CN118813717A - A universal mesenchymal stem cell and its construction method and application - Google Patents

Abstract

The present invention provides a universal mesenchymal stem cell and a construction method and application thereof. A new universal mesenchymal stem cell is constructed by combining electrotransfection and CRISPRoff gene editing methods. Compared with unedited mesenchymal stem cells and mesenchymal stem cells constructed by other transfection methods, the constructed universal mesenchymal stem cells can stably survive in the inflammatory pathological environment of the lungs, continuously exert therapeutic effects, and have a more significant improvement effect on the pathological phenotype and multiple biochemical detection indicators of acute pneumonia, solving the problem of immune rejection of allogeneic stem cells by the human immune system in regenerative therapy based on stem cell transplantation, providing an effective new cell therapy for acute pneumonia, and also providing technical support and new ideas for the clinical treatment of acute pneumonia, and having good application prospects.

Description

A universal mesenchymal stem cell and its construction method and application

Technical Field

The present invention belongs to the field of medicine, and in particular, relates to a universal mesenchymal stem cell and a construction method and application thereof.

Background Art

The description of the background technology is merely a general description for facilitating understanding of the present invention and does not constitute any limitation to the present invention.

Acute lung injury (ALI) is a common life-threatening lung disease, characterized by increased alveolar and capillary permeability, lung tissue structural damage, and lung inflammation. Currently, there is no specific drug to treat ALI. The pathogenesis of ALI is relatively complex. The most recognized mechanism is that various causes induce an increase in proinflammatory factors in the lungs. Therefore, controlling the inflammatory response is a key measure to treat ALI.

At present, the main clinical treatments for ALI are: (1) Mechanical ventilation therapy: It is the classic treatment for ALI. However, improper mechanical ventilation may lead to excessive expansion of alveoli, which in turn causes mechanical ventilation-related lung injury. In clinical practice, personalized ventilation strategies should be adopted for different patients to prevent and reduce mechanical ventilation-related side effects as much as possible. How to avoid respiratory complications while giving full play to the therapeutic effect of mechanical ventilation is still a major problem that needs to be solved in the field of ALI treatment; (2) Chemical drug treatment: Common chemical drugs used to treat acute lung injury include glucocorticoids, antibiotics, traditional Chinese medicine preparations, protease inhibitors, anticoagulants, vasodilators, etc. Although these drugs have certain efficacy in the treatment of ALI, they have side effects.

In recent years, stem cell therapy represented by mesenchymal stem cells (MSCs) has become a potential clinical means of anti-inflammatory treatment. MSCs are a type of self-renewing stem cell with multidirectional differentiation, immunomodulatory and anti-inflammatory abilities. They tend to migrate to the site of inflammation, exert immunomodulatory functions through paracrine and other pathways, promote tissue repair and regeneration, and alleviate multi-organ diseases and lung complications caused by viruses, thereby achieving the effect of disease treatment. However, allogeneic immune rejection is still a bottleneck problem that hinders the widespread clinical application of MSCs. Although MSCs are generally considered to have low immunogenicity, in a pathological inflammatory environment, pro-inflammatory factors such as IFNγ can stimulate MSCs to upregulate HLA-I, increase the immunogenicity of MSCs, and cause MSCs to be immune-rejected and unable to continue to exert their therapeutic effects. In addition, autologous MSCs are limited by bottlenecks such as insufficient cell sources, unstable batches, high prices and long preparation cycles, making it difficult to be widely used in clinical treatment. Although allogeneic MSCs can solve the above bottlenecks to a certain extent, they are limited by allogeneic immune rejection, resulting in MSCs being unable to continue to exert their therapeutic effects in the recipient, resulting in poor efficacy.

In the invention patent with application publication number CN113846063A, the inventor has constructed a universal human stem cell suitable for allogeneic transplantation in preliminary experiments. By partially knocking down or silencing the enhancer gene sequence of the B2M gene in the stem cell, the stem cell can maintain low immunogenicity in an inflammatory environment. However, in the construction method, a lentiviral transfection method is used. On the one hand, the operation procedure of the lentiviral transduction method involved in this method is relatively complicated, has strong selectivity for cell types, and has high operational requirements for R&D personnel, which is difficult to popularize in general laboratories; on the other hand, the viral DNA will be permanently integrated into the MSCs genome, which may bring potential safety risks; and the application and mechanism of action of the universal mesenchymal stem cells in lung injury have not been studied in depth.

Therefore, there is an urgent need to develop a method for constructing universal mesenchymal stem cells that is safe, stable, and has lower immunogenicity, and to clarify the mechanism of action of the universal mesenchymal stem cells in acute lung injury and their related applications, in order to provide new strategies for the clinical treatment of acute lung injury and other diseases.

Summary of the invention

In order to solve the problems existing in the prior art, the present invention provides a universal mesenchymal stem cell and a construction method and application thereof. A new universal mesenchymal stem cell is constructed by combining electrotransfection and CRISPRoff gene editing methods. Compared with unedited mesenchymal stem cells and mesenchymal stem cells constructed by other transfection methods, the constructed universal mesenchymal stem cells can stably survive in the inflammatory pathological environment of the lungs, continue to exert a therapeutic effect, and have a more significant improvement effect on the pathological phenotype and multiple biochemical detection indicators of acute pneumonia, solve the problem of immune rejection of allogeneic stem cells by the human immune system in regenerative therapy based on stem cell transplantation, provide an effective new cell therapy for acute pneumonia, and also provide technical support and new ideas for the clinical treatment of acute pneumonia, and have good application prospects.

In order to achieve the above object, the present invention adopts the following scheme:

In one aspect, the present invention provides a method for constructing universal mesenchymal stem cells, comprising the following steps:

(1) Prepare mesenchymal stem cells;

(2) preparing transfection complexes;

(3) transfecting the plasmid into cells;

In the step (3), the plasmid mixture is transfected into the mesenchymal stem cells by electrofection.

In the invention patents with application publication numbers CN113846063A and CN113801881A, the inventors have constructed and obtained "a universal human stem cell suitable for allogeneic transplantation" in the early stage, but it has not been applied to specific indications such as acute lung injury. This is related to the use of lentiviral transduction in the early stage: on the one hand, the operation procedure of the lentiviral transduction method is relatively complicated, it has strong selectivity for cell types, and has high operating requirements for R&D personnel, so it is difficult to popularize in general laboratories; on the other hand, although mesenchymal stem cells are generally considered to have low immunogenicity, lentiviral vectors and their transgenic products may trigger immune responses, causing the body to reject the transformed cells; at the same time, the lentivirus is randomly inserted into the cell genome, which may destroy important gene functions in the cell and even cause cell malignancy; these may bring potential safety hazards. Therefore, in the research of the present invention, the construction method of the universal mesenchymal stem cell is further optimized, in order to construct a safer and more stable universal mesenchymal stem cell, and it has a good therapeutic effect in the application of various indications.

Therefore, in the present invention, a new universal mesenchymal stem cell is constructed by combining electrotransfection with CRISPRoff. The universal mesenchymal stem cell can maintain low immunogenicity in an inflammatory environment, so that it can avoid immune rejection after transplantation and stably exert its therapeutic effect, significantly improve ALI symptoms, and have a better regeneration and repair effect on damaged lung tissue. Compared with the method of using lentiviral transduction and liposome transfection, the universal mesenchymal stem cell constructed by the present invention has the best transfection efficiency and cell activity. This may be because electroporation causes changes in cell membrane potential through a high-intensity electric field, instantly increases cell membrane permeability, and produces reversible pores on the cell membrane to facilitate the entry of exogenous nucleic acids, which is suitable for almost all types of cells.

Furthermore, in the step (2), the transfection complex includes mesenchymal stem cells and a plasmid mixture; the plasmid mixture includes a CRISPRoff plasmid and a sgRNA plasmid.

Furthermore, in the step (2), the amount of plasmid used is 400-800 ng.

In some embodiments, in order to further improve the transfection efficiency and cell viability in the universal mesenchymal stem cell construction method of the present invention, different plasmid dosages were screened: the experimental results show that as the plasmid dosage increases, the proportion of double-positive cell populations gradually increases from 3.7% to 15.9%. When the plasmid dosage exceeds 800ng, the cell viability decreases significantly, which may be due to the saturation effect or some unexpected effects caused by the interaction of too many plasmids with cells. Therefore, the preferred plasmid dosage of the present invention is 800ng.

Furthermore, in step (2), the mass ratio of the CRISPRoff plasmid to the sgRNA plasmid is 1:1.

Furthermore, in the electrotransfection parameters of step (3), the voltage is 990V-1400V, the duration of the electric pulse is 10ms-40ms, and the number of pulses is 1-3 times.

In some embodiments, in order to achieve good transfection efficiency while minimizing damage to cells and improving cell survival rate, different electroporation parameters were screened. The experimental results showed that under the conditions of 1400V/10ms/3pulse electroporation, a relatively high electroporation efficiency was achieved, and the electroporation efficiency was 93.58%. Therefore, the preferred electroporation parameters of the present invention are 1400V/10ms/3pulse for preparing universal mesenchymal stem cells.

On the other hand, the present invention provides a universal mesenchymal stem cell, which is constructed by the construction method described in any one of the above technical solutions.

In another aspect, the present invention provides a use of universal mesenchymal stem cells for preparing a preparation for improving the repair and regeneration ability of lung injury, wherein the universal mesenchymal stem cells are constructed using the construction method described in any one of the above technical solutions.

In some embodiments, in order to verify that the universal mesenchymal stem cells prepared by the present invention have a more significant therapeutic effect on acute lung injury, an LPS-induced acute pneumonia model is constructed and an in vivo bioluminescence imaging tracking experiment is performed, and HE staining experiments and biochemical index detection experiments of alveolar lavage fluid are also performed.

Among them, the results of the in vitro observation experiment of lung tissue showed that the inflammation and congestion of lung tissue in the ntMSCs treatment group were improved compared with the PBS treatment group. The lung tissues of the GLOBES-treated group still had severe pneumonia symptoms, indicating that the therapeutic effect of ntMSCs on pneumonia was limited; however, the lung tissues of the GLOBES-treated group had a good improvement in pneumonia compared with the PBS-treated group, and The results showed that the GLOBES-1 treatment group and the GLOBES-2 treatment group were closer to the normal lung tissue of the group, indicating that GLOBES has a better therapeutic effect on pneumonia. At the same time, the GLOBES-1 treatment group and the GLOBES-2 treatment group were compared in subsequent experiments. The experimental results showed that compared with the GLOBES-1 and GLOBES-2 treatment groups, the inflammation and congestion of the lung tissue in the GLOBES treatment group were significantly reduced, indicating that the GLOBES treatment group constructed by electrotransfection has a more significant improvement effect on pneumonia.

The results of HE staining experiments showed that compared with The normal lung tissue of the PBS group was treated with LPS, while the lung tissue of the PBS group showed typical acute pneumonia injury. Although ntMSCs treatment alleviated the above acute lung injury to a certain extent, it was still significantly lower than that of the control group. The normal lung tissue of the GLOBES group still clearly showed more lung injury areas. However, the lung tissue sections of the GLOBES treatment group were significantly different from those of the ntMSCs group and were more similar to The normal lung tissue slice morphology of the group indicated that the lung injury was greatly improved and repaired after GLOBES treatment. In subsequent experiments, the group was compared with the GLOBES-1 treatment group and the GLOBES-2 treatment group. The experimental results showed that compared with the GLOBES-1 and GLOBES-2 treatment groups, the lung injury in the GLOBES treatment group was significantly improved and repaired after treatment, which further indicated that the GLOBES treatment group constructed by electrotransfection had a more significant lung injury repair effect.

The results of LDH content detection in bronchoalveolar lavage fluid showed that the LDH content in bronchoalveolar lavage fluid of PBS, ntMSCs, GLOBES-1 and GLOBES-2 groups was significantly higher than that of GLOBES treatment group and group; among them, the LDH content in the ntMSCs treatment group was about 2.05 times that of the GLOBES treatment group; and LDH is only present in the cytoplasm of tissue cells in the body under normal circumstances. The higher the LDH content in the alveolar lavage fluid, the more severe the tissue damage, which indicates that the lung injury has been greatly improved and repaired after GLOBES treatment. In subsequent experiments, the LDH content in the alveolar lavage fluid of the GLOBES-1 treatment group and the GLOBES-2 treatment group was also detected. The experimental results showed that the LDH content in the GLOBES-1 treatment group was about 1.72 times that of the GLOBES treatment group; the LDH content in the GLOBES-2 treatment group was about 1.85 times that of the GLOBES treatment group; compared with the ntMSCs treatment group, the LDH content in the GLOBES-1 and GLOBES-2 treatment groups decreased slightly, but compared with the GLOBES treatment group, it still increased significantly, which further indicated that the GLOBES treatment group constructed by electrotransfection had a more significant lung injury repair effect. Therefore, it can be seen that the detection results of LDH content in alveolar lavage fluid further prove that the GLOBES treatment group constructed by electrotransfection has a more significant effect in repairing acute pneumonia damage.

The results of the albumin content test in the alveolar lavage fluid showed that although the ntMSCs treatment group could reduce the albumin content in the alveolar lavage fluid to a certain extent, the corresponding albumin content was still significantly higher than The albumin content in the alveolar lavage fluid of the ntMSCs treatment group was about 1.43 times that of the GLOBES treatment group; and in subsequent experiments, the LDH content in the alveolar lavage fluid of the GLOBES-1 treatment group and the GLOBES-2 treatment group was detected, and the experiment found that the albumin content in the alveolar lavage fluid of the GLOBES-1 treatment group was about 1.28 times that of the GLOBES treatment group; the albumin content in the alveolar lavage fluid of the GLOBES-2 treatment group was about 1.32 times that of the GLOBES treatment group; compared with the ntMSCs treatment group, the albumin content in the alveolar lavage fluid of the GLOBES-1 and GLOBES-2 treatment groups decreased slightly, but compared with the GLOBES treatment group, it still increased; and the albumin content in the GLOBES treatment group was close to Therefore, the detection results of albumin content in bronchoalveolar lavage fluid further prove that the GLOBES treatment group constructed by electrotransfection has a more significant effect in repairing acute pneumonia damage.

The results of total protein content and total cell count in alveolar lavage fluid showed that the results of total protein content were similar to those of albumin. The total protein content in alveolar lavage fluid of ntMSCs treatment group was about 1.22 times that of GLOBES treatment group. In subsequent experiments, it was also found that the total protein content in alveolar lavage fluid of GLOBES-1 treatment group and GLOBES-2 treatment group was about 1.12 times and 1.15 times that of GLOBES treatment group, respectively. Although the total protein content in alveolar lavage fluid of GLOBES-1 treatment group and GLOBES-2 treatment group was slightly lower than that of ntMSCs treatment group, The total number of cells in the bronchoalveolar lavage fluid of the ntMSCs-treated group was 1.77 times that of the GLOBES-treated group. In subsequent experiments, it was found that the total number of cells in the bronchoalveolar lavage fluid of the GLOBES-1-treated group and the GLOBES-2-treated group were 1.31 times and 1.42 times that of the GLOBES-treated group, respectively. The total number of cells in the bronchoalveolar lavage fluid of the GLOBES-1-treated group and the GLOBES-2-treated group also increased significantly compared with the GLOBES-treated group. There was no statistical difference between the groups.

Therefore, the above results all confirm that giving allogeneic MSCs immune compatibility properties helps MSCs to survive for a long time in pathological environments, thereby stably exerting anti-inflammatory and therapeutic effects and achieving regeneration and repair of damaged tissues. Compared with the universal MSCs constructed by lentiviral transduction and liposome transfection, the universal MSCs constructed by the optimal electrotransfection method in the present invention have the most significant lung injury treatment effect.

In another aspect, the present invention provides a use of universal mesenchymal stem cells for preparing a preparation for reducing allogeneic immune rejection reaction, wherein the universal mesenchymal stem cells are constructed by the construction method described in any one of the above technical solutions.

In some embodiments, to verify that GLOBES can still maintain its low immunogenicity under the pathological environment of lung inflammation, a GLOBES in vivo transplantation recovery experiment was performed, and the experimental results showed that the HLA-I expression of the control ntMSCs (ntMSCs in vivo) in the in vivo pneumonia environment increased by 8.5 times compared with the ntMSCs cultured in vitro (ntMSCs in vitro); while GLOBES in vivo (GLOBES in vivo) still maintained a low HLA-I expression, and the expression was only about 2 times higher than that of GLOBES cultured in vitro (GLOBES in vitro), and the HLA-I expression was only 20.7% of the HLA-I expression in ntMSCs. At the same time, in subsequent experiments, it was also found that the HLA-I expression of the GLOBES-1 group (GLOBES-1in vivo) and the HLA-I expression of the GLOBES-2 group (GLOBES-2in vivo) were significantly increased compared with the in vitro culture group. Therefore, it can be seen that compared with unedited MSCs, lentiviral-transduced GLOBES-1 and liposome-transfected GLOBES-2, the GLOBES constructed by the optimal electrotransfection method in the present invention can still maintain its low immunogenicity under the pathological environment of lung inflammatory disease.

In another aspect, the present invention provides a use of universal mesenchymal stem cells for preparing a preparation for reducing the content of lactate dehydrogenase, albumin and/or total protein in bronchoalveolar lavage fluid, wherein the universal mesenchymal stem cells are constructed using the construction method described in any one of the above technical schemes.

In another aspect, the present invention provides a use of universal mesenchymal stem cells for preparing a preparation for reducing the content of lactate dehydrogenase, albumin and/or total protein in bronchoalveolar lavage fluid, wherein the universal mesenchymal stem cells are constructed using the construction method described in any one of the above technical schemes.

In some embodiments, the survival of transplanted cells in mice in different groups of luc-GLOBES and luc-ntMSCs was tested respectively, and the experimental results showed that compared with unedited MSCs, the GLOBES constructed by the optimal electrotransfection method in the present invention did not induce an immune response of allogeneic MSCs and could survive longer in mice reconstructed with the human immune system.

In subsequent experiments, the same operation was performed on mice injected with equal amounts of luc-GLOBES-1 or luc-GLOBES-2 cells, and the cell survival results were compared. The results showed that although there was no obvious immune rejection-induced cell death in the GLOBES-1 and GLOBES-2 groups, the cell survival was significantly reduced.

Therefore, it is further proved that compared with unedited MSCs, lentiviral-transduced GLOBES-1 and liposome-transfected GLOBES-2, the GLOBES constructed by the optimal electrotransfection method in the present invention does not cause an immune response of allogeneic MSCs and can survive longer in mice reconstructed with the human immune system.

In another aspect, the present invention provides a use of universal mesenchymal stem cells for preparing a preparation for increasing the survival time of mesenchymal stem cells in an inflammatory environment in vivo or maintaining low immunogenicity of mesenchymal stem cells in vivo, wherein the universal mesenchymal stem cells are constructed using the construction method described in any one of the above technical solutions.

The beneficial effects of the present invention are:

1. The present invention uses electrotransfection combined with epigenetic editing CRISPRoff to construct a universal mesenchymal stem cell that can still maintain low immunogenicity in an inflammatory environment, which can not cause immune rejection reaction after intravenous injection into the body; and compared with untreated mesenchymal stem cells, universal mesenchymal stem cells constructed by lentiviral transduction or liposome transfection, on the one hand, it has better pneumonia treatment effect and regeneration and repair effect of damaged lung tissue, and has a more significant improvement effect on the pathological phenotype of acute pneumonia and multiple biochemical detection indicators; on the other hand, it can maintain low immunogenicity in the pathological environment of lung diseases, solves the problem of immune rejection of allogeneic stem cells by the human immune system in regenerative therapy based on stem cell transplantation; at the same time, it can survive longer in the pathological environment of lung diseases, thereby exerting a better anti-inflammatory effect.

2. The present invention uses electrofection combined with epigenetic editing CRISPRoff to construct universal mesenchymal stem cells. In terms of safety, the epigenetic editing method will not affect the genome sequence of genomic cells, and the construction method is safe and simple. At the same time, the universal mesenchymal stem cells gene-edited by the method of the present invention do not have the ability to form tumors in vivo. The universal mesenchymal stem cells that maintain low immunogenicity under an inflammatory environment constructed by epigenetic editing of the present invention are used to treat acute lung injury, which provides a new, safe and effective treatment strategy for the clinical treatment of acute pneumonia.

3. The present invention uses electrofection combined with epigenetic editing CRISPRoff to construct universal mesenchymal stem cells, which provides a new cell source and effective treatment method for mesenchymal stem cells to treat acute lung injury. After transplantation, the universal mesenchymal stem cells can avoid immune rejection and stably exert their therapeutic effects, significantly improve the symptoms of acute lung injury, and provide technical support and new ideas for the clinical treatment of acute pneumonia, and have good application prospects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph showing the transfection efficiency and cell viability under different electroporation parameters.

FIG. 2 is a graph showing the transfection efficiency and cell viability at different plasmid dosages.

FIG. 3A is a schematic diagram of isolated mouse lung tissues after treatment with different groups.

FIG3B is a diagram showing the HE staining results of mouse lung tissue after treatment in different groups.

FIG4A shows the results of LDH content in bronchoalveolar lavage fluid of different groups.

Figure 4B shows the results of albumin content in bronchoalveolar lavage fluid of different groups.

FIG4C shows the results of determination of total protein content in bronchoalveolar lavage fluid of different groups.

FIG4D shows the results of determination of total cell numbers in bronchoalveolar lavage fluid of different groups.

FIG5A is a diagram showing the qualitative fluorescence imaging results of BLI of mice in different groups.

FIG. 5B shows the results of the survival of transplanted cells in mice in different groups.

FIG6 is a flow cytometry result diagram of HLA-I expression in vivo and in vitro in mice of different groups.

DETAILED DESCRIPTION

The present invention will be further described in detail below in conjunction with the examples. It should be pointed out that the examples described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on the present invention.

Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

Example 1 Optimal construction method of universal mesenchymal stem cells (universal MSCs) of the present invention

Given the recognized immunomodulatory and anti-inflammatory capabilities of MSCs, MSCs stem cell therapy has become a potential means of treating ALI. Autologous MSCs are limited by bottlenecks such as insufficient cell sources, unstable batches, high prices, and long preparation cycles, making it difficult to be widely used in clinical treatment. Although allogeneic MSCs can solve the above bottlenecks to a certain extent, they are limited by allogeneic immune rejection, resulting in MSCs being unable to continue to exert their therapeutic effects in the recipient's body, resulting in poor efficacy.

In the invention patents with application publication numbers CN113846063A and CN113801881A, the inventors have previously constructed and obtained "a universal human stem cell suitable for allogeneic transplantation", but it has not been applied to specific indications such as acute lung injury. This may be related to the use of lentiviral transduction: on the one hand, the operation procedures of lentiviral transduction are relatively complicated, it has strong selectivity for cell types, and has high operating requirements for R&D personnel, so it is difficult to popularize in general laboratories; on the other hand, although mesenchymal stem cells are generally considered to have low immunogenicity, lentiviral vectors and their transgenic products may trigger immune responses, causing the body to reject the transformed cells; at the same time, the random insertion of lentivirus into the cell genome may destroy important gene functions in the cell and even cause cell malignancy; all of these may bring potential safety hazards. Therefore, in subsequent studies, the construction method of universal mesenchymal stem cells was further optimized in order to construct a safer and more stable universal mesenchymal stem cell, which has a good therapeutic effect in the application of various indications.

Therefore, in this embodiment, a new universal mesenchymal stem cell is constructed by combining electrotransfection with CRISPRoff. The universal mesenchymal stem cell can maintain low immunogenicity in an inflammatory environment, so that it can avoid immune rejection after transplantation and stably exert its therapeutic effect, significantly improve ALI symptoms, and have a better regeneration and repair effect on damaged lung tissue. The specific construction method is as follows:

1. sgRNA design and recombinant plasmid construction (the specific construction process of the recombinant plasmid, the sequence of the sgRNA and the sequence of the enhancer are all referred to the construction process in the invention patent with application publication number CN113846063A):

① Design sgRNA for the enhancer sequence (SEQ ID NO.1) (Benchling website: https://www.benchling.com/crispr/). The sgRNA sequence is shown in the table below:

Add restriction sites at both ends of the sgRNA, add CACC at the 5' end of the sense strand of the sgRNA sequence, and add AAAC at the 5' end of the antisense strand, so as to form sticky ends complementary to the pLV-U6-gRNA-UbC-eGFP-P2A-Bsr plasmid (Addgene: #83925) after digestion with FastDigest Bbs I. If the first base at the 5' end of the sense strand is not G, add a G after CACC at the 5' end, and add another C at the 3' end of the corresponding antisense strand. pLV-U6-gRNA-UbC-eGFP-P2A-Bsr is an sgRNA backbone expression vector containing the U6 promoter, with a GFP green fluorescent protein gene and ampicillin resistance.

① Use Fast Digest Bbs I to digest pLV-U6-gRNA-UbC-eGFP-P2A-Bsr, and recover the linearized vector after DNA gel electrophoresis.

② Phosphorylate and anneal the sgRNA sequence with T4 PNK; connect the linear pLV-U6-gRNA-UbC-eGFP-P2A-Bsr plasmid vector to the annealed sgRNA double-stranded sequence at room temperature for 1 hour with T4 ligase. Transform the ligation product into competent bacteria Trans 109, ice bath for 30 minutes, 42℃45s, and ice for 2 minutes. Screen clones on ampicillin-resistant LB plates. Pick positive clones and shake them for sequencing. The sequencing primer is the forward primer sequence of the U6 promoter, 5'-GAGGGCCTATTTCCCATGATTCC-3' (SEQ ID NO.18). The clone with correct sequencing is the recombinant plasmid.

2. Collect the mesenchymal stem cells in a centrifuge tube and centrifuge at 400×g for 5 minutes. Aspirate and discard the supernatant, resuspend with PBS, and count; the mesenchymal stem cells are obtained by referring to the method in the Chinese invention patent application with application publication number CN111849885A, and a large number of mature MSCs are obtained by inducing hESC to differentiate into MSCs (wherein the hESC line is WA09, purchased from WiCell Research Center, and signed with relevant regulations);

3. Prepare transfection complex: Use Neon transfection system (Invitrogen, MPK5000) for electroporation. For 10 μL of electroporation system, take 2×10 ⁵ mesenchymal stem cells and resuspend them in 9 μL of electroporation buffer, and add 1 μL of electroporation buffer containing 300 ng of plasmid mixture (including 150 ng of CRISPRoff and 150 ng of sgRNA) (the CRISPRoff is a commercial plasmid purchased);

4. Electrotransfection: Transfer the obtained mixture containing cells and plasmids into a 10-μL tip (Neon) and perform electroporation using the electroporation conditions of "1400V/10ms/3pulses" (here tip is a laboratory pipetting/liquid aspiration tool);

5. After electroporation, transfer the cells to a well plate containing pre-warmed fresh culture medium and place in an incubator at 37°C and 5% CO _2. For a 24-well plate, inoculate 5 reactions (a total of 1x10 ⁶ cells) per well with 0.5 mL of culture medium.

Based on the invention patent with application publication number CN113846063A: By partially knocking down or silencing the enhancer gene sequence of the B2M gene in stem cells, stem cells can maintain low immunogenicity in an inflammatory environment. In this embodiment, based on the super enhancer SE on the B2M gene that responds to IFN-γ stimulation in the aforementioned patent, electrotransfection combined with epigenetic editing CRISPRoff was further used to construct universal mesenchymal stem cells.

In this embodiment, the universal mesenchymal stem cells constructed by electrotransfection combined with epigenetic editing CRISPRoff are: (1) they can still maintain low immunogenicity in an inflammatory environment and will not cause immune rejection after intravenous injection into the body; (2) and compared with unedited MSCs cells, they can survive longer in the body, thereby exerting a better anti-inflammatory effect; (3) in terms of safety, the epigenetic editing CRISPRoff method will not affect the genome sequence of the cells, and the construction method is safe and simple. The experimental data in the following embodiments also prove that MSCs after gene editing using the method of the present invention do not have the ability to form tumors in vivo.

The present invention treats acute lung injury by constructing universal MSCs that maintain low immunogenicity under an inflammatory environment through epigenetic editing, providing a new, safe and effective treatment strategy for the treatment of acute lung injury.

Example 2 Further exploration and optimization of the universal mesenchymal stem cell construction method

1. Screening of transfection methods

In this example, in order to construct a safer, more stable and less immunogenic universal mesenchymal stem cell, different transfection methods were further screened on the basis of the original method for constructing a universal mesenchymal stem cell, including: lentiviral transduction, electrotransfection, and liposome transfection. The specific operations are as follows:

1. Construct universal MSCs 1 by electrotransfection in Example 1;

2. Use lentiviral transduction to construct universal MSCs 2: MSCs cells were seeded into 6-well plates at 2×10 ⁵ /well the day before, so that the fusion degree of MSCs was 70% when infected the next day. Before infection, fresh complete medium was replaced, and the virus particles were diluted with an appropriate amount of fresh complete medium at an infection multiplicity of 0.3, and then dripped into the well plate containing MSCs through a 0.2μm filter; then 2μl 10mg/ml polybrene (final concentration of 10μg/ml) was added; after cross-shaking and mixing, centrifuged at 1300rpm and 37℃ for 45min (the acceleration of both the speed increase and the speed decrease were set to "0"), so that the virus and target cells can better combine. After cell expansion and culture, flow fluorescence sorting was performed to sort out the double-positive cells of mCherry (red fluorescence) and GFP (green fluorescence), and the surviving MSCs were universal MSCs 2.

3. Use liposome transfection to construct universal MSCs 3: One day in advance, MSCs were seeded in 10 cm cell culture dishes at 2×10 ⁶ / dish to ensure that the cell fusion reached 60% on the next day. 12-18 hours after seeding is the best time for transfection. According to the instructions of Lipofectamine 3000 reagent (Invitrogene, catalog number: L3000015), 2.4μg CRISPRoff, 1.2μg sgRNA and 20μl Lipofectamine 3000 were incubated at room temperature for 10 minutes, and then co-transfected with MSCs. After 48 hours, flow fluorescence sorting was performed to sort out the double-positive cells of BFP (blue fluorescence) and GFP (green fluorescence). The surviving MSCs were universal MSCs 3.

The transfection efficiency and cell activity of the different universal MSCs constructed were tested respectively.

The detection steps of the transfection efficiency are as follows: cells are transfected according to different transfection methods, and the fluorescence value is detected on a flow cytometer (FACSAria Fusion SORP, BD, USA). Successfully transfected cells will have red (mCherry) and green (GFP) fluorescence. The position of the control group is taken as the position without fluorescence expression, and a cross gate is drawn to determine the percentage of cell deviation in the experimental group, that is, the transfection rate.

The cell activity detection step is: take 0.5mL cell suspension (1×10 ⁵ cells), add about 10μL 7-aminoactinomycin D (7-AAD) dye, incubate at room temperature for 2 minutes, and then detect the fluorescence intensity under the flow cytometer (FL3 channel) according to its excitation emission wavelength (Ex/Em=545nm/650nm). 7-AAD is a non-permeable fluorescent dye that cannot penetrate the cell membrane of living cells, but can penetrate the cell membrane of dead cells and bind to the DNA inside them, thereby facilitating the detection of the ratio of live cells and dead cells by flow cytometry.

The specific results are shown in Table 1:

Table 1. Transfection efficiency and cell viability of universal MSCs constructed by different transfection methods

As shown in Table 1, the universal mesenchymal stem cells constructed by electrofection have the best transfection efficiency and cell activity. This may be because: (1) There are some disadvantages of lentiviral transduction that cannot be ignored. Its viral DNA will be permanently integrated into the MSCs genome, causing the potential risk of insertion mutation, which may lead to abnormal cell function or even cancer; at the same time, the preparation process of lentivirus is relatively cumbersome, the operation is relatively complex, and the requirements for the experimental environment and operators are relatively strict; (2) The transfection efficiency of liposome transfection is often affected by many factors, such as the ratio of liposomes to nucleic acids, the state of cells, etc., and has a certain degree of instability; and the liposomes themselves may have a certain degree of toxicity to cells, affecting the normal physiological function of cells; (3) However, electrofection shows unique characteristics. Electrofection is to cause changes in cell membrane potential through high-intensity electric fields, instantly increase cell membrane permeability, and create reversible pores on the cell membrane to facilitate the entry of exogenous nucleic acids. It is suitable for almost all types of cells. Compared with the first two, electrofection has the following advantages: it can control the transfection process more accurately, and the transfection efficiency is relatively high and stable; it causes less damage to cells because the electric field action time is short; it is applicable to a wider range of cell types and has greater versatility.

Therefore, in this embodiment, electrofection is preferred to construct universal mesenchymal stem cells.

2. Screening of electrotransfection parameters

Electroporation parameters have a great influence on transfection efficiency, cell survival rate and subsequent growth status. Therefore, in this embodiment, different electroporation parameters were further screened in order to achieve good transfection efficiency while minimizing damage to cells and improving cell survival rate.

Specifically, a negative control (i.e., no electroporation) and three experimental groups were set up. The electroporation parameters in the three experimental groups were as follows: (1) voltage 990 V, electric pulse duration 40 ms, and one electric pulse; (2) voltage 1200 V, electric pulse duration 20 ms, and two electric pulses; (3) voltage 1400 V, electric pulse duration 10 ms, and three electric pulses. The transfection efficiency was determined using a flow cytometer (FACSAria Fusion SORP, BD, USA).

The specific results are shown in Figure 1. It can be seen from Figure 1 that under the electroporation conditions of 1400V/10ms/3pulse (voltage 1400V, electric pulse duration of 10ms, 3 electric pulses), a higher electroporation efficiency was achieved. At this time, the electroporation efficiency was 93.58%. Therefore, in this embodiment, the preferred electroporation parameters are 1400V/10ms/3pulse for preparing universal MSCs.

3. Screening of plasmid dosage

In electrotransfection, the amount of plasmid used also has a great influence on the transfection efficiency and cell viability. Therefore, in this embodiment, different plasmid amounts were further screened: the plasmid amounts were set to three dosage groups of 400 ng, 600 ng and 800 ng, and different universal mesenchymal stem cells were constructed. The optimal amount of plasmid added was determined by comparing the ratio of BFP and GFP fluorescence double-positive cells. The specific results are shown in Figure 2.

As shown in Figure 2, as the amount of plasmid increases, the proportion of double-positive cell populations gradually increases from 3.7% to 15.9%. When the amount of plasmid exceeds 800 ng, the cell viability decreases significantly, which may be due to the saturation effect or some unexpected effects caused by the interaction of too many plasmids with cells. Therefore, the preferred amount of plasmid in this embodiment is 800 ng.

Example 3 Construction of universal mesenchymal stem cells stably expressing luciferase

In this embodiment, in order to better track the universal mesenchymal stem cells in subsequent verification experiments and verify the experimental results, the step of "constructing universal mesenchymal stem cells that stably express luciferase" is added. Among them, the universal mesenchymal stem cells that stably express luciferase are constructed by lentiviral transduction, and the specific construction steps are as follows (wherein, GLOBES is the universal mesenchymal stem cell constructed by the best electrotransfection of the present invention, ntMSCs are untreated mesenchymal stem cells, GLOBES-1 is the universal mesenchymal stem cell constructed by lentiviral transduction in Example 2, and GLOBES-2 is the universal mesenchymal stem cell constructed by liposome transfection in Example 2):

1. Preparation of luciferase lentivirus: Take 5 μg of core plasmid luciferase-neo (Addgene, Catalog No.: 105621), 6 μg of packaging plasmid psPAX2 (Addgene, Catalog No.: 12260) and 6 μg of envelope plasmid pMD2.G (Addgene, Catalog No.: 12259) to prepare luciferase lentivirus;

2. Infect cells: Infect GLOBES, ntMSCs, GLOBES-1 and GLOBES-2 with the prepared luciferase lentivirus respectively;

3. Neomycin drug screening: Neomycin drug screening was performed with a drug screening concentration of 300 μg/μL;

4. Obtain stable expression cells: Obtain GLOBES, ntMSCs, GLOBES-1 and GLOBES-2 cells that stably express luciferase through drug screening and name them luc-GLOBES, luc-ntMSCs, luc-GLOBES-1 and luc-GLOBES-2 respectively;

And in the subsequent examples, the luc-GLOBES, luc-ntMSCs, luc-GLOBES-1 and luc-GLOBES-2 constructed by three different transfection methods in Example 2 were compared and tested for the therapeutic effect of acute lung injury. And in the subsequent verification experiments, the results all showed that the luc-GLOBES constructed by the best electrotransfection method of the present invention has the best lung injury treatment effect, so it does not conflict with the use of lentiviral transduction to construct universal mesenchymal stem cells that stably express luciferase in order to better track universal mesenchymal stem cells. This shows that in subsequent clinical applications, since the effect of the best universal mesenchymal stem cells constructed by electrotransfection in the present invention has been verified, there is no need to construct "universal mesenchymal stem cells that stably express luciferase" again, so there is no need to use the lentiviral transduction method, so in the absence of the step of "constructing universal mesenchymal stem cells that stably express luciferase", the effect of the best universal mesenchymal stem cells constructed by electrotransfection in the present invention in the treatment of lung injury can be further improved.

Example 4: Test of GLOBES's more significant therapeutic effect on acute pneumonia

1. Construction of LPS-induced acute pneumonia model and in vivo bioluminescence imaging tracking experiment

In this example, in order to better compare the therapeutic effects of luc-GLOBES, luc-ntMSCs, luc-GLOBES-1 and luc-GLOBES-2 on acute lung injury, an LPS-induced acute pneumonia model was constructed, and BLI detection (bioluminescence imaging detection) was performed, and alveolar lavage experiments and HE staining experiments were performed in subsequent experiments. The specific operation steps are as follows:

(1) Allogeneic PBMCs were co-cultured with wild-type MSCs 3 days in advance to sensitize PBMCs;

(2) B-NDG hIL15 mice were purchased from Biocytogen. After one week of adaptive feeding, the mice were anesthetized with pentobarbital at a dose of 60 mg/kg and fixed vertically to expose the throat.

(3) Accurately insert the indwelling tube into the mouse trachea, and use a micropipette to drip LPS solution into the mouse lungs through the indwelling tube at a dose of 2 mg/kg;

(4) After the LPS infusion, continue to maintain an upright position for 2 minutes to ensure that the LPS is fully inhaled into the lungs;

(5) After 5 hours of LPS stimulation, the sensitized PBMCs prepared in step 1 were injected into the tail vein of mice at a rate of 1×10 ⁷ /mouse to construct a mouse model of human immune system reconstitution by PBMC reinfusion;

(6) One hour after the PBMCs were reinfused, luc-GLOBES, luc-ntMSCs, luc-GLOBES-1 or luc-GLOBES-2 were injected into the tail vein at a rate of 1×10 ⁶ /mouse for cell therapy, and a PBS group was set up as a negative control;

(7) BLI detection was performed 2 h after injection of luc-GLOBES, luc-ntMSCs, luc-GLOBES-1, or luc-GLOBES-2, which was used as the initial BLI signal value on day 0;

(8) After 3 days, the same amount of LPS was instilled into the trachea again according to the method in step (3) to maintain pneumonia symptoms; and the BLI was tested again according to the method in step (7);

(9) Collect samples 2 days after the second LPS infusion; after taking BLI images, perform alveolar lavage experiments or remove lung tissue for HE staining.

2. HE staining

(1) The lung tissue of mice was removed and fixed with 4% paraformaldehyde for 24 hours;

(2) After dehydration and paraffin embedding, the sections were cut into 7 μm sections;

(3) The sections were dewaxed with xylene in a fume hood and then hydrated with graded ethanol. The duration of each step was as follows: xylene (I) 1 hour, xylene (II) 1 hour, a mixture of equal volumes of xylene and ethanol for 30 minutes, anhydrous ethanol (I) 2 minutes, anhydrous ethanol (II) 2 minutes, 95% ethanol 2 minutes, 80% ethanol 2 minutes, 70% ethanol 2 minutes, and distilled water for 10 minutes.

(4) Stain the nuclei with hematoxylin for 9 min and then rinse them in tap water three times, 5 min each time;

(5) Use a mixture of equal volumes of hydrochloric acid and alcohol to separate the colors for 1 second, and then soak and wash in tap water three times, each time for 5 minutes;

(6) Stain with eosin for 1 second and rinse in tap water three times, 5 minutes each time;

(7) Dehydration: Dehydrate the sections by sequentially placing them in 50% alcohol for 5 seconds, 70% alcohol for 5 seconds, 80% alcohol for 5 seconds, 90% alcohol for 5 seconds, anhydrous ethanol (I) for 5 seconds, and anhydrous ethanol (II) for 5 seconds;

(8) Transparency: Place the dehydrated sections in a solution of equal volumes of xylene and ethanol for 1.5 min; xylene (I) for 2 min; xylene (II) for >2 min;

(9) Add a coverslip and seal the slide with neutral resin. After the resin is completely dry, wipe off the excess resin with 95% alcohol, blow dry, and scan the slide with a slide scanner.

3. Biochemical index detection of bronchoalveolar lavage fluid

(1) Alveolar lavage: After anesthetizing the mouse, lie it flat on the operating table and use surgical instruments to dissect and expose the trachea; use surgical scissors to make a small incision in the trachea, insert the indwelling tube through the small incision, and fix it to the trachea with surgical thread; use a 1 mL syringe to inject 0.5 mL of PBS into the lungs through the indwelling tube, repeatedly blow and aspirate several times, then aspirate it out and transfer it to a 1.5 mL centrifuge tube and store it on ice; then use 0.5 mL of PBS to lavage once more;

(2) Cell number calculation: centrifuge at 400 × g for 10 min at 4°C, resuspend the pellet in 1 mL of PBS, and count the cells using a CountStar instrument;

(3) The supernatant is used to determine various biochemical indicators, including: LDH content detection, albumin content detection and total protein content detection. The specific operations and results are as follows:

①LDH content detection

According to the instructions of LDH Cytotoxicity Assay Kit, the control group and the lactate dehydrogenase (LDH) content in the supernatant of the test culture medium were set up, and 5 replicates were set up for each experimental group, and a "spontaneous LDH group" and a "maximum LDH group" were set up at the same time; at the end of co-culture, 10 μL of the lysis solution in the kit was added to the co-culture well of the "maximum LDH group" and incubated in a 37°C incubator for 45 min; after the lysis was completed, the culture medium of all groups was collected and centrifuged at 400×g for 5 min; 50 μL of the supernatant after centrifugation was taken to a new 96-well plate, 50 μL of the pre-configured Reaction Mixture was added, and the mixture was mixed and incubated at room temperature in the dark for 0.5 h; 50 μL of Stop Solution was added to terminate the reaction, and the absorbance at 490 nm and 680 nm was measured with an enzyme reader; LDH cytotoxicity was calculated according to the formula provided in the kit.

②Albumin content detection

According to the required number of ELISA strips, take out the ELISA strips and reagents 0.5 hours before the experiment and restore them to room temperature; add 100μL of the sample to be tested and the standard working solution to each reaction well, set 5 replicates for each group, seal the plate and incubate in a 37℃ incubator for 90min; discard the liquid in the well plate and shake off the residual liquid; add 100μL of biotin-labeled albumin antibody working solution to each reaction well, seal the plate and incubate in a 37℃ incubator for 60min; discard the liquid in the well plate and shake off the residual liquid; add 350μL of washing solution to each reaction well, soak for 2min and then shake off the washing solution; repeat the washing process 4 times; add 100μL of each reaction well HRP-labeled streptavidin working solution was added, and the plate was sealed and incubated in a 37°C incubator for 30 minutes; 300 μL of washing solution was added to each reaction well, and the plate was soaked for 2 minutes and then the washing solution was dried; the washing was repeated 4 times; 90 μL of color developer was added to each reaction well in the dark, and the plate was sealed and incubated in a 37°C incubator for 15 minutes; 50 μL of stop solution was added to each reaction well, and the OD value at a wavelength of 450 nm was detected using an enzyme reader within 5 minutes; the albumin concentration was calculated using the standard concentration as the horizontal axis and the absorbance OD value as the vertical axis.

③Total protein content detection

A. Preparation of protein standards: a. Take 1.2mL of protein standard preparation solution and add it to a tube of protein standard (30mgBSA). After fully dissolving, prepare a 25mg/mL protein standard solution. It can be used immediately after preparation, or it can be stored for a long time at -20℃. b. Take an appropriate amount of 25mg/mL protein standard and dilute it to a final concentration of 0.5mg/mL. For example, take 20μL of 25mg/mL protein standard and add 980μL of diluent to prepare a 0.5mg/mL protein standard. The standard should be diluted with the same solution as the protein sample. However, for simplicity, the standard can also be diluted with 0.9% NaCl or PBS. The diluted 0.5mg/mL protein standard can be stored for a long time at -20℃.

B. Preparation of BCA working solution: According to the number of samples, prepare an appropriate amount of BCA working solution by adding 50 volumes of BCA reagent A to 1 volume of BCA reagent B (50:1), and mix thoroughly. For example, add 5mL BCA reagent A to 100μL BCA reagent B, mix well, and prepare 5.1mL BCA working solution. BCA working solution is stable at room temperature for 24 hours.

C. Protein concentration test: a. Add 0, 1, 2, 4, 8, 12, 16, 20 μL of the standard to the standard wells of the 96-well plate, and add the standard diluent to make up to 20 μL, which is equivalent to the standard concentration of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 mg/mL. b. Add an appropriate volume of sample to the sample well of the 96-well plate. If the sample is less than 20 μL, add the standard diluent to make up to 20 μL. Please record the sample volume. c. Add 200 μL of BCA working solution to each well and place at 37℃ for 20-30 minutes. d. Use an ELISA reader to measure the absorbance of A562 or wavelengths between 540-595 nm. e. Calculate the protein concentration of the sample based on the standard curve and the sample volume used.

IV. Experimental Results

1. In vitro observation of lung tissue and HE staining experimental results

In this example, the lung tissue was firstly subjected to gross observation and HE staining after ex vivo treatment to identify the improvement of inflammation and the integrity of tissue structure of the lung tissue. The specific results are shown in FIG3A and FIG3B .

From the general picture in Figure 3A, we can see that the lung tissue of the PBS-treated group was significantly higher than that of the normal mice ( The ntMSCs treatment group showed significant lung inflammation and congestion, indicating that the LPS-induced pneumonia model was successfully established. The ntMSCs treatment group showed improved lung inflammation and congestion compared with the PBS treatment group. The lung tissues of the GLOBES-treated group still had severe pneumonia symptoms, indicating that the therapeutic effect of ntMSCs on pneumonia was limited; however, the lung tissues of the GLOBES-treated group had a good improvement effect on pneumonia compared with the PBS-treated group, and The results showed that GLOBES has a better therapeutic effect on pneumonia.

At the same time, the GLOBES-1 treatment group and the GLOBES-2 treatment group were compared in subsequent experiments. The experimental results showed that compared with the GLOBES-1 and GLOBES-2 treatment groups, the inflammation and congestion of the lung tissue in the GLOBES treatment group were significantly reduced, indicating that the GLOBES treatment group constructed by electrotransfection had a more significant improvement effect on pneumonia. This may be because the lentivirus introduced viral DNA, and the liposome transfection method also introduced liposomes. Since both viral DNA and liposomes have certain toxicity, they may cause the sequence of the super enhancer on the B2M gene that responds to IFN-γ stimulation to mutate. This mutation may affect the interaction between the super enhancer and the surrounding regulatory elements, thereby affecting the expression of the B2M gene. The β2-microglobulin encoded by the B2M gene is an important component of the major histocompatibility complex (MHC) class I molecule and plays an important role in the immune response. Therefore, changes in the expression of the B2M gene may affect the function of immune cells, thereby affecting the therapeutic effect on lung injury, and ultimately leading to a significant reduction in the therapeutic effect on lung injury. In this embodiment, the content of “the discovered super enhancer SE on the B2M gene that responds to IFN-γ stimulation” is specifically referred to the description in the invention patent with application publication number CN113846063A.

HE staining in Figure 3B further confirmed the above conclusion. As can be seen from Figure 3B, compared with The normal lung tissue of the LPS-induced mice in the PBS group showed typical acute pneumonia injury, i.e., thickening of alveolar septa, alveolar hemorrhage, and increased neutrophil infiltration. Although ntMSCs treatment alleviated the above acute lung injury to a certain extent, it was still significantly lower than that of the PBS group. The normal lung tissue of the GLOBES group still clearly showed more lung injury areas. However, the lung tissue sections of the GLOBES treatment group were significantly different from those of the ntMSCs group and were more similar to The normal lung tissue slice morphology of the group indicated that the lung injury was greatly improved and repaired after GLOBES treatment. In subsequent experiments, the group was compared with the GLOBES-1 treatment group and the GLOBES-2 treatment group. The experimental results showed that compared with the GLOBES-1 and GLOBES-2 treatment groups, the lung injury in the GLOBES treatment group was significantly improved and repaired after treatment, which further indicated that the GLOBES treatment group constructed by electrotransfection had a more significant lung injury repair effect.

It can be seen that compared with the ntMSCs, GLOBES-1 and GLOBES-2 groups, the GLOBES treatment group constructed by electrotransfection has a more significant therapeutic effect on acute pneumonia.

2. LDH content test results

Figure 4A shows the results of LDH content in the alveolar lavage fluid of different groups. As can be seen from Figure 4A, the LDH content in the alveolar lavage fluid of the PBS and ntMSCs groups was significantly higher than that of the GLOBES treatment group and group; among them, the LDH content in the ntMSCs treatment group was about 2.05 times that of the GLOBES treatment group. Under normal circumstances, LDH only exists in the cytoplasm of tissue cells in the body. The higher the LDH content in the alveolar lavage fluid, the more severe the tissue damage. Therefore, it shows that lung injury has been greatly improved and repaired after GLOBES treatment. In subsequent experiments, the LDH content in the alveolar lavage fluid of the GLOBES-1 treatment group and the GLOBES-2 treatment group was also detected. The experimental results showed that the LDH content in the GLOBES-1 treatment group was about 1.72 times that of the GLOBES treatment group; the LDH content in the GLOBES-2 treatment group was about 1.85 times that of the GLOBES treatment group; compared with the ntMSCs treatment group, the LDH content in the GLOBES-1 and GLOBES-2 treatment groups decreased slightly, but compared with the GLOBES treatment group, it still increased significantly, which further showed that the GLOBES treatment group constructed by electrotransfection had a more significant lung injury repair effect.

It can be seen that the detection results of LDH content in alveolar lavage fluid further prove that the GLOBES treatment group constructed by electrotransfection has a more significant effect in repairing acute pneumonia damage.

3. Albumin content test results

Figure 4B shows the results of albumin content in bronchoalveolar lavage fluid of different groups. It can be seen from Figure 4B that although the ntMSCs treatment group can reduce the albumin content in bronchoalveolar lavage fluid to a certain extent, the corresponding albumin content is still significantly higher than The albumin content in the alveolar lavage fluid of the ntMSCs treatment group was about 1.43 times that of the GLOBES treatment group; and in subsequent experiments, the albumin content in the alveolar lavage fluid of the GLOBES-1 treatment group and the GLOBES-2 treatment group was tested respectively, and the experiment found that the albumin content in the alveolar lavage fluid of the GLOBES-1 treatment group was about 1.28 times that of the GLOBES treatment group; the albumin content in the alveolar lavage fluid of the GLOBES-2 treatment group was about 1.32 times that of the GLOBES treatment group; compared with the ntMSCs treatment group, the albumin content in the alveolar lavage fluid of the GLOBES-1 and GLOBES-2 treatment groups decreased slightly, but compared with the GLOBES treatment group, it still increased; and the albumin content in the GLOBES treatment group was close to Normal albumin content in the group.

Therefore, it can be seen that the detection results of albumin content in bronchoalveolar lavage fluid further prove that the GLOBES treatment group constructed by electrotransfection has a more significant effect in repairing acute pneumonia damage.

4. Total protein content test results

The albumin and total protein content in the alveolar lavage fluid can reflect the permeability of lung tissue. The more protein permeation, the more serious the damage to lung tissue. As shown in Figure 4C, the results of the total protein content determination are similar to those of the albumin determination. The total protein content in the alveolar lavage fluid of the ntMSCs treatment group is about 1.22 times that of the GLOBES treatment group; and in subsequent experiments, it was also found that the total protein content in the alveolar lavage fluid of the GLOBES-1 treatment group and the GLOBES-2 treatment group was about 1.12 times and 1.15 times that of the GLOBES treatment group, respectively; the total protein content in the alveolar lavage fluid of the GLOBES-1 treatment group and the GLOBES-2 treatment group was slightly lower than that of the ntMSCs treatment group, but still increased compared with the GLOBES treatment group.

The total number of cells in the lavage fluid can reflect the degree of inflammatory cell infiltration and permeability of lung tissue. As shown in Figure 4D, although the total number of cells in the alveolar lavage fluid of the ntMSCs treatment group was significantly lower than that of the PBS treatment group, it was still significantly higher than that of the GLOBES treatment group and The total number of cells in the alveolar lavage fluid of the ntMSCs treatment group was 1.77 times that of the GLOBES treatment group; and in subsequent experiments, it was found that the total number of cells in the alveolar lavage fluid of the GLOBES-1 treatment group and the GLOBES-2 treatment group were 1.31 times and 1.42 times that of the GLOBES treatment group, respectively. The total number of cells in the alveolar lavage fluid of the GLOBES-1 treatment group and the GLOBES-2 treatment group also increased significantly compared with the GLOBES treatment group. There was no statistical difference between the groups.

Therefore, the above results all confirm that giving allogeneic MSCs immune compatibility helps MSCs to survive for a long time in pathological environments, thereby stably exerting anti-inflammatory and therapeutic effects and achieving regeneration and repair of damaged tissues. Compared with the universal MSCs constructed by lentiviral transduction and liposome transfection, the universal MSCs constructed by the optimal electrotransfection method in Example 1 have the most significant lung injury treatment effect.

Example 5 GLOBES does not induce immune response in allogeneic MSCs

In this example, mice were injected with equal amounts of luc-GLOBES and luc-ntMSCs cells, and 2 hours later, BLI photography was performed using the IVIS imaging system of the small animal imager as the initial BLI signal value on day 0. Subsequently, BLI detection was performed at the same time points on days 3 and 5 as the BLI signal values on days 3 and 5, and BLI qualitative imaging fluorescence images were recorded respectively. At the same time, the cell survival amounts in the mice on days 0, 3, and 5 were recorded respectively, and the fluorescence signals were quantitatively analyzed using the software provided by the IVIS imaging system of the small animal imager. The specific results are shown in Figures 5A and 5B.

Figure 5A is a qualitative imaging fluorescence result of BLI in mice in different groups, and Figure 5B is a result of the survival of transplanted cells in mice in different groups. As can be seen from Figures 5A and 5B, after the injection of equal amounts of GLOBES and control ntMSCs cells, the unedited ntMSCs showed obvious immune rejection-induced cell death in the inflammatory pathological environment of the lungs. The survival of ntMSCs on the third day was only 45.15% of the initial transplanted amount, and only 12.17% of ntMSCs survived on the fifth day; while GLOBES survived stably in the inflammatory pathological environment of the lungs, with a cell survival rate of 95.82% on the third day, and 85.48% of GLOBES still survived on the fifth day.

At the same time, in subsequent experiments, the same operation was performed on mice injected with equal amounts of luc-GLOBES-1 or luc-GLOBES-2 cells, and the cell survival results were compared. The results showed that although there was no obvious immune rejection-induced cell death in the GLOBES-1 and GLOBES-2 groups, the cell survival rate was significantly reduced. By the fifth day, only 56.48% and 55.65% of GLOBES-1 and GLOBES-2 survived.

Therefore, it can be seen that compared with unedited MSCs, lentiviral-transduced GLOBES-1 and liposome-transfected GLOBES-2, the GLOBES constructed by the optimal electrotransfection method in Example 1 does not cause an immune response from allogeneic MSCs and can survive longer in mice reconstructed with the human immune system.

Example 6 Testing of GLOBES' ability to maintain low immunogenicity under pulmonary inflammatory pathological conditions

In order to verify that GLOBES can still maintain its low immunogenicity under the pathological environment of lung inflammation, an in vivo post-transplantation recovery experiment of GLOBES was conducted in this example. The specific operation is as follows:

1. Preparation of single cell suspension of lung tissue

(1) Humanized mice (mice with human immune system reconstituted) with LPS-induced acute pneumonia were treated with GLOBES, ntMSCs, GLOBES-1, or GLOBES-2 for 48 hours, and then the mice were killed by cervical dislocation. The lungs of the mice were removed in a clean bench and immersed in PBS.

(2) placing 3 mL of mouse lung cell separation solution (DMEM/F12+15 mM HEPES+1% (v/v) penicillin-streptomycin+1×Glutamax, meaning that the mouse lung cell separation solution is prepared by adding 15 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (also called HEPES) buffer, 1% penicillin-streptomycin mixture by volume, and glutamine solution to DMEM/F12 cell culture medium);

(3) Transfer the mouse lungs to the above 35 mm culture dish and cut the lung tissue into 1 ^mm2 pieces using surgical scissors;

(4) Transfer all lung fragments to a 15 mL centrifuge tube, add 4 mL of mouse lung digestion solution (lung cell separation solution + 2 mg/mL collagenase + 0.1 mg/mL DNase I, meaning 2 mg/mL collagenase and 0.1 mg/mL DNase I were added to the lung cell separation solution), and digest in a 37°C water bath for 2 h, shaking and mixing every half an hour;

(6) After digestion, the digestion solution was passed through a 40 μm mesh and centrifuged at 460 rpm for 5 min;

(7) After centrifugation, discard the supernatant, add 3 mL of red blood cell lysis buffer, and lyse at room temperature for 10 min;

(8) After the red blood cell lysis is completed, 9 mL of lung cell separation solution is used to dilute the red blood cell lysis solution, centrifuge at 460 rpm for 5 min, discard the supernatant and resuspend in PBS to obtain a single cell suspension of lung tissue.

(9) Flow cytometry (Beckman Coulter, DxFLEX) was used to detect the expression of HLA-I in vivo and in vitro in mice in different groups, including: blank group (no treatment), GLOBES group, ntMSCs group, GLOBES-1 group, and GLOBES-2 group.

2. Experimental Results

Figure 6 is a flow cytometry result of HLA-I expression in mice in vitro and in vivo in different groups. As can be seen from Figure 6, through the GLOBES in vivo transplantation recovery experiment, the flow cytometry results showed that the HLA-I expression of the control ntMSCs (ntMSCs invivo) in the pneumonia environment in vivo increased by 8.5 times compared with the ntMSCs cultured in vitro (ntMSCs in vitro); while GLOBES in vivo (GLOBES in vivo) still maintained a low HLA-I expression, and the expression was only about 2 times higher than that of GLOBES cultured in vitro (GLOBES in vitro), and the HLA-I expression was only 20.7% of the HLA-I expression in ntMSCs.

At the same time, in subsequent experiments, it was found that the HLA-I expression levels of the GLOBES-1 group (GLOBES-1in vivo) and the GLOBES-2 group (GLOBES-2in vivo) were significantly increased compared with the in vitro culture groups.

Therefore, it can be seen that compared with unedited MSCs, lentiviral-transduced GLOBES-1 and liposome-transfected GLOBES-2, the GLOBES constructed by the optimal electrotransfection method in Example 1 can still maintain its low immunogenicity under the pathological environment of lung inflammatory disease.

Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

SEQUENCE LISTING

SEQ ID NO.1 (enhancer sequence)

aaagccctag cagttatactgc ttttactatt agtggtcgtt tttttctccc ccccgccccccgacaaatca acagaacaaagaaaattacc taaacagcaa ggacataggg aggaacttct tggcacagaactttccaaac actttttcct gaagggatacaagaagcaag aaaggtactc tttcacta gg accttctctgagctgtcctc aggatgcttt tgggactatt tttcttaccc agagaatggagaaaccctgc agggaattcccaagctgtag ttataaacag aagttctcct tctgctaggt agcattcaaa gatcttaatcttctgggtttccgttttctc gaatgaaaaa tgcaggtccg a gcagttaac tggctggggc accattagcaagtcacttag catctctggggccagtctgc aaagcgaggg ggcagcctta atgtgcctcc agcctgaagtcctagaatga gcgcccggtg tcccaagctggggcgcgcac cccagatcgg agggcgccga tgtacagacagcaaactcac ccagtctagt gcatgccttc ttaaacatcacgagactcta agaaaaggaa actgaaaacgggaaagtccc tctctctaac ctggcactgc gtcgctggct tggagacaggtgacggtccc t.

Claims (10)

1. The construction method of the universal mesenchymal stem cells is characterized by comprising the following steps:

(1) Preparing mesenchymal stem cells;

(2) Preparing a transfection complex;

(3) Transfecting the plasmid into a cell;

in the step (3), the plasmid mixture is transfected into mesenchymal stem cells by using an electrotransfection method.

2. The method of claim 1, wherein in step (2), the transfection complex comprises a mixture of mesenchymal stem cells and a plasmid; the plasmid mixture comprises CRISPRoff plasmids and sgRNA plasmids.

3. The method of claim 2, wherein in step (2), the amount of the plasmid is 400 to 800ng.

4. The method of claim 3, wherein in step (2), the mass ratio of CRISPRoff plasmid to sgRNA plasmid is 1:1.

5. The construction method according to claim 4, wherein in the step (3) of the electric transfection parameters, the voltage is 990V-1400V, the electric pulse duration is 10ms-40ms, and the number of pulses is 1-3.

6. A universal mesenchymal stem cell, constructed by the construction method of any one of claims 1-5.

7. Use of a universal mesenchymal stem cell for preparing a preparation for improving the regeneration capacity of lung injury repair, characterized in that the mesenchymal stem cell is constructed by the construction method according to any one of claims 1 to 5.

8. Use of a universal mesenchymal stem cell for preparing a preparation for reducing allograft rejection, characterized in that the mesenchymal stem cell is constructed by the construction method according to any one of claims 1 to 5.

9. Use of a universal mesenchymal stem cell for preparing a formulation for reducing lactate dehydrogenase, albumin and/or total protein content in alveolar lavage fluid, characterized in that it is constructed using the construction method of any one of claims 1 to 5.

10. Use of universal mesenchymal stem cells for the preparation of a formulation for increasing the survival time of mesenchymal stem cells in an in vivo inflammatory environment or for maintaining mesenchymal stem cells with low immunogenicity in vivo, wherein said method uses universal mesenchymal stem cells constructed using the construction method according to any one of claims 1 to 5.