阅读说明：本技术 一种用于角膜移植免疫排斥反应治疗的药物 (Medicine for treating immunological rejection reaction of cornea transplantation ) 是由 接英 李上 臧云晓 潘志强 杨珂 田磊 于 2020-04-29 设计创作，主要内容包括：本发明涉及CD4+LAP+Tregs细胞或能够调控其表达及功能的ROCK2蛋白抑制剂在治疗角膜移植免疫排斥反应中的应用。本发明利用CD4+LAP+Tregs细胞通路治疗角膜移植免疫排斥反应,相较于现有的NKT细胞通路,更加具有针对性,也更为安全、有效。尤其使用ROCK2蛋白抑制剂治疗,可以避免直接细胞治疗过程中所注入的细胞对于器官其他的副作用,具有良好的临床应用价值。(The invention relates to application of CD4+ LAP + Tregs cells or ROCK2 protein inhibitor capable of regulating and controlling expression and functions thereof in treating corneal transplantation immune rejection. The invention utilizes the CD4+ LAP + Tregs cell pathway to treat the corneal transplantation immune rejection, and has more pertinence, safety and effectiveness compared with the existing NKT cell pathway. Particularly, when the ROCK2 protein inhibitor is used for treatment, the injected cells in the direct cell treatment process can be prevented from having other side effects on organs, and the method has good clinical application value.)

1. A pharmaceutical composition for treating immune rejection in corneal transplantation, comprising: the pharmaceutical composition comprises CD4+ LAP + Tregs cells.

2. The pharmaceutical composition of claim 1, wherein the CD4+ LAP + Tregs cells are isolated from the spleen of a Balb/c mouse.

Use of CD4+ LAP + Tregs cells in the preparation of a pharmaceutical composition for the treatment of immune rejection in corneal transplantation.

4. A pharmaceutical composition for treating immune rejection in corneal transplantation, comprising: the pharmaceutical composition comprises a ROCK protein inhibitor.

5. The pharmaceutical composition of claim 4, wherein: the ROCK protein is ROCK2 protein.

6. The pharmaceutical composition according to any one of claims 4 to 5, wherein: the inhibitor is selected from the group consisting of the siRNA, an antibody, an organic compound; preferably KD 025.

Use of a ROCK protein inhibitor for the preparation of a pharmaceutical composition for the treatment of immune rejection in corneal transplantation.

8. The use of claim 7, wherein the ROCK protein is ROCK2 protein.

9. The use according to any one of claims 7 to 8, wherein the inhibitor is selected from the group consisting of the siRNA, an antibody, an organic compound; preferably KD 025.

Technical Field

The invention relates to the field related to immunosuppression in corneal transplantation, in particular to a method for inducing corneal transplantation immune tolerance and inhibiting the generation of corneal transplantation immune rejection by using regulatory T cells or ROCK protein inhibitors.

Background

Corneal blindness is a type of ocular disease in which visual function is lost due to corneal dysfunction; the cornea is usually damaged to different degrees, and turbidity, rupture or infection occurs due to diseases or trauma of the eye. Generally, the causes of corneal blindness are mainly: infectious keratopathy, non-infectious keratitis, corneal degeneration and corneal dystrophy, keratoconus, ocular trauma, eyelid injury, dry eye, etc. Because the cornea lesion or the corneal trauma only loses the transparency of the cornea and becomes turbid, and the internal structure of the eyeball is normal, if the pathological cornea can be replaced by the transparent healthy cornea, the vision of a patient can be obviously improved, and therefore, the corneal blindness can be cured.

According to the statistics of the world health organization, the cornea blind patients in the world can reach 6000 million people, and about 500 million people exist in China, and the cornea blind patients grow on a scale of more than 10 million people every year, and are ranked the second position of the eye disease. Corneal transplantation is still the most important means for treating such disorders; however, clinically, there is a great difference between the supply and demand of corneal graft materials, and corneal sources all over the world are scarce, while China is very rare, and many patients can only wait for donation passively. In order to solve the corneal insufficiency problem, researchers around the world have made various efforts to develop alternative novel graft materials. With the breakthrough of some key technologies, corneal suppression is raising new storm and becoming an ideal corneal transplantation substitute material. Currently, porcine corneas have been successfully used for human eye transplantation; however, when the cornea is used for transplantation, immunological rejection after the operation is a main cause of operation failure.

Although normal corneal tissue has no blood vessels and lymphatic vessels and is in a relatively immune-privileged state, due to the existence of factors such as inflammatory reaction, neovascularization and the like of pathological corneal tissue, rejection reaction is easy to occur after corneal transplantation, and operation failure is caused.

At present, the immunological rejection reaction after the corneal transplantation in clinic is mainly treated by medicaments, and generally needs to be used for a long time. Among them, glucocorticoids and immunosuppressants (such as cyclosporin and tacrolimus) are most widely used. Applications for these drugs include both local and systemic applications. For the whole body application, the long-term use of the medicines can cause serious side effects of liver and kidney injury, infection, tumor and the like; for topical application to the eye, drugs such as glucocorticoids also cause complications such as hormonal glaucoma, cataract, dry eye, and are also not conducive to long-term survival of corneal implants. Therefore, the research of a more targeted treatment mode aiming at the generation mechanism of the corneal transplantation immunological rejection has positive significance. In the field related to corneal transplantation, some research progress has been made. Researches indicate that TGF-beta eye drops secreted by regulatory T cells can effectively prevent and treat corneal transplantation rejection and prolong the survival time of corneal transplants.

However, prior to this application, CD4 was not studied⁺LAP⁺Tregs cells were used for immune rejection suppression of corneal transplantation, and the CD4 was not studied⁺LAP⁺The regulation mechanism of Tregs cells for regulating immune rejection is researched; this application not only validates CD4⁺LAP⁺Tregs cells maintain corneal transplantation immune tolerance and have the function in the aspect of transplantation immune rejection, systematic research is also carried out on the regulation path, and the regulation of ROCK protein (mainly ROCK2 protein) is found to realize effective inhibition on corneal transplantation rejection and prolong the survival time of corneal transplantation.

Disclosure of Invention

Regulatory T cells (Tregs) are a subset of T cells with unique immunoregulatory functions, including CD4⁺CD25⁺T cells, helper T cells type III, etc., which can suppress pathological or physiological immune response activity and play an important role in maintaining autoimmune tolerance and immune homeostasis. The past research has focused on CD4⁺CD25⁺T-cells, CD4, which have recently been used as cell surface markers for latent-associated peptides (LAPs)⁺LAP⁺Tregs cells were identified. Some studies found CD4⁺LAP⁺Tregs cells are considered to be novelHas immunosuppressive effects. LAP acts as a molecular chaperone for TGF-. beta.and can release LTGF-. beta.which is TGF-. beta.bound to LAP on the cell surface. Alessandra Metelli and other researches find that transmembrane protein on the surface of Tregs is combined with LTGF-beta, and active TGF-beta is released through hydrolysis of LAP to promote Tregs to play a negative immune regulation role. Zhang L et al, for example, have found that the occurrence of an immune response can be suppressed by inducing the expression of Tregs surface LAPs in Peripheral Blood Mononuclear Cells (PBMCs) of patients with systemic lupus erythematosus. The research of da Cunha AP and the like finds that the expression of LAP is blocked and CD4 can be reduced in an animal model of experimental autoimmune encephalomyelitis⁺LAP⁺Tregs cells are expressed and antagonize immune tolerance induced by oral CD3 antibody. However, CD4 is not currently available⁺LAP⁺Studies relating to the role of Tregs cells in the field of corneal transplantation.

As is known, corneal tissue has no blood vessels and lymphatic vessels and is in a relative immune-privileged state, but due to the existence of factors such as inflammatory reaction, neovascularization and the like of pathological corneal tissue, rejection reaction can occur after corneal transplantation; that is, rejection of corneal transplantation is distinguished from other transplantation operations. Therefore, agents that can function as transplants related to or cause immune reactions to occur in other organ or tissue transplantation fields are not expected to be equally applicable in the corneal transplantation field.

The present inventors have been working on research and practice in the field of corneal transplantation, and in previous studies, the mechanism of action of natural killer T cells on corneal transplantation immunosuppression was confirmed. However, natural killer T cells can cause strong local side effects in corneal transplantation immunotherapy, and therefore, there is a need to find a new and more targeted corneal transplantation regulatory pathway and drug. After a great deal of research and experiments, the inventor of the application discovers that CD4 is not good when corneal graft rejection occurs by establishing a mouse allogeneic corneal transplantation model⁺LAP⁺The proportion of Tregs cells is obviously reduced, and the quantity of various Tregs cells can be reduced by using the monoclonal antibody of anti-LAP to inject the monoclonal antibody into a mouse body in an intraperitoneal mode. And confirmed in further studies， CD4⁺LAP⁺Corneal transplantation immunosuppressive function of Tregs cells in vitro and in vivo. CD4 as compared to modulation of natural killer T cells⁺LAP⁺Tregs cells have stronger graft immune regulation pertinence, and local side effects of T cell treatment are avoided.

In order to provide more excellent cornea transplantation immune drug selection, the inventor further provides the CD4⁺LAP⁺Regulatory pathways of Tregs cells have been studied. Through extensive research and experiments, the inventors of the present application demonstrated that ROCK protein (especially ROCK2 protein) is capable of regulating CD4⁺LAP⁺A key factor in Tregs cell expression.

Rho proteins are shown to belong to members of subfamilies of the small G protein superfamily, and up to now more than 20 Rho family members have been found, of which Rho associated coiled coil forming protein kinase (ROCK) is one of the most important. ROCK proteins mainly include two proteins, ROCK1 and ROCK 2. ROCK proteins regulate a variety of key cellular functions, including cell proliferation, migration and activity. The inventor of the application proves that ROCK protein inhibitor (especially ROCK2 protein inhibitor) can remarkably enhance CD4 through experiments⁺LAP⁺Tregs cells have the function of inhibiting immune response and prolong the survival time of the implant in corneal transplantation.

Accordingly, it is an object of the present invention to provide a method for using CD4⁺LAP⁺Tregs cells inhibit immunological rejection reaction in the corneal transplantation operation, maintain the corneal transplantation in an immune tolerance state, and improve the survival time of the graft.

Meanwhile, the invention provides a CD4⁺LAP⁺Tregs cell regulates the pathway, and CD4 is realized by regulating different proteins in the pathway⁺LAP⁺The regulation of Tregs cell expression further plays a role in inhibiting immune rejection in corneal transplantation operation, maintaining the immune tolerance state of corneal transplantation and improving the survival time of the implant.

Preferably, the present invention achieves targeting of said CD by using ROCK inhibitors4⁺LAP⁺The high expression regulation of Tregs cells further realizes the effects of inhibiting immune rejection in the corneal transplantation operation, maintaining the corneal transplantation in an immune tolerance state and improving the survival time of the graft; most preferably, the inhibitor is a ROCK2 protein inhibitor.

Accordingly, the present invention also provides a pharmaceutical composition comprising CD4⁺LAP⁺Tregs cells, the composition can be used for inhibiting immunological rejection reaction in corneal transplantation operation, maintaining corneal transplantation in an immune tolerance state and improving survival time of a graft. Preferably, the pharmaceutical composition further comprises physiological saline.

The CD4 of the invention⁺LAP⁺Methods for preparing Tregs cells include, but are not limited to: obtained by sorting from spleens of normal Balb/c mice and can be obtained by further amplification in vitro.

Further, the present invention provides a pharmaceutical composition comprising a ROCK protein inhibitor, which is also capable of modulating CD4⁺LAP⁺Tregs cells are highly expressed, so that immunological rejection in a corneal transplantation operation is inhibited, the immune tolerance state of corneal transplantation is maintained, and the survival time of the implant is prolonged.

The ROCK protein inhibitor provided by the invention comprises but is not limited to antibodies, siRNA, small molecule compounds with inhibitory activity, short peptides and the like, which can reduce and/or prevent the expression and/or activity of the ROCK protein.

Preferably, the ROCK protein inhibitor is a ROCK2 protein inhibitor, more preferably, the ROCK2 protein inhibitor is KD 025. KD025 is a selective oral inhibitor of ROCK2 and has the following molecular structure:

drawings

FIG. 1 ROCK2 protein siRNA pair CD4⁺LAP⁺A comparison graph of the in vitro immunosuppression rate of Tregs cells;

FIG. 2: graph of KD025 treatment of corneal graft rejection in mice: (A) comparison of survival time of post-operative graft, and (B) comparison of post-operative 8-week graft.

Detailed Description

The following examples further illustrate the present invention but should not be construed as limiting the invention and modifications or alterations to the methods, procedures and conditions of the present invention may be made without departing from the spirit and substance of the invention.

Example 1: CD4⁺LAP⁺Preparation of Tregs cells

Spleen of Balb/c mouse is separated aseptically, and is filtered by a 200-mesh filter screen to prepare cell suspension. According to mouse CD4⁺LAP⁺Tregs cell isolation kit for isolating CD4⁺LAP⁺Tregs cells, analyzing the purity of the CD4+ LAP + Tregs cells by a flow cytometer and separating the separated CD4⁺LAP⁺Tregs cell counts. Adjusting the cell concentration to 1X 10⁶Perml, CD3/28 antibody beads (2: 1 cell to bead ratio) were added and IL-2 was added at a concentration of 1000ng/ml for incubation. On days 5-7 of culture, anti-CD4 FITC, anti-LAP PerCP-Cy5.5 staining was performed, and CD4 was sorted using a flow cytometer⁺LAP⁺Tregs cells are purified and tested after being sorted, and the expression of LAP in an in vitro culture amplification group can be (85.76 +/-5.01)%.

Example 2: construction of mouse cornea transplantation rejection reaction model

BALB/C (H-2d) mice were used as recipients, C57BL/6 (H-2b) mice were used as donors, and the donor mice were all male, 8-10 weeks old. A mouse corneal transplantation model was prepared and CD4 was verified as follows⁺LAP⁺Role of Tregs cells:

(1) mouse anesthesia: 4% of hydrated chlorine is adopted for intraperitoneal injection. After anesthesia, foreign bodies in the oral cavity of the mouse are removed, and the horizontal position is kept. Care was taken during the anesthesia to keep the mice warm.

(2) Mouse corneal transplantation: the operation method adopts 11-0 pure intermittent suture. Preparing the implant: one hour before C57 mouse operation, compound tropicamide mydriasis, 2.5mm drill change, take the implant, micro-tweezers cut along the mark, pay attention not to touch the iris. The anterior chamber can be maintained with a viscoelastic. The plants were removed and placed in PBS for use. Preparing a planting bed: 1 hour before Balb/c mice operation, compound tropicamide mydriasis, 2mm trephine is used for taking a graft, a 1ml needle is used for anterior chamber puncture, and viscoelastic agent is injected into the anterior chamber. And (3) removing the implant along the mark by the micro forceps, wherein the transplanting process comprises the following steps: the implant is put in, 8 needles are sewed along the line 11-0 to the implant bed along the edge of the implant, and the cut is watertight/airtight. The eye is spotted with ointment for surgical occlusion, and the eyelid is sutured with 8-0 thread.

The experiment was divided into a group A (homologous corneal transplantation group) and a group B (allogeneic corneal transplantation group). Group a, homologous group: BALB/c (H-2d) mice served as recipients and donors, respectively. Group B homogeneous heterogeneous group: BALB/C (H-2d) mice were used as recipients and C57BL/6 (H-2b) mice were used as donors.

After the operation for 1 day, the eye suture is removed and observed under a slit lamp microscope. The observation was continued for 1 to 3 days after the operation, 1 week, 2 weeks, 3 weeks, and 4 weeks after the operation.

Corneal rejection criteria: (see Holland score criteria) rejection was considered when total score of the graft was 5 points or turbidity score was 2 points. Exclusion criteria: iris incarceration, anterior iris adhesions, loss of implants, dislocation of lens, hyphema, endophthalmitis after surgery. After 28 days of corneal transplantation, corneal graft rejection does not occur, and immune tolerance is considered to be achieved; corneal graft rejection occurs and immune rejection is thought to be achieved.

The specific scoring standard comprises a plant opacity score, a plant edema score and a plant neovascularization growth score; the total score of the three is the total score of the graft, and the specific score is given below (see Holland EJ, Chan CC, Wetzig RP, et al. clinical and biochemical students of the probabilistic evaluation model. Cornea, 1991, 10 (5): 374-) 380.):

(1) corneal implants were divided into 0-5 points according to the cloudiness, as follows:

(2) corneal implants were divided into 0-3 points according to the edema of the implant, as follows:

(3) according to the growth condition of the new blood vessels of the implant, the cornea implant is divided into 0 to 3 parts, and the standard is as follows:

example 3: CD4⁺ LAP⁺Tregs cell treatment for corneal transplantation rejection of mice

After the completion of the corneal transplantation operation in the mice of example 3, 10. mu.l of 5X10 was injected into each group of mice⁵/ml CD4 prepared in example 1⁺LAP⁺Tregs cells; and with no injection of CD4⁺LAP⁺Allogeneic corneal transplant mice with Tregs cells were controls. The results show that CD4 was injected⁺LAP⁺The survival time of the Tregs cell mice of the transplant is obviously prolonged, and compared with the control group, the survival time of the transplant is averagely prolonged by 15 +/-1.32 days.

Example 4: ROCK2 protein siRNA to CD4⁺LAP⁺Tregs cell in-vitro immunosuppression function detection

CD4⁺Effector T cells are T cells involved in transplant immune rejection, which recognize the MHC2 receptor on the surface of an antigen primarily through a direct or indirect pathway, and exert immune functions through secretion of lymphokines or direct killing of cells. CD4⁺LAP⁺Tregs cells are novel regulatory T cells, are related to transplantation immune tolerance and mainly play an immunosuppressive role. CD4⁺LAP⁺Tregs cells for CD4⁺Inhibition of effector T cell proliferation embodies CD4⁺LAP⁺Regulatory capacity of Tregs cells for transplantation. The higher the inhibition rate, the more immune tolerance the graft immunity is. CD4 prepared in example 1 was acted on by siRNA in vitro interference method using ROCK2 protein⁺LAP⁺Tregs cells; observation vs. CD4⁺LAP⁺Tregs cell pair CD4⁺Effect of the proliferation inhibitory function of effector T cells.

CD4 prepared in example 1⁺LAP⁺Tregs cells, concentration adjusted to 1X 10⁵Individual cell with CD4⁺Effector T cells were co-cultured at different ratios (1: 1, 1: 2, 1: 4, 1: 8) for 3 days, to which siRNA against ROCK2 protein was added, and the group to which siRNA was not added served as a control. Cell proliferation was measured by CFSE method, and the inhibition rate was calculated as% = [ (proliferation of effector T cell without siRNA addition-proliferation of effector T cell with siRNA addition)/proliferation of effector T cell without siRNA addition]X 100%, the specific results are shown in FIG. 1. From the results in FIG. 1, it can be seen that the siRNA of ROCK2 regulates CD4 at different cell ratios⁺LAP⁺The inhibition effect of Tregs cells on effector T cells is stronger than that of CD4+ LAP + Tregs cells which are added with siRNA of ROCK2 in the same proportion, and the significant difference is realized (P is<0.05), especially when ROCK2 siRNA modulates CD4⁺LAP⁺When the proportion of the Tregs cells to the effector T cells is 1: 1 and 1: 2, the inhibition effect on the proliferation of the effector T cells is more obvious.

Example 5: ROCK2 protein inhibitor KD025 for treating corneal transplantation rejection of mice

Allogeneic cornea transplantation mice (the mouse model prepared in example 2) are injected with KD025 (150 mg/kg) to the abdominal cavity of the mice after operation as an experimental group, PBS is injected to the abdominal cavity of the mice as a control group, meanwhile, the allogeneic transplantation group is designed to eliminate the influence of operation on the survival of the graft, each group is provided with 10 mice, and the KD025 group is found to obviously prolong the survival time of the graft. The survival time of the mouse graft of each group was analyzed by using Kaplan-meier survival curve as shown in FIG. 2. As shown in fig. 2 (a), the survival rates of the experimental group and the control group decreased gradually with time after the corneal transplantation, but the survival curves of the experimental group and the control group were always above the control group, and the survival rate of the experimental group and the control group was still 50% at 8 weeks. Thus suggesting that injecting KD025 (150 mg/kg) intraperitoneally into mice after surgery has a positive effect on the survival rate of the grafts. The external eye image of the implant at 8 weeks after operation is shown in fig. 2 (B), the PBS control group implant is edematous and thickened, and the implant remains transparent after KD025 treatment; the difference is very significant. The experimental result indicates that KD025 can effectively prolong the survival time of the graft transplanted by the mouse allogeneic cornea.