WO2013113196A1

WO2013113196A1 - Culture medium for primary culture of hippocampus neurons of a neonatal rat and preparation method and use thereof

Info

Publication number: WO2013113196A1
Application number: PCT/CN2012/075162
Authority: WO
Inventors: 孙晶; 刘佳明
Original assignee: Sun Jing; Liu Jiaming
Priority date: 2012-02-02
Filing date: 2012-05-08
Publication date: 2013-08-08
Also published as: CN102533654B; CN102533654A

Abstract

Provided is a culture medium for the primary culture of hippocampus neurons of a neonatal rat. The culture medium is formed by adding 1,6-diphosphate fructose, fructose and nutritional supplements to a basic medium Neurobasal. Also provided is a method for the primary culture of hippocampus neurons of a rat using the abovementioned culture medium, comprising the steps of: (1) preparing the culture medium of hippocampus neurons; (2) sampling and digesting; (3) preparing a single cell suspension; (4) inoculating and culturing. Hippocampus neurons with good growing status and higher purity can be obtained using the abovementioned culture medium.

Description

一种新生大鼠海马神经元原代培养的培养液 Primary culture medium of neonatal rat hippocampal neurons

及其制备方法和应用技术领域 And preparation method and application thereof

本发明属于生物技术领域，具体涉及一种新生大鼠海马神经元原代培养液及其制备方法和应用。 The invention belongs to the field of biotechnology, and particularly relates to a primary culture medium for newborn rat hippocampal neurons, a preparation method thereof and application thereof.

背景技术 Background technique

神经元是神经***结构和功能的基本单位，也是研究神经***生理、病理机制及各种神经递质功能的良好材料。海马组织中神经元数量和分布较为集中，海马神经元体外培养是研究神经 ***结构、功能以及病理、生理变化的重要手段之一。现有的海马神经元培养液中基本都是在 DMEM、 F12培养基添加血清，培养海马神经元。血清增加了胶质细胞等非神经元增殖速度，影响了海马神经元的纯度，对神经细胞有不同程度的毒性，且由于血清中成分复杂，对实验产生一定的影响或干扰。神经元培养的一个重要问题是保证神经元的纯度与活性。 Neurons are the basic unit of the structure and function of the nervous system, and are also good materials for studying the physiological and pathological mechanisms of the nervous system and the functions of various neurotransmitters. The number and distribution of neurons in hippocampus are concentrated. The culture of hippocampal neurons in vitro is one of the important means to study the structure, function and pathological and physiological changes of nervous system. In the existing hippocampal neuron culture medium, serum is added to DMEM and F12 medium to culture hippocampal neurons. Serum increases the proliferation rate of non-neurons such as glial cells, affects the purity of hippocampal neurons, has different degrees of toxicity to nerve cells, and has a certain influence or interference on the experiment due to the complex composition of serum. An important issue in neuronal culture is to ensure the purity and activity of neurons.

中国专利 CN1966674A公开了一种蜘蛛中枢神经细胞培养液该发明所采用的培养液添加了 200ml小牛血清；中国专利 CN1171991C公开了 "人神经干细胞的培养方法"，该发明培养液由 DMEM和 F12以 1 : 1混合而成的基础培养液，添加胰岛素、 L_谷氨酰胺、盐酸丁二胺、硒化钠、人转铁蛋白、氢化可的松、 ***，另外添加 5-20%新鲜血清；因此，以上神经培养液的配方和方法同样存在不能有效减少了胶质细胞的生长，保证神经元纯度的问题。培养液中即使添加了阿糖胞苷等抑制胶质细胞***的药物能提高神经细胞的纯度，但是，阿糖胞苷的加入对神经细胞生长也有一定的抑制作用，严重影响所培养的神经元活性。 Chinese patent CN1966674A discloses a spider central nervous cell culture solution. The culture solution used in the invention is supplemented with 200 ml of calf serum; Chinese Patent CN1171991C discloses "culture method of human neural stem cells", which is prepared by DMEM and F12. 1 : 1 mixed base culture solution, add insulin, L_glutamine, butanediamine hydrochloride, sodium selenide, human transferrin, hydrocortisone, progesterone, and add 5-20% fresh serum Therefore, the above formula and method of the nerve culture solution also have the problem that the growth of the glial cells can not be effectively reduced, and the purity of the neurons is ensured. Even if a drug that inhibits glial cell division, such as cytarabine, can increase the purity of nerve cells, the addition of cytarabine also inhibits the growth of nerve cells, which seriously affects the cultured neurons. active.

尽管无血清培养出现，提高了神经元培养的纯度，减少了胶质细胞的生长，但是所培养的海马神经元仍然具有存活率低、细胞生长缓慢和突触形成少等缺点，不能满足对细胞进一步实验的要求。目前无血清培养液的添加物都含有 Invitrogen ( GIBC0) 公司研发的神经元培养液 B27或（和） N2，其主要成分为多种维生素和微量元素。尽管提高了神经细胞培养的纯度，有效减少了胶质细胞的生长，但是仍然存在不能为海马神经元培养提供足够的能量，在缺氧环境下尤为明显，更容易导致致死性损害，影响了海马神经元的生长状态，从而造成细胞存活率低、细胞生长缓慢和突触形成少等问题。通过增加葡萄糖剂量的方法，结果会更糟糕，葡萄糖作为能源物质在无氧酵解产生大量乳酸堆也会降低培养液的 ρΗ俱增加渗透损害神经细胞，造成培养基中神经细胞生长抑制，同时也增加了神经细胞污染的机会。 Although serum-free culture appeared, the purity of neuron culture was improved and the growth of glial cells was reduced. However, the cultured hippocampal neurons still had shortcomings such as low survival rate, slow cell growth and less synapse formation, which could not satisfy the cells. Further experimental requirements. The current serum-free medium supplements contain the neuronal medium B27 or (and) N2 developed by Invitrogen (GIBC0), whose main components are multivitamins and trace elements. Although the purity of nerve cell culture is improved, and the growth of glial cells is effectively reduced, there is still insufficient energy for hippocampal neuron culture, especially in anoxic environment, which is more likely to cause fatal damage and affect hippocampus. The growth state of neurons causes problems such as low cell survival rate, slow cell growth, and less synapse formation. By increasing the glucose dose, the result will be even worse, as a glucose The production of a large amount of lactic acid heap in the anaerobic glycolysis of the energy substance also reduces the osmotic increase of the culture fluid, which impairs the damage of nerve cells, causes the inhibition of nerve cell growth in the medium, and also increases the chance of nerve cell contamination.

发明内容 Summary of the invention

本发明所要解决的技术问题是提供一种具有纯度高、生长状态好的海马神经元的培养液及其制备方法，并提供了利用这种培养液培养海马神经元的方法。 The technical problem to be solved by the present invention is to provide a culture solution having a high purity and good growth state of hippocampal neurons and a preparation method thereof, and a method for cultivating hippocampal neurons using the culture solution.

为解决上述技术问题，本发明采取的技术方案是，一种海马神经元培养液，该培养液为在基础培养液中加入 1、 6二磷酸果糖、果糖和营养添加剂；每升基础培养液中加入 1-lOmmol的 1、 6二磷酸果糖和 2_20mmol的果糖，其中所述基础培养液为 Neurobasal Medium„ In order to solve the above technical problem, the technical solution adopted by the present invention is a hippocampal neuron culture solution, which is added with fructose 1, 6 diphosphate fructose, fructose and nutritional additives in a basic culture solution; 1-lOmmol of fructose 1, 6 diphosphate and 2-20 mmol of fructose are added, wherein the basic medium is Neurobasal Medium

所述 1、 6二磷酸果糖与果糖质量浓度比为 1 : 2。每升基础培养液中加入如下组分的营养添加剂： 10-20 碱性成纤维细胞生长因子， 6-15mg ATP, 30-50U辅酶 A， 0. l_lg两性霉素 B， 5_50mg胰岛素， 0. 42-0. 83mg硫酸亚铁， 5_50mg人转铁蛋白和 20_60mg维生素混合液。维生素混合液为生物素、氯化胆碱、泛酸钙、叶酸、肌醇、维生素 Bl、维生素 B2、维生素 B6、维生素 B12 和烟酰胺的混合物。其中，本发明所述的商品化基础培养液 Neurobasal Medium ( GIBCO 公司）。之后采用 0. 22 μ πι的滤膜过滤除菌。配置好的培养液置于 4°C储存。 The mass ratio of fructose to fructose of 1,6 diphosphate is 1 : 2 . Each liter of the basic culture solution is supplemented with the following components: 10-20 basic fibroblast growth factor, 6-15 mg ATP, 30-50 U coenzyme A, 0. l_lg amphotericin B, 5_50 mg insulin, 0. 42 -0. 83 mg of ferrous sulfate, 5_50 mg of human transferrin and 20-60 mg of vitamin mixture. The vitamin mixture is a mixture of biotin, choline chloride, calcium pantothenate, folic acid, inositol, vitamin Bl, vitamin B2, vitamin B6, vitamin B12 and nicotinamide. Among them, the commercial basic culture liquid of the present invention is Neurobasal Medium (GIBCO Company). Then, the membrane was sterilized by filtration using a 0.22 μm π filter. The configured culture solution was stored at 4 °C.

本发明的培养液中 1， 6-二磷酸果糖是能在分子水平上调节细胞代谢中若干酶活性，恢复和改善细胞代谢，减轻细胞损伤，能促进血液循环，增加 ATP合成。果糖作为体外培养的能量成分明显优于葡萄糖，果糖酵解与葡萄糖酵解的主要区别在于在果糖酵解过程中，果糖氧化磷酸化为 F-1-P (l-磷酸果糖)一一糖酵解前体物质，能在缺氧状态下提供 ATP，维持细胞膜的离子泵，所以能有效的保护细胞膜的完整性和维持细胞活性的功能。而葡萄糖代谢过程中却不能产生 F-l-P。本发明添加 1， 6-二磷酸果糖和果糖的混合物有较好的效果， 1， 6-二磷酸果糖和果糖有显著的协同作用，可以显著增加增加海马神经元的活力，尤其在缺氧状态下照常提供 ATP 细胞，保证神经元的纯度与活性。 In the culture solution of the present invention, fructose 1,6-diphosphate can regulate several enzyme activities in cell metabolism at the molecular level, restore and improve cell metabolism, reduce cell damage, promote blood circulation, and increase ATP synthesis. Fructose is superior to glucose in energy culture in vitro. The main difference between fructose and glucose glycolysis is that during fructose hydrolysis, fructose is oxidized and phosphorylated to F-1-P (l-phosphate fructose). The solution of the precursor substance, which can provide ATP under hypoxia and maintain the ion pump of the cell membrane, can effectively protect the integrity of the cell membrane and maintain the function of cell activity. However, F-l-P cannot be produced during glucose metabolism. The invention adds a mixture of fructose 1,6-diphosphate and fructose, and has a remarkable synergistic effect, and the synergistic effect of fructose 1,6-diphosphate and fructose can significantly increase the activity of hippocampal neurons, especially in anoxic state. ATP cells are often provided as usual to ensure the purity and activity of neurons.

所述本发明的培养液中营养添加剂包括碱性成纤维细胞生长因子，胰岛素、硫酸亚铁、人转铁蛋白、 ATP、辅酶 A、两性霉素 B和维生素混合液。 The nutritional additive in the culture solution of the present invention includes basic fibroblast growth factor, insulin, ferrous sulfate, human transferrin, ATP, coenzyme A, amphotericin B, and a mixture of vitamins.

本发明中提供的动物细胞无血清低密度培养基中胰岛素是重要的血清替代成分。胰岛素可促进糖元与脂肪酸的合成，同时促进 RNA、蛋白质和脂类的合成。胰岛素可以为重组胰岛素、牛源胰岛素或人源胰岛素，胰岛素类因子也可以使用。本发明中使用的硫酸亚铁为培养基提供细胞生长所需的亚铁离子，同时可以作为转铁蛋白的替代成分。本发明中使用的转铁蛋白主要是细胞获取微量元素的主要渠道，同时具有促进胰岛素发挥作用的功能。在该发明使用的转铁蛋白可以是重组的、人源的或牛源的等，另外柠檬酸铁也可以起到同样的作用。本发明中使用的维生素为细胞的生长代谢提供必要的维生素，促进细胞生长和延长细胞活性，提供营养物质的同时降低代谢产物的堆积。本发明所使用的维生素是无菌包装的维生素混合液。使用浓度参考说市售的产品明书推荐浓度，采用相应培养基稀释即可。 ATP作为能量物质，在分解时放出大量的能量，可以为细胞培养的各个过程都提供充足的能量，改善和提高细胞酶的代谢功能，促进细胞的扩增和传代。辅酶 A能够促进三羧酸的循环，为细胞的扩增与传代提供能量。两性霉素 B的使用浓度在 0. l_lg/L。 In animal cells provided in the present invention, insulin is an important serum replacement component in serum-free low-density medium. Insulin promotes the synthesis of glycogens and fatty acids while promoting the synthesis of RNA, proteins and lipids. Insulin may be recombinant insulin, bovine insulin or human insulin, and insulin factors may also be used. The ferrous sulfate used in the present invention provides the culture medium The ferrous ions required for cell growth can also serve as an alternative to transferrin. The transferrin used in the present invention is mainly a main channel for cells to acquire trace elements, and has a function of promoting insulin action. The transferrin used in the invention may be recombinant, human or bovine, etc., and ferric citrate may also serve the same purpose. The vitamin used in the present invention provides essential vitamins for cell growth and metabolism, promotes cell growth and prolongs cell activity, and provides nutrients while reducing accumulation of metabolites. The vitamin used in the present invention is an aseptically packaged vitamin mixture. Use the concentration reference to refer to the recommended concentration of the commercially available product, and dilute with the corresponding medium. As an energy substance, ATP releases a large amount of energy during decomposition, which can provide sufficient energy for each process of cell culture, improve and enhance the metabolic function of cellular enzymes, and promote cell expansion and passage. Coenzyme A promotes the circulation of tricarboxylic acids and provides energy for cell expansion and passage. L_lg/L。 The amphotericin B is used at a concentration of 0. l_lg / L.

本发明还提供了一种原代培养大鼠海马神经元的方法，具有以下步骤： The invention also provides a method for primary culture of rat hippocampal neurons, having the following steps:

①制备如权利要求 1-5之一所述的海马神经元原代培养的培养液； 1 preparing a culture medium for primary culture of hippocampal neurons according to any one of claims 1 to 5;

②取材、消化：新生大鼠断头取脑，在冰上钝性拨离出脑组织，去除血管膜，完整取出海马组织；剪碎成约 lmm3的小块，加入约与组织等体积的浓度为 2-4 mg/mL的木瓜酶，混匀， 37 °C消化 5_15min。用含 10%胎牛血清 DMEM/F12培养基终止消化，并加入 10 μ 1 DNase I打开絮状黏连，吸管轻轻吹打组织块分散细胞。 2 Materials, digestion: Newborn rats decapitated the brain, bluntly dial out brain tissue on ice, remove the vascular membrane, completely remove the hippocampus tissue; cut into small pieces of about lmm3, add about the same volume of tissue and concentration For the 2-4 mg/mL papain, mix and digest at 37 °C for 5-15 min. The digestion was terminated with DMEM/F12 medium containing 10% fetal bovine serum, and 10 μl of DNase I was added to open the flocculated adhesion, and the pipette was gently pipetted to disperse the cells.

③制备单细胞悬液：将步骤②细胞悬液经 400目尼龙网过滤， lOOOr/min离心 lOmin去上清，力 Π 10%胎牛血清的 DMEM/F12重悬，调细胞密度为 1〜5 X 105个细胞 /mL浓度的单细胞悬液； 3 Preparation of single cell suspension: The cell suspension of step 2 was filtered through a 400 mesh nylon mesh, centrifuged lOOOr/min for 10 min to remove the supernatant, and the DMEM/F12 of 10% fetal bovine serum was resuspended to adjust the cell density to 1~5. a single cell suspension of X 105 cells/mL concentration;

④接种、培养将步骤③制备的单细胞悬液加入多聚 L-赖氨酸的培养板中，置于 37 5%C02 培养箱培养； 24h后将原来含 10%胎牛血清 DMEM/F12培养基更改为步骤①制备的海马神经元原代培养的培养液，每隔 2d换液 1次，每次更换一半培养液。 4 Inoculation and culture The single cell suspension prepared in step 3 was added to a poly-L-lysine culture plate and cultured in a 37 5% CO 2 incubator; after 24 h, the original 10% fetal bovine serum DMEM/F12 was cultured. The base was changed to the culture medium of the primary culture of the hippocampal neurons prepared in the step 1, and the medium was changed once every 2 days, and half of the culture solution was replaced each time.

本发明所能达到的有益效果如下： The beneficial effects that can be achieved by the present invention are as follows:

( 1 )利用本发明的培养液进行海马神经元体外培养，获得了相对单一、纯度高（纯度≡≥90% 以上）的海马神经元。这就是由于 1， 6-二磷酸果糖和果糖为海马神经元培养提供的足够的能量，促进了海马神经元的活性； (1) The hippocampal neurons cultured in vitro using the culture solution of the present invention obtain a relatively single, high purity (purity ≡ ≥ 90% or more) hippocampal neurons. This is due to the fact that fructose 1,6-diphosphate and fructose provide sufficient energy for the culture of hippocampal neurons, which promotes the activity of hippocampal neurons;

( 2 )利用本发明的培养液进行海马神经元体外培养，有效地提高了神经元的存活率，且神经元的生长状态良好、生理特性稳定，表明本培养方法可以满足体外实验的要求； (2) The culture medium of the present invention is used for in vitro culture of hippocampal neurons, thereby effectively improving the survival rate of neurons, and the growth state of the neuron is good and the physiological characteristics are stable, indicating that the culture method can meet the requirements of in vitro experiments;

( 3 )利用本发明的培养液进行海马神经元体外培养，与传统培养方法相比，体外培养的海马神经元存活的时间大大得到延长（高纯度、高活性的海马神经元细胞可以维持 16天以上）;(3) using the culture solution of the present invention to culture hippocampal neurons in vitro, compared with the conventional culture method, the sea cultured in vitro The survival time of horse neurons is greatly prolonged (high-purity, high-activity hippocampal neurons can be maintained for more than 16 days);

( 4) 本发明的培养液海马神经元培养液配制方法简单，可重复性强，操作简便易行。具体实施方式 (4) The preparation method of the culture fluid hippocampal neuron culture medium of the invention is simple, reproducible, and easy to operate. detailed description

实施例 1 海马神经元培养液的配方及制备方法 Example 1 Formulation and preparation method of hippocampal neuron culture solution

按照下表中的配方配置培养液 1-9: Configure the culture solution 1-9 according to the formula in the table below:

培养液 1、 6二磷果糖 ATP 碱性成辅酶两性胰岛硫酸维生素 Culture medium 1, 6 diphosphate fructose ATP alkaline into coenzyme amphoteric islet sulfuric acid vitamin

(mg) 纤维细霉素 (mg) Fibrin

类型 (mmol) 胞生长 A (U) B 亚铁混合液 Type (mmol) cell growth A (U) B ferrous mixture

ol) 因子 (g) (mg) (mg) (mg) (mg) Ol) factor (g) (mg) (mg) (mg) (mg)

( u g) ( u g)

培养液 1 3 5 7 10 35 0. 3 15 0. 55 16 8 Culture medium 1 3 5 7 10 35 0. 3 15 0. 55 16 8

培养液 2 1 3 6 16 30 0. 1 5 0. 45 5 6 Culture medium 2 1 3 6 16 30 0. 1 5 0. 45 5 6

培养液 3 10 20 15 17 50 1 50 0. 83 50 15 Culture medium 3 10 20 15 17 50 1 50 0. 83 50 15

培养液 4 5 10 8 19 40 0. 5 30 0. 61 27 10 Culture medium 4 5 10 8 19 40 0. 5 30 0. 61 27 10

培养液 5 1 2 7 12 32 0. 2 6 0. 42 5 7 Culture medium 5 1 2 7 12 32 0. 2 6 0. 42 5 7

培养液 6 8 13 10 15 38 0. 8 40 0. 77 36 9 Culture medium 6 8 13 10 15 38 0. 8 40 0. 77 36 9

培养液 7 2 8 9 20 44 0. 3 10 0. 51 12 10 Culture medium 7 2 8 9 20 44 0. 3 10 0. 51 12 10

上述培养液 1-7的制备方法如下： The preparation method of the above culture solution 1-7 is as follows:

a.按所述配比取 1、 6二磷酸果糖、果糖加入基础培养液 Neurobasal Medium中，充分搅拌均匀； a. According to the ratio, fructose 1, 6 diphosphate and fructose are added to the basic culture solution, Neurobasal Medium, and stirred well;

b. 加入上述量的碱性成纤维细胞生长因子、胰岛素、硫酸亚铁、人转铁蛋白、两性霉素8、维生素混合液； b. adding the above amount of basic fibroblast growth factor, insulin, ferrous sulfate, human transferrin, amphotericin 8, vitamin mixture;

c以 1-10当量浓度的氢氧化钠调 pH 7. 4-8. 0； l 至优选的优选优选的优选的优选的优选的优选的优选的优选的优选的优选的优选的优选的优选的优选的优选的优选的优选的优选的优选的优选为为为为为为为。

d.层流细胞培养室抽滤消毒， 4°C储存备用，使用前再加入所述量的 ATP以及辅酶。实施例 2 原代培养大鼠海马神经元的方法 d. The laminar flow cell culture chamber is filtered by suction filtration, stored at 4 ° C for use, and the amount of ATP and coenzyme are added before use. Example 2 Method for primary culture of rat hippocampal neurons

具有以下步骤： Has the following steps:

①制备如实施例 1所述的海马神经元原代培养的培养液； 1 preparing a culture medium for primary culture of hippocampal neurons as described in Example 1;

②取材、消化：新生大鼠断头取脑，在冰上钝性拨离出脑组织，去除血管膜，完整取出海马组织；剪碎成约 lmm3的小块，加入约与组织等体积的浓度为 2-4 mg/mL的木瓜酶，混匀， 37 °C消化 5_15min。用含 10%胎牛血清 DMEM/F12培养基终止消化，并加入 10 μ 1 DNase I打开絮状黏连，吸管轻轻吹打组织块分散细胞。 2 Materials, digestion: Newborn rats decapitated the brain, bluntly dial out brain tissue on ice, remove the vascular membrane, completely remove the hippocampus tissue; cut into small pieces of about lmm3, add about the same volume of tissue and concentration For 2-4 mg/mL papain, mix, 37 Digest for 5_15 min at °C. The digestion was terminated with DMEM/F12 medium containing 10% fetal bovine serum, and 10 μl of DNase I was added to open the flocculated adhesion, and the pipette was gently pipetted to disperse the cells.

④接种、培养将步骤③制备的单细胞悬液加入多聚 L-赖氨酸的培养板中，置于 37 5%C02 培养箱培养； 24 h后将原来含 10%胎牛血清 DMEM/F12培养基更改为步骤①制备的海马神经元原代培养的培养液，每隔 2d换液 1次，每次更换一半培养液。 4 Inoculation and culture The single cell suspension prepared in step 3 was added to the poly-L-lysine culture plate and cultured in a 37 5% CO 2 incubator; after 24 h, the original 10% fetal bovine serum DMEM/F12 was contained. The culture medium was changed to the culture medium of the primary culture of hippocampal neurons prepared in the step 1, and the medium was changed once every 2 days, and half of the culture solution was replaced each time.

实施例 3 原代培养大鼠海马神经元的形态学观察和功能试验 Example 3 Morphological observation and functional test of primary cultured rat hippocampal neurons

在倒置显微镜下观察，大鼠海马神经细胞接种 6〜8 h开始贴壁，单个分散，呈圆形。 24 h 后基本完全贴壁，细胞开始变扁平，并开始长出 1〜2个突起。 3 d后，神经元数量进一步增多，突起进一步增长，而神经胶质细胞也开始迅速***增殖，神经元下形成一片支持层。 10 d神经元状态良好，神经元突触发育成熟，形成明显的神经网络。培养至 14 d，虽然细胞数目有所减少，细胞却达到最佳的状态，胞体周围有明显的光晕，且细胞最饱满。培养 17 d，随着培养时间的进一步延长，细胞开始出现退化、脱落。 DMEM/F12+20%小牛血清组和 Neurobasal medium+2%B27组出现神经细胞的死亡，可见凋亡小体。 DMEM/F12+20%小牛血清实验组培养至 24 d，神经细胞全部死亡、退化，可见变性的神经细胞残迹。 Under the inverted microscope, the rat hippocampal neurons were inoculated 6 to 8 h after inoculation, and they were individually dispersed and rounded. After 24 h, it was almost completely adherent, and the cells began to flatten and began to grow 1 to 2 protrusions. After 3 days, the number of neurons increased further, the protrusions further increased, and the glial cells began to rapidly divide and proliferate, forming a support layer under the neurons. On the 10th day, the neurons were in good condition, and the neurons suddenly triggered to mature and form a distinct neural network. After 14 days of culture, although the number of cells was reduced, the cells reached an optimal state, and there was a clear halo around the cell bodies, and the cells were the most full. After 17 days of culture, the cells began to degenerate and fall off as the culture time was further extended. Neuronal death occurred in the DMEM/F12+20% calf serum group and the Neurobasal medium+2%B27 group, and apoptotic bodies were observed. The DMEM/F12+20% calf serum test group was cultured for 24 days, and all the nerve cells died and degenerated, and the degenerated nerve cell remnants were observed.

本发明实验组神经元胞体饱满，核大，突起明显增长，连接密集，形成复杂的神经纤维网络，阳性细胞（纯度）≡≥90%。显著高于 DMEM/F12+20%小牛血清组和 Neurobasal Medium+2%B27 组； DMEM/F12+20%小牛血清组和 Neurobasal Medium+2%B27组细胞数量相对较少，极少数细胞长出突起。用图像分析仪分析，结果见表 1-4。本发明组神经细胞胞体面积、长径、短径均显著高于 DMEM/F12+20%小牛血清组和 Neurobasal Medium+2%B27组（p〈0. 01 )，表明本发明组对神经细胞生长发育有促进作用。 In the experimental group of the present invention, the neurons are full, the nucleus is large, the protrusions are obviously increased, the connections are dense, and a complex nerve fiber network is formed, and the positive cells (purity) ≡ ≥ 90%. Significantly higher than DMEM/F12+20% calf serum group and Neurobasal Medium+2%B27 group; DMEM/F12+20% calf serum group and Neurobasal Medium+2%B27 group were relatively small in number, and very few cells were long. Protrusion. Analysis by image analyzer, the results are shown in Table 1-4. The cell body area, long diameter and short diameter of the nerve cell of the present invention were significantly higher than those of the DMEM/F12+20% calf serum group and the Neurobasal Medium+2% B27 group (p<0.01), indicating that the present invention group was on the nerve cells. Growth and development have a promoting effect.

培养的海马神经元的细胞活力细胞活力（％) Cell viability of cultured hippocampal neurons Cell viability (%)

培养液类型 Culture medium type

I d 3 d 7 d 10 d I d 3 d 7 d 10 d

DMEM/F12+20%小牛血清 96. 23 ± 2. 52 74. 51 ± 3. 28** 42. 57 ± 5. 49** 21. 45 ± 4. 29** Neurobasal Medium+2%B27 96.21 ±1.78 81.24±3.29 63.45 + 8.12** 58.14±3.62** 本发明（培养液 4) 98.21 ±1.24 96.53±3. 24 90. 14±3.28 86. 56±5.12 本发明（培养液 6) 99.21±0.97 98.68±1. 02 95. 08 ±2.65 88. 31 ±4.64 注： **表示 P〈0.01表示差异极显著。 DMEM/F12+20% calf serum 96. 23 ± 2. 52 74. 51 ± 3. 28** 42. 57 ± 5. 49** 21. 45 ± 4. 29** Neurobasal Medium+2%B27 96.21 ±1.78 81.24±3.29 63.45 + 8.12** 58.14±3.62** The present invention (culture solution 4) 98.21 ±1.24 96.53±3. 24 90. 14±3.28 86.56±5.12 The present invention Culture medium 6) 99.21±0.97 98.68±1. 02 95. 08 ±2.65 88. 31 ±4.64 Note: ** means P<0.01 means that the difference is extremely significant.

培养液 4中 5mmol/L 1， 6_二磷酸果糖和 lOmmol/L果糖有最好的效果。 In the culture solution 4, 5 mmol/L 1,6-diphosphate fructose and 10 mmol/L fructose have the best effects.

表 2 培养的海马神经元 MTT Table 2 Cultured hippocampal neurons MTT

MTT (0D) MTT (0D)

培养液类型 Culture medium type

Id 3d 7d 10d Id 3d 7d 10d

DMEM/F12+20%小牛血清 0.698 ±0.042 0. 342 ±0.054 0.178±0.048 0.119±0.081 DMEM/F12+20% calf serum 0.698 ±0.042 0. 342 ±0.054 0.178±0.048 0.119±0.081

Neurobasal Medium+2%B27 0.683 ±0.041 0. 621 ±0.052 0.421 ±0.057 0.361±0.087 本发明（培养液 1) 0.721±0.021 0. 701 ±0.032 0.681±0.035 0.659±0.061 本发明（培养液 5) 0.781±0.031 0. 763 ±0.041 0.711±0.051 0.681±0.053 由表 1可看出，与 DMEM/F12+20%小牛血清组、 eurobasal Medium+2%B27组比较，本发明（培养液 1) 组、本发明（培养液 5) 组 0D值均明显增加，表明本发明培养液可以促进神经细胞的增殖，提高神经活性。 Neurobasal Medium+2%B27 0.683 ±0.041 0. 621 ±0.052 0.421 ±0.057 0.361±0.087 The present invention (culture solution 1) 0.721±0.021 0. 701 ±0.032 0.681±0.035 0.659±0.061 The present invention (culture solution 5) 0.781± 0.031 0. 763 ±0.041 0.711±0.051 0.681±0.053 As can be seen from Table 1, compared with DMEM/F12+20% calf serum group, eurobasal medium+2%B27 group, the present invention (culture medium 1) group, this In the invention (culture solution 5), the 0D values of the group were significantly increased, indicating that the culture solution of the present invention can promote the proliferation of nerve cells and increase the nerve activity.

MTT (0D) MTT (0D)

培养液类型 Culture medium type

Id 3d 7d 10d Id 3d 7d 10d

DMEM/F 12+20%小牛血清 7.10±0.28 37.12±0.48** 82.18±3.25** 98.31 ±2.64** DMEM/F 12+20% calf serum 7.10±0.28 37.12±0.48** 82.18±3.25** 98.31 ±2.64**

Neurobasal Medium+2%B27 6.96±0.24 11.27±0.52** 33.76±0.91** 45.88±1.87** 本发明（培养液 2) 6.72±0.21 5.70±0.39 9.61±0.35 11.45±0.61 本发明（培养液 7) 6.72±0.21 5.52±1.31 9.78±0.41 10.52±0.89 注： **表示 p〈0.01表示差异极显著。 Neurobasal Medium+2%B27 6.96±0.24 11.27±0.52** 33.76±0.91** 45.88±1.87** The present invention (culture solution 2) 6.72±0.21 5.70±0.39 9.61±0.35 11.45±0.61 The present invention (culture solution 7) 6.72±0.21 5.52±1.31 9.78±0.41 10.52±0.89 Note: ** indicates that p<0.01 indicates that the difference is extremely significant.

表 4 组胞体直径和突起长度测量培养液类型胞体直径 (um) 突起长度 /cell (um) Table 4 Measurement of cell body diameter and protrusion length Culture medium type cell body diameter (um) protrusion length / cell (um)

DMEM/F 12+20%小牛血清 13.27±3.37** 32.36 ±14.29** DMEM/F 12+20% calf serum 13.27±3.37** 32.36 ±14.29**

Neurobasal Medium+2%B27 15.43±2.16 38.91 ±15.22** 本发明（培养液 3) 19.32±2.01 49.6 ±3.22 本发明（培养液 4) 21.36±2.45 47.3±3.71 注： **表示 P〈0.01表示差异极显著。 Neurobasal Medium+2%B27 15.43±2.16 38.91 ±15.22** The present invention (culture solution 3) 19.32±2.01 49.6 ±3.22 The present invention (culture solution 4) 21.36±2.45 47.3±3.71 Note: ** means P<0.01 means difference significant.

实施例 4 神经细胞存活率的测定 Example 4 Determination of nerve cell survival rate

本发明神经元培养液对神经细胞的存活起明显的促进作用，培养 14天时神经元存活率，与 The neuron culture liquid of the invention obviously promotes the survival of nerve cells, and the survival rate of neurons after 14 days of culture,

DMEM/F12+20%小牛血清、 Neurobasal Medium+2%B27组相比， DMEM/F12+20%小牛血清组、 Neurobasal Medium+2%B27组存活率分别为 65%、 69%。，而发明 1组和发明 2组分别为 81%、 Compared with DMEM/F12+20% calf serum and Neurobasal Medium+2%B27 group, the survival rates of DMEM/F12+20% calf serum group and Neurobasal Medium+2%B27 group were 65% and 69%, respectively. , and the invention group 1 and the invention group 2 are 81%, respectively.

85%, 本发明明显促进神经细胞的存活，差异极显著（p〈0.01)。 At 85%, the present invention significantly promoted the survival of nerve cells, and the difference was extremely significant (p < 0.01).

实施例 5 不同培养时间神经元纯度的测定 Example 5 Determination of Neuron Purity at Different Culture Time

表 5 不同培养时间神经元纯度（％) 和神经元形态 Table 5 Neuronal purity (%) and neuronal morphology at different culture times

培养时间 Training time

培养液类型 Culture medium type

第 2天第 4天第 6天第 8天第 10天第 12天第 14天第 16天本发明（培 71.06 91.46 92.14 92.29 95.32 96.45 96.54 97.65 养液 1) ±3.21 ±5.32 ±5.43 +4.21 ±2.90 ±1.32 ±1.21 ±0.92 Day 2 Day 4 Day 6 Day 8 Day 10 Day 12 Day 14 Day 16 This invention (cultivation 71.06 91.46 92.14 92.29 95.32 96.45 96.54 97.65 Nutrient 1) ±3.21 ±5.32 ±5.43 +4.21 ±2.90 ±1.32 ±1.21 ±0.92

+++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ +++ ++++ ++++ ++++ ++++ ++++ ++++ ++++

DMEM/F12+2 70.26 79.76 84.14 81.06 81.46 74.14 61.66 51.81DMEM/F12+2 70.26 79.76 84.14 81.06 81.46 74.14 61.66 51.81

0%小牛血清 ±3.41 ±4.72 ±4.43 + 3.21 ±5.32 ±4.53 + 3.92 ±1.06 0% calf serum ±3.41 ±4.72 ±4.43 + 3.21 ±5.32 ±4.53 + 3.92 ±1.06

+++ +++ ++++ +++ +++ ++ ++ + +++ +++ ++++ +++ +++ ++ ++ +

Neurobasal 69.06 87.14 89.06 81.46 76.14 68.06 62.46Neurobasal 69.06 87.14 89.06 81.46 76.14 68.06 62.46

Medium+2%B +4.53 ±5.39 ±5.89 + 3.57 ±5.72 ±5.92 + 2.51 ±1.32Medium+2%B +4.53 ±5.39 ±5.89 + 3.57 ±5.72 ±5.92 + 2.51 ±1.32

27 +++ +++ ++++ ++++ +++ ++ ++ + 注解： 27 +++ +++ ++++ ++++ +++ ++ ++ + Notes:

++++，神经元密度高，分布均匀，胞体粗大 +++，神经元较多，形态典型 ++++, high density of neurons, uniform distribution, large cell body +++, more neurons, typical form

++，神经元散在 ++, neurons are scattered

+，偶尔神经元 +, occasional neurons

结果显示， 12天后 DMEM/F12+20%小牛血清组和 Neurobasal Medium+2%B27组的神神经元大量死亡；而本发明（培养液 2 )组神经元的存活高，生长旺盛，第 16天仍然有很好的神经元纯度和典型的神经元形态。 The results showed that after 12 days, the DMEM/F12+20% calf serum group and the Neurobasal Medium+2%B27 group had a large number of neuronal deaths; while the (culture medium 2) group had high survival and vigorous growth, the 16th. The day still has good neuron purity and typical neuronal morphology.

结论：本发明方法培养的原代海马神经元体外最长存活期达 1月余，神经元形态典型，海马神经元纯度≡≥90%，且神经元的生长状态良好、生理特性稳定，可以满足体外实验的要求，可作为海马神经元原代培养的良好培养液。 Conclusion: The long-term survival of primary hippocampal neurons cultured by the method of the present invention is more than one month, the morphology of neurons is typical, the purity of hippocampal neurons is ≡≥90%, and the growth state of neurons is stable and the physiological characteristics are stable. The requirements of in vitro experiments can be used as a good culture medium for primary culture of hippocampal neurons.

Claims

权利要求书 Claim

1. 一种新生大鼠海马神经元原代培养的培养液，其特征在于：该培养液为在基础培养液中加入 1、 6二磷酸果糖、果糖和营养添加剂；其中，每升基础培养液中加入 1-lOmmol的 1、 6二磷酸果糖、 2_20mmol的果糖，其中所述基础培养液为 Neurobasal Medium。 A culture medium for primary culture of neonatal rat hippocampal neurons, characterized in that: the culture solution is added with fructose 1, 6 diphosphate, fructose and nutritional supplements in the base culture solution; wherein, each liter of the basic culture solution 1-lOmmol of fructose 1, 6 diphosphate, and 2-20 mmol of fructose were added thereto, wherein the basic medium was Neurobasal Medium.

2. 根据权利要求 1所述的新生大鼠海马神经元原代培养的培养液，其特征在于，所述 1、 6二磷酸果糖与果糖质量浓度比为 1 : 2。 The culture medium for primary culture of neonatal rat hippocampal neurons according to claim 1, wherein the ratio of the fructose to fructose of the 1,6-diphosphate is 1 : 2 .

3. 根据权利要求 1所述的新生大鼠海马神经元原代培养的培养液，其特征在于每升基础培养液中加入如下组分的营养添加剂： 10-20 碱性成纤维细胞生长因子， 6-15mg ATP, 30- 50U 辅酶 A 0. 1-lg两性霉素 B 5_50mg胰岛素 0. 42-0. 83mg硫酸亚铁 5_50mg人转铁蛋白和 20_60mg 维生素混合液。 The culture medium for primary culture of neonatal rat hippocampal neurons according to claim 1, characterized in that each of the basal medium is supplemented with a nutritional additive of the following components: 10-20 basic fibroblast growth factor, 6-15 mg ATP, 30-50 U Coenzyme A 0. 1-lg amphotericin B 5_50 mg insulin 0. 42-0. 83 mg ferrous sulfate 5_50 mg human transferrin and 20-60 mg vitamin mixture.

4. 根据权利要求 3所述的新生大鼠海马神经元原代培养的培养液，其特征在于，维生素混合液为生物素、氯化胆碱、泛酸钙、叶酸、肌醇、维生素 Bl、维生素 B2、维生素 B6、维生素 B12 和烟酰胺的混合物。 The culture medium for primary culture of neonatal rat hippocampal neurons according to claim 3, wherein the vitamin mixture is biotin, choline chloride, calcium pantothenate, folic acid, inositol, vitamin B1, vitamin B2, a mixture of vitamin B6, vitamin B12 and nicotinamide.

5. 根据权利要求 4所述的新生大鼠海马神经元原代培养的培养液，其特征在于，所述每升基础培养液中，维生素混合液由如下含量的各组分构成， The culture medium for primary culture of neonatal rat hippocampal neurons according to claim 4, wherein in each liter of the basic culture solution, the vitamin mixture liquid is composed of the following components.

生物素 0. 001-0. 01mg/L Biotin 0. 001-0. 01mg/L

氯化胆碱 5-20mg/L Choline chloride 5-20mg/L

泛酸钙 2-10mg/L Calcium pantothenate 2-10mg/L

叶酸 0-5 mg/L Folic acid 0-5 mg/L

肌醇 5-20 mg/L Inositol 5-20 mg/L

维生素 B1 2-10 mg/L Vitamin B1 2-10 mg/L

维生素 B2 0. 1-0. 5 mg/L Vitamin B2 0. 1-0. 5 mg/L

维生素 B6 2-10mg/L Vitamin B6 2-10mg/L

维生素 B12 0. 2-1. 0 mg/L Vitamin B12 0. 2-1. 0 mg/L

烟酰胺 2-5 mg/L。 Niacinamide 2-5 mg/L.

6. 根据权利要求 1所述的新生大鼠海马神经元原代培养的培养液的制备方法，其特征在于，包括如下步骤， A: 按所述配比取 1、 6二磷酸果糖、果糖加入基础培养液 Neurobasal Medium中，充分搅拌均匀； The method for preparing a culture medium for primary culture of newborn rat hippocampal neurons according to claim 1, comprising the following steps: A: According to the ratio, fructose 1, 6 diphosphate and fructose are added to the basic culture liquid Neurobasal Medium, and fully stirred;

B: 在上述溶液中加入营养添加剂； B: adding a nutritional additive to the above solution;

C: 以 1-10当量浓度的氢氧化钠调 pH 7. 4-8. 0； C: The pH is adjusted with a concentration of 1-10 equivalents of sodium hydroxide 7. 4-8. 0;

D: 层流细胞培养室抽滤消毒， 4°C储存备用； D: Laminar flow cell culture chamber is filtered and sterilized, stored at 4 ° C for use;

E: 最后加入所述量的 ATP和辅酶 A。 E: The amount of ATP and CoA were finally added.

7. 根据权利要求 1所述的新生大鼠海马神经元原代培养的培养液的制备方法，其特征在于，每升基础培养液中加入如下组分的营养添加剂： 10-20 μ g碱性成纤维细胞生长因子， 6-15mg ATP, 30-50U辅酶 A， 0. 1-lg两性霉素 B， 5_50mg胰岛素， 0. 42-0. 83mg硫酸亚铁， 5- 50mg人转铁蛋白和 20-60mg维生素混合液。 The method for preparing a culture medium for primary culture of neonatal rat hippocampal neurons according to claim 1, wherein a nutrient additive of the following components is added per liter of the base culture solution: 10-20 μg of alkaline Fibroblast growth factor, 6-15 mg ATP, 30-50 U coenzyme A, 0.1-lg amphotericin B, 5-50 mg insulin, 0. 42-0. 83 mg ferrous sulfate, 5- 50 mg human transferrin and 20 - 60 mg of vitamin mixture.

8. 新生大鼠海马神经元原代培养的方法，其特征在于具有以下步骤： 8. A method of primary culture of hippocampal neurons in neonatal rats, characterized by the following steps:

②取材、消化：新生大鼠断头取脑，在冰上钝性拨离出脑组织，去除血管膜，完整取出海马组织；剪碎成约 lmm3的小块，加入约与组织等体积的浓度为 2-4 mg/mL的木瓜酶，混匀， 37°C消化 5_15min，用含 10%胎牛血清 DMEM/F12培养基终止消化，并加入 10 μ 1 DNase I打开絮状黏连，吸管轻轻吹打组织块分散细胞； 2 Materials, digestion: Newborn rats decapitated the brain, bluntly dial out brain tissue on ice, remove the vascular membrane, completely remove the hippocampus tissue; cut into small pieces of about lmm3, add about the same volume of tissue and concentration For 2-4 mg/mL papain, mix, digest at 37 °C for 5-15 min, terminate digestion with 10% fetal bovine serum DMEM/F12 medium, add 10 μl DNase I to open flocculent adhesion, pipette light Lightly blow the tissue block to disperse the cells;

③制备单细胞悬液：将步骤②细胞悬液经 400目尼龙网过滤， lOOOr/min离心 lOmin去上清，力口 10%胎牛血清的 DMEM/F12重悬，调细胞密度为 1〜5 X 105个细胞 /mL浓度的单细胞悬液； 3 Preparation of single cell suspension: The cell suspension of step 2 was filtered through a 400 mesh nylon mesh, centrifuged lOOOr/min for 10 min to remove the supernatant, and DMEM/F12 of 10% fetal bovine serum was resuspended to adjust the cell density to 1~5. a single cell suspension of X 105 cells/mL concentration;

④接种、培养：将步骤③制备的单细胞悬液加入多聚 L-赖氨酸的培养板中，置于 37°C、 5%C02 培养箱培养； 24h后将原来含 10%胎牛血清 DMEM/F12培养基更改为步骤①制备的海马神经元原代培养的培养液，每隔 2d换液 1次，每次更换一半培养液。 4 Inoculation, culture: The single cell suspension prepared in step 3 was added to the poly-L-lysine culture plate, and cultured in a 5% CO 2 incubator at 37 ° C; after 10 h, the original 10% fetal bovine serum was contained. The DMEM/F12 medium was changed to the culture medium of the primary culture of hippocampal neurons prepared in the step 1, and the medium was changed once every 2 days, and half of the culture solution was replaced each time.