Chinese Journal of Tissue Engineering Research ›› 2019, Vol. 23 ›› Issue (29): 4735-4742.doi: 10.3969/j.issn.2095-4344.1781
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Li Nan, Lin Zhong
Revised:
2019-04-02
Online:
2019-10-18
Published:
2019-10-18
Contact:
Lin Zhong, MD, Chief physician, Reproductive Medicine Center, Maternal and Child Health Hospital of Liuzhou City, Liuzhou 545001, Guangxi Zhuang Autonomous Region, China
About author:
Li Nan, MD, Associate researcher, Reproductive Medicine Center, Maternal and Child Health Hospital of Liuzhou City, Liuzhou 545001, Guangxi Zhuang Autonomous Region, China
Supported by:
the Natural Science Foundation of Guangxi Zhuang Autonomous Region, No. 2015GXNSFBA139177 (to LN), 2017GXNSFAA198193 (to LZ) and 2017GXNSFAA198199 (to LN); the Self-Financing Research Project of the Health and Family Planning Commission of Guangxi Zhuang Autonomous Region, No. Z20180027 (to LN); Liuzhou Science and Technology Development Plan, No. 2016G020217 (to LN)
CLC Number:
Li Nan, Lin Zhong . In vitro embryo culture is the core of embryo engineering technology [J]. Chinese Journal of Tissue Engineering Research, 2019, 23(29): 4735-4742.
2.1 早期胚胎体外培养系统 目前,胚胎体外培养系统仍无法完全模拟哺乳动物雌性生殖道的物理及环境条件。早期胚胎体外发育的最佳体系不仅受到培养基组成成分的影响,同时还受到多种物理参数的影响(胚胎发育至孵化的环境、饲养细胞层的有无等)。最早在1970年,Bowman等[6]在研究中就发现体外培养的早期胚胎会发生发育阻断现象。体外培养的胚胎经过移植后,会出现大量的胚胎吸收、死亡、流产、死胎、出生后死亡、胎儿过大综合征等问题[7]。目前序贯培养系统的体外胚胎生产的新技术已有了较大的进步,但是仍然无法满足生产及科研的需要,仍然有待进一步完善、优化[8]。体外胚胎培养系统的分类如下: 离体器官培养系统:1949年,Hammond等[9]将生理盐水中加入卵黄,在体外将8-细胞期的BALB/C小鼠胚胎成功培养到囊胚阶段,这打开了早期胚胎体外培养的大门。1962年,Biggers等[10]首次利用离体BALB/C小鼠输卵管膨大部来培养BALB/C小鼠早期胚胎,并且成功获得了囊胚。4年后,Papaioannou等[11]利用离体小鼠输卵管培养系统将小鼠的单细胞胚胎培养至桑椹胚\囊胚阶段,其后采用同样的方法获得大鼠、仓鼠、猪、牛的桑椹胚\囊胚,表明输卵管上皮细胞分泌某些特殊物质能够支持早期胚胎的发育[12-13]。曾经有研究将母兔输卵管作为临时“培养箱”,并获得桑椹胚\囊胚阶段胚胎[14-15],所获得的囊胚经过选择,由胚胎学家装入到移植管中移植到子宫,剩余的囊胚进行冷冻保存,但是要进行输卵管结扎从而避免胚胎的丢失,程序比较繁琐,效率也不是很高。后来随着体外胚胎培养技术的提高而逐渐放弃了该方法。 离体小鼠输卵管培养系统系统在使用CZB基础培养液作为基础培养基时,胚胎发育效果较好。但是更换为其他基础培养基如培养基199、碳酸盐缓冲液、重碳酸盐缓冲液的时候,效果却不尽人意[16]。 简单微滴培养系统:常规培养系统是将胚胎单独在培养液中进行培养,而不添加其他细胞及其分泌因子的一种培养方法,分为微滴法和培养板法[6]。前者是在塑料培养皿中制作30-100 μL的微滴,上面覆石蜡油,将胚胎置于其中培养,一般用于数量较少的胚胎培养; 后者是直接将胚胎置于含500-800 μL 培养液的四孔培养板中培养,一般用于数量较多的胚胎培养[11]。经过胚胎培养液的不断改进和培养条件的逐步完善, 该方法的效率和质量有了很大的提高,因此,成为目前胚胎体外培养的主要方法。 简单微滴培养系统根据所使用的器械的不同分为微滴培养法、平板培养法、微穴培养法。该系统将体外生产的早期胚胎与简单的培养液共同培养。微滴培养法主要针对培养胚胎数量较少的情况,其可创造一个更有利于早期胚胎发育的环境。平板培养法则主要用于培养比较多的胚胎,其操作简单,但效果欠佳,与体内雌性生殖道中的环境相差甚远[17]。近年来随着无透明带核移植技术的发展,在徒手克隆中出现了培养板单孔内进行微穴培养法培养[4]。 细胞共培养系统:体外共培养系统是随着对胚胎发育阻断问题的研究而产生的,该系统主要是指将早期胚胎与辅助细胞或者体细胞在简单培养体系的基础上一起培养,使早期胚胎完成发育。目前用于共培养体系统使用的细胞有卵丘细胞、输卵管上皮细胞、卵泡颗粒细胞、子宫内膜细胞、BRL细胞、非洲绿猴肾细胞等[18]。共培养系统可以提高体外胚胎的囊胚率和胚胎质量,有利于胚胎移植后妊娠。Minami等[12]使用了输卵管上皮细胞与早期羊胚进行共培养。Krisher等[13]将小鼠囊胚与人宫颈癌细胞共培养,发现孵化率有显著提高。目前对共培养的作用机制仍然不是很清楚。Allen等[18]认为胚胎与体细胞的直接接触是主要原因。但是McCaffrey等[19]的试验结果却否定了这个观点。还有研究者认为体细胞主要通过分泌一些有益的细胞因子,如生长因子等,并去除培养液中的重金属物质、细胞毒性物质等,减少过氧化物的产生而发挥作用[20]。研究表明,共培养对克服小鼠早期胚胎发育阻滞作用较为明显,且输卵管上皮细胞共培养的效果优于卵丘细 胞[12]。有研究者建立了小鼠早期胚胎与肿瘤细胞体外共培养模型,结果显示,早期胚胎与肿瘤细胞共培养可显著提高小鼠2-细胞胚胎的体外发育效率[18]。但共培养系统的具体机制仍待研究。而输卵管离体培养因较为烦琐和胚胎发育率不高等原因较少被使用。与体细胞共培养法可以利用体细胞生长过程中产生的有益因子克服发育阻断,从而促进胚胎发育,但随着培养液的不断改良, 实验室更倾向于使用常规培养系统。 序贯培养系统:序贯培养系统是指在对早期胚胎发育特点研究的基础上,针对早期胚胎发育的不同时期对各种物质的不同需求而添加不同的物质到培养基中的连续培养系统[21]。序贯培养系统是在对早期胚胎发育代谢的特点和雌性生殖道中各种物质的认识基础上逐渐发展起来的,其发展对共培养系统等也起到了促进作用。1997年Gardner等[22]利用G1、G2序贯培养基成功地培养了人类的早期胚胎。随着其他科学家的研究及推广,成分确定的序贯培养系统已开始受到了学者们的重视[23]。其他商品化的序贯培养基也得到了开发,如S1、S2;Q1、Q2;P1、P2等序贯培养基[24]。近年来,虽然胚胎培养体系已从静置的微滴法逐渐发展为效果更好的动态微流体培养,这促进了人类辅助生殖技术等的应用和发展[25],但动态培养体系操作较为复杂,限制了其广泛应用,而静置培养体系则因培养效果稳定、易操作而一直占据主导地位,其中微滴法是目前最常用的培养方法。 各种早期胚胎体外培养系统差异见表1。"
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