CN111568927A - Application of MSC (mesenchymal stem cell) in regulating number of memory B cells - Google Patents
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Abstract
The invention specifically discloses an application of MSC (mesenchymal stem cell) in regulating the number of memory B cells, in particular to an application of MSC in regulating the number of memory B cells of a patient with viral infection, relating to the technical field of biomedical engineering. The prepared MSC preparation is incorporated into an MSC treatment group to carry out combined treatment on patients with viral infection, so that the number of memory B cells in the body of the patient after the MSC treatment group is cured is effectively increased, and the secondary immunity of the patient after the MSC treatment group is cured is enhanced.
Description
Technical Field
The invention relates to the technical field of biomedical engineering, in particular to application of MSC (mesenchymal stem cell) in regulating the number of memory B cells.
Background
Immunological memory is one of the main characteristics of adaptive immunity, and plays a powerful role by assisting other lymphocytes through intercellular interaction, secretion of cytokines, chemokines and the like. Through immunological memory, the body can eliminate antigen fast and effectively. B lymphocytes (B cells) are differentiated and matured from lymphoid stem cells in mammalian bone marrow or avian bursa of Fabricius, and are also called bone marrow-dependent lymphocytes (bone marrow dependent lymphocytes) or bursa-dependent lymphocytes (bursa latent lymphocytes). Mainly colonize lymph nodes in the superficial cortical area of the lymph nodes and in the white marrow of the spleen. B cells can differentiate into plasma cells under antigen stimulation, synthesize and secrete immunoglobulins, and mainly perform humoral immunity (humoralimnity) of the body. B cells account for approximately 10% -15% of the total number of lymphocytes in peripheral blood, 50% of the total number of lymphocytes in the spleen and 5% of the total number of lymphocytes in the lymph nodes. B cells are the only cells in the body that produce antibodies, and contain antibody molecules that recognize different antigen specificities, and their diversity is from different B cell clones. In peripheral blood, B cells account for approximately 10% -15% of the total number of lymphocytes. The antigen substance entering the body is processed by mononuclear phagocyte and presented to B lymphocyte, the surface of B cell has membrane immunoglobulin molecule, which is antigen recognition receptor and can recognize conformation determinant of soluble protein antigen molecule, leading the B cell to activate, proliferate and differentiate into plasma cell for synthesizing and secreting various immunoglobulins. B cells survive in vivo for a short period of time, only days to weeks, but their memory cells can persist in vivo for a long period of time.
Viral infection refers to infectious diseases caused by viruses that can parasitize and propagate in the human body and cause diseases. Mainly shows general toxic symptoms such as fever, headache, general malaise and the like and local symptoms caused by inflammatory injury caused by virus hosts and invading tissues and organs. Viral infections of the human body are classified into recessive infections, dominant infections, and lentivirus infections. In most cases, the infection is recessive (after the infection of the human body with the virus, no symptoms appear, but specific antibodies can be produced). A few are dominant infections (meaning that symptoms appear after the human body is infected with viruses). The majority of the dominant infections are acute infections, the onset of diseases is acute, the course of diseases is short, and the minority of the dominant infections are latent infections (such as herpes virus infections) and chronic infections (such as hepatitis B virus infections). At present, the virus infection diseases lack specific treatment, mainly support and symptomatic treatment, and antiviral drugs and hormone treatment cannot play an effective treatment role. And viral infections (such as influenza, SARS, MERS and ebola) spread rapidly in humans, posing a significant threat to human health. Especially, the novel coronavirus which is outbreaked worldwide in 2020 causes serious loss to human health and economy.
The etiology of COVID-19 has been shown to be a novel coronavirus, now known as Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, no specific medicine or vaccine aiming at the virus is successfully developed, and the appearance of SARS-CoV-2 brings a difficult treatment dilemma to clinicians. Most infected patients exhibit non-specific symptoms such as fever, dry cough, and fatigue. The prognosis of most patients is good, and some patients with severe disease can rapidly develop acute respiratory distress syndrome, septic shock, metabolic acidosis, blood coagulation dysfunction and even die. The deterioration of the patient's condition may be due to a cytokine storm in the body, and since SARS-CoV-2 is a novel strain that causes a global pandemic, there is an urgent need for effective targeted therapies, including antiviral and immunotherapy. Currently, although many clinical trials have begun, there is currently no effective antiviral or immunomodulatory therapy for the treatment of SARS-CoV-2.
Clinical studies have found that MSC is an effective tool for treating diseases associated with Human Immunodeficiency Virus (HIV) immune abnormality, Hepatitis B Virus (HBV) chronic hepatitis, influenza virus Acute Lung Injury (ALI), and the like. Mesenchymal Stem Cells (MSCs) refer to a group of pluripotent stem cells with diverse differentiation potential that have differentiated into osteoblasts, chondroblasts and adipoblasts. They are derived from immature embryonic connective tissues of mesoderm and ectoderm in early embryonic development stage, and can differentiate into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium and even blood under specific induction conditions in vitro and in vivo. Clinical studies have found that MSC is an effective tool for treating diseases associated with Human Immunodeficiency Virus (HIV) immune abnormality, Hepatitis B Virus (HBV) chronic hepatitis, influenza virus Acute Lung Injury (ALI), and the like. MSCs have low immunogenicity as well as significant and extensive immune-regulatory functions. MSCs do not express co-stimulatory molecules such as CD40, CD80, and CD86, as well as MHC-class II molecules. The T cell inhibitory effect of MSCs is not restricted by MHC, i.e. the effect on autologous and allogeneic T cells is similar. The antiviral response is critical to eradicate the virus and prevent the development of virus-related diseases. If antiviral specific T cells are allowed to function in the presence of MSCs, they have the ability to maintain the integrity of the host's defense against infection. EB virus-specific cytotoxic T Cells (CTL) or cytomegalovirus CTL cultured together with MSC have been found to maintain the ability to proliferate and produce interferon-in vitro and have a killing effect on virus-infected cells. MSC-derived interferon-is thought to counteract the immunosuppressive effects of MSCs by mediating a partial cytotoxic response during viral infection. Therefore, when using MSCs as regenerative medicine, it is very important to recognize the dual role of MSCs in fighting viral infections of the immune system.
In view of the above, the present invention provides an application of MSC for regulating the number of memory B cells, especially an application of MSC for regulating the number of memory B cells of a patient with viral infection.
Disclosure of Invention
The invention aims to provide application of MSC (mesenchymal stem cell) in regulating the number of memory B cells, in particular application of MSC in regulating the number of memory B cells of a patient with viral infection. The prepared MSC preparation is incorporated into the MSC treatment group, so that the number of memory B cells of the patient after the MSC treatment group is cured can be effectively increased, and the immunity of the patient after the MSC treatment group is cured against secondary virus infection is enhanced.
In the invention, the application of the MSC for regulating the number of memory B cells of a virus infection patient comprises the following specific steps:
s1, preparing an MSC preparation;
s2, screening patients with viral infection and bringing the screened patients with viral infection into an MSC treatment group for combination treatment;
s3, sampling and analyzing the peripheral blood mononuclear cells of the healed patient, and acquiring the number of memory B cells of the healed patient.
Preferably, the preparation method of the MSC preparation comprises:
s11, collecting an umbilical cord, placing the umbilical cord in a culture dish, and then cleaning an umbilical cord tissue through physiological saline;
s12, cutting the cleaned umbilical cord tissue into small tissue blocks and planting the small tissue blocks in a culture dish for culture;
s13, 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 a counting result;
s14, removing the culture solution in the new culture dish in the step S13, then washing with normal saline, adding pancreatin for digestion, adding a stop solution to stop digestion after cell digestion in the culture dish is finished, filtering with a cell sieve, transferring the filtered cell suspension into a centrifuge tube, counting, centrifuging, discarding supernatant, preparing cell preparation suspension, adding cell preparation suspension for cell suspension resuspension, transferring the cell suspension into a transfer bag, and putting the transfer bag into a low-temperature environment for taking, thereby completing preparation of the MSC preparation;
s15, carrying out qualification detection on the MSC preparation prepared in the step S14.
Preferably, the combination therapy in step S2 is: the MSC preparation prepared in step S1 is included in the MSC treatment group and administered in combination to patients with viral infection.
Preferably, the combination in the MSC treatment group specifically comprises: MSC preparation is added in the original treatment scheme as a unique interference factor, and the combined medication condition of the MSC treatment group is tracked and recorded.
Preferably, the patients after healing in step S3 include patients with viral infection cured in the MSC-treated group and patients with viral infection cured in the MSC-untreated group.
Preferably, in step S3, the peripheral blood mononuclear cells are analyzed by single cell sequencing, so as to obtain the number of memory B cells of the patient after healing.
Compared with the prior art, the prepared MSC preparation is incorporated into the MSC treatment group to carry out combined treatment on the virus infection patients, so that the number of memory B cells of the patients after the MSC treatment group is cured is effectively increased, and the immunity of the patients after the MSC treatment group is used for enhancing the secondary virus infection immunity.
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FIG. 1 is a flow chart of an application of MSC for regulating the number of memory B cells of a patient with viral infection according to the present invention,
figure 2 is a flow chart of a method of preparing the MSC formulation of the present invention,
FIG. 3 is a diagram showing the ratio of the number of memory B cells in each group to the total number of corresponding cells in the patients after MSC-treated group healing, patients without MSC-treated group healing and healthy group members in the present invention,
FIG. 4 is a graph showing the fold change of the ratio of the number of memory B cells in each group with respect to the healthy group in the present invention, wherein the ratio of the number of memory B cells in the healthy group is set to 1.
Detailed Description
In order to make the technical solutions of the present invention better understood, the present invention is further described in detail below with reference to the accompanying drawings.
It should be noted that the original treatment plan in the present invention refers to a treatment plan adopted in a novel coronavirus pneumonia diagnosis and treatment plan issued by the office of the national Health and care committee and the office of the national traditional Chinese medicine administration, MSC + represents an MSC treatment group, MSC-represents a non-MSC treatment group, and Health represents a healthy group.
As shown in figure 1, the application of MSC for regulating the number of memory B cells of a virus infection patient comprises the following specific steps:
s1, preparing an MSC preparation;
s2, screening patients with viral infection and bringing the screened patients with viral infection into an MSC treatment group for combination treatment;
s3, sampling and analyzing the peripheral blood mononuclear cells of the healed patient, and acquiring the number of memory B cells of the healed patient.
As shown in fig. 2, the preparation method of the MSC preparation comprises:
s11, collecting an umbilical cord, placing the umbilical cord in a culture dish, and then cleaning an umbilical cord tissue through physiological saline;
s12, cutting the cleaned umbilical cord tissue into small tissue blocks and planting the small tissue blocks in a culture dish for culture;
s13, 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 a counting result;
s14, removing the culture solution in the new culture dish in the step S13, then washing with normal saline, adding pancreatin for digestion, adding a stop solution to stop digestion after cell digestion in the culture dish is finished, filtering with a cell sieve, transferring the filtered cell suspension into a centrifuge tube, counting, centrifuging, discarding supernatant, preparing cell preparation suspension, adding cell preparation suspension for cell suspension resuspension, transferring the cell suspension into a transfer bag, and putting the transfer bag into a low-temperature environment for taking, thereby completing preparation of the MSC preparation;
s15, carrying out qualification detection on the MSC preparation prepared in the step S14.
In this embodiment, the MSC preparation is prepared from Human umbilical cord mesenchymal stem cells (hiuc-MSC), the preparation process is performed in a sterilized clean bench, instruments and consumables and the like required in the preparation process are sterilized with 75% alcohol, and the collection or cells in the culture dish and the specific operation process need to be labeled in detail in the preparation process; meanwhile, the stem cells can be frozen between the step S13 and the step S14, and if the frozen stem cells need to be revived before the step S14 is performed on the stem cell preparation, the stem cell freezing and reviving operations both belong to the prior art, and are not described in detail herein.
In this embodiment, the qualification testing of the MSC preparation further includes quality testing of the collected material, warehousing testing of the primary cell bank of the umbilical cord mesenchymal stem cells, and warehousing testing of the main cell bank of the umbilical cord mesenchymal stem cells, and the step and standard of the qualification testing are consistent with those of the conventional passing testing, which is not repeated here.
The combined treatment in the MSC treatment group refers to that the MSC preparation prepared in the step S1 is brought into the MSC treatment group and is used as a unique interference factor in the original treatment scheme to carry out combined medication on patients with viral infection, and meanwhile, the situation of the combined medication is tracked and recorded in the combined treatment scheme5-5×108Cell number/ml, MSC preparation was administered by peripheral intravenous infusion throughout the MSC treatment group and was used several times. Wherein the number of times of use can be controlled between 1 and 10 times according to actual conditions.
In this embodiment, the selected common new coronary positive patients (i.e., SARS-CoV-2 virus infected patients) are equally divided into two groups and used as samples of MSC treatment group and samples of non-MSC treatment group (i.e., original treatment scheme), and after the virus infected patients in the two groups of samples are cured, the peripheral blood mononuclear cells of the cured patients are sampled, and in order to better explain the working principle and technical effect of the present invention, we select to sample the peripheral blood mononuclear cells of a corresponding number of healthy group personnel at the same time, and then perform the group analysis on the collected peripheral blood mononuclear cell samples respectively by single cell sequencing and process the analysis result, so as to obtain the memory B cell number relationship between the patients after the MSC treatment group is cured, and the patients without the MSC treatment group are cured and the healthy group personnel (see table 1). In other embodiments, other virally infected patients may be selected as samples for the MSC-treated group and the non-MSC-treated group.
TABLE 1 relation table of memory B cell numbers of patients after MSC-treated group recovery, patients after MSC-untreated group recovery and healthy group
Meanwhile, drawing a relationship schematic diagram of the memory B cell number between the healed patients and healthy group personnel in two groups of samples according to the processing result data after single cell sequencing analysis (as shown in fig. 3 and 4, wherein, the first represents Health, the second represents MSC +, and the third represents MSC-), the abscissa in fig. 3 represents the memory B cell, the ordinate represents the proportion of the memory B cell number of each group in the corresponding cell total number, the abscissa in fig. 4 represents the memory B cell, and the ordinate represents the change multiple of the proportion of the memory B cell number of each group relative to the healthy group.
As can be seen from table 1, fig. 3 and fig. 4, the number of memory B cells of patients after the MSC treatment group is cured accounts for 0.491% of the number of all the immune cell subsets, the number of memory B cells of patients without the MSC treatment group is 0.061% of the number of all the immune cell subsets, and the number of memory B cells of healthy people accounts for 0.119% of the number of all the immune cell subsets, and the number of memory B cells of patients after the MSC treatment group is 8.095 times of the number of patients without the MSC treatment group, and the number of memory B cells of patients after the MSC treatment group is 4.131 times of the number of healthy people, because the virus stimulates the memory B cells to differentiate effector B cells when the same virus re-infects patients, and thus a large amount of antibodies generated in the patients are combined with the virus and digested, the increase of the number of memory B cells means that the patients after the treatment have stronger immunity to secondary virus infection, therefore, the MSC treatment group has stronger anti-infection capability after the patients are cured.
The use of an MSC in accordance with the present invention for regulating the number of memory B cells is described in detail above. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the core concepts of the present invention. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
Claims (8)
1. Use of an MSC for modulating the number of memory B cells.
2. The use of MSCs as claimed in claim 1 for modulating the number of memory B cells in a virally infected patient.
3. The use of MSCs as claimed in claim 2 for modulating the number of memory B-cells in a virally infected patient comprising the steps of:
s1, preparing an MSC preparation;
s2, screening patients with viral infection and bringing the screened patients with viral infection into an MSC treatment group for combination treatment;
s3, sampling and analyzing the peripheral blood mononuclear cells of the healed patient, and acquiring the number of memory B cells of the healed patient.
4. The use of the MSCs of claim 3 for modulating the number of memory B-cells, wherein the preparation of MSCs comprises:
s11, collecting an umbilical cord, placing the umbilical cord in a culture dish, and then cleaning an umbilical cord tissue through physiological saline;
s12, cutting the cleaned umbilical cord tissue into small tissue blocks and planting the small tissue blocks in a culture dish for culture;
s13, 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 a counting result;
s14, removing the culture solution in the new culture dish in the step S13, then washing with normal saline, adding pancreatin for digestion, adding a stop solution to stop digestion after cell digestion in the culture dish is finished, filtering with a cell sieve, transferring the filtered cell suspension into a centrifuge tube, counting, centrifuging, discarding supernatant, preparing cell preparation suspension, adding cell preparation suspension for cell suspension resuspension, transferring the cell suspension into a transfer bag, and putting the transfer bag into a low-temperature environment for taking, thereby completing preparation of the MSC preparation;
s15, carrying out qualification detection on the MSC preparation prepared in the step S14.
5. The use of the MSC of claim 4 for modulating the number of memory B cells, wherein the combination therapy of step S2 is: the MSC preparation prepared in step S1 is included in the MSC treatment group and administered in combination to patients with viral infection.
6. The use of the MSC of claim 5 for modulating the number of memory B cells, wherein the combination in the MSC treatment group is selected from the group consisting of: MSC preparation is added in the original treatment scheme as a unique interference factor, and the combined medication condition of the MSC treatment group is tracked and recorded.
7. The use of the MSCs for modulating the number of memory B-cells in accordance with claim 6, wherein the patients after healing in step S3 comprise patients with viral infection cured in the MSC-treated group and patients with viral infection cured in the MSC-untreated group.
8. The use of the MSCs for modulating the number of memory B-cells in claim 7, wherein in step S3 the number of memory B-cells in a patient after recovery is obtained by analyzing a sample of peripheral blood mononuclear cells by single cell sequencing.
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| CN202010444815.4A CN111568927A (en) | 2020-05-23 | 2020-05-23 | Application of MSC (mesenchymal stem cell) in regulating number of memory B cells |
| PCT/CN2020/105751 WO2021237930A1 (en) | 2020-05-23 | 2020-07-30 | Application of mesenchymal stem cells in treatment of patients with viral infection |
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| US20190038742A1 (en) * | 2016-02-04 | 2019-02-07 | Longeveron Llc | Mesenchymal stem cells as vaccine adjuvants and methods for using the same |
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| US20190038742A1 (en) * | 2016-02-04 | 2019-02-07 | Longeveron Llc | Mesenchymal stem cells as vaccine adjuvants and methods for using the same |
| CN110691600A (en) * | 2016-11-11 | 2020-01-14 | 美商生命科学公司 | Method for achieving cellular immunity and humoral immunity by using human mesenchymal stem cells |
| CN110540959A (en) * | 2019-10-08 | 2019-12-06 | 孟明耀 | Umbilical cord mesenchymal stem cell isolation culture amplification method |
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