Bionic glycocalyx layer material, immunosuppression composition and application of biomimetic glycocalyx layer material in endothelial cell protection and organ transplantation immune response inhibition

Abstract

The invention belongs to the technical field of biological medicine and glycopeptide preparation, and particularly relates to a bionic glycocalyx layer material, an immunosuppression composition and application of the biomimetic glycocalyx layer material in endothelial cell protection and organ transplantation immune response inhibition. The bionic glycocalyx layer material (self-assembled glycopeptide) has the effects of maintaining the integrity of cells and preventing immune cell infiltration, and maintains the integrity of endothelial cells, so that the effect of protecting the endothelial cells is achieved. On the basis of the scheme, the bionic glycocalyx layer material in the technical scheme and an immunosuppressive agent are combined for application, and in the using process, the bionic glycocalyx layer material can play a role in packaging the immunosuppressive agent, the solubility of the immunosuppressive agent can be remarkably improved, the systemic toxicity is reduced, and the immunosuppressive agent can be used for preparing the biomimetic glycocalyx layer material. The circulation time of the immunosuppressant is prolonged, so that the accumulation of the immunosuppressant in a disease part is enhanced, the survival time of a graft is prolonged, the dosage of the immunosuppressant is reduced, and the effect of better inhibiting the immune response of organ transplantation is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and glycopeptide preparation, and specifically relates to a bionic glycocalyx layer material, an immunosuppressive composition and applications thereof in endothelial cell protection and suppression of organ transplantation immune response. Background Art

[0002] Endothelial cells (ECs) are a thin layer of epithelial cells, composed of a layer of flat cells, polygonal in shape, with jagged edges and interlocking. Endothelial cells form the inner wall of blood vessels, and are the interface between blood and other blood vessel walls (single layer of squamous epithelium) in the lumen of blood vessels. Endothelial cells are along the entire circulatory system, from the heart to the smallest microvessels.

[0003] Endothelial cells play an important role in cardiac transplantation. They are not only the physical barrier between the transplanted heart and the recipient, but also play a role in multiple key physiological processes such as vascular regulation, inflammation, immune response, coagulation and fibrinolysis. During cardiac transplantation, the cold storage and reperfusion process can cause damage to fragile endothelial cells. Damaged ECs release damage-associated molecular patterns (DAMPs), such as HMGB1, which in turn activate innate immune cells. These immune cells further signal with T (Treg) cells and B (Breg) cells, leading to adaptive immune activation. In addition, ECs themselves act as hapten-presenting cells and participate in the activation of adaptive immunity. In turn, ECs are the first cells to contact the recipient's immune system and become the primary target of immune attack, further exacerbating endothelial damage.

[0004] Although the early and sufficient use of broad-spectrum immunosuppressants can inhibit immune activation, it not only brings about problems such as increased susceptibility to infection, increased cancer risk, and liver and kidney toxicity, but the immunosuppressants themselves can also damage endothelial cells. Studies have shown that immunosuppressants such as tacrolimus, mycophenolate mofetil, and cyclosporine are toxic to the vascular system by reducing the migration of endothelial cells in damaged areas and inhibiting endothelial cell proliferation, leading to endothelial cell dysfunction and inhibiting angiogenesis. Damaged endothelium leads to a series of vascular homeostasis imbalances such as inflammation, complement activation, and coagulation activation. It can be seen that maintaining endothelial cell integrity is crucial to the function of transplanted hearts.

[0005] An alternative to traditional immunosuppressants is to introduce immune regulation using cell therapy, such as the use of regulatory T cells or regulatory B cells. Engineered T and B cells induce immune tolerance in allogeneic transplants. In contrast, combined bone marrow transplantation utilizes a transient mixed chimerism approach that enables transplant recipients to possess both the donor and recipient immune systems and has achieved some success in achieving sustained chimerism. However, these approaches are suboptimal in their current state because they rely on combination therapy, inconsistency in donor-specific tolerance induction, and potential challenges in adapting to different patients. It can be seen that alternatives to immunosuppressants currently also have major challenges.

[0006] However, there is currently no better technology or means to simultaneously inhibit organ transplant immune response and maintain endothelial cell integrity. Summary of the invention

[0007] The object of the present invention is to provide a bionic glycocalyx layer material, an immunosuppressive composition and their use in endothelial cell protection and suppression of organ transplantation immune response. The bionic glycocalyx layer material can effectively suppress the immune response during organ transplantation, thereby protecting endothelial cells, reducing the damage of endothelial cells to the immune response, and maintaining the integrity of endothelial cells.

[0008] The invention provides a bionic glycocalyx layer material. The raw materials for preparing the bionic glycocalyx layer material include a polypeptide SBP1, a self-assembling peptide FFVDF and sialyl lactose. The sialyl lactose is coupled with a peptide bond formed by dehydration condensation of the polypeptide SBP1 and the self-assembling peptide FFVDF through an azide group. The amino acid sequences of the polypeptide SBP1 and the self-assembling peptide FFVDF are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0009] Preferably, the structural formula of the biomimetic glycocalyx layer material is as shown in Formula I:

[0010]

[0011] The present invention also provides a method for preparing the bionic glycocalyx layer material described in the above technical solution, comprising: using polypeptide SBP1, self-assembling peptide FFVDF and sialyl lactose as raw materials to prepare the bionic glycocalyx layer material through solid phase synthesis technology.

[0012] Preferably, the molar ratio of the polypeptide SBP1, the self-assembling peptide FFVDF and sialyllactyl sugar is 1:1:2.

[0013] The present invention also provides an immunosuppressive composition, which comprises independently packaged bionic glycocalyx layer materials and immunosuppressants; the bionic glycocalyx layer material is the bionic glycocalyx layer material described in the above technical solution or the bionic glycocalyx layer material prepared by the preparation method described in the above technical solution.

[0014] Preferably, the immunosuppressant comprises tacrolimus.

[0015] The present invention also provides the use of the bionic glycocalyx layer material described in the above technical solution or the bionic glycocalyx layer material prepared by the preparation method described in the above technical solution or the immunosuppressive composition described in the above technical solution in the preparation of endothelial cell protection products.

[0016] The present invention also provides the use of the bionic glycocalyx layer material described in the above technical solution or the bionic glycocalyx layer material prepared by the preparation method described in the above technical solution or the immunosuppressive composition described in the above technical solution in preparing products that inhibit immune responses in organ transplantation.

[0017] Preferably, the organ transplant comprises a heart transplant.

[0018] Preferably, the product comprises a medicine.

[0019] Beneficial effects:

[0020] The present invention provides a biomimetic glycocalyx layer material, wherein the raw materials for preparing the biomimetic glycocalyx layer material include polypeptide SBP1, self-assembling peptide FFVDF and sialyl lactose, wherein the sialyl lactose is coupled with the polypeptide SBP1 and the self-assembling peptide FFVDF respectively through an azide group; the amino acid sequences of the polypeptide SBP1 and the self-assembling peptide FFVDF are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively. The biomimetic glycocalyx layer material (self-assembling glycopeptide) of the present invention has the function of maintaining cell integrity and preventing immune cell infiltration, maintaining the integrity of endothelial cells, and thus achieving the effect of protecting endothelial cells.

[0021] On the basis of the above scheme, the present invention further provides an immunosuppressive composition, which comprises independently packaged bionic glycocalyx layer materials and immunosuppressants, that is, the bionic glycocalyx layer material described in the above technical scheme is used in combination with the immunosuppressant. During use, the bionic glycocalyx layer material can encapsulate the immunosuppressant, reduce systemic toxicity, prolong the circulation time of the immunosuppressant, so as to enhance its accumulation in the disease site, thereby prolonging the survival time of the transplant, reducing the dosage of the immunosuppressant, and achieving the effect of inhibiting the immune response of organ transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0023] Figure 1 is the mass spectrum of the SBP polypeptide in Example 1;

[0024] Figure 2 The molecular structure formula (A) and electrospray ionization mass spectrometry (B) of EPG in Example 2 are shown;

[0025] Figure 3 The HPLC test result of EPG in Example 2 is shown;

[0026] Figure 4 The molecular structure formula (A) and electrospray ionization mass spectrometry (B) of EP in Example 2 are shown;

[0027] Figure 5 This is a graph showing the high performance liquid chromatography test results of EP in Example 2;

[0028] Figure 6 TEM detection image of EPG and EP in Example 3, the scale is 200 nm;

[0029] Figure 7 The DLS detection graphs of EPG+SLC9A3R2 and EPG at different time points in Example 3;

[0030] Figure 8 is the retention time curve of EPG on the HUVEC cell surface in Example 4;

[0031] Fig. 9 is the intensity curve of FITC-labeled BSA in Example 4;

[0032] Fig.10 CLSM image of the interaction between EPG and HUVEC cells in Example 5, scale bar is 12 μm;

[0033] Fig.11 is a SEM image of the interaction between EPG and HUVEC cells in Example 5;

[0034] Fig.12 This is a flowchart of the EPG-intervention mouse heart transplantation in Example 6;

[0035] Fig.13 This is the survival curve of the control group and the EPG group after transplantation in Example 6, "**" indicates significant difference P < 0.01;

[0036] Fig.14The survival curves of the FK506 group and the FK506+EPG group after transplantation in Example 6, "**" indicates significant difference P < 0.01;

[0037] Fig.15 The H&E and immune cell infiltration images of the myocardial tissues of the control group and the EPG group in Example 6, "**" and "***" indicate significant differences, P < 0.01 and P < 0.001, respectively;

[0038] Fig.16 H&E and immune cell infiltration images of myocardial tissues in the FK506 group and the FK506+EPG group in Example 6, "*", "**" and "***" indicate significant differences (P<0.05, P<0.01 and P<0.001), respectively;

[0039] Fig.17 It is the synthetic reaction formula of sialyllactyl sugar in the present invention. DETAILED DESCRIPTION

[0040] The invention provides a biomimetic glycocalyx layer material (EPG). The raw materials for preparing the biomimetic glycocalyx layer material include a polypeptide SBP1, a self-assembling peptide FFVDF and sialyl lactose (Sia3Lac). The sialyl lactose is coupled with a peptide bond formed by dehydration condensation of the polypeptide SBP1 and the self-assembling peptide FFVDF through an azide group. The amino acid sequences of the polypeptide SBP1 and the self-assembling peptide FFVDF are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0041] In the present invention, as an embodiment, the structural formula of the sialyllactyl sugar is as shown in Formula II:

[0042]

[0043] As an embodiment, the synthesis method of sialyllactyl sugar of the present invention comprises the following steps: a compound represented by the structure of formula III and a compound represented by the structure of formula IV undergo a glycosyl transfer reaction under the action of α-2,3-sialyltransferase (reaction formula: Fig.17 ), to produce a polysaccharide as shown in formula II.

[0044]

[0045] As an embodiment, the reaction system of the transglycosylation reaction of the present invention includes CMP sialic acid (CAS: 3063-71-6), 4-hydroxyethylpiperazineethanesulfonic acid (Hepes) and MgCl2, and the concentrations of CMP sialic acid, 4-hydroxyethylpiperazineethanesulfonic acid (Hepes) and MgCl2 in the reaction system are 50mM, 50mM and 20mM respectively. As an embodiment, the temperature of the transglycosylation reaction is 37°C, the time is 4h, and the pH value is 7.5.

[0046] In the present invention, the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 are as follows:

[0047] SEQ ID NO:1: KQVFKRH; SEQ ID NO:2: RRRFDVFF. The polypeptide SBP1 of the present invention has a high affinity for the characteristic molecule SLC9A3R2 on the surface of endothelial cells, and can specifically bind to SLC9A3R2 to target endothelial cells. The self-assembling peptide FFVDF of the present invention has the function of forming a nanostructure of EPG molecules as an assembly unit.

[0048] As an embodiment, the structural formula of the bionic glycocalyx layer material of the present invention is as shown in Formula I:

[0049]

[0050] The present invention also provides a method for preparing the biomimetic glycocalyx layer material described in the above technical solution, comprising: using polypeptide SBP1, self-assembling peptide FFVDF and sialyl lactose as raw materials to prepare the biomimetic glycocalyx layer material through solid phase synthesis technology. As an embodiment, the molar ratio of the polypeptide SBP1, self-assembling peptide FFVDF and sialyl lactose in the present invention is 1:1:2. The present invention does not specifically limit the specific steps of the solid phase synthesis, and the conventional solid phase synthesis steps in the art can be used.

[0051] The present invention also provides an immunosuppressive composition, which comprises independently packaged biomimetic glycocalyx layer materials and immunosuppressants; the biomimetic glycocalyx layer material is the biomimetic glycocalyx layer material described in the above technical solution or the biomimetic glycocalyx layer material prepared by the preparation method described in the above technical solution. As an embodiment, the immunosuppressant of the present invention can be tacrolimus (FK506).

[0052] The present invention also provides the use of the bionic glycocalyx layer material described in the above technical solution or the bionic glycocalyx layer material prepared by the preparation method described in the above technical solution or the immunosuppressive composition described in the above technical solution in the preparation of endothelial cell protection products.

[0053] The present invention also provides the use of the bionic glycocalyx layer material described in the above technical solution or the bionic glycocalyx layer material prepared by the preparation method described in the above technical solution or the immunosuppressive composition described in the above technical solution in preparing products that inhibit immune responses in organ transplantation.

[0054] In one embodiment, the organ transplant comprises a heart transplant. In one embodiment, the product is a drug.

[0055] The present invention utilizes the inherent advantages of self-assembling glycopeptide nanotechnology to encapsulate traditional immunosuppressants with biomimetic glycocalyx layer materials (self-assembling glycopeptides), thereby reducing the systemic toxicity of immunosuppressants, prolonging the circulation time and enhancing their accumulation at the disease site. This method can be applied in the early acute immune rejection process to enhance graft protection, thereby preventing transplant rejection, and further protecting endothelial cells from damage and maintaining the integrity of endothelial cells.

[0056] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0057] Example 1

[0058] The present invention uses single-cell RNA sequencing analysis of pig allogeneic heart transplants to identify the characteristics of vulnerable endothelial cells after heart transplantation - high expression of SLC9A3R2, a cell membrane expression molecule. Through verification of human, pig, and mouse specimens, it was found that the damage of endothelial cells with high expression of SLC9A3R2 is consistent in different species.

[0059] In this example, a library was constructed by standard solid phase peptide synthesis using all standard amino acids except cysteine. SLC9A3R2 covalently labeled with the fluorescent dye fluorescein isothiocyanate (FITC, green) was used as the screening target. 100,000 beads from the 7-mer linear OBOC peptide library were incubated with FITC-labeled albumin (100 μg / mL) in phosphate buffered saline (PBS) for 12 h. Strong fluorescent beads were then picked up at 488 nm for sequencing under a fluorescence microscope, and the results are shown in Figure 2. Figure 1 As shown, in Figure 1 A2, A3 and A4 respectively represent [M-4H] 4- , [M-3H] 3- , [M-2H] 2- .

[0060] Depend on Figure 1It can be concluded that the obtained sequences were identified as SBPs, including KQVFKRH (SBP1, SEQ ID NO: 1) and WHIQQIE (SBP2, SEQ ID NO: 3). Among them, SBP1 has a high specific binding ability to SLC9A3R2, with the highest binding affinity of 1.3×10 -9 (Table 1).

[0061] Table 1 Amino acid sequence and related information of SLC9A3R2 targeting peptide

[0062]

[0063]

[0064] Table 1 shows the peptide sequences with high affinity to SLC9A3R2 obtained from OBOC screening, as well as the GRAVY value and isoelectric point value. Through specific binding to SLC9A3R2, cardiac endothelial cells can be targeted, effectively improving the efficiency of endothelial cell engineering.

[0065] Example 2

[0066] This example successfully prepared a biomimetic glycocalyx layer material (EPG), and the specific steps were: using solid phase synthesis technology, using Fmoc coupling chemical method to synthesize the polypeptide KQVFRHG-Sia3Lac-RRRFDVFF (EPG) and the negative control molecule KQVFRHG-RRRFDVFF (EP);

[0067] The compound represented by the structure of formula III and the compound represented by the structure of formula IV are subjected to a glycosyl transfer reaction (reaction formula as shown in Fig.17 As shown), Sia3Lac is generated; the reaction system consists of CMP sialic acid with a final concentration of 50mM, 4-hydroxyethylpiperazineethanesulfonic acid (Hepes) with a final concentration of 50mM and 20mM MgCl2, the temperature of the glycosyl transfer reaction is 37°C, the time is 4h, and the pH value is 7.5.

[0068] EPG and EP were characterized by high performance liquid chromatography (HPLC) and electrospray ionization (ESI) mass spectrometry. Figures 2 to 5 As shown, Figure 2 The molecular structure of EPG (A) and the electrospray ionization mass spectrometry detection result (B) are shown. Figure 3 This is a graph showing the results of high performance liquid chromatography testing of EPG; Figure 4 The molecular structure of EP (A) and the electrospray ionization mass spectrometry (B) results are shown. Figure 5 This is the high performance liquid chromatography test result diagram of EP.

[0069] Depend on Figures 2 to 5 It can be concluded that EPG and EP were successfully prepared.

[0070] Example 3

[0071] In order to evaluate the potential of EPG as a biomimetic glycocalyx layer, the self-assembled nanostructure of EPG was analyzed in detail in this example.

[0072] By adding SLC9A3R2 extracellular receptor protein (i.e., SLC9A3R2 protein), expressed in human umbilical vein endothelial cells (Human UmbilicalVein Endothelial Cells, HUVEC, purchased from the Cell Culture Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences), the nanoparticle morphology was transformed into nanofibers and cultured at 37°C for several hours. Specifically, EPG, SLC9A3R2 protein and HUVEC cells in DMEM culture medium were co-incubated at room temperature, and the amount of SLC9A3R2 protein added was 1 / 10 of the amount of EPG substance. Samples were prepared at 0, 0.5, 2, and 4 hours, and the co-incubation solution was used to measure the size (DLS, NanoZS, Malvern) and transmission electron microscopy (Philips CM-120TEM, USA), and TEM samples were stained with uranyl acetate.

[0073] TEM images showed that the diameter of EPG and EP was about 10 nm at 0 h, and then gradually increased to 20 nm. Nanofiber structures with a diameter of about 9 nm were observed, and the number of nanofibers increased within 2 h. After 4 h, the nanofiber network was clearly visible with a wide size distribution ( Figure 6 ). DLS measurements showed the distribution size of EPG and EP aggregates, confirming the structural transformation from nanoparticles to nanofibers. These results indicate that both EPG and EP have stable nanostructures ( Figure 7 ).

[0074] Example 4

[0075] In this example, EPG labeled with Cy7 was perfused into the mouse donor heart at a perfusion volume of 2 mL for transplantation experiments. Fluorescence imaging of small animals was performed at different time points, and the half-life was then calculated using the fluorescence signal. The results are shown in Figure 2. Figure 8 As shown;

[0076] The steps of labeling EPG with Cy7 are as follows: dissolve EPG in 3 mL of PBS buffer, then add 1 mL of 1 / 10 molar amount of Cy7, add an appropriate amount of triethylamine, and adjust the pH to 9. Stir overnight in an ice bath at 4°C. The reaction solution turns fluorescent blue, and then dialyzes with a dialysis bag with a molecular weight of 1000 Diatoms, changing the water once every 1 hour, and dialyzing for 2 days. The obtained retentate is freeze-dried for standby use.

[0077] Depend on Figure 8 It can be concluded that EPG can prolong the half-life and enhance resistance to immune rejection. The half-life of EPG on the cell surface is as long as 21 hours.

[0078] HUVEC cells at 60-80% confluence were treated with trypsin and plated at 100,000 cells / cm 2 The cells were inoculated at different speeds in the presence of tumor necrosis factor TNF-α (TNF-α, TNF-α purchased from Abcam, CAS: 94948-59-1, 2000 U / mL), EPG (EPG, 50 μM), EFG+TNF (EPG, 50 μM, TNF-α 2000 U / mL) (3 μm pore size, 0.33 cm 2 The HUVEC cells were grown in tissue culture inserts (3415, Corning) with a surface area of ​​1.5 μm and incubated at 37°C and 5% CO2 for 3 hours, and a blank control group was set up. The tissue culture inserts were placed in 24-well plates. HUVEC cells were allowed to grow in tissue culture inserts for 1 week before the transendothelial protein channel experiment, and the cell culture medium was changed every 2 days.

[0079] The passage of FITC-labeled BSA (A92771, Sigma) in the monolayer was tracked for 3 hours. First, the culture medium in both compartments was replaced with serum-free medium for 1 hour. Afterwards, 0.5 mg / mL of FITC-BSA culture medium was added to the upper compartment, and 0.5 mg / mL of unlabeled BSA (A-7888, Sigma) culture medium was added to the lower compartment. Over a period of 3 hours, 100 μL aliquots were collected from the lower compartment every hour and replaced with culture medium containing 0.5 mg / mL unlabeled BSA. The fluorescence of the collected culture medium was recorded, and the concentration was calculated based on the standard curve of FITC-labeled BSA. The results are shown in Figure 2. Fig. 9 shown.

[0080] Depend on Fig. 9 It can be concluded that EPG does not affect the passage of BSA through endothelial cells.

[0081] Example 5

[0082] CLSM and SEM verified the structural transformation of EPG on the cell surface. The steps are as follows:

[0083] HUVEC cells were cultured in glass-bottom dishes for 12 h. EPG (50 μM) was incubated with cells in DMEM at 37 °C for 6 or 12 h. For confocal laser scanning microscopy (CLSM, LSM800, ZEISS) imaging, samples were fixed with glutaraldehyde (4%) for 10 min, washed three times with PBS, and examined using a 405 nm laser and a 40× immersion objective. Fig.10 shown.

[0084] To further verify the binding of EPG to SLC9A3R2, the present invention used rabbit anti-SLC9A3R2 (Abcam, USA) monoclonal antibody (Sigma Aldrich, USA) to detect the extracellular domain of SLC9A3R2 on the surface of HUVEC. Specifically, the rabbit anti-SLC9A3R2 monoclonal antibody was co-incubated with the above-mentioned HUVEC cells bound to EPG in DMEM medium in a 5% CO2, 37°C incubator. For scanning electron microscopy (SEM, Philips XL30 TMP, FEI Company, Hillsboro), the cells were fixed with glutaraldehyde (4%) overnight and then gold plated for 2 minutes ( Fig.11 , the first three icons have a scale of 20 μm, and the last icon has a scale of 5 μm).

[0085] Depend on Fig.10 and Fig.11 It can be concluded that EPG has a good binding ability with SLC9A3R2, and EPG on the cell surface can achieve structural transformation.

[0086] Example 6

[0087] In order to further evaluate the protective effect of EPG on endothelial cells (ECs) in vivo, four groups of mouse heart transplantation (HTx) models were constructed in this example: control group, EPG group, FK506 group and EPG+FK506 group.

[0088] Control group: 2 mL of HTK solution was perfused into the donor heart from the inferior vena cava;

[0089] EPG group: 2 mL of HTK solution containing EPG was perfused into the donor heart from the inferior vena cava, and the final concentration of EPG in HTK solution was 1 mg / mL;

[0090] FK506 group: 2 mL of HTK solution was perfused into the donor heart from the inferior vena cava;

[0091] EPG+FK506 group: 2 mL of HTK solution containing EPG was perfused into the donor heart from the inferior vena cava, and the final concentration of EPG in HTK solution was 1 mg / mL.

[0092] The mouse heart transplantation protocol was based on the cervical heterotopic transplantation model (J Vis Exp. 2014 Oct 12; (92): e50753). Briefly, the mouse heterotopic heart transplantation model was established using BALB / c (H-2d) and C57BL / 6 (H-2b) mice (6-8 weeks old). C57BL / 6 mice were anesthetized and placed in a supine position. After preparing the skin, the layers were cut open to expose and release the left external jugular vein and common carotid artery. Then, the blood vessels were cut, slid onto a specially designed cannula, and fixed with sutures. Under deep anesthesia, the chest cavity of the BALB / c mouse was opened, and the ascending aorta and pulmonary trunk were cut after releasing a sufficient length. The remaining blood vessels were ligated and cut in sequence, and the heart was removed. The donor heart was placed in the recipient's neck, and the aorta was connected to the common carotid artery and the pulmonary trunk to the external jugular vein with sutures. The skin was then sutured and the mice were placed in a warm environment to recover. All model mice were housed in an SPF environment with sufficient food and water. The FK506 and EPG+FK506 groups received 0.5 mg / kg of FK506 daily. The flow chart is shown in Fig.12 shown.

[0093] The survival of all groups was monitored by visual and palpation assessment. The Kaplan-Meier analysis was performed to compare the protective effect of EPG on cardiac allografts. The results showed that the median survival time of the control group and the EPG group was 11 days and 7 days, respectively. Compared with the control group, the graft survival time of the EPG group was slightly increased ( Fig.13 ,exist Fig.13 And the following Figures 14 to 16 PBS represents the control group). As a classic immunosuppressant, the survival time of the 0.5 mg / kg / day FK506 treatment group was 12 days. It is worth noting that the EPG+FK506 group achieved a survival time of more than 30 days ( Fig.14 ).

[0094] In order to further evaluate the protective effect of EPG on the endothelium, the mouse heart grafts of the control group and the EPG group were obtained in this example 3 days after surgery. The 3 days are the period of most serious endothelial damage after heart transplantation, and early endothelial protection is crucial for heart grafts. In this example, routine HE pathological examination was performed, and T cell and macrophage surface markers CD3 (ZA-0503, zsbio) and CD68 (ZM-0060, zsbio) were stained to evaluate endothelial integrity and the degree of myocardial inflammatory infiltration. In the control group, obvious endothelial swelling and shedding were observed due to IRI and immune damage, while the EPG endothelium remained intact and tightly attached to the vascular lumen. At the same time, the EPG group had less immune cell infiltration than the control group ( Fig.15 ).

[0095] In order to further evaluate the protective effect of EPG on the long-term survival of heart transplants, the mouse heart transplants of the FK506 group and the FK506+EPG group were obtained in this example 7 days after surgery. The heart transplants that did not receive any treatment were rejected within 7 days, so 7 days is the standard observation time for the therapeutic effect after heart transplantation. This example also performed routine HE pathological examinations, and stained the T cell and macrophage surface markers CD3 (ZA-0503, zsbio) and CD68 (ZM-0060, zsbio) to assess endothelial integrity and the degree of myocardial inflammatory infiltration. The results of 7 days showed that the FK506+EPG group had minimal T cell and macrophage infiltration ( Fig.16 ).

[0096] It can be concluded from the above examples that the bionic glycocalyx layer material of the present invention can effectively inhibit the immune response during organ transplantation, thereby protecting endothelial cells, reducing the damage of immune response to endothelial cells, and maintaining the integrity of endothelial cells.

[0097] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A bionic glycocalyx layer material, characterized in that: The raw materials for preparing the bionic glycocalyx layer material include polypeptide SBP1, self-assembling peptide FFVDF and sialyl lactose, wherein the sialyl lactose is coupled with a peptide bond formed by dehydration condensation of the polypeptide SBP1 and the self-assembling peptide FFVDF via an azide group; the amino acid sequences of the polypeptide SBP1 and the self-assembling peptide FFVDF are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

2. The bionic glycocalyx layer material according to claim 1, characterized in that: The structure of the bionic glycocalyx layer material is shown in Formula I:

3. The method for preparing the bionic glycocalyx layer material according to claim 1 or 2, characterized in that: include: The bionic glycocalyx layer material is prepared by solid phase synthesis technology using polypeptide SBP1, self-assembling peptide FFVDF and sialyl lactyl sugar as raw materials.

4. The preparation method according to claim 3, characterized in that: The feeding molar ratio of the polypeptide SBP1, the self-assembling peptide FFVDF and sialyl lactyl sugar is 1:1:

2.

5. An immunosuppressive composition, characterized in that: The immunosuppressive composition comprises independently packaged bionic glycocalyx layer materials and immunosuppressants; the bionic glycocalyx layer material is the bionic glycocalyx layer material according to claim 1 or 2 or the bionic glycocalyx layer material prepared by the preparation method according to claim 3 or 4.

6. The immunosuppressive composition according to claim 5, characterized in that The immunosuppressant includes tacrolimus.

7. Use of the bionic glycocalyx layer material according to claim 1 or 2, or the bionic glycocalyx layer material prepared by the preparation method according to claim 3 or 4, or the immunosuppressive composition according to claim 5 or 6 in the preparation of endothelial cell protection products.

8. Use of the bionic glycocalyx layer material according to claim 1 or 2, or the bionic glycocalyx layer material prepared by the preparation method according to claim 3 or 4, or the immunosuppressive composition according to claim 5 or 6 in preparing a product that inhibits immune response in organ transplantation.

9. The use according to claim 8, characterized in that: The organ transplant includes a heart transplant.

10. The use according to any one of claims 7 to 9, characterized in that: The products include pharmaceuticals.

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