CN111808960A - Medicine and method for treating acute myelogenous leukemia - Google Patents

Medicine and method for treating acute myelogenous leukemia Download PDF

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CN111808960A
CN111808960A CN202010696955.0A CN202010696955A CN111808960A CN 111808960 A CN111808960 A CN 111808960A CN 202010696955 A CN202010696955 A CN 202010696955A CN 111808960 A CN111808960 A CN 111808960A
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leukemia
beta
cells
bone marrow
sympathetic
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马道新
张琛
杨新雨
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Qilu Hospital of Shandong University
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Qilu Hospital of Shandong University
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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Abstract

The invention provides a medicine and a method for treating acute myelogenous leukemia, and belongs to the technical field of biological medicines. The invention researches the functions of marrow sympathetic nerve and Th subgroup immune imbalance in the generation and development of acute myelogenous leukemia and the related mechanism thereof, finds that the marrow of AML patients is locally damaged by sympathetic nerve and locally damaged by Th subgroup immune imbalance, and proves that the nerve damage can promote the development of the acute myelogenous leukemia; simultaneous sympathetic neurotransmitterCan promote proliferation of primary leukemia cells and leukemia cell line, and inhibit CD4+The T cells are differentiated towards the directions of Th1 and Th17 to inhibit the killing effect on leukemia cells, thereby providing a new idea for treating acute myelogenous leukemia.

Description

Medicine and method for treating acute myelogenous leukemia
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a medicine and a method for treating acute myelogenous leukemia.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Acute Myeloid Leukemia (AML) is a heterogeneous group of malignant diseases characterized by excessive proliferation of leukemic stem and/or progenitor cells causing the accumulation of large numbers of leukemic cells and inhibiting normal hematopoiesis. Even with current clinically-applied intensive chemotherapy regimens and stem cell transplantation, the overall survival of AML remains poor: the overall 5-year survival rate of young AML patients is only 40%. Survival rates are also poor in adults over the age of 60 years, and 60% -80% of patients eventually die from refractory and/or relapsed leukemia. In the elderly 65-70 years of age, their 5-year survival rate is only 10%.
With the continuous and intensive research on leukemia in recent years, the bone marrow microenvironment attracts more and more attention in the role of the development of leukemia, the bone marrow microenvironment may provide a refuge for leukemia cells and can protect AML cells from cell death induced by chemotherapeutic drugs, and the research on the interaction between leukemia and the bone marrow microenvironment in the future may lead to the change of the treatment strategy of leukemia, and the traditional cytotoxic chemotherapy is shifted to the targeted therapy. However, the inventors have found that, since the specific pathogenesis of acute myeloid leukemia is still unclear, it is important to search for the cause and therapeutic strategy of AML.
Disclosure of Invention
In view of the above problems in the prior art, the present invention provides a medicine and a method for treating acute myelogenous leukemia. The invention researches the functions of marrow sympathetic nerve and Th subgroup immune imbalance in the generation and development of acute myelogenous leukemia and the related mechanism thereof, finds that the marrow of AML patients is locally damaged by sympathetic nerve and locally damaged by Th subgroup immune imbalance, and proves that the nerve damage can promote the development of the acute myelogenous leukemia; at the same time, sympathetic neurotransmitter can promoteProliferation of primary leukemia cells and leukemia cell lines by inhibition of CD4+The T cells are differentiated towards the directions of Th1 and Th17 to inhibit the killing effect on leukemia cells, thereby providing a new idea for treating acute myelogenous leukemia.
The technical scheme of the invention is as follows:
in a first aspect of the invention, there is provided a biomarker for detecting acute myeloid leukaemia, the biomarker being selected from any one or more of:
neural specific molecules, adrenoceptors and Th subsets.
Wherein the neural specific molecules include, but are not limited to, Nestin, GFAP, TUB3, MAP2, and S-100;
the adrenoceptors are beta-adrenoceptors, and comprise three different subtypes of the beta-adrenoceptors, namely beta 1-ADR, beta 2-ADR and beta 3-ADR;
the Th subpopulations include, but are not limited to, Th1, Th17, Treg, and Th22 cells; also included are downstream transcription factors from the Th subset T-beta, RORc, GATA3, Foxp3 and AHR; further included are cytokines secreted by the Th subset, such as IFN- γ, IL-17A, TGF- β, IL-10, IL-4, and IL-22.
The biomarkers are obtained locally from the bone marrow of the subject.
The detection of acute myeloid leukemia specifically includes diagnosis or auxiliary diagnosis of acute myeloid leukemia, and more specifically, includes:
(1) local nerve destruction (degree) of the bone marrow, in particular sympathetic nerve destruction;
(2) clinical features of acute myeloid leukemia, such as extramedullary infiltrates;
(3) local Th subsets of bone marrow are immunologically imbalanced.
In a second aspect of the invention, there is provided a use of a substance for detecting the above biomarker in the preparation of a product for diagnosing or aiding in the diagnosis of acute myeloid leukemia.
Wherein, the substance includes but is not limited to a substance for detecting the expression level of the biomarker based on a high-throughput sequencing method and/or a quantitative PCR method and/or a probe hybridization method.
Such products include, but are not limited to, devices (e.g., oligonucleotide probes or their integrations, high-throughput mRNA detection chips on chip substrates or detection substrates, protein chips, and microfluidic detection chips) and kits.
In a third aspect of the invention, there is provided an apparatus comprising:
one or more devices for detecting the biomarkers described above.
In a fourth aspect of the invention, a kit is provided, which comprises the device described above.
In a fifth aspect of the invention, there is provided the use of any one or more of the following in the preparation of a medicament for the treatment of acute myeloid leukaemia.
1) A neuroprotective agent;
2) a sympathetic neurotransmitter inhibitor;
3) TNF-alpha and/or TNFR2 receptor inhibitors.
Wherein the neuroprotective agent is specifically a sympatholytic agent, such as 4-MY;
the sympathetic neurotransmitters include epinephrine and norepinephrine.
The treatment of acute myeloid leukemia specifically comprises:
1) inhibiting leukemia cell proliferation;
2) improving the immune condition of the body, specifically comprising relieving or weakening the suppression effect on the T cell function and reducing the proportion of the Tregs of immunosuppressive Th subgroups; the invention proves that the sympathetic neurotransmitter can inhibit the functions of T cells to a great extent through research, and particularly comprises the inhibition of CD4+T cells are differentiated to Th1 and Th17 subgroups with killing function, and the generation of effector molecules IFN-gamma and IL-17A is greatly inhibited; meanwhile, the research proves that the high TNF-alpha in the acute myelogenous leukemia bone marrow microenvironment can increase the proportion of the Treg of the immunosuppressive Th subgroup through the TNFR2 receptor.
In a sixth aspect of the invention, a medicament for treating acute myelogenous leukemia is provided, wherein the active ingredients of the medicament comprise any one or more of the following components:
1) a neuroprotective agent;
2) a sympathetic neurotransmitter inhibitor;
3) TNF-alpha and/or TNFR2 receptor inhibitors.
Wherein the neuroprotective agent is specifically a sympatholytic agent, such as 4-MY;
the sympathetic neurotransmitters include epinephrine and norepinephrine.
The treatment of acute myeloid leukemia specifically comprises:
1) inhibiting leukemia cell proliferation;
2) improving the immune condition of the body, specifically comprising relieving or weakening the suppression effect on the T cell function and reducing the proportion of the Tregs of immunosuppressive Th subgroups; the invention proves that the sympathetic neurotransmitter can inhibit the function of T cells to a great extent, particularly inhibits the differentiation of CD4+ T cells to Th1 and Th17 subgroups with killing function, and greatly inhibits the generation of effector molecules IFN-gamma and IL-17A; meanwhile, the research proves that the high TNF-alpha in the acute myelogenous leukemia bone marrow microenvironment can increase the proportion of the Treg of the immunosuppressive Th subgroup through the TNFR2 receptor.
The medicine for treating acute myeloid leukemia also comprises at least one pharmaceutically acceptable auxiliary material and/or carrier.
The drug administration dosage form can be a liquid dosage form and a solid dosage form. The liquid dosage form can be true solution, colloid, microparticle, emulsion, or mixed suspension. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, clathrate, landfill, patch, liniment, etc.
The medicament of the present invention may also contain conventional carriers, wherein the pharmaceutically acceptable carriers include, but are not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerol, sorbates, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, lanolin and the like. The carrier may be present in the pharmaceutical composition in an amount of 1% to 98% by weight, typically about 80% by weight. For convenience, the local anesthetic, preservative, buffer, etc. may be dissolved directly in the vehicle.
Oral tablets and capsules may contain excipients such as binding agents, for example syrup, acacia, sorbitol, tragacanth, or polyvinylpyrrolidone, fillers such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, glycine, lubricants such as magnesium stearate, talc, polyethylene glycol, silica, disintegrants such as potato starch, or acceptable wetting agents such as sodium lauryl sulfate. The tablets may be coated by methods known in the art of pharmacy.
The oral liquid can be made into water and oil suspension, solution, emulsion, syrup, or dried product, and supplemented with water or other suitable medium before use. Such liquid preparations may contain conventional additives such as suspending agents, sorbitol, cellulose methyl ether, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gelatin, hydrogenated edible fats and oils, emulsifying agents such as lecithin, sorbitan monooleate, gum arabic; or a non-aqueous carrier (which may comprise an edible oil), such as almond oil, an oil such as glycerol, ethylene glycol, or ethanol; preservatives, e.g. methyl or propyl p-hydroxybenzoates, sorbic acid. Flavoring or coloring agents may be added if desired.
Suppositories may contain conventional suppository bases such as cocoa butter or other glycerides.
For parenteral administration, liquid dosage forms are generally prepared from the compound and a sterile carrier. The carrier is preferably water. The compound can be dissolved in the carrier or made into suspension solution according to the concentration of the carrier and the drug, and the compound is firstly dissolved in water when made into the solution for injection, filtered and sterilized and then filled into a sealed bottle or ampoule.
In a seventh aspect of the present invention, there is provided a method for screening a drug for treating acute myelogenous leukemia, the method comprising: the effect of the candidate drug on the biomarkers before and after use is used to determine whether the candidate drug can be used to treat acute myeloid leukemia.
The beneficial technical effects of one or more technical schemes are as follows:
the technical scheme firstly reports a medicine and a method for diagnosing and treating acute myelogenous leukemia, and concretely, the invention researches the action of marrow sympathetic nerve and Th subgroup immune imbalance in the occurrence and development of the acute myelogenous leukemia and a related mechanism thereof, finds that the marrow of AML patients locally has sympathetic nerve damage and the marrow of AML patients locally has Th subgroup immune imbalance, and proves that the nerve damage can promote the development of the acute myelogenous leukemia; the sympathetic neurotransmitter can promote the proliferation of primary leukemia cells and leukemia cell lines and inhibit CD4+The T cells are differentiated towards the directions of Th1 and Th17 to further inhibit the killing effect on leukemia cells, so that a new idea is provided for the diagnosis and treatment of acute myelogenous leukemia, and the method has good practical application value.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings according to the provided drawings without creative efforts.
FIG. 1 is a graph showing the relative expression levels of mRNA for Nestin, GFAP, beta-TUB 3, MAP2 and S-100 in the local microenvironment of bone marrow in the initial acute myelogenous leukemia patient and the control group in example 1 of the present invention. P <0.05, p <0.01
FIG. 2 is a graph showing the relative mRNA expression levels of three different subtypes, β 1-ADR, β 2-ADR and β 3-ADR, of β -adrenoceptor in the local microenvironment of bone marrow of the initial-diagnosed acute myelogenous leukemia patient in example 1 of the present invention. P < 0.05;
FIG. 3 is a graph showing the relative expression level differences between different subtypes of a-c. beta. -adrenoceptor in example 1 of the present invention among different subtypes of acute myelogenous leukemia patients. d-f. analysis of the relationship between extramedullary infiltrates and different subtypes of beta-adrenoceptors. P < 0.05;
FIG. 4 is a Bayesian network analysis of potential associations between leukemia patients (a) and control groups (b) neural related molecules Nestin, GFAP, β -TUB3, MAP2 and S-100 and three different subtypes of β -adrenoceptors in the bone marrow microenvironment, in example 1 of the present invention;
FIG. 5 is a graph showing the ratio of Th1(a), Th2(b) and Th17(c) in Th subgroups in the microenvironment of bone marrow in the initial acute myelogenous leukemia patient and the control group, and their corresponding representative flow charts in example 1 of the present invention. P <0.01, p < 0.001;
FIG. 6 shows the ratio of Th9(a), Th22(b) and Treg (c) in the Th subpopulation in the bone marrow microenvironment of the naive acute myelogenous leukemia patient and the control group, and the corresponding representative flow charts and the ratio of Th1/Th2(d) and Th17/Treg (e) in example 1 of the present invention. P <0.05, p <0.01, p <0.001, p < 0.000;
FIG. 7 is a graph showing the relative expression levels of T-beta (a), RORc (b), GATA3(c), Foxp3(d) and AHR (e) in the bone marrow microenvironment of the initial acute myelogenous leukemia patient and the control group in example 1 of the present invention. P <0.01, p <0.001, p < 0.000;
FIG. 8 is a graph showing the detection of IFN-. gamma. (a), IL-4(b), IL-17A (c), TGF-. beta.d, IL-22(e), IL-10(f), IL-6(g) and TNF-. alpha.h cytokines in the bone marrow microenvironment of the initial acute myelogenous leukemia patient and the control group in example 1 of the present invention. P <0.05, p <0.001, p < 0.000;
FIG. 9 is a Bayesian network analysis of potential association between specific cytokines IFN-. gamma.IL-4, IL-17A, TGF-. beta., IL-22, IL-10 and IL-6 secreted by a Th subset of myeloid minicircles in leukemia patients (a) and a control group (b) in example 1 of the present invention;
FIG. 10 is a graph showing the effects of nerve damage on the development of leukemia in example 1 of the present invention, which was conducted by using AML model mice to which 6-OHDA was applied as a nerve-damaging agent and 4-MY was applied as a neuroprotective agent, and observing the survival time (a) of mice, the infiltration of leukemia cells in bone marrow pictures (b), and the size of the spleen on at days 10 and 14 (c);
FIG. 11 is a graph showing the effects of nerve damage on the development of leukemia in example 1 of the present invention, which was conducted by using AML model mice to which 6-OHDA was applied as a nerve-damaging agent and 4-MY was applied as a neuroprotective agent, and observing the survival time (a) of mice, the infiltration of leukemia cells in bone marrow pictures (b), and the size of the spleen on at days 10 and 14 (c);
FIG. 12 is a schematic view showing the destruction of local sympathetic nerves of a single femur of a leukemia model mouse in (a) a method of injecting 6-OHDA into a medullary cavity in example 1 of the present invention. The leukemia load in the mouse spleen (b), the weight of the mouse spleen (c), and the tumor load in the bone marrow (d) were examined. P < 0.05;
FIG. 13 shows the proliferation of the sympathetic neurotransmitters epinephrine and norepinephrine on leukemia cell lines MV4-11(a, b), THP-1(c, d) and leukemia primary cells (e, f) after 24 hours and 48 hours in example 2 of the present invention. P <0.05, p <0.01, p <0.001, p < 0.000;
FIG. 14 shows three different subtypes of the beta-adrenoceptor ADR-beta 1(a), ADR-beta 2(b) and ADR-beta 3(c) in human leukemia cell line, glioma and CD4 of example 2 of the present invention+Relative expression of mRNA in T cells. (d) Expression profile of β -adrenoceptors in different tissues of mice;
FIG. 15 is a graph showing that low concentrations of sympathetic neurotransmitters have no significant effect on the ratio of Th subsets (a-f) and the concentration of Th-secreted cytokines (g-h) in example 2 of the present invention;
FIG. 16 shows a graph 10 obtained in example 2 of the present invention-4M adrenaline and noradrenaline can alter the proportion of Th subsets. P<0.01；
FIG. 17 shows example 2 of the present invention 10-4M adrenaline and noradrenaline can alter the ratio of Th subsets and the concentration of Th-secreted cytokines. P<0.05，**p<0.01；
FIG. 18 is a graph of the relative expression levels of downstream transcription factors and the immune balance of Th subsets in mice altered by sympathetic neurotransmitters according to example 2 of the present invention. P <0.05, p <0.01, p < 0.001;
FIG. 19 is an agarose gel electrophoresis showing the genotype of a β 1/2-adrenoceptor knock-out mouse in example 2 of the present invention. The Th subset changes after the sympathetic neurotransmitter acts on beta 1/2-adrenoceptor to knock off CD4+ T cells of mice;
FIG. 20 is a drawing showing TNFR2 of example 2 of the present invention in CD4 of an initial patient suffering from acute myelogenous leukemia+T (a) cells and tregs (b) expression levels on cells. Effect of TNF- α and TNFR2 blockers on Treg ratio (c), and its representative flow diagram (d);
FIG. 21 is a schematic view of example 2 of the present invention (a) showing co-culture with leukemia cells after the addition of sympathetic neurotransmitter to the culture of CD4+ T cells; (b-c) neurotransmitter-supplemented cultured CD4+Proliferation of leukemia cells after co-culture of T and leukemia cells. P<0.05，**p<0.01。
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific embodiments.
Example 1
Research method
1. Sign boardThis collection: the study collected 63 initial acute myelogenous leukemia patients and 16 bone marrow fluids of a control group, who had a visit to the university of Shandong, Qilu Hospital; isolation of mononuclear cells from bone marrow and sorting of CD4 by MACS+T cells, and bone marrow supernatant was collected.
Local presence of nerve destruction in AML patient bone marrow: RT-PCR was used to detect the relative expression levels of the neural specific molecules Nestin, GFAP, TUB3, MAP2 and S-100 in bone marrow cells of patients and controls.
Correlation of AML patients' bone marrow local adrenoceptor expression profile with clinical characteristics: RT-PCR was used to detect the differences in expression of three different subtypes of β 1-ADR, β 2-ADR and β 3-ADR of the bone marrow local β -adrenoceptor in the patients and controls and to analyze their correlation with clinical characteristics.
Immunological imbalance of local Th subpopulations of AML patients bone marrow: flow cytometry to determine the partial Th subgroup ratio of AML patient to control group bone marrow, including Th1, Th2, Th17, Th9, Th22 and Treg; detecting mRNA levels of Th subset specific transcription factors T-beta, RORc, GATA3, AHR and Foxp3 using RT-PCR technology; ELISA method was used to determine the concentration of the cytokines IFN-. gamma.IL-17A, TGF-. beta.IL-22, IL-10 and IL-4 in the bone marrow supernatant.
5. Analysis by systematic biological method: we used bayesian networks in a system biology approach to analyze Th subsets detected in the bone marrow microenvironment of leukemia patients and controls, as well as critical transcription factors of Th subsets.
Effect of sympathetic destruction on leukemia and Th subpopulations in AML animal models: injecting MLL-AF9 cell tail vein into wild female mouse with C57BL/6J background to build AML mouse model; carrying out the intervention of nerve destruction and nerve protection on the mouse by respectively carrying out intraperitoneal injection of 6-Hydroxydopamine hydrobromide (6-Hydroxydopamine hydrobromide, 6-OHDA) and 4-Methylcatechol (4-methycatechol, 4-MY); the proportion of local Th subset of animal model bone marrow is determined by flow cytometry.
7. Effects of local sympathetic nerve destruction of bone marrow on leukemia: the nerve of the local part of the mouse bone marrow is damaged by a method of injecting 6-OHDA in the medullary cavity.
Results of the study
1. Neurological damage in the local microenvironment of the bone marrow of incipient acute myelogenous leukemia patients
To determine whether there was a disruption of nerves in the myeloid microenvironment of acute myelogenous leukemia patients, we used RT-PCR to measure the expression levels of mRNA of neural-related molecules Nestin, GFAP, beta-TUB 3, MAP2 and S-100 in myeloid cells of control and preliminary leukemic groups. Nestin is a molecular marker of intermediate filament protein, which can be specifically expressed on nerve cells. GFAP is a glial fibrillary acidic protein and a marker of astrocyte activation. beta-TUB 3 is a class III beta tubulin, a microtubule element expressed only on neurons, and is a neural tissue-specific marker. Map2 is a specific marker for mature neurons. S-100 is a calcium binding protein, which is primarily present in the cytosol of astrocytes. The expression levels of these five molecules can largely represent whether the nervous system has been disrupted. Relative expression levels of Nestin (0.000372 + -0.000111 vs 0.00145 + -0.000904, p <0.05), GFAP (0.000430 + -0.000160 vs 0.00355 + -0.00189, p <0.05), TUB3(0.000519 + -0.000264 vs 0.00193 + -0.00162, p <0.01) and MAP2(0.00131 + -0.000569 vs 0.00441 + -0.00212, p <0.01) in AML patients were all significantly lower than in the control group. The S-100(0.0172 ± 0.00440vs 0.00261 ± 0.00124, p ═ 0.0620) molecule was expressed higher in AML patient bone marrow than in the control group, but was statistically insignificant. The above results demonstrate the presence of neural destruction in the bone marrow microenvironment of AML patients. (FIG. 1)
Correlation of bone marrow local adrenoceptor expression and clinical characteristics of AML patients and systematic biological analysis
2.1 bone marrow local adrenergic receptor expression of the patient with the initial acute myelogenous leukemia and the control group
The relative expression quantity of mRNA of three different subtypes of beta-adrenaline receptor beta 1-ADR, beta 2-ADR and beta 3-ADR in bone marrow microenvironment of leukemia patients and control groups is detected by RT-PCR technology. We found that the beta 1 subtype (0.001779 + -0.0003832 vs 0.004658 + -0.002386, p)<0.05) in leukemiaThe bone marrow local expression of the patient is lower than that of the control group, the beta 2 subtype (0.02998 +/-0.008986 vs 0.01276 +/-0.002018, p is 0.0656) and the beta 3(0.0002132 +/-8.796 multiplied by 10)- 5vs 0.0001199±6.738×10-5P-0.4032) is highly expressed locally in the bone marrow of leukemia patients. Moreover, the relative expression amount of the mRNA of the beta 2-ADR subtype is far higher than that of the beta 1-ADR and beta 3-ADR subtypes. In conclusion, leukemia patients locally express a certain amount of beta-adrenergic receptors in the bone marrow. (FIG. 2)
2.2 correlation analysis of bone marrow local beta-adrenoceptor expression of leukemia patients with clinical typing and clinical characteristics
The patients with acute myelogenous leukemia at the initial diagnosis are classified according to FAB typing standard and the presence or absence of extramedullary infiltration, and then the relationship between the beta-adrenergic receptor subtype and the leukemia typing and clinical characteristics is further analyzed. First, the relation between the expression level of the beta-adrenergic receptor and clinical typing is analyzed, and the AML-M5 subtype highly expresses the beta 1-ADR receptor and the AML-M3 subtype highly expresses the beta 2-ADR receptor. When analyzing the relationship between the expression level of beta-adrenergic receptors and the extramedullary infiltration, the probability that patients with high expression of beta 2-ADR receptors will have extramedullary infiltration is higher. (FIG. 3)
2.3 systematic biological analysis of neural molecules and their receptor expression in the bone marrow microenvironment
Using a method in systemic biology, the neural-related molecules Nestin, GFAP, beta-TUB 3, MAP2 and S-100 in the bone marrow microenvironment of leukemia patients and control groups, and three different subtypes of beta-adrenoceptor, beta 1-ADR, beta 2-ADR and beta 3-ADR, were analyzed in a Bayesian network in order to infer the role of the sympathetic nerves and their receptors in the progression of leukemia. The analysis result shows that the endpoint of the Bayesian network is changed in the control group and the leukemia patient group, so that the sympathetic nerve and the receptor thereof are inferred to play a certain role in the development process of the acute myelogenous leukemia. (FIG. 4)
Immunological imbalance of Th subsets locally present in AML patient's bone marrow
3.1 the proportion of Th1, Th17, Th9 and Th22 in the bone marrow microenvironment of AML patients is significantly increased
Mononuclear cells in bone marrow of the patient with primary leukemia and the control group are taken, and the proportions of Th1, Th2, Th17, Th22, Th9 and Treg in the Th subgroups are analyzed by flow cytometry. Wherein Th1, Th17 and Th22 belong to immunoprotective Th subgroups, and Th2 and Treg belong to immunosuppressive Th subgroups. The detection shows that compared with the control group, the proportion of Th1(8.42 +/-1.04% vs 16.52 +/-2.03%, p <0.001), Th17(3.52 +/-0.32% vs 7.39 +/-1.59%, p <0.01), Th9(3.29 +/-0.63% vs 6.49 +/-1.79%, p <0.05) and Th22(3.02 +/-0.34% vs 8.86 +/-1.45%, p <0.000) in the bone marrow microenvironment of the leukemia patients is obviously reduced. The proportion of Th2(2.31 ± 0.48% vs 0.68 ± 0.18%, p <0.01) and Treg (2.26 ± 0.76% vs 0.96 ± 0.23%, p ═ 0.1123) in leukemia was also higher than that in the control group, and Treg was not statistically significant. Th1 and Th2 and Th17 and Treg are two pairs of accepted balances, a certain balance exists in the microenvironment of normal people, however, the immune balance can be broken under the disease state, the proportions of Th1/Th2 and Th17/Treg are further analyzed towards immune protection or immune suppression, the proportion of Th1/Th2(21.02 +/-6.04 vs 48.83 +/-11.26, p <0.01) is obviously reduced in a leukemia patient group compared with that of a control group, and the proportion of Th17/Treg is not obviously different. The above results suggest that there is an immune imbalance of Th subpopulations in the myeloid microenvironment of AML patients. (FIGS. 5-6)
3.2 expression of specific transcription factors of Th subsets in AML patient bone marrow
Using RT-PCR technology, we examined mRNA levels of the Th subset specific transcription factors T-beta, GATA3, RORc, Foxp3 and AHR in the bone marrow microenvironment of the initial acute myelogenous leukemia patients and the control group. Wherein T-beta is a specific transcription factor of Th1, GATA3 is a specific transcription factor of Th2, RORc is a specific transcription factor of Th17, AHR is a specific transcription factor of Th22, and Foxp3 is a specific transcription factor of Treg. As a result, T-beta (0.00598 + -0.00321 vs 0.0154 + -0.00874, p) in the microenvironment of leukemia bone marrow was found<0.000)、RORc(0.00372±0.00342vs0.0326±0.0227，p<0.01) and GATA3(0.00247 + -0.000631 vs 0.00477 + -0.00139, p<0.001) and the expression level of mRNA is obviously reduced compared with the control group, while the specific transcription factor Foxp3 of Treg (0.000266 +/-7.68X 10)-5vs0.000146±7.98×10-5P-0.0824) and Th22 are not statistically significant. The above results further confirm the status of immune imbalance of Th subpopulations in the myeloid microenvironment of AML patients. (FIG. 7)
3.3 conditions of Th subpopulation secreted specific cytokines in AML patients' bone marrow
The conditions of specific cytokines IFN-gamma, IL-4, IL-17A, TGF-beta, IL-22, IL-10 and IL-6 secreted by Th subgroups in the bone marrow microenvironment of leukemia patients and control groups are detected by ELISA method. IFN-gamma (4.36 +/-1.52 pg/mL vs 10.56 +/-3.36 pg/mL, p <0.000), IL-17A (1.96 +/-0.41 pg/mL vs6.10 +/-2.45 pg/mL, p <0.05), TGF-beta (0.75 +/-0.069 pg/mL vs 1.65 +/-0.17 pg/mL, p <0.000) and IL-22(3.05 +/-0.24/mL vs 4.58 +/-0.68 pg/mL, p <0.05) in the bone marrow supernatant of leukemia patients are all lower than the control group, while the secretion amounts of IL-10(12.74 + -1.88 pg/mL vs 8.73 + -3.46 pg/mL, p <0.05), IL-4(3.70 + -0.18 pg/mL vs 2.34 + -0.34 pg/mL, p <0.001), and TNF-alpha (12.45 + -3.48 pg/mL vs 10.61 + -0.99 pg/mL, p <0.05) were higher than those of the control group, while IL-6 did not have any significant difference between the two groups. Wherein IFN-gamma is a specific cytokine secreted by Th1 cells, IL-4 and IL-10 are specific cytokines secreted by Th2 cells, IL-17A is a specific cytokine secreted by Th17 cells, and IL-22 is a specific cytokine secreted by Th22 cells. In conclusion, there is an immune imbalance of Th subgroup in the myeloid microenvironment of AML patients and affects the secretion of its potent cytokines. (FIG. 8)
3.4 application of systematic biological methods to speculate key molecules of Th subgroups in the development of leukemia
Using a systematic biological method, the above test results were analyzed by Bayesian network, and it was found that the endpoints of Bayesian network in the control group were directed to IFN-. gamma.and IL-4, and that the endpoints of network in AML patients were changed to IL-17A and IL-10. The two groups respectively represent two pairs of Th immune balances, namely Th1/Th2 and Th17/Treg, and the change of the network endpoint direction indicates that the Th has immune imbalance in the process of the occurrence and the development of leukemia, so that the immune imbalance of the Th subgroup can be presumed to play an important role in the occurrence and the development of the leukemia.
(FIG. 9)
Effect of sympathetic nerve disruption on leukemia and Th subsets in AML animal models
To further observe the effect of neural destruction on the development of leukemia, we used AML model mice to apply a neurolytic and neuroprotective agent to them to observe the development of Th and leukemia in the bone marrow microenvironment for the survival agent of mice.
4.1 the survival time of the nerve-disrupted AML model mice was shorter than that of normal and other control mice
The C57BL/6 mouse CD34+c-Kit+Cells transfected with MLL-AF9 (from Tianjin blood institute) as acute myelogenous leukemia cells were injected into mice via tail vein, CD34+c-Kit+The leukemia cells can home into the bone marrow to proliferate, and the generation and development process of leukemia is well simulated. After the mice are sick, bone marrow cells of the mice are taken out under the aseptic condition and counted as P0 generation MLL-AF9 cells. The cells of P0 generation were cultured at 1X 106The number of the mice is injected into the bodies of the mice by means of tail veins, and the mice take the P1 generation of bone marrow cells after the mice are sick, and the like. We used the more stable P2 generation cells for modeling.
The test pieces were divided into a blank group into which no MLL-AF9 had been injected and a control group molded from MLL-AF 9. The 6-OHDA was applied to leukemia mice for sympathetic nerve destruction to the neurolytic group, and the 4-MY was applied to leukemia to the neuroprotective group. Until the observation period was cut, none of the mice in the blank group died, and the survival time of AML mice in each group was shorter than that in the blank group (p < 0.05). After application of the neurolytic agent, the survival of AML mice was shorter than that of the control group and the neuroprotective agent group, and the neuroprotective agent extended the survival of mice (p < 0.05). (FIG. 10)
4.2 leukemia load was significantly higher in the neurolytic group mice than in the other groups, while leukemia load was significantly lower in the neuroprotective group mice than in the other groups
According to the survival period of the early-stage mice, the mice are judged to be the terminal stage of leukemia development in 14 days, and the leukemia load of the leukemia mice reaches the saturation state no matter whether the leukemia mice are interfered by a nerve damaging agent or a protective agent, so that no obvious difference exists among groups. Thus, we chose the disease progression and observed the spleen size and leukemia burden in the bone marrow of mice on day 10 of tail vein injection of MLL-AF 9. It was found that the bone marrow of the mice with leukemia in the neuro-destructive group had more leukemia cells than the control group and the neuroprotective group, while the bone marrow of the mice with neuroprotective group had less leukemia cells than the control group and the neuroprotective group. The spleen of the mice is taken to compare the sizes of the mice, and the spleen of the leukemia mice in the nerve damage agent group is larger than those of the control group and the nerve protective agent group, while the spleen of the nerve protective agent group is smaller than those of the control group and the nerve protective agent group. The experimental results show that the leukemia load of the mice of the neurolytic agent group is obviously higher than that of the other groups, and the leukemia load of the mice of the neuroprotective agent group is obviously lower than that of the other groups. (FIG. 10)
4.3 neural destruction in the local microenvironment of the bone marrow altering the Th subpopulation ratio
The analysis of Th subgroup by flow cytometry on mouse bone marrow mononuclear cells was found to be significantly lower than that of control group (0.090 + -0.040% vs 0.33 + -0.074%, p <0.05) for immune protective cells Th1, and lower than that of control group (1.09 + -0.27% vs 2.81 + -0.27%, p <0.05) and neuroprotective cells (1.36 + -0.15% vs 2.81 + -0.27%, p <0.05) for Th 17. The immunosuppressive Th2 cells were increased in both the neurolytic (1.99 + -0.11% vs 0.59 + -0.11%, p <0.05) and neuroprotective (1.71 + -0.28% vs 0.59 + -0.11%, p <0.05) groups. The ratio Th1/Th2 was significantly lower in the neurolytic group than in the control group (0.044 + -0.018% vs 0.64 + -0.24%, p <0.05), the ratio Th17/Treg was lower in both the neurolytic group (0.14 + -0.032% vs 0.56 + -0.052%, p <0.01) and the neuroprotective group (0.27 + -0.018% vs 0.56 + -0.052%, p <0.01) than in the control group, and the protective group was higher than the destructive group (0.14 + -0.032% vs 0.27 + -0.018%, p < 0.05). The above results suggest that we have Th immune imbalance in the local microenvironment of bone marrow in AML model mice. (FIG. 11)
5. Effects of local sympathetic nerve destruction of bone marrow on leukemia
Since leukemia is a malignant disease of myeloid origin, studying the effect of neural destruction on leukemia in the bone marrow microenvironment better defines the role of neural destruction in the development of leukemia. Therefore, the local sympathetic nerve of a single femur of a leukemia model mouse is damaged by a method of injecting 6-OHDA into a medullary cavity, and the leukemia load condition in bilateral medullary cavities of the mouse is observed. Taking mouse medullary cavity cells and spleen after mice are sick, and detecting the proportion of GFP positive cells by using a flow cytometry method to indicate the load of leukemia cells in bone marrow and periphery. We found that the spleen of the bone marrow nerve mice on the side of the destruction had a greater tumor burden (68.08 ± 6.2%) than the spleen of the non-destroyed nerve mice (39.32 ± 0.5%) (p <0.05), but the difference between the spleen weight and the tumor burden in the medullary cavity was not statistically significant. (FIG. 12)
Example 2
Research method
1. Collecting a specimen: bone marrow fluid of a patient with initial acute myelogenous leukemia from Qilu hospital of Shandong university is collected in the research; bone marrow mononuclear cells were isolated and sorted for CD4 using MACS+T cells, and bone marrow supernatant was collected.
2. Sympathetic neurotransmitters promote proliferation of primary and leukemic cell lines: selecting acute myelogenous leukemia cell lines MV4-11 and THP-1 and primary cells of a patient with initial acute myelogenous leukemia; CCK8 was used to test the proliferation-promoting effect of various concentrations of epinephrine and norepinephrine at the 24 hour and 48 hour time points.
3. Expression of β -adrenoceptors on different cells: RT-PCR technology is used to detect the expression of beta 1/beta 2/beta 3-adrenoceptor mRNA on different cells.
4. Effect of different concentrations of sympathetic neurotransmitters on Th subsets: setting concentration gradients 0, 10-8M、10-7M、10-6M、10-5M and 10-4M adrenaline and noradrenaline acting on CD4+A T cell; sorting of CD4 Using MACS Immunomagnetic bead method+A T cell; flow cytometry was used to detect the proportion of Th subpopulations; ELISA was performed to determine the concentration of IFN-. gamma.and IL-17A cytokines in the culture supernatant.
5. Sympathetic neurotransmitters influence the immune balance of Th subsets via β -adrenoreceptors: MACS immunomagneticBead method for sorting mice CD4+A T cell; detecting the genotype of a beta-adrenergic receptor knockout mouse by agarose gel DNA electrophoresis; detecting changes of Th subgroups of mice by flow cytometry; ELISA was used to measure the concentration of IFN-. gamma.and IL-17A in the supernatant of cultured cells.
6. Neurotransmitter pair CD4+T differentiation effects in turn affect killing of leukemia cells: MACS immunomagnetic bead sorting of CD4+A T cell; flow cytometry was used to detect the proportion of Th subpopulations; the CCK8 method is used to detect cell proliferation.
Results of the study
1. Sympathetic neurotransmitters promote proliferation of primary and leukemic cell lines
1.1 sympathetic neurotransmitters promote proliferation of leukemia cell lines
In order to determine how the sympathetic neurotransmitter affects the generation and development of leukemia, leukemia cell lines MV4-11 and THP-1 are selected, and the neurotransmitters epine (Epi) and Norepinephrine (NE) are added in logarithmic growth period, and the concentration gradient is set to be 0, 1 × 10-6、1×10-5、2.5×10-5、5×10-5、1×10-4mol/L. Cell proliferation was measured by the CCK8 method at two time points, 24 hours and 48 hours, respectively. As a result of the study, it was found that the degree of cell proliferation gradually increased with increasing neurotransmitter concentration, at 10-4For MV4-11 (p) at M concentration24h<0.05，p48h<0.05) and THP-1 (p)24h<0.001，p48h<0.05) the proliferation-promoting effect is most obvious. And the proliferation effect of the epinephrine promoted leukemia cell line is superior to that of norepinephrine. Taken together, sympathetic neurotransmitters can promote the proliferation of leukemia cell lines (fig. 13).
1.2 sympathetic neurotransmitters promote proliferation of Primary leukemia cells
After it is clear that the sympathetic neurotransmitter can promote the proliferation of the leukemia cell line, in order to further clarify the effect on primary leukemia cells, the bone marrow fluid of a patient with the bone marrow primary cell number of 90% is selected, the mononuclear cells of the patient are separated, and epinephrine and norepinephrine with different concentration gradients are respectively applied. At 24 hours and 48 hoursAt these two time nodes, the effect of the sympathetic neurotransmitter on the leukemia cell proliferation is more obvious when the CCK8 method is applied. Adrenalin (p)24h<0.000，p48h<0.000) and noradrenals (p)24h<0.000，p48h<0.000) element is 10-4The proliferation promoting effect is most obvious when the concentration of M is higher than that of noradrenalin, and the proliferation promoting effect of adrenalin on leukemia cells is better than that of noradrenalin. From this we conclude that sympathetic neurotransmitters can promote the proliferation of leukemia cell lines and primary leukemia cells. (FIG. 13)
2. Expression of beta-adrenoceptor on different cells
2.1 expression of beta-adrenoceptor in leukemia cell lines
The prophase detection of sympathetic neurotransmitters promotes the action of leukemia cell lines, and the specific action mechanism is not clear. We speculate that the sympathetic neurotransmitters epinephrine and norepinephrine act via the β -adrenergic receptors, and that prior assays involved the β -adrenergic receptors in the development of leukemia through systemic biological assays. To this end, we further examined several leukemia cell lines and CD4 in the patient's bone marrow microenvironment+Expression of β -adrenoceptors on T cells. We selected human brain gliomas as controls and the beta 1-adrenoceptor at CD4+T cells and most leukemia cell lines show low expression, but high expression in THP-1 (0.00617 + -4.28X 10)-5vs 0.00449 ± 0.00034). In leukemia cell lines, certain amounts of beta 2-adrenoceptors are expressed, and the beta 2-adrenoceptors are in CD4+The expression level in T cells was higher than that in human gliomas (0.0118. + -. 0.00029vs 0.0147. + -. 0.00091). Beta 3-adrenoceptor in various cell lines and CD4+T cells have certain expression. It is known that a certain amount of beta-adrenoceptors are expressed in immune cells and leukemia cell lines, and therefore, sympathetic neurotransmitters can exert biological effects on immune cells and leukemia cells via beta-adrenoceptors. (FIG. 14)
2.2 beta-adrenoceptor expression in mice
As early experiments prove that nerve damage can change leukemia load and Th subgroup of AML model mice, we simultaneously detect spleen, bone marrow, peripheral blood and sorted CD4 of mice+Beta-adrenoceptor expression in T cells and skeletal muscle cells, mouse skeletal muscle was used as a positive control cell. The expression level of beta 2-adrenergic receptor is far higher than that of other two subtypes, CD4+The expression level of the beta 2-adrenoceptor on T cells approaches the level of expression in skeletal muscle. (FIG. 14)
3. Low concentrations of sympathetic neurotransmitters have no significant effect on the immune balance of Th subsets
3.1 Low concentration of sympathetic neurotransmitters had no significant effect on the Th subpopulation ratio
The previous experimental results demonstrate that there is an immune imbalance of the Th subgroup in the myeloid microenvironment of leukemia patients, and CD4+The surface of T cells highly expresses beta-adrenergic receptors. To further demonstrate whether neurotransmitters can affect CD4+Differentiation of T cells into Th subgroups, we used magnetic bead sorting method to sort out CD4 in marrow of leukemia patients for first diagnosis+T cells, and performing amplification culture on the T cells. In logarithmic phase of growth, 10 are added-8M-10-5Concentration of M adrenaline and noradrenaline in CD4+In a T cell culture system, a flow cytometry method is applied after 3 days to detect the proportion and ratio relation of Th1, Th2, Th17 and Treg subgroups. The study found that 10-8M-10-5The M concentration interval, Th subgroup, Th1/Th2, Th17/Treg and blank control group have no statistical difference. The above experimental results show that 10-8M-10-5The M concentration of sympathetic neurotransmitter does not affect the ratio of Th. (FIG. 15)
3.2 Low concentration of sympathetic neurotransmitters had no significant effect on cytokines secreted by Th subsets
And (3) taking the cell supernatant after the culture, and detecting the concentrations of IFN-gamma and IL-17A cytokines secreted by Th1 and Th17 by using an enzyme-linked immunosorbent assay (ELISA). The experimental results found that 10-8M-10-5The neurotransmitter concentration of M cannot influence secretion of IFN-gamma from Th1 and Th17And the concentration of IL-17A. In summary, 10-8M-10-5The M concentration of sympathetic neurotransmitter cannot affect the proportion of Th and its cytokine secretion.
(FIG. 15)
4. High concentrations of sympathetic neurotransmitters alter the immune balance of Th subsets
4.1 high concentration of sympathetic neurotransmitter alters the Th subpopulation ratio
In definition 10-5After concentrations below M failed to alter the ratio of Th, we further increased the concentration of the sympathetic neurotransmitters epinephrine and norepinephrine to 10-4M, to see if high concentrations of neurotransmitters could affect differentiation of Th subsets. In CD4+Adding 10 into the culture system of T cells respectively-4And D, culturing adrenaline and noradrenaline with M concentration for 3 days, and detecting the proportions and ratio relations of Th1, Th2, Th17 and Treg subgroups by using a flow cytometry method. The study found that both epinephrine and norepinephrine significantly reduced Th1(11.07 + -2.89% vs 23.01 + -4.75%, p)<0.01；11.41±3.44％vs 23.01±4.75％，p<0.01) and Th17 (1.22. + -. 0.34% vs 3.13. + -. 0.56%, p<0.01；1.21±0.23％vs 3.13±0.56％，p<0.01), adrenergic activity significantly increased Treg (5.03 + -1.20% vs 3.50 + -0.94%, p<0.01). (FIG. 16) and both epinephrine and norepinephrine significantly reduced Th1/Th2(9.38 + -3.51 vs 46.52 + -22.71, p)<0.05；14.64±6.89vs 46.52±22.71，p<0.01) and Th17/Treg (0.41 + -0.16 vs 1.55 + -0.60, p)<0.01；0.69±0.33vs 1.55±0.60，p<0.01) the ratio of these two pairs of immune equilibria. The results of the experiments prove that 10-4The sympathetic neurotransmitter of M can inhibit the proportion of protective Th. (FIG. 17)
4.2 high concentrations of sympathetic neurotransmitters affect secreted cytokines from Th subsets
And (3) detecting the cultured cell supernatant by using an ELISA kit, and determining whether the Th subgroup is a functional Th subgroup or not and whether the specific cytokine secreted by the Th subgroup is also changed by sympathetic neurotransmitter or not. The experimental result shows that CD4 is cultured by adrenaline and noradrenaline+IFN-gamma (0.62 +/-0.016) in supernatant of T cellspg/mL，p<0.01；0.83±0.017pg/mL，p<0.05) and IL-17A (0.58. + -. 0.14pg/mL, p<0.01；0.39±0.084pg/mL，p<0.05) the concentration of the cytokine was also significantly reduced compared to the control group. The above results demonstrate that 10-4The M sympathetic neurotransmitter can inhibit the proportion of protective Th and can inhibit the secretion of its cytokines. (FIG. 17)
5. Sympathetic neurotransmitters also alter immune balance of Th subsets in mice
To further clarify the effect of neurotransmitters on mouse Th. We sorted CD4 in mouse bone marrow+After T cells, 10 was added to the culture system-4M concentration of sympathetic neurotransmitter is cultured, and the change condition of Th subgroup is detected by a flow cytometry method. After 3 days of culture with neurotransmitter, the proportion of Th1 and Th17 is obviously reduced and the proportion of Treg is obviously increased compared with the control group containing both epinephrine and norepinephrine (5.14 +/-0.57% vs 0.81 +/-0.16%, p is<0.01). And detecting and finding IL-17A (0.34 +/-0.12, p) caused by epinephrine and norepinephrine by using RT-PCR technology<0.000；0.22±0.050，p<0.001)、T-bet(0.50±0.057，p<0.01；0.50±0.047，p<0.001)、TGF-β(0.41±0.11，p<0.01；0.41±0.12，p<0.05) was significantly lower than the control group, while epinephrine caused Foxp3(4.19 ± 0.73, p) in mice<0.05) significantly increased mRNA levels. It was demonstrated that epinephrine and norepinephrine altered the Th subpopulation of mice from the cellular phenotype and mRNA levels. (FIG. 18)
6. Sympathetic neurotransmitters influencing immune balance of Th subgroups via beta-adrenoreceptors
6.1 detection of genotype of β 1/β 2-adrenoceptor knock-out mouse
The prophase uses the system biology method to speculate that the occurrence and the development of leukemia are possibly related to sympathetic neurotransmitters, which can promote the proliferation of leukemia cells and obviously influence CD4+The proportion of T cells differentiated towards Th subpopulations. Therefore, to further clarify how sympathetic neurotransmitters act on Th, we purchased β 1/β 2-adrenoreceptor knockout mice from the american Jackson laboratory. Extracting somatic cells of wild-type mice and beta 1/beta 2-adrenoceptor knockout miceTaking DNA to carry out gel electrophoresis experiment. The beta 1 genotype of the wild mouse is 151bp, the beta 2 genotype is 225bp, the beta 1 genotype of the knockout mouse is 650bp, and the beta 2 genotype is 410bp, so that the wild mouse accords with the knockout mouse genotype. (FIG. 19)
6.2 failure of sympathetic neurotransmitters to alter the Th subset of beta-adrenoceptor knock-out mice
To further clarify whether the sympathetic neurotransmitter acts through the β 1/β 2-adrenoceptor, we used magnetic bead sorting to sort out CD4 in the bone marrow of mice+T cells were cultured in vitro. And (3) taking the cells in the logarithmic phase of growth, adding adrenaline and noradrenaline into a culture system respectively, and detecting the proportions of Th1, Th2, Th17 and Treg by using a flow cytometry method after 3 days of culture. The previous results show that the proportion of Th1 and Th17 of wild-type mice is obviously inhibited by the sympathetic neurotransmitter, while the proportion of Th of the mice knocked by the adrenergic receptors is not obviously changed, which proves that the sympathetic neurotransmitter plays a role in changing Th subgroups through beta-adrenergic receptors. (FIG. 19)
TNF-alpha promoting the proportion of Tregs in leukemia patients by TNFR2
The experimental results in the previous chapter show that the concentration of TNF-alpha is obviously higher than that of a control group in a patient with leukemia in an initial diagnosis, and in order to determine the effect of TNF-alpha in the occurrence and development of leukemia, the influence of TNF-alpha on Treg and the acting way of the Treg are further researched.
7.1 initial diagnosis of leukemia patient CD4+TNFR2 highly expressed on T cell and Treg cell surface
TNFR2 is the major receptor for TNF-alpha and we used flow cytometry to treat CD4 in naive patients with acute myelogenous leukemia and controls+The expression level of TNFR2 on the surface of T cells and Treg cells was determined. We found that the primary diagnosis of CD4 in leukemia patients was compared to the control group+T cells (14.67. + -. 6.88% vs 6.88. + -. 2.51%, p)<0.01) and Treg cells (9.65. + -. 3.99% vs 3.22. + -. 1.40%, p)<0.01) TNFR2, the main functional receptor of TNF-alpha, is highly expressed on the surface. (FIG. 20)
7.2TNF- α promotes the proportion of Tregs by TNFR2
To further explore the effect of TNF-alpha on Treg, TNF-alpha (20ng/mL) was added to a culture system of CD4+ T cells for culture, and a control group and a group blocking TNFR2 receptor were set to observe the effect of TNF-alpha on the proportion of Treg. It was found that the proportion of Tregs was significantly increased after addition of TNF- α compared to the control group (10.64. + -. 4.14% vs 11.80. + -. 4.62%, p < 0.01). However, compared to the addition of TNF- α, the addition of TNF- α after the addition of TNFR2 antagonist in advance reduced the proportion of Treg (11.80. + -. 4.62% vs 11.02. + -. 4.30%, p < 0.01). In conclusion, we can conclude that the high TNF-alpha level of patients with primary leukemia can increase the proportion of Tregs of immunosuppressive subpopulations, and play a role in suppressing immunity. (FIG. 20)
8. Neurotransmitter pair CD4+Influence of T differentiation in turn affecting killing of leukemia cells
Through preliminary studies, the fact that the sympathetic neurotransmitters adrenaline and noradrenaline can promote the proliferation of leukemia cells and can inhibit the differentiation of protective Th subgroups is found, and whether the killing effect of the leukemia cells is influenced after the proportion of Th1 and Th17 subgroups is inhibited is not clear. Therefore, we will add neurotransmitter cultured CD4+The effect of Th subgroup after immune imbalance on leukemia cells is determined by detecting the number of leukemia cells through co-culturing T and leukemia cells. First sorted CD4+After the T cells, 10 cells were added to the culture system-4Adrenaline and noradrenaline at M concentration, and CD4 was added after 3 days of culture+T cells and leukemia cells are co-cultured, and the proliferation of the leukemia cells is detected by a cck8 method at 24 hours and 48 hours. Both epinephrine and norepinephrine at 24 hours (1.62 + -0.19, p) compared to control without neurotransmitter addition<0.05；1.30±0.083，p<0.05) and 48 hours (1.39. + -. 0.042, p)<0.000；1.09±0.017，p<0.001) and further promote the proliferation of leukemia cells by changing the balance of Th subgroups, wherein the epinephrine effect is more obvious. Taken together, sympathetic neurotransmitters may affect CD4+Differentiation of T in turn affects the killing of leukemia cells. (FIG. 21)
It should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the same. Although the present invention has been described in detail with reference to the examples given, those skilled in the art can modify the technical solution of the present invention as needed or equivalent substitutions without departing from the spirit and scope of the technical solution of the present invention.

Claims (10)

1. A biomarker for detecting acute myeloid leukemia, the biomarker being selected from any one or more of:
neural specific molecules, adrenoceptors and Th subsets.
2. The biomarker of claim 1, wherein the neural specific molecules comprise Nestin, GFAP, TUB3, MAP2, and S-100;
the adrenoceptors are beta-adrenoceptors, and comprise three different subtypes of the beta-adrenoceptors, namely beta 1-ADR, beta 2-ADR and beta 3-ADR;
the Th subpopulation includes Th1, Th17, Treg and Th22 cells;
including downstream transcription factors T-beta, RORc, GATA3, Foxp3 and AHR from the Th subpopulation;
including cytokines secreted by the Th subset, IFN-gamma, IL-17A, TGF-beta, IL-10, IL-4, and IL-22.
Preferably, the biomarker is obtained locally from the bone marrow of the subject.
3. The biomarker of claim 1 or 2, wherein detecting acute myeloid leukemia specifically comprises diagnosing or aiding in diagnosing acute myeloid leukemia; preferably, the method comprises the following steps:
(1) local nerve destruction (degree) of the bone marrow, in particular sympathetic nerve destruction;
(2) clinical features of acute myeloid leukemia, including extramedullary infiltrates;
(3) local Th subsets of bone marrow are immunologically imbalanced.
4. Use of a substance for detecting a biomarker according to any of claims 1 to 3 in the manufacture of a product for the diagnosis or assisted diagnosis of acute myeloid leukemia.
5. An apparatus, characterized in that the apparatus comprises:
one or more devices for detecting a biomarker according to any of claims 1 to 3.
6.A kit comprising the device of claim 5.
7. The application of any one or more of the following substances 1) to 3) in preparing medicines for treating acute myelogenous leukemia.
1) A neuroprotective agent;
2) a sympathetic neurotransmitter inhibitor;
3) TNF-alpha and/or TNFR2 receptor inhibitors.
8. The use as claimed in claim 7, wherein the neuroprotective agent is in particular a sympatholytic agent; the sympathetic neurotransmitter includes epinephrine and norepinephrine; or the like, or, alternatively,
the treatment of acute myeloid leukemia specifically comprises:
1) inhibiting leukemia cell proliferation;
2) improving the immune status of the body, specifically comprising relieving or weakening the suppression effect on the T cell function and reducing the proportion of the Tregs of the immunosuppressive Th subgroup.
9. The medicine for treating acute myelogenous leukemia is characterized in that the effective components of the medicine comprise any one or more of the following components:
1) a neuroprotective agent;
2) a sympathetic neurotransmitter inhibitor;
3) TNF-alpha and/or TNFR2 receptor inhibitors;
preferably, the medicament for treating acute myeloid leukemia further comprises at least one carrier.
10. A method of screening for a drug for the treatment of acute myeloid leukemia, the method comprising: using the effect of the candidate drug on the biomarker of any of claims 1 to 3 before and after use to determine whether the candidate drug can be used to treat acute myeloid leukemia.
CN202010696955.0A 2020-07-20 2020-07-20 Medicine and method for treating acute myelogenous leukemia Pending CN111808960A (en)

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