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Nov 2018

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Bioorthogonal Labeling and Chemoselective Functionalization of Lung Extracellular Matrix
肺细胞外基质的生物正交标记和化学选择性功能化   

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Abstract

Decellularized extracellular matrix (ECM) biomaterials derived from native tissues and organs are widely used for tissue engineering and wound repair. To boost their regenerative potential, ECM biomaterials can be functionalized via the immobilization of bioactive molecules. To enable ECM functionalization in a chemoselective manner, we have recently reported an effective approach for labeling native organ ECM with the click chemistry-reactive azide ligand via physiologic post-translational glycosylation. Here, using the rat lung as a model, we provide a detailed protocol for in vivo and ex vivo metabolic azide labeling of the native organ ECM using N-Azidoacetylgalactosamine-tetraacylated (Ac4GalNAz), together with procedures for decellularization and labeling characterization. Our approach enables specific and robust ECM labeling within three days in vivo or within one day during ex vivo organ culture. The resulting ECM labeling remains stable following decellularization. With our approach, ECM biomaterials can be functionalized with desired alkyne-modified biomolecules, such as growth factors and glycosaminoglycans, for tissue engineering and regenerative applications.

Keywords: Extracellular matrix (细胞外基质), Decellularization (去细胞化), Bioorthogonal (生物正交), Chemoselective functionalization (化学选择性官能化), Click chemistry (点击化学), Lung (肺)

Background

The extracellular matrix (ECM) is a hydrated network scaffold composed of non-cellular components from a given tissue or organ, and plays key roles in supporting the activities of residential cells through its contained bioactive elements, such as fibrous proteins, growth factors and glycosaminoglycans (GAGs) (Theocharis et al., 2016). Decellularized ECM materials have been widely used for tissue engineering due to their inherent biocompatibility, highly preserved tissue architecture and biomechanical properties (Ott et al., 2008 and 2010; Petersen et al., 2010; Wagner et al., 2014). Despite the importance, one critical barrier for applying decellularized ECM to regenerative applications is that the harsh decellularization conditions usually cause dramatic loss or denaturation of key bioactive components embedded within the ECM (Reing et al., 2010; Uhl et al., 2020). To overcome this challenge, emerging efforts are underway to functionalize ECM materials via the immobilization of desired bioactive molecules, such as growth factors and GAGs, to facilitate tissue repair and regeneration.


For ECM biomaterial functionalization, amine-reactive chemistry has been widely used, due to the wide availability of amines in almost all ECM proteins. However, such high abundance of amine ligands within the ECM leads to limited control over reaction specificity and poses potential risk of compromising the desired biochemical and biomechanical properties of ECM biomaterials (Wissink et al., 2001; Chiu and Radisic, 2010; Grover et al., 2012; Davidenko et al., 2015). Chemoselective engineering of ECM materials, via the specific conjugation between two ligands that do not exist in native biological systems, offers a promising solution to this challenge. The azide-to-alkyne click conjugation is one such chemistry that has been widely used for protein engineering and has demonstrated excellent biocompatibility for in vivo and in vitro applications (Laughlin and Bertozzi, 2007; Chang et al., 2010; Sletten and Bertozzi, 2011).


Here, we describe a metabolic labeling approach to covalently incorporate the azide ligand into organ ECM using an azido monosaccharide, azidoacetylgalactosamine-tetraacylated (Ac4GalNAz). Ac4GalNAz can be incorporated as post-translational glycan modification during new ECM protein synthesis both in vivo and during ex vivo organ culture. The resulting click-reactive azide ligands within the ECM remain stable following decellularization and enable further functionalization with desired biomolecules bearing the complementary alkyne ligand using the copper-catalyzed, azide-alkyne cycloaddition (Ren et al., 2018). The protocol described here, using the rat lung as a model, is expected to be applicable to label the ECM of a wide variety of organs, such as the heart, liver, kidney and blood vessel. We also expect our protocol to be applicable to engineer ECM biomaterials derived from other animal sources, such as the mouse and porcine models (Ren et al., 2018).


Materials and Reagents

  1. 27G × 1/2 tuberculin syringe (BD, catalog number: 305620)

  2. Reusable Stainless Steel Dispensing Needle with Luer Lock Connection, Blunt, 18 Gauge (McMaster Carr, catalog number: 6710A44)

  3. Dremel rotary tool kit (Dremel, catalog number: 8220)

  4. Luer-Lok Syringe 1 ml (BD, catalog number: 309628)

  5. Luer-Lok Syringe 10 ml (BD, catalog number: 302995)

  6. Luer-Lok Syringe 30 ml (BD, catalog number: 302832)

  7. Petri Dish, PS, 145/20 mm (Greiner Bio-One, catalog number: 639102)

  8. 2-0 PERMA-HAND Silk Suture (Ethicon, catalog number: LA55G)

  9. Sterile Gauze Sponges (Covidien, catalog number: 2187)

  10. PVDF Transfer Membrane (Thermo Fisher Scientific, catalog number: 88518), storage: RT

  11. Sprague Dawley rats (100-250 gram) (Charles River Laboratories, Strain Code: 400)

  12. N-azidoacetylgalactosamine-tetraacylated (Ac4GalNAz) (Click Chemistry Tools, catalog number: 1086), storage: -20 °C

  13. Dimethyl Sulfoxide (DMSO) (Fisher Scientific, catalog number: BP231-100), storage: room temperature (RT)

  14. Dulbecco′s Phosphate Buffered Saline (DPBS), 1× without calcium and magnesium (Corning, catalog number: 21-031-CV), storage: RT

  15. Dulbecco′s Modification of Eagle′s Medium (DMEM) (Corning, catalog number: 10-013-CV), storage: 2-8 °C

  16. FetalClone I Serum (Cytiva, catalog number: SH30080.03), storage: -20 °C

  17. Antibiotic-Antimycotic (100×) (Thermo Fisher Scientific, catalog number: 15240062), storage: -5 °C to -20 °C

  18. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: 74255), storage: 2-8 °C

  19. Triton X-100 (Sigma-Aldrich, catalog number: T8787), storage: RT

  20. Phosphate Buffered Saline (PBS), 20× (Boston BioProducts, catalog number: BM-220), storage: RT

  21. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148), storage: 2-8 °C

  22. Click-iT Cell Reaction Buffer Kit (Invitrogen, catalog number: C10269), storage: 2-6 °C

  23. Biotin-PEG4-Alkyne (Click Chemistry Tools, catalog number: TA105), storage: -20 °C

  24. Bovine Serum Albumin (BSA) (Fisher Scientific, catalog number: BP9706100), storage: 2-8 °C

  25. Rabbit Polyclonal Laminin Antibody (Novus, catalog number: NB300-144), storage: -20 °C

  26. Streptavidin, Alexa Fluor 647 conjugate (Thermo Fisher Scientific, catalog number: S21374), storage: -5 °C to -30 °C and protect from light

  27. Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor Plus 488 (Thermo Fisher Scientific, catalog number: A32790), storage: 4 °C and protect from light

  28. DAPI Fluoromount-G (SouthernBiotech, catalog number: 0100-20), storage: RT

  29. Urea, ultrapure, 99% (Alfa Aesar, catalog number: J65769), storage: RT

  30. HEPES (1 M) (Thermo Fisher Scientific, catalog number: 15630-080), storage: 2-8 °C

  31. Protease Inhibitor Cocktail (100×) (Thermo Fisher Scientific, catalog number: 87786), storage: 4 °C

  32. BCA Protein Assay Kit (Thermo Fisher Scientific, catalog number: 23225), storage: RT

  33. 4-15% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad Laboratories, catalog number: 4561085), storage: 4 °C

  34. Tris Buffered Saline-Tween (TBST, 10×, with 0.5% Tween-20, pH 7.4) (Boston BioProducts, catalog number: IBB-181), storage: RT

  35. High Sensitivity Streptavidin-HRP (Thermo Fisher Scientific, catalog number: 21130), storage: 4 °C

  36. SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, catalog number: 34580), storage: RT

  37. SYPRO Ruby Protein Gel Stain (Thermo Fisher Scientific, catalog number: S12000), storage: RT

  38. Ac4GalNAz Injection Solution (see Recipes)

  39. Control Injection Solution (see Recipes)

  40. Ex vivo Culture Medium (see Recipes)

  41. Decell Solution (see Recipes)

  42. 2× Decell Solution (see Recipes)

  43. 1× PBS (pH 7.4) (see Recipes)

  44. Fixation Solution (see Recipes)

  45. Biotin-PEG4-Alkyne Click Reaction Mixture (see Recipes)

  46. Urea Extraction Buffer (see Recipes)

  47. 1× TBST (pH 7.4) (see Recipes)

  48. Blocking Buffer (see Recipes)

  49. HRP-Imaging Mixture (see Recipes)

Equipment

  1. Iris Scissors, 11.5 cm, straight (World Precision Instruments, catalog number: 503708-12)

  2. Pulmonary artery (PA) cannula (Figure 1)



    Figure 1. Custom-made PA cannula. A. Diagram. The cannula is composed of a top luer connector (1/8’, for connection with the bioreactor), a middle transparent tubing segment (1/8’, for detecting potential air bubbles), and a bottom curved metal needle (18 gauge, for connection with the PA). A small side hole at the end of the metal needle is generated using Dremel rotary tool with a sanding disc, to avoid potential perfusion blockage upon insertion into the PA. A series of grooves are created using a wire cutter around the tip of the metal needle to help stabilize the suture. B. An actual image of the cannula.


  3. Adson Forceps, straight, serrated (World Precision Instruments, catalog number: 503719-12)

  4. SILASTIC Thin-wall Silicone Laboratory Tubing (Dow Corning, catalog number: 508-009)

  5. Easy-Load Pump Head with 2-Channels for Precision Pump Tubing (Cole Parmer, catalog number: EW-77202-60)

  6. Masterflex L/S Digital Precision Modular Drive with Remote I/O and Benchtop Controller (Cole Parmer, catalog number: UX-07557-00)

  7. Autoclavable three-way valve (Cole Parmer, catalog number: EW-31200-80)

  8. Organ culture bioreactor (Figure 2)



    Figure 2. Organ culture bioreactor. A. Driven by a peristaltic pump, the culture medium is aspirated from the organ chamber, and perfuses through a series of thin-wall silicone tubing (for oxygenation) into the cannula leading to the PA of the lungs in culture. The filter on the organ chamber is for pressure equilibration. B. An actual image of the perfusion bioreactor.


  9. Decellularization chambers (Figure 3)



    Figure 3. Decellularization chambers. A. There are two chambers in the system: the reservoir chamber containing fresh solutions for decellularization; and the collection chamber containing the lungs to be decellularized. The reservoir chamber should be placed approximately 50 cm higher than the collection chamber, which generates the gravity pressure to drive the fluid flow from the reservoir chamber into the lungs through the PA cannula. The filters on both chambers are for pressure equilibration. B. An actual image of the decellularization chambers.


  10. Semi-Automated Rotary Microtome (Leica Biosystems, catalog number: RM2245)

  11. EVOS FL Auto 2 Imaging System (Thermo Fisher Scientific, catalog number: AMAFD2000)

  12. Microcentrifuge (Eppendorf, catalog number: 5415R)

  13. BeadBug Microtube Homogenizer (Benchmark Scientific, catalog number: D1030)

  14. Prefilled 2.0 ml Tubes, with Acid Washed Silica (Glass) Beads, 1.0 mm (Benchmark Scientific, catalog number: D1031-10)

  15. Mini-PROTEAN Tetra Vertical Electrophoresis Cell for Mini Precast Gels, with Mini Trans-Blot Module (Bio-Rad Laboratories, catalog number: 1658036)

  16. ChemiDoc Gel and Western blot imaging system (Bio-Rad Laboratories, catalog number: 12003153)

  17. Reach-in IR CO2 Incubator (Caron, catalog number: 7400-25-1)

Procedure

  1. In vivo metabolic labeling of the native organ ECM and lung harvest

    Note: All animal procedures should be performed according to protocols approved by the Institutional Animal Care and Use Committee. Consultation and/or training is advised with a veterinarian or a researcher experienced with rat intraperitoneal injection, euthanasia, dissection, and blood vessel cannulation.

    Day 1:

    1. Weigh the rat and calculate the injection volume accordingly (0.3 mg of Ac4GalNAz per gram body weight). Use the Ac4GalNAz Injection Solution prepared at 70 mg/ml, the injection volume is 4.29 μl per gram body weight. For control rat, 4.29 μl of Control Injection Solution is administered per gram body weight.

    2. Administer the proper volume of Ac4GalNAz or Control Injection Solution by intraperitoneal injection using a 1-ml syringe with a 27-gauge needle.

    3. House each injected rat individually with the cage marked with the type of injection solution and the date(s) of injection(s).


  2. Day 2, Day 3:
    1. Repeat Steps A1 to A3 for two additional days.


    Day 4:
    1. Sacrifice the rat via CO2 inhalation. Open up the thoracic cavity using sterilized surgical tools to expose the heart and lungs without damaging them.

      Note: This procedure can be performed with beating or non-beating heart.

    2. Make a small incision (2-3 mm) at the right ventricle immediately next to the pulmonary artery (PA) as the infusion inlet and cut open the left atrial appendage as the drainage outlet (Figure 4).



      Figure 4. Surgical procedures during lung harvest. The PA (blue) is connected to the right ventricle. A small incision is made at the right ventricle immediately next to the PA to allow insertion of a blunt-end needle (for flushing) or cannula into the PA. Prior to the initial flushing, the left atrial appendage should be cut open as the drainage outlet.


    3. Prepare a syringe with 10 ml DPBS attached to an 18-gauge blunt-end needle. Insert the blunt-end needle gently into the PA via the small incision at the right ventricle, and use forceps to gently secure the needle in the PA. Infuse 10 ml DPBS over approximately 10 s to flush the remaining blood out of the pulmonary vasculature. Successful flushing is indicated by initial blood-colored fluid coming out of left atrial appendage, which gradually loses the blood color over the infusion course, and by the entire lungs turning white.

      Note: The DPBS flushing step needs to be performed as soon as possible following euthanization of the animal to avoid blood clot formation in the pulmonary vasculature.

    4. Isolate the heart-lung bloc free from surrounding tissues and place it in a Petri dish on top of a piece of gauze pre-wet with ice-cold DPBS.

    5. To cannulate the PA, attach a custom-made cannula (Figure 1) to a 10-ml syringe pre-filled with DPBS, and insert the cannula into the PA via the small incision at the right ventricle (Figure 4) while having the DPBS slowly dripping out of the cannula by gentle syringe injection. This is to avoid accidental introduction of air bubbles into the pulmonary vasculature, which may cause air embolism and non-homogeneous perfusion. The insertion depth should be ~5 mm into the PA to ensure stable suturing. Following cannula insertion into the PA, secure the cannula at the grooves using the 2-0 silk suture. To test the cannulation, infuse DPBS through the PA cannula by gently pushing the syringe. Successful cannulation is indicated by low perfusion resistance and the expansion of the PA upon perfusion.


  3. Ex vivo labeling of the lung ECM

    1. Add 100 ml of pre-warmed Ex Vivo Culture Medium to the organ culture bioreactor (Figure 2), and pre-fill the entire perfusion tubing with the medium using a 30-ml syringe through the three-way valve to remove air bubbles. For metabolic ECM labeling, the Ex Vivo Culture Medium is supplemented with Ac4GalNAz (50 μM in 0.1% DMSO). For the control group, the medium is supplemented with 0.1% DMSO.

    2. Harvest the heart-lung bloc from a rat without prior metabolic labeling, and cannulate the PA following Steps A5 to A9.

      Note: Ex vivo lung culture should start within 1 h following organ harvest to ensure optimal metabolic activity of the organ to be labeled.

    3. In a laminar hood, lift the lid of the organ culture bioreactor to allow access to the interior luer connector (Figure 2). Connect the lung cannula to the interior luer connector while having the medium slowly dripping out of the luer connector (by gentle medium injection using a syringe through the three-way valve) (Figure 2). This is to avoid introduction of air bubbles into the perfusion line. The use of one-time sterile gloves is highly recommended for this step to ensure sterility.

      Note: This procedure requires the cooperation of two persons. One should gently push the medium-filled syringe while the other connect the lung cannula to the luer connector.

    4. Attach the pump tubing of the organ culture bioreactor to a peristaltic pump and verify the perfusion direction (Figure 2). Culture the lungs in a humidified incubator supplemented with 5% CO2 for 24 h under constant-rate perfusion (5 ml/min for lungs harvested from a 100-gram rat and 10 ml/min for lungs harvested from a 200-gram rat).


  4. Whole-lung Decellularization

    1. Pre-fill the reservoir chamber with 1 L of Decell Solution and place it approximately 50 cm above the collection chamber (Figure 3). Pre-fill the tubing connecting between both chambers with the Decell Solution.

    2. Attach the lungs (following in vivo or ex vivo metabolic labeling) to the luer connector inside the collection chamber while having the Decell Solution slowly dripping out of the luer connector. This is to avoid the introduction of air bubbles to the perfusion line.

    3. Have the Decell Solution perfuse through the lungs driven by gravity pressure (determined by the height difference between the reservoir and collection chambers).

      Note: During decellularization, the lungs should gradually expand in size and turn semi-transparent within the initial 5-10 min. Otherwise, it may suggest that the cannula or other parts of the perfusion line is blocked. The entire 1 L of Decell Solution should perfuse through the lungs over 1-2 h. If it takes much less time (such as less than 30 min), it may suggest damage in the lung or leakage in the tubing connection. If it takes much longer time (more than 2 h), it may suggest blockage in the perfusion line or lung vasculature by tissue debris or air bubbles, or it may be due to improper positioning of the cannula in the PA.

    4. Pause the perfusion by turning one of the three-way valves in the perfusion line right before the fluid reservoir runs out. Discard the waste solution in the collection chamber and replace the reservoir chamber with 200 ml of H2O, and perfuse the lungs for 15 min.

    5. Pause the perfusion, replace the reservoir chamber with 200 ml of 1% Triton X-100 in H2O, and perfuse the lungs for an additional 15 min.

    6. Replace the reservoir chamber with 1 L of 1× PBS, and have it completely perfused through the lungs to wash away the detergents.

    7. Repeat Step C6 for two additional times.


  5. Histological characterization of metabolic ECM labeling

    1. To fix the decellularized lungs, infuse 5 ml of the Fixation Solution into the trachea using a blunt-end needle connected to a 10-ml syringe, and ligate the trachea with the 2-0 silk suture. This is to open the alveolar structures for optimal histological assessment. Soak the lungs in a conical tube with an additional 15 ml of Fixation Solution and incubate at 4 °C overnight with gentle agitation.

    2. Process the fixed lung tissues for paraffin embedding, and section the embedded tissue block at the thickness of 5 μm using a microtome.

    3. Following standard deparaffinization and rehydration steps, wash the section with 1× PBS for 3 times, 5 min each.

    4. To detect potential azide labeling in the decellularized lung ECM, perform biotin conjugation by adding the Biotin-PEG4-Alkyne Click Reaction Mixture to the histological section (100-200 μl per section), and incubate for 1 h at RT in a humidified staining chamber.

    5. Wash the section with 1× PBS for 3 times, 5 min each.

    6. Block the section with 1% BSA in 1× PBS for 20 min.

    7. Incubate the section with primary Rabbit anti-Laminin antibody (1:500 dilution in 1% BSA in 1× PBS) at 4 °C overnight in a humidified staining chamber.

    8. Wash the section with 1× PBS for 3 times, 5 min each.

    9. Incubate the section with Streptavidin (Alexa Fluor 647 conjugate, 1:500 dilution in 1% BSA in 1× PBS) and Donkey anti-Rabbit-488 (1:500 dilution) for 45 min at RT.

    10. Wash with 1× PBS for 3 times, 5 min each.

    11. Mount the slide using Fluoromount and let it air dry.

    12. Perform fluorescence imaging using the EVOS FL Auto 2 Imaging System (Figure 5).

      Note: Standard immunofluorescence staining can be performed together with the detection of azide labeling by the click reaction. In such dual staining, it is recommended to perform the Biotin-PEG4-Alkyne click conjugation first, followed by standard antibody staining. As an example, we performed a co-staining of Laminin.


  6. Biochemical characterization of metabolic ECM labeling

    1. Cut a piece of the un-decellularized lung tissue (from in vivo or ex vivo metabolic labeling) and mince it using fine scissors.

      Note: For biochemical detection of azide labeling in the ECM, 50-100 mg of lung tissue is usually sufficient.

    2. Transfer the minced lung tissue into a 2.0-ml tube prefilled with glass beads, add 300 μl H2O, and homogenize on a microtube homogenizer at full speed (4,000 rpm) for 3 min. Repeat this homogenization step if large tissue pieces remain visible.

    3. Add 300 μl of 2× Decell Solution to the tube, and agitate at RT for 30 min. This step will solubilize all cellular components in the homogenized lung tissue.

    4. Centrifuge the tube at 16,100 × g for 20 min at RT. Discard supernatant.

    5. Add 1 ml DPBS to the tube, mix thoroughly by inversion, centrifuge at 16,100 × g for 10 min at RT. Discard supernatant. This is to wash away any remaining cellular components.

    6. Repeat Step E5 for two additional times.

    7. Add 500 μl of the Biotin-PEG4-Alkyne Click Reaction Mixture to the tube, mix thoroughly, and incubate for 1 h at RT with agitation. This is to conjugate biotin to the potential azide ligand in the decellularized lung ECM.

    8. Centrifuge the tube at 16,100 × g for 10 min at RT. Discard supernatant.

    9. Add 1 ml DPBS to the tube and mix thoroughly. This is to wash away any remaining Biotin-PEG4-Alkyne and other click reaction reagents.

    10. Centrifuge the tube at 16,100 × g for 10 min at RT. Discard supernatant.

    11. Repeat E9 and E10 for two additional times.

    12. Add 500 μl of Urea Extraction Buffer to the tube, and gently agitate for 2 h at RT.

    13. Centrifuge the tube at 16,100 × g for 10 min at RT.

    14. Collect the supernatant as the ECM protein sample.

    15. Quantify the ECM protein concentration using BCA assay following manufacturer’s protocol with BSA as the protein standard.

    16. Perform SDS-PAGE electrophoresis with equal protein loading from each sample, such as ECM protein samples extracted from lungs with and without metabolic azide labeling, and transfer proteins from the gel onto a piece of PVDF membrane blot. Block the blot for 20 min using the Blocking Buffer, incubate the blot in 10 ml of Streptavidin-HRP (1:10,000 dilution in Blocking Buffer) with agitation for 1 h at RT, and wash the blot thoroughly with TBST for 3 times, 10 min each.

    17. Incubate the blot in the HRP-imaging Mixture for 3 min, and image using ChemiDoc Gel and Western Blot Imaging System (Figure 6A).

    18. To validate the equal protein loading from all samples, perform SDS-PAGE electrophoresis, and stain total protein in the gel using Sypro Ruby Protein Gel Stain following manufacturer’s protocol. Image the Sypro Ruby-stained gel using ChemiDoc Gel and Western Blot Imaging System (Figure 6B).

Data analysis

  1. Histological characterization of metabolic azide labeling: On a histological section of the decellularized lung tissue, the potential azide ligand was conjugated to biotin using the click chemistry. Streptavidin staining of biotin and Laminin staining of the ECM was imaged using two different fluorophores. Laminin staining served as a control to verify similar ECM content being present in the lung ECM with and without metabolic azide labeling. Care should be taken to ensure consistent imaging parameters for all samples under comparison. Adjust exposure time according to the sample with the strongest fluorescence signal. Compared to the control, the lung ECM derived from the rat receiving Ac4GalNAz metabolic labeling is expected to show more robust azide labeling and biotin conjugation (Figure 5).



    Figure 5. Histological characterization of metabolic azide labeling of the lung ECM. The representative results show two ECM samples derived from rat lungs following three-day in vivo labeling with or Ac4GalNAz or DMSO. The potential azide ligand in the ECM were conjugated to biotin and then detected using fluorophore-conjugated streptavidin (Red). The lung ECM was indicated by Laminin antibody staining (green). Scale bars: 200 μm.


  2. Biochemical characterization of metabolic azide labeling. The potential azide ligand in the extracted lung ECM protein was conjugated to biotin using the click chemistry, followed by gel electrophoresis. Using standard Western blot procedures, biotin was detected using HRP-conjugated streptavidin and chemiluminescence. Compared to the control, the ECM protein sample extracted from lungs receiving Ac4GalNAz metabolic labeling is expected to show more robust azide labeling and biotin conjugation (Figure 6).



    Figure 6. Biochemical characterization of azide labeling in the lung ECM. The representative results show two ECM samples derived from rat lungs following one-day ex vivo labeling with Ac4GalNAz or DMSO. A. Detection of the azide ligand by Western blot analysis of the conjugated biotin. B. Protein loading was verified by in-gel staining of total protein with Sypro Ruby.


Recipes

  1. Ac4GalNAz Injection Solution

    70 mg/ml Ac4GalNAz in DPBS with 70% DMSO

  2. Control Injection Solution

    70% DMSO in DPBS

  3. Ex vivo Culture Medium

    DMEM supplemented with 10% FetalClone I Serum

    1% antibiotic/antimycotic

  4. Decell Solution

    0.1% SDS in autoclaved deionized water

  5. 2× Decell Solution

    0.2% SDS in autoclaved deionized water

  6. 1× PBS (pH 7.4)

    Dilute 20× PBS (pH 7.4) in deionized water, sterilize by autoclave

  7. Fixation Solution

    4% paraformaldehyde (PFA) in 1× PBS (pH 7.4)

  8. Biotin-PEG4-Alkyne Click Reaction Mixture

    Note: Components from the Click-iT Cell Reaction Buffer Kit (Table 1).

    Table1. Recipe for Biotin-PEG4-Alkyne Click Reaction Mixture


  9. Urea Extraction Buffer

    6 M Urea

    10 mM HEPES

    1× protease inhibitor

    Adjust pH to 8.0

  10. 1× TBST (pH 7.4)

    Dilute 10× TBST (pH 7.4) in deionized water

  11. Blocking Buffer

    5% BSA in TBST

  12. HRP-Imaging Mixture

    SuperSignal West Pico PLUS Luminol/Enhancer solution and SuperSignal West Pico PLUS Peroxide solution, 1:1 (freshly prepared)

Acknowledgments

This works is supported by the Department of Biomedical Engineering at Carnegie Mellon University. Z.L. and Y.X. are supported by scholarships from the China Scholarship Council. This protocol is originally adapted from Ren et al. (2018). We thank Pitt Biospecimen Core (University of Pittsburgh) for histological processing. We also thank the Vivarium at Carnegie Mellon University for animal husbandry. Finally, we thank Piyumi Wijesekara and Wai Hoe Ng for their assistance in gathering information regarding materials and reagents.

Competing interests

X.R. has a patent application (US20170362266A1) related to this work.


Ethics

All animal procedures were performed according to the protocol approved by the Institutional Animal Care and Use Committee at Carnegie Mellon University (Approval ID#TR202000006, valid 2018-2022).

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简介

[摘要]源自天然组织和器官的脱细胞细胞外基质(ECM)生物材料被广泛用于组织工程和伤口修复。为了增强其再生潜力,可以通过固定生物活性分子来使ECM生物材料功能化。为了使ECM以化学选择性的方式实现功能化,我们最近报告了一种有效的方法,可通过生理学上的翻译后糖基化,用点击化学反应的叠氮化物配体标记天然器官ECM 。在此,使用大鼠肺为模型,我们提供一种用于详细方案在体内和离体代谢叠氮化物使用N- Azidoacetylgalactosamine-tetraacylated天然器官ECM的标记(AC 4GalNAz),以及用于脱细胞和标记表征的程序。我们的方法可以在体内三天内或离体器官培养期间的一天之内进行特异性而稳定的ECM标记。脱细胞后,所得的ECM标记保持稳定。通过我们的方法,ECM生物材料可以用所需的炔烃修饰的生物分子(例如生长因子和糖胺聚糖)进行功能化,以用于组织工程和再生应用。

关键字:细胞外基质,脱细胞,生物正交,化学选择性功能化,点击化学,肺



[背景]细胞外基质(ECM)是由特定组织或器官的非细胞成分组成的水合网络支架,在通过其所包含的生物活性成分(例如纤维蛋白,生长)支持住宅细胞的活动中起关键作用。因子和糖胺聚糖(GAG)(Theocharis et al。,2016)。由于脱细胞ECM材料固有的生物相容性,高度保存的组织结构和生物力学特性,它们已被广泛用于组织工程(Ott等,2008和2010; Petersen等,2010; Wagner等,2014)。尽管很重要,但将脱细胞ECM应用于再生应用的一个关键障碍是苛刻的脱细胞条件通常会导致ECM中嵌入的关键生物活性成分急剧损失或变性(Reing等人,2010; Uhl等人,2020)。为了克服这一挑战,正在通过固定所需的生物活性分子(例如生长因子和GAG)来使ECM材料功能化的新方法,以促进组织修复和再生。

对于ECM生物材料功能化,由于几乎所有ECM蛋白中胺的广泛利用,胺反应化学已被广泛使用。然而,ECM中如此高的胺配体含量导致对反应特异性的控制有限,并存在损害ECM生物材料所需的生化和生物力学特性的潜在风险(Wissink等,2001 ;Chiu和Radisic,2010;Grover等。 。,2012;达维坚等人,2015) 。通过天然生物系统中不存在的两个配体之间的特异性结合,ECM材料的化学选择性工程化为这一挑战提供了有希望的解决方案。叠氮化物与炔烃的点击偶联是一种已广泛用于蛋白质工程的化学方法,并已证明在体内和体外应用中具有出色的生物相容性(Laughlin和Bertozzi,2007;Chang等,2010;Sletten和Bertozzi, 2011)。

在此,我们描述了使用叠氮单糖,叠氮乙酰基半乳糖胺四酰化(Ac 4 GalNAz)将叠氮化物配体共价掺入器官ECM的代谢标记方法。Ac 4 GalNAz可以在体内和离体器官培养新ECM蛋白合成过程中作为翻译后聚糖修饰而被并入。将得到的点击反应性叠氮化物配位体小号在ECM内保持稳定以下脱细胞和能够与生物分子所需的轴承互补炔配位体使用铜催化,叠氮化物-炔烃环加成进一步官能化(仁等人,2018) 。此处描述的协议以大鼠肺为模型,有望适用于标记各种器官(例如心脏,肝脏,肾脏和血管)的ECM。我们还希望我们的协议适用于工程化从其他动物来源(例如小鼠和猪模型)衍生的ECM生物材料(Ren等,2018)。

关键字:细胞外基质, 去细胞化, 生物正交, 化学选择性官能化, 点击化学, 肺

材料和试剂
27G × 1/2结核菌素syring E(BD,Ç atalog号:305620)
可重复使用的不锈钢点胶针,带鲁尔锁接头,钝头,18号。(麦克马斯特卡尔,Ç atalog号:6710A44)
Dremel旋转工具套件(Dremel,目录号:8220)
鲁尔旋锁注射器1毫升(BD,Ç atalog号:309628)
鲁尔旋锁注射器10毫升(BD,Ç atalog号:302995)
鲁尔旋锁注射器30毫升(BD,Ç atalog号:302832)
培养皿,PS,20分之145毫米(格雷纳生物ø NE,Ç atalog号:639102)
2-0 PERMA-HAND丝线缝合(Ethicon的,Ç atalog号:LA55G)
无菌纱布海绵(Covidien公司,Ç atalog号:2187)
PVDF转移膜(赛默飞世尔科技,Ç atalog号:88518),存储:RT
Sprague Dawley大鼠(100 - 250克)(Charles River实验室,应变代码:400)
的N- azidoacetylgalactosamine-tetraacylated(AC 4 GalNAz)(点击化学工具,Ç atalog编号:1086),存储:-20℃
二甲亚砜(DMSO)(Fisher Scientific公司,Ç atalog号:BP231-100),存储:室温(RT)
Dulbecco氏磷酸盐缓冲盐水(DPBS),1 ×不含钙和镁(康宁,Ç atalog号:21-031-CV ),存储:RT
Eagle氏M的Dulbecco改良的edium(DMEM)中(Corning,Ç atalog号:10-013-CV),存储:2-8℃
FetalClone我血清(Cytiva,Ç atalog号:SH30080.03),存储:-20℃
抗生素-抗真菌(100 × )(赛默飞世尔科技,Ç atalog号:15240062),存储:-5℃至-20℃
十二烷基硫酸钠(SDS)(Sigma-Aldrich公司,Ç atalog号:74255),存储:2-8℃
的Triton X-100(Sigma-Aldrich公司,Ç atalog号码:T8787),存储:RT
磷酸盐缓冲盐水(PBS),20 × (波士顿生物产品,Ç atalog号:BM-220),存储:RT
多聚甲醛(Sigma-Aldrich公司,Ç atalog号:P6148),存储:2-8℃
点击-IT细胞反应缓冲液试剂盒(Invitrogen,Ç atalog号:C10269),存储:2-6℃下
生物素PEG4-炔(点击化学工具,Ç atalog号:TA105),存储:-20℃
牛血清白蛋白(BSA)(Fisher Scientific公司,Ç atalog号:BP9706100),存储:2-8℃
兔多克隆抗体层粘连蛋白(Novus公司,Ç atalog号:NB300-144),存储:-20℃
链亲和素,Alexa氟647缀合物(赛默飞世尔科技,Ç atalog号:S21374),存储:-5℃至-30℃,并从光保护
驴抗兔IgG(H + L)二抗,Alexa氟加上488(赛默飞世尔科技,Ç atalog号:A32790),存储:4℃和避光
DAPI Fluoromount-G(SouthernBiotech,Ç atalog号:0100-20),存储:RT
尿素,超纯,99%(阿法埃莎,Ç atalog号:J65769),存储:RT
HEPES(1 M)(赛默飞世尔科技,Ç atalog号:15630-080),存储:2-8℃
蛋白酶抑制剂混合物(100 × )(赛默飞世尔科技,Ç atalog号:87786),存储:4℃
BCA蛋白测定试剂盒(赛默飞世尔科技,Ç atalog号:23225),存储:RT
4-15%的Mini-PROTEAN TGX预制蛋白凝胶(Bio-Rad实验室,Ç atalog号:4561085),存储:4℃
的Tris缓冲盐水-吐温(TBST,10 × ,用0.5%Tween-20的,pH为7.4)(波士顿生物产品,Ç atalog号:IBB-181)存储:RT
高灵敏度的链霉亲和素-HRP(赛默飞世尔科技,Ç atalog号:21130),存储:4℃
的SuperSignal西微微PLUS化学发光底物(赛默飞世尔科技,Ç atalog号:34580),存储:RT
SYPRO Ruby的蛋白凝胶染色(赛默飞世尔科技,Ç atalog号:S12000 ),存储:RT
Ac 4 GalNAz注射液(请参阅配方)
控制注射液(请参见配方)
离体培养基(请参阅食谱)
Decell解决方案(请参阅食谱)
2 × Decell解决方案(请参阅食谱)
1 × PBS(pH 7.4)(请参阅食谱)
固定解决方案(请参阅食谱)
生物素-PEG4-炔烃单击反应混合物(请参见食谱)
尿素提取缓冲液(请参见配方)
1 × TBST(pH 7.4)(请参阅食谱)
阻塞缓冲区(请参见配方)
HRP成像混合物(请参阅食谱)

设备


虹膜剪刀,11.5厘米,直(World Precision Instruments,目录号503708-12)
肺动脉(PA)套管(图1)

图1.定制的PA套管。一。图表。套管由顶部鲁尔接头(1/8',用于与生物反应器连接),中间透明管段(1/8',用于检测潜在的气泡)和底部弯曲的金属针(18号,用于与PA连接)。使用带打磨盘的Dremel旋转工具在金属针的末端产生一个小侧孔,以避免在插入PA时潜在的灌注阻塞。使用钢丝钳在金属针的尖端周围创建一系列凹槽,以帮助稳定缝合线。乙。套管的实际图像。


斜角肌挤压钳,直,锯齿(世界精密仪器,Ç atalog号:503719-12)
SILASTIC薄壁硅实验室管(道康宁,Ç atalog编号:508-009)
易装泵头2通道的精密泵管(科尔帕默,Ç atalog号:EW-77202-60)
的Masterflex L / S数字精密模块化驱动器与远程I / O和台式控制器(科尔帕默,Ç atalog号:UX-07557-00)
耐高压加热的三通阀(科尔帕默,Ç atalog号:EW-31200-80)
器官培养生物反应器(图2)

图2。器官培养生物反应器。A.在蠕动泵的驱动下,从器官腔中抽出培养基,并通过一系列薄壁硅胶管(用于充氧)将其灌注到插管中,通向培养中的肺PA 。器官腔上的过滤器用于压力平衡。乙。灌注生物反应器的实际图像。


脱细胞室(图3)

图3.去细胞室。A.系统中有两个腔室:储存腔室,其中包含用于脱胶的新鲜溶液;收集腔包含要脱细胞的肺。储液腔应放置在比收集腔高约50厘米的位置,这会产生重力,以驱动流体从储液腔通过PA套管流入肺部。两个腔室上的过滤器均用于压力平衡。乙。脱细胞室的实际图像。

半自动轮转式切片机(Leica Biosystems公司,Ç atalog号:RM2245)
EVOS FL自动2成像系统(赛默飞世尔科技,Ç atalog号:AMAFD2000)
微量仪(Eppendorf,Ç atalog号:5415R)
BeadBug微管混合机(基准科学,Ç atalog号:D1030)
预填充2.0毫升管,用酸洗过的二氧化硅(玻璃)珠,1.0毫米(基准科学,Ç atalog号:D1031-10)
迷你PROTEAN利垂直电泳细胞为小型预制胶,与迷你反式印迹模块(生物- [R广告实验室Ç atalog号:1658036)
ChemiDoc凝胶和Western印迹成像系统(Bio-Rad实验室,Ç atalog号:12003153)
到达式IR CO 2培养箱(Caron,目录号:7400-25-1)

程序


天然器官ECM和肺收获的体内代谢标记
注意:所有动物程序均应按照机构动物护理和使用委员会批准的协议进行。兽医或研究人员应进行咨询和/或培训,他们应具有大鼠腹膜内注射,安乐死,解剖和血管插管的经验。

第一天:

称重大鼠并相应地计算注射量(每克体重0.3毫克Ac 4 GalNAz)。使用以70 mg / ml配制的Ac 4 GalNAz注射溶液,注射量为4.29μl /克体重。对于对照大鼠,每克体重施用4.29μl的对照注射溶液。
通过使用带有27号针头的1 ml注射器腹膜内注射来管理适当体积的Ac 4 GalNAz或对照注射液。
将每个注射的大鼠单独安置在笼子上,并标有注射溶液的类型和注射日期。

第2天,第3天:

再重复两天,重复步骤s A1至A3。

第四天:

通过吸入CO 2牺牲大鼠。使用消毒的外科工具打开胸腔,以暴露心脏和肺部而不会损坏它们。
注:钍是可以用跳动或不跳动的心脏进行的过程。

在靠近肺动脉(PA)的右心室做一个小切口(2-3 mm)作为输液入口,并切开左心耳作为引流出口(图4)。

图4.肺收获过程中的手术程序。PA(蓝色)连接到右心室。在紧邻PA的右心室做一个小切口,以允许将平头针(用于冲洗)或套管插入PA中。在初次冲洗之前,应将左心耳切开作为引流口。


准备一个带有10毫升DPBS的注射器,该注射器连接到18号钝端针上。通过右心室的小切口将平头针轻轻插入PA中,并用镊子将针轻轻地固定在PA中。在大约10秒钟内注入10 ml DPBS,以将剩余的血液冲洗出肺血管。左心耳最初流出的血液是彩色的液体,表示冲洗成功,在整个输注过程中逐渐失去了血液的颜色,整个肺部都变了白色。
注意:对动物实施安乐死后,应尽快执行DPBS冲洗步骤,以避免在肺血管系统中形成血块。

隔离心肺组织,使其与周围组织隔离,然后将其放在用冰冷的DPBS预湿的纱布上的培养皿中。
要插管PA,将定制的插管(图1)连接到预先填充有DPBS的10 ml注射器中,并在有DPBS的情况下通过右心室的小切口将插管插入PA (图4)。通过温和的注射器注射缓慢地从套管中滴出。这是为了避免将气泡意外引入肺血管,以免引起空气栓塞和不均匀的灌注。插入PA的深度应约为5 mm,以确保稳定的缝合。将套管插入PA后,使用2-0丝线将套管固定在凹槽中。为了测试通过所述PA套管的插管,INFUSE DPBS通过gentl ý推动注射器。低灌注阻力和灌注后PA的扩张表明成功插管。

肺ECM的离体标记
向器官培养生物反应器中添加100 ml的预热d Ex Vivo培养基(图2),并使用30 ml注射器通过三通阀将整个灌注管用该培养基预填充,以去除气泡。用于代谢ECM标签,所述离体培养基中补充用Ac 4 GalNAz(DMSO 50μM在0.1%)。对于对照组,培养基中添加了0.1%DMSO。              
在没有事先进行代谢标记的情况下,从大鼠中收获心肺组织,并按照步骤A5至A9插管PA 。
注意:离体肺培养应在器官收获后1小时内开始,以确保要标记器官的最佳代谢活性。

在层流罩中,抬起器官培养生物反应器的盖子,以允许进入内部鲁尔接头(图2)。将肺部套管连接到内部鲁尔接头上,同时使介质缓慢地从鲁尔接头上滴落(通过使用注射器通过三通阀缓慢地注入介质)(图2)。这是为了避免将气泡引入灌注管线。强烈建议在此步骤中使用一次性无菌手套,以确保无菌。
注意:此过程需要两个人的配合。一个应轻推介质填充的注射器,而另一个应将肺套管连接到鲁尔接头。

将器官培养生物反应器的泵管连接至蠕动泵,并验证灌注方向(图2)。在加有5%CO 2的加湿培养箱中以恒定速率灌流培养肺24小时(对于100克大鼠采集的肺为5 ml / min,对于200克大鼠采集的肺为10 ml / min) 。

全肺脱细胞
向储液室中预填充1 L脱细胞溶液,并将其放置在收集室上方约50 cm处(图3)。用Decell Solution预填充两个腔室之间的连接管。
将肺(在体内或体外代谢标记后)连接到收集室内的鲁尔接头上,同时让Decell溶液缓慢地从鲁尔接头上滴落。这是为了避免将气泡引入灌注管线。
让Decell溶液在重力(由储液室和收集室之间的高度差决定)的驱动下通过肺部灌注。
注意:在脱细胞过程中,肺部应逐渐扩大大小,并在最初的5-10分钟内变为半透明。否则,可能表明插管或灌注线的其他部分被阻塞。整个1 L脱细胞溶液应在1-2小时内通过肺灌注。如果花费的时间少得多(例如少于30分钟),则可能表明肺部受损或管路连接泄漏。如果花费更长的时间(超过2小时),则可能表示灌注线或肺血管被组织碎屑或气泡阻塞,或者可能是由于插管在PA中的位置不正确所致。

在储液器用完之前,通过转动灌注管路中的三通阀之一来暂停灌注。丢弃收集室中的废液,并用200 ml H 2 O代替储液室,并灌注肺15分钟。
暂停灌注,用200 ml H 2 O中的1%Triton X-100代替储液腔,然后再灌注肺15分钟。
用1 L的1 × PBS代替储液腔,并通过肺完全灌注以洗去洗涤剂。
再重复两次步骤C6。

代谢ECM标记的组织学表征
为了固定脱细胞肺,注入5米升次的使用连接到10ml的注射器的钝头针头Ê固定溶液进气管,并结扎用2-0丝线缝合气管。这是为了优化组织学评估而开放的牙槽结构。浸泡在锥形管中的肺用另外的15毫升固定溶液并孵育在4℃下过夜温和搅拌。
处理固定的肺组织以进行石蜡包埋,并使用切片机将嵌入的组织块切成5μm的厚度。
按照标准的脱石蜡和补水步骤,用1 × PBS冲洗切片3次,每次5分钟。
要检测脱细胞肺ECM中潜在的叠氮化物标记,请通过向组织学切片中加入Biotin-PEG4-Alkyne Click反应混合物(每节100-200μl)进行生物素偶联,并在湿润的染色室中于室温孵育1 h 。
用1 × PBS清洗切片3次,每次5分钟。
在1 × PBS中用1%BSA封闭切片20分钟。
将切片与兔抗层粘连蛋白一抗(在1 x PBS中的1%BSA中以1:500的稀释度,在1 × PBS中)于4 °C在潮湿的染色室内孵育过夜。
用1 × PBS清洗切片3次,每次5分钟。
在室温下用链霉亲和素(Alexa Fluor 647偶联物,在1%BSA在1 × PBS中以1:500稀释)和驴抗兔488(1:500稀释)孵育切片45分钟。
用1 × PBS洗涤3次,每次5分钟。
使用Fluoromount安装玻片,使其风干。
使用EVOS FL Auto 2成像系统(图5)执行荧光成像。
注意:标准免疫荧光染色可与点击反应检测叠氮化物标记一起进行。在这种双重染色中,建议先进行生物素-PEG4-炔烃的点击偶联,然后再进行标准抗体染色。例如,我们进行了层粘连蛋白的染色。


代谢ECM标记的生化表征
切下一块未脱细胞的肺组织(通过体内或体外代谢标记),然后用细剪刀将其切碎。
注意:对于ECM中叠氮化物标记的生化检测,通常50-100 mg肺组织就足够了。

将切碎的肺组织转移到预装有玻璃珠的2.0 ml管中,加入300μlH 2 O,并在微管均质器上以全速(4,000 rpm)均质3分钟。如果大的组织块仍然可见,请重复此均化步骤。
添加300μ升2 × DECELL解管,并搅拌在室温30分钟。该步骤将溶解均质化的肺组织中的所有细胞成分。
在室温下将试管以16,100 × g离心20分钟。丢弃上清液。
向试管中加入1 ml DPBS,通过颠倒彻底混合,在室温以16,100 × g离心10分钟。丢弃上清液。这是为了洗去所有剩余的细胞成分。
再重复两次步骤E5。
向试管中加入500μlBiotin-PEG4-Alkyne Click反应混合物,充分混合,并在室温搅拌下温育1小时。这是为了使生物素与脱细胞的肺ECM中潜在的叠氮化物配体缀合。
在室温下将试管以16,100 × g离心10分钟。丢弃上清液。
向试管中加入1 ml DPBS并充分混合。这是要洗净任何剩余的生物素-PEG4-炔烃和其他点击反应试剂。
在室温下将试管以16,100 × g离心10分钟。丢弃上清液。
再重复两次E9和E10。
向试管中加入500μl尿素提取缓冲液,在室温下轻轻搅拌2小时。
在室温下将试管以16,100 × g离心10分钟。
收集上清液作为ECM蛋白样品。
使用BSA作为蛋白质标准品,按照制造商的规程,使用BCA分析定量ECM蛋白质浓度。
进行SDS-PAGE电泳,使每个样品的蛋白质负载相等,例如从肺中提取的带有或不带有代谢叠氮化物标记的ECM蛋白样品,然后将蛋白质从凝胶上转移到一块PVDF膜印迹上。使用封闭缓冲液封闭印迹20分钟,将印迹在10 ml的链霉亲和素-HRP(封闭缓冲液中以1:10,000稀释)中孵育,在室温下搅拌1小时,然后用TBST彻底清洗印迹3次,每次10次每分钟。
在HRP成像混合物中孵育印迹3分钟,然后使用ChemiDoc Gel和Western Blot成像系统成像(图6A)。
为了验证所有样品中蛋白质的负载均等,请执行SDS-PAGE电泳,并按照制造商的规程使用Sypro Ruby Protein Gel Stain对凝胶中的总蛋白质进行染色。使用ChemiDoc凝胶和Western Blot成像系统对Sypro Ruby染色的凝胶进行成像(图6B)。

数据分析


代谢叠氮化物标记的组织学表征:在脱细胞肺组织的组织学切片上,使用点击化学将潜在的叠氮化物配体与生物素偶联。使用两种不同的荧光团对生物素的链霉亲和素染色和ECM的层粘连蛋白染色进行成像。层粘连蛋白染色用作对照,以验证在有和没有代谢叠氮化物标记的情况下,肺ECM中存在相似的ECM含量。应注意确保所比较的所有样品的成像参数一致。根据荧光信号最强的样品调整曝光时间。与对照组相比,源自接受Ac 4 GalNAz代谢标记的大鼠的肺ECM有望显示出更强大的叠氮化物标记和生物素结合(图5)。

图5.肺ECM的代谢叠氮化物标记的组织学表征。代表性的结果显示,在用Ac 4 GalNAz或DMSO进行三天体内标记后,从大鼠肺中获得了两个ECM样品。将ECM中潜在的叠氮化物配体与生物素偶联,然后使用荧光团偶联的链霉亲和素(红色)进行检测。层粘连蛋白抗体染色指示肺ECM(绿色)。比例尺s :200μm。


代谢叠氮化物标记的生化表征。使用点击化学方法,将提取的肺ECM蛋白中潜在的叠氮化物配体与生物素偶联,然后进行凝胶电泳。使用标准的蛋白质印迹程序,使用结合了HRP的链霉亲和素和化学发光法检测生物素。与对照组相比,从接受Ac 4 GalNAz代谢标记的肺中提取的ECM蛋白样品有望显示出更强大的叠氮化物标记和生物素结合(图6)。

图6.肺ECM中叠氮化物标记的生化特征。代表性结果显示,在用Ac 4 GalNAz或DMSO进行离体一天的体外标记后,从大鼠肺部得到了两个ECM样品。一。通过共轭生物素的蛋白质印迹分析检测叠氮化物配体。乙。通过用Sypro Ruby对总蛋白进行凝胶内染色来验证蛋白负载量。


菜谱


Ac 4 GalNAz注射液
含70%DMSO的DPBS中的70 mg / m l Ac 4 GalNAz

控制注射液
DPBS中70%的DMSO

离体培养基
DMEM补充了10%FetalClone I血清

1%抗生素/抗真菌药

德赛尔解决方案
高压灭菌去离子水中的0.1%SDS

2 × Decell解决方案
高压灭菌去离子水中的0.2%SDS

1 × PBS(pH 7.4)
用去离子水稀释20 × PBS(pH 7.4),用高压灭菌器灭菌

固定解决方案
1 × PBS(pH 7.4)中的4%多聚甲醛(PFA )

生物素-PEG4-炔烃点击反应混合物
注意:来自Click-iT细胞反应缓冲液试剂盒的组分(表1)。





表1 。生物素-PEG4-炔烃点击反应混合物的配方


尿素提取缓冲液
600万尿素

10毫米HEPES

1 ×蛋白酶抑制剂

调节pH至8.0

1 × TBST(pH 7.4)
用去离子水稀释10 × TBST(pH 7.4)

阻塞缓冲
TBST中5%的BSA

HRP成像混合物
SuperSignal West Pico PLUS Luminol / Enhancer解决方案和SuperSignal West Pico PLUS Peroxide解决方案,1:1(新鲜配制)


致谢


这项工作得到卡内基梅隆大学生物医学工程系的支持。ZL和YX得到了中国奖学金委员会的奖学金支持。该协议最初是从Ren等人改编的。(2018年)。我们感谢匹兹堡生物标本中心(匹兹堡大学)的组织学处理。我们还要感谢卡耐基梅隆大学的畜牧业。最后,我们感谢Piyumi Wijesekara和吴惠和在收集有关材料和试剂的信息方面所提供的帮助。


利益争夺


XR拥有与此工作相关的专利申请(US20170362266A1)。




伦理


所有动物程序均根据卡内基梅隆大学机构动物护理和使用委员会批准的协议(批准ID #TR202000006 ,有效期至20-2022 )进行。


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引用:Ling, Z., Xing, Y., Reinoso Jacome, E., Fok, S. W. and Ren, X. (2021). Bioorthogonal Labeling and Chemoselective Functionalization of Lung Extracellular Matrix. Bio-protocol 11(4): e3922. DOI: 10.21769/BioProtoc.3922.
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