CN116121177A - Pig potential expansion stem cell culture medium and cultivation method thereof - Google Patents
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Abstract
Description
技术领域technical field
本发明属于动物细胞培养技术领域，具体涉及潜能扩展干细胞培养基及其培育方法。The invention belongs to the technical field of animal cell culture, and in particular relates to a potential expansion stem cell culture medium and a cultivation method thereof.
背景技术Background technique
虽然小鼠胚胎干细胞培育技术手段已非常成熟，但是在将近20多年的研究中，研究者们始终没有获得类似小鼠的具有真正全能性的猪胚胎干细胞系（pES）[1, 2]。大多数研究人员发现在通过各种培养手段和体内分离培养出的猪pES细胞系很难维持胚胎干细胞（ES）样状态，pES细胞高表达Nanog、Rex1和TDGF1等直接参与向外胚层干细胞分化的细胞因子，也就是说pES细胞表现出强烈的分化成外胚层ES样状态的趋势[3-5]。甚至Sox2、bFGF、FG·FR1、FGFR2等仅在猪外胚层细胞中表达的细胞因子也在分离培养的pES细胞系中表达[6-8]。究其原因，主要是因为并不能确定可以维持猪胚胎干细胞多能性状态的培养液和细胞信号通路。研究者们针对于这两个主要因素，仿照其他哺乳动物胚胎干细胞建系的成功研究做了一系列的尝试，目前却只能获得具有一定多能性的“猪外胚层样胚胎干细胞系（EpiSC-like pESCs）”[5, 9, 10]。Although mouse embryonic stem cell cultivation techniques are very mature, in nearly 20 years of research, researchers have not obtained a truly totipotent porcine embryonic stem cell line (pES) similar to mice [1, 2]. Most researchers found that it is difficult to maintain the embryonic stem (ES)-like state in porcine pES cell lines isolated and cultured through various culture methods and in vivo. pES cells highly express Nanog, Rex1 and TDGF1, which are directly involved in the differentiation of ectodermal stem cells. Cytokines, that is to say pES cells show a strong tendency to differentiate into an ectodermal ES-like state [3-5]. Even Sox2, bFGF, FG·FR1, FGFR2 and other cytokines expressed only in porcine ectoderm cells are also expressed in isolated and cultured pES cell lines [6-8]. The reason is mainly because the culture medium and cell signaling pathways that can maintain the pluripotent state of porcine embryonic stem cells cannot be determined. Aiming at these two main factors, researchers have made a series of attempts in imitation of the successful research on the establishment of other mammalian embryonic stem cell lines, but so far they can only obtain a certain pluripotent "porcine ectoderm-like embryonic stem cell line (EpiSC -like pESCs)” [5, 9, 10].
除了用传统的方法从内细胞团分离培养ES细胞，近年来，诱导多能干细胞（iPS）技术也被应用到建立pES细胞系中来，但是截至目前，利用iPS技术建立起来的piPS细胞系都显示出启动多能性状态，具有扁平的形态和FGF以及许多与mEpiSCs类似的特征，并且piPS细胞系在形态学、AP活性、信号通路的激活和多能标记物的表达等方面与EpiSC-likepESCs细胞系非常相似，也就是说piPSC类似于“EpiSC-like pESCs”[8, 9, 11]。并且迄今为止，也没有获得嵌合体的成功案例。In addition to the traditional method of isolating ES cells from the inner cell mass, induced pluripotent stem (iPS) technology has also been applied to establish pES cell lines in recent years, but so far, the piPS cell lines established using iPS technology are all Displays an activated pluripotent state with a flattened morphology and FGF and many features similar to mEpiSCs, and the piPS cell line is similar to EpiSC-like pESCs in terms of morphology, AP activity, activation of signaling pathways, and expression of pluripotent markers. The cell lines are very similar, that is, piPSCs are similar to “EpiSC-like pESCs” [8, 9, 11]. And so far, there is no successful case of chimerism.
猪潜能扩展干细胞（EPSCs）比目前的干细胞系具有更大的分化、扩增潜力，它们具有发育胚胎中第一细胞的特征，并且可以发育成任何类型的细胞[12]。其获取阶段在受精卵还只分裂为4或8个细胞时期，这些细胞比从内细胞团发育阶段分离获取的ES细胞更少进入分化进程，实验数据显示其全能性更高，能生成ESCs、EpiSCs等所有的干细胞类型，甚至是滋养层干细胞。其通过靶向调节着床前小鼠胚胎谱系分化的关键分子通路，具有广泛胚外和胚胎谱系分化倾向的扩增潜能干细胞可以成功地衍生出[9, 12]。大量的研究已经确定了丝裂原活化蛋白激酶（MAPKs）通路、Src通路、Hippo通路和核糖基化多聚ADP（PARP）细胞因子在胚胎干细胞分化中的主要作用[10, 13]。特别是Src阻断剂部分阻断了桑葚胚的发育，影响了胚泡中的滋养外胚层和原始内胚层的分化[14]。基因Parp1/2和tankyrase 1/2(Tnks/ Tnks2) 的表达降低分别导致胚胎干细胞的分化和滋养层的分化，而敲除Parg基因会直接影响PARP水解能力，从而导致滋养层干细胞分化能力和滋养外胚层的缺失[15]。另外，Wnt信号在脊椎动物和滋养细胞的早期发育中也起着重要作用。重要的是，通过Hippo通路的关键效应因子Yap1，Wnt和MAPK通路之间存在信号交叉，从而影响胚胎的分化[12,16]。Porcine potential-expanding stem cells (EPSCs) have greater differentiation, expansion potential than current stem cell lines, they have the characteristics of the first cells in the developing embryo, and can develop into any type of cell [12]. In the acquisition stage, the fertilized egg is only divided into 4 or 8 cells. These cells are less likely to enter the differentiation process than ES cells isolated from the inner cell mass development stage. Experimental data show that they are more totipotent and can generate ESCs, EpiSCs and all stem cell types, even trophoblast stem cells. By targeting key molecular pathways regulating preimplantation mouse embryonic lineage differentiation, expansion potential stem cells with broad extraembryonic and embryonic lineage differentiation propensities can be successfully derived [9, 12]. Numerous studies have identified the major roles of the mitogen-activated protein kinases (MAPKs) pathway, Src pathway, Hippo pathway, and ribosylated poly-ADP (PARP) cytokines in ES cell differentiation [10, 13]. In particular, Src blockers partially blocked morula development, affecting the differentiation of trophectoderm and primitive endoderm in blastocysts [14]. The reduced expression of genes Parp1/2 and tankyrase 1/2 (Tnks/ Tnks2) leads to the differentiation of embryonic stem cells and trophoblast differentiation, respectively, while the knockout of Parg gene will directly affect the ability of PARP hydrolysis, resulting in the differentiation ability of trophoblast stem cells and the differentiation of trophoblasts. Loss of ectoderm [15]. In addition, Wnt signaling also plays an important role in the early development of vertebrates and trophoblasts. Importantly, there is a signaling crossover between the Wnt and MAPK pathways through the key effector of the Hippo pathway, Yap1, which affects embryonic differentiation [12,16].
尽管现有技术对猪潜能扩展干细胞进行了大量的理论和实验研究，但尚未建立稳定的猪潜能扩展干细胞培育体系。Although a large number of theoretical and experimental studies have been carried out on porcine potential expansion stem cells in the prior art, a stable porcine potential expansion stem cell cultivation system has not been established yet.
发明内容Contents of the invention
针对上述存在的技术不足，本发明提供了潜能扩展干细胞培养基及其培育方法，具体通过如下技术方案实现：In view of the above-mentioned technical deficiencies, the present invention provides a potential expansion stem cell culture medium and a cultivation method thereof, which are specifically realized through the following technical solutions:
一种用于猪潜能扩展干细胞的培养基，其包括以下成分：DMEM/F-12培养基，KSR，MEM NEAA，谷氨酰胺-青霉素-链霉素，β-巯基乙醇，人LIF蛋白，PD0325901，CHIR99021，JNK抑制剂VIII，SB203580和XAV939。A medium for porcine potential expansion stem cells, which includes the following components: DMEM/F-12 medium, KSR, MEM NEAA, glutamine-penicillin-streptomycin, β-mercaptoethanol, human LIF protein, PD0325901 , CHIR99021, JNK inhibitor VIII, SB203580 and XAV939.
优选地，所述培养基包括以下成分：DMEM/F-12培养基作为基础培养基；基础培养基终体积20%的KSR，1× (vol/vol)的MEM NEAA，1×(vol/vol)的谷氨酰胺-青霉素-链霉素，0.1 mM终浓度的β-巯基乙醇，103 U人LIF蛋白，1.0 μM终浓度的PD0325901，3.0 μM终浓度的CHIR99021，4.0 μM终浓度的JNK抑制剂VIII，10.0 μM终浓度的SB203580，5.0 μM终浓度的XAV939。通过该优选的培养基配方，可以培养得到并维持稳定的猪潜能扩展干细胞体系。Preferably, the medium comprises the following components: DMEM/F-12 medium as basal medium; 20% KSR of basal medium final volume, 1× (vol/vol) MEM NEAA, 1× (vol/vol ), β-mercaptoethanol at 0.1 mM final concentration, 10 3 U human LIF protein, PD0325901 at 1.0 μM final concentration, CHIR99021 at 3.0 μM final concentration, JNK inhibitor at 4.0 μM final concentration Agent VIII, SB203580 at 10.0 μM final concentration, XAV939 at 5.0 μM final concentration. Through the preferred medium formula, a stable porcine potential expansion stem cell system can be cultivated and maintained.
本发明还提供了建立猪潜能扩展干细胞的培育方法，包括以下步骤：将猪8-细胞胚胎加入本发明所述培养基中进行培养，经培养、传代和扩增得到猪潜能扩展干细胞。The present invention also provides a cultivation method for establishing porcine potential-expanding stem cells, comprising the following steps: adding porcine 8-cell embryos to the culture medium of the present invention for culturing, and obtaining porcine potential-extending stem cells through culturing, passage and expansion.
优选地，所述方法包括以下步骤：从猪受精胚胎获取猪8-细胞胚胎，分别移入96孔SNL给料板或凝胶化板的孔中，加入本发明所述培养基，培养所述8-细胞胚胎直到孵化并形成衍生物，将其分离或用口吸管将其机械切成小片，转移到新的96孔SNL给料板或凝胶化板中，继续培养至细胞群落达到80-90%密度，将细胞传代至24孔SNL给料板或凝胶化板中，扩增至6孔板，将所得猪潜能扩展干细胞冷冻于胚胎冷冻液，保存于液氮中。通过该优选的培育方法，可以培养得到并维持稳定的猪潜能扩展干细胞体系。Preferably, the method comprises the following steps: obtaining pig 8-cell embryos from pig fertilized embryos, respectively transferring them into the wells of a 96-well SNL feeding plate or a gelling plate, adding the culture medium described in the present invention, and culturing the 8-cell embryos. -Cell embryos until they hatch and form derivatives, which are detached or mechanically cut into small pieces with a mouth pipette, transferred to a new 96-well SNL feeder plate or gelling plate, and continue to grow until the cell colony reaches 80-90 % density, the cells were subcultured to 24-well SNL feeder plates or gelling plates, expanded to 6-well plates, and the obtained porcine potential expansion stem cells were frozen in embryo freezing solution and stored in liquid nitrogen. Through the preferred cultivation method, a stable porcine potential expansion stem cell system can be obtained and maintained.
本发明还提供了通过本发明所述培育方法得到的猪潜能扩展干细胞。The present invention also provides the porcine potential expansion stem cells obtained by the cultivation method of the present invention.
本发明成功建立了高表达OCT4、SOX2、NANOG的稳定猪EPSC细胞。The invention successfully establishes stable porcine EPSC cells highly expressing OCT4, SOX2 and NANOG.
附图说明Description of drawings
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图进行说明：In order to make the purpose, technical scheme and beneficial effect of the present invention clearer, the present invention provides the following drawings for illustration:
图1显示了使用本发明培养基培育猪胚胎的结果对比图。其中左图为使用普通培养基培养10天时的胚胎发育情况，右图为使用本发明培养基培养10天时成功筛选到的pEPSC克隆。Figure 1 shows a comparison chart of the results of cultivating pig embryos using the medium of the present invention. Among them, the left picture shows the development of embryos when the ordinary medium is used for 10 days, and the right picture shows the successfully screened pEPSC clones when the medium of the present invention is used for 10 days.
图2显示了通过免疫荧光染色检测EPSC关键因子OCT4、SOX2、NANOG表达水平的图。结果显示，EPSC中OCT4、SOX2、NANOG均高表达。Figure 2 shows the graph of detecting the expression levels of key EPSC factors OCT4, SOX2, and NANOG by immunofluorescence staining. The results showed that OCT4, SOX2, and NANOG were highly expressed in EPSCs.
具体实施方式Detailed ways
以下结合具体实施方式对本发明进行更为详细的描述，这些描述仅仅是为了例证性说明本发明的技术方案，并非对本发明的保护范围进行任何方式的限制。在不脱离本发明基本原理和精神的前提下，本领域技术人员可以对本发明的某些具体方案、技术特征、参数等进行各种修改、替换和调整，仍落在本发明的保护范围之内。The present invention will be described in more detail below in conjunction with specific embodiments. These descriptions are only for illustrative purposes to illustrate the technical solution of the present invention, and are not intended to limit the protection scope of the present invention in any way. Without departing from the basic principles and spirit of the present invention, those skilled in the art can make various modifications, replacements and adjustments to certain specific solutions, technical features, parameters, etc. of the present invention, which still fall within the protection scope of the present invention .
实施例1 猪潜能扩展干细胞（pEPSC）的培育方法及初步鉴定。Example 1 The cultivation method and preliminary identification of porcine potential-expanding stem cells (pEPSC).
收集D3.5天的猪体内受精胚胎，将获取的猪8-细胞胚胎分别移入96孔SNL给料板或EPSCM凝胶化板的1孔。将胚胎在EPSC培养基中培养数天，直到孵化并形成衍生物。将其精确分离或用口吸管将其机械切成小片，转移到新的96孔SNL给料板或EPSCM凝胶化板中，继续培养至细胞群落达到80-90%密度。将细胞传代至24孔SNL给料板或凝胶化板，扩增至6孔板。将所得EPSCs冷冻于胚胎冷冻液，保存于液氮中。Pig in vivo fertilized embryos on day D3.5 were collected, and the obtained pig 8-cell embryos were transferred to 1 well of a 96-well SNL feeding plate or an EPSCM gelling plate, respectively. Embryos are cultured in EPSC medium for several days until they hatch and form derivatives. Precisely separate them or mechanically cut them into small pieces with a mouth pipette, transfer them to a new 96-well SNL feeding plate or EPSCM gelling plate, and continue culturing until the cell colony reaches 80-90% density. Cells were passaged to 24-well SNL feeder plates or gelling plates and expanded to 6-well plates. The resulting EPSCs were frozen in embryo freezing solution and stored in liquid nitrogen.
上述潜能扩展干细胞培养基的配方为：DMEM/F-12培养基（高葡萄糖含量的无谷氨酰胺培养基）作为基础培养基，加入基础培养基终体积20%的KSR（作为血清替代物）、1×(vol/vol)的MEM NEAA（提供非必需氨基酸）、1× (vol/vol)的谷氨酰胺-青霉素-链霉素、0.1 mM终浓度的β-巯基乙醇、103 U人LIF蛋白、1.0 μM终浓度的PD032590、3.0 μM终浓度的CHIR99021、4.0 μM终浓度的JNK抑制剂VIII、10.0 μM终浓度的SB203580、5.0 μM终浓度的XAV939。本发明所述的潜能扩展干细胞培养基也简称为EPSC培养基或者EPSCM，其所含有的这些培养基成分都是本领域普通技术人员熟知的且可容易地市购获得。The formula of the above-mentioned potential-expanding stem cell medium is: DMEM/F-12 medium (glutamine-free medium with high glucose content) is used as the basal medium, and 20% of the final volume of the basal medium is added with KSR (as a serum substitute) , 1×(vol/vol) MEM NEAA (providing non-essential amino acids), 1×(vol/vol) glutamine-penicillin-streptomycin, 0.1 mM final concentration of β-mercaptoethanol, 10 3 U human LIF protein, PD032590 at a final concentration of 1.0 μM, CHIR99021 at a final concentration of 3.0 μM, JNK inhibitor VIII at a final concentration of 4.0 μM, SB203580 at a final concentration of 10.0 μM, and XAV939 at a final concentration of 5.0 μM. The potential-expanding stem cell medium of the present invention is also referred to as EPSC medium or EPSCM for short, and the medium components contained therein are well-known to those of ordinary skill in the art and can be easily obtained commercially.
如图1所示，本发明实施例1获得了可长期传代的EPSC细胞系，克隆呈现大而扁平圆形，边缘光滑，细胞群落密集。As shown in FIG. 1 , in Example 1 of the present invention, an EPSC cell line capable of long-term passage was obtained, and the clones were large, flat and round, with smooth edges and dense cell colonies.
如图2所示，通过免疫荧光染色检测本发明实施例1所得EPSC中关键因子OCT4、SOX2、NANOG的表达水平。结果显示，EPSC中OCT4、SOX2、NANOG均高表达。As shown in FIG. 2 , the expression levels of key factors OCT4, SOX2, and NANOG in EPSCs obtained in Example 1 of the present invention were detected by immunofluorescence staining. The results showed that OCT4, SOX2, and NANOG were highly expressed in EPSCs.
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