,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种用于绵羊卵巢皮质组织体外培养的无血清培养液,其特征在于,所述培养液包括基础培养液和添加物,所述基础培养基为α-MEM和DMEM的混合液;所述添加物包括以下含量的组分:3~10 mg/mL牛血清白蛋白、0.10~0.50 mM丙酮酸钠、1~5 mM谷氨酰胺、1~5 mM次黄嘌呤、3~10 μg/mL胰岛素、1~5 μg/mL转铁蛋白、2~8 ng/mL亚硒酸钠、10~200 μg/mL VC、50~200 IU/mL 青霉素和50~200 IU/mL 链霉素,pH 6.9~7.5。
2.根据权利要求1所述用于绵羊卵巢皮质组织体外培养的无血清培养液,其特征在于,所述基础培养液中α-MEM和DMEM的体积比为6~10:1~4。
3.根据权利要求1所述用于绵羊卵巢皮质组织体外培养的无血清培养液,其特征在于,所述基础培养液中α-MEM和DMEM的体积比为4:1。
4.根据权利要求1所述用于绵羊卵巢皮质组织体外培养的无血清培养液,其特征在于,所述添加物包括以下含量的组分:0.2 mM丙酮酸钠、2 mM谷氨酰胺、2 mM次黄嘌呤、8 μg/mL胰岛素、5 μg/mL转铁蛋白、6.0 ng/mL亚硒酸钠、50 μg/mL VC、100 IU/mL青霉素和100 IU/mL链霉素。
5.如权利要求1~4任一项所述无血清培养液在用于绵羊卵巢皮质组织体外培养方面的应用。
6.一种体外培养绵羊卵巢皮质组织的方法,其特征在于,具体包括以下步骤:获取绵羊的卵巢皮质组织并切割成碎块,然后将得到的皮质碎块置于嵌入式细胞培养小室表面,再加入权利要求1~4所述无血清培养液,使无血清培养液的液面没过皮质碎块的下表面但不高于所述皮质碎块的上表面,再将其置于5% CO2,35~38℃的培养箱中进行气液相界面培养。
7.根据权利要求6所述体外培养绵羊卵巢皮质组织的方法,其特征在于,所述皮质碎块大小为0.5~4 mm3,优选为1 mm3。
8.根据权利要求6所述体外培养绵羊卵巢皮质组织的方法,其特征在于,所述培养小室表面的皮质碎块的放置密度为6~20块/小室,优选为12块/小室。
9.根据权利要求6所述体外培养绵羊卵巢皮质组织的方法,其特征在于,所述培养时间为6~10天,每2天换一次新的无血清培养液。
10.根据权利要求6所述体外培养卵巢皮质组织中原始卵泡的方法,其特征在于,所述绵羊选自新疆军垦型细毛羊,亦可是其他品种绵羊或山羊。
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