CN110129257A - 血源性女性自体生殖干细胞的制备方法、试剂盒及应用 - Google Patents

血源性女性自体生殖干细胞的制备方法、试剂盒及应用 Download PDF

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CN110129257A
CN110129257A CN201910402537.3A CN201910402537A CN110129257A CN 110129257 A CN110129257 A CN 110129257A CN 201910402537 A CN201910402537 A CN 201910402537A CN 110129257 A CN110129257 A CN 110129257A
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cell
haematogenous
women
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林雄斌
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Lin Liu Yin
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Lin Liu Yin
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Abstract

本发明涉及血源性女性自体生殖干细胞的制备方法、试剂盒及应用，利用女性自体血液细胞作为原料细胞，通过非转基因的生产血源性女性自体生殖干细胞技术和方法。这种血源性女性自体生殖干细胞的制备方法，包括：将女性自体血液细胞在细胞培养液O1中培养3天；在细胞培养液O2中继续培养6‑9天；然后在细胞培养液O3中继续培养6‑9天；以及收集血源性女性自体生殖干细胞；本发明还提供了一种用于制备血源性女性自体生殖干细胞的试剂盒。使用本发明的方法及试剂盒在血源性女性自体生殖干细胞的取材、生产速度、产量、纯度方面具有显著优势。

Description

血源性女性自体生殖干细胞的制备方法、试剂盒及应用
技术领域
本发明涉及血源性女性自体生殖干细胞的制备方法、试剂盒及应用，属于生物医学技术领域，具体而言，本发明涉及一种使女性的血液细胞逆向分化产生血源性自体生殖干细胞的细胞培养方法、干细胞及应用。
背景技术
在雌性哺乳动物中，生殖腺里的生殖干细胞来源于一种从胚胎传留下来的数量稀少的生殖干细胞。胚胎形成及卵巢形成的过程中，生殖干细胞就定居于卵巢，主导卵巢分化、发育和成熟。生殖干细胞发育依次形成卵原细胞►初级卵泡►次级卵泡►成熟卵泡►卵子。生殖干细胞具有Mvh+和Dazl+阳性的特异细胞表型。卵巢的生殖干细胞在产生卵泡及卵子的过程中是一个缓慢的消耗过程。女性一生中，大约经过400至500次卵泡发育成熟、排卵和月经形成后，卵巢中的生殖干细胞就会逐步消耗枯竭，卵巢不再产生卵细胞和卵泡，雌激素的主要来源也同时发生枯竭，卵巢萎缩变小，卵巢衰老丧失生殖功能，女性进入老年。
国内外学者发明了一些生殖干细胞的制备方法，例如通过自然胚胎干细胞体外分化，产生生殖干细胞，或通过iPS多能干细胞（转基因性干细胞）体外分化，产生生殖干细胞，或通过卵巢组织分离培养制备生殖干细胞，分离培养耗时困难，获取数量极其稀少。
生殖干细胞是卵巢的重要种子细胞，对卵巢的结构具有再生性修复作用，所以，自体生殖干细胞的生产技术在女性生殖系统的疾病和损伤的治疗领域，在女性不孕的修复治疗应用领域，在卵巢抗衰老保健领域以及建立自体生殖干细胞储存库领域的应用，都有着十分重要的价值和意义。
发明内容
本发明所要解决的技术问题是：
1. 应用人自体血液细胞作为更方便更简易的逆向分化生产人自体生殖干细胞的自体原料细胞；
2. 研发一组由各种小分子物质组成的能使人的体细胞逆向分化产生人血源性生殖干细胞的细胞培养液配方；
3. 应用小分子物质组成的细胞培养液配方，在不改变人的体细胞染色体DNA序列，不插入任何外来基因或DNA片段的情况下，快速使人的自体血液细胞逆向分化产生人血源性生殖干细胞，这种新型自体干细胞。
为了解决上述技术问题，本发明的一个目的是：
1. 提供1组使人的体细胞逆向分化产生人血源性自体生殖干细胞的细胞培养液配方;
2. 提供采用该细胞培养液配方使人的体细胞产生血源性生殖干细胞的技术方法;
3.提供通过上述方法获得的生殖干细胞的试剂盒。
本发明的目的是通过以下技术方案来实现的：
将女性自体血液白细胞作为原料细胞以2×106 的密度接种于细胞培养皿中。原料细胞包括但不限于女性的脐带血细胞、胎盘细胞、皮肤细胞、血液有核细胞、脂肪细胞等。
在37℃和5% 的CO2 的环境中，将自体原料细胞在细胞培养液O1（RPMI 1640培养液）中培养3天。培养液O1中含有：
2-10%的血清替代品(KSR)，优选值为5%；
EPO 1-1000 ng/ml，优选值为10ng/ml；
M-GSF 1-1000 ng/ml，优选值为10ng/ml；
G-GSF 1-1000 ng/ml，优选值为100ng/ml；
IL-3 1-1000 ng/ml，优选值为10ng/ml；
IL-6 1-1000 ng/ml，优选值为10ng/ml；
SCF 1-1000 ng/ml，优选值为10ng/ml；
bFGF 1-1000 ng/ml，优选值为100ng/ml；
Nanog 1-1000 ng/ml，优选值为30ng/ml；
Oct4 1-1000 ng/ml，优选值为30ng/ml；
Sox2 1-1000 ng/ml，优选值为20ng/ml；
c-myc 1-1000 ng/ml；优选值为20ng/ml；
在37℃和5% 的CO2 的环境中，将原料细胞在细胞培养液O2（M2培养液）中培养3-6天。培养液O2中含有：
2-10%的血清替代品(KSR)，优选值为5%；
Nanog 1-1000 ng/ml, 优选值为30ng/ml；
Oct4 1-1000 ng/ml, 优选值为30ng/ml；
Sox2 1-1000 ng/ml, 优选值为20ng/ml；
c-myc 1-1000 ng/ml, 优选值为20ng/ml；
DDX4(Mvh) 1-1000 ng/ml, 优选值为50ng/ml；
Dazl 1-1000 ng/ml, 优选值为50ng/ml；
bFGF 1-1000 ng/ml, 优选值为100ng/ml；
EGF 1-1000 ng/ml, 优选值为10ng/ml；
IL-6 1-1000 ng/ml, 优选值为10ng/ml；
Sox17 1-1000 ng/ml, 优选值为50ng/ml；
VASA 1-1000 ng/ml, 优选值为50ng/ml；
SCF 1-1000 ng/ml, 优选值为10ng/ml；
G-GSF 1-1000 ng/ml，优选值为10ng/ml；
在37℃和5% 的CO2 的环境中，将体细胞或单个核细胞在细胞培养液O3（M16 培养液）中培养3-6 天。培养液O3中含有：
2-10%的血清替代品(KSR)，优选值为5%；
RA 1-1000 uM, 优选值为200 uM；
DMC1 1-1000 ng/ml, 优选值为30ng/ml；
PRDM1 1-1000 ng/ml, 优选值为30ng/ml；
PGK2 1-1000 ng/ml, 优选值为30ng/ml；
hCG 1-200 iu/ml，优选值为50 iu/ml；
孕酮1-200 iu/ml，优选值为50 iu/ml；
雌二醇 1-200 iu/ml，优选值为50 iu/ml；
FSH 1-1000 ng/ml, 优选值为100ng/ml；
DDX4(Mvh) 1-1000 ng/ml, 优选值为30ng/ml；
Dazl 1-1000 ng/ml, 优选值为30ng/ml；
Oct4 1-1000 ng/ml, 优选值为10ng/ml；
Sox17 1-1000 ng/ml, 优选值为30ng/ml；
VASA 1-1000 ng/ml, 优选值为50ng/ml；
bFGF 1-1000 ng/ml, 优选值为100ng/ml；
VASA 1-1000 ng/ml, 优选值为50ng/ml；
FGF 2 1-1000 ng/ml, 优选值为10ng/ml；
FGF 7 1-1000 ng/ml, 优选值为10ng/ml；
BMP2 1-1000 ng/ml, 优选值为10ng/ml；
BMP4 1-1000 ng/ml, 优选值为10ng/ml；
IL-6 1-1000 ng/ml, 优选值为10ng/ml；
NT 4 1-1000 ng/ml, 优选值为10ng/ml；
GDNF 1-1000 ng/ml, 优选值为10ng/ml；
Activin A 1-1000 ng/ml, 优选值为10ng/ml；
UB 1-1000 ng/ml, 优选值为100ng/ml；
17α,20β-DHP 1-1000 ng/ml, 优选值为10ng/ml；
AY9944-A-7 1-1000 ng/ml, 优选值为10ng/ml；
EGF 1-1000 ng/ml, 优选值为10ng/ml；
VEGF 1-1000 ng/ml, 优选值为10ng/ml；
SCF 1-1000 ng/ml, 优选值为10ng/ml；
IGF 1-1000 ng/ml, 优选值为10ng/ml；
IGF I 1-1000 ng/ml, 优选值为10ng/ml；
IGF II 1-1000 ng/ml, 优选值为10ng/ml；
GRF 1-1000 ng/ml, 优选值为10ng/ml；
Ifitm3 1-1000 ng/ml, 优选值为10ng/ml；
GH 1-1000 ng/ml, 优选值为10ng/ml；
地塞米松1-1000 ng/ml, 优选值为10ng/ml；
G-GSF 1-1000 ng/ml，优选值为10ng/ml；
轻轻吹打分别在细胞培养液O1、细胞培养液O2 及细胞培养液O3中培养后的细胞，使细胞完全悬浮后，收集细胞于离心管中，进行离心操作，然后使用生理盐水对悬浮的细胞进行清洗操作，重复3次后，将细胞悬浮于生理盐水中，得到血源性女性自体生殖干细胞。
将得到的血源性自体生殖干细胞以1×105/ml 至1×106/ml的细胞密度与等体积的二甲基亚砜和10% 的低分子右旋糖酐的混合液混合，将混合物降温至-80℃，然后转移到-186℃液氮中深低温保存。
在本方法中，所使用的优选培养液为：
培养液O1 是RPMI 1640 培养液，其中含有：
5%的血清替代品(KSR);
EPO 10 ng/ml;
M-GSF 10 ng/ml;
G-GSF 150 ng/ml;
IL-3 10 ng/ml;
IL-6 10 ng/ml;
SCF 10 ng/ml;
bFGF 100 ng/ml;
Nanog 30 ng/ml;
Oct4 30 ng/ml;
Sox2 20 ng/ml;
c-myc 20 ng/ml。
培养液S2是M2培养液，其中含有：
5%的血清替代品(KSR);
Nanog 30 ng/ml;
Oct4 30 ng/ml;
Sox2 10 ng/ml;
c-myc 20 ng/ml;
DDX4(Mvh) 30 ng/ml;
Dazl 30 ng/ml;
bFGF 100 ng/ml;
EGF 10 ng/ml;
IL-6 10 ng/ml;
Sox17 100 ng/ml;
VASA 50 ng/ml；
SCF 10 ng/ml;
G-GSF 10 ng/ml;
培养液S3是M16 培养液，其中含有：
RA 200 uM;
DMC1 50 ng/ml;
PRDM1 50 ng/ml;
PGK2 50 ng/ml;
hCG 50iu/ml；
孕酮50 iu/ml；
雌二醇 50 iu/ml；
FSH 100 ng/ml；
DDX4(Mvh) 50 ng/ml;
Dazl 50 ng/ml,
Oct4 10 ng/ml;
Sox17 50 ng/ml;
VASA 50 ng/ml；
bFGF 100 ng/ml;
FGF 2 100 ng/ml;
FGF 7 100 ng/ml;
BMP2 100 ng/ml;
BMP4 100 ng/ml;
IL-6 10 ng/ml;
NT 4 10 ng/ml;
GDNF 10 ng/ml;
Activin A 10 ng/ml;
UB 100 ng/ml;
17α,20β-DHP 10 ng/ml;
AY9944-A-7 10 ng/ml;
EGF 10 ng/ml;
VEGF 10 ng/ml;
SCF 10 ng/ml;
IGF 10 ng/ml;
IGF I 10 ng/ml;
IGF II 10 ng/ml;
GRF 10 ng/ml;
Ifitm3 10 ng/ml;
GH 10 ng/ml;
地塞米松100 ng/ml;
G-GSF 10 ng/ml。
本发明还提供了一种用于制备血源性女性自体生殖干细胞的试剂盒。试剂盒包括细胞培养液O1、细胞培养液O2和细胞培养液O3。其中：
培养液O1是RPMI 1640培养液，其中含有：
2-10%的血清替代品(KSR)，优选值为5%；
EPO 1-1000 ng/ml，优选值为10ng/ml；
M-GSF 1-1000 ng/ml，优选值为10ng/ml；
G-GSF 1-1000 ng/ml，优选值为100ng/ml；
IL-3 1-1000 ng/ml，优选值为10ng/ml；
IL-6 1-1000 ng/ml，优选值为10ng/ml；
SCF 1-1000 ng/ml，优选值为10ng/ml；
bFGF 1-1000 ng/ml，优选值为100ng/ml；
Nanog 1-1000 ng/ml，优选值为30ng/ml；
Oct4 1-1000 ng/ml，优选值为30ng/ml；
Sox2 1-1000 ng/ml，优选值为20ng/ml；
c-myc 1-1000 ng/ml；优选值为20ng/ml；
培养液O2是M2培养液，其中含有：
2-10%的血清替代品(KSR)，优选值为5%；
Nanog 1-1000 ng/ml, 优选值为30ng/ml；
Oct4 1-1000 ng/ml, 优选值为30ng/ml；
Sox2 1-1000 ng/ml, 优选值为20ng/ml；
c-myc 1-1000 ng/ml, 优选值为20ng/ml；
DDX4(Mvh) 1-1000 ng/ml, 优选值为50ng/ml；
Dazl 1-1000 ng/ml, 优选值为50ng/ml；
bFGF 1-1000 ng/ml, 优选值为100ng/ml；
EGF 1-1000 ng/ml, 优选值为10ng/ml；
IL-6 1-1000 ng/ml, 优选值为10ng/ml；
Sox17 1-1000 ng/ml, 优选值为50ng/ml；
VASA 1-1000 ng/ml, 优选值为50ng/ml；
SCF 1-1000 ng/ml, 优选值为10ng/ml；
G-GSF 1-1000 ng/ml，优选值为10ng/ml；
培养液O3是M16培养液，其中含有：
2-10%的血清替代品(KSR)，优选值为5%；
RA 1-1000 uM, 优选值为200 uM；
DMC1 1-1000 ng/ml, 优选值为30ng/ml；
PRDM1 1-1000 ng/ml, 优选值为30ng/ml；
PGK2 1-1000 ng/ml, 优选值为30ng/ml；
hCG 1-200 iu/ml，优选值为50 iu/ml；
孕酮1-200 iu/ml，优选值为50 iu/ml；
雌二醇 1-200 iu/ml，优选值为50 iu/ml；
FSH 1-1000 ng/ml, 优选值为100ng/ml；
DDX4(Mvh) 1-1000 ng/ml, 优选值为30ng/ml；
Dazl 1-1000 ng/ml, 优选值为30ng/ml；
Oct4 1-1000 ng/ml, 优选值为10ng/ml；
Sox17 1-1000 ng/ml, 优选值为30ng/ml；
VASA 1-1000 ng/ml, 优选值为50ng/ml；
bFGF 1-1000 ng/ml, 优选值为100ng/ml；
VASA 1-1000 ng/ml, 优选值为50ng/ml；
FGF 2 1-1000 ng/ml, 优选值为10ng/ml；
FGF 7 1-1000 ng/ml, 优选值为10ng/ml；
BMP2 1-1000 ng/ml, 优选值为10ng/ml；
BMP4 1-1000 ng/ml, 优选值为10ng/ml；
IL-6 1-1000 ng/ml, 优选值为10ng/ml；
NT 4 1-1000 ng/ml, 优选值为10ng/ml；
GDNF 1-1000 ng/ml, 优选值为10ng/ml；
Activin A 1-1000 ng/ml, 优选值为10ng/ml；
UB 1-1000 ng/ml, 优选值为100ng/ml；
17α,20β-DHP 1-1000 ng/ml, 优选值为10ng/ml；
AY9944-A-7 1-1000 ng/ml, 优选值为10ng/ml；
EGF 1-1000 ng/ml, 优选值为10ng/ml；
VEGF 1-1000 ng/ml, 优选值为10ng/ml；
SCF 1-1000 ng/ml, 优选值为10ng/ml；
IGF 1-1000 ng/ml, 优选值为10ng/ml；
IGF I 1-1000 ng/ml, 优选值为10ng/ml；
IGF II 1-1000 ng/ml, 优选值为10ng/ml；
GRF 1-1000 ng/ml, 优选值为10ng/ml；
Ifitm3 1-1000 ng/ml, 优选值为10ng/ml；
GH 1-1000 ng/ml, 优选值为10ng/ml；
地塞米松1-1000 ng/ml, 优选值为10ng/ml；
G-GSF 1-1000 ng/ml，优选值为10ng/ml；
优选地，试剂盒的组成包括：
培养液O1是RPMI 1640 培养液，其中含有：
5%的血清替代品(KSR);
EPO 10 ng/ml;
M-GSF 10 ng/ml;
G-GSF 150 ng/ml;
IL-3 10 ng/ml;
IL-6 10 ng/ml;
SCF 10 ng/ml;
bFGF 100 ng/ml;
Nanog 30 ng/ml;
Oct4 30 ng/ml;
Sox2 20 ng/ml;
c-myc 20 ng/ml。
培养液O2是M2培养液，其中含有：
5%的血清替代品(KSR);
Nanog 30 ng/ml;
Oct4 30 ng/ml;
Sox2 10 ng/ml;
c-myc 20 ng/ml;
DDX4(Mvh) 30 ng/ml;
Dazl 30 ng/ml;
bFGF 100 ng/ml;
EGF 10 ng/ml;
IL-6 10 ng/ml;
Sox17 100 ng/ml;
VASA 50 ng/ml；
SCF 10 ng/ml;
G-GSF 10 ng/ml;
培养液O3是M16 培养液，其中含有：
RA 200 uM;
DMC1 50 ng/ml;
PRDM1 50 ng/ml;
PGK2 50 ng/ml;
hCG 50iu/ml；
孕酮50 iu/ml；
雌二醇 50 iu/ml；
FSH 100 ng/ml；
DDX4(Mvh) 50 ng/ml;
Dazl 50 ng/ml,
Oct4 10 ng/ml;
Sox17 50 ng/ml;
VASA 50 ng/ml；
bFGF 100 ng/ml;
FGF 2 100 ng/ml;
FGF 7 100 ng/ml;
BMP2 100 ng/ml;
BMP4 100 ng/ml;
IL-6 10 ng/ml;
NT 4 10 ng/ml;
GDNF 10 ng/ml;
Activin A 10 ng/ml;
UB 100 ng/ml;
17α,20β-DHP 10 ng/ml;
AY9944-A-7 10 ng/ml;
EGF 10 ng/ml;
VEGF 10 ng/ml;
SCF 10 ng/ml;
IGF 10 ng/ml;
IGF I 10 ng/ml;
IGF II 10 ng/ml;
GRF 10 ng/ml;
Ifitm3 10 ng/ml;
GH 10 ng/ml;
地塞米松100 ng/ml;
G-GSF 10 ng/ml。
该试剂盒可以应用于血源性女性自体生殖干细胞的制备;该试剂盒制备的血源性女性自体生殖干细胞涉及卵巢再生和修复的疾病治疗、卵巢保健、卵巢抗衰老等领域的应用；
通过本发明的上述方法及试剂盒，创造了一种新的利用自体血液细胞作为原料细胞，通过非转基因式的血源性女性自体生殖干细胞生产技术。本发明在血源性女性自体生殖干细胞的生产速度、产量、纯度方面具有显著优势。
附图说明
图1 是静脉血单个核细胞（PBMC）在显微镜下的示图，
图2 是PBMC 逆向分化后得到的血源性女性自体生殖干细胞在显微镜下的示图，
图3 是示出了血源性女性自体生殖干细胞鉴定结果的DAZL表型检测图，
图4 是示出了血源性女性自体生殖干细胞鉴定结果的MVH表型检测图，
图5 是示出了血源性女性自体生殖干细胞鉴定结果的FRAGILIS表型检测图，
图6示出了血源性女性自体生殖干细胞转化率测定的结果的曲线图。
下面将结合本发明实施例中的附图，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所获得的所有其他实施例，都属于本发明保护的范围。
实施例1试剂盒的制备
在10-100 级洁净度的安全操作台里操作，4-10度的低温条件下配制。
在500ml 的RPMI 640 培养液中，分别加入：
5%的血清替代品(KSR);
EPO 10 ng/ml;
M-GSF 10 ng/ml;
G-GSF 150 ng/ml;
IL-3 10 ng/ml;
IL-6 10 ng/ml;
SCF 10 ng/ml;
bFGF 100 ng/ml;
Nanog 30 ng/ml;
Oct4 30 ng/ml;
Sox2 20 ng/ml;
c-myc 20 ng/ml。
充分溶解，然后经0.22微米孔径的滤器过滤、消毒，配制成细胞培养液O1。
在500ml 的M2培养液中，分别加入：
5%的血清替代品(KSR);
Nanog 30 ng/ml;
Oct4 30 ng/ml;
Sox2 10 ng/ml;
c-myc 20 ng/ml;
DDX4(Mvh) 30 ng/ml;
Dazl 30 ng/ml;
bFGF 100 ng/ml;
EGF 10 ng/ml;
IL-6 10 ng/ml;
Sox17 100 ng/ml;
VASA 50 ng/ml；
SCF 10 ng/ml;
G-GSF 10 ng/ml;
充分溶解，然后经0.22 微米孔径的滤器过滤、消毒，配制成细胞培养液O2。
在500ml 的M16 培养液中，分别加入：
RA 200 uM;
DMC1 50 ng/ml;
PRDM1 50 ng/ml;
PGK2 50 ng/ml;
hCG 50iu/ml；
孕酮50 iu/ml；
雌二醇 50 iu/ml；
FSH 100 ng/ml；
DDX4(Mvh) 50 ng/ml;
Dazl 50 ng/ml,
Oct4 10 ng/ml;
Sox17 50 ng/ml;
VASA 50 ng/ml；
bFGF 100 ng/ml;
FGF 2 100 ng/ml;
FGF 7 100 ng/ml;
BMP2 100 ng/ml;
BMP4 100 ng/ml;
IL-6 10 ng/ml;
NT 4 10 ng/ml;
GDNF 10 ng/ml;
Activin A 10 ng/ml;
UB 100 ng/ml;
17α,20β-DHP 10 ng/ml;
AY9944-A-7 10 ng/ml;
EGF 10 ng/ml;
VEGF 10 ng/ml;
SCF 10 ng/ml;
IGF 10 ng/ml;
IGF I 10 ng/ml;
IGF II 10 ng/ml;
GRF 10 ng/ml;
Ifitm3 10 ng/ml;
GH 10 ng/ml;
地塞米松100 ng/ml;
G-GSF 10 ng/ml。
充分溶解，然后经0.22 微米孔径的滤器过滤、消毒，配制成细胞培养液O3。
将细胞培养液O1、细胞培养液O2以及细胞培养液O3分别置于试剂盒的三个相互隔离的容器内，从而制成用于制备血源性女性自体生殖干细胞的试剂盒。
实施例2血源性女性自体生殖干细胞的制备
抽取女性静脉血液（来源于志愿者）50ml，使用常规Ficoll 离心分离技术对静脉血液进行离心分离，从而获取血液单个核细胞。将获得的单个核细胞以5x106 的密度，用实施例1 中配制的细胞培养液O1，在37℃和5% 的CO2 培养箱中培养3天；然后换用实施例1中配制的细胞培养液O2，继续对单个核细胞以5x106 的密度进行培养6-9天；然后换用实施例1中配制的细胞培养液O3，继续对单个核细胞以5x106的密度进行培养6-9天。轻轻吹打分别在细胞培养液O1、细胞培养液O2 及细胞培养液O3中培养后的细胞，使细胞完全悬浮后，收集细胞于离心管中，进行离心操作，然后使用生理盐水对悬浮的细胞进行清洗操作，重复3次后，将细胞悬浮于生理盐水中，得到血源性女性自体生殖干细胞。将得到的血源性女性自体生殖干细胞以1×105/ml 至1×106/ml 的细胞密度与等体积的二甲基亚砜和10% 的低分子右旋糖酐的混合液混合，将混合物降温至-80℃，然后转移到-186℃液氮中深低温保存。
细胞表型鉴定实验
用已预先制得的聚-D-赖氨酸的载玻片，选择实施例2中制备的血源性女性自体生殖干细胞的最佳的细胞悬液浓度，用细胞制片离心机在1800RCF 下转动抛片2分钟制成玻片并做好标记，抛片上细胞密度（细胞均匀，密度适当）。抛片晾干后存放于-20 度冰箱中冻存。
从-20 度冰箱中取出细胞玻片在室温晾干，在反面用记号笔划定区域。然后使用4% 多聚甲醛（购买自Solarbio）固定，经过2% 的Triton X-100 打孔10分钟以及磷酸盐缓冲液（PBS，pH 值为7.4）的清洗后，使用10% 正常山羊血清在室温的条件下封闭1小时，得到样品。将第一抗体试剂加入样品中，所使用的第一抗体为兔抗人DDX4/MVH 多克隆抗体（1:100，购买自Abcam公司，产品编号ab13840），在4℃的条件下使样品中的抗原与第一抗体反应12小时（过夜）。然后在使用磷酸盐缓冲液（PBS）清洗样品后，将与第一抗体相对应的第二抗体加入到样品中，第二抗体为FITC 标记标记的羊抗兔IgG（1:200 ；购买自Chemicon 公司, 产品编号为AP132F）使样品中的第一抗体与第二抗体反应1 小时。在室温的条件下使用DAPI(1:1000) 进行染核步骤。在使用磷酸盐缓冲液PBS 清洗后，对样品进行封片剂封片操作，所使用封边剂为甘油/PBS 封片剂（甘油：PBS=1 ：9，pH8.0-8.5，美工生物公司制造）。最后使用尼康荧光显微镜观察拍照，在6-8小时内完成各种拍照。
图4 示出了实施例2 制得的细胞在与一抗（兔抗人DDX4/MVH 多克隆抗体）及二抗（FITC 标记标记的羊抗兔IgG）反应后，可以在荧光显微镜观察到荧光部分，从而说明实施例2 中所制得的细胞为血源性女性自体生殖干细胞。
图6 示出了实施例2制得的细胞在与一抗（兔抗鼠DAZL 多克隆抗体）及二抗（FITC标记标记的羊抗兔IgG）反应后，可以在荧光显微镜观察到荧光部分，从而说明实施例3 中所制得的细胞为血源性女性自体生殖干细胞。

Claims (14)

1.一种血源性女性自体生殖干细胞的制备方法，其特征在于，所述方法包括：
将人的体细胞在细胞培养液O1中培养3天，所述细胞培养液O1为RPMI1640、并含有: 2-10%的血清替代品(KSR)；EPO 1-1000 ng/ml；M-GSF 1-1000 ng/ml；G-GSF 1-1000 ng/ml；IL-3 1-1000 ng/ml；IL-6 1-1000 ng/ml；SCF 1-1000 ng/ml；bFGF 1-1000 ng/ml；Nanog1-1000 ng/ml；Oct4 1-1000 ng/ml；Sox2 1-1000 ng/ml；c-myc 1-1000 ng/ml；然后换用细胞培养液O2培养6-9天，所述细胞培养液O2为M2培养液、并含有: 2-10%的血清替代品(KSR)；Nanog 1-1000 ng/ml；Oct4 1-1000 ng/ml；Sox2 1-1000 ng/ml；myc 1-1000 ng/ml；DDX4(Mvh) 1-1000 ng/ml；Dazl 1-1000 ng/ml；bFGF 1-1000 ng/ml；EGF 1-1000 ng/ml；IL-6 1-1000 ng/ml；Sox17 1-1000 ng/ml；VASA 1-1000 ng/ml；SCF 1-1000 ng/ml；G-GSF 1-1000 ng/ml；再换用细胞培养液O3继续培养6-9天，所述细胞培养液O3为M16培养液、并含有: 2-10%的血清替代品(KSR)；RA 1-1000 uM；DMC1 1-1000 ng/ml；PRDM1 1-1000ng/ml；PGK2 1-1000 ng/ml；hCG 1-300iu/ml; 孕酮1-300iu/ml； 雌二醇 1-300iu/ml；FSH1-1000 ng/ml；DDX4(Mvh) 1-1000 ng/ml；Dazl 1-1000 ng/ml；Oct4 1-1000 ng/ml；Sox171-1000 ng/ml；VASA 1-1000 ng/ml；bFGF 1-1000 ng/ml；VASA 1-1000 ng/ml；GF 2 1-1000 ng/ml； FGF 7 1-1000 ng/ml；BMP2 1-1000 ng/ml；BMP4 1-1000 ng/ml；IL-6 1-1000 ng/ml；NT 4 1-1000 ng/ml；GDNF 1-1000 ng/ml；Activin A 1-1000 ng/ml；UB 1-1000 ng/ml；17α,20β-DHP 1-1000 ng/ml；AY9944-A-7 1-1000 ng/ml；EGF 1-1000 ng/ml；VEGF 1-1000 ng/ml；SCF 1-1000 ng/ml；IGF 1-1000 ng/ml；IGF I 1-1000 ng/ml；IGF II1-1000 ng/ml；GRF 1-1000 ng/ml；Ifitm3 1-1000 ng/ml；GH 1-1000 ng/ml；地塞米松1-1000 ng/ml；G-GSF 1-1000 ng/ml；获得人血源性自体生殖干细胞；
培养条件，在5% CO2和37℃条件下进行细胞培养。
2.如权利要求1所述血源性女性自体生殖干细胞的制备的方法，其特征在于，所述人的体细胞包括但不限于人的脐带血细胞、胎盘细胞、皮肤细胞、血液有核细胞、脂肪细胞、血液等；
培养条件，将细胞在细胞培养液O1中培养 72小时，所述细胞培养液O1为RPMI1640、并含有: 5%的血清替代品(KSR);EPO 10 ng/ml;M-GSF 10 ng/ml;G-GSF 150 ng/ml;IL-3 10ng/ml;IL-6 10 ng/ml;SCF 10 ng/ml;bFGF 100 ng/ml;Nanog 30 ng/ml;Oct4 30 ng/ml;Sox2 20 ng/ml;c-myc 20 ng/ml; 再换用细胞培养液O2继续在5% CO2和37℃条件下培养6-9天，所述细胞培养液O2为M2培养液含有:5%的血清替代品(KSR);Nanog 30 ng/ml;Oct430 ng/ml;Sox2 10 ng/ml;c-myc 20 ng/ml;DDX4(Mvh) 30 ng/ml;Dazl 30 ng/ml;bFGF100 ng/ml;EGF 10 ng/ml;IL-6 10 ng/ml;Sox17 100 ng/ml;VASA 50 ng/ml；SCF 10 ng/ml;G-GSF 10 ng/ml;再换用细胞培养液O3继续在5% CO2和37℃条件下培养6-9天，所述细胞培养液O3为M16培养液含有: RA 200 uM;DMC1 50 ng/ml;PRDM1 50 ng/ml;PGK2 50 ng/ml; hCG 30iu/ml; 孕酮30iu/ml; 雌二醇 30iu/ml; FSH 100 ng/ml；DDX4(Mvh) 50 ng/ml;Dazl 50 ng/ml, Oct4 10 ng/ml;Sox17 50 ng/ml;VASA 50 ng/ml；bFGF 100 ng/ml;FGF 2 100 ng/ml;FGF 7 100 ng/ml;BMP2 100 ng/ml;BMP4 100 ng/ml;IL-6 10 ng/ml;NT 4 10 ng/ml;GDNF 10 ng/ml;Activin A 10 ng/ml;UB 100 ng/ml;17α,20β-DHP 10ng/ml;AY9944-A-7 10 ng/ml;EGF 10 ng/ml;VEGF 10 ng/ml;SCF 10 ng/ml; IGF 10 ng/ml;IGF I 10 ng/ml; IGF II 10 ng/ml;GRF 10 ng/ml;Ifitm3 10 ng/ml;GH 10 ng/ml;地塞米松100 ng/ml;G-GSF 10 ng/ml,获得人血源性自体生殖干细胞。
3.如权利要求1-2中任一项所述血源性女性自体生殖干细胞的制备的方法，其特征在于，制备的血源性女性自体生殖干细胞。
4.如权利要求3所述的血源性女性自体生殖干细胞的制备的方法，其特征在于，涉及以用于女性生殖系统再生和修复的疾病治疗、卵巢保健、卵巢抗衰老等领域的应用；
所述疾病为女性不孕、卵巢功能减退、卵巢早衰、卵巢衰老/绝经。
5.如权利要求4所述的血源性女性自体生殖干细胞的制备的方法，其特征在于，用于治疗女性不孕不育、卵巢功能减退、卵巢早衰、卵巢衰老/绝经的药物和用于治疗涉及器官再生再造和修复的疾病药物中的应用。
6.如权利要求4所述的血源性女性自体生殖干细胞的制备的方法，其特征在于，用于生产储存库，体外自体组织器官工程和生产的应用。
7.一组用于使女性的体细胞逆向分化的培养液，其特征在于，所述培养液选自细胞培养液O1、O2和O3，所述细胞培养液O1为RPIM1640、并含有: 2-10%的血清替代品(KSR)；EPO1-1000 ng/ml；M-GSF 1-1000 ng/ml；G-GSF 1-1000 ng/ml；IL-3 1-1000 ng/ml；IL-6 1-1000 ng/ml；SCF 1-1000 ng/ml；bFGF 1-1000 ng/ml；Nanog 1-1000 ng/ml；Oct4 1-1000ng/ml；Sox2 1-1000 ng/ml；c-myc 1-1000 ng/ml；然后换用细胞培养液O2培养6-9天，所述细胞培养液O2为M2培养液、并含有: 2-10%的血清替代品(KSR)；Nanog 1-1000 ng/ml；Oct41-1000 ng/ml；Sox2 1-1000 ng/ml；myc 1-1000 ng/ml；DDX4(Mvh) 1-1000 ng/ml；Dazl1-1000 ng/ml；bFGF 1-1000 ng/ml；EGF 1-1000 ng/ml；IL-6 1-1000 ng/ml；Sox17 1-1000 ng/ml；VASA 1-1000 ng/ml；SCF 1-1000 ng/ml；G-GSF 1-1000 ng/ml；再换用细胞培 养液O3继续培养6-9天，所述细胞培养液O3为M16培养液、并含有: 2-10%的血清替代品(KSR)；RA 1-1000 uM；DMC1 1-1000 ng/ml；PRDM1 1-1000 ng/ml；PGK2 1-1000 ng/ml；hCG1-300iu/ml; 孕酮1-300iu/ml； 雌二醇 1-300iu/ml；FSH 1-1000 ng/ml；DDX4(Mvh) 1-1000 ng/ml；Dazl 1-1000 ng/ml；Oct4 1-1000 ng/ml；Sox17 1-1000 ng/ml；VASA 1-1000ng/ml；bFGF 1-1000 ng/ml；VASA 1-1000 ng/ml；GF 2 1-1000 ng/ml； FGF 7 1-1000 ng/ml；BMP2 1-1000 ng/ml；BMP4 1-1000 ng/ml；IL-6 1-1000 ng/ml；NT 4 1-1000 ng/ml；GDNF 1-1000 ng/ml；Activin A 1-1000 ng/ml；UB 1-1000 ng/ml；17α,20β-DHP 1-1000ng/ml；AY9944-A-7 1-1000 ng/ml；EGF 1-1000 ng/ml；VEGF 1-1000 ng/ml；SCF 1-1000ng/ml；IGF 1-1000 ng/ml；IGF I 1-1000 ng/ml；IGF II 1-1000 ng/ml；GRF 1-1000 ng/ml；Ifitm3 1-1000 ng/ml；GH 1-1000 ng/ml；地塞米松1-1000 ng/ml；G-GSF 1-1000 ng/ml；获得人血源性自体生殖干细胞。
8.如权利要求7所述一组用于使女性的体细胞逆向分化的培养液，其特征在于，细胞培养液O1、O2和O3在培养人的体细胞使之逆向分化产生自体生殖干细胞中的应用。
9.一种用于制备血源性女性自体生殖干细胞的试剂盒，其特征在于，所述试剂盒包括如权利要求8所述的细胞培养液O1、O2和O3；
所述试剂盒进一步包括女性的体细胞；
所述体细胞包括但不限于女性的脐带血细胞、胎盘细胞、皮肤细胞、血液有核细胞、脂肪细胞。
10.权利要求9所述的一种用于制备血源性女性自体生殖干细胞的试剂盒，其特征在于，在制备血源性女性自体生殖干细胞，涉及以用于治疗女性不孕、卵巢功能减退、卵巢早衰、卵巢衰老/绝经的应用。
11.根据权利要求10所述的一种用于制备血源性女性自体生殖干细胞的试剂盒应用，其特征在于，所述疾病包括女性不孕、卵巢功能减退、卵巢早衰、卵巢衰老/绝经。
12.权利要求9所述的一种用于制备血源性女性自体生殖干细胞的试剂盒，其特征在于，在建立女性血源性自体生殖干细胞储存及女性血源性自体生殖干细胞库的应用。
13.权利要求1-6的血源性女性自体生殖干细胞的制备方法，其特征在于，制备的所述血源性女性自体生殖干细胞在卵巢再生和修复的疾病治疗、女性不孕、卵巢功能减退、卵巢早衰、卵巢衰老/绝经等领域的应用。
14.权利要求1-6的血源性女性自体生殖干细胞的制备方法，其特征在于，制备的所述血源性女性自体生殖干细胞在建立女性血源性自体精原干细胞储存及女性血源性自体生殖干细胞库的应用。
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