WO2018028369A1 - Tnfr2的用途 - Google Patents

Tnfr2的用途 Download PDF

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WO2018028369A1
WO2018028369A1 PCT/CN2017/092338 CN2017092338W WO2018028369A1 WO 2018028369 A1 WO2018028369 A1 WO 2018028369A1 CN 2017092338 W CN2017092338 W CN 2017092338W WO 2018028369 A1 WO2018028369 A1 WO 2018028369A1
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tnfr2
bmx
stem cells
cardiac stem
cells
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PCT/CN2017/092338
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English (en)
French (fr)
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王敏
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中山大学附属第一医院
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Priority claimed from CN201710530538.7A external-priority patent/CN107449913A/zh
Priority claimed from CN201710530130.XA external-priority patent/CN107441491A/zh
Application filed by 中山大学附属第一医院 filed Critical 中山大学附属第一医院
Publication of WO2018028369A1 publication Critical patent/WO2018028369A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

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  • the invention relates to the use of TNFR2, in particular to the use of TNFR2 as a target and marker.
  • Cardiovascular disease is one of the most serious diseases that threaten human life worldwide.
  • acute myocardial infarction is the most serious disease in coronary heart disease, with high morbidity and mortality.
  • Myocardial infarction causes a decrease in the number of irreversible functional myocardial cells, resulting in a decrease in myocardial contractile function.
  • the traditional treatment methods for myocardial infarction mainly include drug treatment, interventional therapy and surgical treatment. Although it can improve the symptoms of myocardial ischemia, it can not save the necrotic cardiomyocytes.
  • Stem cells are a kind of cells with self-renewal and multi-directional differentiation potential.
  • CSCs cardiac stem cells
  • the activator known in English as activator, is known as an activator for substances that increase enzyme activity, most of which are ions or simple organic compounds.
  • a ligand any molecule that binds to an anchored protein, is referred to as a ligand, and the ligand is one of the activators.
  • the ligand In receptor-mediated endocytosis, the ligand binds to the cytoplasmic membrane receptor protein and is finally the ligand that is engulfed in the cell.
  • the ligands are divided into four categories: I. Nutrients, such as transferrin, low density lipoprotein (LDL), etc.; II. Harmful substances, such as certain Bacteria; III. Immunological substances, such as immunoglobulins, antigens, etc.; IV. Signal substances, such as insulin and other peptide hormones.
  • TNF- ⁇ is a type II membrane protein secreted mainly by activated mononuclear macrophages and functions as a trimer. It has both soluble TNF-[alpha] and membrane-associated TNF-[alpha] forms. TNF- ⁇ exerts its biological function by binding to the receptor TNFR. There are also two types of TNFR: TNFR1 and TNFR2, both of which are type I membrane proteins. Soluble TNF- ⁇ plays an important role in regulating the inflammatory response and regulating cell survival or death.
  • TNF- ⁇ After TNF- ⁇ binds to its receptor, it first recruits the TRA receptor (TNF receptor-associated death domain) in the cell to bind to the intracellular portion of the TNF- ⁇ receptor, and then activates another two receptor-related proteins, TRAF2 (TNF receptor- Associated factor 2) and FADD (fas-associated deathdomain), thereby activating multiple signaling pathways, such as NF- ⁇ B, JNK, MAPK, and apoptotic signaling pathways to achieve TNF- ⁇ regulation.
  • TRA receptor TNF receptor-associated death domain
  • TRAF2 TNF receptor-associated factor 2
  • FADD fas-associated deathdomain
  • TNFR2 tumor necrosis factor receptor-2
  • TNFR2 can pass -Bmx (bone marrow non-receptor) Tyrosine kinase in X-chromosome) is a bone marrow non-receptor tyrosine kinase on the X chromosome that mediates cell survival, proliferation and migration.
  • TNFR2-Bmx signaling pathway may play an important role in the activation and survival of endogenous CSCs in ischemic myocardium. Accordingly, it is an object of the present invention to provide various uses of TNFR2.
  • the present invention adopts the technical solution of using TNFR2 as a target for screening or preparing a drug for promoting activation, migration or survival of cardiac stem cells.
  • the present invention provides the use of TNFR2 as a target for screening or preparing a drug that promotes differentiation of cardiac stem cells into cardiomyocytes or promotes myocardial repair.
  • the present invention provides the use of TNFR2 as a target for screening or preparing a medicament for treating ischemic heart disease in humans.
  • the invention provides the use of a TNFR2 activator for the manufacture of a medicament for promoting activation, migration or survival of cardiac stem cells.
  • the present invention provides the use of a TNFR2 activator for the preparation of a medicament for promoting differentiation of cardiac stem cells into cardiomyocytes or promoting myocardial repair.
  • the present invention provides the use of a TNFR2 activator for the preparation of a medicament for the treatment of ischemic heart disease in humans.
  • the TNFR2 activator comprises a TNFR2 specific ligand.
  • the TNFR2 specific ligand is R2-TNF.
  • the invention provides the use of a TNFR2 inhibitor or a TNFR2 antagonist for the preparation of a medicament for inhibiting cardiomyocyte differentiation.
  • the invention provides the use of TNFR2 as a marker in the preparation of a reagent or kit for screening or recognizing cardiac stem cells.
  • the beneficial effects of the present invention are that the TNFR2 activator of the present invention activates TNFR2-Bmx, thereby enhancing the expression and activity of Nkx2.5+/Gata4+, mediating activation and migration of c-Kit+ endogenous cardiac stem cells (eCSCs). And the role of survival, and activation of TNFR2, increase the differentiation of hESC/hiPSC-derived cardiomyocytes, facilitate the repair of myocardial infarction, provide a new strategy for the treatment of human heart disease; TNFR2 inhibitor or TNFR2 of the present invention Antagonists can inhibit cardiomyocyte overactivity Differentiation.
  • the present invention provides a cardiac stem cell marker, TNFR2 as a new biomarker of cardiac stem cells, providing a new molecular marker for isolating and recognizing endogenous cardiac stem cells.
  • Figure 1 shows the expression profiles of TNF receptor molecules and downstream effector molecules in human normal and ischemic hearts;
  • A is the immunofluorescence staining of sections, TNFR1/ ⁇ -SA, TNFR2/ ⁇ -SA and Bmx/ ⁇ -SA, respectively.
  • FIG. 2 shows the distribution of cardiac precursor cells in human normal and ischemic hearts
  • A is the immunofluorescence staining of Gata4, NKx2.5 and ⁇ -SA in human normal heart and ischemic heart tissue sections, and the arrow indicates the expression of Gata4 in the nucleus of cardiomyocytes
  • C For human heart and ischemic heart, the cardiac precursor cells are labeled with NKx2.5, Gata4 and TNFR2, and the arrows point to double positive cells with a magnification of 40 and a scale of 50 ⁇ m;
  • Figure 3 is a flow chart showing the method for obtaining iPSC-CSC by using the method of GiWi-heart stem cell differentiation; A.GiWi-heart stem cell differentiation method; B.QPCR detecting the expression of TNFR2, GATA4 and ISL1 in differentiated iPSC-CSC, vertical Coordinates indicate the percentage of target gene relative to internal reference (GAPDH) expression, abscissa indicates hESCs cell line and differentiated CSC cells; C.
  • GPDH internal reference
  • iPSC-CSC immunofluorescence staining images of TNFR2, NKX2.5 and GATA4 (10 ⁇ ); Immunofluorescence staining of TNFR2 and tropomyosin in iPSC-CSC (63 ⁇ ); immunofluorescence staining of NKX2.5 and tropomyosin in E.iPSC-CSC (63 ⁇ ); NKX2 in F.iPSC-CSC .5, immunofluorescence staining of TNFR2 and tropomyosin (63 ⁇ ); immunofluorescence staining of Ki67, TNFR2 and tropomyosin in G.iPSC-CSC (63 ⁇ );
  • Figure 4 shows the colocalization of TNFR2 and cardiac progenitor markers in hiPSC-CM
  • FIG. 6 is a model of a mouse myocardial infarction according to the present invention.
  • a wild-type and TNFR2-KO mouse is modeled in a myocardial infarction model;
  • A is a measure of infarct size;
  • B is a wild type mouse after myocardial infarction 3 -28 days of ischemia;
  • C is myocardial infarction after myocardial infarction can enhance myocardial repair and function, wild type and AIP1-KO mice undergo myocardial infarction modeling, using echocardiogram on days 7, 14, 21, 28
  • D is the ischemic condition of wild-type and AIP1-KO mice after 21 days of my
  • FIG. 7 is a diagram showing the induction of cardiac stem cells in the ischemic mouse heart of the present invention.
  • Example 8 is a technical route diagram for exploring the mechanism of TNFR2-Bmx signaling regulation of CSC by using endo/exogenous cardiac stem cells (mouse TNFR2+eCSCs and hESC/hiPSC-derived CSC) according to Example 5 of the present invention;
  • Figure 9 is a graph showing the results of inhibition of TNFR2 signaling pathway by TNFR2 neutralizing antibody in Example 5 of the present invention.
  • Figure 10 is a diagram showing the results of R2-TNF specific activation of TNFR2 signaling pathway in hiPSCs in Example 5 of the present invention.
  • Figure 11 is a diagram showing the results of R2-TNF specific up-regulation of TNFR2 signaling pathway in Example 5 of the present invention.
  • Figure 12 is a diagram showing that TNFR2 neutralizing antibody inhibits cardiomyocyte differentiation in Example 5 of the present invention.
  • Figure 13 is a diagram showing that R2-TNF promotes cardiomyocyte differentiation in Example 5 of the present invention.
  • Example 1 Establishing a myocardial infarction model
  • Coronary artery ligation After the anesthesia, the mouse guides a hose from the oral cavity into the tracheal external ventilator, and makes a oblique incision from the lower right to the upper left side of the left chest. The scalpel is placed along the lower rib in the fourth intercostal space. The marginal intercostal muscle enters the thoracic cavity, and the heart is gently squeezed out. The proximal edge of the left anterior descending artery is quickly sewed between the left atrial appendage and the pulmonary artery cone. The left anterior descending artery is quickly sewed, and the electrocardiogram changes are observed. The syringe draws out the gas in the chest to restore the negative pressure in the chest. Cardiac ultrasound examination of ejection fraction ⁇ 40% of mice was included in the experiment.
  • Model of diffuse myocardial injury In recent years, the model of diffuse myocardial injury has been reported to be more suitable for myocardial repair experiments. Wild type, TNFR2-KO and Bmx-KO mice received a single subcutaneous injection of 5 mg kg-1 isoproterenol at the loose skin of the neck, respectively. ISO will play a heart pathological process similar to Takotsubo, leading to diffuse subendocardial and apical myocardial necrosis, acute damage will damage 8-10% of the left ventricular myocardium, leading to acute heart failure.
  • this myocardial injury and heart failure play a key role in myocardial regeneration and functional repair by increasing the morphological and functional recovery of eCSC by BradU-positive eCSC after 28 days.
  • Example 2 Increased expression of TNFR2-Bmx in human ischemic heart
  • the experimental method of this example is to detect the expression of TNF receptor molecules and downstream effector molecules by IB or immunohistochemistry using anti-TNFR2 and anti-Bmx antibodies.
  • the experimental samples were paraffin-embedded tissue sections from a heart transplant we obtained from Dr. Bradley, University of Cambridge, UK.
  • TNFR1 is strongly expressed ( ⁇ -SA) in CMs (cardiomyocytes), VECs (vascular endothelial cells) and ICs (interstitial cells) of normal hearts.
  • CMs cardiacocytes
  • VECs vascular endothelial cells
  • ICs interstitial cells
  • TNFR1 and AIP1 are down-regulated in ischemic heart grafts.
  • TNFR2 and Bmx kinases are only weakly expressed in normal human NM but upregulated in ischemic hearts.
  • Example 3 Co-expression of TNFR2 with Nkx2.5+/Gata4+ CSC markers in ischemic hearts
  • Heart ultrasound was performed to detect cardiac function before execution.
  • TNFR2-Bmx expression was detected by IB or immunohistochemistry using anti-TNFR2 and anti-Bmx antibodies.
  • Bmx activation was detected by anti-phospho-Bmx.
  • Mouse eCSCs were evaluated using anti-Gata4, anti-Nkx2.5. The expression of TNFR2 and Bmx and eCSC markers were also tested. Immunological double-labeling was used to detect possible cardiac precursor cells of CSC markers (Gata4, NKx2.5 and ⁇ -SA).
  • hESC or iPS is monolayer cultured on Matrigel, the extracellular matrix is prepared, and then covered with matrigel. This method, like gastrulation, produces N-cadherin-positive mesenchymal cells and promotes EMT.
  • Some growth factors Activin A, BMP4, FGF
  • Matrigel can be used to obtain high-purity (up to 98%) and high-number (up to 11CMs/hESC) cardiomyocytes from a variety of cells. Cardiomyocytes gradually matured in the culture medium for 30 days, and have myofilament expression and mitotic activity.
  • Fig. 3 The experimental results are shown in Fig. 3: as shown by A in Fig. 3, on the 14th day of cell differentiation, the cells began to express cardiomyocyte characteristics, including spontaneous contraction and cardiac-related gene protein expression; As shown in B, we detected the cardiac markers GATA4 and Isl1 by qPCR, and the expression was significantly increased after differentiation. At the same time, as shown by the CG in Fig. 3, immunostaining showed that these cells were positive for TNFR2 and NKX2.5, and TNFR2/NKX2. .5 and TNFR2/Ki67 were positive.
  • Figure 4 shows the colocalization of TNFR2 with cardiac progenitor markers in hiPSC-CM by three staining.
  • Example 4 Detection of TNFR2-Bmx signal in myocardium using TNFR2-KO and BMX-KO mice Role of activation and survival in CSCs in injury models
  • Example 2 As shown by the results of Example 2 and Example 3, the inventors observed that Nkx2.5+/Gata4+ cells were increased in both ischemic human heart samples and mouse hearts, and TNFR2-Bmx promoted activation of eCSCs in ischemic myocardium. Therefore, it is proposed to further utilize TNFR2-KO and BMX-KO mice to examine the role of the TNFR2-Bmx signaling pathway in the activation and survival of eCSCs in a myocardial injury model.
  • a myocardial infarction model and a diffuse myocardial injury model were established by the method shown in Example 1.
  • the experimental results are shown in Fig. 6.
  • the inventors further confirmed the role of the TNFR signaling pathway in the activation of precursor cells in vivo, and established a mouse myocardial infarction model by permanently ligating the coronary artery with the anterior descending artery, as shown in Fig. 6 Shown.
  • the chest is opened on the left side, the chest muscle is laterally cut in the fourth intercostal space, the thoracic cavity is exposed, the thymus is retracted upward, the left lung is partially collapsed, and after the pericardium is opened, the left is positioned.
  • most of the TNFR2-KO and Bmx-KO died in the first week after surgery, consistent with the survival of the TNFR2-Bmx signaling pathway.
  • TNFR1-KO and AIP1-KO mice survived, and cardiac function was better compared with WT on day 7 and 28 after surgery.
  • HE staining on day 14 showed that the ischemic WT infarct area was more severe, but cardiac tissue showed significant regeneration at 21-28, and high magnification showed AIP1-KO heart. The new myocardial tissue is regenerated in the ischemic area.
  • CSCs were induced like ischemic hearts.
  • These cells are CD45- and VEGFR2-, excluding them Blood progenitor cells or endothelial progenitor cells. As shown in A and B of Figure 7, these cells were significantly increased in the AIP1-KO heart.
  • TNFR2 was co-expressed with Nkx2.5 and Gata4 in neo-SA stained regenerated cardiomyocytes. Importantly, TNFR2, Nkx2.5 and Gata4 were significantly increased in the AIP1-KO ischemic heart.
  • Example 5 Study on the mechanism of TNFR2-Bmx signaling regulation of CSC by using endo/exogenous cardiac stem cells (mouse TNFR2+eCSCs and hESC/hiPSC-derived CSC)
  • TNFR2 and Nkx2.5+/Gata4+ were co-localized in Nkx2.5+/Gata4+eCSC, and the inventors of the present invention presumed that TNFR2-Bmx can enhance Nkx2.5+/Gata4+ expression and activity, Mediates the activation, migration and survival of eCSCs.
  • TNFR2 gene silencing or antagonists, BMX gene silencing or inhibitors, and TNFR2-specific ligands to detect the effect of TNFR2-Bmx signaling on Nkx2.5+/Gata4+ expression and activity.
  • the experimental method is specifically: the mouse heart-derived TNFR2+eCSC will be isolated and cultured.
  • eCSC4-24h was treated with TNFR2-specific ligand (R2-TNF, 50 ng/ml; TNFR2-Bmx activation).
  • R2-TNF TNFR2-specific ligand
  • the expression of Nkx2.5/Gata4 mRNA was detected by RT-PCR, and the protein expression was detected by IB.
  • eCSC was treated with or without the TNFR2-specific ligand R2-TNF (20 ng/ml) for 0, 5, 15, 30 and 60 minutes.
  • TNFR2 or Bmx was down-regulated by siRNA, anti-TNFR2 antagonist (TNFR2 neutralizing antibody) (50ng/ml) was used to inhibit TNFR2, and LFM-A13 (10 ⁇ M) inhibited TNFR2-Bmx-induced Nkx2 after inhibiting Bmx. 5/Gata4 mRNA expression and activation.
  • TNFR2-Bmx signaling pathway plays an important role in the activation and survival of endogenous CSCs in ischemic myocardium.
  • TNFR2 activator R2-TNF can activate TNFR2 or TNFR2-Bmx signaling to promote CSCs activation and survival.
  • TNFR2 activator R2-TNF can specifically activate TNFR2 signaling pathway in hiPSCs and promote cardiomyocyte differentiation;
  • TNFR2 neutralizing antibody can inhibit TNFR2 signaling Pathway that inhibits cardiomyocyte differentiation.

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Abstract

TNFR2作为靶标筛选药物例如TNFR2激活剂中的用途，所述药物用于促进心脏干细胞活化、迁移或存活，促进心脏干细胞分化为心肌细胞或促进心肌修复，治疗人类缺血性心脏病。TNFR2也可作为标志物用于制备筛选或识别心脏干细胞的试剂或试剂盒。

Description

TNFR2的用途 技术领域
本发明涉及TNFR2的用途，具体是TNFR2作为靶标和标志物的用途。
背景技术
心血管疾病是世界范围内威胁人类生命的最主要疾病之一，其中急性心肌梗塞是冠心病中最严重的疾病，具有很高的发病率和死亡率。心肌梗死会造成不可逆性功能心肌细胞数量的减少，导致心肌收缩功能降低。目前心梗传统的治疗方法主要包括药物治疗、介入治疗和手术治疗，虽能改善心肌缺血症状，但都无法挽救已坏死的心肌细胞。干细胞是一类具有自我更新和多向分化潜能的细胞，可以替代损伤的心肌细胞，构建新的血管，修复心脏泵血功能，为心梗的治疗提供了一种新的选择。越来越多的证据支持成年心脏干细胞(Cardiac stem cells，CSCs)的存在，并在缺血状态下被激活，在心脏功能修复中发挥重要作用。
激活剂，英文名为activator，凡是能提高酶活性的物质都被称为激活剂，其中大部分是离子或简单的有机化合物。配体，同锚定蛋白结合的任何分子都称为配体，配体属于激活剂的一种。在受体介导的内吞中，配体与细胞质膜受体蛋白结合，最后被吞入细胞的即是配体。根据配体的性质以及被细胞内吞后的作用，将配体分为四大类：I.营养物，如转铁蛋白、低密度脂蛋白(LDL)等；II.有害物质，如某些细菌；III.免疫物质，如免疫球蛋白、抗原等；IV.信号物质，如胰岛素等多种肽类激素等。
TNF-α属II型膜蛋白，主要由活化的单核巨噬细胞分泌产生，以三聚体形式发挥其作用。它具有可溶性TNF-α和膜相关TNF-α两种存在形式。TNF-α通过与受体TNFR结合来发挥其生物学功能。TNFR也有两种类型：TNFR1和TNFR2，均为I型膜蛋白。可溶性TNF-α在调节炎症反应和调节细胞生存或死亡方面起着重要作用。TNF-α与其受体结合后，首先招募细胞中的TRADD(TNF receptor-associated death domain)与TNF-α受体的胞内部分结合，然后再活化另外2个受体相关蛋白TRAF2(TNF receptor-associated factor 2)和FADD(fas-associated deathdomain)，从而激活多条信号通路，如NF-κB、JNK、MAPK及细胞凋亡信号通路来实现TNF-α的调节功能。
发明内容
本申请人经过大量研究发现，TNFR2(tumor necrosis factor receptor-2)即肿瘤坏死因子受体2，其不同于TNFR1所介导的炎症和凋亡功能，TNFR2可通过-Bmx(bone marrow non-receptor tyrosine kinase in X-chromosome)即X染色体上的骨髓非受体酪氨酸激酶来介导细胞存活、增殖和迁移。而且，TNFR2-Bmx信号通路可能对缺血性心肌中内源性CSCs活化和存活起重要作用。因此，本发明的目的在于提供TNFR2的多种用途。
为实现上述目的，本发明采取的技术方案为：TNFR2作为靶标在筛选或制备促进心脏干细胞活化、迁移或存活的药物中的用途。
本发明提供了TNFR2作为靶标在筛选或制备促进心脏干细胞分化为心肌细胞或者促进心肌修复的药物中的用途。
本发明提供了TNFR2作为靶标在筛选或制备治疗人类缺血性心脏病的药物中的用途。
本发明提供了TNFR2激活剂在制备促进心脏干细胞活化、迁移或存活的药物中的用途。
本发明提供了TNFR2激活剂在制备促进心脏干细胞分化为心肌细胞或者促进心肌修复的药物中的用途。
本发明提供了TNFR2激活剂在制备治疗人类缺血性心脏病的药物中的用途。
优选地，所述TNFR2激活剂包含TNFR2特异性配体。
优选地，所述TNFR2特异性配体为R2-TNF。
本发明提供了TNFR2抑制剂或者TNFR2拮抗剂在制备抑制心肌细胞分化的试剂中的用途。
本发明提供了TNFR2作为标志物在制备筛选或识别心脏干细胞的试剂或试剂盒中的用途。
本发明的有益效果在于：本发明所述TNFR2激活剂激活了TNFR2-Bmx，从而增强Nkx2.5+/Gata4+的表达和活性，介导c-Kit+内源性心脏干细胞(eCSCs)的激活、迁移和存活的作用，并且激活了的TNFR2，增加hESC/hiPSC来源的心肌细胞的分化，有利于心肌梗死的修复，为人类心脏疾病治疗提供一种新的策略；本发明所述TNFR2抑制剂或者TNFR2拮抗剂能够抑制心肌细胞的过度 分化。本发明提供了一种心脏干细胞标志物，TNFR2作为心脏干细胞的一种新的生物标志物，为分离和识别内源心脏干细胞提供新的分子标记。
附图说明
图1为TNF受体分子和下游效应分子在人正常心脏和缺血心脏的表达分布图；A为切片免疫荧光染色，分别为TNFR1/α-SA，TNFR2/α-SA和Bmx/α-SA三组(CM-心肌细胞，VEC-血管内皮细胞，IC-间质细胞)；B为正常心脏组织和缺血心脏组织western blot检测相应蛋白表达情况；C-D为磷酸化组蛋白3(pH3s10)与α-SA免疫荧光染色；D为pH3阳性细胞统计图，纵坐标表示高倍视野pH3阳性细胞数，横坐标表示三组细胞(CM-心肌细胞，VEC-血管内皮细胞，IC-间质细胞)，**，p＜0.01。n＝5张切片；E组织切片免疫荧光染色，显示pH3s10与TNFR2存在共定位，其中比例尺为50μm；
图2为人正常心脏和缺血性心脏中心脏前体细胞分布情况；A为人正常心脏和缺血心脏组织切片中Gata4，NKx2.5和α-SA免疫荧光染色，箭头指出Gata4在心肌细胞细胞核表达；B为Gata4+和NKx2.5+细胞统计，纵坐标表示高倍视野阳性细胞数，横坐标表示c-Kit、Gata4和NKx2.5染色组别，**p＜0.01，n＝5张切片；C为人正常心脏和缺血心脏中的心脏前体细胞，用NKx2.5，Gata4和TNFR2进行标记，箭头指向双阳性的细胞，图片放大倍数为40，比例尺为50μm；
图3为利用GiWi-心脏干细胞分化的方法获取iPSC-CSC；A.GiWi-心脏干细胞分化方法的流程图；B.QPCR检测TNFR2，GATA4以及ISL1在已分化的iPSC-CSC中的表达结果，纵坐标表示靶基因相对于内参(GAPDH)表达的百分比，横坐标表示hESCs细胞系和分化的CSC细胞；C.iPSC-CSC中TNFR2，NKX2.5以及GATA4的免疫荧光染色图像(10×)；D.iPSC-CSC中TNFR2以及原肌球蛋白的免疫荧光染色(63×)；E.iPSC-CSC中NKX2.5以及原肌球蛋白的免疫荧光染色(63×)；F.iPSC-CSC中NKX2.5，TNFR2以及原肌球蛋白的免疫荧光染色(63×)；G.iPSC-CSC中Ki67，TNFR2以及原肌球蛋白的免疫荧光染色(63×)；
图4为hiPSC-CM中TNFR2与心脏祖细胞标记物存在共定位现象；
图5为本发明实施例4所述利用TNFR2-KO和Bmx-KO小鼠来检测TNFR2-Bmx信号在心肌损伤模型中对CSCs的激活和生存中的作用的技术路线图；
图6为本发明所述：小鼠心肌梗死模型，将野生型和TNFR2-KO小鼠进行心肌梗死模型造模；A为梗死大小的测量图；B为野生型小鼠在心肌梗死后的3-28天的缺血情况；C为心肌梗死后AIP1-KO能够增强心肌修复和功能，野生型和AIP1-KO小鼠进行心肌梗死造模，利用超声心电图在第7、14、21、28天测量心脏的功能，分别在0天(手术前)、7天以及28天展示缩短的百分比，纵坐标表示左室射血分数缩短率(LVFS)，横坐标表示手术前后时间，N＝10，*p＜0.05；D为野生型和AIP1-KO小鼠在心肌梗死21天后的缺血情况，褐色箭头表示新生成的心肌细胞。比例尺：50μm；
图7为本发明所述在缺血小鼠心脏中诱导心脏干细胞。野生型和AIP1-KO小鼠进行3-28天的心肌梗死造模；A-B为在第21天对心脏部分进行c-kit+以及cKit+/TNFR2+细胞免疫染色，B图为量化图，*，p＜0.05AIP1-KO vs WT.N＝6只小鼠(比例尺：50μm)；纵坐标表示高倍视野c-Kit和TNFR2共同阳性表达细胞数，横坐标表示心脏不同区域(remote zone-远区，border zone-交界区，ischemic zone-缺血区)，C为将TNFR2和Gata4进行共染色；D为Gata4以及α-SA进行共染色；
图8为本发明实施例5所述利用内/外源心脏干细胞(小鼠TNFR2+eCSCs和hESC/hiPSC来源的CSC)探讨TNFR2-Bmx信号调控CSC的机制的技术路线图；
图9为本发明实施例5中TNFR2中和抗体抑制TNFR2信号通路的结果；
图10为本发明实施例5中R2-TNF特异性激活hiPSCs中TNFR2信号通路的结果；
图11为本发明实施例5中R2-TNF特异性上调TNFR2信号通路的结果；
图12为本发明实施例5中TNFR2中和抗体抑制心肌细胞分化；
图13为本发明实施例5中R2-TNF促进心肌细胞分化。
具体实施方式
为更好的说明本发明的目的、技术方案和优点，下面将结合具体实施例及附图对本发明作进一步说明。
实施例1：建立心梗模型
本实施例中所有动物实验均得到动物伦理委员会批准。
实验方法：
1)冠状动脉结扎：小鼠麻醉后从口腔导引一软管进入气管外接呼吸机，在左侧胸部从右下至左上做一斜行切口，在第4肋间以手术刀沿下位肋骨上缘切开肋间肌进入胸腔，将心脏轻轻挤出后，在左心耳与肺动脉圆锥之间平左心耳下缘迅速缝扎左前降支近端，同时观察心电图变化，术后立即关胸并用注射器抽出胸腔内气体，以恢复胸腔负压。心脏超声检测射血分数＜40％小鼠纳入实验。
2)弥漫性心肌损伤模型：近年报道弥漫性心肌损伤模型更适合心肌修复实验。野生型、TNFR2-KO和Bmx-KO小鼠分别接受在颈部疏松皮肤处接受5mg kg-1异丙肾上腺素单次皮下注射。ISO将发挥类似Takotsubo的心脏病理过程，导致弥漫性心内膜下和心尖心肌坏死，急性损伤将损坏8-10％左心室心肌，导致急性心力衰竭。和传统心梗模型相比，这种心肌损伤和心力衰竭通过增加BrdU-阳性eCSC在28天后形态上和功能上自发恢复eCSC在心肌再生和功能修复中发挥关键作用。
实施例2：TNFR2-Bmx在人体缺血心脏中表达增加
本实施例的实验方法为运用anti-TNFR2和anti-Bmx抗体进行IB或免疫组化检测TNF受体分子和下游效应分子的表达。实验样品为我们从英国剑桥大学Dr.Bradley获得的心脏移植术中的石蜡包埋组织切片。
其具体的实验方法为：
1)免疫荧光：组织切片固定，Triton-X100通透处理后，用1％BSA(牛血清蛋白)封闭30分钟，之后PBS(磷酸盐缓冲液)漂洗，相应抗体孵育2小时，PBS漂洗，对应荧光二抗避光孵育1小时，PBS漂洗后甘油封片，荧光显微镜下观察拍照。
2)Western blot：提取心脏组织蛋白定量后，进行SDS-PAGE凝胶电泳，之后PVDF膜进行转膜，之后PVDF膜用5％脱脂奶粉进行封闭1小时，相应一抗4℃孵育过夜，PBS漂洗，对应二抗室温孵育1小时，PBS漂洗，化学发光检测蛋白表达水平。
实验结果如附图1所示，切片显示正常(NM；n＝20)与缺血性心脏病(IHD，n＝58)组织形态并无异常。TNFR1在正常心脏的CMs(心肌细胞)，VECs(血管内皮细胞)和ICs(间质细胞)中均强表达(α-SA)。然而，TNFR1和AIP1在缺血心脏移植物中下调。TNFR2和Bmx激酶只在正常人类心脏(normal human)NM中弱表达，而在缺血心脏中上调。
以上结果表明，TNFR2-Bmx在人体缺血心脏中表达增加。
实施例3：TNFR2在缺血心脏中与Nkx2.5+/Gata4+CSC标志物共表达
将野生型、TNFR2-KO和Bmx-KO小鼠分别接受在颈部疏松皮肤处接受5mg kg-1异丙肾上腺素单次皮下注射，分别于1，3，6，14或28天处死小鼠(每个时间点每组n＝10)。处死前行心脏超声检测心功能。运用anti-TNFR2和anti-Bmx抗体进行IB或免疫组化检测TNFR2-Bmx表达。Bmx活化情况通过anti-phospho-Bmx进行检测。小鼠eCSC利用anti-Gata4、anti-Nkx2.5进行评估。同时检测TNFR2和Bmx与eCSC标志物的表达。免疫双标法检测CSC标志物(Gata4，NKx2.5和α-SA)可能的心脏前体细胞。
实验结果如附图2中的A所示，在正常心脏只有部分游离细胞Nkx2.5和Gata4阳性。而在缺血心脏的缺血区域，如附图2中的A和B所示，三种标志物(Gata4，NKx2.5和α-SA)均阳性。之后我们评估它们的免疫表型，运用免疫荧光双染明确它们和TNFR2的关系，结果如图2中B中的NM所示，正常心肌细胞中Nxk2.5+TNFR2+或Gata4+TNFR2+表达很低，然而在缺血心脏Nkx2.5+或Gata4+前体细胞中TNFR2也呈阳性，如附图2的图C中的IHD所示。
我们采用Dr.Kamp’s课题组建立的基质胶三明治法，培养、分化细胞，从而增加细胞产出。其具体实验方法为：hESC或iPS在Matrigel上单层培养，细胞外基质准备，然后覆盖基质胶。该法如同原肠胚形成一样，产生N-cadherin-阳性间充质细胞，促进EMT发生。将一些生长因子(Activin A，BMP4，FGF)联合基质胶应用，可以从多种细胞得到高纯度(高至98％)和高数量(高至11CMs/hESC)的心肌细胞。心肌细胞在培养液中30天后逐渐成熟，具备肌丝表达和有丝分裂活性。实验结果如附图3所示：如附图3中的A所示，观察到细胞分化第14天，细胞开始表现心肌细胞特征，包括自发收缩及心脏相关基因蛋白表达；如附图3中的B所示，我们通过qPCR检测了心脏标记物GATA4和Isl1，分化后表达均显著增高，同时如附图3中的C-G所示，免疫染色显示这些细胞为TNFR2、NKX2.5阳性，TNFR2/NKX2.5及TNFR2/Ki67共阳性。图4用三染色显示hiPSC-CM中TNFR2与心脏祖细胞标记物存在共定位现象。
以上结果表明，TNFR2在缺血心脏中与Nkx2.5+/Gata4+CSC标志物共表达。
实施例4：利用TNFR2-KO和BMX-KO小鼠来检测TNFR2-Bmx信号在心肌 损伤模型中对CSCs的激活和生存中的作用
如实施例2和实施例3结果所示，本发明人观察到Nkx2.5+/Gata4+细胞在缺血的人体心脏标本和小鼠心脏中均增加，TNFR2-Bmx促进缺血心肌中eCSCs激活。因此，拟进一步利用TNFR2-KO和BMX-KO小鼠来检测TNFR2-Bmx信号通路在心肌损伤模型中对eCSCs的激活和生存中的作用。
实验技术路线如附图5所示。
实验技术路线概括为：
(1)采用实施例1所示的方法建立心梗模型及弥漫性心肌损伤模型。
(2)然后取野生型、TNFR2-KO和Bmx-KO小鼠分别接受在颈部疏松皮肤处接受5mg kg-1异丙肾上腺素单次皮下注射，分别于1，3，6，14或28天处死小鼠(每个时间点每组n＝10)。处死前行心脏超声检测心功能。利用HE染色评估心脏损伤后形态学变化，CD45评价炎性浸润，Ki67或pH3评价增殖TUNEL染色检测凋亡。运用anti-TNFR2和anti-Bmx抗体进行IB或免疫组化检测TNFR2-Bmx表达。Bmx活化情况通过anti-phospho-Bmx进行检测。小鼠eCSC利用anti-Gata4、anti-Nkx2.5进行评估。同时检测TNFR2和Bmx与eCSC标志物的表达。
实验结果如附图6所示，本发明人为进一步在体实验明确TNFR信号通路在前体细胞激活中的作用，运用前降支永久结扎冠状动脉建立小鼠心梗模型，如附图6中A所示。接下来，如附图6中B所示，在左侧行开胸术，在第四肋间隙横向切开胸肌暴露胸腔，胸腺向上缩回，左肺部分塌陷，打开心包膜后，定位左冠状动脉(LCA)，并用6-0丝线结扎2-3mm作为起点，心室左前壁颜色苍白作为成功结扎的标志，野生型(n＝10每组)术后均存活。然而，如附图6中C所示，大部分TNFR2-KO和Bmx-KO在术后第一周死亡，与TNFR2-Bmx信号通路的存活作用一致。相反，TNFR1-KO和AIP1-KO小鼠存活，术后第7天和28天采用心脏超声检测心功能结果与WT比较更佳。与心功能结果一致，如附图6中D所示，第14天HE染色结果显示缺血WT梗死区域更为严重，但心脏组织在21-28显示显著的再生，高倍镜提示AIP1-KO心脏中新的心肌组织在缺血区域再生。
接下来，为明确CSCs在缺血心脏中的激活情况，免疫组化显示像缺血心脏一样，CSCs被诱导产生。这些细胞为CD45-和VEGFR2-，排除了它们是造 血祖细胞或内皮祖细胞。如附图7中A和B所示，这些细胞在AIP1-KO心脏显著增加。如附图7中C和D所示，在新的-SA染色的再生心肌细胞中，TNFR2与Nkx2.5和Gata4共表达。重要的是，TNFR2、Nkx2.5和Gata4在AIP1-KO缺血心脏中显著增加。
实施例5：利用内/外源心脏干细胞(小鼠TNFR2+eCSCs和hESC/hiPSC来源的CSC)探讨TNFR2-Bmx信号调控CSC的机制研究
如实施例3结果所示，Nkx2.5+/Gata4+eCSC中TNFR2和Nkx2.5+/Gata4+共定位，据此本申请发明人推测TNFR2-Bmx能增强Nkx2.5+/Gata4+表达和活性，介导eCSC的激活、迁移和存活。为证实这一假设，我们使用TNFR2基因沉默或拮抗剂，BMX基因沉默或抑制剂，和TNFR2特异性配体等，来检测TNFR2-Bmx信号对Nkx2.5+/Gata4+表达和活性的作用。
其实验技术路线如图8所示。
其实验方法具体为：将分离并培养小鼠心脏来源的TNFR2+eCSC。用TNFR2特异性配体(R2-TNF，50ng/ml；TNFR2-Bmx激活)处理eCSC4-24h。RT-PCR检测Nkx2.5/Gata4mRNA表达，IB检测蛋白表达。Nkx2.5/Gata4激活实验中，eCSC用或不用TNFR2特异性配体R2-TNF(20ng/ml)处理0，5，15，30和60分钟。采用IB法，利用特异的磷酸化抗体检测TNFR2下游Bmx，Akt，STAT3，ERK1/2磷酸化情况。Nkx2.5/Gata4的DNA结合活性采用EMSA方法。分别采用siRNA下调TNFR2或Bmx，anti-TNFR2拮抗剂(TNFR2中和抗体)(50ng/ml)抑制TNFR2作用，LFM-A13(10μM)抑制Bmx作用后，检测是否阻断TNFR2-Bmx诱导的Nkx2.5/Gata4mRNA表达和激活。
实验结果如图9、图10、图11、图12和图13所示，从图9-13的结果明确了TNFR2-Bmx信号通路对缺血性心肌中内源性CSCs活化和存活起重要作用，TNFR2激活剂R2-TNF能激活TNFR2或TNFR2-Bmx信号促进CSCs活化和存活，TNFR2激活剂R2-TNF能特异性激活hiPSCs中TNFR2信号通路，促进心肌细胞分化；TNFR2中和抗体能抑制TNFR2信号通路，抑制心肌细胞分化。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。

Claims (10)

  1. TNFR2作为靶标在筛选或制备促进心脏干细胞活化、迁移或存活的药物中的用途。
  2. TNFR2作为靶标在筛选或制备促进心脏干细胞分化为心肌细胞或者促进心肌修复的药物中的用途。
  3. TNFR2作为靶标在筛选或制备治疗人类缺血性心脏病的药物中的用途。
  4. TNFR2激活剂在制备促进心脏干细胞活化、迁移或存活的药物中的用途。
  5. TNFR2激活剂在制备促进心脏干细胞分化为心肌细胞或者促进心肌修复的药物中的用途。
  6. TNFR2激活剂在制备治疗人类缺血性心脏病的药物中的用途。
  7. 如权利要求4-6任一所述的用途，其特征在于，所述TNFR2激活剂包含TNFR2特异性配体。
  8. 如权利要求7所述的用途，其特征在于，所述TNFR2特异性配体为R2-TNF。
  9. TNFR2抑制剂或者TNFR2拮抗剂在制备抑制心肌细胞分化的试剂中的用途。
  10. TNFR2作为标志物在制备筛选或识别心脏干细胞的试剂或试剂盒中的用途。
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