WO2024041292A1 - Use of umbilical cord mesenchymal stem cells in prevention of lung diseases caused by virus infection - Google Patents

Abstract

Use of umbilical cord mesenchymal stem cells in the preparation of a drug for preventing influenza virus infection or coronavirus infection. Use of umbilical cord mesenchymal stem cells in the preparation of a drug for preventing lung diseases caused by virus infection. The umbilical cord mesenchymal stem cells are applied to preventive administration before virus infection, so that the umbilical cord mesenchymal stem cells can control the spread of viruses and prevent virus infection and are suitable for susceptible population such as medical staff, thus providing an effective and safe new way for preventing viral pneumonia.

Description

Application of umbilical cord mesenchymal stem cells in preventing lung diseases caused by viral infection Technical field

The invention relates to the field of preventive drugs for severe viral pneumonia, and in particular to the application of umbilical cord mesenchymal stem cells in preventing lung diseases caused by viral infection.

Background technique

Viral pneumonia is an inflammation of the lungs caused by an upper respiratory tract infection that spreads downward. Herpes simplex virus, varicella-zoster virus, cytomegalovirus, influenza virus, coronavirus, etc., can all cause severe lung disease. These pneumonia viruses are usually contagious to a certain extent and prone to mutation. The population is generally susceptible and has a high incidence rate. In history, it has caused many outbreaks of viral pneumonia around the world and is an important public health issue of global concern.

Human influenza is mainly caused by influenza A viruses and influenza B viruses. Influenza A viruses often undergo antigenic variation. The influenza virus subtypes that have been confirmed to infect humans are H7N9, H5N1, H9N2, H3N2, H1N1, etc. After influenza virus, coronavirus, etc. infect the body, they rapidly replicate in the respiratory tract, which in turn leads to the body's excessive immune response, damages alveolar epithelium and capillary endothelial cells, and causes clinical manifestations similar to acute lung injury.

Influenza viruses, coronaviruses, etc. can freely shuttle within the cells of the human body. The human lungs, liver, kidneys, brain, immune system, excretory system, and reproductive system are all their targets. The new coronavirus can remain asymptomatic in the human body for 14 days or even longer. The purpose is to infect the people it comes into contact with to the maximum extent possible without anyone noticing. Previously, the most popular treatment for COVID-19 patients was the anti-AIDS drug (Karetra), but subsequent double-blind experiments proved that it was ineffective. The outlook for chloroquine is even less promising. Many patients with rheumatoid arthritis and lupus erythematosus have been taking chloroquine for a long time, but there are no reports that such patients are less likely to get COVID-19 or become severely ill. Some hospitals even use chloroquine for all patients, but it does not reduce the risk of serious illness.

In the fight against viruses, a passive but most widely used method is to produce specific vaccines against viruses. Immunity is usually acquired 2 to 3 weeks after vaccination, and a protective immune response can be initiated when the body is exposed to the virus targeted by the vaccine. Vaccines are divided into inactivated vaccines, live attenuated vaccines, recombinant vaccines, subunit vaccines, etc. Since they retain the characteristics of the virus in stimulating the body's immune system, they induce an immune response after being injected into the human body and produce memory T cells and memory B cells. When the virus When invading the human body, these two cells will rapidly proliferate and differentiate into effector T cells and effector B cells. The effector T cells combine with the target cells to lyse and die. The effector B cells produce antibodies that bind to the antigen and quickly eliminate the antigen.

The new coronavirus is a single-stranded RNA coronavirus that is extremely unstable and prone to mutation. Just before February 12, 2020, there were at least 5 haplotypes in the evolutionary tree of the virus. This feature is This is also seen on other viruses. Scientists originally thought that the new coronavirus mainly established an attack on the human body through angiotensin-converting enzyme (ACE-2). However, the virus subsequently mutated and developed three new attack paths: FURIN, GRP78 and CD 147.

Due to the strong variability of viruses, vaccines that are effective against certain viruses are often ineffective when encountering other mutant strains of the virus, so the preventive and control effect of the vaccine will be greatly reduced. During the global COVID-19 outbreak in 2020, the approved Pfizer mRNA recombinant vaccine was 95% effective in preventing infection within 7 days after the second vaccination. However, according to the top international medical journal "The Willow Leaf" published on June 4, 2021 In the largest study to date on the Pfizer/BioNtech vaccine published in The Knife, people who received the Pfizer COVID-19 vaccine were significantly less likely to develop protective antibodies against the mutated strains first detected in India and South Africa, and over time, Antibody levels gradually decrease. 79% showed a detectable antibody response to the original strain, but this dropped to 50% for the Alpha variant (first discovered in the UK) and 50% for the Delta variant (first discovered in India) and Beta mutant strain (first discovered in South Africa), dropped to 32% and 25% respectively ^[1] .

Inactivated vaccines have better antiviral variability than recombinant vaccines, but the antibody titer in the human body will gradually decrease over time after the vaccine is injected. Regular injections are required, and usually one dose of vaccine injection cannot achieve the desired results. Protective efficacy requires two or even three doses of injections. At the same time, the development cycle of a vaccine takes at least several months or even a year. This speed is far less than the mutation speed of the virus. This means that without the intervention of effective drugs such as traditional Chinese medicine, human beings are actually in danger. Riding a bicycle to chase the high-speed train of the virus.

Mesenchymal stem cells (MSCs) are a type of mesenchymal tissue-derived stem cells with multi-directional differentiation potential. They can be obtained from umbilical cord, bone marrow, fat and other tissues. They are a group of multipotent cells that can differentiate into a variety of tissues. Such as bone, cartilage, muscle, ligament, tendon, fat and stromal cell proliferation and differentiation. Although there are many reports of MSCs treating lung injury caused by influenza virus, there are no unified standards regarding the administration method, dosage, time and frequency of administration of MSCs. More clinical research is needed to explore the best method. MSCs dosing regimen. Regarding the role of MSCs in the treatment of influenza virus infection, existing research results focus on the antiviral therapeutic effect after viral infection and do not involve the preventive effect before viral infection ^[2,3] .

As an ideal source of MSCs, human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have attracted more and more attention, and research reports on hUC-MSCs are also increasing. In recent years, with the increasing research on MSCs in respiratory diseases, their role in repairing damaged alveolar epithelial cells, pulmonary vascular endothelial cells, and reversing lung pathological changes has also been confirmed. Other studies have pointed out that CXCL10-CXCR3 promotes the occurrence of neutrophil-mediated viral and non-viral fulminant lung injury ^[4] . However, these are all based on the repair of existing damage. Whether MSCs can protect the body from or reduce viral infection before viral infection or reduce the damage caused by viruses to the body has not yet been reported.

Contents of the invention

The purpose of the present invention is to provide the application of umbilical cord mesenchymal stem cells in the preparation of drugs for preventing viruses.

Another object of the present invention is to provide an application of umbilical cord mesenchymal stem cells in preventing viral lung diseases. influenza Immune dysfunction caused by the virus can trigger a "cytokine storm", leading to severe pneumonia with multiple organ dysfunction, mainly acute respiratory failure. The present invention evaluates the efficacy of allogeneic human umbilical cord mesenchymal stem cells in preventing severe pneumonia caused by H7N9, H5N1 avian influenza viruses, influenza A H3N2 and H1N1 viruses and its inhibitory effect on the new coronavirus SARS-CoV-2 under in vitro conditions. Provide a new way to prevent severe viral pneumonia.

The object of the present invention can be achieved through the following technical solutions:

The present invention applies umbilical cord mesenchymal stem cells to preventive administration before virus infection. It is found that umbilical cord mesenchymal stem cells are of great significance in controlling the spread of viruses and preventing viral infections, and are especially suitable for providing a kind of medicine to susceptible groups such as medical staff. An effective and safe new way to prevent viral pneumonia.

Application of umbilical cord mesenchymal stem cells in the preparation of drugs to prevent influenza virus infection or coronavirus infection.

As a preference of the present invention, the influenza virus is selected from human or animal influenza viruses.

As a further preference of the present invention, the influenza virus includes but is not limited to influenza A virus, influenza B virus, and influenza C virus.

As a further preference of the present invention, the influenza virus is selected from highly pathogenic H7N9 or H5N1 subtype avian influenza viruses.

As a further preference of the present invention, the influenza virus is selected from influenza A H3N2 or H1N1 influenza virus.

As a further preference of the present invention, the coronavirus is selected from the novel coronavirus SARS-CoV-2.

Application of umbilical cord mesenchymal stem cells in the preparation of drugs to prevent lung diseases caused by viral infection.

As a preference of the present invention, the pulmonary disease caused by viral infection is mainly a pulmonary disease caused by human or animal influenza virus or coronavirus infection.

As a further preference of the present invention, the pulmonary disease caused by influenza virus infection is a pulmonary disease caused by but not limited to influenza A virus, influenza B virus, and influenza C virus.

As a further preference of the present invention, the pulmonary diseases caused by influenza virus infection include pulmonary diseases caused by highly pathogenic H7N9 or H5N1 subtype avian influenza viruses.

As a further preference of the present invention, the pulmonary diseases caused by influenza virus infection include pulmonary diseases caused by influenza A H3N2 or H1N1 influenza viruses.

As a further preference of the present invention, the pulmonary diseases caused by coronavirus infection include pulmonary diseases caused by the new coronavirus SARS-CoV-2.

As a further preference of the present invention, the pulmonary disease is severe pneumonia.

Compared with the existing technology, the present invention has the following advantages:

The innovation of the present invention is to use hUC-MSCs for preventive administration of lung diseases caused by viral infection. Preventive drug therapy is widely used in clinical applications, such as patients with thrombotic diseases taking preventive anticoagulant drugs, patients with recurrent migraines who are common clinically taking preventive drugs, and prophylactic antibacterial drugs used in surgical operations. Compared with the 14 days or longer required by injected vaccines to establish immune protection, it only takes about 7 days to use hUC-MSCs to prevent viral lung diseases. When new strains emerge, there will be a lag in vaccine development, and hUC-MSCs can establish a protective effect faster than vaccines. In addition, the vaccine can only specifically immunize a specific virus, and its immune effect against mutant strains or other strains is greatly reduced. hUC-MSCs have a consistent and effective immune effect against influenza viruses and coronaviruses, and hUC-MSCs A single injection can achieve the desired results. Therefore, the application of hUC-MSCs in the preventive administration of lung diseases caused by viral infection can provide quick and efficient protection for medical staff and front-line workers.

The present invention selects mouse models infected by H7N9 avian influenza virus, H5N2 avian influenza virus, influenza A H3N2 virus, and influenza A H1N1 virus, which have a higher clinical lethality than SARS. Early infusion of umbilical cord mesenchymal stem cells can significantly prolong the survival of mice. average survival period, improve the survival rate of mice, and have a high death protection rate. Among them, at the dose of 1.2×10 ⁶ stem cells/animal, the effect of stem cells for 7 days of early prevention was most significant. At the same time, in vitro inhibition experiments of the new coronavirus SARS-CoV-2 have shown that incubating umbilical cord mesenchymal stem cells with PBMC 24 hours in advance and then inoculating the new coronavirus can significantly inhibit the replication of the new coronavirus, with an inhibition rate of up to 90%; stem cells and PBMC The inhibitory effect on the virus when the ratio is 1:10 is significantly better than that when the ratio is 1:5.

Different from the mechanism of vaccine defense against viruses, our mechanistic study points out that hUC-MSCs achieve the purpose of alleviating viral pneumonia by reducing the recruitment of neutrophils to the lungs of mice after infection and reducing the expression of CXCR3 receptors. . And it is safe to use hUC-MSCs in the preventive administration of lung diseases caused by influenza virus infection. Therefore, by incorporating umbilical cord mesenchymal stem cell preparations into existing prevention programs to prevent severe viral pneumonia, it is expected to provide an effective, safe and fast new way to prevent viral pneumonia for vulnerable groups such as medical staff. And provide theoretical basis and data support for the prevention of similar viral infections.

Description of drawings

Figure 1 is a schematic diagram of the test results of the survival protection efficiency of umbilical cord mesenchymal stem cells on H7N9 infected mice according to the present invention.

Control represents the blank control, Model represents the H7N9 virus infection group, MSC 15, MSC 7, and MSC 5 represent the umbilical cord mesenchymal stem cells injected 15 days, 7 days, and 5 days in advance respectively; the same below.

Figure 2 is a schematic diagram of the effect of umbilical cord mesenchymal stem cells on weight gain in mice infected with H7N9 influenza virus.

Figure 3 is a schematic diagram of the effect of umbilical cord mesenchymal stem cells on pulmonary edema in H7N9-infected mice.

Figure 4 is a schematic diagram of the effect of umbilical cord mesenchymal stem cells on mouse serum antibody titers.

Figure 5 is a schematic diagram of the effect of umbilical cord mesenchymal stem cells on the lung pathological tissue structure of H7N9-infected mice.

Figure 6 is a schematic diagram of the effect of umbilical cord mesenchymal stem cells on the cytokine TNF-α in H7N9-infected mice.

Figure 7 is a schematic diagram of the effect of umbilical cord mesenchymal stem cells on the cytokine GM-CSF in H7N9-infected mice.

Figure 8 is a schematic diagram of the effect of umbilical cord mesenchymal stem cells on cytokine IFN-γ in H7N9-infected mice.

Figure 9 shows the effects of umbilical cord mesenchymal stem cells on cytokines IL-1β, IL-2, IL-4, IL-9, IL-12p70, IL-17A, IL-18, IL-23, and IL-22 in H7N9-infected mice. Schematic diagram of the impact of

Figure 10 is a schematic diagram of the effect of umbilical cord mesenchymal stem cells on the cytokine IP-10 in H7N9-infected mice.

Figure 11 is a schematic diagram of the effect of umbilical cord mesenchymal stem cells on cytokines MIP-1α and MIP-2 in H7N9-infected mice.

Figure 12 is a schematic diagram of the process of cytokine storm after viral infection.

Figure 13 is a schematic diagram of the cytokine storm signaling pathway in COVID-19 patients.

Figure 14 shows the results of the in vitro inhibition experiment of umbilical cord mesenchymal stem cells on the new coronavirus.

Figure 15 shows the results of research on the mechanism of umbilical cord mesenchymal stem cells in preventing viral pneumonia in advance.

Detailed ways

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

The following examples illustrate the effectiveness of the present invention by taking H7N9 avian influenza, H5N1 avian influenza, H3N2 influenza A, H1N1 influenza A and novel coronavirus SARS-CoV-2 in vitro inhibition experiments as examples, but do not limit the present invention. scope of protection.

Example 1 A method for preparing umbilical cord mesenchymal stem cells

The mesenchymal stem cell preparation in the following examples is a human umbilical cord mesenchymal stem cell preparation, which is prepared by collecting human umbilical cord. Collect the human umbilical cord and place it in a petri dish. Then clean the umbilical cord tissue with physiological saline. After the cleaning is completed, Wharton's glue is peeled off and placed in a centrifuge tube.

Add an appropriate amount of complete culture medium, cut Wharton's glue into small tissue pieces and plant them in a petri dish for culture.

Remove the culture medium from the culture dish, then wash it with physiological saline and add trypsin for digestion. When the cells in the culture dish are completely digested, add stop solution to terminate the digestion. Transfer the cell suspension to a centrifuge tube, centrifuge and discard the supernatant. Then resuspend the cells in an appropriate amount of culture medium and count them. Finally, according to the counting results, plant the cell suspension in a new culture dish for culture.

Remove the culture medium in the new culture dish, then wash it with physiological saline and add trypsin for digestion. When the cells in the culture dish are completely digested, add stop solution to terminate the digestion. Then filter with a cell sieve and transfer the filtered cell suspension to Count the centrifuge tube and discard the supernatant. Then prepare the cell preparation suspension and add the cell preparation suspension to resuspend the cells. Finally, the cells are suspended. The liquid is transferred into a transfer bag and placed in a low-temperature environment for collection, thereby completing the preparation of mesenchymal stem cell preparations.

Conduct qualification testing on the prepared mesenchymal stem cell preparations.

In the following examples, the mesenchymal stem cell preparations are all carried out in a sterilized ultra-clean bench, and the utensils and consumables required during the preparation process need to be sterilized with 75% ethanol. During the preparation process, the culture dishes also need to be sterilized. The collected materials and specific operation processes are marked in detail, and production-related information is recorded in the GMP workshop ledger.

At the same time, stem cells can also be cryopreserved. If it is necessary to prepare stem cell preparations, the cryopreserved stem cells need to be resuscitated. The cryopreservation and resuscitation operations of stem cells are existing technologies and will not be repeated here.

Umbilical cord mesenchymal stem cells may not be prepared according to the method of this example. Umbilical cord mesenchymal stem cells prepared according to other methods disclosed in the art or commercially available umbilical cord mesenchymal stem cell preparations are also suitable for use in the present invention.

Example 2 H7N9 avian influenza virus infection prevention test in mice

This example was carried out using the human umbilical cord mesenchymal stem cells obtained in Example 1.

experiment method

Female ICR mice weighing 14-15g were randomly divided into 5 groups, 20 in each group, and were adaptively fed for 5 days. The animals were divided into groups according to the following methods: normal control group; viral infection control group; 15-day stem cell prevention group; stem cell In the 7-day prevention group and the 5-day stem cell prevention group, each mouse was inoculated with 1.2×10 ⁶ stem cells via tail vein injection.

After the mice were adaptively fed for 5 days, on the 15th day before the challenge, the 15-day stem cell prevention group was injected with stem cell preparation; on the 7th day before the challenge, the 7-day stem cell prevention group was injected with the stem cell preparation; on the 5th day before the challenge, the mice were adaptively fed for 5 days. days, and the 5-day stem cell prevention group was injected with stem cell preparations.

On the day of challenge, the mice in the normal control group were intranasally infected with PBS, and the virus-infected control group and the early prevention group were all intranasally infected with H7N9 influenza virus at a dose of 7.9 mg/kg.

The intranasal dose of the virus described in this example is 5 times the median lethal dose LD50.

Animal handling: After the end of the experiment, the mice in each group were killed for sampling and analysis. The mice were fasted for 12 hours after the last dose. After being anesthetized with chloral hydrate, they were weighed. The eyeballs were removed to collect blood. EDTA anticoagulant was used directly. T cell analysis was performed with a flow cytometry sorter. The remaining blood samples were centrifuged at 2500 rpm to collect plasma and frozen at -80 degrees for cytokine detection. The lungs were removed and weighed, and the ratio of lung tissue to body weight was recorded. At the same time, the right large lobe of the lung was removed and placed They were fixed in 10% formalin solution and pathological analysis was performed according to the pathological tissue sectioning process. The remaining tissues were frozen in a -80 degree refrigerator for viral load analysis.

Statistical methods: SPSS12.0 was used for statistical analysis of data. Measurement data were expressed as mean ± standard deviation (X ± SD). Paired sample t test was used for single-factor analysis of variance. P < 0.05 indicated statistical difference.

test results

Results of protective efficacy against death in mice infected with H7N9 influenza virus

During the test, observe the disease of the animals every day, weigh them, and record the number of deaths for a total of 15 days. According to "Death Protection Efficiency = (Number of Deaths in the Virus Control Group - Number of Deaths in the Dosing Group)/Number of Deaths in the Virus Control Group × 100%", " Life extension rate = (average survival days of the drug group - average survival days of the virus control group)/average survival of the virus control group × 100%" to calculate the death protection efficiency and life extension rate.

As shown in Figure 1, the H7N9 virus-infected group began to die on the 8th day after challenge, with a death peak on days 9-11. During the entire observation period, 9 mice died, with a mortality rate of 90%. The death protection efficiencies of 15 days, 7 days, and 5 days of preventive treatment with umbilical cord mesenchymal stem cells against H7N9 influenza virus infection were 55.6%, 66.7%, and 44.4% respectively, and there were statistical differences between them and the virus-infected control group (* indicates P <0.05, ** means P<0.01, *** means P<0.001). In addition, early prophylaxis with umbilical cord mesenchymal stem cells can also effectively prolong the survival time of mice infected with H7N9 influenza virus. The average survival time of 15 days, 7 days, and 5 days of early prophylaxis with umbilical cord blood stem cells was 13.2±2.3 days and 13.7±, respectively. The life extension rates of 2.1 days and 12.3±2.9 days were 51.4%, 64.9%, and 27.0% respectively. There were significant differences in the virus-infected control group (P<0.01 or P<0.05).

Based on the above observation indicators, early prevention of umbilical cord mesenchymal stem cells can significantly extend the average survival period of mice and improve the survival rate of mice. The effect of early prevention of stem cells for 7 days is the most significant.

Mouse weight gain

As shown in Figure 2, the weight growth of mice in each group was affected and began to gradually decline 3 days after H7N9 influenza virus infection. Among them, the weight loss of each group prevented by umbilical cord mesenchymal stem cells was significantly lighter than that of the virus-infected control group. At the same time, compared with other groups, the mice in the umbilical cord mesenchymal stem cell prevention group for 7 days had the smallest weight loss and the fastest weight recovery during the recovery period.

The above results show that umbilical cord mesenchymal stem cells for 7 days in advance can significantly improve the weight loss of mice caused by H7N9 influenza virus infection.

The above-mentioned recovery period refers to the period from virus elimination to complete recovery in mice after viral infection.

Detection of lung related indicators

After the mice in each group were sacrificed, the whole lungs were removed, blood stains were washed with physiological saline, and the general morphology of the organs was observed. Weigh and calculate the lung index and lung wet-dry ratio according to "lung index = mouse lung weight/mouse body weight × 100%" and "wet-to-dry ratio = mouse lung wet weight/mouse lung dry weight × 100%".

Table 1 and Figure 3 show that on the 5th and 6th days after H7N9 influenza virus infection, compared with the normal control group, the lung index of the virus-infected control group of mice increased significantly, and there was a significant difference from the normal control group (P< 0.05), and the lung wet-to-dry ratio was significantly reduced (P<0.05), indicating an increase in pulmonary edema in mice; compared with the virus-infected control group, early prevention of mesenchymal stem cells can effectively reduce the lung index and lung wet-to-dry ratio of mice. , among which the effects of 7 days and 5 days of advance prevention are the most significant, and there is significance The difference (P<0.01 or P<0.05) shows that early prevention of mesenchymal stem cells can improve the degree of pulmonary edema in mice.

Table 1. Effect of umbilical cord mesenchymal stem cells on pulmonary edema in H7N9-infected mice

Detection of antibody titers in mice using hemagglutination inhibition method

After the mice were fasted for 12 hours and anesthetized with chloral hydrate, the eyeballs were removed and whole blood was collected into anticoagulant tubes with EDTA added, and centrifuged at 2500 rpm for 10 min to remove the supernatant. Take 25 μL of the serum sample and add it to the first column of the 96-well well. Then use PBS to make a 2-fold contrast dilution to 10 wells. Add 25 μL of 4 units of H7N9 inactivated virus to each well, incubate at room temperature for 45 min, and then add 25 μL of 1% The red blood cells were incubated at room temperature for 45 minutes, and finally the level of serum antibodies was determined based on the wells of the serum sample that inhibited viral hemagglutination.

As shown in Figure 4, the antibodies of mice in each group increased significantly and abnormally 15 days after virus infection, among which the antibodies of mice in the model group increased most significantly, indicating that the mice were successfully infected. Compared with the virus-infected control group, the antibody titers of mice in the 15-day, 7-day and 5-day umbilical cord mesenchymal stem cell early prevention groups were about 2-3 lower on average. Among them, the antibody titer of the 7-day early prevention group was the lowest, suggesting that the umbilical cord Early prevention by mesenchymal stem cells may be related to early clearance of the virus, resulting in reduced antigens and low antibody titers.

Pathological changes in mouse lung tissue

After the mice were dissected, the lungs were removed and weighed, and the ratio of lung tissue to body weight was recorded. At the same time, the right large lobe of the removed mouse lungs was fixed in 10% formalin solution, and pathological analysis was performed according to the pathological tissue sectioning process.

As shown in Figure 5, H&E staining of the lung tissue showed that the alveolar tissue structure of the normal control mice was intact, there was no obvious inflammatory cell infiltration around the bronchioles and small blood vessel walls, the bronchiolar mucosa was intact, and there was no vasodilation, congestion and inflammation in the alveolar septum. Cellular infiltration. Compared with the normal control group, the pathological changes in the lung tissue of mice in the H7N9 influenza virus-infected group were obvious. Diffuse alveolar wall thickening, dilation of blood vessels in the interstitium of the lungs, and the infiltration of a large number of inflammatory cells were seen in the mice in the H7N9 influenza virus-infected group. Infiltration of mononuclear cells and lymphocytes, and bronchiolar mucosa detachment and necrosis. Compared with the virus-infected control group, early prevention with stem cells can effectively improve the effect of lung inflammation, among which the 7-day early prevention effect is the most significant. The lung structure of mice in this group is basically intact, the lung inflammation is mild, and the lung bronchioles and There was only a small amount of monocytes and lymphocytes infiltrating around the walls of small blood vessels.

The above results show that compared with the virus-infected control group, the degree of lung disease in mice in the 7-day and 5-day mesenchymal stem cell prevention groups was significantly reduced.

Liquid phase chip detection of mouse serum cytokines

The mice were anesthetized before dissection and then removed the eyeballs to collect blood, collect EDTA anticoagulated blood, collect plasma by centrifugation at 2500 rpm, and use Luminex liquid phase chip to detect the expression of relevant cytokines.

Figure 6-11 shows the cytokines TNF-α, GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-12p70, IL-17A, IL- 18. IL-23, IP-10, MIP-1α, and MIP-2 were significantly increased, among which the cytokines in the virus-infected control group mice were most significantly increased. Compared with the virus-infected control group, the levels of the above-mentioned cytokines in mice in the 15-day, 7-day, and 5-day prevention groups with umbilical cord mesenchymal stem cells all decreased. Among them, the 15-day prevention of TNF-α in umbilical cord mesenchymal stem cells was related to virus infection. There was a significant difference compared with the control group (p<0.05).

As shown in Figure 12-13, the massive release of cytokines can cause cytokine storm, which is an important cause of acute lung injury. Clinical observation shows that patients with severe 2019-nCoV infection have significant increases in pro-inflammatory cytokines such as TNF-α and IFN-γ, which are characterized by cytokine storms.

Early prevention of mesenchymal stem cells reduces the secretion of multiple cytokines in H7N9-infected mice, suggesting that it can reduce the possibility of acute lung injury.

Example 3 Preventive test on mice infected with H5N1 avian influenza virus

This example was carried out using the human umbilical cord mesenchymal stem cells obtained in Example 1.

Female ICR mice weighing 14-15g were randomly divided into 5 groups, with 10 mice in each group, and were adaptively fed for 5 days. The animals were grouped according to the following methods: normal control group; viral infection control group; stem cell early prevention 15-day group; stem cell In the 7-day prevention group and the 5-day stem cell prevention group, each mouse was inoculated with 1.2×10 ⁶ stem cells via tail vein injection.

After the mice were adaptively fed for 5 days, on the 15th day before the challenge, the 15-day stem cell prevention group was injected with stem cell preparation; on the 7th day before the challenge, the 7-day stem cell prevention group was injected with the stem cell preparation; on the 5th day before the challenge, the mice were adaptively fed for 5 days. days, and the 5-day stem cell prevention group was injected with stem cell preparations.

On the day of challenge, the mice in the normal control group were intranasally infected with PBS, and the virus-infected control group and the early prevention group were all intranasally infected with H5N1 avian influenza virus at a dose of 7.9 mg/kg.

Record the death of mice, and calculate the death protection efficiency of each group of mice according to "death protection efficiency = (number of deaths in the virus control group - number of deaths in the administration group)/number of deaths in the virus control group × 100%".

The results showed that the H5N1 virus-infected group began to die on the 6th day after challenge, with a death peak on days 7-9. During the entire observation period, 10 mice died, with a mortality rate of 100%. The death protection efficiency of umbilical cord mesenchymal stem cells against H5N1 influenza virus infection for 15 days, 7 days and 5 days in advance was 30.0%, 50.0% and 40.0% respectively.

Table 2. Effect of umbilical cord mesenchymal stem cells on mortality in H5N1-infected mice

Example 4 Preventive test on mice infected with influenza A H3N2 virus

This example was carried out using the human umbilical cord mesenchymal stem cells obtained in Example 1.

Female ICR mice weighing 14-15g were randomly divided into 5 groups, with 7 mice in each group, and were adaptively fed for 5 days. The animals were grouped according to the following methods: normal control group; viral infection control group; 15-day stem cell prevention group; stem cell In the 7-day prevention group and the 5-day stem cell prevention group, each mouse was inoculated with 1.2×10 ⁶ stem cells via tail vein injection.

After the mice were adaptively fed for 5 days, on the 15th day before the challenge, the 15-day stem cell prevention group was injected with stem cell preparation; on the 7th day before the challenge, the 7-day stem cell prevention group was injected with the stem cell preparation; on the 5th day before the challenge, the mice were adaptively fed for 5 days. days, and the 5-day stem cell prevention group was injected with stem cell preparations.

On the day of challenge, the mice in the normal control group were intranasally infected with PBS, and the virus-infected control group and the early prevention group were all intranasally infected with influenza A H3N2 virus at a dose of 7.9 mg/kg.

Record the death of mice, and calculate the death protection efficiency of each group of mice according to "death protection efficiency = (number of deaths in the virus control group - number of deaths in the administration group)/number of deaths in the virus control group × 100%".

The results showed that the H3N2 virus-infected group began to die on the 7th day after challenge, with a death peak on days 8-9. During the entire observation period, 6 mice died, with a mortality rate of 86%. The death protection efficiencies of umbilical cord mesenchymal stem cells against H3N2 influenza virus infection for 15 days, 7 days, and 5 days in advance were 33.3%, 66.7%, and 50.0% respectively.

Table 3. Effect of umbilical cord mesenchymal stem cells on mortality in H3N2-infected mice

Example 5 H1N1 influenza A virus infection prevention test in mice

This example was carried out using the human umbilical cord mesenchymal stem cells obtained in Example 1.

Female ICR mice weighing 14-15g were randomly divided into 5 groups, with 8 mice in each group, and were adaptively fed for 5 days. The animals were grouped according to the following methods: normal control group; viral infection control group; stem cell early prevention 15-day group; stem cell In the 7-day prevention group and the 5-day stem cell prevention group, each mouse was inoculated with 1.2×10 ⁶ stem cells via tail vein injection.

After the mice were adaptively fed for 5 days, on the 15th day before the challenge, the 15-day stem cell prevention group was injected with stem cell preparation; on the 7th day before the challenge, the 7-day stem cell prevention group was injected with the stem cell preparation; on the 5th day before the challenge, the mice were adaptively fed for 5 days. days, and the 5-day stem cell prevention group was injected with stem cell preparations.

On the day of challenge, the mice in the normal control group were intranasally infected with PBS, and the virus-infected control group and the early prevention group were all intranasally infected with H1N1 influenza virus at a dose of 7.9 mg/kg.

Record the death of mice, and calculate the death protection efficiency of each group of mice according to "death protection efficiency = (number of deaths in the virus control group - number of deaths in the administration group)/number of deaths in the virus control group × 100%".

The results showed that the H1N1 virus-infected group began to die on the 7th day after challenge, with a death peak on days 8-9. During the entire observation period, 7 mice died, with a mortality rate of 88%. The death protection efficiencies of umbilical cord mesenchymal stem cells against H1N1 influenza virus infection for 15 days, 7 days, and 5 days in advance were 42.9%, 71.4%, and 57.1% respectively.

Table 4. Effect of umbilical cord mesenchymal stem cells on mortality in H1N1-infected mice

Example 6 In vitro inhibition experiment of novel coronavirus (SARS-COV-2)

This example was carried out using the human umbilical cord mesenchymal stem cells obtained in Example 1.

The positive control drug described in this example is remdesivir, a white powder provided by the Jiangsu Provincial Center for Disease Control and Prevention.

The cells described in this example are African green monkey kidney (Vero-E6) cells, provided by the Jiangsu Provincial Center for Disease Control and Prevention. .

The new coronavirus (SARS-CoV-2) strain described in this example is (BetaCoV/JS02/Human/2019) and is stored at -80°C in the P3 laboratory of the Jiangsu Provincial Center for Disease Control and Prevention.

The PBMC cells described in this example were isolated by density gradient centrifugation.

The amount of virus added was MOI=0.05.

The amount of remdesivir added is 5uM.

Inhibitory effect of stem cells on SARS-COV-2 in vitro

Vero-E6 cells were seeded into two 24-well plates at 5 × 10 ⁴ cells/well.

Cell plating: stem cell + PBMC (incubated for 24 hours in advance) group, stem cell group, PBMC group, the concentration of stem cells is 1×10 ⁵ /well, the concentration of PBMC is 1×10 ⁶ /well, the ratio of stem cells and PBMC is 1:10 ; In another 24-well plate, the stem cell concentration is 1×10 ⁵ /well, the PBMC concentration is 5×10 ⁵ /well, and the ratio of stem cells and PBMC is 1:5.

Place two 24-well plates in a 37°C, 5% _CO2 constant temperature incubator for 18 hours. In the stem cell + PBMC (co-incubated for 6 hours in advance) group, the corresponding number of stem cells and PBMC were inoculated into each well; after continuing to culture for 6 hours, except for the cell control New coronavirus (MOI=0.05) was added to all groups outside the group and placed in a 37°C, 5% CO2 incubator for 1 hour. After the adsorption was completed, the culture medium containing the virus was discarded and new virus growth solution was added. The positive control group was added with virus growth solution containing remdesivir (5 μM); cultured in a 37°C, 5% CO2 incubator for 72 hours. After the culture, the culture supernatant from each well was sucked out, inactivated at 56°C for 30 minutes, and viral nucleic acid was detected. Extraction and genetic testing.

Table 5. Cell plate distribution of each experimental group

Statistical processing method: All data are input into the EXCEL table, ΔCt=Ct _{experimental group} -Ct _{Virus control group} , inhibition rate=(1-2 ^-ΔCt )×100%, calculate the inhibition rate of each group. Experimental data are expressed as x ± s. One-way analysis of variance was performed using the GraphPad Prism 8.0 software package. P < 0.05 was considered a significant difference.

According to Figure 14, incubating umbilical cord mesenchymal stem cells with PBMC 24 hours in advance, and then inoculating the new coronavirus can significantly inhibit the replication of the new coronavirus (PBMC (E6) + Stem cells 24h group, PBMC (E5) + stem cells 24h group and Compared with the virus control group (P<0.001), the inhibition rate reached 90% when the ratio of stem cells to PBMC was 1:10; Co-culture of mesenchymal stem cells and PBMC (incubated in advance) can inhibit the replication of the new coronavirus more effectively than the two alone (the PBMC+Stem cells 24h group is compared with the PBMC group and stem cells group respectively); stem cells and PBMC The inhibitory effect on the virus when the ratio is 1:10 is significantly better than that when the ratio is 1:5 (PBMC(E6)+Stem cells 24h group compared with PBMC(E5)+stem cells 24h group, P<0.001).

Example 7 Research experiment on the mechanism of hUC-MSCs in preventing viral pneumonia in advance

This example was carried out using the human umbilical cord mesenchymal stem cells obtained in Example 1.

Female ICR mice weighing 14-15g were randomly divided into 5 groups, 20 in each group, and were adaptively fed for 5 days. The animals were divided into groups according to the following methods: normal group, model group, 7-day stem cell injection group, and 7-day stem cell injection infection. group and positive drug group.

On the 7th day before challenge, each mouse in the 7-day stem cell injection group and the 7-day stem cell injection infection group was inoculated with 1.2×10 ⁶ stem cells via tail vein injection, and the positive drug group was injected with ribavirin. On the day of challenge, except for the normal group and the 7-day stem cell injection group, which were intranasally infected with PBS, all other groups were intranasally infected with H7N9 influenza virus at a dose of 7.9 mg/kg.

Detection of neutrophils and CXCR3 expression in alveolar lavage fluid: Alveolar lavage fluid was labeled with CD45, CD11b, and Ly6G antibodies, and the expression of CD45 ⁺ CD11b ⁺ Ly6G ⁺ neutrophils was detected by flow cytometry. The lavage fluid was stained with CD11b, Ly6G and CXCR3 antibodies for neutrophils, and CXCR3 levels in CD11b ⁺ Ly6G ⁺ cells were analyzed by flow cytometry.

As shown in Figure 15, CD45 ⁺ CD11b ⁺ Ly6G ⁺ represents the number of neutrophils, and CD45 ⁺ CD11b ⁺ Ly6G ⁺ CD183 ⁺ represents the expression of CXCR3. It can be seen that early prevention of stem cells significantly reduces the number of neutrophils in the alveolar lavage fluid of mice (P < 0.05) (Figure 15, A). At the same time, there is a tendency to significantly reduce the expression of CXCR3, and the effect is better than positive. The drug ribavirin (panel B of Figure 15).

main reference

[1] Wall, E.C., et al., Neutralising antibody activity against SARS-CoV-2VOCs B.1.617.2and B.1.351 by BNT162b2vaccination.Lancet, 2021.397(10):p.2331-2333.

[2]Akbari,A.and J.Rezaie,Potential therapeutic application of mesenchymal stem cell-derived exosomes in SARS-CoV-2pneumonia.Stem Cell Research&Therapy,2020.11(1).p.243-258.

[3]Harrell, C.R., et al., Therapeutic Potential of Mesenchymal Stem Cells and Their Secretome in the Treatment of SARS-CoV-2-Induced Acute Respiratory Distress Syndrome.Analytical Cellular Pathology, 2020.169.p.73-82.

[4]Ichikawa, A., et al., CXCL10-CXCR3 enhances the development of neutrophil-mediated fulminant lung injury of viral and nonviral origin. Am J Respir Crit Care Med, 2013.187(1):p.65-77.

Claims (13)

1. Application of umbilical cord mesenchymal stem cells in the preparation of drugs to prevent influenza virus infection or coronavirus infection.
2. The application according to claim 1, characterized in that the influenza virus is selected from human or animal influenza viruses.
3. The application according to claim 2, characterized in that the influenza virus includes but is not limited to influenza A virus, influenza B virus, and influenza C virus.
4. The application according to claim 2, characterized in that the influenza virus is selected from highly pathogenic H7N9 or H5N1 subtype avian influenza viruses.
5. The application according to claim 2, characterized in that the influenza virus is selected from influenza A H3N2 or H1N1 influenza virus.
6. The application according to claim 1, characterized in that the coronavirus is the novel coronavirus SARS-CoV-2.
7. Application of umbilical cord mesenchymal stem cells in the preparation of drugs to prevent lung diseases caused by viral infection.
8. The application according to claim 7, characterized in that the pulmonary diseases caused by viral infection are mainly pulmonary diseases caused by human or animal influenza virus infection or coronavirus infection.
9. The application according to claim 8, characterized in that the lung disease caused by influenza virus infection is not limited to lung diseases caused by influenza A virus, influenza B virus or influenza C virus.
10. The application according to claim 8, characterized in that the pulmonary diseases caused by influenza virus infection include pulmonary diseases caused by highly pathogenic H7N9 or H5N1 subtype avian influenza viruses.
11. The application according to claim 8, characterized in that the pulmonary diseases caused by influenza virus infection include pulmonary diseases caused by influenza A H3N2 or H1N1 influenza viruses.
12. The application according to claim 8, characterized in that the lung disease caused by coronavirus infection is a lung disease caused by the new coronavirus SARS-CoV-2.
13. The application according to any one of claims 7-12, characterized in that the lung disease is severe pneumonia.