KR20220096051A - Transplant rejection avatar animal model, method of making the same, and use thereof - Google Patents
Transplant rejection avatar animal model, method of making the same, and use thereof Download PDFInfo
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- KR20220096051A KR20220096051A KR1020200188174A KR20200188174A KR20220096051A KR 20220096051 A KR20220096051 A KR 20220096051A KR 1020200188174 A KR1020200188174 A KR 1020200188174A KR 20200188174 A KR20200188174 A KR 20200188174A KR 20220096051 A KR20220096051 A KR 20220096051A
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- animal model
- transplant rejection
- transplantation
- cells
- transplant
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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Abstract
Description
본 발명은 이식 거부 반응 아바타 동물모델, 이의 제작 방법 및 이의 용도에 관한 것이다.The present invention relates to a transplant rejection avatar animal model, a method for manufacturing the same, and a use thereof.
이식(transplantation)이란 한 개체로부터 세포나 조직 혹은 기관, 즉 이식편(graft)를 취하여 다른 개체로 이를 전이시키는 과정을 말한다. 이식편을 제공한 개체를 공여자(donor)라 하며, 이를 받는 개체를 수용자(recipient) 혹은 숙주(host)라고 한다. 이식된 장기의 경우에는 이식편의 세포표면에 있는 조직적합성항원(이식항원)에 대하여 면역학적 반응에 의해 거부반응이 발생한다. 면역 억제되지 않은 수용자에서 이식편이 장기간 생착되는 경우는 조직적합성이 완전히 일치하거나 대부분이 일치할 때에 한하며, 공여자와 수용자간의 유전학적 관계가 이식편의 생착기간을 크게 좌우하는 인자가 된다. 일반적으로 자가이식(autograft)과 동계이식(isograft)에서는 거의 거부반응이 발생하지 않으나, 이종이식(allograft)에서는 거의 대부분에서 거부반응이 발생한다.Transplantation refers to the process of taking cells, tissues, or organs, ie, grafts, from one individual and transferring them to another. The individual who provides the graft is called a donor, and the individual who receives it is called the recipient or host. In the case of transplanted organs, rejection occurs due to an immunological reaction to the histocompatibility antigen (transplant antigen) on the cell surface of the graft. Long-term engraftment of grafts from recipients who are not immunosuppressed is limited to cases with complete or almost identical histocompatibility. In general, rejection rarely occurs in autografts and isografts, but rejection occurs in almost all cases in allografts.
조직이나 기관 등의 이식시 공여자와 수여자 간의 유전적 상이성은 쉽게 숙주 면역 체계에 의해 감지되어 이식 조직에 대한 숙주의 반응(host-versus-graft response) 및/또는 숙주에 대한 이식 조직의 반응(graft-versus-host response)을 유발한다. 이러한 사실은 조직과 기관 이식시 나타나는 거부 반응에 의해 입증되었다(Nash 등, Blood, 80, 1838 - 1845, 1992). 또한, 이식된 세포의 표면에 존재하는 MHC에 대한 면역 반응으로 활성화된 T 세포들에 의해 타가이식된 조직의 거부반응이 일어난다는 보고가 있었다(Benichou 등, J. Exp. Med. 175, 305 - 308, 1992; Benichou 등, J. Immunol. 162, 352 - 358, 1998; Fangmann 등, J. Exp. Med. 175, 1521 - 1529, 1992; Lombardi 등, Proc. Acad. Sci. USA, 86 , 4190 - 4194, 1989).During transplantation of tissues or organs, genetic differences between donors and recipients can be easily detected by the host immune system, resulting in host-versus-graft response and/or response of the graft to the host ( graft-versus-host response). This fact was evidenced by the rejection of tissue and organ transplantation (Nash et al., Blood, 80, 1838 - 1845, 1992). In addition, it has been reported that the rejection of the allografted tissue occurs by activated T cells as an immune response to MHC present on the surface of the transplanted cells (Benichou et al., J. Exp. Med. 175, 305 - 308, 1992; Benichou et al., J. Immunol. 162, 352 - 358, 1998; Fangmann et al., J. Exp. Med. 175, 1521 - 1529, 1992; Lombardi et al., Proc. Acad. Sci. USA, 86, 4190 - 4194, 1989).
한편, 성공적인 장기 이식을 위해서는 이식할 세포 및 장기에 대한 수혜자의면역 거부반응을 극복해야 한다.On the other hand, for successful organ transplantation, it is necessary to overcome the recipient's immune rejection of the cells and organs to be transplanted.
이식면역 거부반응의 주요 매개체는 T 세포로서, 이식편(graft)에 발현되어져 있는 주조직적합성분자(major histocompatibility complex, MHC)를 T 세포 수용체(T cell receptor)가 인지함으로써 면역반응이 유도되어 이식 거부반응이 발생하게 된다.The main mediator of transplant immune rejection is T cells, and T cell receptors recognize major histocompatibility complex (MHC) expressed in the graft, thereby inducing an immune response and rejecting the transplant. reaction will occur.
최근 외과적 시술 및 HLA 유형(type) 판독기법의 발달과 면역억제제의 개발에 의해 이식 성공률은 높아졌지만 여전히 면역거부반응과 면역억제제의 부작용에 의한 사망률이 높아 효과적이고 안전한 새로운 면역억제제의 개발이 요구되고 있다. 기존에 사용하고 있는 모든 면역억제제들의 공통된 목적은 이식편에 대한 T 세포-매개 면역반응을 억제하는 것으로서, 임상적으로 이식 후 T 세포-매개 급성거부반응을 막기 위해 매일 비특이적 면역억제제가 투여된다(Pirsch, J. D., curr. opin. organ. transplant., 2, 76-81, 1997). 일반적으로 사용되는 면역억제제는 글루코코티코스테로이드(glucocoticosteroids)을 포함하여 DNA 합성을 차단하여 T 세포의 증식을 억제하는 아자치오프린(azathioprine)과 미코페놀레이트 모페틸(mycophenolate mofetil)이 있으며, 칼시뉴린 억제제(calcineurin inhibitor)인 사이크로스포린 A(cyclosporine A)와 타크로리무스(tacrolimus) 등이 있다.Although the success rate of transplantation has increased due to the recent development of surgical procedures and HLA type reading techniques and the development of immunosuppressants, there is still a high mortality rate due to immune rejection and side effects of immunosuppressants, so the development of effective and safe new immunosuppressants is required. is becoming The common purpose of all existing immunosuppressants is to suppress the T cell-mediated immune response to the graft. Clinically, non-specific immunosuppressants are administered daily to prevent T cell-mediated acute rejection after transplantation (Pirsch). , J. D., curr. opin. organ. transplant., 2, 76-81, 1997). Commonly used immunosuppressants include glucocoticosteroids, azathioprine and mycophenolate mofetil, which block DNA synthesis to inhibit T cell proliferation, and calcineurin inhibitors. (calcineurin inhibitors) such as cyclosporine A and tacrolimus.
이들 약재는 비록 장기이식을 수혜 받은 환자의 면역거부반응을 극복하는데 많은 발전을 이루었지만 치료효과가 일시적이고 높은 독성이 나타나는 문제점을 가지고 있다. 따라서 이식 거부 반응을 억제하기 위한 면역억제제의 개발도 중요하지만 뚜렷한 치료 효과를 보이는 면역억제제의 개발이 미흡한 현 시점에서 면역억제제의 투여로 인해 발생하는 부작용을 최소화 시킬 수 있는 가장 효과적이면서도 빠른 방법은 환자의 면역체계와 적합한 면역억제제를 투여하는 것이다.Although these drugs have made great strides in overcoming the immune rejection response of patients receiving organ transplantation, they have a problem in that their therapeutic effect is temporary and their toxicity is high. Therefore, although the development of immunosuppressive agents to suppress transplant rejection is important, the most effective and quick way to minimize the side effects caused by the administration of immunosuppressants is to of the immune system and appropriate immunosuppressive agents.
그러나 이식 받은 환자에게, 환자 체내에서의 면역 상태를 확인하여, 면역 상태에 따른 면역억제제를 처리할 수 있는 방법이 개발된다면 면역억제제로 인한 환자의 고통을 감소시킬 수 있을 것이다.However, if a method is developed that can treat the immunosuppressive agent according to the immune status by checking the immune status in the patient's body, it will be possible to reduce the patient's pain due to the immunosuppressant agent.
본 발명의 목적은, 면역결핍 마우스에 이식 거부 반응 환자 유래 PBMC(Peripheral blood mononuclear cell)이 투여된, 인간화된 이식 거부 반응 동물 모델을 제공하는 것이다.It is an object of the present invention to provide a humanized transplant rejection animal model in which immunodeficient mice are administered with PBMCs (peripheral blood mononuclear cells) derived from transplant rejection patients.
본 발명의 다른 목적은, 면역결핍 마우스에 이식 거부 반응 환자로부터 분리한 PBMC를 주입하는 단계를 포함하는 인간화된 이식 거부 반응 동물 모델을 제작하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for preparing a humanized transplant rejection animal model comprising injecting PBMCs isolated from transplant rejection patients into immunodeficient mice.
본 발명의 또 다른 목적은, 상기의 인간화된 이식 거부 반응 동물 모델에, 후보 물질을 처리하는 단계;를 포함하는, 이식 거부 반응 치료 물질을 스크리닝하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for screening a therapeutic agent for transplant rejection, including treating a candidate substance in the humanized animal model for transplant rejection.
본 발명은, 면역결핍 마우스에 이식 거부 반응 환자 유래 PBMC(Peripheral blood mononuclear cell)이 투여된, 인간화된 이식 거부 반응 동물 모델을 제공한다.The present invention provides a humanized transplant rejection animal model in which immunodeficient mice are administered with PBMCs (peripheral blood mononuclear cells) derived from transplant rejection patients.
또한, 본 발명은, 면역결핍 마우스에 이식 거부 반응 환자로부터 분리한 PBMC를 주입하는 단계를 포함하는 인간화된 이식 거부 반응 동물 모델을 제작하는 방법을 제공한다.In addition, the present invention provides a method for preparing a humanized transplant rejection animal model comprising injecting PBMCs isolated from transplant rejection patients into immunodeficient mice.
또한, 본 발명은, 상기의 인간화된 이식 거부 반응 동물 모델에, 후보 물질을 처리하는 단계;를 포함하는, 이식 거부 반응 치료 물질을 스크리닝하는 방법을 제공한다.In addition, the present invention provides a method for screening a transplant rejection therapeutic agent, comprising the step of treating a candidate material in the humanized transplant rejection response animal model.
본 발명의 이식 거부 반응 아바타 동물모델은, 환자의 이식거부 반응 지표인 혈청 크레아티닌의 증가, 인간 CD4 양성세포가 증가하고, 염증성 사이토카인인 IL-17이 동물모델의 신장 조직 내에 침윤한 것을 확인하였다. 또한, 면역억제제의 투여에 따라, 증가된 혈청 크레아티닌, 인간 CD4 양성세포 및 염증성 사이토카인 IL-17의 침윤이 감소하는 것을 확인하였다. 따라서, 효과적으로 환자의 면역상태가 반영된 동물모델이 제작되고, 이에 따른 면역억제제의 효과를 확인할 수 있어, 관련 산업에 유용하게 이용할 수 있다.In the transplant rejection avatar animal model of the present invention, it was confirmed that the increase in serum creatinine and human CD4 positive cells, which are indicators of the patient's transplant rejection response, increased, and the infiltration of IL-17, an inflammatory cytokine, into the kidney tissue of the animal model. . In addition, it was confirmed that the infiltration of increased serum creatinine, human CD4-positive cells and the inflammatory cytokine IL-17 was decreased according to the administration of the immunosuppressant. Therefore, an animal model that effectively reflects the patient's immune status can be produced, and the effect of the immunosuppressant can be confirmed accordingly, which can be usefully used in related industries.
도 1은 본 발명의 마우스 동물모델의 제작 과정을 도식화한 것이다.
도 2a는 본 발명의 마우스 모델에서, 인간세포의 생착을 유세포분석으로 분석한 도이다.
도 2b는 본 발명의 마우스 모델에서, SCR의 수준을 분석한 도이다.
도 3a은 본 발명의 정상인 PBMC 주입한 마우스 모델에서, 신장 조직 손상 정도를 확인한 도이다.
도 3b는 본 발명의 이식 거부 반응 환자 PBMC 주입한 마우스 모델에서, 신장 조직 손상 정도를 확인한 도이다.
도 3c는 본 발명의 정상인 PBMC 주입한 마우스 모델 및 이식 거부 반응 환자 PBMC 주입한 마우스 모델에서 면역억제제인 SD911의 처리에 따른 신장 손상 점수 정량화한 도이다.
도 4a는 본 발명의 정상인 PBMC 주입한 마우스 모델에서 인간 CD4 양성세포 침윤을 면역화학조직염색으로 확인한 도이다.
도 4b는 본 발명의 이식 거부 반응 환자 PBMC 주입한 마우스 모델에서 인간 CD4 양성세포 침윤을 면역화학조직염색으로 확인한 도이다.
도 4c는 본 발명의 정상인 PBMC 주입한 마우스 모델 및 이식 거부 반응 환자 PBMC 주입한 마우스 모델에서 면역억제제인 SD911의 처리에 따른 CD4 양성세포 수를 정량화한 것이다.
도 5a는 본 발명의 본 발명의 정상인 PBMC 주입한 마우스 모델에서 IL-17 양성세포 침윤을 면역화학조직염색으로 확인한 도이다.
도 5b는 본 발명의 본 발명의 이식 거부 반응 환자 PBMC 주입한 마우스 모델에서 IL-17 양성세포 침윤을 면역화학조직염색으로 확인한 도이다.
도 5c는 본 발명의 정상인 PBMC 주입한 마우스 모델 및 이식 거부 반응 환자 PBMC 주입한 마우스 모델에서 면역억제제인 SD911의 처리에 따른 IL-17 양성세포 수를 정량화한 것이다.1 is a schematic diagram of the manufacturing process of the mouse animal model of the present invention.
Figure 2a is a diagram analyzing the engraftment of human cells by flow cytometry in the mouse model of the present invention.
Figure 2b is a diagram analyzing the level of SCR in the mouse model of the present invention.
Figure 3a is a view confirming the extent of damage to the kidney tissue in the normal PBMC-injected mouse model of the present invention.
Figure 3b is a view confirming the extent of damage to the kidney tissue in the mouse model injected with PBMC in transplant rejection patients of the present invention.
3C is a diagram illustrating quantification of kidney damage scores according to treatment with the immunosuppressant SD911 in a mouse model injected with normal PBMCs and a mouse model injected with PBMCs from transplant rejection patients of the present invention.
Figure 4a is a diagram confirming the infiltration of human CD4-positive cells by immunochemical histology staining in a mouse model injected with normal PBMCs of the present invention.
Figure 4b is a diagram confirming the infiltration of human CD4-positive cells by immunochemical histology staining in the mouse model injected with PBMC of transplant rejection patients of the present invention.
Figure 4c shows the quantification of the number of CD4-positive cells according to the treatment with the immunosuppressant SD911 in the mouse model injected with normal PBMCs of the present invention and the mouse model injected with PBMCs from transplant rejection patients.
Figure 5a is a diagram confirming the infiltration of IL-17-positive cells in the mouse model injected with normal PBMC of the present invention by immunochemical tissue staining.
5b is a diagram confirming the infiltration of IL-17-positive cells by immunochemical histology staining in a mouse model injected with PBMC of a transplant rejection patient of the present invention.
Figure 5c shows the quantification of the number of IL-17-positive cells according to the treatment with the immunosuppressant SD911 in the mouse model injected with normal PBMC of the present invention and the mouse model injected with PBMC of a transplant rejection patient.
본 발명은, 면역결핍 마우스에 이식 거부 반응 환자 유래 PBMC(Peripheral blood mononuclear cell)이 투여된, 인간화된 이식 거부 반응 동물 모델을 제공한다.The present invention provides a humanized transplant rejection animal model in which immunodeficient mice are administered with PBMCs (peripheral blood mononuclear cells) derived from transplant rejection patients.
본 발명의 용어, "이식 거부(transplantation rejection)"는 이식 후 이식된 조직을 수여자의 면역체계가 비자기(non-self)로 인식하여 이식된 장기를 공격하여 제거하고자 하는 반응을 장기 이식 거부라 한다. 이식 거부에 관여하는 가장 중요한 요인은 major histocompatibility complex(MHC) 이고, minor histocompatibility complex 또한 관련이 있다고 알려져 있다. 거부반응은 세포매개면역(cellmediated immune response)와 체액 면역(humoral immune response)이 모두 관여한다. 세포매개반응의 경우 수용자의 림프구가 이식장기의 공여자 MHC를 만나(CD4 T cell-type II MHC molecule, 혹은 CD8 T cell-type I MHC molecule) 시작된다. 활성화된 T 세포는 cytokine을 분비, 혈관의 투과성이 증가되고, macrophage 등의 monocytes의 침윤을 일으키게 된다. 그 결과 미세혈관의 손상, 조직 허혈, 이식조직의 및 세포의 파괴가 일어나게 된다.As used herein, the term "transplantation rejection" refers to the rejection of organ transplantation in response to the recipient's immune system recognizing the transplanted tissue as non-self, attacking the transplanted organ and removing it. say The most important factor involved in transplant rejection is the major histocompatibility complex (MHC), and it is known that the minor histocompatibility complex is also involved. Rejection involves both cell-mediated immune response and humoral immune response. In the case of a cell-mediated reaction, the recipient's lymphocytes meet the donor MHC of the transplanted organ (CD4 T cell-type II MHC molecule, or CD8 T cell-type I MHC molecule) to initiate. Activated T cells secrete cytokines, increase vascular permeability, and cause infiltration of monocytes such as macrophages. As a result, damage to microvessels, tissue ischemia, and destruction of graft tissue and cells occur.
상기 이식 거부 반응은 세포, 혈액, 조직 및 장기로 이루어진 군에서 선택된 1종 이상의 이식 거부 반응이고, 바람직하게는 골수 이식, 심장 이식, 각막 이식, 장 이식, 간 이식, 폐 이식, 췌장 이식, 신장 이식 및 피부이식의 거부반응으로 이루어진 군에서 선택된 1종 이상이나, 이에 제한되지 않는다.The transplant rejection reaction is at least one transplant rejection reaction selected from the group consisting of cells, blood, tissue and organs, and preferably bone marrow transplantation, heart transplantation, corneal transplantation, intestinal transplantation, liver transplantation, lung transplantation, pancreatic transplantation, and kidney transplantation. At least one selected from the group consisting of graft and skin graft rejection, but is not limited thereto.
본 발명의 일실시예에 따르면, 상기 이식 거부 반응 환자 유래 PBMC는 1 내지 5 x 106의 농도로 투여하는 것일 수 있으며, 바람직하게는 5 x 106이나, 이에 제한되지는 않는다.According to an embodiment of the present invention, the PBMC derived from the transplant rejection patient may be administered at a concentration of 1 to 5 x 10 6 , preferably 5 x 10 6 , but is not limited thereto.
본 발명에 있어서, 유래되는 마우스는 제한되지 않고, 일반 실험용 마우스, 면역결핍 마우스 등이 이용될 수 있다. In the present invention, the derived mouse is not limited, and general laboratory mice, immunodeficient mice, and the like may be used.
본 발명에서 용어, "면역결핍 마우스"는 하나 또는 그 이상의 하기 목록에 의해 특징되는 마우스를 의미한다: T 세포 및 B 세포와 같은 기능적 면역 세포의 결함; DNA 복구 결함; 림프구에서 항원 특이적 수용체를 코딩하는 유전자의 재배치 상의 결함; 및 IgM, IgG1, IgG2a, IgG2b, IgG3 및 IgA와 같은 면역 기능 분자들의 결함. 일 구현예에서, 면역결핍 마우스는 면역 기능에 관여하는 Rag1 및 Rag2와 같은 유전자의 하나 이상의 결핍에 의해 특징될 수 있으며 (Oettinger et al, Science, 248:1517-1523, 1990; 및 Schatz et al, Cell, 59:1035-1048, 1989), 면역결핍 마우스는 마우스에서 비정상적인 면역 기능을 초래하는 이들 또는 다른 결함을 가질 수 있다.As used herein, the term "immunodeficient mouse" means a mouse characterized by one or more of the following lists: defects in functional immune cells such as T cells and B cells; DNA repair defects; defects in the rearrangement of genes encoding antigen-specific receptors in lymphocytes; and defects in immune function molecules such as IgM, IgG1, IgG2a, IgG2b, IgG3 and IgA. In one embodiment, immunodeficient mice can be characterized by a deficiency in one or more genes involved in immune function, such as Rag1 and Rag2 (Oettinger et al, Science, 248:1517-1523, 1990; and Schatz et al, Cell, 59:1035-1048, 1989), immunodeficient mice may have these or other defects that result in abnormal immune function in the mouse.
특히 유용한 면역결핍 마우스 종자(strains)는 Shultz et al., J Immunol, 174: 6477-6489, 2005에 상세히 기재된 바와 같이 일반적으로 NOD scid 감마 (NSG) 마우스를 의미하는 NOD, Cg-PrkdcscidIl2rgtml Wjl/SzJ 및 일반적으로 NRG 마우스를 의미하는 NOD.Cg-Rag1tmlMomIl2rgtml Wjl/SzJ (Shultz et al, Clin Exp Immunol, 154(2):270-284, 2008)가 있다.Particularly useful immunodeficient mouse strains include NOD, Cg-PrkdcscidIl2rgtml Wjl/SzJ, commonly referred to as NOD scid gamma (NSG) mice, as detailed in Shultz et al., J Immunol, 174: 6477-6489, 2005. and NOD.Cg-Rag1tmlMoml12rgtml Wjl/SzJ, generally referring to NRG mice (Shultz et al, Clin Exp Immunol, 154(2):270-284, 2008).
본 발명의 일실시예에 따르면, 상기 동물 모델은, 혈청 내 크레아티닌(creatinine)이 대조군의 기준치와 비교하여 증가된 것일 수 있다.According to an embodiment of the present invention, in the animal model, serum creatinine may be increased compared to the reference value of the control group.
본 발명의 일실시예에 따르면, 상기 동물 모델은, 인간 CD4 양성세포가 대조군의 기준치와 비교하여 증가된 것일 수 있다.According to an embodiment of the present invention, in the animal model, human CD4-positive cells may be increased compared to the reference value of the control group.
본 발명의 일실시예에 따르면, 상기 동물 모델은, 염증성 사이토카인인 IL-17의 조직 세포 내 침윤이 대조군의 기준치와 비교하여 증가된 것일 수 있다.According to an embodiment of the present invention, in the animal model, the infiltration of the inflammatory cytokine IL-17 into tissue cells may be increased compared to the reference value of the control group.
본 발명의 일실시예에 따르면, 상기 이식 거부 반응은, 신장 이식에 의한 거부반응인 것일 수 있다.According to an embodiment of the present invention, the transplant rejection reaction may be rejection by a kidney transplant.
또한, 본 발명은, 면역결핍 마우스에 이식 거부 반응 환자로부터 분리한 PBMC를 주입하는 단계를 포함하는 인간화된 이식 거부 반응 동물 모델을 제작하는 방법을 제공한다.In addition, the present invention provides a method for preparing a humanized transplant rejection animal model comprising injecting PBMCs isolated from transplant rejection patients into immunodeficient mice.
본 발명의 일실시예에 따르면, 상기 PBMC를 주입하는 단계는, 0 내지 4주간 1 내지 5회 수행되는 것일 수 있다.According to an embodiment of the present invention, the step of injecting the PBMC may be performed 1 to 5 times for 0 to 4 weeks.
또한, 본 발명은, 상기의 인간화된 이식 거부 반응 동물 모델에, 후보 물질을 처리하는 단계;를 포함하는, 이식 거부 반응 치료 물질을 스크리닝하는 방법을 제공한다.In addition, the present invention provides a method for screening a transplant rejection therapeutic agent, comprising the step of treating a candidate material in the humanized transplant rejection response animal model.
본 발명에서 사용하는 용어 “면역억제제”는 신체의 면역체계 활성을 줄이거나 억제하는 약물로서, 크게 스테로이드제, 세포증식 억제제, 항체 제제, 이뮤노필린에 작용하는 약물, 미코페놀레이트, 종양괴사인자(TNF-α) 억제제등으로 분류되는 약물이다.As used herein, the term "immunosuppressant" is a drug that reduces or inhibits the body's immune system activity, largely steroids, cell proliferation inhibitors, antibody preparations, drugs acting on immunophilin, mycophenolate, tumor necrosis factor It is a drug classified as a (TNF-α) inhibitor.
이식 수술 뿐만 아니라 많은 면역질환 환자에 투여되고 있는 면역억제제는 체내에서 각종 부작용을 초래하는 문제점이 발생하고 있으나, 특히 이식과 같은 수술을 받은 환자의 경우, 면역 거부 반응의 억제를 위해 불가피하게 부작용이 일어 날 수 있음을 감안하고서라도 면역억제제를 처방할 수 밖에 없어, 이식 거부 반응에 있어서, 이식 받은 환자의 면역체계에 따른 적절한 면역억제제의 스크리닝이 중요하다.Immunosuppressive drugs administered to patients with many immune diseases as well as transplant surgery have a problem that causes various side effects in the body. Immunosuppressive agents have to be prescribed even in consideration of the possible occurrence. Therefore, in the case of transplant rejection, it is important to screen for an appropriate immunosuppressant agent according to the immune system of the transplant recipient.
본 발명의 일실시예에 따르면, 상기 후보 물질은 면역억제제인 것일 수 있으며, 상기 면역억제제는, SD911, 타크로리무스(tacrolimus), 사이클로스포린 A(cyclosporine A), 프로드니솔론(prednisolone), 메틸프레드니솔론(methylpredisolone), 데플라자코트(deflazacort), 마이코페놀산(mycophenolic acid), 아자티오프린(azathioprine), 미조리빈(mizoribine), 시롤리무스(sirolimus) 및 에베로리무스(everolimus)로 이루어진 군에서 선택되는 어느 하나인 것일 수 있다.According to an embodiment of the present invention, the candidate substance may be an immunosuppressant, and the immunosuppressant is SD911, tacrolimus, cyclosporine A, prodnisolone, methylprednisolone (methylpredisolone). Any selected from the group consisting of ), deflazacort, mycophenolic acid, azathioprine, mizoribine, sirolimus and everolimus may be one.
본 발명의 일실시예에 따르면, 상기 SD911은 하기 화학식 1로 표시되는 것일 수 있다According to an embodiment of the present invention, the SD911 may be represented by the following formula (1).
[화학식 1][Formula 1]
상기 약학적으로 허용가능한 염은 약학적으로 허용 가능한 유리산(free acid)에 의하여 형성된 산 부가염을 포함할 수 있으며, 상기 유리산은 유기산과 무기산을 사용할 수 있다. 상기 유기산은 구연산, 초산, 젖산, 주석산, 말레인산, 푸마르산, 포름산, 프로피온산, 옥살산, 트리플로오로아세트산, 벤조산, 글루콘산, 메타술폰산, 글리콜산, 숙신산, 4-톨루엔술폰산, 글루탐산, 아스파르트산 등을 포함할 수 있으나, 이에 제한되지 않는다. 또한 상기 무기산은 염산, 브롬산, 황산, 인산 등을 포함할 수 있으나, 이에 제한되지 않는다.The pharmaceutically acceptable salt may include an acid addition salt formed by a pharmaceutically acceptable free acid, and the free acid may be an organic acid or an inorganic acid. The organic acids include citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, glutamic acid, aspartic acid, etc. may include, but is not limited to. In addition, the inorganic acid may include, but is not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like.
타크리니무스 등과 같은 종래의 면역억제제는 신장 독성이 있어 장기간 복용에 문제가 있으나, 본 발명의 신규한 화합물은 신장 보호 효능을 가지고 있어, 부작용이 없는 면역억제제로서 사용될 수 있으나, 이에 제한되지 않는다.Conventional immunosuppressive agents such as tacrinimus have kidney toxicity and thus have a problem in long-term administration, but the novel compound of the present invention has renal protective efficacy and can be used as an immunosuppressive agent without side effects, but is not limited thereto.
본 발명의 일실시예에 따르면, 상기 후보 물질은, 혈청 내 크레아티닌(creatinine)을 감소시키는 것일 수 있다.According to an embodiment of the present invention, the candidate substance may decrease creatinine in serum.
본 발명의 일실시예에 따르면, 상기 후보물질은, 인간 CD4 양성세포를 감소시키는 것일 수 있다.According to an embodiment of the present invention, the candidate substance may reduce human CD4-positive cells.
본 발명의 일실시예에 따르면, 상기 후보물질은, 염증성 사이토카인인 IL-17의 조직 세포 내 침윤을 감소시키는 것일 수 있다.According to an embodiment of the present invention, the candidate material may be to reduce the infiltration of the inflammatory cytokine IL-17 into tissue cells.
이하, 본 발명을 실시예에 의하여 더욱 상세하게 설명한다. 이들 실시예는 단지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 국한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail by way of Examples. These examples are merely for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited to these examples.
<실시예 1> 이식 거부 반응 환자 모사 아바타 모델 평가 플랫폼 구축<Example 1> Establishment of an evaluation platform for imitation avatar model of transplant rejection patients
본 발명의 이식 거부 반응 아바타 동물모델을 제작하기 위하여, 8-10주령의 면역결핍 마우스(NSG)에 정상인 또는 이식 거부 반응 환자 유래의 PBMC(Peripheral blood mononuclear cell)을 5x10^6/mice로 혈관 내 주사한 뒤, 3주 후 혈액 내 정상인 또는 이식 거부 반응 환자(신장이식거부 환자) 세포의 생착 확인을 혈액 세포에서 확인하였다. 그 후, 세포이식 4주 후 마우스를 희생하여 조직 내 인간 세포의 침윤 및 조직학적 변화를 확인하였다(도 1).In order to produce the transplant rejection avatar animal model of the present invention, PBMCs (peripheral blood mononuclear cells) derived from normal or transplant rejection patients were intravascularly administered to 8-10 week old immunodeficient mice (NSG) at 5x10^6/mice. After the injection, confirmation of engraftment of normal or transplant-rejected patients (renal transplant-rejected patients) cells in the blood was confirmed in blood cells 3 weeks after injection. Then, 4 weeks after cell transplantation, mice were sacrificed to confirm the infiltration of human cells and histological changes in the tissue (FIG. 1).
<실시예 2> 이식 거부 반응 환자 모사 아바타 모델의 혈액 내 인간 세포 생착 확인<Example 2> Confirmation of engraftment of human cells in blood of a transplant rejection patient imitation avatar model
상기 실시예 1에서 이식 거부 반응 환자 모사 아바타 모델이 제대로 구축되었는지 확인하기 위해, 인간 세포의 생착을 유세포 분석을 통해 분석하였다. 구체적으로 실시예 1의 이식 거부 반응 인간화 마우스 (정상인 PBMC 주입군, HC; 이식거부환자 PBMC 주입군, Patient)로부터 혈액을 수득한 뒤, 인간 항체와 반응하여 양성인 세포를 분석하였다. 또한, 정상인과 이식 거부 반응 환자의 PBMC 주입 마우스의 신장손상을 측정하는 지표로서 혈액 내 크레아틴 (SCR) 농도를 측정하였다. SCR측정은 동물의 혈액내 혈청을 분리하여 quantitative enzymatic colorimetric method 법(Stanbio laboratory, 0430-120)으로 측정하였다.In order to confirm that the avatar model mimicking the transplant rejection response patient in Example 1 was properly constructed, engraftment of human cells was analyzed through flow cytometry. Specifically, blood was obtained from the transplant-rejected humanized mice of Example 1 (normal PBMC injection group, HC; transplant rejection patient PBMC injection group, Patient), and cells reacted with human antibodies to analyze positive cells. In addition, blood creatine (SCR) concentration was measured as an index for measuring kidney damage in PBMC-injected mice of normal subjects and transplant rejection patients. SCR was measured by the quantitative enzymatic colorimetric method (Stanbio laboratory, 0430-120) by separating animal blood serum.
그 결과, 도 2a에 나타낸 바와 같이, 유세포 분석으로 인간세포가 생착 된 것을 확인하였다. As a result, as shown in Fig. 2a, it was confirmed that human cells were engrafted by flow cytometry.
또한, 도 2b에 나타낸 바와 같이, 정상인 PBMC 주입군(정상인)에 비해 이식 거부 반응 환자 PBMC 주입군(신장 이식 거부 환자)에서 SCR수치가 높은 것을 확인할 수 있었다.In addition, as shown in FIG. 2B , it was confirmed that the SCR level was higher in the PBMC-injected group (kidney transplant-rejected patients) of the transplant-rejected patient than in the normal PBMC-injected group (normal).
<실시예 3> 이식 거부 반응 환자 모사 아바타 모델에서의 면역억제제 효과 확인<Example 3> Confirmation of immunosuppressant effect in the imitation avatar model of transplant rejection patients
<3-1> 신장조직 손상 제어 확인<3-1> Confirmation of kidney tissue damage control
상기 실시예 1에서 수립한 이식 거부 반응 인간화 마우스 모델을 구체적으로, 1) 정상인 PBMC 주입군, 2) 정상인 PBMC 주입군에 면역억제제를 처리한 군, 3) 이식 거부 반응 환자 PBMC 주입군 및 4) 이식 거부 반응 환자 PBMC 주입군에 면역억제제를 처리한 군으로 분류하였으며, 면역억제제로는 SD911을 사용하였다. 구체적으로, 상기 실시예 1의 동물모델에, PBMC를 생착시킨 후 3주째에, SD911을 처리하였으며, 대조군으로는 동량의 생리식염수를 처리하였다. 약물 처리후 1주일 후 마우스를 희생하여, 신장을 적출한 뒤, 조직의 손상정도를 확인하였다. 구체적으로 사구체의 손상 확인 지표인 GN score(membranous glomerulonephritis score), 신장의 면역세포 침윤 지표인 IN score(renal interstitial nephritis score) 및 혈관 주위의 면역세포 침윤정도를 확인하는 지표인 Vasculitis를 각각 측정하였다.Specifically, the transplant rejection humanized mouse model established in Example 1 was described as 1) a normal PBMC-injected group, 2) a normal PBMC-injected group with an immunosuppressive agent, 3) a transplant-rejected patient PBMC-injected group, and 4) Transplant rejection patients were classified into a group treated with an immunosuppressant in the PBMC injection group, and SD911 was used as the immunosuppressant. Specifically, in the animal model of Example 1, 3 weeks after PBMC engraftment, SD911 was treated, and as a control group, the same amount of physiological saline was treated. One week after drug treatment, the mice were sacrificed, the kidneys were removed, and the degree of tissue damage was checked. Specifically, GN score (membranous glomerulonephritis score), which is an indicator of glomerular damage, IN score (renal interstitial nephritis score), which is an indicator of renal immune cell invasion, and Vasculitis, an indicator that confirms the degree of infiltration of immune cells around blood vessels, were measured, respectively.
그 결과, 이식 거부 반응 환자 PBMC가 주입된 군에서는 신장 조직의 손상이 정상인 PBMC가 주입된 군보다, 유의적으로 증가한 것을 확인하였다(도 3a 및 도 3b). 또한, 면역억제제인 SD911을 이식 거부 반응 환자 PBMC가 주입된 군 투여하면, 신장 조직의 손상이 감소하는 것을 확인하였다(도 3b 및 도 3c).As a result, it was confirmed that the group injected with PBMCs of transplant rejection patients significantly increased kidney tissue damage than the group injected with normal PBMCs ( FIGS. 3A and 3B ). In addition, it was confirmed that when the immunosuppressant SD911 was administered to the transplant-rejected patient group injected with PBMCs, the damage to the kidney tissue was reduced ( FIGS. 3B and 3C ).
<3-2> 신장 조직 내 병인 T 세포 침윤 제어 확인<3-2> Confirmation of etiological T cell infiltration control in kidney tissue
상기 실시예 3-1에서 적출된 신장조직내 인간의 면역세포아형인 CD4+T(CD4+) 가 침윤 되었는지 확인하기 위하여, 면역 조직 화학 염색을 수행하였다. 적출한 조직은 포르말린으로 고정한 후, 파라핀에 임베딩하여 5㎛ 두께의 절편을 생성하였다. 조직 내 면역세포를 관찰하기 위해 상기 절편 슬라이드에서 인간 CD4 항체와 반응시켜 면역조직화학분석을 수행하였다.In order to confirm whether CD4+T (CD4 + ), a human immune cell subtype, was infiltrated in the kidney tissue extracted in Example 3-1, immunohistochemical staining was performed. The excised tissue was fixed with formalin and then embedded in paraffin to produce a 5 μm thick section. In order to observe immune cells in the tissue, immunohistochemical analysis was performed by reacting with human CD4 antibody on the section slide.
그 결과, 실시예 1의 정상인과 루푸스 환자의 PBMC를 주입한 마우스 신장 조직 내 인간 CD4+T 세포가 검출되어 인간 세포가 잘 생착된 것을 확인하였으며, SD911을 처리하면, 본 이식 거부 반응 환자 PBMC를 주입한 군에서, CD4 양성세포의 침윤이 유의적으로 감소하였으며, 정상인 PBMC를 주입한 군보다 감소하는 것을 확인하였다(도 4a 내지 도 4c).As a result, human CD4+ T cells were detected in the mouse kidney tissue injected with PBMCs from normal subjects and lupus patients of Example 1, confirming that human cells were well engrafted. In the injected group, the infiltration of CD4-positive cells was significantly reduced, and it was confirmed that it was decreased compared to the group injected with normal PBMCs ( FIGS. 4a to 4c ).
<3-3> 신장 조직 내 IL-17 침윤 제어 확인<3-3> Confirmation of IL-17 invasion control in kidney tissue
상기 실시예 3-1에서 적출된 신장조직내 염증성 사이토카인인 IL-17이 침윤 되었는지 확인하기 위하여, 면역 조직 화학 염색을 수행하였다. 적출한 조직은 포르말린으로 고정한 후, 파라핀에 임베딩하여 5㎛ 두께의 절편을 생성하였다. 조직 내 면역세포를 관찰하기 위해 상기 절편 슬라이드에서 인간 IL-17 항체와 반응시켜 면역조직화학분석을 수행하였다.In order to confirm whether IL-17, an inflammatory cytokine, in the kidney tissue extracted in Example 3-1 was infiltrated, immunohistochemical staining was performed. The excised tissue was fixed with formalin and then embedded in paraffin to produce a 5 μm thick section. In order to observe immune cells in the tissue, immunohistochemical analysis was performed by reacting with human IL-17 antibody on the section slide.
그 결과, 실시예 1의 정상인과 신장이식거부 환자의 PBMC를 주입한 마우스 신장 조직 내 인간 IL-17 세포가 검출되어 인간 세포가 잘 생착된 것을 확인하였으며, SD911을 처리하면, 본 이식 거부 반응 환자 PBMC를 주입한 군에서, IL-17 양성세포의 침윤이 유의적으로 감소하였으며, 정상인 PBMC를 주입한 군보다 감소하는 것을 확인하였다(도 5a 내지 도 5c).As a result, it was confirmed that human IL-17 cells were detected in the mouse kidney tissue injected with PBMCs of normal persons and kidney transplant rejection patients of Example 1, confirming that human cells were well engrafted. In the PBMC-injected group, the infiltration of IL-17-positive cells was significantly reduced, and it was confirmed that it was decreased compared to the normal PBMC-injected group ( FIGS. 5a to 5c ).
따라서, 본 발명의 이식 거부 반응 아바타 동물모델은, 환자의 이식거부 반응의 지표인 혈청 크레아티닌의 증가, 인간 CD4 양성세포가 증가하고, 염증성 사이토카인인 IL-17이 동물모델의 신장 조직 내에 침윤한 것을 확인하여, 인간화 된 것을 확인하였다. 또한, 면역억제제의 투여에 따라, 증가된 혈청 크레아티닌, 인간 CD4 양성세포 및 염증성 사이토카인 IL-17의 침윤이 감소하는 것을 확인하여, 효과적으로 환자의 면역상태가 반영된 동물모델이 제작되고, 이에 따른 면역억제제의 효과를 확인하였다.Therefore, in the transplant rejection avatar animal model of the present invention, an increase in serum creatinine, an increase in human CD4 positive cells, which are indicators of a patient's transplant rejection response, and IL-17, an inflammatory cytokine, infiltrated into the kidney tissue of the animal model. By confirming that, it was confirmed that it was humanized. In addition, according to the administration of the immunosuppressant, it was confirmed that the increased serum creatinine, the infiltration of human CD4-positive cells and the inflammatory cytokine IL-17 decreased, so that an animal model that effectively reflected the patient's immune status was produced, and the immunity The effect of the inhibitor was confirmed.
Claims (17)
상기 이식 거부 반응 환자 유래 PBMC는 1 내지 5 x 106의 농도로 투여하는 것인, 동물 모델.The method of claim 1,
The animal model, wherein the transplant rejection patient-derived PBMC is administered at a concentration of 1 to 5 x 10 6 .
상기 동물 모델은, 혈청 내 크레아티닌(creatinine)이 대조군의 기준치와 비교하여 증가된 것을 특징으로 하는 동물 모델.The method of claim 1,
The animal model is an animal model, characterized in that serum creatinine (creatinine) is increased compared to the reference value of the control group.
상기 동물 모델은, 인간 CD4 양성세포가 대조군의 기준치와 비교하여 증가된 것을 특징으로 하는 동물 모델.The method of claim 1,
The animal model is an animal model, characterized in that human CD4-positive cells are increased compared to the reference value of the control group.
상기 동물 모델은, 염증성 사이토카인인 IL-17의 조직 세포 내 침윤이 대조군의 기준치와 비교하여 증가된 것을 특징으로 하는 동물 모델.The method of claim 1,
The animal model is an animal model, characterized in that the infiltration of the inflammatory cytokine IL-17 into tissue cells is increased compared to the reference value of the control group.
상기 이식 거부 반응은 세포, 혈액, 조직 및 장기로 이루어진 군에서 선택된 1종 이상의 이식 거부 반응인 것인, 동물 모델.According to claim 1,
The transplant rejection reaction is one or more types of transplant rejection reaction selected from the group consisting of cells, blood, tissue and organs, animal model.
상기 이식 거부 반응은 골수 이식, 심장 이식, 각막 이식, 장 이식, 간 이식, 폐 이식, 췌장 이식, 신장 이식 및 피부이식으로 이루어진 군에서 선택된 1종 이상의 이식 거부 반응인 것인, 동물 모델.7. The method of claim 6,
The transplant rejection reaction is one or more transplant rejection reaction selected from the group consisting of bone marrow transplantation, heart transplantation, corneal transplantation, intestinal transplantation, liver transplantation, lung transplantation, pancreatic transplantation, kidney transplantation and skin transplantation, animal model.
상기 이식 거부 반응은, 신장 이식에 의한 거부반응인 것을 특징으로 하는 동물 모델.8. The method of claim 7,
The transplant rejection reaction is an animal model, characterized in that the rejection reaction by the kidney transplant.
상기 PBMC를 주입하는 단계는, 0 내지 4주간 1 내지 5회 수행되는 것을 특징으로 하는 방법.10. The method of claim 9,
The step of injecting the PBMC is characterized in that it is performed 1 to 5 times for 0 to 4 weeks.
상기 후보 물질은 면역억제제인 것을 특징으로 하는 방법.12. The method of claim 11,
The method, characterized in that the candidate substance is an immunosuppressant.
상기 면역억제제는, SD911, 타크로리무스(tacrolimus), 사이클로스포린 A(cyclosporine A), 프로드니솔론(prednisolone), 메틸프레드니솔론(methylpredisolone), 데플라자코트(deflazacort), 마이코페놀산(mycophenolic acid), 아자티오프린(azathioprine), 미조리빈(mizoribine), 시롤리무스(sirolimus) 및 에베로리무스(everolimus)로 이루어진 군에서 선택되는 어느 하나인 것을 특징으로 하는 방법.13. The method of claim 12,
The immunosuppressant is SD911, tacrolimus, cyclosporine A, prodnisolone, methylprednisolone, deflazacort, mycophenolic acid, azathioprine (azathioprine), mizoribine (mizoribine), sirolimus (sirolimus) and everolimus (everolimus) method, characterized in that any one selected from the group consisting of.
[화학식 1]
[Formula 1]
상기 후보 물질은, 혈청 내 크레아티닌(creatinine)을 감소시키는 것을 특징으로 하는 방법.12. The method of claim 11,
The candidate substance, a method characterized in that it reduces the creatinine (creatinine) in the serum.
상기 후보물질은, 인간 CD4 양성세포를 감소시키는 것을 특징으로 하는 방법.12. The method of claim 11,
The candidate substance, a method characterized in that it reduces human CD4-positive cells.
상기 후보물질은, 염증성 사이토카인인 IL-17의 조직 세포 내 침윤을 감소시키는 것을 특징으로 하는 방법.12. The method of claim 11,
The candidate material, the method characterized in that it reduces the infiltration of the inflammatory cytokine IL-17 into tissue cells.
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| KR20000029575A (en) * | 1996-08-01 | 2000-05-25 | 다께다 가즈히꼬 | Method for examining chronic rejection reactions following organ transplantation and method for determining urine components |
| KR20200019460A (en) * | 2018-08-14 | 2020-02-24 | 가톨릭대학교 산학협력단 | A humanized animal model of liver disease or liver transplantation and uses therof |
| KR20200042685A (en) * | 2018-10-16 | 2020-04-24 | 가톨릭대학교 산학협력단 | A humanized animal model of autoimmune disease and uses therof |
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| KR20000029575A (en) * | 1996-08-01 | 2000-05-25 | 다께다 가즈히꼬 | Method for examining chronic rejection reactions following organ transplantation and method for determining urine components |
| KR20200019460A (en) * | 2018-08-14 | 2020-02-24 | 가톨릭대학교 산학협력단 | A humanized animal model of liver disease or liver transplantation and uses therof |
| KR20200042685A (en) * | 2018-10-16 | 2020-04-24 | 가톨릭대학교 산학협력단 | A humanized animal model of autoimmune disease and uses therof |
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