WO2023024664A1 - Application of abt-263 in preparation of drug for inhibiting corneal transplant immune rejection - Google Patents
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/5375—1,4-Oxazines, e.g. morpholine
- A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
Definitions
- the invention belongs to the technical field of chemical drugs, and in particular relates to the application of ABT-263 in the preparation of drugs for inhibiting immune rejection of corneal transplantation.
- Corneal disease is one of the eye diseases with a high rate of blindness.
- Penetrating Keratoplasty is an important means of restoring vision for patients with corneal blindness, and it is also the last choice for preserving vision.
- PK Penetrating Keratoplasty
- immune rejection after PK is still the main reason for corneal transplantation failure. Therefore, it has important clinical significance and value to prevent and treat immune rejection after PK surgery and maintain the transparency of corneal grafts. Due to the lack of blood vessels and lymphatic vessels, normal corneal tissue is in a relatively "immune privileged state", so the incidence of immune rejection after non-high-risk keratoplasty is low.
- Cell senescence is characterized by irreversible decline in cell function and structure.
- corneal parenchymal cells especially corneal endothelial cells
- corneal sutures especially corneal sutures
- surgical trauma showing characteristics of senescent cells.
- premature senescence and aging of key cells in tissues and organs are closely related to the decline in the function of the tissues and organs, and cellular senescence plays an important role in pathophysiological processes such as immune rejection and chronic dysfunction of solid organ transplants.
- corneal endothelial cells will undergo superphysiological rapid loss, and some degenerative changes will occur prematurely and excessively.
- the number of corneal endothelial cells falls below a certain critical value, the remaining cells cannot fully compensate for normal tissue function, which can lead to corneal edema and bullous keratopathy.
- corneal parenchymal cells especially corneal endothelial cells
- timely and effective prevention of corneal endothelial cell decompensation is the key to preventing corneal immune rejection and maintaining good vision.
- drugs that can effectively inhibit the aging and functional decline of corneal parenchymal cells and prevent decompensation of corneal endothelial cells in a timely and effective manner.
- the purpose of the present invention is to provide an application of ABT-263 in the preparation of a drug for inhibiting immune rejection of corneal transplantation.
- the invention provides the application of ABT-263 in the preparation of medicine for inhibiting immune rejection of corneal transplantation.
- the corneal transplantation includes allogeneic corneal transplantation.
- said suppression of immune rejection of corneal transplantation includes prolonging the survival time of the donor corneal graft and reducing the inflammatory response in the corneal graft.
- prolonging the survival time of the donor corneal graft is to eliminate senescent cells through ABT-263, delay corneal graft immune rejection, and maintain the transparency of the corneal graft.
- said reducing the inflammatory response in the corneal graft is reducing the expression levels of inflammation-related cytokines in the corneal graft.
- the inflammation-related cytokines include IL-1 ⁇ , IL-17A, TNF- ⁇ and IFN- ⁇ .
- the medicine includes injections.
- the concentration of ABT-263 in the injection is 1-10 mg/ml.
- the concentration of the ABT-263 is 1-5 mg/ml.
- the invention provides the application of ABT-263 in the preparation of medicine for inhibiting immune rejection of corneal transplantation.
- the corneal stroma and endothelium have obvious aging phenotypes: the number of positive cells stained with SA- ⁇ -Gal increased significantly; the expression levels of aging-related genes (p16, p21, etc.) Significantly increased; the allogeneic mouse corneal transplantation model was constructed by using aging gene p16-deficient mice as corneal donors, and it was found that the aging-related gene p16 deletion can effectively prolong the survival time of corneal grafts and delay the immune rejection of corneal transplantation.
- the present invention can significantly inhibit the immune rejection of corneal transplantation by intraperitoneal injection of anti-aging drug ABT-263 (such as 1.25 mg/ml), prolong the survival time of the donor corneal graft, reduce the inflammatory reaction in the corneal graft, and maintain the corneal graft transparency. Therefore, ABT-263 can be used in the prevention and treatment of immune rejection after corneal transplantation.
- ABT-263 anti-aging drug
- Figure 1 is the corneal aging phenotype analysis of normal mice (Nor), syngeneic corneal transplantation (Syn) and allogeneic corneal transplantation (Allo), in which (A) SA- ⁇ of different groups of mouse corneal smears -Gal staining analysis; (B) Frozen section analysis of SA- ⁇ -Gal staining in corneal grafts; (C) Western Blot analysis of the expression of aging-related genes (p16 and p21) in corneal grafts.
- Figure 2 shows the effect of aging gene p16 knockout mice as corneal donors on immune rejection after PK, in which (A) observation of corneal transparency of mice after keratoplasty; (B) corneal transplantation of different groups of mice Survival curve analysis of slices; (C) Immunofluorescence analysis of CD45 + cell infiltration in corneal grafts of different groups of mice.
- Figure 3 is the effect of anti-aging drug ABT-263 on the immune rejection of corneal transplantation after PK, wherein (A) after the anti-aging drug ABT-263 intervention, the mouse cornea transparency analysis; (B) after the ABT-263 intervention Survival curve analysis of mouse corneal grafts; (C) SA- ⁇ -Gal staining analysis of the effect of ABT-263 on eliminating senescent cells; (D) Real-time PCR analysis of the effect of ABT-263 intervention on the expression of aging-related genes in corneal grafts ; (E) Real-time PCR analysis of the effect of ABT-263 intervention on the expression of major inflammatory factors in corneal grafts.
- the invention provides the application of ABT-263 in the preparation of medicine for inhibiting immune rejection of corneal transplantation.
- the structural formula of the ABT-263 is preferably as shown in formula I:
- the molecular formula of ABT-263 is C47H55ClF3N5O6S3, the molecular weight is 974.6127096, and the CAS accession number is 923564-51-6.
- the present invention has no special limitation on the source of the ABT-263, and the source of ABT-263 well known in the art can be used. In the embodiment of the present invention, the ABT-263 was purchased from Selleck Company.
- the corneal transplantation preferably includes allogeneic corneal transplantation.
- allogeneic corneal transplantation After keratoplasty in allogeneic mice, obvious aging phenotypes appeared in the corneal stroma and endothelium, including a significant increase in the number of positive cells stained with SA- ⁇ -Gal; the expression levels of aging-related genes (p16, p21, etc.) obviously increase.
- ABT-263 was injected, compared with the model control group, it significantly inhibited the immune rejection of corneal grafts and maintained the transparency of corneal grafts.
- the suppression of immune rejection of the corneal graft preferably includes prolonging the survival time of the donor corneal graft and reducing the inflammatory response in the corneal graft.
- the prolonging of the survival time of the donor corneal graft is preferably to use ABT-263 to clear the senescent cells in the allogeneic cornea, delay the immune rejection of the corneal graft, and maintain the transparency of the corneal graft.
- the allogeneic cornea preferably includes corneal stroma and endothelial cells. Said reducing the inflammatory response in the corneal graft is preferably reducing the expression level of inflammation-related cytokines in the corneal graft.
- the inflammation-related cytokines preferably include IL-1 ⁇ , IL-17A, TNF- ⁇ and IFN- ⁇ .
- the drug preferably includes injections.
- the injection preferably includes ABT-263 and pharmaceutically acceptable excipients.
- the dressing included 2-hydroxypropyl-beta-cyclodextrin and corn oil.
- the concentration of ABT-263 in the injection is preferably 1-10 mg/ml, more preferably 1-5 mg/ml, most preferably 1.25 mg/ml.
- the injection amount of the ABT-263 is 50mg/kg body weight of mice.
- the injection frequency of the ABT-263 is preferably 1 time/2d.
- ABT-263 provided by the present invention in the preparation of drugs for inhibiting immune rejection of corneal transplantation will be described in detail below in conjunction with the examples, but they should not be construed as limiting the protection scope of the present invention.
- the penetrating keratoplasty model was constructed as follows: the right eye of all animals was selected as the operated eye. Before the operation, a slit lamp examination was performed to understand the condition of the operated eye, and levofloxacin eye drops were given to the operated eye to prevent infection. After the donor mouse was anesthetized by intraperitoneal injection of pentobarbital sodium, the 2.25mm corneal central full-thickness graft was taken under the operating microscope, and the 2.25mm central corneal penetrating implant bed of the recipient mouse was immediately implanted. 8 stitches were sutured intermittently with 11-0 Japanese MANI nylon thread. After the operation, a small amount of sterile air was injected into the anterior chamber to form the anterior chamber.
- mice were sacrificed, and the eyeballs of the mice in the above three groups were removed, and then the corneal cups were carefully cut along the corneoscleral limbus, and the senescence associated ⁇ -galactosidase (SA- ⁇ -galactosidase, SA- ⁇ - In situ histochemical staining was carried out with the Gal) kit (Biyuntian), incubated overnight at 37°C, and the corneal endothelium was stained with the corneal endothelium facing up, and the staining was observed through a microscope.
- Gal Gal
- the corneal endothelial cells in the Allo group were significantly positive for ⁇ -Gal staining, and were mainly distributed in the center of the corneal graft and the junction between the corneal graft and the implant bed (Fig. 1 A).
- the stained corneal grafts of the three groups were embedded in OCT, and frozen sections were made with a thickness of 7 ⁇ m. Then, the ⁇ -Gal staining of corneal tissue in each group was observed under a phase contrast microscope.
- mice were killed, and the eyeballs of the above-mentioned 3 groups of mice were taken, 3 samples in each group, and the corneal grafts of the mice in each group were cut out, and RIPA lysate and PMSF protease inhibitor were added according to the ratio specified by Western Blotting.
- the protein was electrotransferred from the gel to the PVDF membrane, the band was cut, and the rabbit anti-mouse p16 antibody (1:1000 dilution), rabbit Anti-mouse p21 antibody (diluted 1:1000) and mouse anti-mouse ⁇ -actin antibody (diluted 1:2000), after incubation and washing the membrane, then add goat anti-rabbit IgG-HRP and goat anti-mouse IgG- After the HRP secondary antibody was incubated and washed, the Bio-Rad fast luminescence camera was used to perform luminescence with the ECL luminescence kit.
- Cell senescence is mainly realized through two signaling pathways, p16INK4a/Rb and p19ARF/p53/p21Cipl.
- the present invention uses p16-deleted C57BL/6 (Frederick National Laboratory for Cancer Research (FNL)/SAIC-Frederick, Inc) mice as corneal donors, Balb/C small Rats were used to construct penetrating keratoplasty models for corneal recipients to evaluate the effect of aging on immune rejection of corneal transplantation.
- a penetrating keratoplasty model was established, and the operation method was as described in Example 1.
- mice were sacrificed, and the eyeballs of the above two groups were removed. After OCT embedding, frozen sections were made with a thickness of 7 ⁇ m. Immunofluorescence staining was performed with anti-CD45 antibody, and photographs were taken under an immunofluorescence microscope. Observe the staining results.
- the penetrating keratoplasty model was established according to the method described in Example 1.
- mice were killed, and the eyeballs of the above two groups were removed, and then the corneal cup was carefully cut along the corneoscleral limbus, and the in situ histochemistry was performed using the aging-related SA- ⁇ -Gal kit (Beiyuntian) For staining, incubate overnight at 37°C. The endothelium of the cornea is stained upwards, and the corneal slices are spread, and the staining is observed under a microscope.
- SA- ⁇ -Gal kit Beiyuntian
- mice were killed, and the eyeballs of the mice in the normal control group (Nor), the non-medication group (Allo) and the drug intervention group (Allo+ABT-263) were removed respectively, and 3 samples were cut from each group.
- Nor normal control group
- Allo non-medication group
- Allo+ABT-263 drug intervention group
- the Ct value (Threshold cycle) shown in the result is defined as the number of cycles required to reach the detection threshold of the fluorescent signal, and the resulting data is analyzed using ROTOR-GeneTMQ Series Software (v1.2.0.) of QIAGEN Company, with 2 - ⁇ Ct was calculated using GAPDH as an internal reference.
- the expression levels of senescence-related genes (p16 and p21) in corneal grafts were significantly decreased after ABT-263 intervention, suggesting that ABT-263 intervention can significantly clear senescent cells. It was further found that after ABT-263 intervention, the expression levels of inflammation-related cytokines in corneal grafts were significantly lower than those in the Allo group, and the difference was statistically significant.
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Abstract
The present invention relates to the technical field of chemical drugs and provides an application of ABT-263 in the preparation of a drug for inhibiting corneal transplantation immune rejection. The present invention provides an application of a small-molecule Bcl-2 inhibitor Navitoclax (ABT-263), which is capable of inducing apoptosis, in the preparation of a product for inhibiting immune rejection after a corneal transplant. According to the present invention, researches show that the Bcl-2 inhibitor ABT-263 can prolong the survival time of a corneal implant after a corneal transplant, reduce the immune inflammatory response in the corneal implant, maintain the transparency of the corneal implant, reduce the immune rejection, and can be used in prevention and treatment of diseases such as immune rejection and corneal endothelial cell decompensation after a corneal transplant.
Description
本发明属于化学药物技术领域，具体涉及ABT-263在制备抑制角膜移植免疫排斥反应的药物中的应用。The invention belongs to the technical field of chemical drugs, and in particular relates to the application of ABT-263 in the preparation of drugs for inhibiting immune rejection of corneal transplantation.
角膜病是致盲率极高的眼部疾病之一，穿透性角膜移植术(PenetratingKeratoplasty,PK)是角膜盲患者复明的重要手段，也是保存患者视力的最后选择。尽管随着显微手术技术的提高，角膜移植术的成功率有了很大提高，但PK(尤其是高危角膜移植)术后的免疫排斥反应仍是导致角膜移植失败的最主要原因。因此，防治PK术后的免疫排斥反应，维持角膜植片的透明性具有重要的临床意义和价值。正常角膜组织因缺乏血管和***，处于相对的“免疫赦免状态”，因而非高危角膜移植术后的免疫排斥发生率较低。然而，许多患者在PK术后没有发生以角膜新生血管与***为特征的明显免疫排斥反应的情况下，角膜植片的内皮细胞和基质细胞也会逐渐出现水肿浑浊、功能丧失等病理性改变。目前这已成为导致角膜移植术后角膜植片功能丧失、发生免疫排斥反应的重要原因之一。Corneal disease is one of the eye diseases with a high rate of blindness. Penetrating Keratoplasty (PK) is an important means of restoring vision for patients with corneal blindness, and it is also the last choice for preserving vision. Although the success rate of keratoplasty has been greatly improved with the improvement of microsurgical techniques, immune rejection after PK (especially high-risk keratoplasty) is still the main reason for corneal transplantation failure. Therefore, it has important clinical significance and value to prevent and treat immune rejection after PK surgery and maintain the transparency of corneal grafts. Due to the lack of blood vessels and lymphatic vessels, normal corneal tissue is in a relatively "immune privileged state", so the incidence of immune rejection after non-high-risk keratoplasty is low. However, many patients do not experience obvious immune rejection characterized by corneal neovascularization and lymphatic vessels after PK, and the endothelial cells and stromal cells of the corneal graft will gradually show pathological changes such as edema, turbidity, and loss of function . At present, this has become one of the important reasons leading to the loss of corneal graft function and immune rejection after keratoplasty.
细胞衰老的特征是细胞功能与结构等发生不可逆转衰退。在角膜移植手术过程中，角膜实质细胞(尤其是角膜内皮细胞)受炎症细胞、角膜缝线以及手术创伤等的刺激后，表现出衰老细胞特征。研究表明，组织器官中关键细胞的早衰和老化与该组织器官的功能下降密切有关，细胞衰老在实质性器官移植物的免疫排斥与慢性失功等病理生理过程中发挥重要作用。一旦发生排斥反应，角膜的免疫赦免状态发生偏离，在内外因素的刺激下，角膜内皮细胞会出现超生理性快速丢失，过早和过多地产生一些退行性改变。当角膜内皮细胞数量低于某一临界值后，残余细胞无法完全代偿正常组织功能，则可导致角膜水肿和大泡性角膜病变。Cell senescence is characterized by irreversible decline in cell function and structure. During keratoplasty, corneal parenchymal cells (especially corneal endothelial cells) are stimulated by inflammatory cells, corneal sutures, and surgical trauma, showing characteristics of senescent cells. Studies have shown that premature senescence and aging of key cells in tissues and organs are closely related to the decline in the function of the tissues and organs, and cellular senescence plays an important role in pathophysiological processes such as immune rejection and chronic dysfunction of solid organ transplants. Once rejection occurs, the state of immune pardon of the cornea deviates, and under the stimulation of internal and external factors, the corneal endothelial cells will undergo superphysiological rapid loss, and some degenerative changes will occur prematurely and excessively. When the number of corneal endothelial cells falls below a certain critical value, the remaining cells cannot fully compensate for normal tissue function, which can lead to corneal edema and bullous keratopathy.
角膜移植术后，能否通过抑制角膜实质细胞(尤其是角膜内皮细胞)的衰老和功能的减退，及时有效地防止角膜内皮细胞失代偿，是预防角膜免疫排斥反应、维持良好视力的关键。目前，尚未有关于有效抑制角膜实质细胞的衰老和功能减退，及时有效防止角膜内皮细胞失代偿的药物报道。After keratoplasty, whether the corneal parenchymal cells (especially corneal endothelial cells) can be inhibited from aging and functional decline, and timely and effective prevention of corneal endothelial cell decompensation is the key to preventing corneal immune rejection and maintaining good vision. At present, there are no reports about drugs that can effectively inhibit the aging and functional decline of corneal parenchymal cells and prevent decompensation of corneal endothelial cells in a timely and effective manner.
发明内容Contents of the invention
有鉴于此，本发明的目的在于提供一种ABT-263在制备抑制角膜移植免疫排斥反应的药物中的应用。In view of this, the purpose of the present invention is to provide an application of ABT-263 in the preparation of a drug for inhibiting immune rejection of corneal transplantation.
本发明提供了ABT-263在制备抑制角膜移植免疫排斥反应的药物中的应用。The invention provides the application of ABT-263 in the preparation of medicine for inhibiting immune rejection of corneal transplantation.
优选的，所述ABT-263的结构式如式I所示：Preferably, the structural formula of the ABT-263 is shown in formula I:
优选的，所述角膜移植包括同种异体角膜移植。Preferably, the corneal transplantation includes allogeneic corneal transplantation.
优选的，所述抑制角膜移植免疫排斥反应包括延长供体角膜植片的存活时间和降低角膜植片中的炎症反应。Preferably, said suppression of immune rejection of corneal transplantation includes prolonging the survival time of the donor corneal graft and reducing the inflammatory response in the corneal graft.
优选的，所述延长供体角膜植片的存活时间是通过ABT-263清除衰老细胞，延缓角膜移植免疫排斥反应，维持角膜植片的透明性。Preferably, prolonging the survival time of the donor corneal graft is to eliminate senescent cells through ABT-263, delay corneal graft immune rejection, and maintain the transparency of the corneal graft.
优选的，所述降低角膜植片中的炎症反应是降低角膜植片中炎症相关细胞因子的表达水平。Preferably, said reducing the inflammatory response in the corneal graft is reducing the expression levels of inflammation-related cytokines in the corneal graft.
优选的，所述炎症相关细胞因子包括IL-1β、IL-17A、TNF-α和IFN-γ。Preferably, the inflammation-related cytokines include IL-1β, IL-17A, TNF-α and IFN-γ.
优选的，所述药物包括注射剂。Preferably, the medicine includes injections.
优选的，所述注射剂中ABT-263的浓度为1～10mg/ml。Preferably, the concentration of ABT-263 in the injection is 1-10 mg/ml.
优选的，所述ABT-263的浓度为1～5mg/ml。Preferably, the concentration of the ABT-263 is 1-5 mg/ml.
本发明提供了ABT-263在制备抑制角膜移植免疫排斥反应的药物中的应用。实验证明，在同种异体小鼠角膜移植术后，角膜基质及内皮出现明显的衰老表型：SA-β-Gal染色阳性细胞的数目显著增多；衰老相关基因(p16、p21等)的表达水平明显增加；通过衰老基因p16缺失小鼠为角膜供体构建同种异体小鼠角膜移植模型发现，衰老相关基因p16缺失能有效延长角膜植片存活时间，延缓角膜移植免疫排斥反应。本发明通过腹腔注射抗衰老药物ABT-263(如1.25mg/ml)能显著抑制角膜移植免疫排斥反应，延长供体角膜植片的存活时间，减少角膜植片中的炎症反应，维持角膜植片的透明性。因此，ABT-263可用于角膜移植手术后免疫排斥反应的防治与治疗。The invention provides the application of ABT-263 in the preparation of medicine for inhibiting immune rejection of corneal transplantation. Experiments have shown that after keratoplasty in allogeneic mice, the corneal stroma and endothelium have obvious aging phenotypes: the number of positive cells stained with SA-β-Gal increased significantly; the expression levels of aging-related genes (p16, p21, etc.) Significantly increased; the allogeneic mouse corneal transplantation model was constructed by using aging gene p16-deficient mice as corneal donors, and it was found that the aging-related gene p16 deletion can effectively prolong the survival time of corneal grafts and delay the immune rejection of corneal transplantation. The present invention can significantly inhibit the immune rejection of corneal transplantation by intraperitoneal injection of anti-aging drug ABT-263 (such as 1.25 mg/ml), prolong the survival time of the donor corneal graft, reduce the inflammatory reaction in the corneal graft, and maintain the corneal graft transparency. Therefore, ABT-263 can be used in the prevention and treatment of immune rejection after corneal transplantation.
图1为正常小鼠(Nor)、同系角膜移植(Syn)以及同种异体角膜移植(Allo)术后的角膜衰老表型分析，其中(A)不同组别小鼠角膜铺片的SA-β-Gal染 色分析；(B)利用冰冻切片分析角膜植片中的SA-β-Gal染色情况；(C)Western Blot分析角膜植片中衰老相关基因(p16与p21)的表达情况。Figure 1 is the corneal aging phenotype analysis of normal mice (Nor), syngeneic corneal transplantation (Syn) and allogeneic corneal transplantation (Allo), in which (A) SA-β of different groups of mouse corneal smears -Gal staining analysis; (B) Frozen section analysis of SA-β-Gal staining in corneal grafts; (C) Western Blot analysis of the expression of aging-related genes (p16 and p21) in corneal grafts.
图2为衰老基因p16敲除小鼠作为角膜供体对PK术后免疫排斥反应的影响，其中(A)角膜移植术后小鼠角膜透明情况观察；(B)不同组别小鼠的角膜植片生存曲线分析；(C)免疫荧光分析不同组别小鼠角膜植片中CD45
+细胞的浸润情况。
Figure 2 shows the effect of aging gene p16 knockout mice as corneal donors on immune rejection after PK, in which (A) observation of corneal transparency of mice after keratoplasty; (B) corneal transplantation of different groups of mice Survival curve analysis of slices; (C) Immunofluorescence analysis of CD45 + cell infiltration in corneal grafts of different groups of mice.
图3为抗衰老药物ABT-263对PK术后角膜移植免疫排斥反应的影响，其中(A)抗衰老药物ABT-263干预后，小鼠角膜透明情况分析；(B)ABT-263干预后小鼠角膜植片生存曲线分析；(C)SA-β-Gal染色分析ABT-263清除衰老细胞的效果；(D)Real-time PCR分析ABT-263干预对角膜植片中衰老相关基因表达的影响；(E)Real-time PCR分析ABT-263干预对角膜植片中主要炎症因子表达的影响。Figure 3 is the effect of anti-aging drug ABT-263 on the immune rejection of corneal transplantation after PK, wherein (A) after the anti-aging drug ABT-263 intervention, the mouse cornea transparency analysis; (B) after the ABT-263 intervention Survival curve analysis of mouse corneal grafts; (C) SA-β-Gal staining analysis of the effect of ABT-263 on eliminating senescent cells; (D) Real-time PCR analysis of the effect of ABT-263 intervention on the expression of aging-related genes in corneal grafts ; (E) Real-time PCR analysis of the effect of ABT-263 intervention on the expression of major inflammatory factors in corneal grafts.
本发明提供了ABT-263在制备抑制角膜移植免疫排斥反应的药物中的应用。The invention provides the application of ABT-263 in the preparation of medicine for inhibiting immune rejection of corneal transplantation.
在本发明中，所述ABT-263的结构式优选如式I所示：In the present invention, the structural formula of the ABT-263 is preferably as shown in formula I:
所述ABT-263的分子式为C47H55ClF3N5O6S3，分子量为974.6127096，CAS登录号为923564-51-6。本发明对所述ABT-263的来源没有特殊限制，采用本领域所熟知的ABT-263来源即可。在本发明实施例中，所述ABT-263购自Selleck公司。The molecular formula of ABT-263 is C47H55ClF3N5O6S3, the molecular weight is 974.6127096, and the CAS accession number is 923564-51-6. The present invention has no special limitation on the source of the ABT-263, and the source of ABT-263 well known in the art can be used. In the embodiment of the present invention, the ABT-263 was purchased from Selleck Company.
在本发明中，所述角膜移植优选包括同种异体角膜移植。在同种异体小鼠角膜移植术后，角膜基质及内皮中出现明显的衰老表型，其中包括SA-β-Gal染色阳性细胞的数目显著增多；衰老相关基因(p16、p21等)的表达水平明显增加。当注射ABT-263后，与模型对照组相比，显著抑制角膜移植免疫排斥反应，维持角膜植片的透明性。所述抑制角膜移植免疫排斥反应优选包括延长供体角膜植片的存活时间和降低角膜植片中的炎症反应。所述延长供体角膜植片的存活时间优选是通过ABT-263清除所述同种异体角膜中的衰老细胞，延缓角膜移植免疫排斥反应，维持角膜植片的透明性。 所述同种异体角膜优选包括角膜基质和内皮细胞。所述降低角膜植片中的炎症反应优选是降低角膜植片中炎症相关细胞因子的表达水平。所述炎症相关细胞因子优选包括IL-1β、IL-17A、TNF-α和IFN-γ。In the present invention, the corneal transplantation preferably includes allogeneic corneal transplantation. After keratoplasty in allogeneic mice, obvious aging phenotypes appeared in the corneal stroma and endothelium, including a significant increase in the number of positive cells stained with SA-β-Gal; the expression levels of aging-related genes (p16, p21, etc.) obviously increase. When ABT-263 was injected, compared with the model control group, it significantly inhibited the immune rejection of corneal grafts and maintained the transparency of corneal grafts. The suppression of immune rejection of the corneal graft preferably includes prolonging the survival time of the donor corneal graft and reducing the inflammatory response in the corneal graft. The prolonging of the survival time of the donor corneal graft is preferably to use ABT-263 to clear the senescent cells in the allogeneic cornea, delay the immune rejection of the corneal graft, and maintain the transparency of the corneal graft. The allogeneic cornea preferably includes corneal stroma and endothelial cells. Said reducing the inflammatory response in the corneal graft is preferably reducing the expression level of inflammation-related cytokines in the corneal graft. The inflammation-related cytokines preferably include IL-1β, IL-17A, TNF-α and IFN-γ.
在本发明中所述药物优选包括注射剂。所述注射剂优选包括ABT-263和医药领域可接受的辅料。所述敷料包括2-羟丙基-β-环糊精和玉米油。所述注射剂中ABT-263的浓度优选为1～10mg/ml，更优选为1～5mg/ml，最优选为1.25mg/ml。所述ABT-263的注射量为50mg/kg体重小鼠。所述ABT-263的注射频率优选为1次/2d。In the present invention, the drug preferably includes injections. The injection preferably includes ABT-263 and pharmaceutically acceptable excipients. The dressing included 2-hydroxypropyl-beta-cyclodextrin and corn oil. The concentration of ABT-263 in the injection is preferably 1-10 mg/ml, more preferably 1-5 mg/ml, most preferably 1.25 mg/ml. The injection amount of the ABT-263 is 50mg/kg body weight of mice. The injection frequency of the ABT-263 is preferably 1 time/2d.
下面结合实施例对本发明提供的ABT-263在制备抑制角膜移植免疫排斥反应的药物中的应用进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。The application of ABT-263 provided by the present invention in the preparation of drugs for inhibiting immune rejection of corneal transplantation will be described in detail below in conjunction with the examples, but they should not be construed as limiting the protection scope of the present invention.
实施例1Example 1
同种异体角膜移植后免疫排斥反应实验Immune rejection experiment after allogeneic corneal transplantation
1、将18只BALB/c小鼠(6～8周龄)随机分为如下3组：1. Randomly divide 18 BALB/c mice (6-8 weeks old) into the following 3 groups:
A.正常组(Nor)；A. Normal group (Nor);
B.同系角膜移植组(Syn-transplantation，Syn)；B. Syngeneic corneal transplantation group (Syn-transplantation, Syn);
C.同种异体角膜移植(供体为同周龄C57BL/6小鼠)组(Allo-transplantat ion，Allo)。C. Allo-transplantation (Allo-transplantation, Allo).
穿透性角膜移植模型构建如下：所有动物均选择右眼为术眼，手术前，行裂隙灯检查，了解术眼情况，并给予术眼滴用左氧氟沙星滴眼液预防感染。供体小鼠腹腔内注射戊巴比妥钠麻醉后，常规消毒铺巾，在手术显微镜下取2.25mm角膜中央全层植片，立即植入2.25mm受体小鼠角膜中央穿透植床，用11-0日本MANI尼龙线间断缝合8针，术毕前房内注入少量无菌空气，形成前房，术后2d局部常规应用氧氟沙星眼膏，每日1次。术后每天裂隙灯观察，观察期30d，将术后出现虹膜前粘、白内障或虹膜阻塞等其他眼部并发症的小鼠剔除出组。记录各组排斥反应发生的时间，排斥反应发生后不再进行观察。The penetrating keratoplasty model was constructed as follows: the right eye of all animals was selected as the operated eye. Before the operation, a slit lamp examination was performed to understand the condition of the operated eye, and levofloxacin eye drops were given to the operated eye to prevent infection. After the donor mouse was anesthetized by intraperitoneal injection of pentobarbital sodium, the 2.25mm corneal central full-thickness graft was taken under the operating microscope, and the 2.25mm central corneal penetrating implant bed of the recipient mouse was immediately implanted. 8 stitches were sutured intermittently with 11-0 Japanese MANI nylon thread. After the operation, a small amount of sterile air was injected into the anterior chamber to form the anterior chamber. On the second day after the operation, ofloxacin eye ointment was routinely applied locally, once a day. Slit lamp observation was performed every day after the operation, and the observation period was 30 days. Mice with iris anterior adhesion, cataract or iris obstruction and other ocular complications after operation were excluded from the group. The time when rejection occurred in each group was recorded, and no further observation was performed after rejection occurred.
2、第10d时，处死小鼠，分别取上述3组小鼠眼球，然后沿角巩膜缘小心剪取角膜杯，采用衰老相关的β-半乳糖苷酶(senescence associatedβ-galactosidase,SA-β-Gal)试剂盒(碧云天)行原位组织化学染色，在37℃下孵育过夜，角膜内皮面向上做染色后角膜铺片，通过显微镜观察染色情况。2. On the 10th day, the mice were sacrificed, and the eyeballs of the mice in the above three groups were removed, and then the corneal cups were carefully cut along the corneoscleral limbus, and the senescence associated β-galactosidase (SA-β-galactosidase, SA-β- In situ histochemical staining was carried out with the Gal) kit (Biyuntian), incubated overnight at 37°C, and the corneal endothelium was stained with the corneal endothelium facing up, and the staining was observed through a microscope.
结果显示：与Nor组和Syn组相比，Allo组中角膜内皮细胞呈明显的β-Gal染色阳性，且主要分布于角膜植片中央、以及角膜植片与植床的交界处(图1中A)。The results showed that compared with the Nor group and the Syn group, the corneal endothelial cells in the Allo group were significantly positive for β-Gal staining, and were mainly distributed in the center of the corneal graft and the junction between the corneal graft and the implant bed (Fig. 1 A).
将3组染色后的角膜植片进行OCT包埋后，行冰冻切片，切片厚度为7μm，然后在相差显微镜下观察各组角膜组织的β-Gal染色情况。The stained corneal grafts of the three groups were embedded in OCT, and frozen sections were made with a thickness of 7 μm. Then, the β-Gal staining of corneal tissue in each group was observed under a phase contrast microscope.
与Nor组和Syn组相比，Allo组角膜基质层存在大量β-Gal染色阳性的细胞(图1中B)。Compared with the Nor group and the Syn group, there were a large number of cells positive for β-Gal staining in the corneal stroma of the Allo group (B in Figure 1).
上述结果说明，角膜移植免疫排斥过程中，角膜植片的基质层和内皮层呈衰老表型。The above results indicated that during the immune rejection of corneal transplantation, the stroma and endothelial layer of the corneal graft exhibited an aging phenotype.
3、第10d时，处死小鼠，另取上述3组小鼠眼球，每组3个样本，剪取各组小鼠角膜植片，按照Western Blotting说明比例加入RIPA裂解液、PMSF蛋白酶抑制剂，研磨超声后提取各组样本蛋白，进行SDS-PAGE电泳结束后，将蛋白从凝胶电转移至PVDF膜上，剪取条带，分别加入兔抗小鼠p16抗体(1：1000稀释)、兔抗小鼠p21抗体(1：1000稀释)和小鼠抗小鼠β-actin抗体(1：2000稀释)，孵育、洗膜后，再分别加入羊抗兔IgG-HRP和羊抗小鼠IgG-HRP二抗，孵育、洗膜后，采用Bio-Rad快速发光照相仪，以ECL发光试剂盒进行发光。3. On the 10th day, the mice were killed, and the eyeballs of the above-mentioned 3 groups of mice were taken, 3 samples in each group, and the corneal grafts of the mice in each group were cut out, and RIPA lysate and PMSF protease inhibitor were added according to the ratio specified by Western Blotting. After grinding and ultrasonically extracting the protein of each group of samples, after SDS-PAGE electrophoresis, the protein was electrotransferred from the gel to the PVDF membrane, the band was cut, and the rabbit anti-mouse p16 antibody (1:1000 dilution), rabbit Anti-mouse p21 antibody (diluted 1:1000) and mouse anti-mouse β-actin antibody (diluted 1:2000), after incubation and washing the membrane, then add goat anti-rabbit IgG-HRP and goat anti-mouse IgG- After the HRP secondary antibody was incubated and washed, the Bio-Rad fast luminescence camera was used to perform luminescence with the ECL luminescence kit.
结果显示：在β-actin作为标准内参整齐的条件下，Allo组的角膜植片中p16和p21的蛋白表达量较Nor组和Syn组明显升高(图1中C)。鉴于p16和p21均属于细胞周期蛋白依赖激酶抑制因子(cyclin-dependent kinase inhibitor,CDKI)，是细胞衰老的关键标记物。它们的高表达提示角膜植片存在明显的衰老表型。The results showed that: under the condition that β-actin was used as a standard internal reference, the protein expression levels of p16 and p21 in the corneal grafts of the Allo group were significantly higher than those of the Nor group and Syn group (C in Figure 1). Since both p16 and p21 belong to the cyclin-dependent kinase inhibitor (cyclin-dependent kinase inhibitor, CDKI), they are key markers of cell senescence. Their high expression suggested that corneal grafts had obvious aging phenotype.
可见，角膜移植术后，同种异体角膜植片呈明显衰老表型。It can be seen that after keratoplasty, allogeneic corneal grafts showed obvious aging phenotype.
实施例2Example 2
角膜供体缺失衰老基因p16明显延长角膜植片存活时间。Deletion of aging gene p16 in corneal donors significantly prolongs the survival time of corneal grafts.
细胞衰老过程主要是通过p16INK4a/Rb和p19ARF/p53/p21Cipl两条信号通路实现的。为明确衰老在角膜移植免疫排斥中的关键作用，本发明以p16缺失的C57BL/6(Frederick National Laboratory for Cancer Research(FNL)/SAIC-Frederick,Inc)小鼠为角膜供体、Balb/C小鼠为角膜受体构建穿透性角膜移植模型，评估衰老对角膜移植免疫排斥反应的影响。Cell senescence is mainly realized through two signaling pathways, p16INK4a/Rb and p19ARF/p53/p21Cipl. In order to clarify the key role of aging in corneal transplant immune rejection, the present invention uses p16-deleted C57BL/6 (Frederick National Laboratory for Cancer Research (FNL)/SAIC-Frederick, Inc) mice as corneal donors, Balb/C small Rats were used to construct penetrating keratoplasty models for corneal recipients to evaluate the effect of aging on immune rejection of corneal transplantation.
1、将12只BALB/c小鼠(6-8周龄)随机分为如下2组：1. Randomly divide 12 BALB/c mice (6-8 weeks old) into the following 2 groups:
A.以野生型C57B/L6
WT为供体的同种异体角膜移植组(WT)；
A. Allogeneic corneal transplantation group (WT) with wild-type C57B/L6 WT as the donor;
B.以p16敲除小鼠C57B/L6
p16KO为植片供体的同种异体角膜移植组(p16KO)。
B. Allogeneic keratoplasty group (p16KO) using p16 knockout mouse C57B/L6 p16KO as graft donor.
建立穿透性角膜移植模型，手术方法如实施例1记载。A penetrating keratoplasty model was established, and the operation method was as described in Example 1.
术后每天裂隙灯观察，记录各组排斥发生的时间并绘制角膜植片排斥生存曲线。Slit lamp observation was performed every day after operation, the time of rejection in each group was recorded and the survival curve of corneal graft rejection was drawn.
研究发现：与WT组相比，p16KO组角膜植片的存活时间明显延长(图2中A和B)，初步表明角膜植片衰老可明显促进角膜移植术后的免疫排斥反应。The study found that compared with the WT group, the survival time of the corneal grafts in the p16KO group was significantly prolonged (A and B in Figure 2), preliminarily indicating that corneal graft aging can significantly promote immune rejection after keratoplasty.
2、第30d时，处死小鼠，分别取下上述2组小鼠眼球，OCT包埋后，行冰冻切片，切片厚度为7μm，然后采用抗CD45抗体进行免疫荧光染色，在免疫荧光显微镜下照相观察染色结果。2. On the 30th day, the mice were sacrificed, and the eyeballs of the above two groups were removed. After OCT embedding, frozen sections were made with a thickness of 7 μm. Immunofluorescence staining was performed with anti-CD45 antibody, and photographs were taken under an immunofluorescence microscope. Observe the staining results.
结果显示：与WT角膜移植组相比，p16KO组角膜植片中CD45
+免疫细胞的浸润明显减少(图2中C)。这些结果进一步表明p16基因敲除可有效延缓角膜移植免疫排斥反应。
The results showed that: compared with the WT corneal transplantation group, the infiltration of CD45 + immune cells in the corneal grafts of the p16KO group was significantly reduced (Figure 2, C). These results further indicated that knockout of p16 gene can effectively delay the immune rejection of corneal transplantation.
实施例3Example 3
抗衰老药物ABT-263干预治疗明显抑制角膜移植免疫排斥反应Intervention of anti-aging drug ABT-263 significantly inhibits immune rejection of corneal transplantation
1、将12只BALB/c小鼠(6-8周龄)随机分为如下2组：1. Randomly divide 12 BALB/c mice (6-8 weeks old) into the following 2 groups:
A.同种异体角膜移植组(Allo)；A. Allogeneic corneal transplantation group (Allo);
B.同种异体角膜移植并ABT-263注射组(Allo+ABT-263)。B. Allogeneic corneal transplantation and ABT-263 injection group (Allo+ABT-263).
按照实施例1记载方法建立穿透性角膜移植模型。The penetrating keratoplasty model was established according to the method described in Example 1.
术后3d，根据分组不同应用不同药物注射：A组PBS溶液，B组ABT-263(50mg/kg，1次/2d)，分别在术后第2、4、6、8、10天行腹腔注射，共注射5次。在此基础上，术后每天裂隙灯观察，并记录各组排斥反应发生的时间，绘制角膜植片生存曲线。3 days after operation, different drugs were injected according to different groups: group A PBS solution, group B ABT-263 (50mg/kg, 1 time/2d), intraperitoneal injections were performed on the 2nd, 4th, 6th, 8th, and 10th days after operation, respectively. Injection, a total of 5 injections. On this basis, slit lamp observation was performed every day after operation, and the time of rejection in each group was recorded, and the survival curve of corneal grafts was drawn.
通过统计发现：与Allo组相比，ABT-263注射组(Allo+ABT-263)角膜植片排斥时间明显延长(图3中A和B)，提示抗衰老药物ABT-263可以有效延长角膜植片存活时间。Through statistics, it was found that compared with the Allo group, the corneal graft rejection time of the ABT-263 injection group (Allo+ABT-263) was significantly prolonged (A and B in Figure 3), suggesting that the anti-aging drug ABT-263 can effectively prolong the corneal graft rejection time. slice survival time.
2、第30d时，处死小鼠，分别取下上述2组小鼠眼球，然后沿角巩膜缘小心剪取角膜杯，采用衰老相关SA-β-Gal试剂盒(碧云天)行原位组织化学染色，37℃孵育过夜，角膜内皮面向上做染色后角膜铺片，在显微镜下观察染色情况。2. On the 30th day, the mice were killed, and the eyeballs of the above two groups were removed, and then the corneal cup was carefully cut along the corneoscleral limbus, and the in situ histochemistry was performed using the aging-related SA-β-Gal kit (Beiyuntian) For staining, incubate overnight at 37°C. The endothelium of the cornea is stained upwards, and the corneal slices are spread, and the staining is observed under a microscope.
结果见图3中A和B。结果显示：与Allo组相比，经ABT-263干预治疗后，ABT-263注射组中角膜植片内皮层β-Gal染色阳性明显减少。通过冰冻切片并结合相差显微镜观察，结果表明：经ABT-263干预治疗后，角膜基质层中SA-β-Gal染色阳性的细胞数目明显低于Allo组。The results are shown in Figure 3, A and B. The results showed that compared with the Allo group, after ABT-263 intervention, the positive staining of β-Gal in the endothelial layer of corneal grafts in the ABT-263 injection group was significantly reduced. The results of frozen section combined with phase-contrast microscope observation showed that after ABT-263 intervention, the number of cells positive for SA-β-Gal staining in the corneal stroma was significantly lower than that in the Allo group.
以上结果表明ABT-263可有效清除角膜植片中的衰老细胞。The above results indicate that ABT-263 can effectively remove senescent cells in corneal grafts.
3、第30d时，处死小鼠，分别取下正常对照组(Nor)、未用药组(Allo)和药物干预组(Allo+ABT-263)小鼠眼球，每组3个样本，剪取各组小鼠角膜植片，Trizol法分别提取各组角膜组织的总mRNA，逆转录试剂盒将mRNA逆转录为cDNA，将H
2O、SYBRPrimix Ex TaqⅡ和引物按照比例混匀，加入各组cDNA，荧光定量PCR检测各组角膜植片中衰老相关基因(p16、p21)以及炎症相关因子(IL-1β、IL-17A、TNF-α和IFN-γ)的表达情况(详见附表1～3)。
3. On the 30th day, the mice were killed, and the eyeballs of the mice in the normal control group (Nor), the non-medication group (Allo) and the drug intervention group (Allo+ABT-263) were removed respectively, and 3 samples were cut from each group. Group mouse corneal grafts, Trizol method to extract the total mRNA of corneal tissue of each group, reverse transcription kit reverse mRNA into cDNA, mix H 2 O, SYBRPrimix Ex Taq Ⅱ and primers according to the proportion, add the cDNA of each group, The expressions of aging-related genes (p16, p21) and inflammation-related factors (IL-1β, IL-17A, TNF-α, and IFN-γ) in the corneal grafts of each group were detected by fluorescent quantitative PCR (see attached tables 1-3 for details). ).
表1小鼠RT-qPCR引物核苷酸序列Table 1 Nucleotide sequences of mouse RT-qPCR primers
表2PCR反应体系表Table 2 PCR reaction system table
表3PCR反应程序Table 3 PCR reaction program
结果所显示的Ct值(Threshold cycle)定义为达到超出荧光信号的检测阈值时所需要的循环数，结果的数据利用QIAGEN公司的ROTOR-GeneTMQ Series Software(v1.2.0.)进行分析，以2
-ΔΔCt的方法计算，以GAPDH作为内参。
The Ct value (Threshold cycle) shown in the result is defined as the number of cycles required to reach the detection threshold of the fluorescent signal, and the resulting data is analyzed using ROTOR-GeneTMQ Series Software (v1.2.0.) of QIAGEN Company, with 2 - ΔΔCt was calculated using GAPDH as an internal reference.
统计学分析Statistical analysis
利用GraphPad Prism 8.0软件进行统计学分析，数据以均数±标准差表示，多组比较使用单因素方差(One-WayAnova)分析，*P<0.05，**P<0.01,***P<0.001视为有显著性差异。GraphPad Prism 8.0 software was used for statistical analysis, and the data were expressed as mean ± standard deviation. Multiple groups were compared using One-Way Anova analysis, *P<0.05, **P<0.01, ***P<0.001 considered to be significantly different.
与Nor和Allo组相比，经ABT-263干预治疗后，角膜植片中衰老相关基因(p16与p21)表达水平明显下降，提示ABT-263干预治疗可明显清除衰老细胞。进一步发现，经ABT-263干预治疗后，角膜植片中炎症相关细胞因子的表达水平均明显低于Allo组，差异有统计学意义。Compared with the Nor and Allo groups, the expression levels of senescence-related genes (p16 and p21) in corneal grafts were significantly decreased after ABT-263 intervention, suggesting that ABT-263 intervention can significantly clear senescent cells. It was further found that after ABT-263 intervention, the expression levels of inflammation-related cytokines in corneal grafts were significantly lower than those in the Allo group, and the difference was statistically significant.
以上结果表明清除衰老细胞可有效延缓角膜移植免疫排斥反应。The above results indicated that removing senescent cells could effectively delay the immune rejection of corneal transplantation.
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.
Claims (10)
- ABT-263在制备抑制角膜移植免疫排斥反应的药物中的应用。Application of ABT-263 in preparation of medicine for inhibiting immune rejection of corneal transplantation.
- 根据权利要求1所述应用，其特征在于，所述ABT-263的结构式如式I所示：According to the described application of claim 1, it is characterized in that, the structural formula of described ABT-263 is as shown in formula I:
- 根据权利要求1所述应用，其特征在于，所述角膜移植包括同种异体角膜移植。The use according to claim 1, characterized in that the corneal transplantation comprises allogeneic corneal transplantation.
- 根据权利要求1或3所述应用，其特征在于，所述抑制角膜移植免疫排斥反应包括延长供体角膜植片的存活时间和降低角膜植片中的炎症反应。The use according to claim 1 or 3, characterized in that said inhibiting corneal graft immune rejection includes prolonging the survival time of the donor corneal graft and reducing the inflammatory response in the corneal graft.
- 根据权利要求4所述应用，其特征在于，所述延长供体角膜植片的存活时间是通过ABT-263清除衰老细胞，延缓角膜移植免疫排斥反应，维持角膜植片的透明性。The application according to claim 4, characterized in that, prolonging the survival time of the donor corneal graft is to remove senescent cells through ABT-263, delay corneal graft immune rejection, and maintain the transparency of the corneal graft.
- 根据权利要求4所述应用，其特征在于，所述降低角膜植片中的炎症反应是降低角膜植片中炎症相关细胞因子的表达水平。The application according to claim 4, characterized in that said reducing the inflammatory response in the corneal graft is reducing the expression levels of inflammation-related cytokines in the corneal graft.
- 根据权利要求6所述应用，其特征在于，所述炎症相关细胞因子包括IL-1β、IL-17A、TNF-α和IFN-γ。The use according to claim 6, wherein the inflammation-related cytokines include IL-1β, IL-17A, TNF-α and IFN-γ.
- 根据权利要求1～3和5～7任意一项所述应用，其特征在于，所述药物包括注射剂。The use according to any one of claims 1-3 and 5-7, characterized in that the medicine includes injections.
- 根据权利要求8所述应用，其特征在于，所述注射剂中ABT-263的浓度为1～10mg/ml。The application according to claim 8, characterized in that the concentration of ABT-263 in the injection is 1-10 mg/ml.
- 根据权利要求9所述应用，其特征在于，所述ABT-263的浓度为1～5mg/ml。The application according to claim 9, characterized in that the concentration of the ABT-263 is 1-5 mg/ml.
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