WO2022183600A1 - 增强tbx1表达在修复心脏组织损伤中的用途 - Google Patents

增强tbx1表达在修复心脏组织损伤中的用途 Download PDF

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WO2022183600A1
WO2022183600A1 PCT/CN2021/094932 CN2021094932W WO2022183600A1 WO 2022183600 A1 WO2022183600 A1 WO 2022183600A1 CN 2021094932 W CN2021094932 W CN 2021094932W WO 2022183600 A1 WO2022183600 A1 WO 2022183600A1
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tbx1
cardiac
protein
lymphatic
myocardial infarction
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PCT/CN2021/094932
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English (en)
French (fr)
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张臻
张敏
王文峰
丁晓宁
王晔
杨俊杰
魏璐
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上海交通大学医学院附属上海儿童医学中心
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Priority to EP21928681.2A priority Critical patent/EP4302774A1/en
Priority to US18/279,765 priority patent/US20240139283A1/en
Publication of WO2022183600A1 publication Critical patent/WO2022183600A1/zh

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Definitions

  • the invention relates to the field of cardiovascular disease treatment, in particular to the use of TBX1 in repairing cardiac tissue damage.
  • Cardiovascular disease is the leading cause of death in my country, and the mortality rate is still on the rise in recent years.
  • Mammalian cardiomyocytes are proliferative only for a brief window after birth, and exit the cell cycle after the myocardium matures. Therefore, myocardial necrosis is often irreversible, has a poor prognosis, and can lead to severe consequences, e.g., up to 60% of ischemia.
  • Patients with myocardial infarction develop heart failure within six years of their initial onset.
  • the treatment for myocardial infarction mainly reduces myocardial ischemia and necrosis through vascular recanalization, and slows the adverse remodeling of the ventricle through the regulation mechanism of drug regulation of neurohumoral.
  • these treatments do not completely block ventricular remodeling after MI, and a substantial proportion of patients progress to heart failure in a relatively short period of time without continued ischemic injury.
  • the purpose of the present invention is to provide a new active ingredient for effectively treating heart disease and/or lymphatic vessel abnormality related diseases related to heart damage and its use.
  • a first aspect of the present invention provides a use of an active ingredient for the preparation of a preparation or medicine, and the preparation or medicine is used for:
  • the active ingredient includes: TBX1 protein, its coding sequence, or its promoter, or its combination.
  • the coding sequence includes DNA, cDNA and mRNA.
  • the "promoting the transformation of M1 macrophages to M2 macrophages” comprises: promoting M1 macrophages (promoting inflammatory responses) to M2 macrophages (promoting tissue fix) conversion.
  • the "heart disease associated with cardiac tissue damage” is a heart disease associated with an immune response (especially an autoimmune response).
  • the cardiac disease associated with cardiac tissue damage is selected from the group consisting of myocardial infarction, myocarditis, idiopathic dilated cardiomyopathy, Chagas' cardiomyopathy, rheumatic heart disease, novel coronavirus pneumonia ( heart damage due to COVID-19), or a combination thereof.
  • the disease associated with abnormal lymphatic vessels is selected from the group consisting of primary lymphedema caused by lymphatic dysplasia, secondary lymphedema caused by rupture or obstruction of lymphatic vessels, Turner syndrome, Lymphangioedema presented by genetic syndromes such as thyroid syndrome, Hennekam syndrome, or a combination thereof.
  • the second aspect of the present invention provides the use of a TBX1 protein or its coding sequence or its detection reagent for preparing a diagnostic reagent or a diagnostic kit, the diagnostic reagent or diagnostic kit is used for damage to cardiac tissue Related heart disease or lymphatic vessel abnormalities-related diseases for prognostic judgment.
  • the detection reagent is selected from the group consisting of primers, probes, chips, antibodies, or a combination thereof.
  • the diagnosed subject has or is suspected to have a heart disease related to cardiac tissue damage or a disease related to abnormal lymphatic vessels.
  • the method for prognosing a heart disease related to cardiac tissue damage or a disease related to abnormal lymphatic vessels includes: providing a subject sample, detecting the expression level T1 of TBX1 protein in the subject sample, and Compared with the average expression level T0 of TBX1 protein in cardiac lymphatic vessels of patients with myocardial infarction;
  • T1 is greater than T0, it indicates that the subject has a good prognosis
  • T1 is less than T0, the subject has a poor prognosis.
  • the method for prognosing a heart disease related to cardiac tissue damage or a disease related to abnormal lymphatic vessels includes: providing a subject sample, and detecting whether the subject sample has TBX1 encoding and/or For regulatory sequence variants, if the TBX1 variant is a variant that causes loss or decline of TBX1 function, it indicates that the subject has a poor prognosis; if the TBX1 variant is a variant that results in enhanced TBX1 function, it indicates that the subject has a good prognosis.
  • the function of TBX1 refers to the promotion of lymphatic vessel regeneration and the formation of an immunosuppressive microenvironment.
  • the cardiac disease related to cardiac tissue damage includes myocardial infarction.
  • the subject suffers from myocardial infarction.
  • the subject is a human or a non-human mammal.
  • the sample includes a heart tissue sample, a heart cell sample, a lymphatic endothelial cell sample, a lymphatic fluid sample, a peripheral blood sample cell, or a combination thereof.
  • the sample includes a cell sample, a DNA sample, an RNA sample, or a combination thereof.
  • the kit further includes detection reagents for detecting additional markers selected from the group consisting of Top2a, Mki67, Cenpe, Aurkb, or a combination thereof.
  • the expression levels (or expression levels) of these additional markers increase, indicating that the TBX1 mutation is a mutation that leads to enhanced TBX1 function.
  • the expression levels (or expression levels) of these additional markers are increased compared with the normal population or controls, it is suggested that the diagnosed subject has a good prognosis.
  • the expression levels (or expression levels) of these additional markers are decreased compared with the normal population or controls, it indicates that the diagnosed subject has a poor prognosis.
  • the third aspect of the present invention provides an active ingredient that can be used for repairing cardiac tissue damage and treating diseases related to abnormal lymphatic vessels, the active ingredient comprising: TBX1 protein, its coding sequence, or its promoter, or a combination thereof.
  • the active ingredient is a promoter of TBX1 protein.
  • the promoter promotes the expression of TBX1 protein in the lymphatic vessels of the heart and other parts of the body.
  • the promoter upregulates the function of TBX1 protein in the heart and lymphatic vessels in other parts of the body.
  • the fourth aspect of the present invention provides an expression vector, which contains an expression cassette for expressing TBX1 protein.
  • the expression vector is selected from the group consisting of DNA, RNA, plasmid, eukaryotic expression vector, prokaryotic expression vector, lentiviral vector, adenovirus vector, adeno-associated virus vector, retroviral vector, transfection seat, or a combination thereof.
  • the expression vector specifically infects lymphatic endothelial cells.
  • the expression cassette has the structure of formula I from the 5' end to the 3' end:
  • each "-" is independently a chemical bond or a nucleotide linking sequence
  • Z0 is none, or 5'UTR sequence
  • Z1 is the nucleotide sequence encoding the TBX1 protein
  • Z2 is none, or a 3'UTR sequence.
  • the expression cassette further comprises a lymphatic vessel endothelium-specific promoter operably linked to the coding sequence of the TBX1 protein.
  • each nucleotide linking sequence is 1-30nt, preferably 1-15nt, more preferably 3-6nt.
  • the nucleotide sequence encoding the TBX1 protein is derived from human or non-human mammals.
  • nucleotide sequence is shown in SEQ ID NO.1.
  • nucleotide sequence is shown in SEQ ID NO.2.
  • the fifth aspect of the present invention provides a host cell containing the expression vector of the fourth aspect of the present invention.
  • the host cell is a eukaryotic cell or a prokaryotic cell.
  • the host cell is selected from the group consisting of bacterial cells, yeast cells, or mammalian cells.
  • the host cells are lymphatic endothelial cells.
  • the host cells are cardiac lymphatic endothelial cells.
  • the sixth aspect of the present invention provides a pharmaceutical preparation comprising (a) the active ingredient described in the third aspect of the present invention, or the expression vector described in the fourth aspect of the present invention, or the fifth aspect of the present invention The cells, and (b) a pharmaceutically acceptable carrier, excipient or diluent.
  • the dosage form of the pharmaceutical preparation is selected from the group consisting of freeze-dried preparation, liquid preparation, or a combination thereof.
  • the pharmaceutical preparation is an injection.
  • the pharmaceutical preparation further comprises: additional active ingredients;
  • the additional active ingredients include: BMP4, CCL28, anti-CD8 antibody, or a combination thereof.
  • the seventh aspect of the present invention provides an active ingredient described in the third aspect of the present invention, or the expression vector described in the fourth aspect of the present invention, or the cell described in the fifth aspect of the present invention, or the first aspect of the present invention.
  • the heart disease related to heart injury is selected from the group consisting of myocardial infarction, myocarditis, idiopathic dilated cardiomyopathy, Chagas' cardiomyopathy, rheumatic heart disease, novel coronavirus pneumonia (COVID-19) -19) induced cardiac damage, or a combination thereof.
  • the disease associated with abnormal lymphatic vessels is selected from the group consisting of primary lymphedema caused by lymphatic dysplasia, secondary lymphedema caused by rupture or obstruction of lymphatic vessels, Turner syndrome, Lymphangioedema presented by genetic syndromes such as thyroid syndrome, Hennekam syndrome, or a combination thereof.
  • the eighth aspect of the present invention provides a method for screening a promoter of TBX1 protein, comprising the steps of: administering a candidate drug to lymphatic endothelial cells cultured in vitro and/or experimental animals, if the lymphatic endothelial cells cultured in vitro or If the level of TBX1 mRNA or TBX1 protein in the lymphatic vessel tissue of experimental animals is increased relative to the control group without the drug candidate, or the function of TBX1 protein is enhanced relative to the control group without the drug candidate, the candidate drug can act as a promoter of TBX1 protein .
  • the TBX1 protein promoter can be used to treat cardiac diseases related to cardiac tissue damage.
  • the promoter of TBX1 protein can be used to treat diseases related to abnormal lymphatic vessels.
  • the ninth aspect of the present invention provides a method for treating heart disease related to cardiac tissue damage, the method comprising: mixing the active ingredient described in the third aspect of the present invention or the pharmaceutical preparation described in the sixth aspect of the present invention Apply to those in need.
  • the subject in need suffers from cardiac disease associated with cardiac tissue damage.
  • the heart disease related to heart injury is selected from the group consisting of myocardial infarction, myocarditis, idiopathic dilated cardiomyopathy, Chagas' cardiomyopathy, rheumatic heart disease, novel coronavirus pneumonia (COVID-19) -19) induced cardiac damage, or a combination thereof.
  • the tenth aspect of the present invention provides a method for treating diseases related to abnormal lymphatic vessels, the method comprising: administering the active ingredient described in the third aspect of the present invention or the pharmaceutical preparation described in the sixth aspect of the present invention to a patient in need Object.
  • the subject in need suffers from a heart disease associated with abnormal lymphatic vessels.
  • the disease associated with abnormal lymphatic vessels is selected from the group consisting of: primary lymphedema caused by lymphatic dysplasia, secondary lymphedema caused by rupture or obstruction of lymphatic vessels, Turner syndrome, jaundice Lymphangioedema presented by genetic syndromes such as thyroid syndrome, Hennekam syndrome, or a combination thereof.
  • FIG. 1 shows the activated expression of Tbx1 in lymphatic endothelium after myocardial infarction.
  • FIG. 2 shows that endothelial cell-specific knockout of Tbx1 leads to abnormal lymphatic regeneration and repair impairment after myocardial infarction.
  • A Schematic diagram of the construction of Tbx1 deficient mice, the functional genome sequence was knocked out under the activation of Cre;
  • B ventral view of the heart 28 days after myocardial infarction.
  • Black arrow aneurysm in the anterior wall of the left ventricle.
  • Green arrow postoperative adhesion
  • Vegfr3 staining showed the regeneration of cardiac lymphatic endothelium at the site of myocardial infarction, the gross map and histological section (F) of the heart at 7 days after myocardial infarction, and the statistics of the number of lymphatic endothelium at the site of myocardial infarction and distal uninjured sites (G), **, P ⁇ 0.01, Mann-Whitney U test;
  • H Endothelium-specific transcriptome differential genes and analysis of Tbx1 downstream target genes by chromatin immunoprecipitation combined with high-throughput sequencing (ChIP-seq) ;
  • FIG. 3 shows that Tbx1-depleted hearts significantly altered the expression of lymphatic vessel growth-related genes.
  • A Transcriptome analysis showed that the lymphatic vessel growth-related genes Dtx1, Dtx3, Sema4c and Foxc2 were significantly decreased in cardiac endothelial cells after myocardial infarction.
  • B, C Immunofluorescence staining analysis of the distribution and quantitative quantification of lymphatic endothelial cells expressing Dtx1 and Notch1 in the heart at day 7 after myocardial infarction.
  • FIG. 4 shows that Tbx1-depleted hearts neutralize immune regulation-related genes significantly altered in lymphatic endothelial cells.
  • A, B Immunofluorescence staining analysis of the distribution and quantitative quantification of lymphatic endothelial cells expressing Ccl21, Ccl28 and Icam1 in the heart on day 7 after myocardial infarction.
  • Figure 5 shows that autoimmune CD8+ T cells are significantly increased in Tbx1 deficient hearts.
  • B T cell subset cell proliferation analysis shows CD8 + T cells Highly proliferative, data shown as mean ⁇ SD, **, p ⁇ 5.6x10-14 , ⁇ p ⁇ 4.3x10-11 , Student's t-test;
  • E F
  • Figure 6 shows a decrease in inflammatory M1 macrophages in Tbx1-deficient hearts and a significant increase in repairing M2 macrophages.
  • A,B Changes in the number of macrophages in Tbx1Cko hearts on day 7 after myocardial infarction by flow cytometry, *, p ⁇ 0.05, **, p ⁇ 0.01, Mann-Whitney U test.
  • C, D Immunohistochemical staining decreased the number of M2 macrophages in Tbx1Cko myocardial infarction sites on day 7 after myocardial infarction, **, p ⁇ 0.01, Mann-Whitney U test.
  • Figure 7 shows a schematic diagram of the construction of the mouse Rosa26 STOP-Tbx1-ZG allele.
  • FIG. 8 shows that the mRNA expression levels of genes related to cell proliferation in cardiac endothelial cells were significantly increased in the Tbx1 overexpression (Tbx1OE) group compared with the control group, ***P ⁇ 1x10 -3
  • FIG. 9 shows that overexpression of Tbx1 in cardiac lymphatic endothelial cells improves myocardial repair after myocardial infarction.
  • C Masson staining showed cardiac scar tissue (blue) 60 days after myocardial infarction, and the heart was shown as a transverse section.
  • FIG. 10 shows that lymphatic endothelial overexpression of Tbx1 reduces the proportion of cytotoxic CD8 + T cells in the heart.
  • TBX1 protein in the lymphatic endothelial cells of cardiac lymphatic vessels can promote the proliferation of lymphatic endothelial cells.
  • TBX1 can also play an immunoregulatory role, promote the establishment of an immunosuppressive microenvironment in the myocardium after myocardial infarction, inhibit the autoimmune response after cardiac tissue injury, alleviate the concurrent cardiac tissue inflammation, and promote the repair and regeneration of damaged cardiac tissue.
  • the term “about” may refer to a value or composition within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.
  • the expression “about 100” includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the terms "containing” or “including (including)” can be open, semi-closed, and closed. In other words, the term also includes “consisting essentially of,” or “consisting of.”
  • the terms "subject”, “subject in need” refer to any mammal or non-mammalian. Mammals include, but are not limited to, humans, vertebrates such as rodents, non-human primates, cattle, horses, dogs, cats, pigs, sheep, goats.
  • the present invention provides an active ingredient that can be used to repair cardiac tissue damage, the active ingredient comprising: TBX1 protein, its coding sequence, or its promoter, or its combination.
  • the TBX1 gene encodes a T-box transcription factor and is located in the 22q11.2 region of human chromosomes, and is the main causative gene for 22q11.2 microdeletion syndrome.
  • the incidence of this disease in newborns is as high as 1:2000. 75% of children have congenital heart disease, which is accompanied by birth defects such as abnormal head and face development and absence of thymus.
  • Tbx1 is expressed in a subset of cardiac progenitor cells with differentiation potential and is also critical for the development of the cardiac lymphatic system, however, Tbx1 expression in the heart is extremely low in adulthood.
  • the gene ID of the TBX1 gene in the NCBI database is 6899, and its expressed mRNA sequence has 4 isoforms: NM_005992.1, NM_080646.2, NM_080647.1 and NM_001379200.1.
  • the gene ID of the mouse Tbx1 gene in the NCBI database is 21380, and its expressed mRNA sequence has 4 isoforms: NM_011532.2, NM_001285472.1, NM_001285476.1 and NM_001373938.1.
  • the coding sequence for a TBX1 protein can be any of the above sequences or variants thereof, so long as the sequence is capable of efficiently expressing a normal or increased TBX1 protein. The function is to promote lymphatic regeneration and the formation of an immunosuppressive microenvironment.
  • the coding sequence of the TBX1 protein is shown in SEQ ID NO.1.
  • the coding sequence of the Tbx1 protein is the CDS sequence used in the examples:
  • cardiac tissue damage refers to cardiac tissue damage including cardiomyocyte degeneration, necrosis, and cardiac angiolymphatic degeneration and necrosis. Inflammation and an autoimmune response can occur when heart tissue is damaged.
  • TBX1 protein, TBX1 mRNA or its promoter can be used to repair cardiac tissue damage. After overexpression of TBX1 in cardiac lymphatic vessels, it can not only promote the proliferation of lymphatic endothelial cells, but also regulate autoimmune responses and alleviate cardiac inflammation, which can be used to treat cardiac diseases related to cardiac tissue damage.
  • the "cardiac disease associated with cardiac tissue damage” includes ischemia, acute myocardial damage caused by hypoxia, autoimmune heart disease, and other diseases that cause myocardial damage.
  • the cardiac disease associated with cardiac tissue damage is selected from the group consisting of myocardial infarction, myocarditis, idiopathic dilated cardiomyopathy, Chagas' cardiomyopathy, rheumatic heart disease, novel coronavirus pneumonia ( heart damage due to COVID-19), or a combination thereof.
  • the present invention provides an expression vector for expressing TBX1 protein, which contains the coding sequence of the TBX1 protein of the present invention.
  • sequence information the skilled artisan can use available cloning techniques to generate nucleic acid sequences or vectors suitable for transduction into cells.
  • the expression vector is selected from the group consisting of DNA, RNA, plasmid, eukaryotic expression vector, prokaryotic expression vector, lentiviral vector, adenovirus vector, adeno-associated virus vector, retroviral vector, transfection seat, or a combination thereof.
  • the nucleic acid sequence comprising the TBX1 protein is provided as a vector, preferably as an expression vector.
  • it can be provided as a gene therapy vector preferably suitable for transduction and expression in target cells (eg cochlear supporting cells).
  • Vectors can be viral or non-viral (eg, plasmids).
  • Viral vectors include those derived from adenovirus, adeno-associated virus (AAV) including mutated forms, retrovirus, lentivirus, herpes virus, vaccinia virus, MMLV, GaLV, simian immunodeficiency virus (SIV) , HIV, poxvirus and SV40.
  • the viral vector is replication deficient, or may be replication deficient, replication capable or conditionally replicable.
  • Viral vectors can generally remain in an extrachromosomal state without integrating into the target cell's genome.
  • a preferred viral vector for introducing into target cells the nucleic acid sequence encoding the TBX1 protein is the AAV vector.
  • Selective targeting can be achieved using specific AAV serotypes (AAV serotype 2 to AAV serotype 12) or modified versions of any of these serotypes.
  • Viral vectors have the ability to enter cells.
  • non-viral vectors such as plasmids can be complexed with agents to facilitate uptake of the viral vector by target cells.
  • agents include polycationic agents.
  • delivery systems such as liposome-based delivery systems can be used.
  • the carrier for use in the present invention is preferably suitable for use in vivo or in vitro, and preferably for use in humans.
  • the vector will preferably contain one or more regulatory sequences to direct expression of the nucleic acid sequence in the target cell. Regulatory sequences can include promoters, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, and homologous recombination sites operably linked to the nucleic acid sequence.
  • the vector may also include a selectable marker, eg, to determine the expression of the vector in a growth system (eg, bacterial cells) or in target cells.
  • operably linked means that a nucleic acid sequence is functionally related to the sequences to which it is operably linked, such that they are linked in a manner such that they affect the expression or function of each other.
  • a nucleic acid sequence operably linked to a promoter will have an expression pattern affected by the promoter.
  • the present invention also provides a host cell for expressing TBX1 protein.
  • Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, T cells, NK cells, etc.
  • the present invention provides a pharmaceutical preparation or composition containing (a) the active ingredient described in the second aspect of the present invention, or the carrier described in the third aspect of the present invention, or the fourth aspect of the present invention the host cell, and (b) a pharmaceutically acceptable carrier, excipient or diluent.
  • the pharmaceutical preparation is used to repair cardiac tissue damage.
  • the pharmaceutical preparation is used to treat cardiac diseases related to cardiac tissue damage.
  • the pharmaceutical preparation is used to treat diseases related to abnormal lymphatic vessels.
  • the “active ingredient” in the pharmaceutical preparation of the present invention refers to the TBX1 protein, its coding sequence, or its promoter, or a combination thereof, or the expression vector of the present invention, such as a viral vector.
  • the "active ingredients", preparations and/or compositions of the present invention can be used to treat cardiac diseases and/or lymphatic vessel abnormalities related diseases associated with cardiac tissue damage.
  • a “safe and effective amount” refers to an amount of the active ingredient sufficient to significantly improve the condition or symptoms without causing serious side effects.
  • “Pharmaceutically acceptable carrier or excipient” means: one or more compatible solid or liquid filler or gel substances, which are suitable for human use and which must be of sufficient purity and sufficiently low toxicity .
  • “Compatibility” as used herein means that the components of the composition can be blended with the active ingredients of the present invention and with each other without significantly reducing the efficacy of the active ingredients.
  • the formulations and/or compositions of the present invention may also contain BMP4, CCL28, anti-CD8 antibodies and other active ingredients that are functionally similar to TBX1 molecules.
  • the formulations and/or compositions may be used in combination with other drugs known in the art for the treatment of cardiac disease and/or lymphatic vessel abnormalities associated with cardiac tissue damage.
  • compositions may be liquid or solid, such as powders, gels or pastes.
  • the composition is a liquid, preferably an injectable liquid. Suitable excipients will be known to those skilled in the art.
  • the carrier is preferably provided as an injectable liquid.
  • the injectable liquid is provided as a capsule or syringe.
  • Examples of pharmaceutically acceptable carrier moieties include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid) , magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween) ), wetting agents (such as sodium lauryl sulfate), colorants, flavors, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • cellulose and its derivatives such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.
  • gelatin such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate
  • compositions may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • the present invention also provides a method for screening a promoter of TBX1 protein, the method comprising the steps of: administering a candidate drug to lymphatic endothelial cells cultured in vitro and/or experimental animals, if the lymphatic endothelial cells cultured in vitro or experimental animals.
  • the TBX1 mRNA level, TBX1 protein level or TBX1 protein function of the lymphatic vessel tissue in the animal is increased compared to the control group not administered the candidate drug, so the candidate drug can act as a promoter of TBX1 protein.
  • the "drug candidates” include, but are not limited to, small molecule compounds, nucleic acid drugs, or protein drugs.
  • the drug candidates can directly act on TBX1 or indirectly act on TBX1, but ultimately increase the TBX1 protein function, level or TBX1 mRNA level in lymphatic endothelial cells cultured in vitro or in lymphatic tissue in experimental animals.
  • the TBX1 protein promoter can be used to treat cardiac diseases related to cardiac tissue damage.
  • the present invention proposes a control method for this pathological mechanism for the first time, and is expected to bring a new treatment method to heart disease caused by this related pathological mechanism.
  • the present invention proposes a brand-new mechanism of endogenous repair and regeneration of the heart.
  • the active ingredient (or combination) of the present invention strengthens the repair and regeneration of the heart after injury from two different levels: inhibiting autoimmune responses and promoting lymphatic vessel proliferation, So it has a good effect. It provides a new strategy and a new solution for the regeneration and repair of human myocardial infarction injury.
  • the active ingredient (or combination) of the present invention can also create favorable conditions for cell transplantation to repair and regenerate myocardial infarction by inhibiting the favorable microenvironment created by autoimmune responses.
  • mice After tracheal intubation, the mice were connected to a ventilator (Harvard apparatus). The anesthesia concentration of isoflurane was maintained at 3%-5%, the ventilation frequency was 100 times/min, and the tidal volume was 350ul. The left anterior descending coronary artery of the mouse was ligated to induce myocardial infarction. . The sham operation group was the same as the experimental group except that the coronary artery was not ligated. After the animals were awake, buprenorphine (0.1 mg/kg) was injected subcutaneously immediately, and then again after 24 hours.
  • buprenorphine 0.1 mg/kg
  • Tbx1 Cardiac lymphatic endothelial cell-specific knockout of Tbx1 results in impaired repair after myocardial infarction
  • Tbx1 lymphatic endothelial cell-specific knockout mice were constructed (Fig. 2A), and myocardial infarction was induced in both the control and knockout groups. Compared with controls, Tbx1-null mice had worse repair, severe tissue adhesions, and ventricular aneurysm formation after MI (Fig. 2B). Histological examination showed that inflammation resolution was slowed in the heart (Fig. 2C). Cardiac ejection was reduced and left ventricular end-diastolic volume increased ( Figures 2D-2E). Cytological examination revealed marked impairment of cardiac lymphangiogenesis ( Figures 2F-2G).
  • Endothelial transcriptome sequencing (Fig. 3A) and immunofluorescence staining (Fig. 3B) showed that Tbx1 deletion can significantly alter the expression of lymphangiogenesis genes: in the heart one week after myocardial infarction, the genes Dtx1, Dtx3, Sema4c, Gene expression such as Foxc2 was decreased in cardiac endothelial cells. At the same time, the gene Notch1, which inhibits lymphatic endothelial growth, was elevated (Fig. 3C).
  • Tbx1 regulates autoimmune responses after myocardial infarction
  • the endothelium-specific transcriptome showed that Tbx1 deletion resulted in a significant decrease in the levels of mRNAs that regulate immune function, such as Icam1, Ccl21, and Ccl28, in endothelial cells.
  • Icam1 is a cell surface adhesion protein that plays an important role in leukocyte migration and inhibiting leukocyte autoimmune activity.
  • Ccl21 and Ccl28 are cytokines that regulate the immune microenvironment. High concentrations of Ccl21 and Ccl28 can attract cells with immunosuppressive functions such as regulatory T cells into the inflammatory site and reduce autoimmune responses.
  • Tbx1 Loss of Tbx1 leads to increased numbers of inflammatory macrophages after myocardial infarction
  • Tbx1 Lymphatic endothelial overexpression of Tbx1 activates lymphatic endothelial cell proliferation
  • Lymphatic endothelial cells are more likely to play an important role in the repair of myocardial infarction injury.
  • Tbx1 drives the transcriptome changes of lymphatic endothelial cells, which can not only promote the regeneration of cardiac lymphatic vessels, but also endow them with additional immune regulation, thereby inhibiting the autoimmune response after myocardial infarction.
  • the Rosa26 Stop-Tbx1-ZG allele was first constructed (Tbx1OE mice, Figure 7). Lymphatic vessel-specific Prox1Cre strain mice were crossed with Tbx1OE mice to obtain double heterozygous mice. Infusion of Tamoxifen induces lymphatic endothelial Tbx1 overexpression in adult two-heterozygous mice. Transcriptome sequencing technology was used to analyze the changes of gene expression profiles of lymphatic endothelial cells in the heart, and to detect the molecular biological effects and potential target genes of enhanced Tbx1 expression.
  • Lymphatic vessel-specific Prox1Cre strain mice were crossed with Tbx1OE mice to obtain double heterozygous mice.
  • the overexpression of Tbx1 in the lymphatic endothelium was induced by perfusion of Tamoxifen in adult two-heterozygous mice, followed by coronary ligation to model myocardial infarction one week later. Cardiac function was measured by echocardiography 7, 14, and 60 days after modeling.
  • Lymphatic endothelial overexpression of Tbx1 reduces the proportion of cytotoxic CD8+ T cells in the heart
  • Lymphatic vessel-specific Prox1Cre strain mice were crossed with Tbx1OE mice to obtain double heterozygous mice.
  • the overexpression of Tbx1 in the lymphatic endothelium was induced by perfusion of Tamoxifen in adult two-heterozygous mice, followed by coronary ligation to model myocardial infarction one week later.
  • CD8 + T cell subset numbers were analyzed by flow cytometry on day 7 after modeling.
  • lymphatic endothelial cells are more likely to play an important role in the repair of myocardial infarction injury.
  • Tbx1 drives the transcriptome changes of lymphatic endothelial cells, which can not only promote the proliferation of lymphatic endothelial cells, but also endow them with additional immune regulation, thereby inhibiting the autoimmune response after myocardial infarction.
  • Tbx1 has the functions of regulating the regenerative microenvironment and coordinating and promoting the repair and regeneration of myocardial infarction in cardiac lymphatic endothelial cells.
  • Tbx1 can assist in the repair of injury after myocardial infarction through lymphatic endothelial cells.
  • Another feature of the present invention is to reveal for the first time an immune regulation repair regeneration mechanism for the initial stage of myocardial infarction repair.
  • This mechanism has a special stage and cell type specificity, which is different from the previous immune intervention in myocardial infarction repair.
  • Regeneration Research Previous studies have shown that indiscriminate inflammation suppression is not conducive to myocardial infarction repair, and current clinical studies of immune intervention in the treatment of myocardial infarction have not shown a clear therapeutic benefit. Therefore, the present invention will provide a new dimension for the orderly regulation mechanism of repair and regeneration after myocardial infarction, and by enhancing this new endogenous repair and regeneration mechanism, a new strategy and a new scheme for promoting regeneration and repair after myocardial infarction injury are formed.
  • the good microenvironment created by the inhibition of autoimmune response can also create good conditions for cell transplantation to repair and regenerate myocardial infarction.
  • the heart is an autoimmune-prone organ.
  • Certain cardiomyocyte-specific proteins are usually not expressed in the thymus, so that autoreactive T cells targeting cardiomyocyte-specific proteins cannot be cleared by central tolerance mechanisms and are thus released into the peripheral blood.
  • Myocardial autoantigens are released after cardiac injury, thereby activating autoreactive T cells, leading to an autoimmune response.
  • other diseases that lead to myocardial damage such as myocarditis, some idiopathic dilated cardiomyopathy, Chagas' cardiomyopathy, and rheumatic heart disease, are all related to persistent autoimmunity. Reaction related. Therefore, the research of the present invention suggests that overexpression of Tbx1 can also be used to treat other autoimmune-related cardiac diseases besides myocardial infarction.
  • the mechanism presented in the present invention is mainly based on the cardiac injury model
  • the genes regulated by Tbx1 and the lymphatic endothelial growth-related genes are all genes commonly expressed by lymphatic endothelial cells.
  • a similar function is also found in endothelial cells of the ductal endothelium. Therefore, similar mechanisms can be used to treat disorders related to lymphatic abnormalities in various tissues, such as primary lymphedema due to lymphatic dysplasia, secondary lymphedema due to rupture or obstruction of lymphatic vessels, Turner syndrome, Lymphangioedema presented by genetic syndromes such as yellow nail syndrome and Hennekam syndrome.

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Abstract

一种活性成分在修复心脏组织损伤中的用途。具体地，一种可用于修复心脏组织损伤和治疗淋巴管异常相关疾病的活性成分，所述活性成分包括：TBX1蛋白、其编码基因、或其促进剂、或其组合。TBX1蛋白可促进心脏淋巴管内皮细胞增生，发挥免疫调节作用，促进心脏组织损伤后心肌内免疫抑制微环境的建立，抑制心脏组织损伤后的自身免疫反应，缓解并发的心脏组织炎症，促进损伤的心脏组织修复再生。

Description

增强TBX1表达在修复心脏组织损伤中的用途 技术领域
本发明涉及心血管疾病治疗领域，尤其涉及TBX1在修复心脏组织损伤中的用途。
背景技术
心血管疾病在我国是首要致死原因，且近年来死亡率仍呈上升态势。哺乳动物的心肌细胞仅在出生后短暂的窗口期内具有增殖能力，心肌成熟后便退出细胞周期，因此，心肌坏死通常不可逆，预后差，会导致严重后果，例如，高达60％的缺血性心肌梗死病人在初次发病后六年内会发展成心脏衰竭。目前，针对心梗治疗主要通过血管再通减少心肌缺血坏死，以及通过药物调控神经体液的调节机制减缓心室的不良重塑。然而，这些治疗方法不能完全阻断心梗后的心室重塑，相当一部分病人在没有继续缺血损伤的情况下在较短的时间内进展到心脏衰竭。
因此本领域急需发展出新的心脏疾病疗法。
发明内容
本发明的目的在于提供一种新的有效治疗与心脏损伤相关的心脏疾病和/或淋巴管异常相关疾病的活性成分及其用途。
本发明的第一方面，提供了一种活性成分的用途，用于制备一制剂或药物，所述的制剂或药物用于：
(i)激活心脏淋巴管内皮细胞增生；
(ii)抑制心脏自身免疫反应；
(iii)促进M1型巨噬细胞向M2型巨噬细胞转化；
(iv)修复心脏组织损伤；
(v)治疗与心脏组织损伤相关的心脏疾病；和
(vi)治疗淋巴管异常相关疾病；
其中，所述活性成分包括：TBX1蛋白、其编码序列、或其促进剂、或其组合。
在另一优选例中，所述的编码序列包括DNA、cDNA、mRNA。
在另一优选例中，所述的“促进M1型巨噬细胞向M2型巨噬细胞转化”包括：促进心肌组织中M1型巨噬细胞(促进炎症反应)向M2型巨噬细胞(促进组织修复)转化。
在另一优选例中，所述的“与心脏组织损伤相关的心脏疾病”是与免疫反应(尤其是自身免疫反应)相关的心脏疾病。
在另一优选例中，所述与心脏组织损伤相关的心脏疾病选自下组：心肌梗死、心肌炎、特发性扩张性心肌病、Chagas'心肌病、风湿性心脏病、新型冠状病毒肺炎(COVID-19)引起的心脏损伤、或其组合。
在另一优选例中，所述淋巴管异常相关疾病选自下组：淋巴管发育不良造成的原发性淋巴水肿、由于淋巴管破裂或阻塞造成的继发性淋巴水肿、Turner综合征、黄甲综合征、Hennekam综合征等遗传综合征所呈现的淋巴管水肿、或其组合。
本发明的第二方面，提供了一种TBX1蛋白或其编码序列或其检测试剂的用途，用于制备一诊断试剂或诊断试剂盒，所述的诊断试剂或诊断试剂盒用于对心脏组织损伤相关的心脏疾病或淋巴管异常相关疾病进行预后判断。
在另一优选例中，所述的检测试剂选自下组：引物、探针、芯片、抗体、或其组合。
在另一优选例中，与正常人群或对照相比，如果TBX1表达量和/或活性上升，则提示被诊断的对象的预后好。
在另一优选例中，与正常人群或对照相比，如果TBX1表达量和/或活性下降，则提示被诊断的对象的预后差。
在另一优选例中，所述被诊断的对象患有或疑似患有心脏组织损伤相关的心脏疾病或淋巴管异常相关疾病。
在另一优选例中，所述对心脏组织损伤相关的心脏疾病或淋巴管异常相关疾病进行预后的方法包括：提供一受试者样本，检测受试者样本中TBX1蛋白的表达水平T1，并与心肌梗死患者心脏淋巴管中TBX1蛋白的平均表达水平T0比较；
若T1大于T0，则表明所述受试者的预后好；
若T1小于T0，则表明所述受试者的预后差。
在另一优选例中，所述的对心脏组织损伤相关的心脏疾病或淋巴管异常相关疾病进行预后的方法包括：提供一受试者样本，检测受试者样本中是否有TBX1编码和/或调节序列变异，如果TBX1变异是导致TBX1功能丧失或下降的变异，则提示所述受试者的预后差；如果TBX1变异是导致TBX1功能增强的变异，则提示所述受试者的预后好。
在另一优选例中，所述TBX1功能是指促进淋巴管再生及免疫抑制微环境的形成。
在另一优选例中，所述的心脏组织损伤相关的心脏疾病包括心肌梗死。
在另一优选例中，所述受试者患有心肌梗死。
在另一优选例中，所述受试者为人类或非人类哺乳动物。
在另一优选例中，所述样本包括心脏组织样本、心脏细胞样本、淋巴管内皮细胞样本、淋巴液样本、外周血样本细胞、或其组合。
在另一优选例中，所述的样本包括细胞样本、DNA样本、RNA样本、或其组合。
在另一优选例中，所述的试剂盒还包括检测额外标志物的检测试剂，所述的额外标志物选自下组：Top2a、Mki67、Cenpe、Aurkb、或其组合。
在另一优选例中，这些额外标志物的表达水平(或表达量)上升，则提示TBX1变异是导致TBX1功能增强的变异。
在另一优选例中，与正常人群或对照相比，如果这些额外标志物的表达水平(或表达量)上升，则提示则提示所述被诊断的对象的预后好。
在另一优选例中，与正常人群或对照相比，如果这些额外标志物的表达水平(或表达量)下降，则提示所述被诊断的对象的预后差。
本发明的第三方面，提供了一种可用于修复心脏组织损伤和治疗淋巴管异常相关疾病的活性成分，所述活性成分包括：TBX1蛋白、其编码序列、或其促进剂、或其组合。
在另一优选例中，所述活性成分为TBX1蛋白的促进剂。
在另一优选例中，所述促进剂促进TBX1蛋白在心脏及机体其他部位的淋巴管内表达。
在另一优选例中，所述促进剂上调TBX1蛋白在心脏及机体其他部位的淋巴管内的功能。
本发明的第四方面，提供了一种表达载体，所述的表达载体含有用于表达TBX1蛋白的表达盒。
在另一优选例中，所述表达载体选自下组：DNA、RNA、质粒、真核表达载体、原核表达载体、慢病毒载体、腺病毒载体、腺相关病毒载体、逆转录病毒载体、转座子、或其组合。
在另一优选例中，所述的表达载体特异性感染淋巴管内皮细胞。
在另一优选例中，所述表达盒从5'端-3'端具有式I结构：
Z0-Z1-Z2    (I)
式中，
各“-”独立地为化学键或核苷酸连接序列；
Z0为无、或5'UTR序列；
Z1为编码TBX1蛋白的核苷酸序列；和
Z2为无、或3'UTR序列。
在另一优选例中，所述表达盒还包括在与TBX1蛋白的编码序列可操作性连接的淋巴管内皮特异性启动子。
在另一优选例中，各个核苷酸连接序列的长度为1-30nt，较佳地1-15nt，更佳地3-6nt。
在另一优选例中，所述编码TBX1蛋白的核苷酸序列来源于人或非人类哺乳动物。
在另一优选例中，所述核苷酸序列如SEQ ID NO.1所示。
在另一优选例中，所述核苷酸序列如SEQ ID NO.2所示。
本发明的第五方面，提供了一种宿主细胞，所述宿主细胞含有本发明第四方面所述的表达载体。
在另一优选例中，所述宿主细胞为真核细胞或原核细胞。
在另一优选例中，所述宿主细胞选自下组：细菌细胞、酵母细胞、或哺乳动物细胞。
在另一优选例中，所述宿主细胞为淋巴管内皮细胞。
在另一优选例中，所述宿主细胞为心脏淋巴管内皮细胞。
本发明的第六方面，提供了一种药物制剂，所述制剂含有(a)本发明第三方面所述的活性成分，或本发明第四方面所述的表达载体，或本发明第五方面所述的细胞，以及(b)药学上可接受的载体、赋形剂或稀释剂。
在另一优选例中，所述药物制剂的剂型选自下组：冻干制剂、液体制剂、或其组合。
在另一优选例中，所述药物制剂为注射液。
在另一优选例中，所述药物制剂还包含：额外的活性成分；
较佳地，所述的额外的活性成分包括：BMP4、CCL28、抗CD8抗体、或其组合。
本发明的第七方面，提供了一种本发明第三方面所述的活性成分，或本发明第四方面所述的表达载体，或本发明第五方面所述的细胞，或如本发明第六方面所述的药物制剂在制备用于治疗与心脏损伤相关的心脏疾病和/或淋巴管异常相关疾病的药物中的用途。
在另一优选例中，所述与心脏损伤相关的心脏疾病选自下组：心肌梗死、心肌炎、特发性扩张性心肌病、Chagas'心肌病、风湿性心脏病、新型冠状病毒肺炎(COVID-19)引起的心脏损伤、或其组合。
在另一优选例中，所述淋巴管异常相关疾病选自下组：淋巴管发育不良造成的原发性淋巴水肿、由于淋巴管破裂或阻塞造成的继发性淋巴水肿、Turner综合征、黄甲综合征、Hennekam综合征等遗传综合征所呈现的淋巴管水肿、或其组合。
本发明的第八方面，提供了一种筛选TBX1蛋白的促进剂的方法，包括步骤：将候选药物施用于体外培养的淋巴管内皮细胞和/或实验动物，若体外培养的淋巴管内皮细胞或实验动物体内淋巴管组织的TBX1mRNA水平或TBX1蛋白水平相对于未施用候选药物的对照组升高，或TBX1蛋白功能相对于未施用候选药物的对照组增强，则候选药物可以作为TBX1蛋白的促进剂。
在另一优选例中，所述TBX1蛋白的促进剂可用于治疗与心脏组织损伤相关的心脏疾病。
在另一优选例中，所述TBX1蛋白的促进剂可用于治疗淋巴管异常相关疾病。
本发明的第九方面，提供了一种治疗与心脏组织损伤相关的心脏疾病的方法，所述方法包括：将本发明第三方面所述的活性成分或本发明第六方面所述的药物制剂施用于有需要的对象。
在另一优选例中，所述有需要的对象患有与心脏组织损伤相关的心脏疾病。
在另一优选例中，所述与心脏损伤相关的心脏疾病选自下组：心肌梗死、心肌炎、特发性扩张性心肌病、Chagas'心肌病、风湿性心脏病、新型冠状病毒肺炎(COVID-19)引起的心脏损伤、或其组合。
本发明的第十方面，提供一种治疗淋巴管异常相关疾病的方法，所述方法包括：将本发明第三方面所述的活性成分或本发明第六方面所述的药物制剂施用于有需要的对象。
在另一优选例中，所述有需要的对象患有淋巴管异常相关的心脏疾病。
在另一优选例中，所述淋巴管异常相关疾病选自下组：淋巴管发育不良造成的原发性淋巴水肿、由于淋巴管破裂或阻塞造成的继发性淋巴水肿,Turner综合征、黄甲综合征、Hennekam综合征等遗传综合征所呈现的淋巴管水肿、或其组合。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了心梗后Tbx1在淋巴内皮激活表达。(A)心梗后3及7天Tbx1在心脏的表达。未损伤心脏(baseline，n＝2)、心梗后第3天(n＝2)、第4天(n＝2)及第7天(n＝6)。(B)心梗后第7天X-gal染色心脏切片Vegfr3免疫染色(n＝3)。显示Tbx1主要在淋巴管内皮细胞表达。(C)心梗后第14(n＝3)及28天(n＝3)整体心脏X-gal染色。dpMI：心梗后天数。
图2显示了内皮细胞特异性敲除Tbx1导致心梗后淋巴管再生异常及修复障碍。(A)Tbx1功能缺失小鼠构建示意图，功能性基因组序列在Cre的激活下被敲除；(B)心梗后28天心脏腹侧图。黑色箭头：左室前壁室壁瘤。绿色箭头：术后黏连；(C)心梗14天后心脏切片HE染色(n＝3)，绿色箭头：单核浸润炎症细胞，蓝色箭头：出血点；(D)心梗后对照组及Tbx1Cko组射血分数(EF)变化(n＝7)，数据显示为均值±标准误，*，P<0.05。Mann-Whitney U检验；(E)心梗后对照组及Tbx1Cko组左室舒张末期容积(LVEDV)变化(n＝7)，数据显示为均值±标准误，*，p<0.05，**，p<0.01，Mann-Whitney U检验。(F，G)Vegfr3染色显示心脏淋巴管内皮在心梗部位再生状况，心梗后7天心脏大体图及组织切片(F)，以及心梗部位及远端未受损部位淋巴管内皮数量统计(G)，**，P<0.01，Mann-Whitney U检验；(H)内皮特异转录组差异基因及用染色质免疫沉淀反应结合高通量测序法(ChIP-seq)分析Tbx1下游靶点基因；(I)Tbx1靶点基因功能分析。
图3显示了Tbx1缺失心脏中和淋巴管生长相关基因表达显著改变。(A)转录组分析表明与淋巴管生长相关基因Dtx1、Dtx3、Sema4c及Foxc2在心梗后心脏内皮细胞中显著下降。(B，C)免疫荧光染色分析心梗后第7天心脏中表达Dtx1和Notch1的淋巴内皮细胞在梗死区分布及计算定量。
图4显示了Tbx1缺失心脏中和免疫调控相关基因在淋巴管内皮细胞中表达显著改变。(A，B)免疫荧光染色分析心梗后第7天心脏中表达Ccl21、Ccl28及Icam1的淋巴内皮细胞在梗死区分布及计算定量。
图5显示了Tbx1缺失心脏中自身免疫性CD8+T细胞显著增加。(A)单细胞测序结果中CD8 +T细胞数量分布，**p＝3x10 -3，§§p＝5x10 -15，卡方检验；(B)T细胞亚群细胞增殖分析显示CD8 +T细胞具有高度增殖性，数据显示为均值±标准差，**，p<5.6x10 -14，§§p<4.3x10 -11，Student's t检验；(C，D)流式分析心梗后第7天内皮特异敲除(F-Cko)及淋巴内皮特异敲除(P-Cko)小鼠心脏中Cd8 +T细胞数目改变，数据显示为均值±标准误(n＝5)，*，p<0.05，**，p<0.01，Mann-Whitney U检验；(E，F)免疫荧光染色分析心梗后第7天心脏中CD8 +T细胞在梗死区分布及计算定量，数据显示为均值±标准误(n＝5)，*，p<0.05，Mann-Whitney U检验；(G)流式分析CD8 +T细胞中分泌的细胞杀伤因子，数据显示为均值±标准误(n＝3)，n.s.，统计没有显著意义，Mann-Whitney U检验；(H，I)流式分析Myh6多肽-MHC I四聚体复合物染色。心梗后第7天F-Cko组自身反应性CD8 +T细胞显著增多， 数据显示为均值±标准误(n＝4)，*，p<0.05，Mann-Whitney U检验；(J)流式分析阴性对照多肽-MHC I四聚体复合物染色心梗后样本在CD8 +T细胞中的阳性率，数据显示为均值±标准误(n＝4)，n.s.，统计没有显著意义，Mann-Whitney U检验。
图6显示了Tbx1缺失心脏中炎性M1巨噬细胞减少，具有修复作用M2巨噬细胞显著增加。(A,B)流式分析心梗后第7天Tbx1Cko心脏中巨噬细胞细胞数目改变,*，p<0.05，**，p<0.01，Mann-Whitney U检验。(C,D)免疫组化染色心梗后第7天Tbx1Cko心脏梗死部位M2巨噬细胞细胞数目减少，**，p<0.01，Mann-Whitney U检验。
图7显示了小鼠Rosa26 STOP-Tbx1-ZG等位基因构建示意图。
图8显示了Tbx1过表达(Tbx1OE)组和对照组相比，心脏内皮细胞中与细胞增殖相关基因的mRNA表达水平显著上升，***P<1x10 -3
图9显示了心脏淋巴管内皮细胞过表达Tbx1具有改善心梗后心肌修复的作用。(A，B)心脏超声检测对照组(n＝8)和Tbx1OE组(n＝10)心梗后射血分数(A)和左心室舒张末容积(B)，统计方法采用非参数检验Mann-Whitney U-test。n.s：无统计学差别，*P<0.05；(C)马森染色显示心梗后60天心脏瘢痕组织(蓝色)，心脏显示为横切面。(D)Tbx1OE组(n＝5)心脏缺血组织的相对面积明显小于对照组(n＝4)。统计方法采用非参数检验Mann-Whitney U-test,*P<0.05。
图10显示了淋巴管内皮过表达Tbx1降低心脏中细胞毒性作用CD8 +T细胞比例。(A,B)流式分析心梗后第7天Tbx1OE心脏中Cd8 +T细胞数目改变，数据显示为均值±标准误(n＝5)，*，p<0.05,Mann-Whitney U检验；PD-1 +TNFα -耗竭性CD +8T细胞数量在Tbx1OE组中明显增加,Foxp3 +CD25 +调节性CD8 +T细胞(Treg)数量在Tbx1OE组中明显升高。
具体实施方式
本发明人经过广泛而深入的研究，首次意外地研发了一种可用于治疗与心脏损伤相关的心脏疾病的活性成分。本发明人发现，在心脏淋巴管的淋巴管内皮细胞中过表达TBX1蛋白，可促进淋巴管内皮细胞增生。此外，TBX1还可以发挥免疫调控作用，促进心梗后心肌内免疫抑制微环境的建立，抑制心脏组织损伤后的自身免疫反应，缓解并发的心脏组织炎症，促进损伤的心脏组织修复再生。实验证明，在心肌梗死小鼠模型中，在心脏淋巴管内皮过表达TBX1，明显改善了心梗后的心脏功能，促进了心梗后心脏组织修复。在此基础上，完成了本发明。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整 个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
如本文使用的，术语“受试者”、“需要的对象”指任何哺乳动物或非哺乳动物。哺乳动物包括但不限于人类、脊椎动物诸如啮齿类、非人类灵长类、牛、马、狗、猫、猪、绵羊、山羊。
TBX1
本发明提供了一种可用于修复心脏组织损伤的活性成分，所述活性成分包括：TBX1蛋白、其编码序列、或其促进剂、或其组合。
TBX1基因编码T盒转录因子，位于人类染色体22q11.2区域，是22q11.2微缺失综合征的主要致病基因。该病在新生儿中发病率高达1:2000。75％患儿有先天性心脏病，伴有头面部发育异常、胸腺缺如等先天缺陷。在胚胎发育早期，Tbx1在一部分具有分化潜能的心脏祖细胞中表达，对心脏淋巴管系统的发育也至关重要，然而成年后Tbx1在心脏中的表达水平极低。
TBX1基因在NCBI数据库中的基因ID为6899，其表达的mRNA序列有4种变异体(isoform)：NM_005992.1、NM_080646.2、NM_080647.1和NM_001379200.1。小鼠Tbx1基因在NCBI数据库中的基因ID为21380，其表达的mRNA序列有4种变异体(isoform)：NM_011532.2、NM_001285472.1、NM_001285476.1和NM_001373938.1。如本文所用，TBX1蛋白的编码序列可以为上述任一序列或其变体，只要所述序列能够有效表达功能正常或增加的TBX1蛋白。所述功能为促进淋巴管再生及免疫抑制微环境的形成。
在另一优选例中，所述TBX1蛋白的编码序列如SEQ ID NO.1所示。
Figure PCTCN2021094932-appb-000001
Figure PCTCN2021094932-appb-000002
在另一优选例中，所述Tbx1蛋白的编码序列如实施例中所用CDS序列：
Figure PCTCN2021094932-appb-000003
Figure PCTCN2021094932-appb-000004
心脏组织损伤
如本文所用，术语“心脏组织损伤”是指包括心肌细胞变性、坏死以及心脏血管淋巴管变性、坏死在内的心脏组织损伤。心脏组织损伤后，可引起炎症以及自身免疫反应。
本发明发现，TBX1蛋白、TBX1mRNA或其促进剂可用于修复心脏组织损伤。在心脏淋巴管中过表达TBX1后，不仅可促进淋巴管内皮细胞增生，还可以调控自身免疫反应，缓解心脏炎症，从而可用于治疗与心脏组织损伤相关的心脏疾病。
所述“与心脏组织损伤相关的心脏疾病”包括缺血、缺氧引起的急性心肌损伤、自身免疫性心脏疾病以及其它一些导致心肌损伤的疾病。在另一优选例中，所述与心脏组织损伤相关的心脏疾病选自下组：心肌梗死、心肌炎、特发性扩张性心肌病、Chagas'心肌病、风湿性心脏病、新型冠状病毒肺炎(COVID-19)引起的心脏损伤、或其组合。
表达载体及宿主细胞
本发明提供了一种表达载体，用于表达TBX1蛋白，它含有本发明的TBX1蛋白的编码序列。
通过提供的序列信息，熟练的技术人员可以使用可用的克隆技术以产生适于 转导进入细胞的核酸序列或载体。
在另一优选例中，所述表达载体选自下组：DNA、RNA、质粒、真核表达载体、原核表达载体、慢病毒载体、腺病毒载体、腺相关病毒载体、逆转录病毒载体、转座子、或其组合。
优选地，包含编码TBX1蛋白的核酸序列作为载体，优选地作为表达载体被提供。优选地，其可作为优选地适用于在靶细胞(例如耳蜗支持细胞)中转导和表达的基因治疗载体被提供。载体可以是病毒的或非病毒的(例如质粒)。病毒载体包括源自以下的那些病毒载体:腺病毒、包括突变的形式的腺相关病毒(AAV)、逆转录病毒、慢病毒、疱疹病毒、牛痘病毒、MMLV、GaLV、猿猴免疫缺陷病毒(SIV)、HIV、痘病毒和SV40。优选地，病毒载体是复制缺陷的(replication defective)，或者可以是复制缺乏的(replication deficient)、能够复制或条件性复制的。病毒载体通常可以保持染色体外状态而不整合进入靶细胞的基因组。用于向靶细胞引入编码TBX1蛋白的核酸序列的优选的病毒载体是AAV载体。使用特定的AAV血清型(AAV血清型2到AAV血清型12)或这些血清型中的任何一个的修饰的版本可以实现选择性靶向。
病毒载体具有进入细胞的能力。然而，非病毒载体诸如质粒可与试剂复合以有利于病毒载体被靶细胞的摄取。此类试剂包括聚阳离子剂。可选地，递送系统诸如基于脂质体的递送系统可被使用。用于在本发明中使用的载体优选地适于在体内或体外使用，并且优选地适于在人类中使用。
载体将优选地包含一个或多个调节序列以指导核酸序列在靶细胞中的表达。调节序列可以包括与核酸序列可操作地连接的启动子、增强子、转录终止信号、多腺苷酸化序列、复制起点、核酸限制性位点、和同源重组位点。载体还可包括选择性标记，例如来确定载体在生长系统(例如细菌细胞)中或在靶细胞中的表达。
“可操作地连接”意指，核酸序列在功能上与其可操作地连接的序列相关，以使得它们以使得它们影响彼此的表达或功能的方式连接。例如，与启动子可操作地连接的核酸序列将具有被启动子影响的表达模式。
本发明还提供了一种宿主细胞，用于表达TBX1蛋白。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞、T细胞、NK细胞的动物细胞等。
药物制剂
本发明提供一种药物制剂或组合物，所述药物制剂或组合物含有(a)本发明第二方面所述的活性成分、或本发明第三方面所述的载体、或本发明第四方面所 述的宿主细胞，以及(b)药学上可接受的载体、赋形剂或稀释剂。
在另一优选例中，所述药物制剂用于修复心脏组织损伤。
在另一优选例中，所述药物制剂用于治疗与心脏组织损伤相关的心脏疾病。
在另一优选例中，所述药物制剂用于治疗淋巴管异常相关疾病。
本发明所述药物制剂中的“活性成分”是指TBX1蛋白、其编码序列、或其促进剂、或其组合，或本发明所述的表达载体，例如病毒载体。本发明所述的“活性成分”、制剂和/或组合物可用于治疗与心脏组织损伤相关的心脏疾病和/或淋巴管异常相关疾病。“安全有效量”指的是：活性成分的量足以明显改善病情或症状，而不至于产生严重的副作用。“药学上可接受的载体或赋形剂”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的活性成分以及它们之间相互掺和，而不明显降低活性成分的药效。
本发明的制剂和/或组合物还可以包含BMP4、CCL28、抗CD8抗体以及其它与TBX1分子功能类似的活性成分。所述制剂和/或组合物可与本领域已知的其它治疗与心脏组织损伤相关的心脏疾病和/或淋巴管异常相关疾病的药物组合使用。
组合物可以是液体或固体，例如粉末、凝胶或糊剂。优选地，组合物是液体，优选地可注射液体。合适的赋形剂将是本领域技术人员己知的。
在任一种施用模式中，优选地，载体作为可注射液体被提供。优选地，可注射液体作为胶囊或注射器被提供。
药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如吐温
Figure PCTCN2021094932-appb-000005
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。
组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。
筛选TBX1蛋白的促进剂的方法，
本发明还提供了一种筛选TBX1蛋白的促进剂的方法，所述方法包括步骤：将候选药物施用于体外培养的淋巴管内皮细胞和/或实验动物，若体外培养的淋巴管内皮细胞或实验动物体内淋巴管组织的TBX1mRNA水平、TBX1蛋白水平或TBX1蛋白功能相对于未施用候选药物的对照组升高，则候选药物可以作为TBX1蛋白的促进剂。
在本发明中，所述“候选药物”包括但不限于：小分子化合物、核酸药物、 或蛋白药物。所述候选药物可直接作用于TBX1或间接作用于TBX1，但最终都使得体外培养的淋巴管内皮细胞或实验动物体内淋巴管组织的TBX1蛋白功能、水平或TBX1mRNA水平升高。
在另一优选例中，所述TBX1蛋白的促进剂可用于治疗与心脏组织损伤相关的心脏疾病。
本发明的主要优点在于：
(1)心脏损伤后倾向于发生自身免疫的损伤，但目前针对这种病理机制没有很好的控制手段，导致在相当一部分病人在心脏损伤后导致心衰的发生。本发明首次提出针对这一病理机制的控制手段，有望给这一相关病理机制导致心脏疾病带来新的治疗方法。
(2)本发明提出了一种全新的心脏内源性修复再生机制，本发明的活性成分(或组合)从抑制自身免疫反应以及增进淋巴管增生两个不同层面加强心脏损伤后的修复再生，因此具有很好的效果。为人类心梗损伤后再生修复提供了新策略与新方案。
(3)此外，本发明的活性成分(或组合)通过抑制自身免疫反应创造的良好微环境，对细胞移植进行心梗修复再生也能创造良好的条件。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York：Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例中所用的材料、试剂、仪器等，如无特殊说明，均可从商业途径得到。
实验方法
(1)构建Rosa26-STOP-Tbx1/STOP-Tbx1(Tbx1OE)品系小鼠：将LoxP-STOP-LoxP-Tbx1cDNA-P2A-ZsGreen元件插入Rosa26基因位点，其中Tbx1cDNA为包含含有编码TBX1蛋白质的开放阅读框的核酸序列，如SEQ ID NO:2所示(图7)。在Cre介导的LoxP位点重组后，STOP元件被切除，Tbx1得以表达。该Rosa26-STOP-Tbx1/STOP-Tbx1(Tbx1OE)品系小鼠可健康存活。
(2)转录测序和分析：使用
Figure PCTCN2021094932-appb-000006
试剂(ThermoFisher science)提取RNA，并按照Illumina平台建库测序方法，使用1μg全RNA进行高通量测序。每个样本生成至少1500万条原始配对序列(paired-end reads)。使用STAR(2.5.2b，MIT license)将原始序列与小鼠基因组(Gencode，GRCm38，Open Access)进行比对。 基因表达水平以单个基因为单位进行统计。用R语言分析包DESeq2(1.20.0版，LGPL license≥3)鉴定差异表达的基因(鉴定标准按倍数变化≥1.5且p值≤0.05)。然后用Metascape(https://metascape.org/gp/index.html#/menu/term_service)对进行差异基因进行功能分析。
(3)心肌梗死动物模型：心肌梗死实验选用8-12周龄小鼠，方法参照之前的文献描述并稍作修改。简要实验步骤如下：
小鼠气管插管后连接呼吸机(Harvard apparatus)，异氟烷维持麻醉浓度为3％-5％，通气频率100次/分钟，潮气量350ul.结扎小鼠心脏冠状动脉左前降支诱发心肌梗死。假手术组除了没有结扎冠状动脉外其他手术步骤与实验组一致。动物苏醒后，迅速皮下注射丁丙诺啡(0.1mg/kg)，24小时后再注射一次。
(4)测量心脏功能：在心脏冠状动脉左前降支结扎7天、14天、28天后行经胸廓的心脏超声检测。使用Vevo 2100系统(Visual Sonics)，所用探头频率为30MHz，帧率为30帧/秒。测量是采集图像为左心室心动周期中的长轴图像横截面，使用以下公式计算左心室收缩末期容积(LVESV)，舒张末期容积(LVEDV)和左心室射血分数(LVEF)。
Figure PCTCN2021094932-appb-000007
Figure PCTCN2021094932-appb-000008
实施例1
心肌梗死后Tbx1在淋巴内皮激活表达
通过构建小鼠心梗模型诱导成年小鼠心梗后，Tbx1在心脏内皮细胞中表达量持续增高，且90％的Tbx1表达阳性细胞是淋巴管内皮细胞(图1A-1C)。
实施例2
心脏淋巴管内皮细胞特异性敲除Tbx1导致心梗后修复障碍
构建了Tbx1淋巴管内皮细胞特异性敲除小鼠(图2A)，同时诱导对照组和敲除组小鼠心梗。与对照组相比，Tbx1缺失小鼠在心梗后修复变差，组织粘连严重，室壁瘤形成(图2B)。组织学检验表明，心脏中炎症消退减慢(图2C)。心脏射血功能减低及左室舒张末期容积变大(图2D-2E)。细胞学检验发现心脏淋巴管再生明显障碍(图2F-2G)。
内皮转录组测序(图3A)及免疫荧光染色(图3B)显示，Tbx1缺失能影响淋巴管增生基因表达显著改变：在心梗后一周心脏,促进淋巴管内皮生长的基因Dtx1、 Dtx3、Sema4c、Foxc2等基因表达在心脏内皮细胞中下降。同时,抑制淋巴管内皮生长的基因Notch1达升高(图3C)。
实施例3
Tbx1调控心梗后自身免疫反应
用免疫沉淀反应结合高通量测序法(ChIP-seq)检测Tbx1作为转录因子在心脏内皮细胞基因组上的结合位点，与自身免疫耐受相关的基因明显富集，表明Tbx1可能在心梗后调控自身免疫反应(图2H-2I)。
内皮特异转录组显示，Tbx1缺失导致内皮细胞中与调控免疫功能的mRNA水平显著降低，例如Icam1、Ccl21、Ccl28等。Icam1是细胞表面黏附蛋白，对白细胞迁移，抑制白细胞自身免疫活性具有重要作用。Ccl21和Ccl28是调控免疫微环境的细胞因子，高浓度Ccl21和Ccl28能吸引调节性T细胞等具有免疫抑制功能的细胞进入炎症部位，减少自身免疫反应。同时，免疫荧光实验表明，小鼠心梗造模7天后,Tbx1缺失小鼠的淋巴管内皮细胞表达的Icam1、Ccl21和Ccl28的蛋白水平明显减少(图4A-B)。
单细胞RNA测序分析发现CD8 +T细胞在Tbx1缺失小鼠心脏中增殖活跃，细胞数目显著升高(图5A-5F)，而且具有与对照组CD8 +T细胞相同毒性(图5G)。MHC I四聚体染色进一步显示F-Cko组中针对α-心肌球蛋白的CD8 +T细胞显著升高(图5H-5J)，提示Tbx1敲除后导致心梗后自身免疫反应升高。
实施例4
Tbx1缺失导致心梗后炎症巨噬细胞数量增加
在成年Tbx1缺失小鼠中行冠脉结扎心梗建模。在建模后第7天用流式细胞分析巨噬细胞亚群数量。结果发现，高表达F4/80、低表达Ly6c的M2型巨噬细胞(这类细胞具有促进再生、介导炎症消退的功能)在Tbx1缺失组中明显减少；同时，低表达F4/80、高表达Ly6c的M1型巨噬细胞(这类细胞的富集通常表明组织处于炎症期，长期累积会导致慢性炎症及自身免疫反应)数量在Tbx1缺失组中显著增加(图6A-B)。
免疫组化染色也显示，心梗后第7天Tbx1缺失组的心脏梗死部位M2巨噬细胞细胞数目减少(图6C-D)。
实施例5
淋巴管内皮过表达Tbx1基因激活淋巴管内皮细胞增生
淋巴管内皮细胞更有可能作为心梗损伤修复的重要功能细胞发挥作用。在心梗急性炎症期向修复期转变阶段，Tbx1驱动淋巴管内皮细胞转录组改变，不仅能 促进心脏淋巴管的再生，而且可以赋予其额外的免疫调控作用，从而抑制心梗后自身免疫反应，防止自身反应性T细胞持续攻击自身组织，造成慢性炎症。因此，在本实施例中，以淋巴管内皮为靶细胞，通过过表达Tbx1，研究针对调控心脏免疫微环境来治疗心梗的可行性。
首先构建了Rosa26 Stop-Tbx1-ZG等位基因(Tbx1OE小鼠，图7)。淋巴管特异Prox1Cre品系小鼠与Tbx1OE小鼠杂交获得双杂合小鼠。在成年双杂合小鼠中灌注Tamoxifen诱导淋巴管内皮Tbx1过表达。通过转录组测序技术分析心脏中淋巴管内皮基因表达谱改变，检测增强Tbx1表达后的分子生物学效应及潜在靶点基因。
结果发现，在成年小鼠心脏淋巴管中激活Tbx1表达一周后，收集Tbx1OE小鼠心脏CD31+内皮细胞，行转录组分析。
结果显示，激活Tbx1能增强心脏内皮细胞中增殖相关基因的表达，例如，Top2a、Mki67、Cenpe、Aurkb等表达水平均增加2倍以上(n＝3，P<1x10 -5)(图8)。
实施例6
淋巴管内皮过表达Tbx1对心梗后修复的影响
淋巴管特异Prox1Cre品系小鼠与Tbx1OE小鼠杂交获得双杂合小鼠。在成年双杂合小鼠中灌注Tamoxifen诱导淋巴管内皮Tbx1过表达，一周后行冠脉结扎心梗建模。在建模后7、14、60天后用心脏超声法测量心脏功能。
结果表明，Tbx1过表达的小鼠在心梗2周后心脏射血分数显著优于对照组(图9A)，且可持续到心梗后2月，左心室舒张末期容积相应减少(图9B)。同时，组织学分析显示心脏心梗后60天Tbx1OE小鼠瘢痕组织面积明显减少(图9C-D)。
实施例7
淋巴管内皮过表达Tbx1降低心脏中细胞毒作用CD8+T细胞比例
淋巴管特异Prox1Cre品系小鼠与Tbx1OE小鼠杂交获得双杂合小鼠。在成年双杂合小鼠中灌注Tamoxifen诱导淋巴管内皮Tbx1过表达，一周后行冠脉结扎心梗建模。在建模后第7天用流式细胞术分析CD8 +T细胞亚群数量。
结果表明，表达细胞表面抗原PD-1的耗竭性CD +8T细胞数量在Tbx1OE组中明显增加，且Tbx1OE组中细胞因子TNFα的表达水平在CD8 +T中明显降低(图10A)。同时，具有免疫抑制功能的Foxp3 +CD25 +调节性CD8 +T细胞数量在Tbx1OE组中明显升高(图10B)。
讨论
心梗后急性炎症发生及损伤修复是自我驱动的复杂过程，包括了各类免疫细 胞、心肌细胞、血管内皮细胞及成纤维细胞等多种细胞类型参与，心梗后修复再生的研究大多围绕这些细胞类型开展。
本发明的结果提示，淋巴管内皮细胞更有可能作为心梗损伤修复的重要功能细胞发挥作用。在心梗急性炎症期向修复期转变阶段，Tbx1驱动淋巴管内皮细胞转录组改变，不仅可以促进淋巴管内皮细胞增殖，更能赋予其额外的免疫调控作用，从而抑制心梗后自身免疫反应，防止自身反应性T细胞持续攻击自身组织，造成慢性炎症。本发明首次意外地发现：Tbx1在心脏淋巴内皮细胞中具有调控再生微环境、协调促进心梗修复再生的作用。Tbx1可通过淋巴管内皮细胞，对心梗后损伤修复起到协助作用。
本发明的另一特色是首次揭示了针对心梗修复期起始阶段出现的免疫调控修复再生机制，这一机制具有特殊的阶段性和细胞类型特异性，有别于既往的免疫干预心梗修复再生研究。既往的研究表明不加选择的炎症抑制并不利于心梗修复，目前的免疫干预治疗心梗临床研究都没有显示出明确的治疗效益。因此，本发明将为心梗后修复再生的有序调控机制提供新的维度，通过增强这一全新内源性修复再生机制，形成促进心梗损伤后再生修复的新策略与新方案。此外，抑制自身免疫反应创造的良好微环境，对细胞移植进行心梗修复再生也能创造良好的条件。
心脏是有自身免疫倾向的器官。某些心肌细胞特异的蛋白通常在胸腺中不表达，使得针对心肌特异蛋白的自身反应性T细胞不能通过中枢耐受机制被清除，因而被释放到外周血液中。心脏损伤后心肌自身抗原被释放,从而激活自身反应性T细胞,导致自身免疫反应。除了缺血、缺氧引起的急性心肌损伤外,其它一些导致心肌损伤的疾病：例如心肌炎、一些特发性扩张性心肌病、Chagas'心肌病、以及风湿性心脏病等都与持续的自身免疫反应相关。因此，本发明的研究提示，过表达Tbx1也可用来治疗除心梗外的其它与自身免疫反应相关的心脏疾病。
同时，尽管本发明中呈现的机制主要基于心脏损伤模型，但是，Tbx1所调节的和淋巴管内皮生长相关基因都是淋巴管内皮细胞普遍表达的基因，这些Tbx1调控的下游基因在其他组织的淋巴管内皮内皮细胞中也有相似的功能。因此，相似的机制能用于治疗各组织的淋巴管异常相关疾病，例如，淋巴管发育不良造成的原发性淋巴水肿、由于淋巴管破裂或阻塞造成的继发性淋巴水肿，Turner综合征、黄甲综合征、Hennekam综合征等遗传综合征所呈现的淋巴管水肿。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种活性成分的用途，其特征在于，用于制备一制剂或药物，所述的制剂或药物用于：
    (i)激活心脏淋巴管内皮细胞增生；
    (ii)抑制心脏自身免疫反应；
    (iii)促进M1型巨噬细胞向M2型巨噬细胞转化；
    (iv)修复心脏组织损伤；
    (v)治疗与心脏组织损伤相关的心脏疾病；和
    (vi)治疗淋巴管异常相关疾病；
    其中，所述活性成分包括：TBX1蛋白、其编码序列、或其促进剂、或其组合。
  2. 如权利要求1所述的用途，其特征在于，所述与心脏组织损伤相关的心脏疾病选自下组：心肌梗死、心肌炎、特发性扩张性心肌病、Chagas'心肌病、风湿性心脏病、新型冠状病毒肺炎(COVID-19)引起的心脏损伤、或其组合。
  3. 如权利要求1所述的用途，其特征在于，所述淋巴管异常相关疾病选自下组：淋巴管发育不良造成的原发性淋巴水肿、由于淋巴管破裂或阻塞造成的继发性淋巴水肿、Turner综合征、黄甲综合征、Hennekam综合征等遗传综合征所呈现的淋巴管水肿、或其组合。
  4. 如权利要求1所述的用途，其特征在于，所述的“促进M1型巨噬细胞向M2型巨噬细胞转化”包括：促进心肌组织中M1型巨噬细胞(促进炎症反应)向M2型巨噬细胞(促进组织修复)转化。
  5. 一种TBX1蛋白或其编码序列或其检测试剂的用途，其特征在于，用于制备一诊断试剂或诊断试剂盒，所述的诊断试剂或诊断试剂盒用于对心脏组织损伤相关的心脏疾病或淋巴管异常相关疾病进行预后判断。
  6. 如权利要求5所述的用途，其特征在于，所述对心脏组织损伤相关的心脏疾病或淋巴管异常相关疾病进行预后的方法包括：提供一受试者样本，检测受试者样本中TBX1蛋白的表达水平T1，并与心肌梗死患者心脏淋巴管中TBX1蛋白的平均表达水平T0比较；
    若T1大于T0，则表明所述受试者的预后好；
    若T1小于T0，则表明所述受试者的预后差。
  7. 如权利要求5所述的用途，其特征在于，所述的对心脏组织损伤相关的心脏疾病或淋巴管异常相关疾病进行预后的方法包括：提供一受试者样本，检测受试者样本中是否有TBX1编码和/或调节序列变异，如果TBX1变异是导致TBX1功能丧失或下降的变异，则提示所述受试者的预后差；如果TBX1变异是导致 TBX1功能增强的变异，则提示所述受试者的预后好。
  8. 一种可用于修复心脏组织损伤和治疗淋巴管异常相关疾病的活性成分，其特征在于，所述活性成分包括：TBX1蛋白、其编码序列、或其促进剂、或其组合。
  9. 一种表达载体，其特征在于，所述的表达载体含有用于表达TBX1蛋白的表达盒。
  10. 如权利要求9所述的表达载体，其特征在于，所述表达盒从5'端-3'端具有式I结构：
    Z0-Z1-Z2  (I)
    式中，
    各“-”独立地为化学键或核苷酸连接序列；
    Z0为无、或5'UTR序列；
    Z1为编码TBX1蛋白的核苷酸序列；和
    Z2为无、或3'UTR序列。
  11. 一种宿主细胞，其特征在于，所述宿主细胞含有如权利要求9或10所述的表达载体。
  12. 一种药物制剂，其特征在于，所述制剂含有(a)如权利要求8所述的活性成分，或如权利要求9或10所述的表达载体，或如权利要求11所述的细胞，以及(b)药学上可接受的载体、赋形剂或稀释剂。
  13. 如权利要求12所述的药物制剂，其特征在于，述药物制剂还包含：额外的活性成分；
    所述的额外的活性成分包括：BMP4、CCL28、抗CD8抗体、或其组合。
  14. 一种如权利要求8所述的活性成分，如权利要求9或10所述的表达载体，如权利要求11所述的宿主细胞，或如权利要求12或13所述的药物制剂在制备用于治疗与心脏损伤相关的心脏疾病和/或淋巴管异常相关疾病的药物中的用途。
  15. 一种筛选TBX1蛋白的促进剂的方法，其特征在于，包括步骤：
    将候选药物施用于体外培养的淋巴管内皮细胞和/或实验动物，若体外培养的淋巴管内皮细胞或实验动物体内淋巴管组织的TBX1 mRNA水平或TBX1蛋白水平相对于未施用候选药物的对照组升高，或TBX1蛋白功能相对于未施用候选药物的对照组增强，则候选药物可以作为TBX1蛋白的促进剂。
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