搜索

VLA-4 Affinity Assay for Murine Bone Marrow-derived Hematopoietic Stem Cells
鼠骨髓源性造血干细胞的VLA-4亲和力测定   

引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
Clinical Cancer Research
Jun 2015

Abstract

Hematopoietic stem cells (HSCs) are defined by their functional ability to self-renew and to differentiate into all blood cell lineages. The majority of HSC reside in specific anatomical locations in the bone marrow (BM) microenvironment, in a quiescent non motile mode. Adhesion interactions between HSCs and their supporting BM microenvironment cells are critical for maintaining stem cell quiescence and protection from DNA damaging agents to prevent hematology failure and death. Multiple signaling proteins play a role in controlling retention and migration of bone marrow HSCs. Adhesion molecules are involved in both processes regulating hematopoiesis and stem- and progenitor-cell BM retention, migration and development. The mechanisms underlying the movement of stem cells from and to the marrow have not been completely elucidated and are still an object of intense study. One important aspect is the modification of expression and affinity of adhesion molecules by stem and progenitor cells which are required both for stem cell retention, migration and development. Adhesion is regulated by expression of the adhesion molecules, their affinity and avidity. Affinity regulation is related to the molecular binding recognition and bond strength. Here, we describe the in vitro FACS assay used in our research to explore the expression, affinity and function of the integrin α4β1 (also termed VLA-4) for murine bone marrow retained EPCR+ long term repopulation HSC (LT-HSC) (Gur-Cohen et al., 2015).

Keywords: Hematopoietic stem cells, HSC mobilization, Bone marrow retention, VLA-4, aPC/EPCR/PAR1 signaling

Background

Integrins are type I transmambrane glycoprotein receptors that mediate cell-cell and cell-matrix adhesion interactions, signaling and communication. All integrins are heterodimers of non-covalently associated α and β subunits. In humans integrin heterodimers are formed from 9 types of β subunits and 24 types of α subunits. This diversity is further increased by alternative splicing of some integrin RNAs. Each heterodimer consists of a large extracellular domain which binds proteins in the extracellular environment, a single transmembrane domain, and an intracellular cytoplasmic tail domain. The largest integrin subfamily is composed by integrin β1 (CD29) that is able to associates with 12 different α subunits (α1-11 and αv). Integrin β1 together with integrin chains α4, α5, α6 and α9 are expressed by murine hematopoietic stem and progenitor cells (HSPCs) and play important roles in regulating their BM retention, migration and development.

Integrins, like other transmembrane receptors, display an ‘outside-in signaling’, i.e., to transduce the signal intracellularly after the binding with their ligand. Moreover, integrins have a peculiar feature: they are able to shift between high- and low-affinity conformation states for ligand binding (‘inside-out’ signaling) (Takagi and Springer, 2002). According to the cell type, integrins can be either basically activated or basally inactive. In the inactive state, the integrin extracellular domains are not bounded to the ligands, and are in a bent conformation. Following intracellular activation signals, the extracellular domain is straightened, stabilizing the extended active conformation. Thus, the external ligand binding site, is now exposed to the ligand binding, allowing the transmission of the signals from the outside to the inside (Luo et al., 2007).

Very Late Antigen-4 (VLA-4, also known as CD49d/CD29 or α4β1) is a member of the integrin α4 family together with α4β7. Within the integrin family, VLA-4 has some unique features. In contrast to related members of β1 subfamily, VLA-4 is predominantly expressed on hematopoietic lineage cells (Hemler, 1990) and is functionally involved in both cell-cell and cell-ECM adhesive interactions. Moreover, despite sequence homology with other integrin α subunits, the α4 strand because of the lack of the inserted I-domain doesn’t undergo post-translational cleavage near the transmembrane region. Finally, the α4 chain contains a trypsin-like cleavage site, constitutively expressed on most leukocytes and on hematopoietic stem and progenitor cells (Hynes, 1992).

VLA-4 plays a major role in the regulation of immune cell recruitment to inflamed endothelia and sites of inflammation through its interactions with two alternative ligands, vascular cell adhesion molecule-1 (VCAM-1) and the alternatively spliced connecting segment 1 (CS-1) of fibronectin (Hemler, 1990; Papayannopoulou et al., 1998). It also participates in many cellular events and is crucial for BM retention and mobilization of immature stem and progenitors cells from the bone marrow (Lapidot and Petit, 2002; Peled et al., 2000).

Migration of hematopoietic stem cells to the bone marrow is a regulated multistep process that requires precise regulation and activation of various molecules including chemoattractants, selectins and integrins. While the initial steps of hematopoietic stem and progenitor cells tethering and rolling along BM blood vessel endothelium are primarily regulated by selectins, various integrins have been shown to be involved in the next stages of this process. VLA-4 plays an important role in homing, lodgment and retention of HSCs within the marrow microenvironment (Rettig et al., 2012). Previous studies demonstrate that treatment of donor BM cells with a neutralizing anti α4 integrin antibody before injection into lethally irradiated recipients, inhibits their homing to the femurs of recipient mice, increasing the number of HSPCs in the peripheral blood and spleen. Moreover, recipient mice pretreated with neutralizing antibodies against VCAM-1 gave similar results, adding evidences to the important role of the VLA-4/VCAM-1 axis in HSPC homing to the bone marrow (Papayannopoulou et al., 1995).

Recently, factors traditionally related to coagulation and inflammation have been shown to independently control long term (LT) HSCs retention in the bone marrow and their recruitment to the blood (Aronovich et al., 2013; Gur-Cohen et al., 2015). Adult murine BM LT-HSCs, endowed with the highest repopulation and self-renewal potential, express endothelial protein C receptor (EPCR) which is used as a marker to isolate them (Balazs et al., 2006).

Protease-activated receptor-1 (PAR1) is functionally expressed by bone marrow stromal and endothelial cells as well as HSC and immature and maturing leukocytes (Gur-Cohen et al., 2016). Activated protein C ([aPC], the major ligand for EPCR)-EPCR/PAR1 signaling facilitate LT-HSC BM repopulation, retention, survival, and chemotherapy resistance by restricting nitric oxide (NO) production. Inhibition of NO generation by aPC/EPCR/PAR1 signaling on LT-HSC, inhibits downstream CDC42 activity and induces CDC42 polarity, as well as increasing VLA4 expression, affinity and adhesion. Conversely, acute stress and clinical mobilization up-regulate thrombin generation and activate different PAR1 signaling leading to NO generation that overcomes BM EPCR+LT-HSC retention, inducing TACE mediated EPCR and VLA-4 shedding, up-regulation of CXCR4 and PAR1 on LT-HSC, stromal PAR1 mediated CXCL12 secretion, resulting in stem and progenitor cell recruitment to the blood stream (Gur-Cohen et al., 2015). VLA-4 is expressed at a higher level by bone marrow EPCR+LT-HSC together with higher affinity to its ligands, inducing their BM retention and protection from DNA damaging agents. The restriction of NO by EPCR/PAR1 signaling increase the affinity of VLA-4 regulating anchorage and bone marrow retention of LT-HSC and chemotherapy resistant (Gur-Cohen et al., 2015).

Multiple small molecules have been developed in an attempt to regulate integrin dependent adhesion. The affinity states of human VLA-4 can be recognized by monoclonal antibodies sensitive to its molecular conformation (Masumoto and Hemler, 1993). Importantly, changes in VLA-4 affinity can be detected in real-time and on a physiologically relevant time frame using a ligand mimicking LDV-containing fluorescent small molecule (LDV-FITC) by FACS (Chigaev et al., 2001). VLA-4 recognize with high affinity a peptide sequence within fibronectin, which comprises 25 amino acid, termed CS-1 (Hynes, 1992). LDV (leu-asp-val) is the tripeptide identified as the minimal sequence for specific VLA-4 recognition of CS-1 segment of fibronectin. Here we describe a method to detect VLA-4 affinity monitoring mean fluorescent intensity through flow cytometry using LDV-FITC.

Materials and Reagents

  1. Syringe with needle 1 ml 25 G x 5/8 in. (0.5 x 16 mm) (BD, catalog number: 300014 )
  2. Dishes 35 x 10 (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 153066 )
  3. 70 μm nylon strainer (Sinun Tech, catalog number: Polymer Screens )
  4. FACS tubes (Corning, FalconTM, catalog number: 352054 )
  5. Cells of interest (here murine bone marrow cells obtained from 8 weeks old mice)
  6. Dulbecco’s phosphate-buffered saline (PBS+/+) (Biological Industries, catalog number: 02-020-1A )
  7. EDTA (5 mM final concentration in water, pH 7.4) (Avantor® Performance Materials, J.T.Baker, catalog number: 8993-01 )
  8. Wet ice
  9. Antibodies to detect LT-HSCs by flow cytometry:
    1. Sca-1 PEcy7 (clone D7) (Biolegend, catalog number: 108114 )
    2. c-kit APC (clone 2B8) (Biolegend, catalog number: 105812 )
    3. CD150 Brilliant Violet (clone TC15-12F12.2) (Biolegend, catalog number: 115922 )
    4. CD48 Pasific Blue (clone HM48-1) (Biolegend, catalog number: 103418 )
    5. EPCR PE (clone eBio1560) (Affymetrix, eBioscience, catalog number: 12-2012-82 )
    6. Lineage: CD4 (clone GK 1.5), CD8a (clone 53-6.7), GR1 (clone RB6-8C5), B220 (clone RA3-682), Ter119 (clone TER-119), CD11b (clone M1/70)
  10. Calcium chloride dihydrate (EMD Millipore, catalog number: 102382 )
  11. Magnesium chloride hexahydrate (EMD Millipore, catalog number: 105833 )
  12. HEPES buffer solution (Biological Industries, catalog number: 03-025-1B )
  13. Hank’s balanced salt solution (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14175095 )
  14. Bovine serum albumin solution (10%, BSA) (Biological Industries, catalog number: 03-010-1B )
  15. 2.4-((N’-2-methylphenyl)ureido)-phenylacetyl-L-leucyl-L-aspartyl-L-valyl-L-prolyl-L-alanyl-L-alanyl-L-lysine (LDV-FITC) (R&D System, catalog number: 4577 )
  16. Deionized water (DDW)
  17. Gentian violet (Sigma-Aldrich, catalog number: 548-62-9 )
  18. Acetic acid (Sigma-Aldrich, catalog number: 64-19-7 )
  19. Paraformaldehyde (PFA) (Sigma-Aldrich, catalog number: 30525-89-4 )
  20. LDV medium (see Recipes)
  21. LDV-FITC stock and working solutions (see Recipes)
  22. Turk’s solution (see Recipes)
  23. 4% PFA (see Recipes)

Note: Flurochrome should be chosen according to the flow cytometry machine.

Equipment

  1. Forceps (Kent Scientific, catalog number: INS700100-2 )
  2. Scissors (Kent Scientific, catalog number: INS750046 )
  3. Centrifuge (Eppendorf, model: 5810R )
  4. Centrifuge swing-bucket rotor A-462 4 x 250 ml rectangular buckets (Eppendorf, catalog number: 5810709008 )
  5. Adapters (Eppendorf, catalog number: 5810752000 )
  6. Inverted light microscope (Olympus, model: CHK2-F-GS )
    Note: This product has been discontinued by the manufacturer.
  7. Hemocytometer (Sigma-Aldrich, Bright-LineTM, catalog number: Z359629 )
  8. Incubator 37 °C, 5% CO2 (Thermo Fisher Scientific, Thermo ScientificTM, model: 150i )
  9. Cold room or refrigerator (4 °C)
  10. Flow cytometer (i.e., Macs Quant instrument [Miltenyi, BergischGladbach, Germany] or BD LSR II flow cytometer)
  11. 2 L glass flask or bottle (Kimble Chase Life Science and Research Products, catalog number: 26500-2000 )
  12. Plastic weigh boat (Sigma-Aldrich, catalog number: Z186856 )

Note: Color combinations can be adjusted to match the laser combinations available.

Software

  1. FlowJo V10 software (Tree Star, optional)

Procedure

  1. Obtain murine bone marrow cells
    Note: All animal experiments have to be approved by local animal care and ethics authorities.
    1. Sacrifice desired mice strain by CO2 euthanization or cervical dislocation.
    2. Extract the femurs, tibias and iliac crest bones using forceps and sharp scissors. Place the bones in a small dish supplemented with PBS+/+ on ice.
    3. Flush total bone marrow cells from the bone cavity with 1-5 ml ice cold PBS (per mouse) using a 1 ml syringe and 25 G needle. Obtain single cell suspension by simply resuspending the cell solution using the same syringe.


      Figure 1. Process of flushing bone marrow out of murine bones. A. Femurs, tibias and iliac crest bones before flushing; B. Femurs, tibias and iliac crest bones after flushing; C. 1 ml syringe with its 25 G needle inserted in the tibia bone cavity.

    4. Filter the cells by passing the cell solution through 70 μm nylon strainer to obtain a uniform single-cell suspension.
    5. Centrifuge the cells (289.5 x g, 5 min) and resuspend in 1 ml LDV medium.
    6. Count the cells by diluting the cells 1:10 with Turk’s solution. Count the cells under the inverted light microscope using the hemacytometer. From one mouse typically one will get 60-90 million cells from both femurs and tibias and iliac crest bones.

  2. Determination of VLA-4 affinity using flow cytometry (LDV assay)
    VLA-4 affinity can be examined on fresh bone marrow cells obtained from treated animals. Alternatively, cells can be obtained from untreated animals and treated in vitro with a desired reagent, in the presence of LDV medium, followed by the abovementioned detailed protocol.
    Note: It is recommended to continue with the staining after isolation of the bone marrow cells.
    1. Place 5 million bone marrow cells in a total volume 100 μl of LDV medium (see Recipes) in a FACS tube.
    2. Add LDV-FITC to achieve a final concentration of 10 nM (see Recipes) and mix gently by pipetting, do not vortex.
    3. To determine LDV-FITC non-specific binding, add EDTA to each LDV-FITC sample at a final concentration of 5 mM.
    4. Incubate all the samples for 30 min at 37 °C.
    5. After incubation, put the tubes immediately on wet ice and fix the cells by adding ice cold 4% PFA for 10 min (i.e., the reaction is in 100 µl of cells, thus add 4% PFA [1-2 ml] directly to the tube). Since that moment, cells need to be kept under cold conditions on wet ice. Avoid vortex along the procedure.
    6. Wash the cells with 2 ml ice cold PBS+/+.
    7. Centrifuge the cells (289.5 x g, 5 min) and gently discard the supernatant.
    8. Add 100 µl of ice cold PBS+/+ and perform staining of the cell surface markers Lineage (1-2 µl per sample of each antigen), Sca-1 (2 µl/sample), c-Kit (2 µl/sample), CD150 (2 µl/sample), CD48 (1 µl/sample) and EPCR (2 µl/sample). These cell surface markers are only suggestive antigens to detect LT-HSCs, however, this can be adjusted according to the research question needs. Color combination should fit with FITC as LDV peptide is conjugated with the FITC fluorochrome.
    9. Add the required antibodies into the cell samples and mix gently by pipetting (do not vortex), and incubate for 30 min on ice and protected from light.
    10. Wash the cells with 1 ml ice cold PBS+/+, centrifuge (289.5 x g, 5 min) and gently discard the supernatant.
    11. Resuspend the cells in 300 μl PBS+/+, do not vortex.
    12. Read the samples immediately by flow cytometry and determine LDV binding to LT-HSC by analyzing the intensity of the FITC fluorochrome on gated Lineage-/c-Kit+/Sca-1+/CD150+/CD48-/EPCR+ cells.

Data analysis



Figure 2. VLA4 affinity measured by LDV probe binding to bone marrow EPCR+ SK (Sca1+ ckit+) SLAM (CD150+ CD48-) cells. In black is the control cells treated with EDTA; in blue the EPCR+ cells gated on SLAM/SK; in pink the EPCR-cells gated on SLAM/SK.

For further analysis information concerning gating strategies and statistical analysis you can consult the article Gur-Cohen et al., 2015 at the following link: http://www.nature.com/nm/journal/v21/n11/full/nm.3960.html.

Recipes

  1. LDV medium
    1 mM CaCl2
    1 mM MgCl2
    20 mM HEPES, containing 1% BSA
    Note: It is recommended to prepare stock solutions of 1 M CaCl2 (dilute 1:1,000 directly in the medium) as well as 100 mM MgCl2 (dilute 1:100 directly in the medium).
  2. LDV-FITC stock and working solutions
    Dissolve 1 mg LDV-FITC powder in 1 ml DDW (ddH2O) according to the manufacturer's instructions (giving a stock concentration of 0.73 mM)
    Note: It is recommended to divide the solution into small aliquots, avoiding repeated freeze/thaw cycles (the solution can be stored at -20 °C for up to 1 year).
    To prepare the working LDV-FITC solution, dilute stock solution with DDW, reaching to a final concentration of 10 nM (for 10 nM working solution dilute 1:730 with DDW and take 1 µl into 100 µl cells supplemented with LDV medium)
  3. Turk solution
    50 mg gentian violet
    5 ml acetic acid
    495 ml DDW
    Dissolve gentian violet in acetic acid and DDW
  4. 4% PFA
    Weight out 40 g of powdered paraformaldehyde into a plastic weigh boat
    Pour into a 2 L glass flask or bottle
    Add 1 L DDW, a stir bar and allow to gently agitate while warming to 65 °C, for 5 min
    Add one drop of 10 N KOH or 10 N NaOH base, the solution should then become clear
    Allow the solution to cool to room temperature and adjust the pH to 7.3
    Note: Smaller volumes can be stored at -20 °C for up to one year.

Acknowledgments

This study was supported by the Israel Science Foundation (851/13), the Ernest and Bonnie Beutler Research Program of Excellence in Genomic Medicine and FP7-HEALTH-2010 (CELL-PID 261387) (T.L.) and the DKFZ, Germany.

References

  1. Aronovich, A., Nur, Y., Shezen, E., Rosen, C., ZlotnikovKlionsky, Y., Milman, I., Yarimi, L., Hagin, D., Rechavi, G., Martinowitz, U., Nagasawa, T., Frenette, P. S., Tchorsh-Yutsis, D. and Reisner, Y. (2013). A novel role for factor VIII and thrombin/PAR1 in regulating hematopoiesis and its interplay with the bone structure. Blood 122(15): 2562-2571.
  2. Balazs, A. B., Fabian, A. J., Esmon, C. T. and Mulligan, R. C. (2006). Endothelial protein C receptor (CD201) explicitly identifies hematopoietic stem cells in murine bone marrow. Blood 107(6): 2317-2321.
  3. Chigaev, A., Blenc, A. M., Braaten, J. V., Kumaraswamy, N., Kepley, C. L., Andrews, R. P., Oliver, M. J., Edwards, S. B., Prossnitz, S. R., Larson, S. R. and Sklar, L. A. (2001). Real time analysis of the affinity regulation of α4-Integrin: the physiologically activated receptor is intermediate in affinity between resting and Mn2+ or antibody activation. JBC 276(52): 48670-48678.
  4. Gur-Cohen, S., Itkin, T., Chakrabarty, S., Graf, C., Kollet, O., Ludin, A., Golan, K., Kalinkovich, A., Ledergor, G., Wong, E., Niemeyer, E., Porat, Z., Erez, A., Sagi, I., Esmon, C. T., Ruf, W. and Lapidot, T. (2015). PAR1 signaling regulates the retention and recruitment of EPCR-expressing bone marrow hematopoietic stem cells. Nat Med 21(11): 1307-1317.
  5. Gur-Cohen, S., Kollet, O., Graf, C., Esmon, C. T., Ruf, W. and Lapidot, T. (2016). Regulation of long-term repopulating hematopoietic stem cells by EPCR/PAR1 signaling. Ann N Y AcadSci 1370(1): 65-81.
  6. Hemler, M. E. (1990). VLA proteins in the integrin family: structures, functions, and their role on leukocytes. Ann Rev Immunol 8(1): 365-400.
  7. Hynes, R. O. (1992). Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69(1): 11-25.
  8. Lapidot, T. and Petit, I. (2002). Current understanding of stem cell mobilization: the roles of chemokines, proteolytic enzymes, adhesion molecules, cytokines, and stromal cells. Exp Hematol 30(9): 973-981.
  9. Luo, B. H., Carman, C. V. and Springer, T. A. (2007). Structural basis of integrin regulation and signaling. Annu Rev Immunol 25: 619-647.
  10. Masumoto, A. and Hemler, M. E. (1993). Multiple activation states of VLA-4. Mechanistic differences between adhesion to CS1/fibronectin and to vascular cell adhesion molecule-1. J Biol Chem 268(1): 228-234.
  11. Papayannopoulou, T., Craddock, C., Nakamoto, B., Priestley, G. V. and Wolf, N. S. (1995). The VLA4/VCAM-1 adhesion pathway defines contrasting mechanisms of lodgement of transplanted murine hemopoietic progenitors between bone marrow and spleen. Proc Natl Acad Sci U S A 92(21): 9647-9651.
  12. Papayannopoulou, T., Priestley, G. V. and Nakamoto, B. (1998). Anti-VLA4/VCAM-1-induced mobilization requires cooperative signaling through the kit/mkit ligand pathway. Blood 91(7): 2231-2239.
  13. Peled, A., Kollet, O., Ponomaryov, T., Petit, I., Franitza, S., Grabovsky, V., Slav, M. M., Nagler, A., Lider, O., Alon, R., Zipori, D. and Lapidot, T. (2000). The chemokine SDF-1 activates the integrins LFA-1, VLA-4, and VLA-5 on immature human CD34+ cells: role in transendothelial/stromal migration and engraftment of NOD/SCID mice. Blood 95(11): 3289-3296.
  14. Rettig, M. P., Ansstas, G. and Di Persio, J. F. (2012). Mobilization of hematopoietic stem and progenitor cells using inhibitors of CXCR4 and VLA-4. Leukemia 26(1): 34-53.
  15. Takagi, J. and Springer, T. A. (2002). Integrin activation and structural rearrangement. Immunol Rev 186: 141-163.

简介

造血干细胞(HSC)由其自我更新的功能能力定义,并分化成所有血细胞谱系。大多数HSC位于骨髓(BM)微环境中的特定解剖位置,处于静止非运动模式。 HSCs与其支持的BM微环境细胞之间的粘附相互作用对于维持干细胞静止和保护免受DNA损伤因子阻止血液学失败和死亡至关重要。多重信号蛋白在控制骨髓HSCs的保留和迁移中起重要作用。粘附分子参与调节造血和干细胞和祖细胞BM保留,迁移和发育的两个过程。干细胞从骨髓移动到骨髓的机制尚未完全阐明,仍然是深入研究的对象。一个重要的方面是修饰干细胞保留,迁移和发育所需的干细胞和祖细胞的粘附分子的表达和亲和力。粘附力通过粘附分子的表达,亲和力和亲合力来调节。亲和力调节与分子结合识别和结合强度有关。在这里,我们描述了在我们的研究中使用的体外 FACS测定来研究整联蛋白α4亚类β1亚单位的表达,亲和力和功能也称为VLA-4)用于小鼠骨髓保留EPCR +长期复制HSC(LT-HSC)(Gur-Cohen等人,2015)。

背景整合素是介导细胞和细胞 - 基质粘附相互作用,信号传导和通讯的I型跨膜糖蛋白受体。所有整合素是非共价相关α和β亚基的异源二聚体。在人类中,整合素异源二聚体由9种β亚基和24种α亚基形成。这种多样性通过某些整合素RNA的选择性剪接进一步增加。每个异二聚体由结合胞外环境中的蛋白质,单个跨膜结构域和细胞内细胞质尾区结构的大的细胞外结构域组成。整合素亚类由整合素β1(CD29)组成,能够与12个不同的α亚基(α1 1-11)。整合素β1与整联蛋白链α4,α5,α6和α9结合,由鼠造血干细胞和祖细胞(HSPC)表达,在调节其BM保留,迁移和发育中起重要作用。
 与其他跨膜受体一样,整合素在与其配体结合后显示“外部信号”,即,以在细胞内转导信号。此外,整合素具有特殊的特征:它们能够在配体结合的高亲和力构象状态(“内向外”信号传递)之间转移(Takagi和Springer,2002)。根据细胞类型,整合素可以基本上被激活或基本无活性。在非活性状态下,整合素胞外结构域不与配体结合,并且呈弯曲构象。在细胞内激活信号后,细胞外结构域被拉直,稳定了延长的活性构象。因此,外部配体结合位点现在暴露于配体结合,允许信号从外部传递到内部(Luo等人,2007)。
&nbsp;非常晚的抗原-4(VLA-4,也称为CD49d / CD29或α4亚型β1)是整联蛋白α4的成员, 系列与α<4>β7 。在整合素家族中,VLA-4具有一些独特的功能。与β1亚类的相关成员相反,VLA-4主要在造血谱系细胞上表达(Hemler,1990),并且在功能上参与细胞和细胞 - ECM粘附相互作用。此外,尽管与其他整合素α亚基序列同源性,由于缺少插入的I结构域,α4链不会在跨膜区附近经历翻译后切割。最后,α4链含有在大多数白细胞和造血干细胞和祖细胞上组成型表达的胰蛋白酶样切割位点(Hynes,1992)。
&nbsp; VLA-4在通过与两种替代配体,血管细胞粘附分子-1(VCAM-1)和可变剪接的连接片段1(VCAM-1)的相互作用的调节免疫细胞募集炎症内皮和炎症部位中发挥主要作用CS-1)纤连蛋白(Hemler,1990; Papayannopoulou等,1998)。它还参与许多细胞事件,并且对骨髓中保留和动员未成熟的干细胞和祖细胞至关重要(Lapidot和Petit,2002; Peled等人,2000)。
&nbsp;造血干细胞迁移到骨髓是一个调节的多步骤过程,需要精确调节和活化各种分子,包括化学反应物,选择素和整合素。虽然造血干细胞和祖细胞沿BM血管内皮的束缚和滚动的初始步骤主要由选择素调节,但已经显示出各种整合素参与该过程的下一阶段。 VLA-4在骨髓微环境中的归巢,保留和保留HSC中起重要作用(Rettig等人,2012)。以前的研究表明,在注射到致死辐射的受体之前,用中和的抗α4A整联蛋白抗体治疗供体BM细胞,抑制它们归巢于受体小鼠的股骨,增加外周血中HSPC的数量和脾此外,用针对VCAM-1的中和抗体预处理的受体小鼠得到类似的结果,增加VLA-4 / VCAM-1轴在HSPC归巢到骨髓中的重要作用的证据(Papayannopoulou等人, ,1995)。
&nbsp;最近传统上与凝血和炎症相关的因素已被证明可以独立控制长期(LT)HSCs在骨髓中的保留及其对血液的募集(Aronovich等人,2013; Gur -Cohen等人,2015)。成年鼠BM LT-HSCs具有最高的再生潜力和自我更新潜力,表达内皮蛋白C受体(EPCR),其被用作标记物以分离它们(Balazs等人,2006)。
&nbsp;蛋白酶激活受体-1(PAR1)在功能上由骨髓基质和内皮细胞以及HSC和未成熟和成熟的白细胞(Gur-Cohen等人,2016)表达。活化蛋白C([aPC],EPCR的主要配体)-EPCR / PAR1信号通过限制一氧化氮(NO)产生促进LT-HSC BM的重新增殖,保留,存活和化疗抗性。通过LT-HSC上的aPC / EPCR / PAR1信号传导抑制NO产生,抑制下游CDC42活性并诱导CDC42极性,以及增加VLA4表达,亲和力和粘附。相反,急性应激和临床动员上调凝血酶产生并激活不同的PAR1信号,导致NO产生,克服了BM EPCR + LT-HSC保留,诱导TACE介导的EPCR和VLA-4脱落,调节LT-HSC上的CXCR4和PAR1,基质PAR1介导的CXCL12分泌,导致干细胞和祖细胞募集到血液中(Gur-Cohen等人,2015)。通过骨髓EPCR + LT-HSC在更高水平上表达VLA-4,同时对其配体具有更高的亲和力,诱导其BM保留和对DNA损伤剂的保护。通过EPCR / PAR1信号传导的NO限制增加了VLA-4调节锚定和LT-HSC和抗化学疗法的骨髓保留的亲和力(Gur-Cohen等人,2015)。
&nbsp;已经开发了多个小分子来试图调节整联蛋白依赖性粘附。人VLA-4的亲和力状态可以被对其分子构象敏感的单克隆抗体识别(Masumoto和Hemler,1993)。重要的是,VLA-4亲和力的变化可以通过FACS使用模拟含有LDV的荧光小分子(LDV-FITC)的配体在实时和生理相关的时间框架下检测(Chigaev等人, ,2001)。 VLA-4以高亲和力识别纤连蛋白内的肽序列,其包含25个氨基酸,称为CS-1(Hynes,1992)。 LDV(leu-asp-val)是鉴定为纤连蛋白CS-1区段的特异性VLA-4识别的最小序列的三肽。在这里,我们描述了使用LDV-FITC通过流式细胞术检测VLA-4亲和力监测平均荧光强度的方法。

关键字

材料和试剂

  1. 针注射器1 ml 25 G x 5/8 in。(0.5 x 16 mm)(BD,目录号:300014)
  2. 菜肴35 x 10(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:153066)
  3. 70μm尼龙过滤器(Sinun Tech,目录号:Polymer Screens)
  4. FACS管(Corning,Falcon TM ,目录号:352054)
  5. 感兴趣的细胞(这里是从8周龄小鼠获得的鼠骨髓细胞)
  6. Dulbecco的磷酸盐缓冲盐水(PBS +/+ )(Biological Industries,目录号:02-020-1A)
  7. EDTA(5mM终浓度在水中,pH7.4)(Avantor,Performance Materials,J.T.Baker,目录号:8993-01)
  8. 湿冰
  9. 通过流式细胞术检测LT-HSC的抗体:
    1. Sca-1 PEcy7(克隆D7)(Biolegend,目录号:108114)
    2. c-kit APC(克隆2B8)(Biolegend,目录号:105812)
    3. CD150 Brilliant Violet(克隆TC15-12F12.2)(Biolegend,目录号:115922)
    4. CD48 Pasific Blue(克隆HM48-1)(Biolegend,目录号:103418)
    5. EPCR PE(克隆eBio1560)(Affymetrix,eBioscience,目录号:12-2012-82)
    6. 谱系:CD4(克隆GK1.5),CD8a(克隆53-6.7),GR1(克隆RB6-8C5),B220(克隆RA3-682),Ter119(克隆TER-119),CD11b(克隆M1/70) />
  10. 氯化钙二水合物(EMD Millipore,目录号:102382)
  11. 六水合氯化镁(EMD Millipore,目录号:105833)
  12. HEPES缓冲溶液(Biological Industries,目录号:03-025-1B)
  13. Hank的平衡盐溶液(HBSS)(Thermo Fisher Scientific,Gibco TM,目录号:14175095)
  14. 牛血清白蛋白溶液(10%,BSA)(Biological Industries,目录号:03-010-1B)
  15. 2.4 - (((N,2-甲基苯基)脲基) - 苯基乙酰基-L-亮氨酰-L-天冬氨酰基-L-缬氨酰-L-脯氨酰基-L-丙氨酰-L-丙氨酰-L-赖氨酸(LDV-FITC)(R& D系统,目录号:4577)
  16. 去离子水(DDW)
  17. 龙胆紫(Sigma-Aldrich,目录号:548-62-9)
  18. 乙酸(Sigma-Aldrich,目录号:64-19-7)
  19. 多聚甲醛(PFA)(Sigma-Aldrich,目录号:30525-89-4)
  20. LDV培养基(参见食谱)
  21. LDV-FITC库存和工作解决方案(见配方)
  22. 土耳其语的解决方案(见食谱)
  23. 4%PFA(见食谱)

注意:荧光灯应根据流式细胞仪选择。

设备

  1. 镊子(Kent Scientific,目录号:INS700100-2)
  2. 剪刀(Kent Scientific,目录号:INS750046)
  3. 离心机(Eppendorf,型号:5810R)
  4. 离心机摇摆转子A-462 4×250毫升矩形桶(Eppendorf,目录号:5810709008)
  5. 适配器(Eppendorf,目录号:5810752000)
  6. 倒置光学显微镜(Olympus,型号:CHK2-F-GS)
    注意:本产品已经被制造商停产。
  7. 血细胞计数器(Sigma-Aldrich,Bright-Line TM,目录号:Z359629)
  8. 培养箱37℃,5%CO 2(Thermo Fisher Scientific,Thermo Scientific,supers TM,型号:150i)
  9. 冷藏室或冰箱(4°C)
  10. 流式细胞仪(即,ems,即,Macs Quant仪器[Miltenyi,BergischGladbach,德国]或BD LSR II流式细胞仪)
  11. 2升玻璃瓶或瓶(Kimble Chase Life Science and Research Products,目录号:26500-2000)
  12. 塑料称重船(Sigma-Aldrich,目录号:Z186856)

注意:可以调整颜色组合以匹配可用的激光组合。

软件

  1. FlowJo V10软件(Tree Star,可选)

程序

  1. 获取小鼠骨髓细胞
    注意:所有动物实验必须经当地动物护理和道德管理当局批准。
    1. 通过CO 2安全化或子宫颈脱位来牺牲所需的小鼠株。
    2. 使用镊子和锋利的剪刀提取股骨,胫骨和髂骨骨骼。将骨头放在冰上加上PBS +/+ 的小盘子上。
    3. 使用1ml注射器和25G针将1-5ml冰冷PBS(每只小鼠)从骨腔中冲洗总骨髓细胞。通过使用相同的注射器简单地重悬细胞溶液来获得单细胞悬液。


      图1.从小鼠骨骼中冲洗骨髓的过程。 A.冲洗前的股骨,胫骨和髂骨骨骼; B.潮红后的股骨,胫骨和髂骨骨头; C. 1 ml注射器,其25 G针插入胫骨骨腔
    4. 通过使细胞溶液通过70μm尼龙过滤器过滤细胞,以获得均匀的单细胞悬浮液
    5. 离心细胞(289.5×g,5分钟),并重悬于1ml LDV培养基中。
    6. 用Turk的溶液稀释细胞1:10计数细胞。使用血细胞计数器在倒置光学显微镜下计数细胞。一只老鼠通常会从股骨和胫骨和髂骨骨骼获得60-90万个细胞。

  2. 使用流式细胞术(LDV测定)测定VLA-4亲和力 可以从获自治疗动物的新鲜骨髓细胞上检查VLA-4亲和力。或者,可以从未处理的动物获得细胞,并在LDV培养基的存在下用所需的试剂在体外处理细胞,随后用上述详细方案。
    注意:建议在分离骨髓细胞后继续染色。
    1. 将500万个骨髓细胞在FACS管中放入总体积为100μl的LDV培养基(参见食谱)。
    2. 加入LDV-FITC以达到10nM的最终浓度(参见食谱),并通过移液轻轻混合,不要旋涡。
    3. 为了确定LDV-FITC非特异性结合,在每个LDV-FITC样品中加入EDTA至终浓度为5 mM。
    4. 在37℃下孵育所有样品30分钟。
    5. 孵育后,将管子立即放在湿冰上,加入冰冷的4%PFA 10分钟(即反应在100μl细胞中)固定细胞,从而加入4%PFA [ 1-2 ml]直接加入管中)。从那时起,细胞需要保持在寒冷的条件下在湿冰上。沿程序避免涡旋。
    6. 用2ml冰冷PBS +/+ 洗涤细胞。
    7. 离心细胞(289.5×g,5分钟),轻轻丢弃上清液。
    8. 加入100μl冰冷的PBS +/+ ,并进行细胞表面标志物的染色谱系(每个抗原每个样品1-2μl),Sca-1(2μl/样品),c-试剂盒(2μl/样品),CD150(2μl/样品),CD48(1μl/样品)和EPCR(2μl/样品)。这些细胞表面标志物只是用于检测LT-HSC的提示抗原,但是这可以根据研究问题的需要进行调整。颜色组合应适合FITC,因为LDV肽与FITC荧光染料缀合。
    9. 将所需的抗体添加到细胞样品中,通过移液(不要旋涡)轻轻混合,并在冰上孵育30分钟并防止光照。
    10. 用1ml冰冷的PBS,将离心机(289.5×g,5分钟)洗涤细胞,轻轻丢弃上清液。
    11. 将细胞重悬在300μlPBS/sup + + +/+,不要旋涡。
    12. 通过流式细胞术立即读取样品,并通过分析门控谱系上的FITC荧光染料的强度来确定LDV与LT-HSC的结合。/c-Kit +/sup>/Sca-1 + /CD150 + /CD48 - /EPCR +

数据分析



图2.通过LDV探针结合骨髓EPCR测量的VLA4亲和力 + SK(Sca1 < strong> + ckit + )SLAM(CD150 + CD48 - 细胞用EDTA;在蓝色的EPCR +电池门控SLAM/SK;在SLAM/SK上的EPCR细胞粉红色。

有关门控策略和统计分析的进一步分析信息,您可以在以下链接中查阅文章Gur-Cohen等:。 http://www.nature.com/nm/journal/v21/n11/full/nm.3960.html

食谱

  1. LDV介质
    1mM CaCl 2
    1mM MgCl 2
    含有1%BSA的20mM HEPES 注意:建议制备1 M CaCl 2 2(直接在培养基中稀释1:1,000)以及100 mM MgCl 2(稀释)的储备溶液1:100直接在媒体中)。
  2. LDV-FITC库存和工作解决方案
    根据制造说明书(给出0.73mM的库存浓度),将1mg LDV-FITC粉末溶于1ml DDW(ddH 2 O)中。
    注意:建议将溶液分成小等分试样,避免反复冻融循环(溶液可在-20°C储存长达1年)。
    为了制备工作的LDV-FITC溶液,用DDW稀释储备溶液,达到10nM的终浓度(对于10nM工作溶液,用DDW稀释1:730,并将1μl加入到补充有LDV培养基的100μl细胞中) />
  3. 土耳其语解决方案
    50毫升龙胆紫色
    5 ml乙酸
    495 ml DDW
    将龙胆紫溶解于乙酸和DDW中
  4. 4%PFA
    将40克粉状多聚甲醛重量放入塑料称重船中 倒入2升玻璃瓶或瓶子
    加入1L DDW,搅拌棒,温热至65°C,缓慢搅拌5分钟 加入一滴10N KOH或10N NaOH碱,溶液应该变得清晰
    让溶液在室温下冷却,并将pH调节至7.3
    注意:较小的体积可以在-20°C保存长达一年。

致谢

这项研究得到了以色列科学基金会(851/13),欧内斯特和邦妮·贝特勒基因组医学优秀研究计划和FP7-HEALTH-2010(CELL-PID 261387)(T.L.)和德国DKFZ的支持。

参考文献

  1. Aronovich,A.,Nur,Y.,Shezen,E.,Rosen,C.,ZlotnikovKlionsky,Y.,Milman,I.,Yarimi,L.,Hagin,D.,Rechavi,G.,Martinowitz, Nagasawa,T.,Frenette,PS,Tchorsh-Yutsis,D.和Reisner,Y。(2013)。因子VIII和凝血酶/PAR1在调节造血及其与骨骼结构相互作用方面的新作用 122(15): 2562-2571。
  2. Balazs,AB,Fabian,AJ,Esmon,CT和Mulligan,RC(2006)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/16304059 "target ="_ blank">内皮蛋白C受体(CD201)明确识别小鼠骨髓中的造血干细胞。血液 107(6):2317-2321。
  3. Chigaev,A.,Blenc,AM,Braaten,JV,Kumaraswamy,N.,Kepley,CL,Andrews,RP,Oliver,MJ,Edwards,SB,Prossnitz,SR,Larson,SR和Sklar,LA(2001) ; α 276(52) :48670-48678。
  4. Gur-Cohen,S.,Itkin,T.,Chakrabarty,S.,Graf,C.,Kollet,O.,Ludin,A.,Golan,K.,Kalinkovich,A.,Ledergor,G.,Wong,E 。,Niemeyer,E.,Porat,Z.,Erez,A.,Sagi,I.,Esmon,CT,Ruf,W.and Lapidot,T。(2015)。< a class ="ke-insertfile" href ="http://www.ncbi.nlm.nih.gov/pubmed/26457757"target ="_ blank"> PAR1信号调节EPCR表达骨髓造血干细胞的保留和募集。 > Nat Med 21(11):1307-1317。
  5. Gur-Cohen,S.,Kollet,O.,Graf,C.,Esmon,CT,Ruf,W.和Lapidot,T。(2016)。通过EPCR/PAR1信号传导调控长期重建造血干细胞。 Ann Ann AcadSci 1370(1):65-81。
  6. Hemler,ME(1990)。< a class ="ke-insertfile"href ="http://www.annualreviews.org/doi/abs/10.1146/annurev.iy.08.040190.002053?journalCode=immunol"target ="_ blank">整合素家族中的VLA蛋白:结构,功能及其在白细胞上的作用。 Ann Rev Immunol 8(1):365-400。
  7. Hynes,RO(1992)。整合:多功能性,调制,以及细胞粘附中的信号。细胞 69(1):11-25。
  8. Lapidot,T.和Petit,I.(2002)。目前对干细胞动员的了解:趋化因子,蛋白水解酶,粘附分子,细胞因子和基质细胞的作用。 30(9):973-981。 >
  9. Luo,BH,Carman,CV and Springer,TA(2007)。  VLA-4的多种激活状态。与CS1 /纤连蛋白和血管细胞粘附分子-1的粘附之间的机械差异.J Biol Chem 268(1):228-234。
  10. Papayannopoulou,T.,Craddock,C.,Nakamoto,B.,Priestley,GV and Wolf,NS(1995)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm。 niH.gov/pubmed/7568190"target ="_ blank"> VLA4/VCAM-1粘附途径定义了移植的鼠造血祖细胞在骨髓和脾脏之间递送的对比机制。 Proc Natl Acad Sci USA 92(21):9647-9651。
  11. Papayannopoulou,T.,Priestley,GV和Nakamoto,B。(1998)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/9516120"target ="_ blank">抗VLA4/VCAM-1诱导的动员需要通过试剂盒/mkit配体途径进行协同信号传递。血液 91(7):2231-2239。 >
  12. Peled,A.,Kollet,O.,Ponomaryov,T.,Petit,I.,Franitza,S.,Grabovsky,V.,Slav,MM,Nagler,A.,Lider,O.,Alon,R.,Zipori ,D.和Lapidot,T。(2000)。趋化因子SDF-1在未成熟的人CD34细胞上激活整合素LFA-1,VLA-4和VLA-5:在经内皮/基质迁移和NOD/SCID小鼠植入中的作用。一个> 血液 95(11):3289-3296。
  13. Rettig,MP,Ansstas,G.and Di Persio,JF(2012)。  使用CXCR4和VLA-4抑制剂动员造血干细胞和祖细胞。白血病26(1):34-53。 />
  14. Takagi,J.和Springer,TA(2002)。整合素激活和结构重排。 Immunol Rev 186:141-163。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Avemaria, F., Gur-Cohen, S., Avci, S. and Lapidot, T. (2017). VLA-4 Affinity Assay for Murine Bone Marrow-derived Hematopoietic Stem Cells. Bio-protocol 7(4): e2134. DOI: 10.21769/BioProtoc.2134.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要谷歌账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。