CN116622624A - 一种促进体外人类精子发生的培养液及应用 - Google Patents
一种促进体外人类精子发生的培养液及应用 Download PDFInfo
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Abstract
本发明提供了一种促进体外人类精子发生的培养液及应用,所述培养液按照体积分数计包括:80‑95%αMEM和5‑20%血清替代物,及50‑100ng/mL转化生长因子β家族激活素A、10‑50ng/mL干细胞生长因子、10‑50ng/mL骨形态发生蛋白、10‑6M维甲酸、10‑50mM睾酮、100‑200ng/mL促卵泡激素和50‑100mg/mL垂体提取物。将人类生殖细胞减数分裂阻滞的睾丸组织分离成小块,在促进体外人类精子发生的培养液中培养10‑30天,可以获得人类精子细胞。本发明的培养液促进了人类男性生殖细胞体外成熟,为研究人类精子发生提供基础,为临床男性不育治疗提供技术支持。
Description
技术领域
本发明属于生物医学领域,具体涉及一种促进体外人类精子发生的培养液及应用。
背景技术
精子发生是男性生育力维持的基础,指从精原细胞发育成为成熟精子的一个连续而复杂的生物学过程,包括了精原细胞的自我更新、精母细胞的减数分裂和精子形成三个阶段。精子发生容易受到遗传突变、内分泌紊乱以及环境内分泌干扰物的影响导致男性不育。
男性不育约占人类不孕不育的50%,其中20%是无精子症。约8-12%的无精子症患者生殖细胞存在成熟阻滞。除此之外,儿童肿瘤发病率为每1000名儿童中有1.0至2.5例,经过治疗后约三分之一的男性面临无精症的风险。因此,亟需建立一个符合“黄金标准”的人类男性生殖细胞体外减数分裂培养方法,将睾丸内不成熟的生殖细胞转变为精子细胞。
体外人类睾丸精子发生方法的建立为研究生殖细胞和睾丸体细胞形成发育的遗传、表观遗传和环境因素,以及各种精子发生障碍的病理机制提供了研究模型,为生殖细胞成熟阻滞患者以及青春期前男性肿瘤患者生育力保存的建立提供技术储备。
发明内容
本发明的目的在于提供一种促进体外人类精子发生的培养液及应用,建立临床精子发生障碍患者睾丸组织的体外培养系统,针对精原细胞或精母细胞成熟阻滞的类型,通过体外培养修复精子发生障碍,完成减数分裂,获得具有功能性的单倍体细胞。应用形态学分析、多组学测序、遗传学分析、功能验证等检测体外诱导获得的单倍体精子细胞。
为达到上述目的,本发明提供如下技术方案:
一种促进体外人类精子发生的培养液,所述培养液按照体积分数计包括:80-95%αMEM和5-20%血清替代物,以及50-100ng/mL转化生长因子β家族激活素A、10-50ng/mL干细胞生长因子、10-50ng/mL骨形态发生蛋白、10-6M维甲酸、10-50mM睾酮、100-200ng/mL促卵泡激素和50-100mg/mL垂体提取物。
一种促进体外人类精子发生的培养方法,采用上述培养液,包括以下步骤:
将生殖细胞成熟阻滞的人类睾丸组织分离成小块,置于含有所述培养液的培养皿中,于32-37℃,4-5%二氧化碳环境中进行培养10-30天。
优选的,所述生殖细胞成熟阻滞的人类睾丸是减数分裂阻滞的人类睾丸。
本发明的有益效果在于:培养减数分裂阻滞的人类睾丸组织,通过在培养液中添加各类细胞因子以及激素,调节睾丸微环境,促进睾丸内生殖细胞与体细胞建立有效的配受体关系,诱导睾丸再生并获得功能性配子。该培养液为体外男性精子成熟提供技术可能,为开发男性不育治疗提供一个平台。
该培养液可以促使成熟阻滞的生殖细胞完成减数分裂,获得精子细胞,另外在体外培养后精原细胞和精母细胞持续存在,说明该体系可多次诱导产生精子细胞。
上述说明仅是本发明技术方案的概述,为了能够更清楚了解本发明的技术手段,并可依照说明书的内容予以实施,以下以本发明的较佳实施例详细说明如后。
附图说明
图1减数分裂阻滞的人类睾丸培养前的鉴定结果:其中A.睾丸组织切片后各细胞类型特异蛋白免疫荧光染色图;B为临床睾丸组织未培养前流式细胞倍体分析(FACS)图,1C:1倍DNA含量;2C:2倍DNA含量;4C:4倍DNA含量。
图2为对比例1中体外培养10天后各细胞类型特异蛋白免疫荧光染色图。
图3实施例1中体外培养后结果,其中A为培养10天后,各细胞类型特异蛋白免疫荧光染色图;B为培养18天后流式倍体分析图。
图4实施例1诱导获得的精子细胞,滴片后PNA和细胞核Hoechst染色鉴定图。
图5实施例1诱导获得的精子细胞基因组CNV测序图。
具体实施方式
下面结合实施例,对本发明的具体实施方式作进一步详细描述。以下实施例用于说明本发明,但不用来限制本发明的范围。
本发明公开了一种促进体外人类精子发生的培养液及应用,本发明提供的促进体外精子成熟培养液所用试剂、仪器、细胞系等均可由市场购得。
收集临床上减数分裂阻滞的人类睾丸组织进行体外3维培养,针对生殖细胞所处的不同阶段,重建精子发生过程。根据不同的成熟阻滞类型对培养条件进行进一步改良,通过对关键时间点的组织块进行免疫荧光染色,对精原细胞,精母细胞和精子细胞及体细胞特异标志蛋白进行鉴定,如PLZF,SYCP3,PNA,SOX9等检测培养睾丸组织中生殖细胞和体细胞的状态。利用流式分选(FACS),检测体外培养的睾丸组织不同关键时间点细胞的DNA含量,检测睾丸组织体外培养过程中精母细胞和单倍体精子细胞的产生。通过FACS获得的单倍体细胞,进一步利用荧光染色、CNV测序等方法鉴定单倍体精子细胞遗传和表观遗传的建立。
培养前准备
(一)减数分裂阻滞的人类睾丸中细胞分型鉴定,
组织固定方法:取2mm左右的减数分裂阻滞的人类睾丸组织用4%PFA室温固定1h,随后转移至梯度酒精中逐步脱水,70%、80%、90%和100%中各40min,100%的酒精:二甲苯(1:1)内6min,二甲苯20s,随后组织浸蜡包埋切片。
将减数分裂阻滞的人类睾丸中的各类细胞类型用特异蛋白免疫荧光染色,其中生殖细胞采用DDX4、支持细胞采用SOX9、精原细胞采用PLZF、精母细胞采用SYCP3、精子细胞采用PNA和细胞核DNA采用hoechst 33342进行染色。
结果如图1中所示,由图1中的A可以看出,免疫荧光染色显示该标本表达生殖细胞标志蛋白DDX4,精原细胞标志蛋白PLZF以及精母细胞标志蛋白SYCP3,不表达精子细胞标志物PNA,说明该例组织生殖细胞阻滞于精母细胞阶段。
由图1中的B可以看出,此时不可见单倍体细胞存在,而四倍体比例高达15%,说明该例标本内存在精母细胞而无精子细胞,结果与免疫荧光结果相互印证。
实施例1
将减数分裂阻滞的人类睾丸在本发明的培养液中进行诱导培养
促进体外人类精子发生的培养方法:将减数分裂阻滞的人类睾丸组织分割至3mm大小的组织块,置于培养皿中,加入1mL培养液于34℃,5%CO2培养。
实验中采用的培养液具体成分如下:
90%(v/v)αMEM,10%(v/v)血清替代物(KSR),100ng/mL转化生长因子β家族激活素A(Activin A,R&D Systems)、20ng/mL干细胞生长因子(SCF,R&D Systems)、20ng/mL骨形态发生蛋白(BMP 4/7,R&D Systems)、10-6M维甲酸(RA)、10mM睾酮(testosterone,AcrosOrganics)、200ng/mL促卵泡激素(FSH,Sigma)和50mg/mL垂体提取物(BPE,Corning LifeSciences)。
组织培养至10天后取材,置于4%的PFA固定,用PNA、SYCP3进行染色标记,结果如图3中A所示,在体外诱导下,睾丸组织培养10天后出现了PNA阳性的细胞即精子细胞,由此表明该优化的诱导体系可以促使减数分裂阻滞的精母细胞完成减数分裂,获得精子细胞,另外在体外培养后精母细胞依然存在,说明该体系可多次诱导产生精子细胞;
对优化培养液中培养18天后的组织进行倍体流式分选,结果如图3中B显示,其中1C:1倍DNA含量;2C:2倍DNA含量;4C:4倍DNA含量。此时可以获得5%左右的单倍体细胞,将纯化的单倍体细胞收集进行后续的验证实验。
对比例1
将减数分裂阻滞的人类睾丸在常规培养液中进行诱导培养
促进体外精子发生的培养方法:将减数分裂阻滞的人类睾丸组织分割至3mm大小的组织块,置于培养皿中,加入1mL所述常规培养液于34℃,5%CO2培养;
实验中采用的所述常规培养液具体成分如下:
90%(v/v)αMEM,10%(v/v)血清替代物(KSR),100ng/mL转化生长因子β家族激活素A(Activin A,R&D Systems)、20ng/mL干细胞生长因子(SCF,R&D Systems)、20ng/mL骨形态发生蛋白(BMP 4/7,R&D Systems)、20ng/mL碱性成纤维细胞生长因子(bFGF,R&DSystems)、20ng/mL表皮生长因子(EGF,R&D Systems)、10mM睾酮(testosterone,AcrosOrganics)、200ng/mL促卵泡激素(FSH,Sigma)和50mg/mL垂体提取物(BPE,Corning LifeSciences)。
和实施例1相比,对比例1中加入了20ng/mL碱性成纤维细胞生长因子(bFGF,R&DSystems)和20ng/mL表皮生长因子(EGF,R&D Systems),缺少了维甲酸。
组织培养至10天后取材,置于4%的PFA固定,用PLZF和SYCP3进行染色标记,结果如图2所示,在常规培养液体外诱导下,睾丸组织培养10天后精原细胞明显增殖,精母细胞不可见,由此表明该诱导体系所提供的微环境会促使睾丸组织内精原细胞增殖而精母细胞凋亡,无法达到预期的体外精子发生效果。由此可见,碱性成纤维细胞生长因子和表皮生长因子的去除,可以避免生精小管内精原细胞的大量增殖以及减数分裂抑制,同时加入维甲酸刺激可促使减数分裂启动并促进完成,获得更优的体外精子发生诱导效果。
培养后检测
将实施例1中睾丸组织体外诱导获得精子细胞特异标记物检测
流式分选纯化单倍体细胞,制备细胞悬液滴片,具体步骤如下:
在体视镜下挑取培养18天的睾丸组织,随后放置到3.5cm的培养皿中,采用两步酶消化法制备单细胞悬液,随后进行细胞核Hoechst 33342染色,37℃,20分钟;进一步进行流式细胞仪倍体分选,收集DNA含量为1C处的细胞,收集管离心弃上清,用50ul培养基重悬沉淀后滴片。
结果如图4所示,细胞滴片后PNA和细胞核Hoechst染色鉴定。
将实施例1中睾丸组织体外诱导获得精子细胞遗传特性鉴定
流式分选纯化单倍体细胞,具体步骤如下:
在体视镜下挑取培养18天的睾丸组织,随后放置到3.5cm的培养皿中,采用两步酶消化法制备单细胞悬液,随后进行细胞核Hoechst 33342染色,37℃,20分钟;进一步进行流式细胞仪倍体分选,收集DNA含量为1C处的细胞,将细胞悬液平铺在培养皿中进行单细胞挑选。
显微镜下挑选的单细胞测序:
在显微镜下进行单个细胞挑选,放入单管无菌无酶的PCR中,随后进行单细胞扩增建库,进一步采用CNV测序验证获得的单倍体细胞染色体完整性,如图5所示,体外诱导可获得基因组完整的X、Y的单精子细胞。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (3)
1.一种促进体外人类精子发生的培养液,其特征在于,所述培养液按照体积分数计包括:80-95%αMEM和5-20%血清替代物,以及50-100ng/mL转化生长因子β家族激活素A、10-50ng/mL干细胞生长因子、10-50ng/mL骨形态发生蛋白、10-6M维甲酸、10-50mM睾酮、100-200ng/mL促卵泡激素和50-100mg/mL垂体提取物。
2.一种促进体外人类精子发生的培养方法,其特征在于,采用权利要求1中所述培养液,包括以下步骤:
将生殖细胞成熟阻滞的人类睾丸组织分离成小块,置于含有所述培养液的培养皿中,于32-37℃,4-5%二氧化碳环境中进行培养10-30天。
3.根据权利要求2所述的一种促进体外人类精子发生的培养方法,其特征在于,所述生殖细胞成熟阻滞的人类睾丸是减数分裂阻滞的人类睾丸。
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