CN101173247A - A method for cultivating osteoclasts using mesenchymal stem cells combined with cytokines - Google Patents

本发明公开了一种利用间充质干细胞联合细胞因子培养破骨细胞的方法。主要是采用小鼠骨实质来源的间充质干细胞与小鼠脾脏单个核细胞共同培养，再添加核因子κB受体活化子配体和巨噬细胞集落刺激因子培养破骨细胞的方法。本发明方法操作简单，方便实用，可以在最早时间获得破骨细胞，缩短了培养时间；其次本发明方法所得破骨细胞数量多，解决了破骨细胞难以大量制备的问题，可以满足一般细胞生物学实验的需要；还有本发明方法所得的破骨细胞成熟度高，骨质吸收能力强，有利于开展功能研究；另外，本发明方法所需要的细胞因子少，可以节约经费开支。The invention discloses a method for cultivating osteoclasts by using mesenchymal stem cells combined with cytokines. The method mainly adopts the co-cultivation of mesenchymal stem cells derived from mouse bone parenchyma and mouse spleen mononuclear cells, and then adds nuclear factor κB receptor activator ligand and macrophage colony-stimulating factor to culture osteoclasts. The method of the present invention is simple to operate, convenient and practical, can obtain osteoclasts at the earliest time, and shortens the culture time; secondly, the number of osteoclasts obtained by the method of the present invention is large, which solves the problem that osteoclasts are difficult to prepare in large quantities, and can meet the requirements of general cell biology. The needs of scientific experiments; in addition, the osteoclasts obtained by the method of the present invention have high maturity and strong bone absorption capacity, which is beneficial to carry out functional research; in addition, the method of the present invention requires less cytokines, which can save money.

一种利用间充质干细胞联合细胞因子培养破骨细胞的方法 A method for cultivating osteoclasts using mesenchymal stem cells combined with cytokines

技术领域technical field

本发明属于破骨细胞的培养方法，具体地说涉及一种利用间充质干细胞联合细胞因子培养破骨细胞的方法。The invention belongs to a method for culturing osteoclasts, in particular to a method for cultivating osteoclasts by using mesenchymal stem cells combined with cytokines.

背景技术Background technique

破骨细胞(osteoclasts，OC)是人和动物体内吸收骨组织的主要细胞，在生理情况下，破骨细胞与成骨细胞相互协助，参与贯穿生命全过程的骨质重建活动，维持骨骼系统的动态平衡；在肿瘤、炎症、骨质疏松症和自身免疫性疾病等病理情况下，破骨细胞参与疾病的发生、进展，影响疾病的转归和预后。Osteoclasts (OC) are the main cells that absorb bone tissue in humans and animals. Under physiological conditions, osteoclasts and osteoblasts cooperate with each other to participate in bone reconstruction activities throughout the whole life process and maintain the integrity of the skeletal system. Dynamic balance; in pathological conditions such as tumors, inflammation, osteoporosis and autoimmune diseases, osteoclasts participate in the occurrence and progression of diseases, and affect the outcome and prognosis of diseases.

破骨细胞由造血干细胞分化发育而来，在体内可以分解骨组织，在体外培养的破骨细胞可以在骨片、象牙或者人工骨组织培养上形成吸收性陷窝，成熟时变为含有多个细胞核的巨大细胞，破骨细胞的主要特征为呈特异性的抗酒石酸的酸性磷酸酶(TRAP+)组化阳性，以及F-actin染色阳性，表达降钙素受体、组蛋白酶K(cathepsin-k)、整合素αvβ3(integrinsαvβ3)等(Walsh MC等，AnnuRev Immunol.2006；24：33-63)。Osteoclasts are differentiated and developed from hematopoietic stem cells, and can decompose bone tissue in vivo. Osteoclasts cultured in vitro can form resorptive lacunae on bone slices, ivory, or artificial bone tissue cultures. When mature, they become containing multiple Huge cells with nuclei and osteoclasts are mainly characterized by specific tartrate-resistant acid phosphatase (TRAP+) histochemical positive staining, positive staining for F-actin, expression of calcitonin receptor, histone K (cathepsin-k ), integrin αvβ3 (integrinsαvβ3), etc. (Walsh MC et al., AnnuRev Immunol.2006; 24:33-63).

在体外培养破骨细胞是研究多种激素、细胞因子与破骨细胞和骨吸收关系的基础，也是研究肿瘤、炎症、骨质疏松症和自身免疫性疾病发病机制和药物治疗方法的基础。近年来，根据从破骨细胞研究中获得的发现，人们使用其调控因子作为动员剂，作为造血干细胞移植前采集造血干细胞的手段之一(KolletO，Nat Med.2006 Jun；12(6)：657-64.Epub 2006 May 21)；开发出了破骨细胞分泌因子的抗体、拮抗剂和抑制剂，用于治疗骨疾病如骨质疏松症和癌症如恶性肿瘤转移(Abe M等，Leukemia.2006 Jul；20(7)：1313-5.Epub 2006 Apr 13)；将破骨细胞引入组织工程领域，将其与成骨细胞共同种植于生物材料上，改善组织工程化骨的三维结构等等(韩大庆等天津生物医学工程2006学术年会论文摘要)。综上所述，建立破骨细胞的体外培养方法，深入研究破骨细胞的发育、分化、成熟和发挥功能的规律以及其分子调控机制，有利于寻找造血、自身免疫疾病、肿瘤和骨代谢疾病方面新的诊断技术和治疗药物。The culture of osteoclasts in vitro is the basis for studying the relationship between various hormones, cytokines, osteoclasts and bone resorption, and is also the basis for studying the pathogenesis and drug treatment of tumors, inflammation, osteoporosis and autoimmune diseases. In recent years, according to the findings obtained from osteoclast research, people use its regulatory factors as mobilizers, as one of the means of collecting hematopoietic stem cells before hematopoietic stem cell transplantation (KolletO, Nat Med.2006 Jun; 12(6): 657 -64.Epub 2006 May 21); Antibodies, antagonists and inhibitors of osteoclast-secreted factors have been developed for the treatment of bone diseases such as osteoporosis and cancers such as malignant tumor metastasis (Abe M et al., Leukemia.2006 Jul; 20(7): 1313-5.Epub 2006 Apr 13); Introduce osteoclasts into the field of tissue engineering, co-plant them with osteoblasts on biomaterials, improve the three-dimensional structure of tissue-engineered bone, etc. ( Han Daqing et al Tianjin Biomedical Engineering 2006 Academic Annual Conference abstract). In summary, the establishment of an in vitro culture method for osteoclasts and the in-depth study of the development, differentiation, maturation, and functioning of osteoclasts and their molecular regulation mechanisms are beneficial to the search for hematopoietic, autoimmune diseases, tumors, and bone metabolism diseases. new diagnostic techniques and therapeutics.

到目前为止，培养破骨细胞的方法(吕明波等国际骨科学杂志，2006年9月，第27卷，第5期)主要有：(1)、成熟破骨细胞分离培养法1984年Chamber等率先从兔子四肢长骨中原代分离出了破骨细胞，然而，破骨细胞在动物体内数量少、体积大、紧贴骨髓腔内表面生长、胞体分离时容易损伤，分离后不易培养；另外，分离法获得的是终末期破骨细胞，难以用于进行生长、增殖和分化方面的研究；(2)、骨巨细胞瘤破骨细胞分离法骨巨细胞瘤是一种以溶骨性骨破坏为特征的骨肿瘤，Zambonin-Zallon A等从骨巨细胞瘤瘤体中分离出大量的破骨细胞。不过，骨巨细胞瘤发病率低，组织来源少，无法长期稳定提供破骨细胞来源；并且，该法获得的破骨细胞具有肿瘤相关特性，与正常破骨细胞还有区别，不能作为常规细胞来源；(3)、骨髓诱导培养法Burger等报道用骨髓加各种诱导剂(维生素D3、白介素6、肿瘤坏死因子、粒细胞-巨噬细胞集落刺激因子、地塞米松等等)来诱导培养出破骨细胞。该法采用骨髓细胞作为培养细胞，在一定程度上可以模拟骨髓微环境对破骨细胞分化发育的影响。但是，该方法所用的骨髓实际包括内皮细胞、脂肪细胞、成纤维细胞等多种成份，不利于开展精确的发育调控机制的研究，另外，骨髓细胞的获得需剥离动物肌肉，分离出长骨，然后反复冲洗骨髓，操作费时费力；(4)、外周血单核细胞培养法Fujikawa等用人的外周血分离出单核细胞，然后诱导培养，获得破骨细胞。但是，外周血中造血干祖细胞细胞数目极少，而造血干祖细胞是破骨细胞的早期发育来源，该法仅用单核细胞进行培养，破骨细胞的获得效率较低；(5)、脾脏细胞诱导培养法是目前常用的破骨细胞培养方法。Udagawa首先用大鼠脾细胞培养获得大量的破骨细胞并证实具有骨吸收能力。与骨髓细胞培养法相比，脾脏细胞诱导培养法取材方便，打开动物腹部即可摘除脾脏，而无须经剥离肌肉，分离多根长骨，反复多次冲洗骨髓；而且排除了骨髓中复杂的细胞环境的影响；鼠类等脾脏具有终身造血能力，脾脏内存在着造血干细胞，采用脾脏细胞比采用单核细胞具有更好的生长增殖潜力。建立一种稳定实用、经济简便、高效的破骨细胞培养方法是开展破骨细胞相关研究的迫切需要。So far, the methods for culturing osteoclasts (International Journal of Orthopedics such as Lu Mingbo, September 2006, Volume 27, No. 5) mainly include: (1), the mature osteoclast separation and culture method was pioneered by Chamber in 1984. Osteoclasts were primary isolated from the long bones of rabbit limbs. However, osteoclasts are small in number, large in size, grow close to the inner surface of the bone marrow cavity, and are easily damaged when the cell body is separated, and difficult to culture after separation. In addition, the separation method Obtained end-stage osteoclasts are difficult to use for growth, proliferation and differentiation research; (2), giant cell tumor of bone osteoclast separation method giant cell tumor of bone is a kind of bone tumor characterized by osteolytic bone destruction Zambonin-Zallon A et al. isolated a large number of osteoclasts from giant cell tumors of bone. However, the incidence of giant cell tumor of bone is low and there are few tissue sources, so it cannot provide a stable source of osteoclasts for a long time; moreover, the osteoclasts obtained by this method have tumor-related characteristics, which are different from normal osteoclasts and cannot be used as conventional cells. Source; (3), bone marrow induction culture method Burger et al. reported that bone marrow plus various inducers (vitamin D3, interleukin 6, tumor necrosis factor, granulocyte-macrophage colony-stimulating factor, dexamethasone, etc.) were used to induce culture Out of osteoclasts. This method uses bone marrow cells as cultured cells, which can simulate the effect of bone marrow microenvironment on the differentiation and development of osteoclasts to a certain extent. However, the bone marrow used in this method actually includes multiple components such as endothelial cells, adipocytes, and fibroblasts, which is not conducive to the study of precise developmental regulation mechanisms. In addition, the bone marrow cells need to be stripped from the animal muscles, and the long bones are separated. Repeated flushing of the bone marrow is time-consuming and laborious; (4) Peripheral blood mononuclear cell culture method Fujikawa et al. isolated mononuclear cells from human peripheral blood, and then induced and cultured them to obtain osteoclasts. However, the number of hematopoietic stem and progenitor cells in peripheral blood is very small, and hematopoietic stem and progenitor cells are the source of early development of osteoclasts. This method only uses monocytes for culture, and the acquisition efficiency of osteoclasts is low; (5) 1. Spleen cell induction culture method is a commonly used osteoclast culture method at present. Udagawa first cultured a large number of osteoclasts with rat splenocytes and confirmed that they have bone resorption capacity. Compared with the bone marrow cell culture method, the spleen cell induction culture method is convenient to obtain materials, and the spleen can be removed by opening the abdomen of the animal without stripping the muscles, separating multiple long bones, and washing the bone marrow repeatedly; Impact: The spleen of mice and other species has a lifelong hematopoietic ability, and there are hematopoietic stem cells in the spleen. The use of spleen cells has better growth and proliferation potential than monocytes. Establishing a stable, practical, economical, simple, and efficient osteoclast culture method is an urgent need for osteoclast-related research.

除了所选用的诱导细胞不同，诱导剂的选择对于破骨细胞的体外培养也很重要，研究发现：核因子κB受体活化子配体(RANKL)和其拮抗分子骨保护素(OPG)通过RANKL-RANK-TRAF6信号通路实现对破骨细胞发育分化成熟进行决定性调控，而巨噬细胞集落刺激因子(M-CSF)则对于通路起到协同作用(Lacey DL等，Cell.1998 Apr 17；93(2)：165-76.Kong YY等，Nature.1999 Jan28；397(6717)：315-23)。因此，核因子κB受体活化子配体和巨噬细胞集落刺激因子共同诱导破骨细胞的方法迅速建立起来，并成为破骨细胞体外培养的主要诱导剂。In addition to the different induced cells, the choice of inducer is also very important for the in vitro culture of osteoclasts. Studies have found that: receptor activator of nuclear factor kappa B ligand (RANKL) and its antagonist molecule osteoprotegerin (OPG) through RANKL -RANK-TRAF6 signaling pathway realizes the decisive regulation of osteoclast development, differentiation and maturation, while macrophage colony-stimulating factor (M-CSF) plays a synergistic effect on the pathway (Lacey DL et al., Cell.1998 Apr 17; 93( 2): 165-76. Kong YY et al., Nature. 1999 Jan28; 397(6717): 315-23). Therefore, the method of co-inducing osteoclasts with nuclear factor κB receptor activator ligand and macrophage colony-stimulating factor was quickly established and became the main inducer of osteoclasts in vitro.

本发明发明人在专利《一种从骨实质中分离间充质干/祖细胞的方法》(专利号：ZL200510055261.4)和Guo Z等(Guo Z等，Stem Cells.2006Apr；24(4)：992-1000)，公开了一种从小鼠骨实质中分离间充质干/祖细胞的方法。小鼠骨实质来源的间充质干细胞具有向多种组织分化的能力，能够在体内和体外调节免疫细胞的功能。以小鼠骨实质来源的间充质干细胞做滋养层，可以支持骨髓来源的造血干细胞扩增。The inventor of the present invention is in the patent "a method for isolating mesenchymal stem/progenitor cells from bone parenchyma" (patent number: ZL200510055261.4) and Guo Z et al. (Guo Z et al., Stem Cells.2006Apr; 24 (4) :992-1000), discloses a method for isolating mesenchymal stem/progenitor cells from mouse bone parenchyma. Mesenchymal stem cells derived from mouse bone parenchyma have the ability to differentiate into various tissues and can regulate the function of immune cells in vivo and in vitro. Using mouse bone parenchyma-derived mesenchymal stem cells as a trophoblast can support the expansion of bone marrow-derived hematopoietic stem cells.

经检索，到目前为止，还没有发现能提供数量多和纯度高的破骨细胞的培养方法。After searching, so far, no culture method that can provide osteoclasts with a large number and high purity has been found.

发明内容Contents of the invention

本发明的目的是提供一种利用小鼠骨实质来源的间充质干细胞与小鼠脾脏细胞共同培养，并添加细胞因子培养破骨细胞的方法，利用本方法可获得数量多、纯度高的破骨细胞。The purpose of the present invention is to provide a method for co-culturing mesenchymal stem cells derived from mouse bone parenchyma and mouse spleen cells, and adding cytokines to culture osteoclasts. This method can obtain osteoclasts with a large number and high purity. bone cells.

本发明通过以下技术方案实现：The present invention is realized through the following technical solutions:

本发明提供一种利用间充质干细胞联合细胞因子培养破骨细胞的方法，包括如下步骤：The invention provides a method for cultivating osteoclasts by using mesenchymal stem cells combined with cytokines, comprising the following steps:

(1)从小鼠骨实质内分离、培养间充质干细胞，并进行传代培养，然后将传代培养的第4～6代间充质干细胞铺于培养板内，在35～38℃、5％CO₂条件下培养12～24小时；(1) Isolate and culture mesenchymal stem cells from the bone parenchyma of mice, and carry out subculture, and then spread the subcultured 4th to 6th generation mesenchymal stem cells in the culture plate, at 35 ~ 38 ° C, 5% CO Cultivate under ₂ conditions for 12 to 24 hours;

(2)从小鼠脾脏分离单个核细胞，并种于步骤(1)的培养板内；(2) mononuclear cells are isolated from the spleen of the mouse, and planted in the culture plate of step (1);

(3)向步骤(2)中的培养板内添加含细胞因子的培养基共同培养，每隔两天半量换液，在35～38℃、5％CO₂条件下培养3～15天，即可得到破骨细胞；(3) Add cytokine-containing medium to the culture plate in step (2) for co-cultivation, change the medium every two days and a half, and cultivate at 35-38°C and 5% _CO2 for 3-15 days, that is, Osteoclasts can be obtained;

其中，步骤(3)所述的培养基为含有1～2mmol/L谷氨酰胺，10～15％体积胎牛血清，10～20ng/ml重组小鼠巨噬细胞刺激因子，10～20ng/ml重组小鼠核因子κB受体活化子配体的a-MEM培养基。Wherein, the culture medium described in step (3) contains 1～2mmol/L glutamine, 10～15% volume of fetal bovine serum, 10～20ng/ml recombinant mouse macrophage stimulating factor, 10～20ng/ml a-MEM medium for recombinant mouse nuclear factor kappa B receptor activator ligand.

上述步骤(1)所述的小鼠骨实质来源的间充质干细胞可根据本发明发明人在专利《一种从骨实质中分离间充质干/祖细胞的方法》(专利号ZL200510055261.4)或者Guo Z等(Stem Cells.2006 Apr；24(4)：992-1000)所公开的方法分离培养获得。The mouse bone parenchyma-derived mesenchymal stem cells described in the above step (1) can be obtained according to the inventor's patent "A Method for Isolating Mesenchymal Stem/Progenitor Cells from Bone Parenchyma" (Patent No. ZL200510055261.4 ) or the method disclosed by Guo Z et al. (Stem Cells.2006 Apr; 24(4):992-1000).

上述细胞因子是指重组小鼠巨噬细胞刺激因子和重组小鼠核因子κB受体活化子配体，均可以在市场上购买。The above cytokines refer to recombinant mouse macrophage stimulating factor and recombinant mouse nuclear factor kappa B receptor activator ligand, both of which can be purchased in the market.

上述步骤(1)所用培养板可以是48孔培养板，用适合细胞培养的生物塑料材料制成。The culture plate used in the above step (1) can be a 48-well culture plate made of bioplastic material suitable for cell culture.

上述步骤(1)铺培养板所用的间充质干细胞数量为2000～10000个/孔。The number of mesenchymal stem cells used in the above step (1) spreading the culture plate is 2000-10000 cells/well.

上述步骤(2)所述的小鼠脾脏单个核细胞从2～3周龄小鼠分离获得。The mouse spleen mononuclear cells described in the above step (2) are isolated from 2-3 week old mice.

上述步骤(2)所种的小鼠脾脏单个核细胞为1800～2200个/孔。The number of mouse spleen mononuclear cells planted in the above step (2) is 1800-2200/well.

上述步骤(3)配制培养基所用的细胞因子来源为浓度(重量百分比)：重组小鼠巨噬细胞刺激因子为10～20ng/ul，重组小鼠核因子κB受体活化子配体为10～20ng/ul，溶剂为含0.1％牛血清白蛋白的磷酸盐缓冲液。The source of the cytokines used in the preparation of the medium in the above step (3) is the concentration (percentage by weight): the recombinant mouse macrophage stimulating factor is 10-20 ng/ul, and the recombinant mouse nuclear factor kappa B receptor activator ligand is 10-20 ng/ul. 20ng/ul, the solvent is phosphate buffer containing 0.1% bovine serum albumin.

本发明一种利用间充质干细胞联合细胞因子培养破骨细胞的方法，其详细的培养步骤包括：The present invention is a method for cultivating osteoclasts using mesenchymal stem cells combined with cytokines, the detailed culturing steps comprising:

(1)小鼠骨实质的间充质干细胞分离和培养取2～3周龄健康C57BL/6雌性小鼠，脱颈处死，75％酒精浸泡3～5分钟后，无菌条件下分离小鼠股骨和胫骨，去净表面组织，以注射器吸取含5～15％体积胎牛血清的磷酸盐缓冲液冲净骨髓腔，剪碎为大小1～2mm²细小骨片，置于重量百分浓度为0.1～0.15％、溶剂为含有15％～20％体积胎牛血清的a-MEM培养基的二型胶原酶溶液中，在37℃，消化1小时后种骨片于含有1～2mmol/L谷氨酰胺、10～15％体积胎牛血清a-MEM培养基内，在35～38℃、5％CO₂条件下培养72小时后首次换液，去除悬浮细胞，保留骨片，当骨片中生长出的细胞长满70～80％培养瓶面积后消化传代，连续传代，取第4～6代细胞做培养用；(1) Isolation and culture of mesenchymal stem cells from mouse bone parenchyma Take healthy C57BL/6 female mice aged 2 to 3 weeks, kill them by neck dislocation, soak them in 75% alcohol for 3 to 5 minutes, and isolate the mice under aseptic conditions Femur and tibia, remove the surface tissue, use a syringe to absorb phosphate buffer solution containing 5-15% fetal bovine serum to rinse the bone marrow cavity, cut into small bone pieces with a size of ^1-2mm2 , and place them in a weight percent concentration of 0.1-0.15%, the solvent is the type II collagenase solution of a-MEM medium containing 15%-20% fetal bovine serum, at 37°C, digest the bone slices for 1 hour in a solution containing 1-2mmol/L grain Aminoamide, 10-15% volume of fetal bovine serum a-MEM medium, cultured at 35-38°C, 5% CO ₂ for 72 hours, then changed the medium for the first time, removed the suspended cells, kept the bone slices, when the bone slices The grown cells cover 70-80% of the area of the culture flask, then digested and passaged, continuously passaged, and the 4th to 6th generation cells are used for culture;

(2)按照每孔2000～10000个将步骤(1)所得间充质干细胞铺在48孔细胞培养板内，每孔加入500～800μl含有1～2mmol/L谷氨酰胺、10％体积胎牛血清的a-MEM培养基，在35～38℃、5％CO₂条件下培养12～24小时；(2) Spread the mesenchymal stem cells obtained in step (1) in a 48-well cell culture plate according to 2000-10000 cells per well, and add 500-800 μl containing 1-2 mmol/L glutamine, 10% volume of fetal bovine Serum a-MEM medium, cultured at 35-38°C, 5% CO ₂ for 12-24 hours;

(3)小鼠脾脏单个核细胞的分离将2～3周龄健康C57BL/6雌性小鼠脱颈处死，75％酒精浸泡3～5分钟后，无菌条件下取出脾脏，将脾脏置于200目滤网上，以玻璃注射器柄碾磨，磷酸盐缓冲液冲洗，获得脾脏单细胞悬液；取小鼠淋巴细胞分离液4～5毫升于试管内，吸取脾脏单细胞悬液4～5毫升小心加入试管，注意保持界面完整，以1500～1800转/分钟离心15～20分钟，吸取单个核细胞层，再加入磷酸盐缓冲液以1000～1200转/分钟8～10分钟洗涤两遍，细胞计数留用；(3) Isolation of mouse spleen mononuclear cells. Healthy C57BL/6 female mice aged 2 to 3 weeks were killed by decapitation. After soaking in 75% alcohol for 3 to 5 minutes, the spleen was removed under aseptic conditions and placed in 200 Mesh filter, grind with a glass syringe handle, wash with phosphate buffer to obtain spleen single-cell suspension; take 4-5 ml of mouse lymphocyte separation solution in a test tube, draw 4-5 ml of spleen single-cell suspension carefully Add to the test tube, pay attention to keep the interface intact, centrifuge at 1500-1800 rpm for 15-20 minutes, absorb the mononuclear cell layer, then add phosphate buffer, wash twice at 1000-1200 rpm for 8-10 minutes, and count the cells retain;

(4)将分离得到的脾脏单个核细胞种入预先铺过了小鼠骨实质来源间充质干细胞的培养板内，每个48孔培养板单孔的密度为1800～2200个脾脏单个核细胞；每孔加培养基500μl，每隔两天半量换液，在35～38℃、5％CO₂条件下培养3～15天，可得破骨细胞；(4) Plant the isolated spleen mononuclear cells into a culture plate pre-coated with mouse bone parenchyma-derived mesenchymal stem cells, and the density of each well of a 48-well culture plate is 1800-2200 spleen mononuclear cells Add 500 μl of medium to each well, change the medium every two days and a half, and culture at 35-38°C and 5% CO ₂ for 3-15 days to obtain osteoclasts;

其中步骤(4)所述的的培养基为含有1～2mmol/L谷氨酰胺，10～15％体积胎牛血清，10～20ng/ml重组小鼠巨噬细胞刺激因子，10～20ng/ml重组小鼠核因子κB受体活化子配体的a-MEM培养基。Wherein the culture medium described in step (4) contains 1～2mmol/L glutamine, 10～15% volume fetal bovine serum, 10～20ng/ml recombinant mouse macrophage stimulating factor, 10～20ng/ml a-MEM medium for recombinant mouse nuclear factor kappa B receptor activator ligand.

上述配制培养基所用的细胞因子溶液组成及浓度(重量百分比)为：重组小鼠巨噬细胞刺激因子为10～20ng/ul，重组小鼠核因子κB受体活化子配体为10～20ng/ul，溶剂为含0.1％牛血清白蛋白的磷酸盐缓冲液。The composition and concentration (percentage by weight) of the cytokine solution used in the above preparation medium are: recombinant mouse macrophage stimulating factor is 10-20 ng/ul, recombinant mouse nuclear factor kappa B receptor activator ligand is 10-20 ng/ul ul, the solvent is phosphate buffer saline containing 0.1% bovine serum albumin.

上述每隔两天半量换液的液是指含有1～2mmol/L谷氨酰胺，10～15％体积胎牛血清，10～20ng/ml重组小鼠巨噬细胞刺激因子，10～20ng/ml重组小鼠核因子κB受体活化子配体的a-MEM培养基。The above-mentioned solution for half-quantity replacement every two days refers to the solution containing 1-2mmol/L glutamine, 10-15% volume of fetal bovine serum, 10-20ng/ml recombinant mouse macrophage-stimulating factor, 10-20ng/ml a-MEM medium for recombinant mouse nuclear factor kappa B receptor activator ligand.

上述步骤(1)中所述的磷酸盐缓冲液的组分及浓度(重量百分比)为：氯化钠8g/L，磷酸氢二钠2.9g/L，氯化钾0.2g/L，磷酸二氢钾0.24g/L，溶剂为去离子水。The composition and concentration (percentage by weight) of the phosphate buffer described in above-mentioned steps (1) are: sodium chloride 8g/L, disodium hydrogen phosphate 2.9g/L, potassium chloride 0.2g/L, phosphoric acid di Potassium hydrogen 0.24g/L, solvent is deionized water.

上述传代培养是指将所得间充质干细胞利用低血清体系进行传代培养，细胞体外增殖旺盛，以满足一般细胞生物学实验的需要。The above-mentioned subculture refers to the subculture of the obtained mesenchymal stem cells in a low-serum system, and the cells proliferate vigorously in vitro, so as to meet the needs of general cell biology experiments.

上述培养板、试剂以及培养基的组分均可以从市场上购买。The components of the above-mentioned culture plate, reagents and culture medium can be purchased from the market.

所得破骨细胞的检测方法，主要有：(1)、将所得到的破骨细胞进行特异性的抗酒石酸的酸性磷酸酶(TRAP+)组化染色，结果应为强阳性；(2)、光学显微镜下观察，单细胞具有多个细胞核(≥3个细胞核)；(3)、象牙片吸收试验显示具有骨质吸收能力。The detection method of gained osteoclast mainly contains: (1), the obtained osteoclast is carried out specific tartrate-resistant acid phosphatase (TRAP+) histochemical staining, and the result should be strongly positive; (2), optical Observed under a microscope, the single cell has multiple nuclei (≥3 nuclei); (3), the resorption test of ivory slices shows that it has bone resorption capacity.

本发明方法可以从每2000个脾脏单个核细胞培养得到400个以上的破骨细胞，可完全满足一般细胞生物学实验数量需求。The method of the invention can obtain more than 400 osteoclasts from every 2,000 spleen mononuclear cells, which can fully meet the quantity requirements of general cell biology experiments.

本发明所具有的优点和有益效果：(1)本发明方法操作简单，方便实用，可以在最早时间获得破骨细胞，可缩短培养时间2～3天；(2)本发明方法所得破骨细胞数量大，解决了破骨细胞难以大量制备的问题，可以满足相关细胞生物学和分子生物学实验的需要；(3)本发明方法培养所得的破骨细胞成熟度高，骨质吸收能力强，有利于开展功能研究；(4)本发明方法所需要的细胞因子少，所需费用低，可以节约费用。The advantages and beneficial effects of the present invention: (1) The method of the present invention is simple to operate, convenient and practical, and can obtain osteoclasts at the earliest time, which can shorten the culture time by 2 to 3 days; (2) The osteoclasts obtained by the method of the present invention The number is large, which solves the problem that osteoclasts are difficult to prepare in large quantities, and can meet the needs of relevant cell biology and molecular biology experiments; (3) the osteoclasts cultivated by the method of the present invention have high maturity and strong bone absorption capacity, It is beneficial to carry out functional research; (4) The method of the present invention requires less cytokines and lower costs, which can save costs.

附图说明Description of drawings

图1不同培养体系获得的破骨细胞抗酒石酸的酸性磷酸酶(TRAP+)组化Figure 1 Histochemistry of tartrate-resistant acid phosphatase (TRAP+) in osteoclasts obtained from different culture systems

结果照片。(A，B，C，D，E，F分组见实施例1)Results photo. (A, B, C, D, E, F group see embodiment 1)

图2不同培养体系获得的成熟破骨细胞数量与培养时间的关系示意图。Figure 2 is a schematic diagram of the relationship between the number of mature osteoclasts obtained in different culture systems and the culture time.

(A，B，C，D，E，F分组见实施例1))(A, B, C, D, E, F group see embodiment 1))

图3成熟破骨细胞在TRAP+细胞中的比例柱形图。Figure 3 Histogram of the proportion of mature osteoclasts in TRAP+ cells.

(A，B，C，D，E，F分组见实施例1)(A, B, C, D, E, F group see embodiment 1)

图4成熟破骨细胞在象牙骨片上形成的骨吸收陷窝照片。Fig. 4 Photographs of bone resorption lacunae formed by mature osteoclasts on ivory bone slices.

(图A(D组)和图B(F组))(Figure A (Group D) and Figure B (Group F))

具体实施方式Detailed ways

实施例1Example 1

(1)小鼠骨实质间充质干细胞的分离和培养(1) Isolation and culture of mouse bone parenchymal mesenchymal stem cells

根据本发明发明人的专利从小鼠骨实质内分离培养获得间充质干细胞(专利号ZL200510055261.4)和Guo Z等(Stem Cells.2006 Apr；24(4)：992-1000)所公开的方法进行小鼠骨实质间充质干细胞的分离和培养，取2周龄健康C57BL/6雌性小鼠(由军事医学科学动物中心提供)，脱颈处死，75％酒精浸泡5分钟后，无菌条件下分离小鼠股骨和胫骨，去净表面组织，以注射器吸取含10％体积胎牛血清(从Hyclone公司购得)的磷酸盐缓冲液(重量体积比为：氯化钠8g/L，磷酸氢二钠2.9g/L，氯化钾0.2g/L，磷酸二氢钾0.24g/L，溶剂为去离子水)冲净骨髓腔，小心剪碎为大小为2mm²细小骨片，置重量百分浓度为0.15％、溶剂为含有15％体积胎牛血清的a-MEM培养基的二型胶原酶(从Sigma公司购得)溶液中，在37℃，消化1小时后种骨片于含有2mmol/L谷氨酰胺(从Sigma公司购得)，10％体积胎牛血清的a-MEM培养基(从Hyclone公司购得)内，在为37℃、5％CO₂条件下72小时后首次换液，去除悬浮细胞，保留骨片，从骨片中生长出的细胞长满80％培养瓶面积后消化传代，连续传代，取第4代细胞做培养用。According to the inventor's patent of the present invention, mesenchymal stem cells were isolated and cultured from mouse bone parenchyma (Patent No. ZL200510055261.4) and the method disclosed by Guo Z et al. (Stem Cells.2006 Apr; 24(4):992-1000) For the isolation and cultivation of mouse bone parenchymal stem cells, 2-week-old healthy C57BL/6 female mice (provided by the Animal Center of Military Medical Sciences) were taken, killed by neck dislocation, soaked in 75% alcohol for 5 minutes, and kept under sterile conditions Separate the mouse femur and tibia, remove the clean surface tissue, draw the phosphate buffer saline (weight to volume ratio: sodium chloride 8g/L, hydrogen phosphate) that contains 10% volume fetal bovine serum (purchase from Hyclone Company) Disodium 2.9g/L, Potassium chloride 0.2g/L, Potassium dihydrogen phosphate 0.24g/L, the solvent is deionized water) rinse the bone marrow cavity, carefully cut into small bone fragments with a size of ^2mm2 , put the weight in 100 Concentration is 0.15%, solvent is the type II collagenase (purchased from Sigma company) solution of a-MEM medium containing 15% volume fetal bovine serum, at 37 ℃, after digesting for 1 hour, the kind of bone piece is in the solution containing 2mmol /L glutamine (purchased from Sigma), in a-MEM medium (purchased from Hyclone) with 10% volume of fetal bovine serum, changed for _the first time after 72 hours at 37°C and 5% CO Remove the suspended cells and retain the bone slices. The cells grown from the bone slices cover 80% of the culture flask area and then digested and passaged for continuous passage. The 4th generation cells were used for culture.

(2)将步骤(1)小鼠骨实质来源间充质干细胞种在48孔细胞培养板(从Corning公司购得)内，每个单孔面积0.8cm²，每孔种2000个或10000个小鼠骨实质来源间充质干细胞，每孔加入500μl含有2mmol/L谷氨酰胺，10％体积胎牛血清的a-MEM培养基内，在37℃、5％CO₂条件下培养24小时。(2) Plant the mouse bone parenchyma-derived mesenchymal stem cells in step (1) in a 48-well cell culture plate (purchased from Corning Company), each single well has an area of 0.8 cm ² , and plant 2,000 or 10,000 cells per well Add 500 μl of a-MEM medium containing 2 mmol/L glutamine and 10% volume of fetal bovine serum to each well of mouse bone parenchyma-derived mesenchymal stem cells, and culture at 37°C and 5% CO ₂ for 24 hours.

(3)小鼠脾脏单个核细胞分离(3) Isolation of mouse spleen mononuclear cells

取2周龄健康C57BL/6雌性小鼠(由军事医学科学院实验动物中心提供)，本发明所有动物实验严格尊重军事医学科学实验动物指南进行。将小鼠脱颈处死，75％酒精浸泡5分钟后，无菌条件下取出脾脏，以玻璃注射器柄在200目滤网上碾磨制取单细胞悬液。小鼠淋巴细胞分离液(天津灏洋生物购得)4毫升于试管内，吸取脾脏单细胞悬液4毫升小心加入试管，注意保持界面完整，以1500转/分钟离心15分钟，吸取单个核细胞层，再加入磷酸盐缓冲液以1000转/分钟离心10分钟洗涤两遍，细胞计数留用。2-week-old healthy C57BL/6 female mice (provided by the Experimental Animal Center of the Academy of Military Medical Sciences) were taken, and all animal experiments of the present invention were carried out in strict respect of the Guidelines for Experimental Animals of Military Medical Sciences. The mice were killed by neck dislocation, soaked in 75% alcohol for 5 minutes, the spleen was removed under aseptic conditions, and the single cell suspension was prepared by grinding on a 200-mesh filter with a glass syringe handle. Put 4 ml of mouse lymphocyte separation solution (purchased by Tianjin Haoyang Biology) into a test tube, draw 4 ml of spleen single cell suspension and carefully add to the test tube, pay attention to keep the interface intact, centrifuge at 1500 rpm for 15 minutes, and absorb mononuclear cells Then add phosphate buffered saline and centrifuge at 1000 rpm for 10 minutes to wash twice, and keep the cells for counting.

(4)脾脏单个核细胞种于培养板内(4) Spleen mononuclear cells are planted in the culture plate

小鼠骨实质来源的间充质干细胞铺板24小时后，在显微镜下观察间充质干细胞贴壁良好，吸出单个孔内含有2mmol/L谷氨酰胺，10％体积胎牛血清的a-MEM培养基500ul弃之。以含有2mmol/L谷氨酰胺，10％体积胎牛血清，重组小鼠巨噬细胞刺激因子10ng/ml或者20ng/ml，重组小鼠核因子κB受体活化子配体10ng/ml或者20ng/ml的a-MEM培养基重悬脾脏单个核细胞，按照每个48孔培养板单孔内单个核细胞2000个脾脏单个核细胞的密度，培养基500ul，种下脾脏单个核细胞。分为6组，分别是：After the mouse bone parenchyma-derived mesenchymal stem cells were plated for 24 hours, it was observed under a microscope that the mesenchymal stem cells adhered well, and the a-MEM culture containing 2mmol/L glutamine and 10% fetal bovine serum was aspirated from a single well The base 500ul is discarded. Containing 2mmol/L glutamine, 10% volume of fetal bovine serum, recombinant mouse macrophage stimulating factor 10ng/ml or 20ng/ml, recombinant mouse nuclear factor κB receptor activator ligand 10ng/ml or 20ng/ml Spleen mononuclear cells were resuspended in ml of a-MEM medium, and the spleen mononuclear cells were planted according to the density of 2000 spleen mononuclear cells in a single well of each 48-well culture plate, and 500ul of medium. Divided into 6 groups, namely:

A组：Sp MNC(2000个/孔)+M-CSF(10ng/ml)+RANKL(10ng/ml)；Group A: Sp MNC (2000/well) + M-CSF (10ng/ml) + RANKL (10ng/ml);

B组：Sp MNC(2000个/孔)+MSC(2000个/孔)+M-CSF(10ng/ml)+RANKL(10ng/ml)；Group B: Sp MNC (2000/well) + MSC (2000/well) + M-CSF (10ng/ml) + RANKL (10ng/ml);

C组：Sp MNC(2000个/孔)+MSC(10000个/孔)+M-CSF(10ng/ml)+RANKL(10ng/ml)；Group C: Sp MNC (2000/well) + MSC (10,000/well) + M-CSF (10ng/ml) + RANKL (10ng/ml);

D组：Sp MNC(2000个/孔)+M-CSF(20ng/ml)+RANKL(20ng/ml)；Group D: Sp MNC (2000/well) + M-CSF (20ng/ml) + RANKL (20ng/ml);

E组：Sp MNC(2000个/孔)+MSC(2000个/孔)+M-CSF(20ng/ml)+RANKL(20ng/ml)；Group E: Sp MNC (2000/well) + MSC (2000/well) + M-CSF (20ng/ml) + RANKL (20ng/ml);

F组：Sp MNC(2000个/孔)+MSC(10000个/孔)+M-CSF(20ng/ml)+RANKL(20ng/ml)；Group F: Sp MNC (2000/well) + MSC (10,000/well) + M-CSF (20ng/ml) + RANKL (20ng/ml);

(注：Sp MNC：小鼠脾脏单个核细胞，MSC：小鼠骨实质来源的间充质干细胞，M-CSF：重组巨噬细胞集落刺激因子，RANKL：重组小鼠核因子κB受体活化子配体)。(Note: Sp MNC: mouse spleen mononuclear cells, MSC: mouse bone-derived mesenchymal stem cells, M-CSF: recombinant macrophage colony-stimulating factor, RANKL: recombinant mouse nuclear factor-κB receptor activator Ligand).

共设3天、6天、9天、12天、15天五个检测时间点，每组每个检测时间点均设3复孔。在37℃，5％CO₂条件下培养，每隔两天半量换液，可得破骨细胞。A total of five detection time points of 3 days, 6 days, 9 days, 12 days, and 15 days were set up, and each detection time point in each group was set with 3 replicate wells. Culture at 37°C, 5% CO ₂ , and change the medium every two days and a half to obtain osteoclasts.

实施例2  抗酒石酸的酸性磷酸酶测定Embodiment 2 The acid phosphatase assay of anti-tartaric acid

分别在培养的第3天，第6天，第9天，第12天和第15天，按照抗酒石酸的酸性磷酸酶测定试剂盒(Sigma-Aldrich产品)说明书提供的操作建议对各组培养孔内的细胞进行染色，每组每次染3个孔，深酒红色的细胞判为TRAP(+)细胞，≥3个细胞核以上TRAP(+)细胞判定为成熟破骨细胞，光学显微镜下观察形态拍照，并分别对TRAP(+)细胞和成熟破骨细胞计数进行统计学分析。On the 3rd day, the 6th day, the 9th day, the 12th day and the 15th day of culturing respectively, according to the operating suggestion provided by the tartrate-resistant acid phosphatase assay kit (Sigma-Aldrich product) instructions, the culture wells of each group were The cells inside were stained, and each group stained 3 wells each time. The deep wine red cells were judged as TRAP(+) cells, and the TRAP(+) cells with more than 3 nuclei were judged as mature osteoclasts. The morphology was observed and photographed under an optical microscope. , and the counts of TRAP (+) cells and mature osteoclasts were analyzed statistically.

结果(见图1)本发明方法获得的破骨细胞与细胞因子法获得的破骨细胞进行比较，前者形态极为活跃，细胞核可多达几十个，胞体巨大，抗酒石酸的酸性磷酸酶深染。Result (see Fig. 1) the osteoclast obtained by the method of the present invention is compared with the osteoclast obtained by the cytokine method, the former is extremely active in morphology, with up to dozens of nuclei, huge cell body, and deep staining of tartrate-resistant acid phosphatase .

本发明方法获得成熟破骨细胞的最早时间为3天，早于细胞因子法中成熟破骨细胞出现的最早时间，后者为6天(见图2)；The earliest time for the method of the present invention to obtain mature osteoclasts is 3 days, which is earlier than the earliest time for mature osteoclasts to appear in the cytokine method, and the latter is 6 days (see Figure 2);

本发明方法获得的成熟破骨细胞的绝对数量多于细胞因子培养破骨细胞法获得的成熟破骨细胞的绝对数量，分别对A组和B组、A组和C组、D组和E组、D组和F组之间成熟破骨细胞在第6，9，12，15天的绝对数量进行统计学T测验，各组P＜0.05(见图2，表1)；The absolute number of mature osteoclasts obtained by the method of the present invention is more than the absolute number of mature osteoclasts obtained by the method of culturing osteoclasts with cytokines, respectively for group A and group B, group A and group C, group D and group E 1. The absolute quantity of mature osteoclasts between group D and group F was carried out statistical T test on the 6th, 9th, 12th and 15th day, each group P＜0.05 (seeing Fig. 2, table 1);

表1  各组间成熟破骨细胞数量差异的统计学分析结果Table 1 The statistical analysis results of the differences in the number of mature osteoclasts among the groups

 各组间P值P value between each group  6天 6 days  9天 9 days  12天 12 days  15天 15 days  A和BA and B  0.04381445410.0438144541  0.00381822060.0038182206  0.00018278960.0001827896  0.00237205570.0023720557  A和CA and C  0.00000192080.0000019208  0.00014251340.0001425134  0.00003863410.0000386341  0.00001837540.0000183754  D和ED and E  0.00926169680.0092616968  0.00202748830.0020274883  0.00001670750.0000167075  0.02039989710.0203998971  D和FD and F  0.00000099970.0000009997  0.00001104120.0000110412  0.00001190390.0000119039  0.00001724520.0000172452

结果(见图3)本发明培养破骨细胞的方法获得的成熟破骨细胞在TRAP(+)细胞中的比例高于细胞因子培养破骨细胞法获得的成熟破骨细胞在TRAP(+)细胞中的比例。Result (see Fig. 3) the mature osteoclast obtained by the method for culturing osteoclasts of the present invention has a higher ratio in TRAP (+) cells than the mature osteoclasts obtained by the method of culturing osteoclasts with cytokines in TRAP (+) cells ratio in .

实施例3  破骨细胞象牙片吸收试验Example 3 Osteoclast ivory sheet absorption test

将直径约15mm，厚度3μm的象牙片(复旦大学医学院金慰芳教授提供)，75％酒精浸泡两小时，超净台内紫外线照射12小时消毒，按照以上培养分组，在D组种上单个核细胞之前预先置于24孔板孔底，在F组铺间充质干细胞之前预先置于24孔板孔底，再依次种上单个核细胞或间充质干细胞和单个核细胞。在第十五天取出象牙片，以甲苯胺兰染色在光学显微镜下观察象牙片表面陷窝形成情况并拍照。Ivory slices with a diameter of about 15 mm and a thickness of 3 μm (provided by Professor Jin Weifang, School of Medicine, Fudan University) were soaked in 75% alcohol for two hours, and sterilized by ultraviolet radiation in a clean bench for 12 hours. According to the above culture groups, mononuclear cells were planted in group D It was pre-placed at the bottom of the 24-well plate, and placed at the bottom of the 24-well plate before mesenchymal stem cells were laid in group F, and then mononuclear cells or mesenchymal stem cells and mononuclear cells were planted in sequence. Take out the ivory sheet on the 15th day, observe the lacuna formation situation on the surface of the ivory sheet under an optical microscope with toluidine blue staining and take pictures.

结果本发明方法获得的成熟破骨细胞在象牙片上形成的骨吸收陷窝(见图4B)较细胞因子培养破骨细胞法获得的成熟破骨细胞在象牙片上形成的骨吸收陷窝(见图4A)大，证实本发明获得的破骨细胞具有骨吸收能力，且功能强于细胞因子法获得的破骨细胞。Results The mature osteoclasts obtained by the method of the present invention formed bone resorption lacunae on the ivory sheet (see Figure 4B) compared with the bone resorption lacuna formed on the ivory sheet by the mature osteoclasts obtained by the method of culturing osteoclasts with cytokines (see Figure 4B). 4A) is large, confirming that the osteoclasts obtained by the present invention have bone resorption capacity, and the function is stronger than that of the osteoclasts obtained by the cytokine method.