Probe HGT for cell integrin to perceive environmental ligand height change, kit and application

Abstract

The invention provides a high gain detector (HGT) for sensing environmental ligand height change by cell integrin, a kit and application, the height of a double-stranded DNA probe is changed to 1-bp, 18-bp, 24-bp and 30-bp, and the length range is 0.5-10 nm. The two probes with different heights are subjected to a combined experiment to detect whether the cells can sense the height change of the ligand or not. According to the invention, the combination of 1bp and 18bp, the combination of 18bp and 30bp and the combination of 24bp and 30bp are utilized to find that the integrin can sense the height of the ligand and the sensitivity is 1-2nm. In addition, through experimental verification, the cell perception ligand height property has universality, and the cell skeleton hardness and the integrin state can adjust the integrin ligand height preference.

Description

Technical Field

[0001] The present invention relates to the technical field of cell integrins, and in particular, to a probe height gauge tether (HGT) for cell integrins to sense the height change of environmental ligands, a kit, and an application thereof. Background Art

[0002] Integrins, as force-sensitive receptors on the cell membrane, play a key role in the interaction between cells and the extracellular matrix (ECM). They can not only sense the mechanical signals in the ECM, but also transmit these signals to the nucleus through the cytoskeleton, thereby regulating cell adhesion, spreading, migration, differentiation and other behaviors.

[0003] Current research mainly focuses on the perception of ligand horizontal spacing by integrins (such as integrin clustering when ligand spacing is less than 70 nm), but there is no systematic research on how the change in ligand vertical height affects integrin perception and organization form. The ECM not only has a complex spatial distribution in the horizontal direction, but also has height changes in the vertical direction (such as fiber networks with different thicknesses, ligand distributions at different depths, etc.). Existing experimental techniques are difficult to precisely control and simulate this ligand height change, resulting in an inability to comprehensively understand the integrin perception mechanism in the vertical direction. Tissue staining reveals that cell adhesion ligands are three-dimensionally distributed in the microscopic network, and their regulation of cell function is complex. Given the wide application of ligands in the design of biomaterials, we need to consider the spatial distribution information of the pairing when designing biomaterial models, which has guiding significance for precise biomedical engineering regulation.

[0004] Although existing technologies such as atomic force microscopy can operate cells at the nanoscale, they cannot measure the height perception of integrins. Integrins, as key molecules for cell mechanical perception, their function in sensing ligand height changes has not been studied. This research breaks new ground and fills the gap by designing the HGT probe, which can be used to analyze the new characteristics of cell integrins and is beneficial for detecting the biological functions of different cells. Summary of the Invention

[0005] The purpose of the present invention is to provide a probe, a kit, and an application for cell integrins to sense the height change of environmental ligands, so as to solve the technical problem of detecting the height change of cell integrins sensing environmental ligands.

[0006] To achieve the above purpose, the present invention provides a probe HGT for cell integrins to sense the height change of environmental ligands, a kit, and an application thereof.

[0007] 1. Preparation method of probes for cell integrin to sense the height change of environmental ligands: Select ssDNA top strands corresponding to different heights, mix and react with cyclic peptide cRGDfk and azide polyethylene glycol modified N-hydroxysuccinimide N3-PEG-NHS to obtain ssDNA upper strands with cRGD, and then mix them with ssDNA bottom strands and anneal at room temperature (25 °C) to obtain the probes; the heights of the probes are 1-bp, 18-bp, 24-bp and 30-bp, and the nucleotide sequences of ssDNA are shown in SEQ ID NO.1-9. Moreover, the 5' or 3' end of all ssDNA top strands has alkyne coupling; See Table 1 for details.

[0008] In Table 1, DBCO (Dibenzocyclooctyne) BHQ2 is Black Hole Quencher 2.

[0009] Bio represents DNA modification with biotin coupling at the 5' or 3' end.

[0010] Cy3 and cy5 represent fluorescent groups.

[0011] Preferably, the length range of the probes is 0.5-10 nm.

[0012] Preferably, the molar ratio of ssDNA top strand, cyclic RGD (cRGDfk), and azide PEG NHS ester (N3-PEG-NHS) is 1:30:3; the molar ratio of ssDNA top strand to ssDNA bottom strand is 1.1:1.

[0013] Preferably, the mixing reaction conditions are: reacting at room temperature for 2 hours or reacting at 4 °C for 12 hours; the annealing time at room temperature is 2 hours.

[0014] 2. Probes for cell integrin to sense the height change of environmental ligands, obtained by the aforementioned preparation method.

[0015] 3. Application of the aforementioned probes in the preparation of kits for cell integrin to sense the height change of environmental ligands.

[0016] Preferably, select a combination of two probes with different heights, and the combination is selected from any one of the following: the combination of 1bp and 18bp, the combination of 18bp and 24bp, the combination of 24bp and 30bp.

[0017] 4. Kits for cell integrin to sense the height change of environmental ligands, containing the aforementioned probes.

[0018] 5. Use of the aforementioned probe or kit in detecting highly variable environmental ligands sensed by cell integrins.

[0019] 6. A method for detecting highly variable environmental ligands sensed by cell integrins, the specific steps are as follows: (1) Coat the surface of the glass dish with biotinylated bovine serum albumin BSA-biotin; (2) Add avidin Avidin to bind biotin biotin; (3) Label two probes with different heights with biotin to obtain biotin-labeled probes with different heights, and bind them to avidin Avidin on the surface of the glass dish; (4) Inoculate cells into the glass dish prepared in step (3) and culture for 2 hours; (5) Capture integrin mechanical signals at different heights and analyze the force signal intensity.

[0020] Preferably, in step (1), the concentration of BSA-biotin is 100 μg / mL.

[0021] Preferably, in step (1), after coating, wash 3 times with PBS to remove non-specific binding.

[0022] Preferably, in step (3), the concentration of both biotin-labeled probes is 0.2 μM.

[0023] Preferably, in step (3), the two probes with different heights are selected from any one of the following: a combination of 1 bp and 18 bp, a combination of 18 bp and 24 bp, a combination of 24 bp and 30 bp.

[0024] Preferably, in step (4), the method for preparing cells is as follows: (4-1) Culture 3T3L1 cells in a 35 mm cell culture dish using DMEM medium containing 10% fetal bovine serum (FBS) by volume and 1% penicillin-streptomycin (Penicillin-Streptomycin, PS) by volume, and use when the cell density reaches 60-80%; (4-2) Wash the cells 4 times with ethylenediaminetetraacetic acid (EDTA) solution; (4-3) Add 1 mL of EDTA solution and incubate the cells in a 37 °C incubator for 5 minutes; (4-4) Terminate digestion with 1 mL of medium; (4-5) Centrifuge the cell suspension at 200 g for 5 min, remove the supernatant, and resuspend the cells with fresh medium to obtain the cells.

[0025] Further preferably, the EDTA solution is prepared by the following method: 100 mL × Hank’s balanced salt solution HBSS (Hank's balanced salt solution) + 10 mL × 1 M 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (pH 7.6) + 10 mL of 7.5% sodium bicarbonate by mass concentration + 2.4 mL of 500 mM EDTA, and make up to 1 L with H2O.

[0026] Preferably, in step (5), a Nikon Ti2E microscope is used to capture integrin mechanical signals at different heights, and MatLab software is used to analyze the force signal intensity.

[0027] The present invention has the following beneficial effects: The present invention can accurately regulate the ligand height at the nanoscale, providing a good tool for precisely studying the perception of ligand height by cell integrins.

[0028] Based on the mechanical probes reported in previous articles, the present invention modifies and combines them, changes the height of the double-stranded DNA probes to 1-bp, 18-bp, 24-bp, and 30-bp, and the length range is 0.5 - 10 nm. The combination experiments of two probes with different heights are carried out to detect whether the cells can perceive the change of ligand height.

[0029] Using the combinations of 1bp and 18bp, 18bp and 30bp, and 24bp and 30bp, the present invention finds that integrins can perceive ligand height and the sensitivity is between 1 and 2 nm.

[0030] In addition, through experimental verification, the present invention shows that the property of cells perceiving ligand height is universal, and the cytoskeleton hardness and integrin state can regulate the preference of integrin ligand height.

[0031] In addition to the purposes, features, and advantages described above, the present invention has other purposes, features, and advantages. The following will refer to the drawings for a further detailed description of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The drawings constituting a part of this application are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings: Figure 1 It is a schematic diagram of the height gauge tether (HGT). Different lengths of dsDNA fragments modified with integrin ligand cyclic RGD peptides are fixed on the glass surface. Cells are inoculated on the HGT surface for a period of time, and height signals are collected.

[0033] Figure 2The efficacy of HGT was confirmed through different combinations and time records. A. HGTs of different heights, such as 1bp, 18bp, and 24bp, were divided into schematic diagrams of 1bp - 18bp and 18bp - 24bp. B. Statistics of the fluorescence signal intensities generated by integrin pulling force in the 1bp and 18bp channels. C. Statistics of the fluorescence signal intensities generated by integrin pulling force in the 18bp and 30bp channels. D. Statistics of the fluorescence signal intensities generated by integrin pulling force in the 18bp and 24bp channels. E. The multiple relationship of the high - probe signal to the low - probe signal in three groups of probe combinations with different heights. F. Pictures of each channel in the probe combination of 18bp and 24bp. G. Fluorescence intensities of two channels along the white line in Figure F.

[0034] Figure 3 Platelets without cell nuclei have the property of sensing ligand height. Among them, A are pictures of each channel of platelets in the probe combination of 18bp and 24bp, B is the fluorescence intensity of two channels along the white line in Figure A, and C is the fluorescence signal intensity generated by integrin pulling force in the 18bp and 30bp channels.

[0035] Figure 4 Fast - moving cells - keratinocytes also show a high degree of preference during migration; among them, A shows a wheel - shaped integrin force pattern, and the 24bp HGT has stronger fluorescence intensity; B compares the fluorescence intensities along the white line; C records the fluorescence signals during the migration of keratinocytes in real - time; D compares the broken HGT signals within the rectangle at different times.

[0036] Figure 5 The preference of integrin ligand height is regulated by cytoskeleton stiffness and integrin status. Among them, A shows that the HGT signal decreases after treatment with blebbistatin; B compares the HGT signals of the Ctrl group and the blebbistatin - intervened group. N = 30. Fewer 24bp HGTs are broken in the cells treated with blebbistatin, while more are broken in the Ctrl group; C shows a decrease in the multiple change of HGT in the blebbistatin - intervened group. Detailed implementation mode

[0037] The following describes the embodiments of the present invention in detail with reference to the accompanying drawings. However, the present invention can be implemented in many different ways defined and covered by the claims.

[0038] Embodiment Embodiment 1: Preparation of ligand - height - sensing probe (HGT) and construction of the device 1) Order modified DNA primers from Sangon, and the sequences are shown in Table 1.

[0039] 2) Prepare a 10 mM cRGDfk (Selleckchem, Cat: S7834, Lot: S783403) solution. Dissolve the RGDfK polypeptide in PBS buffer (pH 7.2), dissolve 30 nM N3-PEG-NHS (aladdin, A163301) in deionized water, and dissolve 10 nM ssDNA top strand in deionized water. After mixing them in a molar ratio of cRGD: N3-PEG-NH: ssDNA top = 30:3:1, react at room temperature for 2 h or at 4 °C for 12 h.

[0040] (The azide group reacts with alkyne (DBCO) in an aqueous solution catalyzed by copper. The cyclic peptide RGD undergoes an amino reaction with NHS. PEGylation can improve the solubility and stability of polypeptides and proteins, reduce immunogenicity. It can also inhibit the non-specific binding of charged molecules to the modified surface and enhance the accessibility of RGD to integrins on the cell membrane.) 3) Dissolve the complementary bottom strand of ssDNA in deionized water, and then anneal the synthesized ssDNA top strand with cRGD and the complementary bottom strand at a molar ratio of 1.1:1 (top: bottom) at room temperature for 2 h. Ensure that all bottom strands hybridize with the top strand.

[0041] 4) Coat the surface of a cell culture confocal glass dish (FCFC016, BeyoGold™ 35 mm confocal dish, 14 mm diameter glass dish) with 100 μg / ml biotinylated-bovine serum albumin BSA-biotin (Bovine serum albumin, A8549, Sigma-Aldrich), and wash it 3 times with 1XPBS (Phosphate Buffered Saline, phosphate buffer, pH 7.2 - 7.4) to remove non-specific binding; 5) Add avidin (31000, Thermo Fisher Scientific Inc.) to bind biotin; 6) Bind the biotin-labeled biomechanical probes with different heights (0.2 μM: 0.2 μM) to the avidin on the surface of the glass dish; The schematic diagram of the detection mechanism of the present invention is shown in Figure 1 。

[0042] Example 2 Cultivation and Experiments of Cells 1) Mouse embryonic fibroblasts (3T3L1) were purchased from Wuhan Pusai Biotechnology Co., Ltd. (Wuhan, China). MSCs cells were inoculated and cultured in DMEM medium supplemented with 10% fetal bovine serum (v / v) and 1% penicillin-streptomycin (C0222, Beyotime). During the experiment, cells were dissociated with ETDA solution [100 mL 10× HBSS + 10 mL 1 M HEPES (pH 7.6) + 10 mL 7.5% sodium bicarbonate (w / v) + 2.4 mL 500 mM EDTA + 1 L H2O]. Cells were seeded into the device prepared in Example 1 at a density of 50,000 / cm 2 and placed in a carbon dioxide incubator at 37 °C for 2 h.

[0043] 2) Microscopic imaging: A Nikon Ti2 fully automatic inverted fluorescence microscope was used. The FITC filter channel was selected. The filters for fluorescence imaging were as follows: Cy3 channel: excitation filter Ex: 554 / 23 nm, emission filter EM: 609 / 54 nm, dichroic mirror DM 573 nm. Cy5 channel: excitation filter Ex: 618 / 50 nm, emission filter EM: 698 / 70 nm, dichroic mirror DM 573 nm.

[0044] 3) Result analysis: Using NIS-Viewer software, the gray values of the fluorescence intensity along the line were recorded. Then, the values of the Cy3 and Cy5 channels were plotted to Figure 2 F. The normalized gray value was calculated as y = ΔI / I total , and in the experiment, the percentage change in the gray value of each channel was used for normalization, and then the normalized values were compared Using combinations of 1 bp and 18 bp; 18 bp and 30 bp; 24 bp and 30 bp, it was found that integrin could sense ligand height with a sensitivity of 1 to 2 nm; Figure 2 The consistency of ligand height perception was verified through different combinations. Figure 2 In A, HGTs of different heights, such as 1 bp, 18 p, 24 bp, 30 bp, were divided into 1 bp - 18 bp, 18 bp - 24 bp, 28 bp - 30 bp, Figure 2 In B - D, the fluorescence intensity generated by integrin pulling force in different channels was analyzed, showing that the higher the HGT, the stronger the single-cell fluorescence signal. Figure 2 In E, the fold change between HGTs was shown. F shows the images of each channel in the probe combination of 18 bp and 24 bp. G shows the fluorescence intensity of the two channels along the white line in Figure F.

[0045] Example 3 Platelet experiment 1) Platelet preparation: 1.8 ml of blood was drawn from the ear vein of a dog and placed in a 3-ml syringe containing 0.2 ml of acid citrate dextrose (ACD) buffer (ACD buffer: 85.3 mM sodium citrate, 41.6 mM citric acid, 136 mM dextrose), and then transferred to a 15-ml falcon tube containing 2 ml of buffered saline glucose citrate (BSGC: 129 mM NaCl, 14 mM sodium citrate, 11 mM dextrose, 10 mM NaH2PO4, pH 7.3). The mixture was centrifuged at 180 g for 8 minutes at room temperature. The supernatant was collected as platelet-rich plasma (PRP) and stored at room temperature until future use. During the purification process, 0.5 ml of PRP was transferred to a new eppendorf tube and mixed with 0.5 ml of buffer 1 (formulation: 113 mM NaCl, 4.3 mM K2HPO4, 4.2 mM Na2HPO4, 24.3 mM NaH2PO4, 5.5 mM dextrose, pH 6.3), and 0.02 U / ml of adenosine triphosphate and 1 μM of prostaglandin E1 were incorporated into the buffer. After centrifugation at 800 g for 8 minutes, the supernatant was removed, and the platelet pellet was resuspended in serum-free F-12 medium (HFL05, Caisson Laboratories). At this time, the platelets were in a quiescent state and could be used for subsequent experiments.

[0046] 2) Probe surface preparation, the same as in Example 1, with the probe height selected as 18 bp and 24 bp.

[0047] 3) 10 μM ADP was added to the platelet suspension, and then it was diluted 200 times and inoculated on the highly sensitive probe surface, and the height signal of the ligand was recorded ( Figure 3 ). Figure 3 In A, the platelets were inoculated on the HGT surface and imaged, Figure 3 In B, compared with the HGT signal along the line in A, the 24-bp HGT had a stronger fluorescence signal. Figure 3 Statistical analysis in C showed that compared with 18-bp HGT, 24-bp HGT ruptured more. N = 40.

[0048] Example 4 Goldfish scale keratinocyte experiment Cell Preparation: Remove the fish scales from the fish and gently press them onto the bottom of a glass culture dish so that the inner side of the scales touches the glass surface. Wait for 1 - 2 minutes for the scales to adhere to the glass surface. Then add a culture medium (DMEM, purchased from BioSharp) containing 20% fetal bovine serum by volume concentration and 1% penicillin by volume concentration to the culture dish. Incubate the scales at room temperature for about 12 - 24 hours to allow keratinocytes to migrate out of the scales. In the experiment, an EDTA solution (with the same formula as above) was used to isolate the keratinocytes from the culture dish. Rinse the culture dish containing corneal cells once with the EDTA solution and then incubate it in the EDTA solution at room temperature for 5 minutes. Use a pipette to disperse the isolated cells in the EDTA solution. Add the DMEM culture medium to the EDTA solution at a volume ratio of 5:1. Seed the keratinocytes on the HGT surface in the wells of the culture dish at a cell density of 2.0×10 5 cells / mL. This relatively low cell density provides more space for cell migration. Incubate the cells on the HGT surface for about 15 minutes. The keratinocytes will polarize and migrate. Subsequently, HGT imaging is performed.

[0049] In Example 4, the fish scale keratinocytes migrated rapidly, and the tracks they passed through demonstrated the effect of integrin on ligands of different heights. It can be seen that the fast - moving cells - keratinocytes also showed a high degree of preference during migration ( Figure 4 ). Figure 4 In A, a wheel - shaped integrin force pattern is presented, while the 24bp HGT has a stronger fluorescence intensity. Figure 4 In B, the fluorescence intensities along the white line are compared. Figure 4 In C, the fluorescence signals during the migration of keratinocytes are recorded in real - time. Figure 4 In D, the broken HGT signals within the rectangle at different times are compared.

[0050] Examples 3 and 4 prove that both fast - crawling cells and slow - crawling cells can sense the ligand height.

[0051] In Example 5, the cytoskeleton stiffness and integrin status can regulate the preference for integrin ligand height.

[0052] Cell Culture: 3T3L1 cells were used in this part, and the culture was the same as in Example 1.

[0053] Cell Experiment: Suspend the 3T3L1 cells and add blebbistatin at a final concentration of 25 μM (a small - molecule inhibitor specific for myosin II. Treating cells can hinder cell force transmission and regulate the integrin state. This is a motor protein crucial for various cellular processes such as muscle contraction, cell movement, and cytokinesis), then disperse the cells on the 18 - 24bp HGT probe surface and culture them in a standard cell culture incubator at 37 °C for 2 hours.

[0054] Microscopic imaging: the same as above.

[0055] Results: Treatment with bryostatin led to a decrease in the high sensitivity of integrin ( Figure 5 ). Figure 5 Image A shows a decrease in the HGT signal after treatment with bryostatin. Figure 5 Image B compares the HGT signals of the Ctrl group and the bryostatin intervention group. N = 30. There were fewer 24bp HGT breaks in the cells treated with bryostatin, while there were more in the Ctrl group. Figure 5 Image C shows a decrease in the fold change of HGT in the bryostatin intervention group.

[0056] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Preparation method of probe HGT for cell integrin to sense the height change of environmental ligands. Select ssDNA top strands corresponding to different heights, mix and react with cRGDfk and N3-PEG-NHS to obtain ssDNA upper strands with cRGD, and then mix them with ssDNA bottom strands and anneal at room temperature to obtain the probe. The heights of the probes are 1-bp, 18-bp, 24-bp and 30-bp. The nucleotide sequences of the ssDNAs are as shown in SEQ ID NO.1-9. Moreover, the 5' or 3' ends of all ssDNA top strands have alkyne coupling.

2. The preparation method according to claim 1, characterized in that, The length range of the probe is 0.5-10 nm.

3. The preparation method according to claim 1, characterized in that, The molar ratio of ssDNA top strand, cRGDfk and N3-PEG-NHS is 1:30:3; the molar ratio of ssDNA top strand to ssDNA bottom strand is 1.1:

1.

4. The preparation method according to claim 1, characterized in that, The reaction conditions are: reacting at room temperature for 2 hours or reacting at 4°C for 12 hours; the annealing time at room temperature is 2 hours.

5. The preparation method according to claim 1, characterized in that, The ssDNA top strand and the ssDNA bottom strand are annealed at a concentration ratio of 1.1:1 at room temperature for 2 hours.

6. A probe for a cell integrin to sense a large change in the environmental ligand, characterized in that, It is obtained by the preparation method described in any one of claims 1-5.

7. Use of the probe described in claim 6 in the preparation of a kit for cell integrin to sense the height change of environmental ligands.

8. A kit for a cell integrin to sense a high change in an environmental ligand, characterized in that, It contains the probe described in claim 6.

9. Use of the probe described in claim 6 or the kit described in claim 8 in detecting the height change of cell integrin to sense environmental ligands.

10. A method for detecting the high change of environmental ligands sensed by cell integrin, characterized in that, The specific steps are as follows: (1) Coat the surface of the glass dish with biotinylated bovine serum albumin; (2) Add avidin Avidin to bind to biotin biotin; (3) Label two probes with different heights with biotin to obtain biotin-labeled probes with different heights, and bind them to avidin Avidin on the surface of the glass dish; (4) Inoculate cells into the glass dish prepared in step (3) and culture for 2 hours; (5) Capture integrin mechanical signals with different heights and analyze the force signal intensity.

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