Method for constructing artificial red blood cells based on enucleated mesenchymal stem cells and application of artificial red blood cells

Abstract

The invention relates to the technical field of biology, in particular to a method for constructing novel artificial erythrocytes based on enucleated mesenchymal stem cells. A novel artificial red blood cell construction method is to be established, hemoglobin alpha and beta subunits are subjected to fusion expression, bone marrow mesenchymal stem cells are taken as a carrier, a stably transfected cell strain capable of stably expressing hemoglobin tetramer is constructed in a lentivirus infection mode, and the stable transfected cell strain has oxygen carrying and oxygen releasing functions. And removing the cell nucleus of the stably transfected cell strain by adopting a centrifugal enucleation method to obtain the artificial red blood cell which is similar to the mature red blood cell in size and has an oxygen transportation function. The artificial red blood cells constructed by the method can be used as a blood substitute to replace red blood cells in blood in a short time to play a role in conveying oxygen under the emergency conditions of insufficient blood reserves or massive hemorrhage and the like at present, so that ischemic symptoms and complications of patients are relieved, and precious rescue time and extra blood source waiting time are won for the patients.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for constructing novel artificial red blood cells based on enucleated bone marrow mesenchymal stem cells and its application. Background Art

[0002] Patients with massive hemorrhage are critically ill and progress rapidly. Acute, massive blood loss leads to a sharp drop in blood volume, a decrease in effective circulating blood volume, and circulatory system failure. This makes it impossible to maintain normal blood pressure, blood flow, and blood perfusion, affecting oxygenation and causing hypoxia in vital organs such as the kidneys, heart, and brain, leading to metabolic acidosis and the release of inflammatory factors. Therefore, early intervention for hemorrhagic shock is extremely important.

[0003] For interventional treatment of hemorrhagic shock, the first step is to stop bleeding, followed by resuscitation. Large volumes of intravenous blood infusion to replenish blood components, such as red blood cells and plasma, are crucial and are a core measure in the treatment of hemorrhagic shock and the first choice for clinical care. Resuscitation is often accompanied by the use of crystalloid solutions (such as saline and Ringer's solution) or colloid solutions (such as albumin and hydroxyethyl starch) to maintain fluid volume and restore blood flow.

[0004] However, existing blood transfusion treatment strategies have many limitations. First, cross-matching is required, which leads to untimely blood transfusions in emergency situations. Second, plasma has been in a state of long-term supply shortage, and the global blood supply is unable to meet demand. At the same time, the aging population makes the blood shortage situation more serious in the future. In addition, the transportation of blood-borne pathogens such as human immunodeficiency virus (HIV), hepatitis, Creutzfeldt-Jakob disease or West Nile virus carried by plasma is also a key issue in blood transfusion therapy. Blood products have a short lifespan of only 42 days, and the storage conditions are also very strict. Over time, the red blood cell (RBC) membrane loses its elasticity, the cells leak potassium, and become more susceptible to hemolysis.

[0005] Blood transfusions are subject to numerous limitations, including imbalances in blood supply and demand, cross-matching, and pathogen contamination. Using blood substitutes (BMS) to transport oxygen, replenish blood volume, and maintain circulatory stability is another approach to treating acute massive bleeding. Currently, this approach primarily utilizes hemoglobin-based oxygen carriers (HBOCs).

[0006] Hemoglobin oxygen carrier (HBOC) is an artificial blood produced by extracting and processing hemoglobin (Hb) and chemically modifying or encapsulating it. This improves the stability of hemoglobin, prolongs its half-life in the blood, and reduces its toxicity. Technical Principle: HBOC is generally prepared using hemoglobin purified from expired blood, bovine hemoglobin, porcine hemoglobin, etc., through chemical modification and encapsulation. Typical chemically modified HBOCs include the early use of cross-linking agents such as fumaric acid (3,5-dibromosalicylate) (DBBF) to link monomeric Hb to form cross-linked HBOCs. Polymeric HBOCs are currently prepared by reacting glutaraldehyde with lysine side chains to form Schiff bases, such as the human-derived Polyheme produced by Northfield Corporation in the United States and the bovine-derived HBOC201 (Hemopure) produced by Biopure.

[0007] Encapsulating hemoglobin within liposomes or micro-nanocarriers, whose structure and function are closer to natural red blood cells, more accurately simulates the physiological encapsulation state of Hb in red blood cells, can avoid or reverse the conversion of Hb to metHb, enhance storage stability, prevent rapid uptake by macrophages, and significantly prolong circulation retention time. In addition, some studies have wrapped natural red blood cell membranes around particles containing hemoglobin to obtain artificial red blood cells (ARTIFICIAL RED BLOOD CELLS) or rebuilt red blood cells (REBBCs), which are more similar to red blood cells. This RRBC is made by fixing purified red blood cells and silicifying them to obtain silica-red blood cell replicas. These are then coated with positive chitosan polymers and negative alginate polymers. After etching, the purified red blood cell ghosts are covered on these replicas to obtain RRBC particles. These particles can be loaded with hemoglobin or other components, such as drugs, to perform functions such as oxygen or drug delivery.

[0008] HBOC-coated hemoglobin is easily broken down in the body, releasing free iron. This is toxic to cells and can also promote peroxidation (free radical generation), leading to oxidative damage and potentially triggering a series of inflammatory responses, leading to acute kidney injury. Compared to natural red blood cells, HBOCs are more likely to bind NO (nitric oxide) in the body, potentially causing vasoconstriction and leading to hypertension. The extraction, modification, and purification of hemoglobin in HBOC production is complex and requires strict quality control and purification, resulting in high costs. Artificial red blood cells, prepared by encapsulating red blood cell ghosts on nanoparticles, cannot avoid cross-matching during intravenous injection due to the presence of natural red blood cell membranes. The hemoglobin and red blood cell membranes used are extracted and purified from exogenous sources, limiting their sources. The preparation process is complex and time-consuming, and the exogenous proteins may also cause an immune response in the body.

[0009] Artificial induction of stem cells to differentiate into red blood cells in vitro is also one of the current ways to prepare blood substitutes. Technical principle: Stem cells from various sources, including adult peripheral blood (PB), umbilical cord blood (CB) and pluripotent stem cells (PSCs), can be induced to differentiate into red blood cells and have been used as the source of in vitro red blood cell culture systems. However, these cells have limited proliferation capacity, which limits the number of red blood cells that can be obtained and most cells produce hemoglobin as HbF instead of HbA. In 2017, a team developed a new strategy to culture immortalized human adult erythrocyte lines (BEL-A) from early immature red blood cells, which can continue to proliferate. These immortalized cell lines can be cultured in the laboratory and effectively differentiated into mature, functional red blood cells.

[0010] Inducing stem cells to differentiate into red blood cells in vitro is expensive, and the production of artificial red blood cells is low. Furthermore, after differentiation, the cells normally express red blood cell surface antigens, necessitating cross-matching. This cross-matching requirement necessitates a large number of specialized donors for specific blood types, further increasing time and cost, and making emergency supplies impossible. Furthermore, because ordinary stem cells have a very limited capacity for proliferation, the need for long-term, large-scale stem cell donations is unavoidable. Summary of the Invention

[0011] The present invention proposes a novel method for constructing artificial red blood cells (ARBCs). This method involves fusion expression of the α and β subunits of hemoglobin, using bone marrow mesenchymal stem cells as a vector. Through lentiviral infection, a stably transfected cell line expressing hemoglobin tetramers is constructed, exhibiting both oxygen-carrying and oxygen-releasing capabilities. Centrifugal enucleation is then used to remove the nuclei of the transfected cell line, yielding artificial RBCs similar in size to mature RBCs and capable of oxygen transport. The artificial RBCs constructed using this method could serve as a blood substitute in emergency situations such as insufficient blood supplies or severe hemorrhage, replacing the oxygen transport function of RBCs in the blood for a short period of time. This could alleviate the patient's ischemic symptoms and complications, buying valuable time for rescue and additional waiting time for a blood source.

[0012] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0013] A two-subunit fusion expression system comprises a first expression group and a second expression group, wherein the first expression group and the second expression group are connected via a self-cleaving short peptide fragment; the first expression group comprises a first subunit fragment and a first tag protein fragment; the second expression group comprises a second subunit fragment and a second tag protein fragment.

[0014] Preferably, in the first expression group, the first subunit fragment is fused with the first tag protein fragment at the N-terminal; and in the second expression group, the second subunit fragment is fused with the second tag protein fragment at the N-terminal.

[0015] Preferably, the double subunit fusion expression system is expressed by recombination with a backbone plasmid to form a recombinant expression plasmid; the backbone plasmid comprises a promoter, a fluorescent protein expression sequence, and a resistance gene; and the fluorescent protein expression sequence is connected to the first expression group through the self-cleavage short peptide fragment.

[0016] Preferably, the self-cleavage short peptide fragment comprises a P2A fragment; the first subunit fragment comprises a hemoglobin alpha subunit fragment; the first tag protein fragment comprises a Myc tag protein fragment; the second subunit fragment comprises a hemoglobin beta subunit fragment; the second tag protein fragment comprises an HA tag protein fragment; the backbone plasmid is a pLenti-GFP plasmid; the promoter comprises a CM promoter; the fluorescent protein expression sequence comprises an EGFP fluorescent protein expression sequence; and the resistance gene comprises a blasticidin resistance gene.

[0017] The present application utilizes BMSC-Hb (bone marrow mesenchymal stem cells) stably expressing hemoglobin, and fuses hemoglobin alpha and beta subunits through P2A to express in vivo, releases through self-cleavage in the cell body, and assembles into hemoglobin tetramer with oxygen carrying function. After the BMSC-Hb is enucleated, the hemoglobin is coated in the natural stem cell membrane, which retains the low immunogenicity of stem cells, has a diameter close to the size of natural red blood cells, and has the function of red blood cell oxygen transport. The present application can produce a large amount of oxygen carriers similar to natural red blood cells, i.e. artificial red blood cells, in a short time, which have low immunogenicity and do not need to be matched, and can be used as a new type of blood substitute.

[0018] Preferably, the sequence of the double subunit fusion expression system is shown in SEQ ID NO: 3.

[0019] The present application also provides an application of the double subunit fusion expression system, which is used to construct one or more of artificial red blood cells, enucleated artificial red blood cells.

[0020] Preferably, the application comprises the following steps:

[0021] A. linearizing a backbone plasmid by enzyme digestion and recovering the linearized plasmid by gel recovery, and then obtaining the double subunit fusion expression system by PCR reaction, and homologously recombining the double subunit fusion expression system with the linearized plasmid to obtain a recombinant expression plasmid;

[0022] B. transfecting the recombinant expression plasmid into ordinary cells, and culturing, purifying, and concentrating to obtain a lentivirus packaging expression body;

[0023] C. Infect stem cells with the lentiviral packaged expression vector, screen and culture, and obtain stably transfected cell lines, thereby forming artificial red blood cells.

[0024] Compared to technologies such as HBOC, which use purified exogenous hemoglobin as the primary material, the purification process is complex, and the exogenous protein may cause an immune response. The present invention constructs a new type of artificial red blood cell (ARBC) by fusion-expressing the α and β subunits of hemoglobin. Using a third-generation lentiviral packaging system, infectious lentiviral particles expressing the α and β subunits of hemoglobin are successfully packaged. The lentiviral particles are then collected and infected with bone marrow mesenchymal stem cells (BMSCs), and a stably transfected BMSC-Hb cell line expressing the α and β subunits is screened. Within the BMSC-Hb cells, the α and β subunits are released through P2A auto-cleavage and autonomously assemble into hemoglobin tetramers with oxygen-carrying and oxygen-releasing functions. By removing the cell nucleus through centrifugation, the resulting hemoglobin-encapsulated cytoplasmic vesicles are closer in size to natural RBCs, while retaining the stem cell's low immunogenicity and oxygen transport function. Artificial RBCs constructed in this manner omit the complex hemoglobin purification and processing steps required for HBOCs. Cells transiently transduced with plasmids will experience plasmid loss during cell passage, whereas stable cell lines screened through lentiviral transduction consistently express hemoglobin, allowing for long-term storage and large-scale expansion. Using BMSCs as a vector avoids blood cross-matching and exhibits low immunogenicity.

[0025] Preferably, the method further comprises the following steps:

[0026] D. The stably transfected cell line is subjected to enucleation treatment to obtain enucleated artificial red blood cells.

[0027] The various cross-linking agents used in technologies such as HBOC to cross-link or embed hemoglobin will produce certain toxicity. However, particles coated with hemoglobin ghosts still need to be cross-matched due to the presence of proteins on their membranes, and the step of coating the cell membrane further prolongs the preparation time. The present invention uses bone marrow-derived mesenchymal stem cells BMSC as a carrier of hemoglobin, which has similar oxygen-carrying and oxygen-releasing functions as red blood cells. After removing the cell nucleus, the size is close to that of natural red blood cells and there is no carcinogenic risk. At the same time, hemoglobin is wrapped by the stem cell membrane, which retains the low immunogenicity of stem cells, has low cytotoxicity, and reduces the time for cross-matching.

[0028] Preferably, the method comprises the following steps:

[0029] A1. Enzyme digestion of the backbone plasmid and gel extraction to obtain linearized plasmid;

[0030] A2. obtaining the dual-subunit fusion expression system by PCR reaction;

[0031] A3. homologously recombining the dual-subunit fusion expression system with the linearized plasmid to obtain a recombinant expression plasmid; the sequence of the recombinant expression plasmid is shown in SEQ ID NO: 5;

[0032] B1. Add DMEM high glucose culture medium to a 10 cm cell culture dish and inoculate 4×10 6 293T cells were cultured in a 37°C incubator. Before transfection, the 293T cells were replaced with fresh DMEM high-glucose culture medium to obtain 293T culture medium for packaging lentivirus.

[0033] B2. Add the plasmid composition for a 10 cm dish to 400 μL of Optical-MEM, mix well, and incubate at room temperature for 5 minutes to obtain the incubated plasmid; the plasmid composition for a 10 cm dish comprises: the recombinant expression plasmid: 12 μg, pMDL: 8 μg, VSVG: 3.2 μg, REV: 4.4 μg, and Total plasmid: 27.6 μg;

[0034] B3. Mix the transfection reagent PEI with 400 μL Opti-MEM and incubate at room temperature for 5 minutes to obtain the incubated transfection reagent; the mass ratio of the transfection reagent PEI to the plasmid composition for the 10 cm dish is 1.5:1;

[0035] B4. Mix the incubated plasmid and the incubated transfection reagent, and incubate at room temperature for 15 minutes to obtain a transfection solution;

[0036] B5. Add the transfection solution to the 293T culture medium and, after transfection for 4 to 6 hours, replace it with fresh DMEM high-glucose culture medium;

[0037] B6. 16 hours after transfection, the culture medium was replaced with collection medium, and the cells were continued to be cultured in an incubator. The collection medium was prepared by adding 0.5 g of BSA and a HEPES buffer having a final concentration of 10-15 mM to 50 mL of complete culture medium without antibiotics, adjusting the pH to 7.4, and filtering through 0.22 μm to obtain the collection medium.

[0038] B7. Collect cell culture fluid 45 h, 54 h, 64 h, 72 h, and 96 h after transfection;

[0039] B8. Centrifuge the cell culture medium at 800 g for 10 min, filter the supernatant with a 0.45 μm filter membrane, add PEG to a final PEG concentration of 10%, mix well, and incubate at 4°C with slow shaking for more than 4 h. Centrifuge at 2000 g for 30 min at 4°C, remove the supernatant, and resuspend in 1 / 20 volume of DMEM high-glucose culture medium to obtain lentiviral packaging expression vectors. Store at 4°C for a short time or at -80°C for a long time.

[0040] C1. The lentiviral expression vector was packaged with polybrene at a ratio of 1000:1 and then added to a 96-well plate to infect BMSC cells. After 24 hours, the medium was replaced with fresh DMEM high-glucose culture medium. After green fluorescence was observed under a microscope, the primary infected cell line was obtained.

[0041] C2. Digest the primary infected cell line, count, and dilute to a cell density of 10-500 cells / mL. Take 100 μL and add it to a 96-well plate, ensuring that each well contains 1-5 cells. After the cells have attached and grown to an appropriate density, observe the fluorescence under a microscope again and look for wells with green fluorescent cells. If any non-fluorescent cells remain, scrape them off. Repeat this operation 1-3 times until only green fluorescent secondary infected cells remain in the wells.

[0042] C3. Expanding the secondary infected cells to obtain a stable cell line, i.e., forming artificial red blood cells;

[0043] D1. Separate the 24-well plate and wash the separated individual 24 wells. After cleaning, soak them in 75% alcohol. Remove them from the alcohol before use, dry them in a clean bench, and sterilize them by ultraviolet irradiation to obtain a clean 24-well plate.

[0044] D2, inoculating African green monkey kidney fibroblasts cos-7 in the clean 24-well plate, and when the cell density of the African green monkey kidney fibroblasts cos7 reached about 90%, adding ammonia water and letting it stand at room temperature for about 5-10 minutes to lyse the African green monkey kidney fibroblasts cos7 until no cell structure was observed under a microscope, removing the liquid in the wells, adding PBS buffer and washing three times, and then using sterilized ddH2O to wash again three times, and finally adding about 500 μL PBS to keep the 24 wells moist to prepare 24 wells coated with ECM;

[0045] D3, 5×10 5 The artificial red blood cells are cultured in a cell culture incubator at 37° C. for 24 to 48 hours, and the pre-enucleated cells are obtained when the cell density reaches about 90%.

[0046] D4. Place the pre-enucleated cells upside down in a 50 mL centrifuge tube containing 10 mL of preheated enucleation solution, and centrifuge at 35°C, 4500 g, for 1 hour to perform enucleation. Each 10 mL of the enucleation solution contains: 8 mL of calcium-free DMEM, 100 μL of 1 mg / mL cytochalasin B, 5 μL of 10 mg / mL colchicine, 2 mL of 10% sucrose, 40 μL of 500 mM N-acetylcysteine, and 10 μL of 100 μM calcium chloride solution.

[0047] D5. After the enucleation operation is completed, the cells are taken out, the enucleation solution is removed, and fresh DMEM high-glucose culture medium is added. The cells are placed in a 37°C incubator for recovery for 2 hours to obtain enucleated artificial red blood cells.

[0048] A product obtained by the above application includes one or more of artificial red blood cells and enucleated artificial red blood cells.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] 1. Low toxicity: The bone marrow mesenchymal stem cells (BMSCs) used in the present invention have been reported to have very low immunogenicity and therefore will not cause a severe immune response in the body.

[0051] 2. Wide range of applications: Since natural BMSC cells do not require strict cross-matching when injected, they can be used even for rare blood types. In the case of blood shortage, the present invention can be used as an emergency blood substitute in the short term.

[0052] 3. Easy to preserve: The storage of stably transfected cells BMSC-Hb only requires the use of ultra-low temperature refrigerators or liquid nitrogen, which makes it easy to revive and has a long shelf life.

[0053] 4. Simple preparation method: The method for preparing a blood substitute of the present invention is simple and can be completed by simply resuming BMSC-Hb and then centrifuging and removing the nucleus in a nucleation solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 A flow chart was constructed for the BMSC-Hb stabilization of hemoglobin.

[0055] Figure 2 This is the map of pLenti-GFP.

[0056] Figure 3 Design of pLentil-Hb map and hemoglobin subunit gene fusion expression, including: (A) plasmid pLenti-Hb map; (B) schematic diagram of hemoglobin subunit fusion expression design.

[0057] Figure 4For the construction of pLenti-Hb plasmid, PCR was performed to obtain Figure 3 Schematic diagram of the designed target gene fusion expression fragment bands.

[0058] Figure 5 pLenti-GFP and pLenti-Hb were transfected into HEK293 cells to verify plasmid expression. (A) Ad293 cells were transfected with pLenti-GFP and pLenti-Hb, respectively, and immunoblotting analysis was performed using HA antibodies (A) and Myc antibodies (B), respectively, as primary antibodies; (C-D) Ad293 cells were transfected with pLenti-GFP and pLenti-Hb, respectively, and immunofluorescence staining was performed using HA antibodies (C) and Myc antibodies (D), respectively, as primary antibodies. Scale bar: 200 μm.

[0059] Figure 6 To verify the effectiveness of lentivirus infection in HEK293 cells, (A) HEK293 cells were infected with lentiviruses LV-GFP and LV-Hb, and immunofluorescence staining was performed using a Myc tag antibody as the primary antibody; scale bar, 100 μm; (B) HEK293 cells were infected with lentiviruses LV-GFP and LV-Hb, and immunofluorescence staining was performed using a HA tag antibody as the primary antibody; scale bar, 100 μm.

[0060] Figure 7 Flowchart for stable cell screening.

[0061] Figure 8 The results of immunoblotting detection of different BMSC-Hb stable cell lines.

[0062] Figure 9 This is an immunofluorescence analysis of hemoglobin expression in different stably transfected BMSC-Hb cell lines.

[0063] Figure 10 (A) Native-PAGE protein gel stained with Coomassie Brilliant Blue; M: Marker; Lanes 1-4 represent BMSC-GFP, BMSC-Hb5, BMSC-7.2, and bovine hemoglobin (BHb), respectively. The red box indicates the newly emerged BMSC-Hb band near the location of bovine serum albumin, and the black arrow indicates the tetrameric hemoglobin band. (B) Immunoblotting analysis of hemoglobin tetramer expression in the stably transfected cell lines using Hb-β and HA as primary antibodies, respectively.

[0064] Figure 11UV-visible spectral analysis under oxygenated and deoxygenated states, including (AC) UV-visible spectral analysis of red blood cells (A), BMSC-GFP (B), and BMSC-Hb (C) in the blood of C57BL / 6 mice under oxygenated (red curve) and deoxygenated (blue curve) states at 365-650 nm.

[0065] Figure 12 Flowchart for enucleated cell preparation.

[0066] Figure 13 Morphological observation of enucleated BMSC-Hb cells. (A) Confocal microscopy of BMSC-Hb cell morphology before and after enucleation. Dil was used to label the cell membrane, and Hoechst 33342 was used to label the nucleus. Scale bar, 10 μm. (B) Diameter of BMSC-Hb cells before enucleation. (C) Diameter of BMSC-Hb cells after enucleation.

[0067] Figure 14 Activity detection of enucleated BMSC-Hb cells. (A) Fluorescence microscopy observation of fluorescent protein expression at different times after enucleation of BMSC-Hb cells. Scale bar, 200 μm. (B) Cell survival rate curve of stably transfected BMSC-Hb cell line at different times after enucleation.

[0068] Figure 15 Figure 3: Cytotoxicity analysis of enucleated BMSC-Hb on HUVEC and HEK293t cells. (A) CCK8 assay was used to detect the cytotoxicity of different concentrations of enucleated BMSC-Hb cells on HUVEC cells; (B) CCK8 assay was used to detect the cytotoxicity of different concentrations of enucleated BMSC-Hb cells on HEK293t cells. DETAILED DESCRIPTION

[0069] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be described in further detail below with reference to the accompanying drawings.

[0070] Example 1

[0071] Technical solution improvement design such as Figure 1 shown.

[0072] 1. Construction of plasmid pLenti-GFP-3Myc-Hba-3HA-Hbb (hereinafter referred to as pLenti-Hb)

[0073] The present invention uses pLenti-GFP plasmid as the backbone plasmid ( Figure 2 ), the plasmid contains CMV promoter, EGFP fluorescent protein expression sequence, and bleomycin resistance gene. After transfection into cells, the expression of green fluorescent protein can be used to determine whether the transfection is successful. The present invention designs the following on the basis of pLenti-GFP:Figure 3 The hemoglobin α and β double subunit fusion expression system shown. The hemoglobin gene is expressed in series, and Myc and HA tag proteins are fused to the N-termini of the α and β subunits respectively. The self-cleavage effect of the short peptide P2A is used to allow the two subunits to be cut apart after expression and become independent protein monomers. The sequence of the hemoglobin α subunit Hba-a1 is shown in SEQ ID NO: 1, and the sequence of the β subunit Hbb-b1 is shown in SEQ ID NO: 2 (both can be obtained from the website https: / / www.ncbi.nlm.nih.gov / nucleotide, NM_008218.2, NM_001278161.1). Lenti-GFP was cut with BsrGI enzyme, and the linearized plasmid was obtained by gel cutting and recovery. Then, through PCR reaction, the target band of 1.3kb (3XMyc-Hba-3XHA-Hbb) was obtained ( Figure 4 ), the sequence is shown in SEQ ID NO: 3.

[0074] The recovered target fragment was homologously recombined with the pLenti-GFP vector linearized by the endonuclease BsrGI, and transformed into Escherichia coli. Single clones were selected for colony PCR verification, and sequencing confirmed that the target sequence was consistent with the expected sequence to obtain the recombinant plasmid pLenti-Hb. pLenti-GFP (control) and the operational plasmid pLenti-Hb were then extracted using a plasmid extraction kit, transfected into HEK293 cells by Ployjet, and cultured for 24 hours before protein immunoblotting and immunofluorescence staining. The sequence of the control pLenti-GFP is shown in SEQ ID NO: 4, and the sequence of the operational plasmid pLenti-Hb is shown in SEQ ID NO: 5.

[0075] like Figure 5 As shown in AB, the control group pLenti-GFP does not express Myc and HA, while the cells transfected with pLenti-Hb have signal bands of Myc and HA, and the band sizes are consistent with expectations, indicating that both subunits of hemoglobin can be successfully expressed. To further verify the expression of the target protein, the present invention subsequently performed a cell immunofluorescence staining experiment. The transfected HEK293 cells showed obvious green fluorescence, indicating that both plasmids pLenti-GFP and pLenti-Hb can be effectively transfected into the cells. Compared with the control group, the HEK293 cells transfected with the pLenti-Hb plasmid had obvious HA signals and also expressed Myc protein ( Figure 5 CD), indicating that the fusion expression plasmid was successfully constructed and both subunits α and β of hemoglobin were successfully expressed.

[0076] 2. Lentiviral Packaging

[0077] A. Day 1:

[0078] DMEM high glucose medium was added to a 10 cm cell culture dish and 4 × 10 6 293T cells were cultured in a 37°C incubator.

[0079] B. Day 2:

[0080] Transfection:

[0081] 15:00-16:00 Replace with fresh DMEM high-glucose culture medium;

[0082] 17:00 Add plasmid and transfection reagent to the dish;

[0083] Table 1. Plasmids used for lentiviral packaging

[0084]

[0085] 1) Add the plasmids described in Table 1 to 400 μL Optical-MEM, mix well, and incubate at room temperature for 5 minutes;

[0086] 2) Mix PEI with 400 μL Opti-MEM at a ratio of 1.5 μL per μg of plasmid and incubate at room temperature for 5 minutes.

[0087] 3) Mix the plasmid and PEI solution and incubate at room temperature for 15 minutes;

[0088] 4) Add the transfection solution to a 10 cm dish and mix gently;

[0089] 21:00-23:00: 4-6 hours after transfection, replace the culture medium;

[0090] C. Day 3:

[0091] 16 h after transfection, the culture medium was replaced with collection medium and the cells were continued to be cultured in the incubator;

[0092] Collection medium:

[0093] 0.5 g BSA and HEPES buffer (pH = 7.4) with a final concentration of 10-15 mM were added to 50 mL complete culture medium (without antibiotics) and filtered through 0.22 μm.

[0094] D. Subsequently, the cell culture medium was collected 45 h, 54 h, 64 h, 72 h, and 96 h after transfection (the viral particles produced after the four plasmids were packaged were released into the culture medium);

[0095] E. Lentivirus purification and concentration:

[0096] 1) Centrifuge the cell culture medium (virus solution) at 800 g for 10 min and filter the supernatant through a 0.45 μm filter membrane;

[0097] 2) Add PEG to the filtered supernatant to a final PEG concentration of 10% and mix thoroughly;

[0098] 3) Place the virus at 4°C and shake slowly for more than 4 hours;

[0099] 4) Centrifugation at 2000 g for 30 min at 4°C;

[0100] 5) Remove the supernatant and resuspend the virus in DMEM (1 / 20 the volume of the collected lentiviral solution);

[0101] Lentivirus can be stored temporarily at 4°C or at -80°C.

[0102] The collected lentivirus was used to infect HEK293 cells, and the culture medium was changed to DMEM after 12 hours. Immunofluorescence experiments were performed 24 hours later to verify the effectiveness of the lentivirus. Since the α and β subunits of hemoglobin were tagged with Myc and HA proteins respectively when constructing the plasmid, the present invention uses these two tags as signals to characterize the expression of the two subunits of hemoglobin. Figure 6 As shown, the infected Ad293 produced green fluorescence. After immunofluorescence staining using antibodies to the tag proteins Myc and HA, respectively, the cells were observed and photographed under an inverted fluorescence microscope. The results showed that the HEK293 cells that expressed green fluorescence signals also expressed red fluorescence signals, indicating that after the lentiviral particles successfully infected the HEK293 cells, the cells expressed hemoglobin, indicating that the virus was successfully packaged.

[0103] 3. Construction of Stable BMSC-GFP and BMSC-Hb Cells

[0104] On the one hand, the efficiency of transient transfection of ordinary plasmids into BMSC cells is low, so lentiviral infection is used; on the other hand, lentiviral infection can integrate exogenous fragments into the host cell genome to obtain stable cell lines. The collected lentiviral solution is mixed with polybrene at a ratio of 1000:1 and added to 96 wells to infect BMSC cells. After 24 hours, it is replaced with fresh DMEM high-glucose culture medium and then fluorescence observation is performed. It usually takes 2-3 days for BMSC cells to begin to show green fluorescence. Figure 7As shown, after observing green fluorescence under a microscope, the cells are digested, counted, and diluted to a cell density of 10-500 cells / mL. 100 μL is then added to a 96-well plate to ensure that each well contains 1-5 cells. After the cells adhere and grow to an appropriate density, the fluorescence is observed again under a microscope to find wells with green fluorescent cells. If there are still non-fluorescent cells, these cells are scraped off to leave only green fluorescent BMSCs in the wells. This process may need to be repeated 1-3 times to ensure that all cells in a well are green fluorescent BMSCs. After the cells are expanded and verified, stably transfected BMSC-GFP and BMSC-Hb are constructed.

[0105] Using green fluorescence as a signal screening, the present invention obtained multiple monoclonal BMSC-Hb stable cell lines and tested whether hemoglobin was stably expressed by WB. The WB results showed that all five monoclonal clones had HA signals ( Figure 8 ), suggesting that these cell lines can express hemoglobin β subunit. Next, the present invention further verified the expression of hemoglobin by immunofluorescence staining. Figure 9 After staining with Myc and HA antibodies, the five monoclonal cell lines all expressed Myc and HA, indicating that the obtained cell lines can successfully express hemoglobin α and β subunits. Figure 8 Immunoblotting and Figure 9 Based on the results of immunofluorescence and cell status, the present invention selected Hb5 and Hb7.2 cell lines with higher expression levels and better status for subsequent functional experiments.

[0106] 4. Stable BMSC-Hb cell line can express tetrameric hemoglobin

[0107] Hemoglobin can only transport oxygen normally in the form of tetramers. Immunofluorescence results showed that the constructed BMSC-HB stable cell line can successfully express the α and β subunits (such as Figure 9 ), but whether these subunits can assemble into a tetrameric form with oxygen-carrying function inside BMSCs cells requires further testing and analysis. The present invention uses the Native-PAGE method to detect whether BMSC-Hb can express tetrameric hemoglobin, and uses a protein solution prepared from finished bovine hemoglobin powder as a positive control. After electrophoresis, the protein gel was stained with Coomassie Brilliant Blue staining solution. Compared with the negative control BMSC-GFP cells, new bands appeared in the BMSC-Hb5 and BMSC-Hb7.2 cell samples at a position close to the positive control bovine hemoglobin band, as shown in Figure 2. Figure 10 The red box, however, whether it is tetrameric hemoglobin requires further analysis.

[0108] To verify whether the band in the red box is hemoglobin tetramer, hemoglobin β subunit antibody and HA antibody were used as primary antibody for immunoblotting detection, respectively. After electrophoresis, the protein gel was transferred to the membrane. The hemoglobin β subunit antibody was used as the primary antibody, and the signals of BMSC-Hb and bovine hemoglobin were detected. The position of the signal of bovine hemoglobin was consistent with the result of the staining, indicating that the bands expressed by the stable cell strains BMSC-Hb5 and BMSC-Hb7.2 were likely to be tetrameric hemoglobin Figure 10 Next, HA antibody was used for further detection. Compared with the control group BMSC-GFP which did not express hemoglobin and the bovine hemoglobin sample without a label protein, HA was detected in BMSC-Hb5 and BMSC-Hb7.2, and the position of the HA signal was the same as that detected by the hemoglobin β subunit antibody Figure 10 B), and consistent with the band of the staining result Figure 10 The black arrow indicates), indicating that the band was indeed tetrameric hemoglobin. The results of Native-PAGE and immunoblotting proved that the stable cell strains BMSC-Hb5 and BMSC-Hb7.2 could successfully express the α and β subunits of hemoglobin, and after expression, they could successfully self-assemble into tetramers, which had the potential to transport oxygen.

[0109] 5. Oxygen carrying capacity of stable cell BMSC-Hb

[0110] Hemoglobin has completely different ultraviolet-visible absorption spectra in deoxygenated and oxygenated states. By analyzing the changes in ultraviolet-visible light (350-650 nm) in the oxidized and deoxygenated states, the reversible binding capacity of BMSC-Hb to oxygen was detected. To completely deoxygenate hemoglobin, carbon dioxide was first introduced into the cell suspension to replace oxygen. After aeration for at least 3 h, sodium dithionite (Na2S2O4) was added (concentration less than 0.1% w / v) to completely deoxygenate hemoglobin in the cells, and the ultraviolet-visible light absorption spectrum was obtained by scanning with a microplate reader.

[0111] C57BL / 6 mouse blood was used as a positive control to analyze the oxygen dissolving and releasing capacity of the control cell strain BMSC-GFP which did not express hemoglobin and the BMSC-Hb which stably expressed hemoglobin. As Figure 11 shown, C57BL / 6 mouse blood in the oxygenated state showed characteristic peaks at 540 nm and 575 nm (red curve), and the two absorption peaks were characteristic peaks produced by the two subunits of hemoglobin. A characteristic peak also appeared at 415 nm, which was a characteristic peak produced by the porphyrin ring, known as the Soret peak Figure 11 A). Figure 11B shows that oxygenated and deoxygenated BMSC-GFP have a characteristic peak near 415 nm. This absorption peak may be due to the presence of other porphyrin ring structures in the cell (such as cytochrome C in mitochondria), but there is no characteristic peak of hemoglobin subunits between 500-600 nm. Figure 11 C shows that the oxygenated stably transfected cell line BMSC-Hb has double characteristic peaks at 550nm and 580nm, and also has an absorption peak at 415nm, which is similar to natural red blood cells, proving that the hemoglobin in BMSC-Hb cells can bind to oxygen.

[0112] After CO2 was introduced into the cells for at least 3 h, sodium bisulfite (Na2S2O4), a reducing agent, was added at a concentration not exceeding 0.1% w / v to completely deoxygenate the hemoglobin. The double peak of natural red blood cells in the blood of C57BL / 6 mice was converted to a single peak at 555 nm ( Figure 11 A, blue curve), the absorption peak shifted to 430nm, successfully converted to a deoxygenated state; BMSC-GFP showed no significant changes; similar to natural red blood cells, the double absorption peak of BMSC-Hb also changed to a single absorption peak at 565nm, and the absorption peak at 430nm shifted to 415nm ( Figure 11 (C, blue curve). Thus, in contrast to BMSC-GFP, BMSC-Hb cells express hemoglobin and possess oxygen-binding capacity. Upon exposure to air, hypoxic red blood cells and BMSC-Hb can once again carry oxygen, with the single peak transforming into a double absorption peak, and the Soret absorption peak shifting back to 415 nm. This demonstrates that the oxygen binding and release process of BMSC-Hb is reversible.

[0113] 6. BMSC-Hb cell enucleation

[0114] The stably transfected cell line BMSC-Hb constructed by the present invention can express recombinant hemoglobin with oxygen transport function. However, natural mature red blood cells do not have nuclei, and enucleated cells have the advantages of low immunogenicity and no risk of cancer. Therefore, BMSC-Hb is subsequently enucleated to simulate the state of natural mature red blood cells in vivo. Figure 12 The formula of the enucleation solution is shown in Table 2.

[0115] Table 2. Nucleation Solution Formula

[0116]

[0117] First, separate the 24-well plates and clean the individual wells. Once clean, soak them in 75% alcohol. Remove from the alcohol before use, air-dry in a clean bench, and sterilize with UV irradiation. Then, prepare the ECM-coated 24-well plates. African green monkey kidney fibroblasts (cos-7) are seeded into the clean 24-well plates. When the COS7 cell density reaches approximately 90%, add ammonia and let stand at room temperature for approximately 10 minutes to lyse the COS7 cells until no cellular structures are visible under a microscope. Remove the liquid from the wells and wash three times with PBS buffer. Then, wash again three times with sterile ddH2O. Finally, add approximately 500 μL of PBS to keep the 24-well plates moist. The resulting ECM-coated 24-well plates can be used immediately or stored at 4°C for approximately one week.

[0118] Approximately 5 × 10 cells were seeded in 24-well ECM-coated plates. 5 BMSC cells are cultured in a 37°C cell culture incubator for approximately 24-48 hours. When the cell density reaches approximately 90%, enucleation can be performed. The 24-well plate seeded with BMSCs is placed upside down in a 50mL centrifuge tube containing 10mL of preheated enucleation solution. Centrifuge at 35°C, 4500g, for 1 hour to enucleate the cells. The enucleation efficiency can reach over 95%. After centrifugation, remove the 24-well plate from the 50mL tube, remove the enucleation solution, add fresh DMEM medium, and place in a 37°C incubator to recover for 2 hours.

[0119] After the cells recovered, the non-enucleated cells and the enucleated cells were stained with Dil (cell membrane dye) and Hoechst 33342 (nuclear dye). Compared with the non-enucleated BMSC-Hb, the enucleated cells had no Hoechst signal ( Figure 13 A), proving that the cells were successfully enucleated. After enucleation, the expressed protein will be wrapped in the cell membrane and present a round vesicle structure in suspension. At the same time, the diameter is significantly smaller than that of non-enucleated cells. ZEN 2 software was used to measure and count the cell diameters, and it was found that the size of BMSC-Hb cells before enucleation was approximately between 14-20μm ( Figure 13 B), after enucleation, due to the loss of cytoplasm to varying degrees, the cell diameter further decreased, ranging from 5 to 10 μm, mainly concentrated in the range of 7 to 9 μm ( Figure 13 C). Natural red blood cells have a diameter of approximately 7 μm, and the size of enucleated cells approaches that of red blood cells. Therefore, through enucleation, BMSCHb transforms into a round vesicle structure close to the size of a natural red blood cell, encapsulating hemoglobin and creating an "erythrocyte-like" structure with the potential to transport oxygen in the body.

[0120] 7. Most enucleated BMSC-Hb cells can survive for more than 24 hours

[0121] In order to evaluate the potential application of the "erythroid cells" obtained by the present invention in vivo, the present invention tested the survival time of the stably transfected cell line BMSC-Hb that overexpresses hemoglobin. After enucleation of BMSC-Hb, the number of green fluorescent cells gradually decreased over time. About 40% of the cells died after 24 hours, and almost all of the cells died after 48 hours. Figure 14 This suggests that enucleation affects BMSC-Hb cell survival, but more than half of the cells still survive. This suggests that the BMSC-Hb of the present invention may be used as a blood substitute in emergency situations, transporting oxygen within 24 hours, buying valuable time for patients with traumatic hemorrhage.

[0122] 8. Cytotoxicity Assay of Enucleated BMSC-Hb Cells

[0123] As a potential oxygen carrier, the cytotoxicity of enucleated BMSC-Hb is particularly important for transporting oxygen in the body. CCK-8 assay was used to assess the cytotoxicity of enucleated BMSC-Hb to other cells. Approximately 5,000 HUVEC and HEK293 cells were seeded into 96-well plates and cultured in a 37°C cell culture incubator. Experiments were performed after adherence. The enucleated cells were digested from the wells, counted, diluted to various concentrations, and co-cultured with the two cell lines. Cytotoxicity was assessed using CCK-8 assays after 24 hours of culture.

[0124] like Figure 15 As shown in the results, low and high concentrations of enucleated BMSC-Hb cells had no significant effect on the cell activity of HUVEC and HEK293t cells, indicating that they have extremely low cytotoxicity, laying a good foundation for the in vivo application of BMSC-Hb.

[0125] The above disclosure is merely a preferred embodiment of the present invention and certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A two-subunit fusion expression system, characterized in that: It includes a first expression group and a second group, wherein the first expression group and the second expression group are connected via a self-cleaving short peptide fragment; the first expression group includes a first subunit fragment and a first tag protein fragment; the second expression group includes a second subunit fragment and a second tag protein fragment.

2. The dual-subunit fusion expression system according to claim 1, characterized in that: In the first expression group, the first subunit fragment is fused to the first tag protein fragment via the N-terminus; in the second expression group, the second subunit fragment is fused to the second tag protein fragment via the N-terminus.

3. The dual-subunit fusion expression system according to claim 1, wherein: The dual-subunit fusion expression system is expressed by recombining with a backbone plasmid to form a recombinant expression plasmid; the backbone plasmid includes a promoter, a fluorescent protein expression sequence, and a resistance gene; the fluorescent protein expression sequence is connected to the first expression group through the self-cleaving short peptide fragment.

4. The dual-subunit fusion expression system according to claim 3, characterized in that: The self-cleaving short peptide fragment includes a short peptide P2A fragment; the first subunit fragment includes a hemoglobin α subunit fragment; the first tag protein fragment includes a Myc tag protein fragment; the second subunit fragment includes a hemoglobin β subunit fragment; the second tag protein fragment includes an HA tag protein fragment; the backbone plasmid is a pLenti-GFP plasmid; the promoter includes a CMV promoter; the fluorescent protein expression sequence includes an EGFP fluorescent protein expression sequence; and the resistance gene includes a bleomycin resistance gene.

5. The dual-subunit fusion expression system according to claim 1, wherein: The sequence of the dual-subunit fusion expression system is shown in SEQ ID NO:

3.

6. A use of the dual-subunit fusion expression system according to claim 1, characterized in that: Used to construct one or more of artificial red blood cells and enucleated artificial red blood cells.

7. The use according to claim 6, characterized in that The steps include: A. Enzyme digestion of the backbone plasmid, gel excision and recovery to obtain a linearized plasmid, followed by PCR reaction to obtain the dual-subunit fusion expression system, and homologous recombination of the dual-subunit fusion expression system with the linearized plasmid to obtain a recombinant expression plasmid; B. transfecting the recombinant expression plasmid into ordinary cells, culturing, purifying, and concentrating to obtain a lentiviral packaging expression vector; C. Infect stem cells with the lentiviral packaged expression vector, screen and culture, and obtain stably transfected cell lines, thereby forming artificial red blood cells.

8. The use according to claim 7, characterized in that The following steps are also included: D. The stably transfected cell line is subjected to enucleation treatment to obtain enucleated artificial red blood cells.

9. The use according to claim 6, characterized in that The steps include: A1. Enzyme digestion of the backbone plasmid and gel extraction to obtain linearized plasmid; A2. obtaining the dual-subunit fusion expression system by PCR reaction; A3. homologously recombining the dual-subunit fusion expression system with the linearized plasmid to obtain a recombinant expression plasmid; the sequence of the recombinant expression plasmid is shown in SEQ ID NO: 5; B1. Add DMEM high glucose culture medium to a 10 cm cell culture dish and inoculate 4×10 6 293T cells were cultured in a 37°C incubator. Before transfection, the cells were replaced with fresh DMEM high-glucose culture medium to obtain 293T culture medium for packaging lentivirus. B2. Add the plasmid composition for a 10 cm dish to 400 μL of Optical-MEM, mix well, and incubate at room temperature for 5 minutes to obtain the incubated plasmid; The plasmid composition for a 10 cm dish includes: the recombinant expression plasmid: 12 μg, pMDL: 8 μg, VSVG: 3.2 μg, REV: 4.4 μg, and Total plasmid: 27.6 μg; B3. Mix the transfection reagent PEI with 400 μL Opti-MEM and incubate at room temperature for 5 minutes to obtain the incubated transfection reagent; the mass ratio of the transfection reagent PEI to the plasmid composition for the 10 cm dish is 1.5:1; B4. Mix the incubated plasmid and the incubated transfection reagent, and incubate at room temperature for 15 minutes to obtain a transfection solution; B5. Add the transfection solution to the 293T culture medium and, after transfection for 4 to 6 hours, replace it with fresh DMEM high-glucose culture medium; B6. 16 hours after transfection, the culture medium was replaced with collection medium, and the cells were continued to be cultured in an incubator. The collection medium was prepared by adding 0.5 g of BSA and a HEPES buffer having a final concentration of 10-15 mM to 50 mL of complete culture medium without antibiotics, adjusting the pH to 7.4, and filtering through 0.22 μm to obtain the collection medium. B7. Collect cell culture fluid 45 h, 54 h, 64 h, 72 h, and 96 h after transfection; B8. Centrifuge the cell culture medium at 800 g for 10 min, filter the supernatant with a 0.45 μm filter membrane, add PEG to a final PEG concentration of 10%, mix well, and incubate at 4°C with slow shaking for more than 4 h. Centrifuge at 2000 g for 30 min at 4°C, remove the supernatant, and resuspend in 1 / 20 volume of DMEM high-glucose culture medium to obtain lentiviral packaging expression vectors. Store at 4°C for a short time or at -80°C for a long time. C1. The lentiviral expression vector is packaged with polybrene at a ratio of 1000:1 and then added to a 96-well plate to infect stem cells. After 24 hours, the medium is replaced with fresh DMEM high-glucose culture medium. After green fluorescence is observed under a microscope, a primary infected cell line is obtained; the stem cells include BMSC cells; C2. Digest the primary infected cell line, count, and dilute to a cell density of 10-500 cells / mL. Take 100 μL and add it to a 96-well plate, ensuring that each well contains 1-5 cells. After the cells have attached and grown to an appropriate density, observe the fluorescence under a microscope again and look for wells with green fluorescent cells. If any non-fluorescent cells remain, scrape them off. Repeat this operation 1-3 times until only green fluorescent secondary infected cells remain in the wells. C3. Expanding the secondary infected cells to obtain a stable cell line, i.e., forming artificial red blood cells; D1. Separate the 24-well plate and wash the separated individual 24 wells. After cleaning, soak them in 75% alcohol. Remove them from the alcohol before use, dry them in a clean bench, and sterilize them by ultraviolet irradiation to obtain a clean 24-well plate. D2, inoculating African green monkey kidney fibroblasts cos-7 in the clean 24-well plate, and when the cell density of the African green monkey kidney fibroblasts cos7 reached 90%, adding ammonia water and letting it stand at room temperature for 5-10 minutes to lyse the African green monkey kidney fibroblasts cos7 until no cell structure was observed under a microscope, removing the liquid in the wells, adding PBS buffer and washing three times, and then using sterilized ddH2O to wash again three times, and finally adding about 500 μL PBS to keep the 24 wells moist to prepare 24 wells coated with ECM; D3, 5×10 5 The artificial red blood cells are cultured in a cell culture incubator at 37° C. for 24 to 48 hours, and the pre-enucleated cells are obtained when the cell density reaches about 90%. D4. Place the pre-enucleated cells upside down in a 50 mL centrifuge tube containing 10 mL of preheated enucleation solution, and centrifuge at 35°C, 4500 g, for 1 hour to perform enucleation. Each 10 mL of the enucleation solution contains: 8 mL of calcium-free DMEM, 100 μL of 1 mg / mL cytochalasin B, 5 μL of 10 mg / mL colchicine, 2 mL of 10% sucrose, 40 μL of 500 mM N-acetylcysteine, and 10 μL of 100 μM calcium chloride solution. D5. After the enucleation operation is completed, the cells are taken out, the enucleation solution is removed, and fresh DMEM high-glucose culture medium is added. The cells are placed in a 37°C incubator for recovery for 2 hours to obtain enucleated artificial red blood cells.

10. A product obtained by the application according to claim 6, characterized in that: Including one or more of artificial red blood cells and enucleated artificial red blood cells.

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