生物技术通报 ›› 2023, Vol. 39 ›› Issue (5): 130-141.doi: 10.13560/j.cnki.biotech.bull.1985.2022-1371
曾虹1(), 曾睿琳1, 付伟1, 吉文汇1, 兰道亮2,3()
收稿日期:
2022-11-07
出版日期:
2023-05-26
发布日期:
2023-06-08
通讯作者:
兰道亮,男,博士,教授,研究方向:高原动物生殖生理与繁殖;E-mail: landaoliang@163.com作者简介:
曾虹,女,硕士研究生,研究方向:动物生殖与遗传、动物干细胞;E-mail: zenghong3000@163.com
基金资助:
ZENG Hong1(), ZENG Rui-lin1, FU Wei1, JI Wen-hui1, LAN Dao-liang2,3()
Received:
2022-11-07
Published:
2023-05-26
Online:
2023-06-08
摘要:
诱导性多能干细胞(induced pluripotent stem cells,iPSCs)是指通过导入特定的转录因子将终末分化的体细胞重编程为可以无限增殖更新并具有分化为三胚层多种细胞类型的一类干细胞系。目前对人与小鼠的iPSCs研究已经取得了很多重要成果,但其他动物,如牛等经济型有蹄类家畜iPSCs的研究始终没有突破性的进展。如何将外源转录因子通过重编程载体高效安全地导入体细胞中并持续表达是生产牛诱导多能干细胞(bovine induced pluripotent stem cells,biPSCs)的主要瓶颈。本文就biPSCs建立中重编程系统的选择、诱导因子的选择、小分子化合物的添加等方面进行综述,以期为进一步完善biPSCs及牛胚胎干细胞系的建立提供参考。
曾虹, 曾睿琳, 付伟, 吉文汇, 兰道亮. 牛诱导多能干细胞的建立及应用研究进展[J]. 生物技术通报, 2023, 39(5): 130-141.
ZENG Hong, ZENG Rui-lin, FU Wei, JI Wen-hui, LAN Dao-liang. Research Progress in the Application and Establishment of Bovine Induced Pluripotent Stem Cells[J]. Biotechnology Bulletin, 2023, 39(5): 130-141.
非整合型方法 Approach of non-integrating | 优点 Pros | 缺点 Cons |
---|---|---|
腺病毒 | 基因组整合频率低,载体容量大 | 重编程效率低,具有免疫原性,对某些细胞需多次感染 |
仙台病毒 | 无基因组整合,载体容量大,转染效率高,稳定转染, 重编程效率高 | 多个转录因子需多个病毒携带,具有免疫原性,需传代10次以上才可完全清除病毒基因组,只能加工RNA |
质粒转染 | 基因组整合频率低,操作简单,免疫原性低,无病毒组分 | 不能自我复制,重编程效率极低,需要多次转染 |
转座子 | 不涉及病毒,可精确切除,只需一次转染,载体容量大,免疫原性低 | 可能导致突变和染色体重排,重编程效率低,需要用转座酶进行切除 |
微环DNA载体 | 基因组整合频率低,操作简单,转染效率高,免疫原性极低 | 不能自我复制,需多次转染,重编程效率极低 |
小分子化合物 | 具有瞬时控制能力,易操作,能促进iPSCs的生成 | 对毒性效率存在争议,确定或非特异的效果具有不确定性或非特异性 |
表1 部分非整合型基因传递方法的优缺点
Table 1 Pros and cons of partial non-integrating gene delivery approaches
非整合型方法 Approach of non-integrating | 优点 Pros | 缺点 Cons |
---|---|---|
腺病毒 | 基因组整合频率低,载体容量大 | 重编程效率低,具有免疫原性,对某些细胞需多次感染 |
仙台病毒 | 无基因组整合,载体容量大,转染效率高,稳定转染, 重编程效率高 | 多个转录因子需多个病毒携带,具有免疫原性,需传代10次以上才可完全清除病毒基因组,只能加工RNA |
质粒转染 | 基因组整合频率低,操作简单,免疫原性低,无病毒组分 | 不能自我复制,重编程效率极低,需要多次转染 |
转座子 | 不涉及病毒,可精确切除,只需一次转染,载体容量大,免疫原性低 | 可能导致突变和染色体重排,重编程效率低,需要用转座酶进行切除 |
微环DNA载体 | 基因组整合频率低,操作简单,转染效率高,免疫原性极低 | 不能自我复制,需多次转染,重编程效率极低 |
小分子化合物 | 具有瞬时控制能力,易操作,能促进iPSCs的生成 | 对毒性效率存在争议,确定或非特异的效果具有不确定性或非特异性 |
组别 Group | Naïve状态 Naïve state | Primed状态 Primed state |
---|---|---|
培养条件 | LIF/血清或2i | Activin A/bFGF或CHIR/IWR1 |
FGF2依赖性 | 不依赖 | 依赖 |
LIF依赖性 | 依赖 | 不依赖 |
BMP4依赖性 | 依赖 | 不依赖 |
Wnt激活 | 自我更新 | 分化 |
克隆形态 | 隆起 | 扁平 |
XX失活情况 | XaXa | XaXi |
Nanog表达 | 高 | 低 |
Fgf5表达 | 低 | 高 |
OCT4增强子 | 远端 | 近端 |
H3K24me3表达 | 低 | 高 |
嵌合小鼠胚胎 | 能 | 不能 |
表2 小鼠胚胎干细胞Naïve与Primed状态多能性的比较
Table 2 A comparison of naïve and primed state pluripotence
组别 Group | Naïve状态 Naïve state | Primed状态 Primed state |
---|---|---|
培养条件 | LIF/血清或2i | Activin A/bFGF或CHIR/IWR1 |
FGF2依赖性 | 不依赖 | 依赖 |
LIF依赖性 | 依赖 | 不依赖 |
BMP4依赖性 | 依赖 | 不依赖 |
Wnt激活 | 自我更新 | 分化 |
克隆形态 | 隆起 | 扁平 |
XX失活情况 | XaXa | XaXi |
Nanog表达 | 高 | 低 |
Fgf5表达 | 低 | 高 |
OCT4增强子 | 远端 | 近端 |
H3K24me3表达 | 低 | 高 |
嵌合小鼠胚胎 | 能 | 不能 |
培养基 Medium | 主要添加成分 Main component | 参考文献 Reference |
---|---|---|
CTRF | TeSR1培养基(无FGF2和TGFb)、FGF2和IWR1 | [ |
LCDM | hLIF、CHIR99021、(S)-(+)-dimethindene maleate、minocycline hydrochloride | [ |
TiF培养基 | mTeSR plus培养基、PD0325901、CHIR-99021、hLIF、IWR1、iDOT1L、Forskolin | [ |
bEPSC培养基 | mTeSR1培养基、CHIR99021、WH-4-023、XAV939、IWR-1、维生素C、LIF、Activin A | [ |
表3 部分用于获得牛多能干细胞的培养基
Table 3 Part of the medium used to obtain bovine pluripotent stem cells
培养基 Medium | 主要添加成分 Main component | 参考文献 Reference |
---|---|---|
CTRF | TeSR1培养基(无FGF2和TGFb)、FGF2和IWR1 | [ |
LCDM | hLIF、CHIR99021、(S)-(+)-dimethindene maleate、minocycline hydrochloride | [ |
TiF培养基 | mTeSR plus培养基、PD0325901、CHIR-99021、hLIF、IWR1、iDOT1L、Forskolin | [ |
bEPSC培养基 | mTeSR1培养基、CHIR99021、WH-4-023、XAV939、IWR-1、维生素C、LIF、Activin A | [ |
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