CN111304157A - Method for obtaining bovine initial state induced pluripotent stem cells - Google Patents
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Abstract
Description
技术领域technical field
本发明属于干细胞培养领域，具体地涉及一种获得牛初始态诱导多能干细胞的方法。The invention belongs to the field of stem cell culture, in particular to a method for obtaining bovine initial state induced pluripotent stem cells.
背景技术Background technique
胚胎干细胞(Embryonic Stem Cells，ESCs)是由内细胞团(Inner cell mass，ICM)分离而来的一种具有可分化为三胚层各种类型细胞的多能性干细胞。小鼠ESCs可直接从胚胎中获取，将获得后的ESCs重新打入胚胎并移植到母体内会产生嵌合子代。对于大家畜牛而言，利用相同的培养基只能获得形态类似于小鼠表皮干细胞(Epiblast stemcells,EpiSCs)的一类干细胞，而这类细胞并不能够形成嵌合胚胎。近年来，科研人员在人、猪和猴等动物中尝试利用不同的小分子抑制剂联合筛选，获得了形态和生物学特点类似小鼠ESCs的克隆。但都是从类似EpiSCs形态的转化而来，并未从胚胎中直接获取得到。可见不同物种间，对于小分子的依赖性是不同的，换句话说，不同物种间的通路调控大相径庭。目前为止，在大家畜特别是牛中，已有报道获得了体内外可分化发育为三个胚层组织和细胞的干细胞。这些干细胞与饲养层细胞有明显的边界并且表达多能性基因如Oct4、Nanog等，但其形态仍类似于EpiSCs且不能够承受单细胞传代。Embryonic Stem Cells (ESCs) are a kind of pluripotent stem cells isolated from the inner cell mass (ICM) that can differentiate into various types of cells in the three germ layers. Mouse ESCs can be obtained directly from embryos, and re-injection of the obtained ESCs into embryos and transplantation into mothers will produce chimeric progeny. For large cattle, only one type of stem cells with morphology similar to mouse epiblast stem cells (EpiSCs) can be obtained using the same medium, and these cells cannot form chimeric embryos. In recent years, researchers have tried combined screening of different small molecule inhibitors in animals such as humans, pigs and monkeys, and obtained clones with morphological and biological characteristics similar to mouse ESCs. However, they were all transformed from EpiSCs-like morphology, and were not directly obtained from embryos. It can be seen that the dependence on small molecules is different between different species. In other words, the pathway regulation between different species is very different. So far, in large animals, especially cattle, it has been reported that stem cells that can differentiate and develop into three germ layer tissues and cells in vitro and in vivo have been obtained. These stem cells have distinct borders with feeder cells and express pluripotency genes such as Oct4, Nanog, etc., but their morphology is still similar to EpiSCs and cannot withstand single-cell passage.
诱导多能干细胞(induced pluripotent stem cells,iPSCs)是将体细胞重编程为一种形态特点类似ESCs的一类细胞，不仅表达与ESCs相似水平的多能性基因而且具有多向分化发育的潜能。在牛上，iPSCs大多呈EpiSCs形态，具有多能性基因的表达，接受机械传代方式。这与真正的ESCs还相差很远，所以，为了获得生物学特性更接近早期胚胎的牛iPSCs，首先需要获得形态特点类似于小鼠iPSCs/ESCs的克隆。Induced pluripotent stem cells (iPSCs) are reprogrammed from somatic cells into a class of cells with morphological characteristics similar to ESCs. They not only express pluripotent genes at similar levels to ESCs, but also have the potential for multi-directional differentiation and development. In cattle, iPSCs are mostly EpiSCs, have pluripotency gene expression, and undergo mechanical passaging. This is far from true ESCs. Therefore, in order to obtain bovine iPSCs with biological characteristics closer to early embryos, it is first necessary to obtain clones with morphological characteristics similar to mouse iPSCs/ESCs.
发明内容SUMMARY OF THE INVENTION
针对现有技术中的不足，本发明的目的是提供一种获得牛初始态诱导多能干细胞的方法，解决了现有牛诱导多能干细胞的多能性不完全等问题。In view of the deficiencies in the prior art, the purpose of the present invention is to provide a method for obtaining bovine induced pluripotent stem cells in the initial state, which solves the problems of incomplete pluripotency of the existing bovine induced pluripotent stem cells.
本发明的目的是通过以下技术方案实现的：一种获得牛初始态诱导多能干细胞的方法，其特征在于，所述方法包括如下步骤：The object of the present invention is achieved through the following technical solutions: a method for obtaining bovine initial state induced pluripotent stem cells, characterized in that the method comprises the following steps:
步骤1、培养牛诱导多能干细胞：Step 1. Cultivate bovine induced pluripotent stem cells:
将牛诱导多能干细胞置于二氧化碳培养箱中，在含有5％二氧化碳，温度37℃的条件下培养，每天定时更换诱导培养基，培养3～4天，将诱导培养基弃掉后，再用PBS缓冲液洗净，用1mg/mL的dispase分散酶37℃下孵育8min～10min，观察克隆边缘与饲养层细胞分离后，吸取消化液，加入诱导培养基，从饲养层细胞上刮取牛诱导多能干细胞，作为培养牛初始态诱导多能干细胞的牛始发态诱导多能干细胞；The bovine induced pluripotent stem cells were placed in a carbon dioxide incubator, and cultured under the conditions of 5% carbon dioxide and a temperature of 37 °C. The induction medium was changed regularly every day for 3 to 4 days. Wash with PBS buffer, incubate with 1 mg/mL dispase dispase at 37°C for 8 min to 10 min, observe the separation of the clone edge from the feeder layer cells, aspirate the digestion solution, add the induction medium, and scrape the cattle induction medium from the feeder layer cells. Pluripotent stem cells, as bovine primary induced pluripotent stem cells for culturing bovine primary induced pluripotent stem cells;
步骤2、培养牛初始态诱导多能干细胞；Step 2, culturing bovine initial state induced pluripotent stem cells;
将所述步骤1中得到的牛诱导多能干细胞转化为牛初始态诱导多能干细胞过程如下：将牛诱导多能干细胞用0.05％的胰酶在37℃的条件下消化2min，加入1mL转化培养基重悬至1.5mL离心管中，在1000rpm转速条件下离心5min，离心后，接种至新鲜的饲养层上，并更换转化培养基进行培养，直至出现圆拱形，类似小鼠胚胎干细胞ESCs样的克隆，即为牛初始态诱导多能干细胞。The process of transforming the bovine induced pluripotent stem cells obtained in the step 1 into bovine initial state induced pluripotent stem cells is as follows: the bovine induced pluripotent stem cells are digested with 0.05% trypsin at 37° C. for 2 min, and 1 mL of transformation culture is added. The cells were resuspended in a 1.5mL centrifuge tube and centrifuged at 1000rpm for 5min. After centrifugation, they were inoculated onto a fresh feeder layer, and the transformation medium was replaced for culture until a round arch appeared, similar to mouse embryonic stem cell ESCs. The clones are bovine naive induced pluripotent stem cells.
进一步，所述诱导培养基的成分及含量为：基础培养基87％Knockout DMEM，10％KSR，Lif(10000x)，1％链霉素盘尼西林，1％谷氨酰胺，1％非必需氨基酸，巯基乙醇(1000x)，3μM CHIR99021，1μM PD0325901，8ng/mL碱性成纤维细胞生长因子。Further, the components and contents of the induction medium are: basal medium 87% Knockout DMEM, 10% KSR, Lif (10000x), 1% streptomycin penicillin, 1% glutamine, 1% non-essential amino acids, sulfhydryl Ethanol (1000x), 3 μM CHIR99021, 1 μM PD0325901, 8 ng/mL basic fibroblast growth factor.
进一步，步骤1中刮取牛诱导多能干细胞的方式为：采用枪头或细胞刮刀以Z型从左到右，从上到下将牛诱导多能干细胞从饲养层细胞上刮下。Further, the method of scraping the bovine induced pluripotent stem cells in step 1 is as follows: using a pipette tip or a cell scraper to scrape the bovine induced pluripotent stem cells from the feeder layer cells in a Z shape from left to right and from top to bottom.
所述步骤1还包括：将刮取得到的牛诱导多能干细胞接种到新鲜饲养层细胞上，且接种到新鲜饲养层细胞上的牛诱导多能干细胞占刮取得到的牛诱导多能干细胞总量的1/6。The step 1 further includes: inoculating the bovine induced pluripotent stem cells obtained by scraping onto fresh feeder layer cells, and the bovine induced pluripotent stem cells inoculated on the fresh feeder layer cells account for the total amount of bovine induced pluripotent stem cells obtained by scraping. 1/6 of the amount.
进一步，步骤2中所述转化培养基是在诱导培养基基础上添加12.5mg/mL胰岛素，(10x)N2supplement，1ng/mL TGF-b1，5mM SB202190,10mM SP600125,10mM SB203580，50mg/mL Vitamin C。Further, the transformation medium described in step 2 is to add 12.5mg/mL insulin on the basis of induction medium, (10x) N2supplement, 1ng/mL TGF-b1, 5mM SB202190, 10mM SP600125, 10mM SB203580, 50mg/mL Vitamin C .
通过上述设计方案，与现有技术相比本发明可以带来如下有益效果：Through the above-mentioned design scheme, compared with the prior art, the present invention can bring the following beneficial effects:
第一，本发明充分利用了始发态和初始态，这两种状态的特点来区别牛诱导多能干细胞iPSCs的不同的多能性阶段；First, the present invention makes full use of the priming state and the initial state, the characteristics of these two states to distinguish different pluripotency stages of bovine induced pluripotent stem cell iPSCs;
第二，本发明获得了更接近于早期胚胎的牛诱导多能干细胞。Second, the present invention obtains bovine induced pluripotent stem cells that are closer to early embryos.
附图说明Description of drawings
此处所说明的附图用来提供对本发明的进一步理解，构成本申请的一部分，本发明示意性实施例及其说明用于理解本发明，并不构成本发明的不当限定，在附图中：The accompanying drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The illustrative embodiments of the present invention and their descriptions are used to understand the present invention and do not constitute improper limitations of the present invention. In the accompanying drawings:
图1为牛诱导多能干细胞，培养过程中诱导多能干细胞的形态图；Figure 1 is a morphological diagram of bovine induced pluripotent stem cells and induced pluripotent stem cells during the culture process;
图2为牛初始态iPSCs，诱导后4天形成与周围饲养层细胞界限明显的圆拱形克隆形态图。Figure 2 shows the morphology of bovine iPSCs in the initial state, forming a dome-shaped clone with a clear boundary with the surrounding feeder layer cells 4 days after induction.
具体实施方式Detailed ways
以下结合优选实施例，进一步阐述本发明。本实施例仅用于说明本发明而不用于限制本发明的范围。本实施例中未注明具体条件的实验方法，通常按照常规条件。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的优选实施方法与材料仅作示范之用。The present invention will be further described below with reference to the preferred embodiments. This embodiment is only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not specify specific conditions in this example are generally in accordance with conventional conditions. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be used in the methods of the present invention. The methods and materials of preferred implementations described herein are provided for illustrative purposes only.
本发明提出的一种获得牛初始态诱导多能干细胞的方法包括培养牛诱导多能干细胞和培养牛初始态诱导多能干细胞两个阶段：A method for obtaining bovine initial state induced pluripotent stem cells provided by the present invention includes two stages of culturing bovine induced pluripotent stem cells and culturing bovine initial state induced pluripotent stem cells:
一、培养牛诱导多能干细胞：1. Cultivate bovine induced pluripotent stem cells:
牛诱导多能干细胞的培养，具体操作如下：将牛诱导多能干细胞置于二氧化碳培养箱中，在含有5％二氧化碳，温度37℃的条件下培养，每天定时更换诱导培养基，所述诱导培养基的成分及含量为：基础培养基87％Knockout DMEM，10％KSR，Lif(10000x)，1％链霉素盘尼西林，1％谷氨酰胺，1％非必需氨基酸，巯基乙醇(1000x)，3μM CHIR99021，1μMPD0325901，8ng/mL碱性成纤维细胞生长因子，培养3～4天，根据克隆长势及状态对其进行传代；将诱导培养基弃掉后，再用PBS缓冲液洗净以防培养基中成分影响消化的效果，用1mg/mL的dispase分散酶37℃下孵育9min，观察克隆边缘与饲养层细胞分离后，吸取消化液，加入诱导培养基，用枪头或细胞刮刀以“Z”型从左到右，从上到下将细胞刮下，从而保证细胞充分消化；将刮取得到的牛诱导多能干细胞接种到新鲜饲养层细胞上，且接种到新鲜饲养层细胞上的牛诱导多能干细胞占刮取得到的牛诱导多能干细胞总量的1/6，从而保证牛诱导多能干细胞稳定繁殖，详见图1，图1示出牛诱导多能干细胞，培养过程中诱导多能干细胞的形态图；The cultivation of bovine induced pluripotent stem cells is as follows: the bovine induced pluripotent stem cells are placed in a carbon dioxide incubator, cultured under the conditions of 5% carbon dioxide and a temperature of 37°C, and the induction medium is changed regularly every day. The composition and content of the base are: basal medium 87% Knockout DMEM, 10% KSR, Lif (10000x), 1% streptomycin penicillin, 1% glutamine, 1% non-essential amino acids, mercaptoethanol (1000x), 3 μM CHIR99021, 1μMPD0325901, 8ng/mL basic fibroblast growth factor, cultured for 3 to 4 days, and passaged according to the clone growth and state; after discarding the induction medium, wash it with PBS buffer to prevent the medium The middle ingredients affect the digestion effect. Incubate with 1 mg/mL dispase dispase at 37°C for 9 min, observe the separation of the clone edge from the feeder layer cells, aspirate the digestion solution, add the induction medium, use a pipette tip or a cell scraper to mark "Z" Type from left to right, and scrape the cells from top to bottom to ensure that the cells are fully digested; inoculate the bovine induced pluripotent stem cells obtained by scraping onto fresh feeder cells, and inoculate the bovine induced pluripotent stem cells on the fresh feeder cells. Pluripotent stem cells account for 1/6 of the total amount of bovine induced pluripotent stem cells obtained by scraping, so as to ensure the stable reproduction of bovine induced pluripotent stem cells. See Figure 1 for details. Figure 1 shows bovine induced pluripotent stem cells. Morphology of stem cells;
二、培养牛初始态诱导多能干细胞：2. Cultivate bovine initial state induced pluripotent stem cells:
牛诱导多能干细胞转化为牛初始态诱导多能干细胞过程如下：将牛诱导多能性干细胞用0.05％的胰酶在37℃的条件下消化2min，加入1mL转化培养基重悬至1.5mL离心管中，转化培养基是在诱导培养基基础上添加12.5mg/mL胰岛素，(10x)N2supplement，1ng/mLTGF-b1，5mM SB202190,10mM SP600125,10mM SB203580，50mg/mL Vitamin C，在1000rpm转速条件下离心5min，离心后，接种至新鲜的饲养层上，并更换转化培养基进行培养，直至出现圆拱形，类似小鼠ESCs样的克隆，即为牛初始态诱导多能干细胞。详见图2，图2示出牛初始态iPSCs，诱导后4天形成与周围饲养层细胞界限明显的圆拱形克隆形态图。The process of transforming bovine induced pluripotent stem cells into bovine initial state induced pluripotent stem cells is as follows: digest bovine induced pluripotent stem cells with 0.05% trypsin at 37°C for 2 min, add 1 mL of transformation medium and resuspend to 1.5 mL and centrifuge In the tube, the transformation medium was supplemented with 12.5mg/mL insulin, (10x) N2supplement, 1ng/mL TGF-b1, 5mM SB202190, 10mM SP600125, 10mM SB203580, 50mg/mL Vitamin C, at 1000rpm on the basis of induction medium Centrifuge for 5 min, inoculate on fresh feeder layer after centrifugation, and replace the transformation medium for culture until a round arch appears, similar to mouse ESCs-like clones, which are bovine initial state induced pluripotent stem cells. See Fig. 2 for details. Fig. 2 shows the morphological diagram of bovine naive iPSCs, which formed a circular arch-shaped clone with a clear boundary with the surrounding feeder cells 4 days after induction.
上述说明并非对本发明的限制，本发明也并不限于上述举例。本技术领域的普通技术人员在本发明的实质范围内，作出的变化、改型、添加或替换，也应属于本发明的保护范围，本发明的保护范围以权利要求书为准。The above description does not limit the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by those of ordinary skill in the art within the essential scope of the present invention should also belong to the protection scope of the present invention, which is subject to the claims.
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