WO2021237930A1

WO2021237930A1 - Application of mesenchymal stem cells in treatment of patients with viral infection

Info

Publication number: WO2021237930A1
Application number: PCT/CN2020/105751
Authority: WO
Inventors: 王文军; 王健; 郑春兵; 李莉; 黄险峰; 王成; 王明; 文乐; 康禹; 欧阳智华; 王程; 李学林; 李珊; 邹彩霞
Original assignee: 湖南源品细胞生物科技有限公司
Priority date: 2020-05-23
Filing date: 2020-07-30
Publication date: 2021-12-02

Abstract

Specifically disclosed in the present invention is an application of mesenchymal stem cells in treatment of patients with viral infection. The patients with viral infection are grouped, and treatment effects of healed patients in a mesenchymal stem cell treatment group into which a mesenchymal stem cell preparation is incorporated and in a reference treatment group are compared and analyzed by using a single cell sequencing technology. The mesenchymal stem cells can not only regulate the number of CD4+T cells, memory B cells, NK cells, effector CD8+T cells, and plasma cells in the healed patients, but also regulate gene expression of effector CD4+T cells, NK cells, and effector CD8+T cells in the healed patients, and can also be used for reducing the impact of cytokine storm in the healed patients and inhibiting virus replication in the healed patients. Therefore, the mesenchymal stem cells can effectively enhance the recovery ability of the healed patients, and can also inhibit virus replication in the healed patients and reduce the impact of cytokine storm at the same time, so that the healed patients have stronger immune function.

Description

Application of mesenchymal stem cells in the treatment of patients with viral infections

This application claims the priority of a Chinese patent application filed with the Chinese Patent Office on May 23, 2020. The Chinese patent application includes: Application No. 202010444837.0, and the title of the invention is "A method for treating patients with viral infections by mesenchymal stem cells "; The application number is 202010444838.5, and the title of the invention is "A kind of MSC used to regulate the gene expression of human-effect CD4+T memory cells"; the application number is 202010444840.2, and the title of the invention is "A kind of MSC is used to regulate the number of CD4+ T cells The application number is 202010444815.4, and the invention title is "An application of MSC for regulating the number of memory B cells"; the application number is 202010444831.3, and the invention title is "An application of MSC for regulating the number of NK cells"; application The number is 202010444832.8, and the name of the invention is "an application of MSC for regulating gene expression of NK cells"; the application number is 202010444827.7, and the name of the invention is "an application of MSC for regulating the number of effector CD8+ T cells"; the application number is 202010444826.2, the title of the invention is "An application of MSC for regulating gene expression of effector CD8+ T cells"; the application number is 202010444829.6, and the title of the invention is "An application of MSC for regulating the number of plasma cells"; the application number is 202010444836.6, The title of the invention is "an application of MSC to reduce the impact of cytokine storm"; the application number is 202010444839.X, and the title of the invention is "an application of MSC to inhibit virus replication", all or part of which is incorporated by reference In this application.

Technical field

The present invention relates to the technical field of biomedicine, in particular to the application of mesenchymal stem cells for treating patients with viral infections.

Background technique

Viral infection refers to an infectious disease caused by a virus that can parasitize and multiply in the human body and can cause disease. The main manifestations are fever, headache, general malaise and other symptoms of systemic poisoning, as well as local symptoms caused by viral hosts and invasion of tissues and organs that cause inflammation and damage. Human viral infections are divided into recessive infections, dominant infections, and lentivirus infections. In most cases, the infection is recessive (referring to the human body being infected with the virus, without symptoms, but can produce specific antibodies). A few are overt infections (referring to symptoms after the body is infected with the virus). Among the dominant infections, most viral infections are acute infections, with acute onset and short course of disease, and a few are latent infections (such as herpes virus infections) and chronic infections (such as hepatitis B virus infections, etc.).

At present, there is no specific treatment for viral infections, and support and symptomatic treatment are the main ones. Neither antiviral drugs nor hormone treatments can be effective. In addition, viral infections (such as influenza, SARS, MERS and Ebola) spread rapidly among humans, posing a huge threat to human health. In particular, the new type of coronavirus (COVID-19 virus), which broke out all over the world in 2020, has caused serious losses to human health and the economy. The pathogen of COVID-19 has been confirmed to be a new type of coronavirus, now known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, neither specific drugs nor vaccines for the virus have been successfully developed. The emergence of SARS-CoV-2 has brought a difficult treatment dilemma for clinicians. Most infected patients show non-specific symptoms such as fever, dry cough, and fatigue. Most patients have a good prognosis, and some severe cases can quickly develop into acute respiratory distress syndrome, septic shock, metabolic acidosis, blood coagulation dysfunction, and even death. The deterioration of the patient’s condition may be due to the cytokine storm in the body. In view of the fact that SARS-CoV-2 is a new strain that causes a global pandemic, there is an urgent need for effective targeted therapy, including antiviral and immunotherapy. At present, although many clinical trials have started, there is currently no effective antiviral or immunomodulatory therapy to treat SARS-CoV-2.

Clinical studies have found that MSC has become an effective tool for treating human immunodeficiency virus (HIV) immune abnormalities, hepatitis B virus (HBV) chronic hepatitis, influenza virus acute lung injury (ALI) and other virus-related diseases. Mesenchyma stem cells (MSC) refer to a group of pluripotent stem cells that differentiate into osteoblasts, chondrocytes and adipocytes and have multiple differentiation potentials. They are derived from the immature embryonic connective tissue of the mesoderm and ectoderm in the early stages of embryonic development, and can differentiate into fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, myocardium, A variety of tissue cells such as endothelium and even blood. MSC has low immunogenicity and significant and extensive immunomodulatory functions. MSC does not express CD40, CD80, CD86 and other costimulatory molecules and MHC-II molecules. The inhibitory effect of MSC on T cells is not limited by MHC, that is, the effect on autologous and foreign T cells is similar. The antiviral response is essential to eradicate the virus and prevent the development of virus-related diseases. If antiviral-specific T cells are allowed to function in the presence of MSCs, they have the ability to maintain the integrity of the host's defense against infection. Studies have found that Epstein-Barr virus-specific cytotoxic T cells (CTL) or cytomegalovirus CTL co-cultured with MSC maintain the ability to proliferate and produce interferon-in vitro, and have a killing effect on virus-infected cells. MSC-derived interferon-is believed to offset the immunosuppressive effect of MSC by mediating part of the cytotoxic reaction during viral infection. Therefore, when using MSC as regenerative medicine, it is very important to recognize the dual role of MSC on the immune system against viral infections.

In view of this, the present invention provides an application of mesenchymal stem cells for treating patients with viral infections.

Summary of the invention

The purpose of the present invention is to provide an application of mesenchymal stem cells for the treatment of patients with viral infections, by incorporating mesenchymal stem cell preparations into existing treatment programs for combined treatment of patients with viral infections, so that the patients will have It has stronger immune function, and enhances the recovery ability of patients after recovery, at the same time inhibits virus replication in patients after recovery, and greatly reduces the damage of cytokine storm to tissues in patients after recovery.

In order to achieve the purpose of the present invention, the present invention provides an application of mesenchymal stem cells for treating patients with viral infections.

Preferably, the mesenchymal stem cells are used to regulate the gene expression of human-effect CD4+T memory cells.

Preferably, the mesenchymal stem cells are used to regulate the number of CD4+ T cells.

Preferably, the mesenchymal stem cells are used to regulate the number of memory B cells.

Preferably, the mesenchymal stem cells are used to regulate the number of NK cells.

Preferably, the mesenchymal stem cells are used to regulate NK cell gene expression.

Preferably, the mesenchymal stem cells are used to regulate the number of effector CD8+ T cells.

Preferably, the mesenchymal stem cells are used to regulate gene expression of effector CD8+ T cells.

Preferably, the mesenchymal stem cells are used to regulate the number of plasma cells.

Preferably, the mesenchymal stem cells are used to alleviate the effects of cytokine storm.

Preferably, the mesenchymal stem cells are used to inhibit virus replication.

Compared with the prior art, the present invention combines mesenchymal stem cell preparations into the existing treatment plan for combined treatment of virally infected patients in the mesenchymal stem cell treatment group, so as to adjust the CD4+T in the patients after recovery. The number of cells, memory B cells, NK cells, effector CD8+T cells and plasma cells, and the regulation of the gene expression of effector CD4+T memory cells, NK cells and effector CD8+T cells in patients after healing, and also It can be used to reduce the impact of cytokine storm in patients after recovery and to inhibit virus replication in patients after recovery, so that patients after recovery have a higher chemotactic effect, and enhance the recovery ability of patients after recovery, and at the same time inhibit recovery The virus replication in the patient's body greatly reduces the damage to the tissues of the patient's body by the cytokine storm.

Description of the drawings

Figure 1 is a flow chart of an application method of mesenchymal stem cells used in the treatment of patients with viral infections of the present invention.

Figure 2 is a flow chart of the preparation method of the mesenchymal stem cell preparation of the present invention,

Fig. 3 is a schematic diagram of the proportion of TEM cells in the total number of cells in the corresponding group and the multiple change relative to the healthy group in the present invention, where the proportion of the healthy group in the total number of cells is set to 1.

Figure 4 is a schematic diagram of the ratio of effector CD4+T memory cells in the total number of cells in each group in the present invention.

Figure 5 is a schematic diagram of the differentially expressed genes of the effector CD4+T memory cells of the present invention between the mesenchymal stem cell treatment group and the reference treatment group.

Fig. 6 is a schematic diagram of the proportion of the number of NK cells, effector CD8+ T cells and plasma cells in the total number of cells in the corresponding group in the present invention.

Figure 7 is a schematic diagram of the fold change of the ratio of the number of NK cells, effector CD8+ T cells and plasma cells in the total number of cells in the corresponding group relative to the healthy group in the present invention, where NK cells, effector CD8+ T cells and plasma cells in the healthy group The ratio of the number to the total number of cells in the corresponding group is set to 1,

Figure 8 is a schematic diagram of the differentially expressed genes of the effector CD8+ T cells of the present invention between the mesenchymal stem cell treatment group and the reference treatment group.

Figure 9 is a schematic diagram of the differentially expressed genes of NK cells between the mesenchymal stem cell treatment group and the reference treatment group in the present invention.

Figure 10 is a schematic diagram of the differentially expressed genes of plasma cells of the present invention between the mesenchymal stem cell treatment group and the reference treatment group.

Figure 11 is a schematic diagram of the comparison of the cell gene expression of each group of persons in the present invention.

Figure 12 is a schematic diagram of the comparison of the expression levels of ribosomal genes in each group of people in the body of the present invention.

Figure 13 is a flow chart of the mesenchymal stem cell preparation of the present invention inhibiting virus replication and inhibiting the AP-1/JNK pathway.

Detailed ways

In order to enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

It should be noted that the original treatment plan mentioned in the present invention refers to the treatment plan adopted in the new coronavirus pneumonia diagnosis and treatment plan issued by the General Office of the National Health Commission and the Office of the State Administration of Traditional Chinese Medicine. MSC+ means mesenchymal Stem cell treatment group, MSC- means the reference treatment group, and Health means the healthy group. Among them, the English name of the effector CD4+T memory cell is Effector memory CD4+T cell, abbreviated as TEM cell.

An application of mesenchymal stem cells for the treatment of patients with viral infections. The application method of the mesenchymal stem cells for treating patients with viral infections includes the following steps:

S1. Preparation of mesenchymal stem cell (mesenchymal stem cells, MSC) preparations. The mesenchymal stem cell preparations in this example are human umbilical cord mesenchymal stem cells (hUC-MSC) preparations by collecting human mesenchymal stem cells. Umbilical cord preparation;

S2. Group the virally infected patients to be treated into groups, including at least the mesenchymal stem cell treatment group and the reference treatment group. It should be noted that in other embodiments, the grouping can also be expanded, such as three groups, four groups Etc.; In this embodiment, the virally infected patient refers to a SARS-CoV-2 virally infected patient or a SARS-CoV-2 virus highly homologous patient;

S3. Treat the patients with viral infections in the reference treatment group according to the original treatment plan, and at the same time include the prepared mesenchymal stem cell preparations into the mesenchymal stem cell treatment group. Combination therapy for infected patients;

S4. Use single-cell sequencing technology to sample and analyze the peripheral blood mononuclear cells of patients in the reference treatment group and mesenchymal stem cell treatment group after viral infection.

In this embodiment, as shown in Figure 2, the preparation method of the mesenchymal stem cell preparation includes:

S11. Collect the umbilical cord and place it in a petri dish, and then wash the umbilical cord tissue with normal saline;

S12. Cut the cleaned umbilical cord tissue into small tissue pieces and plant them in a petri dish for culture;

S13. Remove the culture medium in the culture dish, wash with saline and add trypsin for digestion until the cells in the culture dish are digested and add stop solution to stop the digestion, transfer the cell suspension to a centrifuge tube for centrifugation and discard the supernatant Then resuspend the cells in an appropriate amount of culture medium and count them, and finally plant the cell suspension in a new petri dish for culture according to the counting results;

S14. Remove the culture solution in the new culture dish in step S13, then wash it with saline and add trypsin for digestion until the cells in the culture dish are digested and add stop solution to stop the digestion, then filter with a cell sieve and filter the filtered Transfer the cell suspension to a centrifuge tube to count and centrifuge to discard the supernatant, then configure the cell preparation suspension and add the cell preparation suspension to resuspend the cells, and finally transfer the cell suspension to the transfer bag and place it in a low temperature environment for collection. So as to complete the preparation of mesenchymal stem cell preparations;

S15. Perform a qualification test on the mesenchymal stem cell preparation prepared in step S14.

Wherein, the mesenchymal stem cell preparations are all carried out in a sterilized ultra-clean table, and the utensils and consumables needed in the preparation process need to be sterilized with 75% alcohol, and the collected objects in the petri dish need to be sterilized during the preparation process. Or the cells and the specific process of the operation will be marked in detail; at the same time, the stem cells can also be cryopreserved between step S13 and step S14. If necessary, the cryopreserved stem cells need to be resuscitated before the preparation of the stem cell preparation in step S14, wherein the stem cells are cryopreserved Both the recovery operation and the recovery operation belong to the existing technology, and will not be repeated here.

Wherein, the qualification test of the mesenchymal stem cell preparation also includes the quality test of the collected material, the test of the primary cell bank of umbilical cord mesenchymal stem cells and the test of the main cell bank of umbilical cord mesenchymal stem cells, the steps and standards of the qualified test All of them are consistent with the existing common tests, so I won’t repeat them here.

Wherein, the combination therapy in step S3 refers to: using the prepared mesenchymal stem cell preparation as the only interference factor in the original treatment plan to treat patients with viral infections, and at the same time to treat the virus in the mesenchymal stem cell treatment group. The combined medication of patients with sexual infection is tracked and recorded.

Wherein, the mesenchymal stem cell preparation adopts peripheral intravenous infusion.

Wherein, the mesenchymal stem cell preparation is used 2-6 times, and the frequency of use is 2-5 days/time, and the mesenchymal stem cell concentration of the mesenchymal stem cell preparation is 0.1×10 ⁵ -1.2×10 ⁸ Number of cells/ml. In this embodiment, the use frequency of the mesenchymal stem cell preparation is 4 times, and the use frequency is 3 days/time.

Wherein, in the step S4, the peripheral blood mononuclear cells of the recovered patient are analyzed by single-cell sequencing technology (such as single-cell transcriptome combined immune repertoire (TCR/BCR) sequencing), and mesenchymal stem cells are obtained according to the analysis results The therapeutic effect of the preparation on patients with viral infections. In this embodiment, the indicators of the therapeutic effect include: symptom improvement after four uses of the mesenchymal stem cell preparation, inflammation absorption, and the speed of negative viral nucleic acid markers, and the treatment period The changes of lymphocyte subsets, the trough and peak value of lung function FEV1 during treatment, the annual rate of change of lung function FEV1, FVC and FEV1/FVC during treatment, the patient's quality of life, the patient's symptom score, the length of hospital stay and the drop rate.

In this example, by incorporating the prepared mesenchymal stem cell preparation into the existing treatment plan for the combined treatment of patients with viral infections, the cured patients can have higher chemotactic effects and can also enhance the healing. The recovery ability of the post-patients can also inhibit the virus replication in the post-cure patients, and greatly reduce the damage of the cytokine storm to the tissues of the post-cure patients.

An application of mesenchymal stem cells for the treatment of patients with viral infections. By using mesenchymal stem cells to regulate the gene expression of CD4+T memory cells in patients with viral infections, the cured patients have higher resistance to secondary infections At the same time, it can also improve the production potential of CD4+T memory cell cytokines in patients after recovery, so as to provide longer-term effect CD4+T memory cell immune balance for patients after recovery.

An application of mesenchymal stem cells for the treatment of patients with viral infections. By applying mesenchymal stem cells to regulate the number of CD4+ T cells in patients with viral infections, the number of CD4+ T cells in patients after virus invasion can be greatly increased. This allows the recovered patient to respond quickly when resisting the virus attack, thereby enhancing the body's anti-infection ability of the recovered patient.

An application of mesenchymal stem cells for the treatment of patients with viral infections. By applying mesenchymal stem cells to regulate the number of memory B cells in patients with viral infections, it can effectively increase the number of memory B cells in patients after healing. Thereby enhancing the secondary immunity of patients after recovery.

An application of mesenchymal stem cells for the treatment of patients with viral infections. By applying mesenchymal stem cells to regulate the number of NK cells in patients with viral infections, it can effectively increase the number of NK cells in patients after recovery, thereby providing treatment for patients after recovery. After the recovery period, the inflammatory response has a positive effect.

An application of mesenchymal stem cells for the treatment of patients with viral infections. By applying mesenchymal stem cells to regulate NK cell gene expression, it can effectively increase the gene expression of DDIT4 gene in NK cells in patients after M healing, thereby making After healing, NK cells in patients play an active role in participating in the antiviral process mediated by mTORC1.

An application of mesenchymal stem cells for the treatment of patients with viral infections. By applying mesenchymal stem cells to regulate the number of effector CD8+ T cells, it can effectively increase the number of effector CD8+ T cells in the patient after healing, thereby participating in the healing process. The inflammatory response in the post-recovery stage makes the post-cure patients have a stronger ability to recover.

An application of mesenchymal stem cells for the treatment of patients with viral infections. By using mesenchymal stem cells to regulate the gene expression of CD8+ T cells, it can effectively increase the gene expression of CD8+ T cells in patients after healing, thereby promoting the effect. The proliferation of CD8+ T cells makes the patients have stronger anti-inflammatory ability and higher chemotaxis.

An application of mesenchymal stem cells for the treatment of patients with viral infections. By applying mesenchymal stem cells to regulate the number of plasma cells, it can effectively increase the number of plasma cells in patients after healing, so that patients have stronger Anti-inflammatory ability.

An application of mesenchymal stem cells for the treatment of patients with viral infections. By applying mesenchymal stem cells to reduce the impact of cytokine storms in patients with viral infections, it can effectively inhibit the activation of AP-1/JNK pathways in patients after healing , Which reduces the production of cytokines, thereby inhibiting the formation of cytokine storms, thereby effectively reducing the damage of cytokine storms to tissues in patients with viral infections.

An application of mesenchymal stem cells for the treatment of patients with viral infections. By applying mesenchymal stem cells to inhibit virus replication in patients with viral infections, the expression of ribosomal genes in the patients’ body can be restored to a level close to that of healthy people. This can effectively inhibit virus replication in patients after recovery.

In order to better understand the working principle and beneficial technical effects of the present invention, we will take 4 cases of normal neocorona positive patients (ie SARS-CoV-2 viral infection patients) in Hunan as an example for illustration.

As shown in Figure 3-13, first, we put 4 patients with ordinary new crown positive into the mesenchymal stem cell treatment group and the reference treatment group, and included the prepared mesenchymal stem cell preparations into the mesenchymal stem cell treatment In the group, two viral infection patients in the mesenchymal stem cell treatment group were treated in combination, and then after the viral infection patients in the two treatment groups were cured, the peripheral blood mononuclear cells of the cured patients were sampled. At the same time, we also chose A corresponding number of normal people were taken as the healthy group and the peripheral blood mononuclear cells of the healthy group were sampled, and then the collected peripheral blood mononuclear cell samples were grouped and analyzed through single-cell sequencing (in this example, we introduced The R language software performs corresponding analysis and processing on the data), so as to obtain the grouped analysis data of the patients and the healthy group.

As shown in Table 1, Figure 3, Figure 4 and Figure 5, Table 1 shows the relationship between the functional gene expression of patients in the mesenchymal stem cell treatment group and the reference treatment group. Figure 3a shows that TEM cells are in Corresponding to the proportion of the total number of cells in the group, Figure 3b shows the proportion of TEM cells in each group relative to the multiple of the healthy group. From Table 1, Figure 3 and Figure 4, it can be seen that Effective memory CD4+ T cells ( TEM cells) are almost all newly appeared in the cured patients in the mesenchymal stem cell treatment group and the reference treatment group. Among them, the effector CD4+T memory cells of the mesenchymal stem cell treatment group in the cured patients are KLRB1 gene and IL7R gene The genes of KLRB1 and CXCR4 are much higher than those in the reference treatment group. The KLRB1 gene is a powerful gene for the effector CD4+T memory cells to secrete pro-inflammatory factors after being stimulated by the antigen, and the KLRB1 gene can increase IL-17A and pro-inflammatory factors. The production of TNF-α and IFN-γ 12, and the IL7/IL7R pathway is a key pathway that regulates the long-term survival of memory T cells, and is also a key pathway to maintain the balance of antiviral CD4+T memory cell proliferation. The CXCR4 gene is the chemokine CXCL12 Receptor, therefore, the up-regulation of the gene expression of effector CD4+T memory cells means that patients after recovery can clear antigens more quickly and immediately produce cytokine-driven cytotoxic immune responses against recurring infections. At the same time, CD4+T Cells are the main immune cell type against SARS virus, so the role of this subgroup of cells in the process of anti-virus is very important.

Table 1 The relationship table of functional gene expression between patients in the mesenchymal stem cell treatment group and the reference treatment group

In Figure 5, the dots on the right represent up-regulated genes, the dots on the left represent down-regulated genes, and the volcano graph in the middle part represents the differential gene expression between patients in the mesenchymal stem cell treatment group and patients in the reference treatment group (we Here, the false positive rate (FDR) is combined with the avg.logFC value to distinguish significantly different genes. The criteria for distinguishing significantly different genes are: the absolute value of avg.logFC is greater than 1 and the false positive rate (FDR) is less than 0.05), from In Figure 5, we can also see that the function enhancement of effector CD4+ T memory cells is through highly expressed genes that secrete powerful genes for pro-inflammatory factors. Analysis of differentially expressed genes shows that in the newly added effector CD4+ T memory cells, Highly expressed genes in the mesenchymal stem cell therapy group include KLRB1, CXCR4, IL7R and other genes, especially the KLRB1 gene is a potent gene for the effector CD4+T memory cells to secrete pro-inflammatory factors after being stimulated by the antigen, indicating that the mesenchymal stem cell therapy group is After healing, the patient has a higher ability to resist secondary infections.

It can be seen from Figure 6 and Figure 7 that the number of NK cells, effector CD8+ T cells and plasma cells can be observed in the cured patients of the mesenchymal stem cell treatment group and the reference treatment group, compared with the reference treatment In terms of the group, the increase in the mesenchymal stem cell treatment group has increased to a certain extent, given that patients with viral infections still have a certain degree of inflammation after the virus turns negative, and NK cells, effector CD8+ T cells and plasma cells can all participate The proliferation of these immune cells makes the mesenchymal stem cell treatment group and the reference treatment group have virus killing ability, and the patients in the mesenchymal stem cell treatment group have stronger virus killing ability after healing.

As shown in Table 2, Table 3, Figure 8, Figure 9 and Figure 10, Table 2 shows the relationship between the NK cell expression of DDIT4 gene in the mesenchymal stem cell treatment group and the reference treatment group. Table 3 Shows the relationship between the CXCR4 gene expression in the effector CD8+ T cells between the mesenchymal stem cell treatment group and the reference treatment group. It can be seen from Figure 8, Figure 9 and Figure 10, compared with the reference treatment group NK cells in the mesenchymal stem cell treatment group up-regulated the expression of the DDIT4 gene, and the effector CD8+ T cells highly expressed the CXCR4 gene. From Table 1 and Table 2, it can be seen that the cells expressing the DDIT4 gene in the mesenchymal stem cell treatment group accounted for all 60.6% of NK cells, while the cells expressing the DDIT4 gene in the reference treatment group accounted for 11.8% of all NK cells. Compared with the patients in the reference treatment group, the DDIT4 gene in the NK cells of the patients in the mesenchymal stem cell treatment group recovered The expression level was increased by 3.290 times. Because the DDIT4 gene can participate in the antiviral process mediated by mTORC1, the patient still has a certain degree of inflammation during the recovery period after the virus becomes negative. Therefore, the mesenchymal stem cell treatment group The post-curing patients are more conducive to the recovery of the post-curing recovery period and have stronger antiviral ability; at the same time, the cells expressing the CXCR4 gene in the mesenchymal stem cell treatment group accounted for 57.1% of all effector CD8+T cells, and The CXCR4 gene-expressing cells in the reference treatment group accounted for 7.2% of all effector CD8+ T cells. The CXCR4 gene expression in the effector CD8+ T cells of the patients in the mesenchymal stem cell treatment group was 3.161 of the patients in the reference treatment group. Since the CXCR4 gene in effector CD8+ T cells can promote the proliferation of effector CD8+ T cells, and effector CD8+ T cells can participate in the inflammatory response of patients in the post-healing stage, it can be concluded that the treatment of mesenchymal stem cells After the group is cured, the patients with viral infection have stronger anti-inflammatory ability, but also stronger killing ability.

Table 2 Relationship between DDIT4 gene expression in NK cells between patients in the mesenchymal stem cell treatment group and the reference treatment group

Table 3 Relationship between CXCR4 gene expression in effector CD8+ T cells between patients in the mesenchymal stem cell treatment group and the reference treatment group

As shown in Table 4, Table 5, Figure 11 and Figure 13, Table 4 shows the relationship between gene expression levels between the healthy group and the patients in the mesenchymal stem cell treatment group. Table 5 shows the healthy group and the With reference to the relationship between the gene expression levels of patients in the treatment group, Figure 11 shows the expression levels of genes in each cell between the patients in the mesenchymal stem cell treatment group and the reference treatment group, as well as the people in the healthy group (color in the figure) The deeper it is, the higher the gene expression level). From Table 4, Table 5, Figure 11 and Figure 13, it can be seen that the JUN gene and FOS gene of the patients in the mesenchymal stem cell treatment group were significantly down-regulated, while in the mesenchymal stem cell treatment group. The expression of JUN gene in the mesenchymal stem cell treatment group was lower. At the same time, the DUSP2 gene expression in the mesenchymal stem cell treatment group and the healthy group remained almost at the same level, but the DUSP2 gene expression in patients in the reference treatment group was down-regulated, while the reference treatment group The expression of JUN gene is up-regulated in patients after healing. Because JUN gene and FOS gene are the subunits of the transcription factor AP-1, and AP-1 is mainly involved in the transcription of multiple cytokines, mesenchymal stem cell preparations can affect AP-1 /JNK pathway is inhibited. Therefore, in the mesenchymal stem cell treatment group, the AP-1/JNK pathway was down-regulated to reduce the impact of cytokine storm, thereby reducing the damage to the tissues of the patients after recovery; Figure 13 is based on SARS-CoV- 2 Virus as an example, the process of mesenchymal stem cell preparations inhibiting the AP-1/JNK pathway will be explained in detail, because the transcription factor AP-1 can inhibit virus replication and regulate IL-6, IL-8 and other pro-inflammatory cells Factor transcription, ACE2 is the receptor of SARS-CoV-2, and AngII is the substrate of ACE2 which can induce the activation of AP-1. Therefore, the combination of SARS-CoV-2 virus and ACE2 can induce the accumulation of AngII, thereby activating the AP-1/JNK pathway and eventually leading to a cytokine storm. Therefore, mesenchymal stem cell preparations can inhibit cytokine transcription to reduce excessive cells in the body. Factors to achieve effective treatment of SARS-CoV-2.

Table 4 The relationship table of gene expression levels between the healthy group and the patients in the mesenchymal stem cell treatment group

Table 5 The relationship table of gene expression between the healthy group and the reference treatment group after recovery

As shown in Table 6, Table 7, Figure 12 and Figure 13, Table 6 shows the relationship of ribosomal gene expression between the healthy group and the mesenchymal stem cell treatment group, and Table 7 shows the healthy group and the reference treatment group The relationship between ribosomal gene expression among people in the group can be seen from Table 6, Table 7, Figure 12, and Figure 13. Compared with the people in the healthy group, the ribosomal gene RPS4Y1 of the patients in the reference treatment group was significantly up-regulated and ribosome The somatic gene RPS26 was significantly down-regulated, and the ribosomal genes RPS4Y1 and RPS26 in the cured patients of the mesenchymal stem cell treatment group were closer to the normal level of the healthy group than the patients in the reference treatment group, and the ribosomal genes RPS4Y1 and RPS26 were both involved The formation of the 40S subunit is rich in the viral mRNA translation pathway (R-HSA-192823). Therefore, in the reference treatment group, the expression of the ribosomal genes RPS4Y1 and RPS26 of the cured patients is unregulated and contributes to virus replication. The ribosomal genes RPS4Y1 and RPS26 of the cured patients in the mesenchymal stem cell treatment group were close to the normal levels of the healthy group, so it can effectively inhibit the virus replication in the cured patients.

Table 6 The relationship between ribosomal gene expression between the healthy group and the patients in the mesenchymal stem cell treatment group

Table 7 The relationship between ribosomal gene expression between the healthy group and the patients in the reference treatment group

In this example, in order to further understand the therapeutic effect of mesenchymal stem cell preparations on patients with viral infections, we have also developed a research flow chart (see Table 8, where the "X" corresponding to each follow-up in Table 8 represents this time Follow-up patient information that should be collected), follow-up patients with viral infections according to the research flow sheet (follow-up respectively within 2 years of receiving treatment, including the 1, 4, 7, and 10 days of the treatment period, and 3 days after the end of treatment, 1 week, 2 weeks, 1 month, 3 months, 6 months, 12 months, 24 months for follow-up), the first follow-up should record the patient’s age, gender, race, weight (barefoot, light clothing) (Kg) and height (cm ), collect patient diary cards, physical examination, inquire about the occurrence of adverse events, medications, changes in symptoms, smoking status, electrocardiogram, biochemical examination, blood routine, inflammatory factors, lymphocyte subsets during each follow-up; during V1-V5 Check lung CT, simple lung function, and tumor markers every 0-3 weeks (preferably 1 week), and detect secretory nucleic acid at each visit. During V6-V12, check lung CT at each visit , Simple lung function, tumor markers, V6-V8 three visits need to detect secreted nucleic acid. After the above follow-up content is over, the patient can use the medication on the day of the follow-up.

Table 8 Research process table

The application of a mesenchymal stem cell provided by the present invention for treating patients with viral infections has been described in detail above. Specific examples are used in this article to illustrate the principle and implementation of the present invention. The description of the above examples is only used to help understand the core idea of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

An application of mesenchymal stem cells for the treatment of patients with viral infections.
The use of the mesenchymal stem cells in the treatment of patients with viral infections according to claim 1, wherein the mesenchymal stem cells are used to regulate the gene expression of human-effect CD4+T memory cells.
The use of mesenchymal stem cells for the treatment of patients with viral infections according to claim 1, wherein the mesenchymal stem cells are used to regulate the number of CD4+ T cells.
The use of the mesenchymal stem cells for the treatment of patients with viral infections according to claim 1, wherein the mesenchymal stem cells are used to regulate the number of memory B cells.
The use of mesenchymal stem cells for treating patients with viral infections according to claim 1, wherein the mesenchymal stem cells are used to regulate the number of NK cells.
The use of the mesenchymal stem cells for the treatment of patients with viral infections according to claim 1, wherein the mesenchymal stem cells are used for regulating gene expression of NK cells.
The use of mesenchymal stem cells for the treatment of patients with viral infections according to claim 1, wherein the mesenchymal stem cells are used to regulate the number of effector CD8+ T cells.
The use of the mesenchymal stem cells for the treatment of patients with viral infections according to claim 1, wherein the mesenchymal stem cells are used for regulating gene expression of effector CD8+ T cells.
The use of mesenchymal stem cells for treating patients with viral infections according to claim 1, wherein the mesenchymal stem cells are used to regulate the number of plasma cells.
The use of the mesenchymal stem cells in the treatment of patients with viral infections according to claim 1, wherein the mesenchymal stem cells are used to alleviate the effects of cytokine storm.
The use of the mesenchymal stem cells for the treatment of patients with viral infections according to claim 1, wherein the mesenchymal stem cells are used to inhibit virus replication.