CN117487743B - 一种诱导鸡胚成纤维细胞为鸡多能干细胞的化学诱导剂及诱导方法 - Google Patents
一种诱导鸡胚成纤维细胞为鸡多能干细胞的化学诱导剂及诱导方法 Download PDFInfo
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Abstract
本发明公开了一种诱导鸡胚成纤维细胞为鸡多能干细胞的化学诱导剂及诱导方法。属于多能干细胞制备技术领域。该化学诱导剂包括XAV‑939、Y‑27632、Resveratrol、SF1670、CHIR‑99021、Menthone、β‑Alanine和Ferrostatin‑1。本发明利用小分子化合物诱导鸡胚成纤维细胞，以期在短时间内获得大量鸡iPSC，为其进行离体保种、基因编辑研究等奠定良好基础。
Description
技术领域
本发明涉及多能干细胞制备技术领域，更具体的说是涉及一种诱导鸡胚成纤维细胞为鸡多能干细胞的化学诱导剂及诱导方法。
背景技术
诱导多能干细胞（Induced pluripotent stem cells，iPSC）是日本科学家TAKAHASHI在2006年将表达四种多能性转录因子OCT4、SOX4、KLF4和c-MYC (统称为OSKM)的逆转录病毒载体导入小鼠成纤维细胞，使成纤维细胞转化为具有多向分化潜能的多能性细胞。iPSC具有无限增殖和自我更新的能力，在适当条件下可被诱导分化为多种细胞组织，开启了生命科学的新时代。该技术的诞生，不仅给人类带来了新的、大量的干细胞来源，也为珍稀动物保种、转基因动物的生产等研究开辟了新思路。2013年，第三代基因编辑技术CRISPR问世后，开启了基因编辑技术井喷式发展的时代。利用iPSC进行相对高效安全的基因组编辑，在基础研究和临床应用上有巨大的优势。尽管小鼠和人等多个物种中iPSC的诱导非常成熟，但家禽体细胞重编程研究进程仍较为缓慢。
目前获得鸡iPSC的方法主要利用OSKM四因子及其它转录因子的不同组合在慢病毒过表达载体进行诱导，但效率较低，细胞形态从第6天开始发生变化，到15天时开始形成与多能干细胞形态类似的iPSC克隆，限制了其应用。
近年来，小分子化合物诱导的方法已经成为操控细胞命运的重要手段，利用小分子化合物可以实现细胞重编程、分化和转分化。由于OSKM因子会带来病毒感染及其它外源性细胞成分，非常不安全，需要寻求更安全、高效的诱导方法。近年来研究表明小分子化合物诱导可以避免OSKM因子带来的问题，同时，小分子化合物靶点相对清晰、作用相对可控，对体细胞重编程机制的研究起了很大的推动作用。目前已经在小鼠、人和猪等物种上实现了小分子化合物诱导体细胞为多能干细胞或胚胎干细胞。虽然小分子化合物在体细胞重编程中具有独特优势，但从上千甚至上万的小分子化合物中筛选出合适的诱导组合，绝非易事。目前还没有使用小分子化合物诱导鸡胚成纤维细胞重编程为iPSC的报道。不同起始细胞和目标细胞决定了转分化过程中所需要的小分子化合物不同，因此在小分子化合物的选择上要经过详尽的调研和筛选。
综上，如何提供一种化学诱导鸡胚成纤维细胞为鸡多能干细胞的方法是本领域技术人员亟需解决的问题。
发明内容
有鉴于此，本发明提供了一种诱导鸡胚成纤维细胞为鸡多能干细胞的化学诱导剂及诱导方法。
发明人前期挖掘到影响鸡原始生殖细胞自我更新的核心细胞群和新的信号通路5个，通过添加小分子化合物、抑制剂等调控这5个信号通路，显著提高了鸡原始生殖细胞体外建系效率。因此，发明人结合鸡原始生殖细胞这5个信号通路，开展鸡iPSC的化学诱导体系筛选，从众多的小分子化合物中筛选出合适的诱导组合。
本发明利用小分子化合物诱导鸡胚成纤维细胞，以期在短时间内获得大量鸡iPSC，为其进行离体保种、基因编辑研究等奠定良好基础。
为了实现上述目的，本发明采用如下技术方案：
一种诱导鸡胚成纤维细胞为鸡多能干细胞的化学诱导剂，包括如下组分：XAV-939、Y-27632、Resveratrol、SF1670、CHIR-99021、Menthone、β-Alanine和Ferrostatin-1；
且上述物质的质量浓度比为(5~40):(10~30):(6~100):(0.5~10):(6~40):(5~100):(20~100):(0.1~3)。
所取得的有益效果：小分子化合物组合可以将鸡胚成纤维细胞诱导为表达多能性蛋白的iPSC。
进一步的，一种诱导鸡胚成纤维细胞为鸡多能干细胞的化学诱导剂，包括如下组分：XAV-939、Y-27632、Resveratrol、SF1670、CHIR-99021、Menthone、β-Alanine和Ferrostatin-1；
且上述物质的质量浓度比为20:10:6:1:6:10:20:2。
一种诱导鸡胚成纤维细胞为鸡多能干细胞的方法，使用上述任一的化学诱导剂。
进一步的，在鸡原始生殖细胞培养液中加入所述化学诱导剂，诱导培养鸡胚成纤维细胞。
进一步的，培养过程中所述化学诱导剂各成分的终质量浓度如下：
20 µM XAV-939、10 µM Y-27632、6 µM Resveratrol、1 µM SF1670、6 µM CHIR-99021、10 µM Menthone、20 µM β-Alanine和2 µM Ferrostatin-1。
进一步的，所述鸡原始生殖细胞培养液包括如下组分：基础培养基DMEM、双抗、B27补充液、GlutaMAX、非必需氨基酸、β-巯基乙醇、丙酮酸钠、氨基酸溶液、鸡血清、白蛋白、肝素钠、human Activin A和human FGF。
进一步的，培养过程中，所述鸡原始生殖细胞培养液包括如下组分：在基础培养基DMEM上添加如下质量浓度的组分：1%双抗、2% B27补充液、2 mM GlutaMAX、1%非必需氨基酸、0.1 mM β-巯基乙醇、1 mM丙酮酸钠、1%氨基酸溶液、0.2%鸡血清、20%白蛋白、50mg/mL肝素钠、25 µg/mL human Activin A和25 µg/mL human FGF。
进一步的，诱导培养时间为96 h。
经由上述的技术方案可知，与现有技术相比，本发明取得的有益效果为：本发明的小分子化合物组合在96小时可以将鸡胚成纤维细胞诱导为表达多能性蛋白的iPSC。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据提供的附图获得其他的附图。
图1附图为本发明E组细胞培养24、72和96小时的细胞形态；
图2附图为本发明鸡iPSC的RT-PCR鉴定结果，为Pou5f3、NANOG基因表达情况；
图3附图为本发明鸡iPSC免疫荧光鉴定结果，A为PAS糖原染色结果，B、C、D为E组细胞培养96小时分别表达SSEA1、OCT4蛋白的情况。
具体实施方式
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。
本发明所需药剂为常规实验药剂，采购自市售渠道；未提及的实验方法为常规实验方法，在此不再一一赘述。
实施例1
1.材料与方法
1.1材料和试剂
种蛋来源于广东省农业科学院动物科学研究所原种鸡场，在38.5℃、相对湿度60%孵化至所需日龄。
小分子化合物CHIR-99021、Y-27632、Menthone、β-Alanine、Ferrostatin-1、XAV-939和SF1670购自selleck公司。
Human Activin A购自PeproTech公司。
Resveratrol、β-巯基乙醇购自sigma公司。
DMEM、鸡血清、PBS溶液、双抗、非必需氨基酸、GlutaMAXTM、丙酮酸钠、B27补充液、氨基酸溶液、白蛋白、肝素钠和胰酶购自Gibco公司。
human FGF购自R&D systems公司。
糖原PAS染色液购自北京百奥莱博公司。
RNA抽提试剂盒、cDNA反转试剂盒和PCR扩增试剂盒购自TAKALA公司。
抗体OCT4和SSEA1购自Abcam公司。
1.2鸡胚成纤维细胞的分离
准备孵育至4~18天的种蛋若干，灭菌处理过的剪刀、普通镊子等，提前开紫外照射细胞房30min。用酒精棉球擦拭种蛋尖端，普通镊子敲开一个口子，用镊子小心将胚胎取出，放置于装有PBS溶液的培养皿中，除去胚胎表面的蛋黄颗粒。将清洗好的胚胎转移至新的PBS溶液中，左手拿镊子夹住腿部，右手用剪刀剪取部分腿肌后转移至装有300µl PBS溶液的1.5ml离心管中，加入100µl的0.25% Trypsin/EDTA溶液放置于37℃培养箱中消化25min。加入400µl完全培养基中和，之后1000g常温离心5min，弃掉上清；用300µl的完全培养基重悬细胞沉淀，转移至48孔板进行培养，培养箱条件37℃、5% CO2。细胞每2天半量换液1次，每3~4天传代1次。
1.3试验分组
将分离的鸡胚成纤维细胞随机分组：
A组为对照组，全程用鸡原始生殖细胞培养液：
鸡原始生殖细胞培养液组成：在基础培养基DMEM上添加如下质量浓度的组分：1%双抗、2% B27补充液、2 mM GlutaMAX、1%非必需氨基酸、0.1 mM β-巯基乙醇、1 mM丙酮酸钠、1%氨基酸溶液、0.2%鸡血清、20%白蛋白、50mg/mL肝素钠、25 µg/mL human Activin A和25 µg/mL human FGF。
B组全程在A组基础上添加10 µM XAV-939、5 µM Y-27632、3 µM Resveratrol。
C组全程在B组基础上添加0.5 µM SF1670、3 µM CHIR-99021、5 µM Menthone。
D组全程在C组基础上添加10 µM β-Alanine、1 µM Ferrostatin-1。
E组全程在D组基础上调整小分子化合物的添加量，添加量为D组的2倍：20 µMXAV-939、10 µM Y-27632、6 µM Resveratrol、1 µM SF1670、6 µM CHIR-99021、10 µMMenthone、20 µM β-Alanine、2 µM Ferrostatin-1。
1.4 RT-PCR鉴定
收集1×105个细胞，用RNA抽提试剂盒提取细胞总RNA。采用反转录试剂盒cDNA合成第一链cDNA，以合成的cDNA为模板进行普通PCR实验。
引物为：
Pou5f3-F：5'-GGCTCAATGAGGCAGAGAAC-'3，SEQ ID NO:1；
Pou5f3-R：5'-GGACTGGGCTTCACACATTT-'3，SEQ ID NO:2；
NANOG-F：5'-AGCAGACCTCTCCTTGACCA-'3，SEQ ID NO:3；
NANOG-R：5'-TTCCTTGTCCCACTCTCACC-'3，SEQ ID NO:4。
PCR程序为：
94℃ 5 min，之后30个循环（94℃ 30 s, 57℃ 45 s, 72℃ 30 s），最后72℃延伸5 min。
PCR产物用1%琼脂糖凝胶进行电泳。
1.5鸡iPSC的鉴定
PAS糖原染色：细胞离心后吸去上清，加入10μl PBS重悬。重悬液滴加入载玻片中制成细胞涂片，风干后加入PAS固定液（试剂A）固定12min。纯水冲洗、晾干。加入氧化剂（试剂B），室温氧化16min，纯水冲洗2次每次30s，纯水浸洗两次，每次3min。加入Schiff染色液（试剂C），阴暗处浸染12min。纯水流水冲洗5min，加入Mayer苏木素染色液（试剂E），染色1~2min，水洗、晾干。
免疫荧光鉴定：细胞离心去掉上清后，转移到24孔板中用4％多聚甲醛固定10min。用PBS洗涤3次、每次5 min（以下PBS洗涤均如此），再用0.5％ Triton X-100通透5min。PBS洗涤，然后用含1％（v/v）BSA的PBS封闭液固定细胞45 min。PBS洗涤，在4℃的湿盒内与一抗OCT4（1:50）、SSEA1（1:50）孵育过夜。PBS洗涤，孵育二抗（1:400）。PBS洗涤后，用DAPI复染细胞核，晾干后滴加DAPI染液，室温避光孵育10min。最后通过共聚焦显微镜进行分析。
2.结果与分析
A~D组细胞培养7天，细胞有快速增殖，但形态基本一致，处于贴壁状态。E组细胞在D组基础上增加了小分子化合物的浓度，E组细胞培养72小时形态开始发生变化，小部分细胞由贴壁变为悬浮，并变圆而亮，96小时大部分细胞都是悬浮状态（图1）。而且细胞增殖较快，24孔板一个孔的细胞量由最初约1.6×104到72小时后增殖为1.2×105左右，此时1个孔的细胞传到2个孔；96小时后几乎所有细胞都是悬浮状态，此时每个孔细胞量约为3.1×105。
取E组0、24、48、72和96小时细胞进行RT-PCR鉴定，发现细胞在0和24小时不表达iPSC标记基因Pou5f3和NANOG，在48小时开始表达Pou5f3和NANOG（图2）。
培养96小时的E组悬浮细胞PAS糖原染色呈阳性，表明其为干细胞。免疫荧光结果表明（图3B、图3C、图3D），E组悬浮细胞培养96小时均表达iPSC标志蛋白SSEA1和OCT4。上述结果表明E组的鸡胚成纤维细胞在培养96小时后完全重编程为鸡iPSC。
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。对这些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 (7)
1.一种诱导鸡胚成纤维细胞为鸡多能干细胞的化学诱导剂，包括如下组分：XAV-939、Y-27632、Resveratrol、SF1670、CHIR-99021、Menthone、β-Alanine和Ferrostatin-1；
且上述物质的摩尔浓度比为20:10:6:1:6:10:20:2。
2.一种诱导鸡胚成纤维细胞为鸡多能干细胞的方法，其特征在于，使用权利要求1所述的化学诱导剂。
3.如权利要求2所述的方法，其特征在于，在鸡原始生殖细胞培养液中加入所述化学诱导剂，诱导培养鸡胚成纤维细胞。
4.如权利要求3所述的方法，其特征在于，培养过程中所述化学诱导剂各成分的终摩尔浓度如下：
20μM XAV-939、10μMY-27632、6μM Resveratrol、1μM SF1670、6μMCHIR-99021、10μMMenthone、20μMβ-Alanine和2μM Ferrostatin-1。
5.如权利要求3所述的方法，其特征在于，所述鸡原始生殖细胞培养液包括如下组分：基础培养基DMEM、双抗、B27补充液、GlutaMAX、非必需氨基酸、β-巯基乙醇、丙酮酸钠、氨基酸溶液、鸡血清、白蛋白、肝素钠、human ActivinA和human FGF。
6.如权利要求3所述的方法，其特征在于，所述鸡原始生殖细胞培养液包括如下组分：在基础培养基DMEM上添加如下组分：质量浓度为1％的双抗、质量浓度为2％的B27补充液、2mM GlutaMAX、质量浓度为1％的非必需氨基酸、0.1mMβ-巯基乙醇、1mM丙酮酸钠、质量浓度为1％的氨基酸溶液、质量浓度为0.2％的鸡血清、质量浓度为20％的白蛋白、50mg/mL肝素钠、25μg/mL humanActivinA和25μg/mLhuman FGF。
7.如权利要求3所述的方法，其特征在于，诱导培养时间为96h。
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