CN115089612B

CN115089612B - Application of umbilical cord mesenchymal stem cells in preventing lung diseases caused by virus infection

Info

Publication number: CN115089612B
Application number: CN202211002364.4A
Authority: CN
Inventors: 王广基; 郭薇; 孙银花
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2022-08-22
Filing date: 2022-08-22
Publication date: 2022-11-22
Anticipated expiration: 2042-08-22
Also published as: CN115089612A; WO2024041292A1

Abstract

The invention discloses application of umbilical cord mesenchymal stem cells in preventing lung diseases caused by influenza virus infection. Application of umbilical cord mesenchymal stem cells in preparing a medicament for preventing influenza virus infection or coronavirus infection. Application of umbilical cord mesenchymal stem cells in preparing a medicament for preventing viral lung diseases. The umbilical cord mesenchymal stem cells are applied to preventive administration before virus infection, so that the significance of the umbilical cord mesenchymal stem cells in controlling the spread of the virus and preventing the virus infection is great, and the method is particularly suitable for providing an effective and safe new way for preventing viral pneumonia for susceptible people such as medical workers.

Description

Application of umbilical cord mesenchymal stem cells in preventing lung diseases caused by virus infection

Technical Field

The invention relates to the field of viral severe pneumonia prevention medicines, and particularly relates to application of umbilical cord mesenchymal stem cells in prevention of lung diseases caused by virus infection.

Background

Viral pneumonia is an inflammation of the lungs caused by upper respiratory tract infection, spreading downward. Herpes simplex virus, varicella-zoster virus, cytomegalovirus, influenza virus, coronavirus, etc., all cause serious lung diseases. The pneumonia viruses are generally infectious to a certain extent, are easy to mutate, are generally susceptible to people, have high incidence rate, historically cause multiple viral pneumonia outbreaks all over the world, and are important public health problems concerned all over the world.

Human influenza is mainly caused by influenza a virus and influenza b virus, the influenza a virus often has antigenic variation, and the subtypes of the influenza virus infecting human have been proved to be H7N9, H5N1, H9N2, H3N2, H1N1 and the like. After the body is infected by influenza virus, coronavirus and the like, the virus can be rapidly replicated in the respiratory tract, so that the excessive immune response of the body is caused, alveolar epithelium and capillary endothelial cells are damaged, and the clinical manifestation similar to acute lung injury appears.

Influenza virus, coronavirus and the like can freely shuttle in human cells, and human lungs, livers, kidneys, brains, immune systems, excretory systems and reproductive systems are all targets of attack. The new coronavirus can be latent without symptoms in human body for 14 days or even longer, and aims to maximally transmit to people who can be contacted with the new coronavirus in the unconsciousness and unconsciousness. Anti-aids (krestin) was the most highly called prior treatment for new coronary patients, but it was not effective as demonstrated by the following double blind experiment. The prospect of chloroquine is less optimistic. Many patients with rheumatoid arthritis and lupus erythematosus take chloroquine for a long time, but there is no report that such patients do not easily get new crowns or become severe. Even some hospitals use chloroquine for all patients, and the effect of reducing serious illness is not achieved.

One passive but most widely used approach in human fight against viruses is to produce specific vaccines against the viruses. Immunity is usually achieved 2 to 3 weeks after vaccination and a protective immune response is initiated when the organism comes into contact with the virus against which the vaccine is directed. The vaccine is divided into inactivated vaccine, attenuated live vaccine, recombinant vaccine, subunit vaccine and the like, because the characteristic that the virus stimulates the immune system of the organism is kept, the immune response is caused after the injection into a human body, memory T cells and memory B cells are generated, when the virus invades the human body, the two cells can be rapidly proliferated and differentiated into effector T cells and effector B cells, the effector T cells are combined with target cells to be cracked and killed, and the effector B cells generate antibodies to be combined with antigens to rapidly remove the antigens.

The new coronavirus is a single-stranded RNA coronavirus which is extremely unstable and very easy to generate variation, only before 2020, 2, month and 12 days, at least 5 haplotypes exist in the evolutionary tree of the virus, and the characteristic is also embodied in other viruses. Scientists originally thought that the new coronavirus mainly establishes an attack path to human bodies through angiotensin converting enzyme (ACE-2), but then the virus has mutation, and three new attack paths FURIN, GRP78 and CD 147 are developed.

Because of the strong viral variability, vaccines that are effective against certain viruses tend to be ineffective when encountering other mutant strains of the virus, and the prophylactic control effect of the vaccine will be greatly reduced. For global new crown pneumonia, the efficacy of the pfeiri mRNA recombinant vaccine approved for marketing to prevent infection within 7 days after the second vaccination can reach 95%, but according to one of the largest studies on the pfeiri/BioNtech vaccine published on international top-grade medical journal lancet at 6/4/2021, after the pfeiri mRNA vaccine was inoculated, people generated a large discount on protective antibodies against the variant strain originally detected in india and south africa, and the antibody level gradually decreased with time. 79% of the people show a detectable antibody response to the original strain, but for the Alpha variant strain (first found in the uk), this ratio drops to 50%; whereas for Delta variant strains (first found in India) and Beta variant strains (first found in south Africa), the reduction was 32% and 25%, respectively ^[1] 。

Inactivated vaccines have better antiviral variability than recombinant vaccines, but the antibody titers in humans decrease over time after vaccine injection, requiring regular injections, and often the desired protective efficacy is not achieved with one vaccine injection, requiring two or even three injections. Meanwhile, the development cycle of the vaccine requires a period of several months or even one year at least, and the speed is far less than the virus mutation speed, namely, if effective medicines such as traditional Chinese medicine are not introduced, human beings are actually riding a high-speed train for chasing the viruses.

Mesenchymal Stem Cells (MSCs) are mesenchymal tissue-derived stem cells having multipotential differentiation potential, which can be obtained from various tissues such as umbilical cord, bone marrow, adipose, etc., and are a group of multipotential cells capable of expressing multipotential stem cells to various tissues such as bone, cartilage, muscle, ligament, tendon, adipose, etcAnd the proliferation and differentiation of stromal cells. Although few reports of the MSCs for treating lung injury caused by influenza virus exist at present, the MSCs dosage mode, dosage time, dosage frequency and the like have no unified standard, and more clinical studies are needed to search for the optimal MSCs dosage scheme. Aiming at the function of MSCs in treating influenza virus infection, the existing research results focus on the antiviral treatment function after virus infection, and do not relate to the prevention function before virus infection ^{[2, 3]} 。

Human umbilical cord mesenchymal stem cells (hUC-MSCs) are increasingly regarded as ideal sources of MSCs, and research reports on the hUC-MSCs are increasing. With the recent increase of research on MSCs in respiratory diseases, the functions of repairing and damaging alveolar epithelial cells and pulmonary vascular endothelial cells and reversing pathological changes of lungs are also proved. Another study has indicated that CXCL10-CXCR3 promotes the development of neutrophil-mediated viral and non-viral fulminant lung injury ^[4] . However, these are based on the repair of existing lesions and it has not been reported whether MSCs could protect the body from or reduce viral infection or reduce viral damage prior to viral infection.

Disclosure of Invention

The invention aims to provide application of umbilical cord mesenchymal stem cells in preparing a medicament for preventing viruses.

Another object of the present invention is to provide an application of umbilical cord mesenchymal stem cells in the prevention of viral lung diseases. Influenza virus-induced immune dysfunction causes "cytokine storm" leading to severe pneumonia with multiple organ dysfunction, mainly acute respiratory failure. The invention evaluates the curative effect of the allogeneic human umbilical cord mesenchymal stem cells on preventing severe pneumonia caused by H7N9, H5N1 avian influenza virus, A type H3N2 and H1N1 influenza virus and the inhibition effect on novel coronavirus SARS-CoV-2 under the in vitro condition, and provides a new way for preventing the severe viral pneumonia.

The purpose of the invention can be realized by the following technical scheme:

most of the existing reports about the oncogenic effect of the MSCs take bone marrow MSCs as a research object, and umbilical cord mesenchymal stem cells (hUC-MSCs) as a research object are less reported. The umbilical cord mesenchymal stem cells are applied to preventive administration before virus infection, the significance of the umbilical cord mesenchymal stem cells in controlling virus transmission and preventing virus infection is found to be great, and the umbilical cord mesenchymal stem cells are particularly suitable for providing an effective and safe new way for preventing viral pneumonia for susceptible people such as medical workers and the like.

Application of umbilical cord mesenchymal stem cells in preparing medicines for preventing influenza virus infection or coronavirus infection.

Preferably, the influenza virus is selected from human or animal influenza viruses.

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

As a further preference of the invention, the influenza virus is selected from highly pathogenic avian influenza viruses of subtype H7N9 or H5N 1.

As a further preferred aspect of the present invention, said influenza virus is selected from influenza a H3N2 or H1N1 virus.

As a further preferred aspect of the present invention, said coronavirus is selected from the group consisting of novel coronavirus SARS-CoV-2.

The application of umbilical cord mesenchymal stem cells in preparing a medicament for preventing lung diseases caused by virus infection.

As a preferable mode of the invention, the lung diseases caused by the virus infection are mainly lung diseases caused by human or animal influenza virus or coronavirus infection.

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

As a further preferred aspect of the present invention, the pulmonary disease caused by influenza virus infection includes pulmonary disease caused by highly pathogenic H7N9 or H5N1 subtype avian influenza virus.

As a further preferred aspect of the present invention, the pulmonary disease caused by influenza virus infection includes pulmonary diseases caused by influenza A virus H3N2 or H1N 1.

As a further preferred aspect of the present invention, the pulmonary disease caused by coronavirus infection includes pulmonary disease caused by the novel coronavirus SARS-CoV-2.

In a further preferred embodiment of the present invention, the pulmonary disease is severe pneumonia.

Compared with the prior art, the invention has the following advantages:

the innovation of the invention is that the hUC-MSCs are used for preventive administration of lung diseases caused by virus infection. The preventive medicine has wide clinical application, for example, patients with thrombotic diseases take anticoagulant medicines preventively, patients with recurrent migraine frequently take preventive medicines clinically, and patients with recurrent migraine in surgery take antibacterial medicines preventively. Compared with the vaccine injection which requires about 14 days or even longer time to establish the immune protection effect, the hUC-MSCs are used for preventing the viral lung diseases for about 7 days. When a novel strain appears, the development of the vaccine can have a hysteresis phenomenon, and the hUC-MSCs can establish a protective effect faster than the vaccine. In addition, the vaccine can only specifically immunize a certain specific virus, the immune effect on a variant strain or other strains of the specific virus is greatly reduced, the hUC-MSCs have consistent effective immune effect on influenza viruses, coronaviruses and the like, and the expected effect can be achieved by single injection of the hUC-MSCs. Therefore, the application of the hUC-MSCs in the preventive administration of lung diseases caused by virus infection can provide quick and efficient protection for medical care personnel, first-line workers and the like.

The invention selects H7N9 avian influenza virus, H5N2 avian influenza virus, influenza A H3N2 virus and influenza A H1N1 virus with higher clinical lethality than SARS to infect a mouse model, and the umbilical cord mesenchymal stem cells are infused in advance to obviously prolong the average survival period of the mouse and improve the survival rate of the mouse, thereby having very high death protection rate. Wherein, the ratio is 1.2 multiplied by 10 ⁶ The effect of stem cell prevention in advance for 7 days is most remarkable at each dose of stem cells. At the same time, the experiment of in vitro inhibition of novel coronavirus SARS-CoV-2The umbilical cord mesenchymal stem cells and PBMC are incubated together 24 hours in advance, and then the new coronavirus is inoculated, so that the replication of the new coronavirus can be obviously inhibited, and the inhibition rate can reach 90%; the inhibitory effect on the virus was significantly better when the ratio of stem cells to PBMCs was 1.

Unlike the mechanism of vaccine defense against viruses, our mechanistic studies indicate that hUC-MSCs achieve viral pneumonia reduction by reducing neutrophil recruitment into the mouse lung following infection and by reducing CXCR3 receptor expression. And the application of the hUC-MSCs in the preventive administration of lung diseases caused by influenza virus infection is safe. Therefore, the umbilical cord mesenchymal stem cell preparation is brought into the existing prevention scheme to prevent the viral severe pneumonia, a new way for effectively, safely and quickly preventing the viral pneumonia is hopefully provided for susceptible people such as medical workers, and theoretical basis and data support are provided for preventing similar viral infection diseases.

Drawings

FIG. 1 is a schematic diagram of the survival protection efficiency pre-test result of the umbilical cord mesenchymal stem cells on H7N9 infected mice in the invention.

Control represents blank Control, model represents H7N9 virus infection group, MSC15, 7, MSC5 represent umbilical cord mesenchymal stem cell prevention treatment 15 days, 7 days, 5 days respectively; the same is applied below.

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

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

FIG. 4 is a graph showing the effect of umbilical cord mesenchymal stem cells on the serum antibody titer of mice.

FIG. 5 is a schematic diagram of the effect of umbilical cord mesenchymal stem cells on the lung pathological tissue structure of H7N 9-infected mice.

FIG. 6 is a schematic diagram showing the effect of umbilical cord mesenchymal stem cells on the H7N 9-infected mouse cytokine TNF- α.

FIG. 7 is a schematic diagram showing the effect of umbilical cord mesenchymal stem cells on the H7N 9-infected mouse cytokine GM-CSF.

FIG. 8 is a schematic diagram showing the effect of umbilical cord mesenchymal stem cells on the cytokine IFN-gamma of H7N 9-infected mice.

FIG. 9 is a schematic diagram of the effect of umbilical cord mesenchymal stem cells on H7N 9-infected mouse cytokines IL-1 beta, IL-2, IL-4, IL-9, IL-12p70, IL-17A, IL-18, IL-23, IL-22.

FIG. 10 is a schematic diagram showing the effect of umbilical cord mesenchymal stem cells on the cytokine IP-10 of H7N 9-infected mouse.

FIG. 11 is a schematic diagram showing the effect of umbilical cord mesenchymal stem cells on H7N 9-infected mouse cytokines MIP-1 alpha and MIP-2.

FIG. 12 is a schematic representation of the cytokine storm generation process following viral infection.

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

FIG. 14 shows the results of in vitro inhibition experiments of novel coronavirus by umbilical cord mesenchymal stem cells.

FIG. 15 shows the results of the mechanism of prevention of viral pneumonia by umbilical cord mesenchymal stem cells.

Detailed Description

In order to make the technical solution better understood by those skilled in the art, the present invention is further described in detail below with reference to the accompanying drawings.

The following examples illustrate the effectiveness of the present invention in vitro inhibition experiments of H7N9 avian influenza, H5N1 avian influenza, H3N2 influenza a, H1N1 influenza a and novel coronaviruses, but do not limit the scope of the present invention.

Example 1 preparation method of umbilical cord mesenchymal stem cells

The mesenchymal stem cell preparation in the following examples is a human umbilical cord mesenchymal stem cell preparation, and is prepared by collecting a human umbilical cord. Collecting human umbilical cord, placing the human umbilical cord in a culture dish, cleaning umbilical cord tissue by using normal saline, and stripping Wharton's jelly in a centrifuge tube after cleaning.

Adding proper amount of complete culture solution, shearing Wharton's jelly into small tissue blocks, and culturing in culture dish.

Removing the culture solution in the culture dish, cleaning the culture dish by using normal saline, adding pancreatin for digestion, adding a stop solution to stop digestion until the cells in the culture dish are digested, transferring the cell suspension into a centrifuge tube for centrifugation, discarding supernatant, resuspending the cells by using a proper amount of culture solution, counting, and finally planting the cell suspension into a new culture dish for culture according to the counting result.

Removing the culture solution in the new culture dish, then washing with normal saline, adding pancreatin for digestion, adding a stop solution to stop digestion until the cells in the culture dish are digested, filtering with a cell sieve, transferring the filtered cell suspension to a centrifuge tube, counting, centrifuging and discarding the supernatant, preparing a cell preparation suspension, adding a cell preparation suspension to resuspend cells, transferring the cell suspension to a transfer bag, and placing the transfer bag in a low-temperature environment for taking, thereby completing the preparation of the mesenchymal stem cell preparation.

And (4) carrying out qualification detection on the prepared mesenchymal stem cell preparation.

In the following examples, the mesenchymal stem cell preparations are all performed in a sterilized clean bench, instruments, consumables and the like required in the preparation process need to be sterilized with 75% ethanol, collected materials in a culture dish and specific operation processes need to be labeled in detail in the preparation process, and production related information is recorded in a GMP workshop ledger.

Meanwhile, the stem cells can be frozen, and the frozen stem cells need to be revived before the stem cell preparation is prepared, wherein the stem cell freezing and reviving operations belong to the prior art, and are not described in detail herein.

Umbilical cord mesenchymal stem cells may also be prepared without following the method of this example, umbilical cord mesenchymal stem cells prepared according to other methods known in the art or commercially available preparations of umbilical cord mesenchymal stem cells are also suitable for use in the present invention.

Example 2H 7N9 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.

Test method

Female ICR mice weighing 14-15g were randomly divided into 5 groups20 animals per group were fed ad hoc for 5 days and were grouped as follows: a normal control group; virus infection control group; the stem cells are prevented for 15 days in advance; the group with stem cells prevented for 7 days in advance and the group with stem cells prevented for 5 days in advance were inoculated with 1.2X10 of the injection into the tail vein of each mouse ⁶ And (4) stem cells.

After 5 days of adaptive feeding, on the 15 th day before toxin counteracting, the stem cells are injected into a group for 15 days in advance for stem cell prevention; on the 7 th day before the toxin is attacked, the stem cells are injected into the group for 7 days in advance for prevention; on the 5 th day before the challenge, the stem cells were injected 5 days in advance.

On the day of challenge, mice in the normal control group were treated with PBS nasal drops, and both the virus-infected control group and the pre-prevention group were infected with H7N9 influenza virus at a dose of 7.9 mg/kg.

The nasal drops of virus described in this example were administered at 5 times the median lethal dose LD 50.

Animal treatment: after the test is finished, killing each group of mice for sampling analysis, fasting the mice for 12 hours after the last administration, weighing the mice after the anesthesia by adopting chloral hydrate, picking eyeballs, collecting blood, performing EDTA anticoagulation, directly performing T cell analysis by adopting a flow cytometry classifier, and centrifugally collecting blood plasma of the rest blood samples at 2500rpm, and performing cryopreservation at the temperature of-80 ℃ for cytokine detection; picking and weighing lung, recording the ratio of lung tissue to body weight, fixing the picked lung right large leaf in 10% formalin solution, performing pathological analysis according to the pathological tissue section flow, and freezing the rest tissues in a refrigerator at-80 ℃ for virus load analysis.

The statistical method comprises the following steps: statistical analysis of data was performed using SPSS12.0, and the data was measured using mean. + -. Standard deviation (X ±SD) It is shown that P <0.05 is statistically different using paired sample t test single factor analysis of variance.

Test results

Death protection efficacy results of mice infected with H7N9 influenza virus

The animals were observed daily for the duration of the test, weighed, and the number of deaths recorded for a total of 15 days, and the mortality protection efficiency and the life prolongation rate were calculated as "mortality protection efficiency = (number of deaths in the virus control group-number of deaths in the administered group)/number of deaths in the virus control group × 100%", "life prolongation rate = (mean number of survival days in the administered group-mean number of survival days in the virus control group)/mean survival days in the virus control group × 100%".

As shown in fig. 1, the H7N9 virus-infected group died from day 8 after challenge, with a peak of death occurring on days 9-11, and 9 mice died throughout the observation period, with a mortality rate of 90%. The death protection efficiency of umbilical cord mesenchymal stem cells for prevention and treatment of H7N9 influenza virus infection was 55.6%, 66.7%, and 44.4% for 15 days, 7 days, and 5 days, respectively, and there was a statistical difference from the virus infection control group (p <0.05, p <0.01, p < 0.001). In addition, the survival time of the mice infected with H7N9 influenza virus can be effectively prolonged by the advanced prevention of the umbilical cord mesenchymal stem cells, the average survival times of the umbilical cord mesenchymal stem cells for 15 days, 7 days and 5 days are respectively 13.2 +/-2.3 days, 13.7 +/-2.1 days and 12.3 +/-2.9 days, the life prolonging rates are respectively 51.4%, 64.9% and 27.0%, and the virus infection control group has obvious difference (p is less than 0.01 or p is less than 0.05).

By combining the observation indexes, the early prevention of the umbilical cord mesenchymal stem cells can obviously prolong the average survival period of the mice, improve the survival rate of the mice and have the most obvious effect of preventing the stem cells for 7 days.

Weight gain in mice

As shown in fig. 2, the weight gain of each group of mice was affected after 3 days of H7N9 infection with influenza virus and began to gradually decrease, wherein umbilical cord mesenchymal stem cells prevented the weight decrease of each group in advance to be significantly less than that of the virus-infected control group. Meanwhile, compared with other groups, the weight of the mice in the 7-day prevention group is prevented by the umbilical cord mesenchymal stem cells in advance, so that the weight is reduced to the minimum extent, and the recovery period is the fastest.

The results show that the umbilical cord mesenchymal stem cells prevent the weight loss of mice caused by the infection of the H7N9 influenza virus for 7 days in advance, and have obvious improvement effect.

The recovery period refers to the period from the virus elimination in the mice to the complete recovery after the virus infection.

Detection of pulmonary organ-related index

After each group of mice died, the whole lung was taken, blood stain was washed with physiological saline, and the general morphology of the organs was observed. Weighing, and calculating the lung index and the lung wet-to-dry ratio according to "lung index = mouse lung weight/mouse body weight × 100%", "wet-to-dry ratio = mouse lung wet weight/mouse lung dry weight × 100%".

Table 1 and fig. 3 show that 5 days and 6 days after H7N9 influenza virus infection, the lung index of mice in the virus-infected control group was significantly increased compared to the normal control group, which was significantly different from the normal control group (p < 0.05), and the lung wet-dry ratio was significantly decreased (p < 0.05), indicating that pulmonary edema of the mice was increased; compared with a virus infection control group, the early prevention of the mesenchymal stem cells can effectively reduce the lung index and the lung-to-wet-dry ratio of the mouse, wherein the effect of the early prevention for 7 days and 5 days is most obvious, and the significant difference (p <0.01 or p < 0.05) exists, which indicates that the early prevention of the mesenchymal stem cells can improve the pulmonary edema degree of the mouse.

TABLE 1 Effect of umbilical cord mesenchymal Stem cells on pulmonary edema in H7N 9-infected mice

Group of	Wet to dry ratio (%)	Day 5 pulmonary index	Day	6 pulmonary index	Pulmonary index inhibition rate
						Normal control group	4.42±0.41	0.83±0.08	0.69±0.16	--
Control group of viral infection	5.60±0.26	1.34±0.097	2.12±0.83	--
					MSC-15	5.20±0.21	1.21±0.17	1.75±0.73 ^*	25.9 ^*
MSC-7	4.99±0.42 ^*	1.22±0.15	1.43±0.39 ^**	48.5 ^**
					MSC-5	4.99±0.17 ^*	1.12±0.17	1.36±0.39 ^**	53.1 ^**

Hemagglutination inhibition method for detecting antibody titer in mice

After the mice are fasted for 12h and are anesthetized by chloral hydrate, whole blood is taken out of eyeballs and put into an anticoagulant tube added with EDTA, the whole blood is centrifuged for 10min at 2500rpm, and the supernatant is removed. Taking 25 mu L of a serum sample, adding the serum sample into a first row of 96-well holes, then diluting the serum sample to 10-well holes by using PBS (phosphate buffer solution) for 2-fold comparison, adding 25 mu L of 4 units of H7N9 inactivated virus into each hole, acting for 45min at room temperature, adding 25 mu L of 1% red blood cells, acting for 45min at room temperature, and finally judging the level of a serum antibody according to the hole with the virus hemagglutination inhibition effect of the serum sample.

As shown in fig. 4, the antibodies of each group of mice were significantly and abnormally increased 15 days after virus infection, with the most significant increase in antibodies of the model group of mice indicating successful infection of the mice. Compared with a virus infection control group, the average antibody titer of the mice in the groups of umbilical cord mesenchymal stem cells for preventing 15 days, 7 days and 5 days in advance is about 2-3 lower, wherein the antibody titer for preventing 7 days in advance is the lowest, which indicates that the umbilical cord mesenchymal stem cells can prevent the umbilical cord mesenchymal stem cells from eliminating the virus early to cause antigen reduction and is related to the lower antibody titer.

Pathological changes of lung tissue in mice

Dissecting a mouse, picking the lung, weighing, recording the ratio of lung tissue to body weight, fixing the picked right large leaf of the mouse lung in 10% formalin solution, and performing pathological analysis according to the pathological tissue section flow.

As shown in FIG. 5, H & E staining of lung tissue showed intact alveolar tissue structure, no significant inflammatory cell infiltration around bronchioles and small vessel walls, intact bronchioles mucosa, no vasodilation in alveolar septum, congestion and inflammatory cell infiltration in mice of normal control group. Compared with a normal control group, the H7N9 influenza virus infected group mice have obvious pathological changes of lung tissues, diffuse alveolar wall thickening, pulmonary interstitial intravascular dilation and a large amount of inflammatory cell infiltration can be seen, and bronchioles and tissues around the walls of the small blood vessels can see a large amount of mononuclear cells and lymphocyte infiltration and the mucosa of the bronchioles is exfoliated and necrotized. Compared with a virus infection control group, the effect of preventing stem cells in advance can effectively improve the lung inflammation, wherein the effect of preventing the stem cells in advance for 7 days is most remarkable, the lung structure of the mice in the group is basically complete, the lung inflammation is light, and only a small amount of mononuclear cells and lymphocytes are infiltrated around lung bronchioles and small blood vessel walls.

The results show that compared with a virus infection control group, the mesenchymal stem cells prevent the lung lesion of the mice for 7 days and 5 days in advance, and the degree of the lung lesion of the mice is obviously reduced.

Liquid phase chip for detecting mouse serum cell factor

The mouse is anesthetized before dissection and then the eyeball is picked to take blood, EDTA anticoagulation blood is collected, plasma is collected by centrifugation at 2500rpm, and the expression quantity of relevant cytokines is detected by using a Luminex liquid phase chip.

FIGS. 6-11 show that the cytokines TNF-alpha, GM-CSF, IFN-gamma, IL-1 beta, IL-2, IL-4, IL-12p70, IL-17A, IL-18, IL-23, IP-10, MIP-1 alpha and MIP-2 in each group of mice are obviously increased 15 days after virus infection, wherein the cytokine increase of the mice in the virus infection control group is most obvious. Compared with a virus infection control group, umbilical cord mesenchymal stem cells prevent the level of the cytokines from being reduced in the mice of 15 days, 7 days and 5 days in advance, wherein the level of TNF-alpha of the umbilical cord mesenchymal stem cells which are prevented for 15 days in advance is significantly different from that of the virus infection control group (p is less than 0.05).

As shown in fig. 12-13, the release of large amounts of cytokines can cause cytokine storm, which is a significant cause of acute lung injury. Clinical observation shows that the 2019-novel coronavirus infected severe patients have the phenomenon of obviously increased proinflammatory cytokines such as TNF-alpha, IFN-gamma and the like, and have the characteristic of cytokine storm.

The early prevention of the mesenchymal stem cells reduces the secretion of various cytokines of the H7N9 infected mouse, and the suggestion is that the possibility of acute lung injury occurrence can be reduced.

Example 3 H5N1 avian influenza Virus-infected mouse prevention test

Female ICR mice, weighing 14-15g, were randomly divided into 5 groups of 10 animals, were fed ad hoc for 5 days, and animals were grouped as follows: a normal control group; virus infection control group; the stem cells are prevented for 15 days in advance; the group with stem cells prevented for 7 days in advance and the group with stem cells prevented for 5 days in advance were inoculated with 1.2X10 of the injection into the tail vein of each mouse ⁶ And (4) stem cells.

After 5 days of adaptive feeding, on the 15 th day before toxin counteracting, the stem cells are injected into a group for 15 days in advance for stem cell prevention; on the 7 th day before the toxin is attacked, the stem cells are prevented for 7 days in advance and injected with a stem cell preparation; on day 5 before challenge, the group 5 days before stem cell prevention was injected with the stem cell preparation.

On the day of challenge, mice in the normal control group were treated with PBS by nasal drip, and the virus-infected control group and the pre-prevention group were infected with avian influenza virus H5N1 at a dose of 7.9mg/kg by nasal drip.

Mice were scored for mortality and the mortality protection efficiency of each group of mice was calculated as "mortality protection efficiency = (number of deaths in virus control group-number of deaths in administered group)/number of deaths in virus control group x 100%".

The results showed that death occurred in the H5N1 virus-infected group from day 6 after challenge, peak death occurred at days 7-9, and 10 mice died throughout the observation period, with a mortality rate of 100%. The death protection efficiency of the umbilical cord mesenchymal stem cells for preventing the H5N1 influenza virus infection for 15 days, 7 days and 5 days in advance is respectively 30.0%, 50.0% and 40.0%.

TABLE 2 Effect of umbilical cord mesenchymal Stem cells on mortality of H5N1 infected mice

Group of	Total number (only)	Death number (only)	Mortality rate	Death protection efficiency
					Normal control group	10	0	0	——
Control group of viral infection	10	10	100%	——
					MSC-15	10	7	70%	30.0%
MSC-7	10	5	50%	50.0%
					MSC-5	10	6	60%	40.0%

Example 4 prevention of influenza A H3N2 Virus infection in mice

Female ICR mice, weighing 14-15g, were randomly divided into 5 groups of 7 mice each, were fed ad hoc for 5 days, and animals were grouped as follows: a normal control group; virus infection control group; the stem cells are prevented for 15 days in advance; the group with stem cells prevented for 7 days in advance and the group with stem cells prevented for 5 days in advance were inoculated with 1.2X10 of the injection into the tail vein of each mouse ⁶ And (4) stem cells.

After 5 days of adaptive feeding of mice, 15 days before toxin counteracting, the stem cells are injected into a group for 15 days in advance for prevention; on the 7 th day before the toxin is attacked, the stem cells are prevented for 7 days in advance and injected with a stem cell preparation; on day 5 before challenge, the group 5 days before stem cell prevention was injected with the stem cell preparation.

On the day of challenge, mice in the normal control group were treated with PBS nasal drops, and both the virus-infected control group and the pre-prevention group were infected with influenza a H3N2 virus at a dose of 7.9 mg/kg.

The results showed that death occurred in the H3N2 virus-infected group starting at day 7 after challenge, with a peak of death occurring at days 8-9, and 6 mice died throughout the observation period, with a mortality rate of 86%. The death protection efficiency of the umbilical cord mesenchymal stem cells for preventing H3N2 influenza virus infection for 15 days, 7 days and 5 days in advance is 33.3%, 66.7% and 50.0% respectively.

TABLE 3 Effect of umbilical cord mesenchymal Stem cells on mortality of H3N2 infected mice

Group of	Total number (only)	Death number (only)	Mortality rate	Death protection efficiency
					Normal control group	7	0	0	——
Control group of viral infection	7	6	86%	——
					MSC-15	7	4	57%	33.3%
MSC-7	7	2	29%	66.7%
					MSC-5	7	3	43%	50.0%

Example 5 H1N1 influenza A Virus-infected mouse prophylactic test

Female ICR mice, weighing 14-15g, were randomly divided into 5 groups of 8 mice each, were fed ad hoc for 5 days, and animals were grouped as follows: a normal control group; virus infection control group; the stem cells are prevented for 15 days in advance; the group with stem cells prevented for 7 days in advance and the group with stem cells prevented for 5 days in advance were inoculated with 1.2X10 of the injection into the tail vein of each mouse ⁶ And (4) stem cells.

After 5 days of adaptive feeding of mice, 15 days before toxin counteracting, the stem cells are injected into a group for 15 days in advance for prevention; on the 7 th day before the toxin is attacked, the stem cells are injected into the group for 7 days in advance for prevention; on day 5 before challenge, the group 5 days before stem cell prevention was injected with the stem cell preparation.

On the day of challenge, mice in the normal control group were treated with PBS by nasal drip, and both the virus-infected control group and the pre-prevention group were infected with H1N1 influenza virus at a dose of 7.9mg/kg by nasal drip.

The results showed that death occurred in the H1N1 virus-infected group from day 7 after challenge, peak death occurred at days 8-9, and 7 mice died throughout the observation period with a mortality rate of 88%. The death protection efficiency of the umbilical cord mesenchymal stem cells for preventing the H1N1 influenza virus infection for 15 days, 7 days and 5 days in advance is 42.9%, 71.4% and 57.1% respectively.

TABLE 4 Effect of umbilical cord mesenchymal Stem cells on mortality of H1N1 infected mice

Group of	Total number (only)	Death number (only)	Mortality rate	Death protection efficiency
					Normal control group	8	0	0	——
Control group of viral infection	8	7	88%	——
					MSC-15	8	4	50%	42.9%
MSC-7	8	2	25%	71.4%
					MSC-5	8	3	38%	57.1%

EXAMPLE 6 in vitro inhibition assay of novel coronavirus (SARS-COV-2)

The positive control drug in this example is Reidesciclovir, white powder, provided by Jiangsu province disease prevention and control center.

The cells described in this example were African green monkey kidney (Vero-E6) cells, provided by the Jiangsu provincial disease prevention and control center.

The novel coronavirus (SARS-CoV-2) strain described in this example was (BetaCoV/JS 02/Human/2019) and was stored at-80 ℃ in the P3 laboratory of the Jiangsu province disease prevention and control center.

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

The amount of virus added was MOI =0.05.

The addition amount of Reidesciclovir was 5uM.

In vitro inhibition of SARS-COV-2 by stem cells

Vero-E6 cells were plated at 5X 10 ⁴ One/well was seeded in two 24-well plates.

Cell plating: stem cell + PBMC (24 h preincubation), stem cell group, PBMC group with stem cell concentration of 1 × 10 ⁵ Per well, PBMC concentration 1X 10 ⁶ Number/well, stem cell and PBMC ratio 1; the stem cell concentration of the other 24-well plate is 1X 10 ⁵ Per well, PBMC concentration 5X 10 ⁵ Number/well, stem cell and PBMC ratio 1.

Placing two 24-well plates at 37 ℃ and 5% CO ₂ After culturing for 18h in a constant-temperature incubator, inoculating a corresponding number of stem cells and PBMCs in each hole of a stem cell + PBMC (pre-incubation for 6 h); after further 6h of culture, the novel coronavirus (MOI = 0.05) was added to all groups except the cell control group, placed at 37 ℃,5% ₂ Adsorbing in an incubator for 1 hour, discarding a culture medium with viruses after adsorption is finished, adding a new virus growth solution again, and adding a virus growth solution containing Reidesvir (5 mu M) into a positive control group; at 37 ℃ C, 5% CO ₂ Culturing in an incubator for 72h, sucking culture supernatant of each well after the culture is finished, inactivating for 30min at 56 ℃, and extracting viral nucleic acid and detecting genes.

TABLE 5 cell plate distribution for each experimental group

The statistical processing method comprises the following steps: all data are input into EXCEL sheetΔ Ct = Ct _{Experimental group} －Ct _{Virus control group} Inhibition rate = (1-2) ^－ΔCt ) X 100%, the inhibition rate of each group was calculated. Experimental data onx ±sIt shows that the single-factor variance analysis is carried out by adopting a software package GraphPad Prism 8.0, and the difference of significance is shown when P is less than 0.05.

According to fig. 14, umbilical cord mesenchymal Stem cells and PBMCs were incubated 24h in advance, and then inoculated with the new coronavirus, which can significantly inhibit the replication of the new coronavirus (P < 0.001 in PBMC (E6) + Stem cells 24h group, PBMC (E5) + Stem cells 24h group compared with virus control group), and the inhibition rate reached 90% when the ratio of Stem cells and PBMCs was 1; under the co-culture condition of the umbilical cord mesenchymal Stem cells and the PBMCs (pre-incubation) compared with the two alone, the replication of the novel coronavirus can be more effectively inhibited (compared with the PBMC group and the Stem cells group in the PBMC + Stem cells 24h group); the inhibitory effect on the virus at a Stem cell and PBMC ratio of 1.

Example 7 mechanism study experiment of hUC-MSCs for preventing viral pneumonia in advance

Female ICR mice, weighing 14-15g, were randomly divided into 5 groups of 20 mice each, were fed ad hoc for 5 days, and animals were grouped as follows: a normal group, a model group, a stem cell 7-day injection infection group and a positive medicine group.

On day 7 before challenge, the stem cell 7-day injection group and the stem cell 7-day injection infected group were inoculated with 1.2x10 per mouse by tail vein injection ⁶ The stem cells and the positive medicine group are injected with ribavirin medicine. On the day of virus challenge, except for the normal group and the 7-day stem cell injection group, PBS is dripped into the nose, and the other groups are infected with H7N9 influenza virus at the dose of 7.9mg/kg through nasal dripping.

Detection of neutrophil and CXCR3 expression in alveolar lavage fluid: alveolar lavage fluid labeled with CD45, CD11b, ly6G antibody, and CD45 detected by flow cytometry ⁺ CD11b ⁺ Ly6G ⁺ Neutral particlesExpression of the cell. The lavage fluid was stained for neutrophils with CD11b, ly6G and CXCR3 antibodies, and CD11b was analyzed by flow cytometry ⁺ Ly6G ⁺ CXCR3 levels in cells.

As shown in fig. 15, CD45 ⁺ CD11b ⁺ Ly6G ⁺ Indicating the number of neutrophils, CD45 ⁺ CD11b ⁺ Ly6G ⁺ CD183 ⁺ Represents the expression of CXCR 3. It can be seen that the early prevention of stem cells significantly reduces the number of neutrophils (P < 0.05) in mouse alveolar lavage fluid (figure 15, panel A), and at the same time, the expression of CXCR3 is reduced significantly, and the effect is better than that of ribavirin, which is a positive drug (figure 15, panel B).

Primary references

[1] Wall, E.C., et al., Neutralising antibody activity against SARS- CoV-2 VOCs B.1.617.2 and B.1.351 by BNT162b2 vaccination. 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-2 pneumonia. 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 RespirCrit Care Med, 2013. 187(1): p. 65-77.

Claims

1. The application of the umbilical cord mesenchymal stem cells in preparing a medicament for preventing virus infection, wherein the virus is influenza virus.

2. Use according to claim 1, characterized in that the influenza virus is selected from human or animal influenza viruses.

3. The use according to claim 2, wherein said influenza virus comprises influenza a, influenza b, influenza c.

4. Use according to claim 2, characterized in that said influenza virus is selected from the highly pathogenic subtype avian influenza viruses H7N9 or H5N 1.

5. Use according to claim 2, characterized in that the influenza virus is selected from influenza a H3N2 or H1N1 virus.