WO2006069481A1

WO2006069481A1 - Target enrichment method of stem cells and magnetic enrichment device for target localization

Info

Publication number: WO2006069481A1
Application number: PCT/CN2004/001560
Authority: WO
Inventors: Xuetao Pei; Yunfang Wang; Xue Nan; Yanhua Li; Wen Yue; Fang Yan
Original assignee: Beijing Institute Of Transfusion Medicine
Priority date: 2004-12-29
Filing date: 2004-12-29
Publication date: 2006-07-06

Abstract

The present invention discloses a target enrichment method of stem cells and a magnetic enrichment device for target localization. The target enrichment of stem cells said in the invention includes the following steps: 1) labeling stem cells with immunomagnetic beads of 30-70nm to obtain labeled stem cells; 2) injecting labeled stem cells into circulatory system of target tissue; 3) locating the body surface region which corresponds to the target tissue to face two mutually attracting magnetic poles, and enriching said stem cells into target tissue during the circulation of circulatory system. The magnetic intensity of said two mutually attracting magnetic poles is 9200-24500Gs. According to the present invention, the stem cells are labeled with immunomagnetic beads, and the magnetic enrichment device for target localization is used to create high intensity and gradient magnetic field by utilizing skillfully magnetic nature of superparamagnetic beads in order to enrich given cells into target tissue. The invention has the advantages of high enrichment efficiency, convenience and so on and it is applied broadly.

Description

一种干细胞靶向定位富集的方法及磁靶向定位富集仪技术领域 Method for targeting and enrichment of stem cells and magnetic targeting and enrichment instrument

本发明涉及一种干细胞靶向定位富集的方法及磁靶向定位富集仪。 The invention relates to a method for targeting and enriching stem cells and a magnetic targeting and enrichment instrument.

背景技术 Background technique

随着器官移植学、移植免疫学的发展及免疫抑制剂的开发和应用，器官移植已经成为治疗临床终末期疾病的有效手段。对于各种原因（包括毒物、药物中毒、遗传代谢性疾病等）引发的终末期肝衰竭来讲，原位肝移植是目前唯一有效的治疗手段。然而，由于供肝的极度短缺、手术及移植本身相关的较高死亡率、终生服用免疫抑制剂及其所带来的严重甚至致命的并发症使得肝移植在临床的广泛应用受到极大限制。 With the development of organ transplantation, transplantation immunology and the development and application of immunosuppressive agents, organ transplantation has become an effective means of treating end-stage diseases. For the end-stage liver failure caused by various causes (including poisons, drug poisoning, genetic metabolic diseases, etc.), orthotopic liver transplantation is currently the only effective treatment. However, the widespread use of liver transplantation in clinical settings is severely limited by the extreme shortage of donor liver, the high mortality associated with surgery and transplantation itself, and the lifetime use of immunosuppressants and the serious or even fatal complications they cause.

随着干细胞生物学研究的不断深入，以干细胞为种子细胞的细胞治疗在糖尿病、肝衰竭、心肌梗塞、早老性痴呆、帕金森病、视网膜损伤、癌症、血友病、脊髓损伤、先天性免疫性疾病等难治性、终末期疾病的治疗中越来越显示出良好的应用前景。研究中发现，哺乳动物体内多种组织（包括骨髓、脐带血等）来源的成体干细胞均具有 "可塑性" （即具有向多种组织细胞分化潜能）的特点，比如，其中的 CD34+Lin—细胞向造血***各系细胞分化； c-Met+p₂M—细胞可以向成熟的肝细胞分化；间充质干细胞可以向神经元、心肌细胞或胰岛 β细胞分化等等。 With the deepening of stem cell biology research, cell therapy with stem cells as seed cells in diabetes, liver failure, myocardial infarction, Alzheimer's disease, Parkinson's disease, retinal damage, cancer, hemophilia, spinal cord injury, innate immunity The treatment of refractory and end-stage diseases such as sexual diseases has increasingly shown good application prospects. The study found that adult stem cells derived from various tissues (including bone marrow, cord blood, etc.) in mammals have the characteristics of "plasticity" (ie, having the potential to differentiate into various tissue cells), for example, CD34+Lin-cells. Differentiating into cells of various hematopoietic systems; c-Met+p ₂ M-cells can differentiate into mature hepatocytes; mesenchymal stem cells can differentiate into neurons, cardiomyocytes or islet β cells, and the like.

干细胞移植后有一个趋化、迁移并定居于靶组织，最终在该微环境中增殖，并向靶组织细胞分化、成熟，乃至发挥功能代偿作用的过程。一般说来，只有在靶组织中富集足够数量的干细胞，才能发挥理想的功能。目前，干细胞移植的常用方法有靶组织直接注入和外周静脉输注法等。前者虽然可以保证干细胞能相对集中富集于靶组织中，但需通过开放性的手术实现，操作复杂，受者创伤大，并发症多；后者方法简单，易于操作，但是目的干细胞随血液循环流至全身各个器官，而易于被某些富含网状内皮***的组织吞噬、滞留，使得定位于靶组织的目的细胞数量相对减少，严重影响其代偿功能的发挥。而寻找一种高效、简单的干细胞靶向定位富集方法具有非常重要的意义。 After stem cell transplantation, there is a process of chemotaxis, migration and colonization in the target tissue, which eventually colonizes the microenvironment and differentiates, matures, and even functions compensatory. In general, only a sufficient number of stem cells are enriched in the target tissue to perform the desired function. Currently, common methods for stem cell transplantation include direct injection of target tissue and peripheral intravenous infusion. Although the former can ensure that stem cells can be relatively concentrated and concentrated in the target tissue, it needs to be realized through open surgery. The operation is complicated, the recipient is traumatic and has many complications. The latter method is simple and easy to operate, but the target stem cells follow the blood circulation. It flows to various organs of the body, and is easily phagocytized and retained by certain tissues rich in reticuloendothelial system, so that the number of cells targeted to target tissues is relatively reduced, which seriously affects the function of compensatory functions. It is very important to find an efficient and simple method for stem cell targeting and enrichment.

发明公开 Invention disclosure

本发明的目的是提供一种高效、简单的干细胞靶向定位富集的方法以及 T N2004/001560 磁靶向定位富集仪。 The object of the present invention is to provide an efficient and simple method for targeting and enriching stem cells. T N2004/001560 Magnetic Targeting and Enrichment Instrument.

本发明所提供的干细胞靶向定位富集的方法，包括如下步骤： 1 )将干细胞用粒径为 30— 70nm的免疫磁珠标记，得到标记后的细胞； 2)将所述标记后的干细胞注入靶组织的循环***内； 3)将所述靶组织正对放置于两个异性磁极之间，在所述循环***的循环过程中，将所述干细胞定位富集于所述靶组织内；所述两个异性磁极间的磁场强度为 9200— 24500Gs。 The method for targeting and enriching stem cells provided by the invention comprises the following steps: 1) labeling stem cells with immunomagnetic beads having a particle diameter of 30-70 nm to obtain labeled cells; 2) using the labeled stem cells Injecting into the circulatory system of the target tissue; 3) placing the target tissue directly between the two opposite magnetic poles, and concentrating the stem cells in the target tissue during the circulation of the circulatory system; The magnetic field strength between the two opposite magnetic poles is 9200-24500 Gs.

其中，所述干细胞为具有特定细胞表面标志的、具有多向分化潜能的干细胞，可以来源于人或其它哺乳动物骨髓、动员的外周血、脐带血或其他组织等。如以大鼠骨髓 c-Met M—细胞为例，采用免疫磁珠标记所述干细胞的过程如下：将针对所述干细胞表面标志性抗原的特异性抗体与所述干细胞孵育，经清洗后再与偶联有抗所述特异性抗体的免疫磁珠孵育，清洗得到所述标记后的细胞。所述特异性抗体为兔抗大鼠 c-Met抗体和 β₂Μ—抗体；所述免疫磁珠偶联有山羊抗兔 IgG。 Wherein, the stem cell is a stem cell having a multi-directional differentiation potential with a specific cell surface marker, and may be derived from human or other mammalian bone marrow, mobilized peripheral blood, cord blood or other tissues. For example, in the case of rat bone marrow c-Met M-cells, the process of labeling the stem cells with immunomagnetic beads is as follows: the specific antibody against the stem cell surface marker antigen is incubated with the stem cells, and after washing, The immunomagnetic beads conjugated to the specific antibody are incubated and washed to obtain the labeled cells. The specific antibody is a rabbit anti-rat c-Met antibody and a β ₂ Μ-antibody; the immunomagnetic beads are coupled with a goat anti-rabbit IgG.

为了提高富集的效率，可在本发明的磁靶向定位富集仪中进行富集，磁极间的距离可调整为 5— 25mm。此时，在两个磁极之间形成高强度、高梯度的磁场，磁场的高强度使免疫磁珠标记的目的细胞具有良好的富集效果，磁场的高梯度则使免疫磁珠标记的细胞能选择性地滞留于靶组织而不是靶组织以外的其他组织。 In order to improve the efficiency of enrichment, enrichment can be carried out in the magnetic targeting and enrichment apparatus of the present invention, and the distance between the magnetic poles can be adjusted to 5 - 25 mm. At this time, a high-intensity, high-gradient magnetic field is formed between the two magnetic poles. The high intensity of the magnetic field enables the target cells labeled by the immunomagnetic beads to have a good enrichment effect, and the high gradient of the magnetic field enables the cells labeled with the immunomagnetic beads to Selectively retained in the target tissue rather than other tissues than the target tissue.

本发明所提供的磁靶向定位富集仪，它包括有一支架，所述支架内上下相对设置有二磁极，下磁极垂向设置在所述支架的底面，上磁极与所述下磁极相对应地设置在所述支架的顶面。 The magnetic targeted positioning enrichment apparatus provided by the present invention comprises a bracket, wherein the bracket is vertically disposed opposite to the second magnetic pole, the lower magnetic pole is vertically disposed on the bottom surface of the bracket, and the upper magnetic pole corresponds to the lower magnetic pole Placed on the top surface of the bracket.

上述的磁靶向定位富集仪中，所述上磁极可移动地设置在所述支架的顶面。 In the magnetic targeting concentrator described above, the upper magnetic pole is movably disposed on a top surface of the bracket.

上述的磁靶向定位富集仪中，所述支架的顶面与所述下磁极相对应处设置有一螺孔，所述螺孔内穿设一螺杆，所述螺杆下端连接所述上磁极，螺杆上端设置有转动把手。 In the above magnetic target positioning and enrichment apparatus, a screw hole is disposed in a top surface of the bracket corresponding to the lower magnetic pole, a screw is disposed in the screw hole, and the lower end of the screw is connected to the upper magnetic pole. A rotating handle is provided at the upper end of the screw.

上述的磁靶向定位富集仪中，所述磁极成圆柱状，且两磁极靠近相对的一端直径缩小。 In the magnetic targeting concentrating apparatus described above, the magnetic poles are cylindrical, and the diameters of the two magnetic poles near the opposite end are reduced.

上述的磁靶向定位富集仪中，所述支架及磁极的材料均选用永磁性材料，例如铷铁硼。 In the above magnetic targeting and enrichment analyzer, the material of the bracket and the magnetic pole are selected from a permanent magnet material, such as neodymium iron boron.

附图说明 0 图 1为本发明磁靶向定位富集仪的结构示意图； DRAWINGS 0 is a schematic structural view of a magnetic targeting and enrichment analyzer of the present invention;

图 2为图 1的俯视图； Figure 2 is a plan view of Figure 1;

图 3为免疫磁珠标记的干细胞的结构示意图； Figure 3 is a schematic view showing the structure of immunomagnetic bead-labeled stem cells;

图 4为磁场筛分细胞的示意图； Figure 4 is a schematic view of a magnetic field screening cell;

图 5为免疫磁珠标记细胞的体外富集操作流程示意图； Figure 5 is a schematic diagram showing the in vitro enrichment operation procedure of immunomagnetic bead labeled cells;

图 6为免疫磁珠标记细胞在大鼠肺、肝中的富集照片。 Figure 6 is an enrichment photograph of immunomagnetic bead labeled cells in rat lung and liver.

实施发明的最佳方式 The best way to implement the invention

本发明主要提供干细胞靶向定位富集的方法，以及用于该方法的磁靶向定位富集仪，下面结合具体的实验来阐述本发明方法和设备。 The present invention mainly provides a method for stem cell targeted localization enrichment, and a magnetic targeting localization enrichment apparatus for use in the method, and the method and apparatus of the present invention are described below in conjunction with specific experiments.

具体以 c-Met⁺p₂MT细胞的靶向定位富集为例，来描述本发明的操作步骤。本实施例以肝损伤大鼠模型的肝脏作为靶组织，也可以其他组织作为靶组织。所用免疫磁珠购于天津大学材料学院，该磁珠偶联了山羊抗兔 IgG, 粒径在 50nm左右，具有良好的超顺磁性。 The procedure of the present invention is specifically described by taking targeted localization enrichment of c-Met ⁺ p ₂ MT cells as an example. In the present embodiment, the liver of the rat model of liver injury is used as a target tissue, and other tissues may be used as a target tissue. The immunomagnetic beads used were purchased from the School of Materials, Tianjin University. The magnetic beads were coupled with goat anti-rabbit IgG with a particle size of about 50 nm and good superparamagnetism.

1、大鼠骨髓单个核细胞的获得 1. Acquisition of rat bone marrow mononuclear cells

断颈处死的大鼠经 75%乙醇消毒后，用剖腹的方法取出股骨，以含 10% 胎牛血清的 DMEM培养液作为冲洗液，用 5ml注射器反复冲洗骨髓腔，用 100目筛网过滤冲出的细胞制成大鼠骨髓单细胞悬液， lOOOrpm离心后，将细胞重悬于 PBS中，经 FicolK相对密度 1.083g/ml)密度梯度离心后收集大鼠骨髓单个核细胞，洗涤后重悬于 PBS中。 The rats sacrificed by cervical dislocation were disinfected with 75% ethanol, and the femur was removed by laparotomy. The DMEM medium containing 10% fetal bovine serum was used as the rinsing solution. The medullary cavity was repeatedly washed with a 5 ml syringe and filtered through a 100-mesh sieve. The cells were made into a single cell suspension of rat bone marrow. After centrifugation at 1000 rpm, the cells were resuspended in PBS, and the rat bone marrow mononuclear cells were collected by density gradient centrifugation at a relative density of 1.083 g/ml, washed and resuspended. In PBS.

2、用两步间接免疫磁珠分离方法富集并标记大鼠骨髓来源的 c-Met⁺p₂M2. Enrich and label rat bone marrow-derived c-Met ⁺ p ₂ M by two-step indirect immunomagnetic separation.

—细胞 - cells

如图 3所示，将步骤 1所得细胞 21先与兔抗大鼠 β₂Μ抗体 22于 4°C孵育 30min，经 PBS缓冲液洗涤后，再将其与偶联了山羊抗兔 IgG 24的免疫磁珠 23于 4°C孵育 30min，再经 PBS洗涤，这样，凡是表达 β₂Μ的细胞则被免疫磁珠所标记。 As shown in Fig. 3, the cells 21 obtained in the step 1 were first incubated with rabbit anti-rat β ₂ Μ antibody 22 at 4 ° C for 30 min, washed with PBS buffer, and then coupled with goat anti-rabbit IgG 24 . The immunomagnetic beads 23 were incubated at 4 ° C for 30 min and then washed with PBS, so that cells expressing β ₂ Μ were labeled with immunomagnetic beads.

如图 4所示，将该群细胞流过位于两个磁极 29之间的塑料软管 27，其中被免疫磁珠所标记的表达 β₂Μ的细胞 25 (即阳性细胞）滞留于磁场内，而未被标记的细胞 26 (即阴性细胞）流出磁场，用 Eppendorf管 28收集细胞 26；而滞留于磁场内的细胞 25在去掉磁场后用缓冲液冲洗收集。 As shown in FIG. 4, the cells are flowed through a plastic hose 27 between the two magnetic poles 29, wherein the cells 25 (ie, positive cells) expressing the β ₂标记 labeled by the immunomagnetic beads are retained in the magnetic field. The unlabeled cells 26 (i.e., negative cells) flow out of the magnetic field, and the cells 26 are collected by the Eppendorf tube 28; and the cells 25 retained in the magnetic field are collected by buffer washing after removing the magnetic field.

按照前述操作将收集到的 β₂Μ—细胞群 26先后与兔抗大鼠 c-Met抗体及免疫磁珠标记的山羊抗兔 IgG孵育，再经 PBS洗涤。这样，在 β₂Μ—的细胞群中，凡是表达 c-Met的 c-Met M—细胞均被免疫磁珠所标记，流经磁场时，该细胞滞留于磁场内，将塑料软管移离磁场后，用 PBS将细胞冲出，获得目的细胞——免疫磁珠标记的 c-Met+p₂M—细胞。为了有效检测移植细胞在受者体内的富集效率，该群细胞以荧光染料 DAPI标记并充分洗漆。 The collected β ₂ Μ-cell population 26 was incubated with rabbit anti-rat c-Met antibody and immunomagnetic bead-labeled goat anti-rabbit IgG, and washed with PBS. In this way, cells in β ₂ Μ In the group, all c-Met M-cells expressing c-Met are labeled by immunomagnetic beads. When flowing through a magnetic field, the cells are retained in the magnetic field. After the plastic hose is removed from the magnetic field, the cells are washed out with PBS. , to obtain the target cell - immunomagnetic bead labeled c-Met + p ₂ M - cells. In order to effectively detect the enrichment efficiency of transplanted cells in the recipient, the cells were labeled with fluorescent dye DAPI and fully washed.

3、近交系 F344大鼠的饲养和肝损伤模型的制备 3. Preparation of inbred F344 rat feeding and liver injury model

雄性一级近交系 F344大鼠，体重 180〜200g，室温 20〜22°C饲养，光线 12小时明暗交替，自由饮水、进食。将丙烯醇按 0.62 m_Ol/ g体重经腹膜下注射至 F344大鼠体内，制备药物所致大鼠肝损伤模型，常规饲养 3天后备用。 Male first inbred F344 rats, weighing 180~200g, were housed at room temperature 20~22°C, light and alternating for 12 hours, free to drink and eat. The propylene alcohol was intraperitoneally injected into the F344 rat at a dose of 0.62 m _O l / g body weight to prepare a drug-induced rat liver injury model, which was routinely reared for 3 days.

4、 c-Met+p₂Ivr细胞的定位富集 4. Localization and enrichment of c-Met+p ₂ Ivr cells

将大鼠固定于磁靶向定位富集仪中，使肝脏所对应的体表投射区正对上下两个磁极，然后通过大鼠外周静脉（尾静脉或股静脉）将步骤 2所得目的细胞——荧光素标记的携带免疫磁珠的 οΜ ⁺β₂Μ—细胞输注入体内，这样，标记了免疫磁珠的目的干细胞可在磁力的弓 I导下沿血液循环向分布于肝脏内的各级血管富集，随着大鼠在磁场内停留时间的延长，目的干细胞可穿越血管内皮细胞进入肝组织内整合入肝细胞板，从而使 c-Met+p₂M—细胞靶向定位富集于肝组织。 The rat is fixed in a magnetic targeting localization enrichment instrument, so that the body surface projection area corresponding to the liver faces the upper and lower magnetic poles, and then the target cells obtained in step 2 are obtained through the peripheral vein (tail vein or femoral vein) of the rat- - fluorescein-labeled οΜ ⁺ β ₂ Μ-cells carrying immunomagnetic beads are injected into the body, so that the target stem cells labeled with immunomagnetic beads can be distributed along the blood circulation to each other distributed in the liver under the guidance of magnetic force. Stage vascular enrichment, with the prolongation of the residence time of the rat in the magnetic field, the target stem cells can penetrate into the liver tissue through the vascular endothelial cells and integrate into the hepatocyte plate, so that c-Met+p ₂ M-cells can be targeted and enriched. In liver tissue.

其中，如图 1、图 2所示，在上述操作中所用的磁靶向定位富集仪，它包括有一支架 1，支架 1内上下相对设置有二磁极，下磁极 2垂向设置在支架 1的底面，上磁极 3与下磁极 2相对应的设置在支架 1的顶面。 As shown in FIG. 1 and FIG. 2, the magnetic targeting positioning enrichment instrument used in the above operation includes a bracket 1 in which the upper and lower sides of the bracket 1 are oppositely disposed with two magnetic poles, and the lower magnetic pole 2 is vertically disposed on the bracket 1 On the bottom surface, the upper magnetic pole 3 is disposed on the top surface of the bracket 1 corresponding to the lower magnetic pole 2.

在本实施例中，上磁极 3是可移动的设置在支架 1的顶面的；其具体方法是在支架 1的顶面与下磁极 2相对应的地方设置有一螺孔 11，螺孔 11内穿设一螺杆 12，螺杆 12下端连接上磁极 3，而螺杆 12上端设置有转动把手 13。通过旋转转动把手 13，可以使螺杆 12在螺孔 11内转动，从而使螺杆相对支架 1的顶面沿垂向移动，也就可以带动上磁极 3沿垂向移动，借此来改变上、下磁极之间的距离。 In the embodiment, the upper magnetic pole 3 is movably disposed on the top surface of the bracket 1; the specific method is to provide a screw hole 11 in the top surface of the bracket 1 corresponding to the lower magnetic pole 2, and the screw hole 11 is A screw 12 is disposed, the lower end of the screw 12 is connected to the upper magnetic pole 3, and the upper end of the screw 12 is provided with a rotating handle 13. By rotating the rotating handle 13, the screw 12 can be rotated in the screw hole 11, so that the screw moves in the vertical direction with respect to the top surface of the bracket 1, so that the upper magnetic pole 3 can be moved in the vertical direction, thereby changing the upper and lower sides. The distance between the poles.

在本实施例中，二磁极成圆柱状，为了保证两磁极间的磁场梯度，两磁极靠近相对的一端直径缩小，且直径缩小的越多，两磁极间的磁场梯度越高。此外，支架及二磁极的材料均选用铷铁硼，铷铁硼是很好的永磁性物质，支架也选用和磁极一样的材料，可以有效的提高磁极间的磁场强度。 In the present embodiment, the two magnetic poles are formed in a cylindrical shape. In order to ensure the magnetic field gradient between the two magnetic poles, the diameter of the two magnetic poles near the opposite end is reduced, and the more the diameter is reduced, the higher the magnetic field gradient between the two magnetic poles. In addition, the material of the bracket and the two magnetic poles are all made of neodymium iron boron. The neodymium iron boron is a very good permanent magnet material, and the bracket is also made of the same material as the magnetic pole, which can effectively improve the magnetic field strength between the magnetic poles.

本磁靶向定位富集仪在使用的时候，具体操作如下： When the magnetic target positioning and enrichment meter is used, the specific operation is as follows:

以富集大鼠内的目的细胞为例，首先将大鼠固定在支架 1上，为了使大 004 001560 鼠能够更好的固定在支架 1上，在支架内部可以放置一垫块 4，并使垫块 4 与下磁极 2保持在同一平面；将大鼠的靶器官所对应的体表投射区正对上下两个磁极，通过旋转转动把手 13调整磁极间的距离，这样一来是适应大鼠的体型的要求，二来是可以得到一个比较理想的磁场强度；然后通过大鼠外周静脉（尾静脉或股静脉）将标记过免疫磁珠的细胞输注入体内，这样，标记了免疫磁珠的目的细胞可在磁力的引导下沿血液循环向分布于靶器官内的血管富集，随着大鼠在磁场内停留时间的延长，目的细胞可穿越血管内皮细胞进入组织内整合入目的器官，并在该组织提供的适宜的微环境内向该组织特异性的细胞增殖、分化、成熟，最终发挥功能补偿作用。经体外富集和体内富集实验证实，采用本发明方法能达到良好的富集效果。 Taking the target cells in the rat as an example, the rat is first fixed on the stent 1 in order to make it large. 004 001560 The mouse can be better fixed on the bracket 1, and a spacer 4 can be placed inside the bracket, and the spacer 4 and the lower magnetic pole 2 are kept in the same plane; the body surface projection area corresponding to the target organ of the rat Directly facing the upper and lower magnetic poles, the distance between the magnetic poles is adjusted by rotating the rotating handle 13, so that it is adapted to the requirements of the body shape of the rat, and secondly, an ideal magnetic field strength can be obtained; and then the rat peripheral vein (tail) is passed. Intravenous or femoral veins) Inject cells labeled with immunomagnetic beads into the body, so that the cells labeled with the immunomagnetic beads can be enriched along the blood circulation to the blood vessels distributed in the target organs under the guidance of magnetic force. The prolongation of the residence time of the rat in the magnetic field, the target cells can penetrate into the tissue into the target organ through the vascular endothelial cells, and proliferate, differentiate, mature, and finally play the specific cells in the appropriate microenvironment provided by the tissue. Functional compensation. It has been confirmed by in vitro enrichment and in vivo enrichment experiments that the method of the present invention can achieve a good enrichment effect.

1、体外富集 1, in vitro enrichment

按照 c-Met⁺p₂]Vr细胞的靶向定位富集实验中步骤 1一 2的操作得到免疫磁珠标记的 β₂Μ+细胞， PBS洗涤。然后配制含 20%葡聚糖的生理盐水作为流动相，将所得细胞悬浮于其中。调节蠕动泵流速为 0.1〜15cm/s，使磁珠标记的细胞流过磁场（调节两磁极间的距离 1.6~ 2.5cm，使场强波动于 The immunomagnetic bead labeled β ₂ Μ+ cells were obtained by the procedure of step 1 - 2 in the targeted localization enrichment experiment of c-Met ⁺ p ₂ ]Vr cells, and washed with PBS. Then, physiological saline containing 20% dextran was prepared as a mobile phase, and the obtained cells were suspended therein. Adjust the peristaltic pump flow rate to 0.1~15cm/s, so that the magnetic bead-labeled cells flow through the magnetic field (adjust the distance between the two magnetic poles by 1.6~2.5cm, so that the field strength fluctuates

1.24-0.98T)，显微镜下计数流出和流入细胞的比值，得到磁场对磁珠标记细胞的富集效率。操作过程如图 5所示，图中 15为加有免疫磁珠标记的 β₂Μ+ 细胞的 20%葡聚糖生理盐水溶液； 16为蠕动泵； 17为磁场的两个磁极； 18 为富集得到的细胞。 1.24-0.98T), the ratio of the outflow to the inflowing cells was counted under a microscope, and the enrichment efficiency of the magnetic field on the magnetic bead-labeled cells was obtained. The operation process is shown in Fig. 5. In the figure, 15 is a 20% dextran physiological saline solution with β ₂ Μ+ cells labeled with immunomagnetic beads; 16 is a peristaltic pump; 17 is a magnetic pole of two magnetic poles; Collect the resulting cells.

结果表明，随着磁场强度的增加， β₂Μ+细胞富集效果逐渐加强，当磁场强度在 0.9 Te以上时，均能使磁珠标记的细胞在磁极对应的管道局部很好地富集，其效率达到 90%以上。 The results show that with the increase of magnetic field strength, the β ₂ Μ+ cell enrichment effect is gradually strengthened. When the magnetic field strength is above 0.9 Te, the magnetic bead-labeled cells can be well enriched in the pipeline corresponding to the magnetic pole. Its efficiency is over 90%.

2、体内富集 2, body enrichment

取近交系 F344大鼠，雌雄不限，按实施例 1步骤 4的方法制备肝损伤模型。动物分为 3组： Gl，外周静脉（股静脉）移植 +磁场； G2，开腹经门静脉移植； G3，外周静脉移植（对照组，不加磁场）。其中， G1组的操作如下： Inbred F344 rats were taken, male or female, and a liver injury model was prepared as in Example 1 step 4. Animals were divided into 3 groups: Gl, peripheral vein (femoral vein) transplantation + magnetic field; G2, open abdominal portal vein transplantation; G3, peripheral vein transplantation (control group, no magnetic field). Among them, the operation of the G1 group is as follows:

1 )先制备免疫磁珠标记的 c-Met+p₂M—细胞，然后加入 DAPI染液室温避光染色 30 min，用 PBS洗 4次，按 4X 10⁷/mL将细胞重悬于 DMEM F12培养基中； 1) Prepare immunomagnetic bead-labeled c-Met+p ₂ M-cells, then add DAPI staining solution to avoid staining for 30 min at room temperature, wash 4 times with PBS, and resuspend the cells in DMEM F12 at 4×10 ⁷ /mL. Training Nutrient

2)大鼠常规麻醉后置于磁靶向定位仪中四肢固定，使其肝脏体表投影部位正对上下两个磁极； 2) After routine anesthesia, the rats are placed in the magnetic targeting locator and fixed in the limbs, so that the projection surface of the liver surface faces the upper and lower magnetic poles;

3)于腹股沟切开皮肤暴露股静脉，经股静脉缓慢注入处理好的细胞悬液，术后缝皮，灯照保暖，术后不同时间将大鼠移离磁场，正常饲养； 3) In the inguinal incision, the skin is exposed to the femoral vein, and the treated cell suspension is slowly injected through the femoral vein. After the operation, the skin is sewn and the lamp is kept warm. The rats are removed from the magnetic field at different times after surgery, and are normally raised;

4)术后分别于 3 d和 10 d活杀大鼠，取肝和肺组织制备冰冻切片，于荧光显微镜下观察荧光标记细胞在受体大鼠肝和肺内的分布。 4) The rats were sacrificed on the 3rd and 10th day after operation, and the frozen sections were prepared from the liver and lung tissues. The distribution of fluorescently labeled cells in the liver and lung of the recipient rats was observed under a fluorescence microscope.

结果如图 6所示，其中图 6A和图 6B分别为经股静脉移植细胞后，大鼠固定于磁场内 20 h和 2 h，细胞在肺内的截留；图 6C为经门静脉移植细胞后，细胞在肺内的滞留；图 6D和图 6E分别为经股静脉移植细胞后，大鼠固定于磁场内 20 h移离磁场， 3 d和 10 d后活杀大鼠观察到细胞在肝脏的富集；图 6F为经门静脉移植后细胞在肝脏的富集；图 6G和图 6H分别为经股静脉移植细胞后，大鼠固定于磁场内 2 h移离磁场， 3 d和 10 d后细胞在肝脏的富集。 The results are shown in Fig. 6. In Fig. 6A and Fig. 6B, after the cells were transplanted through the femoral vein, the rats were fixed in the magnetic field for 20 h and 2 h, and the cells were trapped in the lung; Fig. 6C is the transplantation of the cells through the portal vein. The cells were retained in the lungs; Figure 6D and Figure 6E show that after the transfemoral cells were transplanted, the rats were immobilized in a magnetic field for 20 h to remove the magnetic field, and after 3 d and 10 d, the rats were observed to be rich in the liver. Figure 6F shows the enrichment of cells in the liver after portal vein transplantation; Figure 6G and Figure 6H show that after transfemoral transplantation, the rats were fixed in a magnetic field for 2 h and moved away from the magnetic field. After 3 d and 10 d, the cells were Enrichment of the liver.

结果表明，经股静脉移植 DAPI-免疫磁珠标记的细胞后，大鼠固定于磁场内 20 h与 2 h者相比，标记细胞在肺内的截留明显减少，而在肝内的停留明显增多；初步统计结果表明，滞留于肝脏内的细胞数，依次为磁场内 20 h> 经门静脉移植〉磁场内 2 h和股静脉移植（无磁场，对照组），标记细胞在肺内的滞留与此相反。表明采用本发明方法能有效地将细胞定向富集于靶组织内。工业应用 The results showed that after transplantation of DAPI-immunomagnetic beads labeled cells in the femoral vein, the rats were fixed in the magnetic field for 20 h and 2 h, the entrapment of labeled cells in the lungs was significantly reduced, and the retention in the liver was significantly increased. Preliminary statistical results showed that the number of cells retained in the liver was 20 h in the magnetic field> through the portal vein> 2 h in the magnetic field and femoral vein graft (no magnetic field, control group), the retention of labeled cells in the lungs in contrast. It is shown that the method of the present invention can effectively concentrate cells in target tissues. Industrial application

本发明将直径在 50纳米左右的免疫磁珠通过抗原抗体亲和反应来特异性标记细胞，由于该免疫磁珠的超顺磁性，使得目的细胞在磁场的作用下定向富集于磁场中的靶组织，而且当磁场消失后，免疫磁珠失去磁性，在靶组织内细胞不会因为相互吸引聚集成球而导致血管阻塞。在本发明中，免疫磁珠的主要制备原料均可以被生物体吸收、 ***，最终以卟啉铁的形式被机体吸收代谢，对机体无毒副作用。 In the present invention, an immunomagnetic bead having a diameter of about 50 nm is specifically labeled by an antigen-antibody affinity reaction. Due to the superparamagnetism of the immunomagnetic bead, the target cell is directionally enriched in a target in a magnetic field under the action of a magnetic field. Tissue, and when the magnetic field disappears, the immunomagnetic beads lose their magnetism, and the cells in the target tissue do not block the blood vessels due to mutual attraction and aggregation. In the present invention, the main raw materials for the preparation of the immunomagnetic beads can be absorbed and excreted by the organism, and finally absorbed and metabolized by the body in the form of iron porphyrin, which has no toxic and side effects on the body.

本发明的磁靶向定位富集仪的二磁极成圆柱状，且二磁极靠近相对的一端直径缩小，再者磁极和支架的材料均选用了铷铁硼，可以提供髙梯度、高强度的磁场。并能通过旋转转动把手来调整二磁极之间的距离，磁极间距离的调整可以改变二磁极之间的距离，从而使磁极间磁场强度可以调节，以满 The magnetic pole positioning and concentrating instrument of the invention has two magnetic poles in a cylindrical shape, and the diameters of the two magnetic poles near the opposite end are reduced, and the materials of the magnetic pole and the bracket are all made of neodymium iron boron, which can provide a 髙 gradient and a high intensity magnetic field. . And by rotating the handle to adjust the distance between the two magnetic poles, the adjustment of the distance between the magnetic poles can change the distance between the two magnetic poles, so that the magnetic field strength between the magnetic poles can be adjusted to

6 足不同场合的需要。本发明巧妙地根据超顺磁性磁珠的磁特性，利用磁靶向定位富集仪产生的高强度、高梯度的磁场，使目的细胞在靶组织的定位富集，具有富集效率高、方法简便等优点，具有广泛的应用前景。 6 The need for different occasions. The invention skillfully according to the magnetic characteristics of the superparamagnetic magnetic beads, utilizes the high-strength and high-gradient magnetic field generated by the magnetic targeting and enrichment instrument to make the target cells locate and enrich in the target tissue, and has high enrichment efficiency and method. It has the advantages of simplicity and so on, and has broad application prospects.

Claims

权利要求 Rights request

1、一种干细胞靶向定位富集的方法，包括如下步骤： 1 )将干细胞用粒径为 30— 70nm的免疫磁珠标记，得到标记后的干细胞； 2)将所述标记后的干细胞注入靶组织的循环***内； 3)将所述靶组织相对应的体表部位正对两个异性磁极，在所述循环***的循环过程中，将所述干细胞定位富集于所述靶组织内；所述两个异性磁极间的磁场强度为 9200— 24500Gs。 A method for targeting and enriching stem cells, comprising the steps of: 1) labeling stem cells with immunomagnetic beads having a particle size of 30-70 nm to obtain labeled stem cells; 2) injecting the labeled stem cells Within the circulatory system of the target tissue; 3) aligning the body surface portion corresponding to the target tissue with two opposite magnetic poles, and concentrating the stem cells in the target tissue during circulation of the circulatory system The magnetic field strength between the two opposite magnetic poles is 9200-24500 Gs.

2、根据权利要求 1所述的方法，其特征在于：所述干细胞来源于人或其它晡乳动物骨髓、动员的外周血、脐带血或其他组织。 2. A method according to claim 1 wherein: said stem cells are derived from bone marrow of human or other lactating animal, mobilized peripheral blood, cord blood or other tissue.

3、根据权利要求 1所述的方法，其特征在于，所述免疫磁珠标记所述干细胞的过程如下：将针对所述干细胞表面标志性抗原的特异性抗体与所述干细胞孵育，经清洗后再与偶联有抗所述特异性抗体的免疫磁珠孵育，清洗得到所述标记的干细胞。 The method according to claim 1, wherein the immunomagnetic beads mark the stem cells as follows: the specific antibody against the stem cell surface marker antigen is incubated with the stem cells, after washing The cells are then incubated with immunomagnetic beads conjugated to the specific antibody, and the labeled stem cells are washed.

4、根据权利要求 3所述的方法，其特征在于：所述干细胞为来源于大鼠骨髓的 c-Met M—细胞;所述特异性抗体为兔抗大鼠 c-Met抗体和 β₂Μ抗体；所述免疫磁珠偶联有山羊抗兔 IgG。 The method according to claim 3, wherein: the stem cells are c-Met M-cells derived from rat bone marrow; and the specific antibodies are rabbit anti-rat c-Met antibodies and β ₂ Μ The antibody is conjugated with goat anti-rabbit IgG.

5、根据权利要求 1至 4任一所述的方法，其特征在于：步骤 3)所述富集是在磁靶向定位富集仪中进行的。 5. A method according to any one of claims 1 to 4, characterized in that: step 3) said enrichment is carried out in a magnetic targeting locator.

6、一种磁靶向定位富集仪，其特征在于：它包括有一支架，所述支架内上下相对设置有二磁极，下磁极垂向设置在所述支架的底面，上磁极与所述下磁极相对应地设置在所述支架的顶面。 A magnetic targeted positioning enrichment apparatus, comprising: a bracket, wherein the bracket is vertically disposed opposite to the second magnetic pole, and the lower magnetic pole is vertically disposed on a bottom surface of the bracket, the upper magnetic pole and the lower The magnetic poles are correspondingly disposed on the top surface of the bracket.

7、根据权利要求 6所述的磁靶向定位富集仪，其特征在于：所述上磁极可移动地设置在所述支架的顶面。 7. The magnetic targeting concentrating apparatus according to claim 6, wherein: said upper magnetic pole is movably disposed on a top surface of said bracket.

8、根据权利要求 7所述的磁靶向定位富集仪，其特征在于：所述支架的顶面与所述下磁极相对应处设置有一螺孔，所述螺孔内穿设一螺杆，所述螺杆下端连接所述上磁极，螺杆上端设置有转动把手。 The magnetic targeting positioning enrichment apparatus according to claim 7, wherein: a screw hole is disposed in a top surface of the bracket corresponding to the lower magnetic pole, and a screw is disposed in the screw hole. The lower end of the screw is connected to the upper magnetic pole, and the upper end of the screw is provided with a rotating handle.

9、根据权利要求 6或 7或 8所述的磁靶向定位富集仪，其特征在于：所述磁极成圆柱状，且两磁极靠近相对的一端直径缩小。 The magnetic targeting positioning enrichment apparatus according to claim 6 or 7 or 8, wherein the magnetic poles are cylindrical, and the diameters of the two magnetic poles near the opposite end are reduced.

10、根据权利要求 6或 7或 8所述的磁靶向定位富集仪，其特征在于：所述支架及磁极的材料为永磁性材料。 The magnetic targeting positioning enrichment apparatus according to claim 6 or 7 or 8, wherein the material of the bracket and the magnetic pole is a permanent magnet material.

11、根据权利要求 10所述的磁靶向定位富集仪，其特征在于：所述永磁性材料为铷铁硼。 11. The magnetic targeting positioning enrichment apparatus according to claim 10, wherein: said permanent magnet The material is NdFeB.

12、根据权利要求 11所述的磁靶向定位富集仪，其特征在于：所述二磁极间的距离为 5— 25mm。 The magnetic targeting positioning enrichment apparatus according to claim 11, wherein the distance between the two magnetic poles is 5-25 mm.

13、根据权利要求 9所述的磁靶向定位富集仪，其特征在于：所述支架及磁极的材料为永磁性材料。 13. The magnetic targeting positioning enrichment apparatus according to claim 9, wherein: the material of the bracket and the magnetic pole is a permanent magnet material.

14、根据权利要求 13所述的磁靶向定位富集仪，其特征在于：所述永磁性材料为铷铁硼。 The magnetic targeting positioning enrichment apparatus according to claim 13, wherein the permanent magnetic material is neodymium iron boron.

15、根据权利要求 14所述的磁靶向定位富集仪，其特征在于：所述二磁极间的距离为 5— 25mm。 The magnetic targeting positioning enrichment apparatus according to claim 14, wherein the distance between the two magnetic poles is 5 - 25 mm.