KR20230112561A - Liver transplant patient intestinal flora analysis and transplant status monitoring and immunomodulatory Theragnosis composition - Google Patents
Liver transplant patient intestinal flora analysis and transplant status monitoring and immunomodulatory Theragnosis composition Download PDFInfo
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Abstract
본 발명은 간이식 환자 특이 진단과 치료테라그노시스를 위한 마이크로바이옴 대사체 조성물에 관한 것으로서, 본 발명은, 간이식 환자군에서, 장내 균총의 다양성이 감소된 것을 확인하였으며, 특히 간이식 환자군에서, 장내 유익균으로 알려진 비피도박테리움 속 마이크로바이옴인 B. bifidum 및 B. longum, 페칼리박테리움 속 마이크로바이옴인 F. prausnitzii, 아커멘시아 속 균주인 A. muciniphilla가 유의적으로 감소한 것을 확인하였다. 또한, 상기의 마이크로바이옴 균주 또는 균주의 대사체인 소듐 부티레이트를 간이식 환자 유래의 PBMC에 처리하면, 간이식 환자군의 면역세포인 Th17 및 Treg의 발현을 조절하는 것을 확인하였다. 또한, 염증성 사이토카인 또는 케모카인인 IL-17, GM-CSF, IL-6, IL-15, IFN-α, IL-2, IL-1β TNF-α 및 IL-8의 발현을 억제하고, 항염증성 사이토카인인 IL-10 및 T 세포 조절 사이토카인인 TGF-β의 발현을 증가시키는 것을 확인하였다. 따라서, 간이식 환자군의 장내 균총 조성비 및 이상을 진단하고, 이에 따라 본 발명의 유효물질 처리에 따른 유용한 장내 균총의 증가를 확인하여, 개인별 맞춤의학 실현이 가능한 것을 확인하였다.The present invention relates to a microbiome metabolite composition for specific diagnosis and treatment of theragnosis in liver transplant patients. The present invention confirms that the diversity of the intestinal flora is reduced in the liver transplant patient group. It was confirmed that mucinipilla was significantly reduced. In addition, it was confirmed that when the above microbiome strain or sodium butyrate, a metabolite of the strain, was treated with liver transplant patient-derived PBMC, the expression of Th17 and Treg, which are immune cells in the liver transplant patient group, was regulated. In addition, it was confirmed that the expression of inflammatory cytokines or chemokines IL-17, GM-CSF, IL-6, IL-15, IFN-α, IL-2, IL-1β, TNF-α and IL-8 was inhibited, and the expression of anti-inflammatory cytokine IL-10 and T cell regulatory cytokine TGF-β was increased. Therefore, it was confirmed that personalized medicine can be realized by diagnosing the intestinal flora composition ratio and abnormality of the liver transplant patient group and confirming the increase in useful intestinal flora according to the treatment of the active substance of the present invention.
Description
본 발명은 간이식환자 장내균총 분석과 이를 통한 이식상태 모니터링과 면역조절 테라그노시스 조성물에 관한 것이다.The present invention relates to an analysis of the intestinal flora of a liver transplant patient, monitoring of transplantation status through the analysis, and an immunoregulatory theragnosis composition.
장기, 조직 또는 세포 이식은 다양한 종류의 질병을 앓고 있는 환자의 생명을 구하는 데 이용될 수 있다. 인간의 신장, 간, 심장, 신장, 폐 및 췌장 등의 장기와 피부 등의 조직, 골수 등 세포 동종이식(allotransplantation)은 말기 장기 부전 등 난치성 질환을 치료하는 방법으로 병원에서 이미 일반적으로 시행되고 있다. 또한, 인간 이외의 포유동물을 공여자로하는 이종이식(xenotransplantation)도 동종이식 공여자 부족을 대체하는 방법으로 활발히 연구되고 있는 실정이다. 특히, 최근에는 자기 자신을 영구히 재생할 수 있고, 적절한 조건에서 신체를 구성하는 다양한 종류의 세포로 분화 가능한 줄기세포의 이식이 다양한 난치성 질환의 세포대체 치료법의 하나로 대두되고 있다. 국립장기이식센터에 따르면 국내에서 한해 2만 여명에 달하는 환자들이 장기이식을 기다리고 있으나, 기증 건수는 수요의 10%에도 못 미치는 것으로 보고되었다. 또한 미국의 경우 장기를 필요로 하는 대기자는 16분에 한명씩 추가 되고 있으나, 하루에 대기자 중 11명은 수술 받지도 못하고 사망하는 실정이다. 이러한 상황과 더불어 생명과학기술의 발달은 이종 장기이식 기술개발의 배경이 된다. 면역억제 요법 및 이식 수술 전- 및 후 환자 치료에서 현저한 개선이 이루어졌지만, 이식편 거부는 여전히 이식받은 개인의 대략 60%에 영향을 미치며 따라서 상기 이식편 거부는 이식 후 첫해 내에 이식받은 개인의 40% 이하에서 이식편 거부의 관찰과 함께 다수의 이식편 상실의 위험인자이다. 급성 거부가 또한 만성거부로 진행하는 것으로 공지된 위험 인자이며 따라서 가능한 한 빠른 급성 거부 에피소드의 검출 및 치료가 이식편 손상을 최소화하고 하류 거부 에피소드를 저지하기 위한 주요 목표이다. 대부분의 경우에, 이식된 조직에 대한 적응성 면역 응답이 성공적인 이식에 주요 장애이다. 거부는 상기 이식편상의 동종항원에 대한 면역 응답에 의해 유발되며, 상기 동종항원은 종내 개인에 따라 변하고 따라서 수용자에 의해 이질물로서 인식되는 단백질이다.Organ, tissue or cell transplantation can be used to save the lives of patients suffering from various types of diseases. Allotransplantation of human organs such as kidney, liver, heart, kidney, lung, and pancreas, tissue such as skin, and bone marrow is already commonly performed in hospitals as a method for treating intractable diseases such as end-stage organ failure. In addition, xenotransplantation using a non-human mammal as a donor is also being actively researched as a method to replace the shortage of donors for allotransplantation. In particular, recently, transplantation of stem cells capable of regenerating themselves permanently and differentiating into various types of cells constituting the body under appropriate conditions has emerged as one of the cell replacement treatments for various intractable diseases. According to the National Organ Transplantation Center, about 20,000 patients a year are waiting for organ transplantation in Korea, but the number of donations is reported to be less than 10% of the demand. In addition, in the case of the United States, one person on the waiting list is added every 16 minutes, but 11 people on the waiting list die every day without being able to undergo surgery. In addition to this situation, the development of life science technology serves as a background for the development of xenotransplantation technology. Although significant improvements have been made in immunosuppressive therapy and patient care pre- and post-transplantation, graft rejection still affects approximately 60% of transplanted individuals and is thus a risk factor for multiple graft loss, with graft rejection observed in up to 40% of transplanted individuals within the first year after transplantation. Acute rejection is also a known risk factor for progression to chronic rejection and thus detection and treatment of acute rejection episodes as early as possible is a major goal to minimize graft damage and arrest downstream rejection episodes. In most cases, an adaptive immune response to the transplanted tissue is a major obstacle to successful transplantation. Rejection is caused by an immune response to alloantigens on the graft, which are proteins that vary from person to person within the species and are therefore recognized as foreign by the recipient.
지난 10년에 걸쳐 수술 기법, 면역억제요법 및 감염성 모니터링 및 치료의 진보는 환자 및 이식편 생존을 혁신시켰다. 그러나, 이러한 성공에도 불구하고, 이 식물 수용자는 여전히 일반적인 집단보다 훨씬 더 높은 이환율 및 사망률을 나타낸다. 이는 부분적으로 만성적인 동종이식편 손상의 영향에 기인하지만, 주요 원인은 만성적인 면역억제성 약물 사용에 의해 영향을 받는 동반이환(comorbid)이다.Over the past decade, advances in surgical techniques, immunosuppressive therapies, and infectious monitoring and treatment have revolutionized patient and graft survival. However, despite these successes, these plant recipients still exhibit much higher morbidity and mortality rates than the general population. This is due in part to the effects of chronic allograft injury, but the main cause is a comorbid effected by chronic immunosuppressive drug use.
면역억제 관련된 독성들은 유의수준일 수 있다. 예를 들어, 성인 간 이식 수용자에서 다수의 연구는 면역억제 요법에의 노출에 따른 신장 기능의 시간-의존적인 연속적 감퇴를 입증하였다. 장기간 면역억제의 다른 중요한 합병증은 이식후 당뇨병의 새로운 발병(NODAT), 고혈압, 고지질혈증 및 스타틴 요법의 필요성을 포함한다. 이러한 상황을 시정하기 위해서, 기관 이식에 있어서 연구 우선순위가 신규의 강력한 면역억제 약물의 탐색에서 면역억제를 최소화하기 위한 전략으로 이동하고 있으며, 지속적인 면역억제없이 이식된 조직이 장시간동안 유지되도록 하는 것이 목표가 되고 있다.Immunosuppression-related toxicities may be significant. For example, a number of studies in adult liver transplant recipients have demonstrated a time-dependent serial decline in renal function following exposure to immunosuppressive therapy. Other important complications of long-term immunosuppression include new onset of post transplant diabetes (NODAT), hypertension, hyperlipidemia and the need for statin therapy. To rectify this situation, research priorities in organ transplantation are shifting from the search for new potent immunosuppressive drugs to strategies to minimize immunosuppression, with the goal being to maintain the transplanted tissue for a long time without prolonged immunosuppression.
한편, 마이크로바이옴(Microbiome)이란 인체에 서식하는 미생물을 일컫는 말로 장내 미생물이 이에 해당된다. 마이크로바이옴의 수는 순수한 인체의 세포수보다 두 배 이상 많고 유전자 수는 100배 이상 많다. 따라서 미생물을 빼놓고 인간의 유전자를 논할 수 없을 정도이기에 제2의 게놈(Second Genome)이라 부르기도 한다. 마이크로바이옴은 유익균과 유해균이 생성되는 원리와 질병간의 연관성 등을 분석할 수 있어 신약 개발 및 불치병 치료법 연구에 폭넓게 활용될 수 있는 분야다. 또한 마이크로바이옴은 식품, 화장품, 치료제 개발에 쓰인다.On the other hand, the microbiome refers to microorganisms living in the human body, and includes the microorganisms in the intestine. The number of microbiome is more than twice the number of pure human cells, and the number of genes is more than 100 times. Therefore, it is also called the Second Genome because it is impossible to discuss human genes without mentioning microorganisms. The microbiome is a field that can be widely used in the development of new drugs and research on treatments for incurable diseases, as it can analyze the relationship between the principle of production of beneficial and harmful bacteria and diseases. The microbiome is also used in the development of food, cosmetics, and therapeutics.
차세대 염기서열 분석(Next Generation Sequencing, NGS)의 기술이 발전함에 따라, 인체의 생리와 면역조절에 있어서, 장내미생물의 중요성이 부각되고 있어, 인간의 장내 마이크로바이옴의 구조를 직접 조절할 수 있는 프로바이오틱스(probiotics)의 중요성이 대두되고 있다. 최근 많은 연구들을 통해 프로바이오틱스는 인간 마이크로바이옴과 깊은 연관성이 있는 것으로 알려지면서, 장내 환경을 변화시키는 조절제로서의 기능에 대해 주목을 받고 있다. 프로바이오틱스의 섭취는 마이크로바이옴 조절기능을 통해 소화 촉진은 물론, 염증성 장질환, 감염성 질환이나 유해세균의 억제 기능을 하는 것으로도 알져여 있으며, 특히 숙주의 면역시스템을 증진시켜, 아토피, 류마티스 등을 포함하는 다양한 면역관련 질환에도 효능이 있는 것으로 밝혀지고 있다.As the technology of Next Generation Sequencing (NGS) develops, the importance of intestinal microbes in physiology and immune regulation of the human body is emerging, and probiotics that can directly control the structure of the human intestinal microbiome are emerging. The importance of probiotics is emerging. Recently, probiotics have been known to be deeply related to the human microbiome through many studies, and their function as a regulator that changes the intestinal environment is attracting attention. Ingestion of probiotics is known to promote digestion through microbiome regulation, as well as suppress inflammatory bowel disease, infectious diseases, and harmful bacteria.
또한, 프로바이오틱스 분야가 크게 성장하면서, 프리바이오틱스(prebiotics), 신바이오틱스(synbiotics) 및 포스트바이오틱스(postbiotics)에 대해서도 많은 관심이 집중되고 있다, 프리바이오틱스는 "숙주의 미생물 중에서 건강에 도움을 주는 유익균에 의해 선택적으로 이용되는 물질"로 정의되며, 유산균의 먹이가 되는 식이섬유와 올리고당이 대표적인 예이다. 신바이오틱스는 프로바이오틱스와 프리바이오틱스가 함께 포함되어 있는 형태를 말한다. 한편 최근 주목받고 있는 포스트바이오틱스는 프로바이오틱스가 생산하는 유용한 대사산물과 미생물의 구성성분을 포함하는 소재로서 "숙주의 건강에 유익한 미생물의 살아있지 않은 형태 또는 그 미생물의 성분이 포함된 제형"으로 명확하게 정의되었으며, 포스트바이오틱스는 기존 프로바이오틱스 소재가 갖는 안전성(safety), 기능성(function), 안정성(stability)의 한계를 극복할 수 있는 새로운 대안 소재로 주목받고 있다.In addition, as the field of probiotics has grown significantly, much attention has been focused on prebiotics, synbiotics, and postbiotics. Prebiotics are defined as "substances that are selectively used by beneficial bacteria conducive to health among host microorganisms", and dietary fiber and oligosaccharides serving as food for lactic acid bacteria are representative examples. Synbiotics are a combination of probiotics and prebiotics. On the other hand, postbiotics, which has recently been attracting attention, is a material containing useful metabolites produced by probiotics and components of microorganisms, and is clearly defined as "a non-living form of microorganisms beneficial to the host's health or a formulation containing components of the microorganisms".
이에 본 발명자들은 장기 이식 치료를 받은 환자군에서, 군집의 다양성 및 분류군이 감소된 마이크로바이옴을 확인하고 선별하였으며, 선별된 균주들이 장기 이식 치료를 받은 환자군의 면역을 개선시키고, 균주의 대사산물 또한 면역반응을 개선시키는 것을 확인하여 본 발명을 완성하였다.Accordingly, the present inventors identified and selected a microbiome with reduced diversity and taxa in a patient group who received organ transplant treatment, and the selected strains improved the immunity of the patient group who received organ transplant treatment, and the metabolites of the strain also improved the immune response, thereby completing the present invention.
본 발명의 목적은 소듐 부티레이트(Sodium Butyrate) 또는 마이크로바이옴을 유효성분으로 포함하는, 이식거부반응의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.An object of the present invention is to provide a pharmaceutical composition for preventing or treating transplant rejection, comprising sodium butyrate or microbiome as an active ingredient.
본 발명의 다른 목적은 소듐 부티레이트(Sodium Butyrate) 또는 마이크로바이옴을 유효성분으로 포함하는, 이식거부반응의 예방 또는 개선용 식품조성물을 제공하는 것이다.Another object of the present invention is to provide a food composition for preventing or improving transplant rejection, containing sodium butyrate or microbiome as an active ingredient.
본 발명의 다른 목적은 소듐 부티레이트(Sodium Butyrate) 또는 마이크로바이옴을 유효성분으로 포함하는 조성물을 개체에 투여하는 단계;를 포함하는, 간이식 환자군의 페칼리박테리움 속(Faecalibacterium sp.) 균주를 증가시키고, 박테로이데스 속(Bacteroides sp.) 균주를 감소시키는 방법을 제공하는 것이다.Another object of the present invention is to administer a composition comprising sodium butyrate or a microbiome as an active ingredient to an individual; including, Faecalibacterium sp. in a liver transplant patient group. It is to provide a method for increasing strains and reducing Bacteroides sp. strains.
본 발명의 다른 목적은 소듐 부티레이트(Sodium Butyrate) 또는 마이크로바이옴을 유효성분으로 포함하는, 이식거부반응의 예방 또는 치료용 조성물로서,Another object of the present invention is a composition for preventing or treating transplant rejection, comprising sodium butyrate or microbiome as an active ingredient,
상기 조성물은The composition
1) Treg의 발현을 증가시키고,1) increase Treg expression,
2) Th17의 발현을 감소시키며,2) reducing the expression of Th17;
3) IL-17, GM-CSF, IL-6, IL-15, IFN-α, IL-2, IL-1β TNF-α 및 IL-8으로 이루어진 군에서 선택된 염증성 사이토카인 또는 케모카인의 발현 억제시키고, 3) inhibiting the expression of an inflammatory cytokine or chemokine selected from the group consisting of IL-17, GM-CSF, IL-6, IL-15, IFN-α, IL-2, IL-1β, TNF-α and IL-8;
4) 항염증성 사이토카인인 IL-10 또는 T 세포 조절 사이토카인인 TGF-β의 발현을 증가시키는 것인, 조성물을 제공하는 것이다.4) to provide a composition that increases the expression of anti-inflammatory cytokine IL-10 or T cell regulatory cytokine TGF-β.
상기의 목적을 달성하기 위하여, 본 발명은 소듐 부티레이트(Sodium Butyrate) 또는 마이크로바이옴을 유효성분으로 포함하는, 이식거부반응의 예방 또는 치료용 약학적 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating transplant rejection, comprising sodium butyrate or microbiome as an active ingredient.
또한, 본 발명은 소듐 부티레이트(Sodium Butyrate) 또는 마이크로바이옴을 유효성분으로 포함하는, 이식거부반응의 예방 또는 개선용 식품조성물을 제공한다.In addition, the present invention provides a food composition for preventing or improving transplant rejection, containing sodium butyrate or microbiome as an active ingredient.
또한, 본 발명은 소듐 부티레이트(Sodium Butyrate) 또는 마이크로바이옴을 유효성분으로 포함하는 조성물을 개체에 투여하는 단계;를 포함하는, 간이식 환자군의 페칼리박테리움 속(Faecalibacterium sp.) 균주를 증가시키고, 박테로이데스 속(Bacteroides sp.) 균주를 감소시키는 방법을 제공한다.In addition, the present invention includes the step of administering a composition containing sodium butyrate or a microbiome as an active ingredient to an individual; Faecalibacterium sp. Increases the strain of liver transplant patients, and provides a method for reducing the strain of Bacteroides sp .
또한, 본 발명은 소듐 부티레이트(Sodium Butyrate) 또는 마이크로바이옴을 유효성분으로 포함하는, 이식거부반응의 예방 또는 치료용 조성물로서,In addition, the present invention is a composition for preventing or treating transplant rejection, comprising sodium butyrate or microbiome as an active ingredient,
상기 조성물은The composition
1) Treg의 발현을 증가시키고,1) increase Treg expression,
2) Th17의 발현을 감소시키며,2) reducing the expression of Th17;
3) IL-17, GM-CSF, IL-6, IL-15, IFN-α, IL-2, IL-1β TNF-α 및 IL-8으로 이루어진 군에서 선택된 염증성 사이토카인 또는 케모카인의 발현 억제시키고, 3) inhibiting the expression of an inflammatory cytokine or chemokine selected from the group consisting of IL-17, GM-CSF, IL-6, IL-15, IFN-α, IL-2, IL-1β, TNF-α and IL-8;
4) 항염증성 사이토카인인 IL-10 또는 T 세포 조절 사이토카인인 TGF-β의 발현을 증가시키는 것인, 조성물을 제공한다.4) to increase the expression of anti-inflammatory cytokine IL-10 or T cell regulatory cytokine TGF-β, provides a composition.
본 발명은, 간이식 환자군에서, 장내 균총의 다양성이 감소된 것을 확인하였으며, 특히 간이식 환자군에서, 장내 유익균으로 알려진 비피도박테리움 속 마이크로바이옴인 B. bifidum 및 B. longum, 페칼리박테리움 속 마이크로바이옴인 F. prausnitzii, 아커멘시아 속 균주인 A. muciniphilla가 유의적으로 감소한 것을 확인하였다. 또한, 상기의 마이크로바이옴 균주 또는 균주의 대사체인 소듐 부티레이트를 간이식 환자 유래의 PBMC에 처리하면, 간이식 환자군의 면역세포인 Th17 및 Treg의 발현을 조절하는 것을 확인하였다. 또한, 염증성 사이토카인 또는 케모카인인 IL-17, GM-CSF, IL-6, IL-15, IFN-α, IL-2, IL-1β TNF-α 및 IL-8의 발현을 억제하고, 항염증성 사이토카인인 IL-10 및 T 세포 조절 사이토카인인 TGF-β의 발현을 증가시키는 것을 확인하였다. 따라서, 간이식 환자군의 장내 균총 조성비 및 이상을 진단하고, 이에 따라 본 발명의 유효물질 처리에 따른 유용한 장내 균총의 증가를 확인하여, 개인별 맞춤의학 실현이 가능한 바, 관련 산업에 유용하게 이용할 수 있다.The present invention confirmed that the diversity of intestinal flora was reduced in the liver transplant patient group, and in particular, in the liver transplant patient group, it was confirmed that B. bifidum and B. longum , a microbiome of the genus Bifidobacterium known as intestinal beneficial bacteria , F. prausnitzii , a microbiome of the genus Faecalibacterium, and A. mucinipilla , a strain of the genus Acamencia, were significantly reduced. In addition, it was confirmed that when the above microbiome strain or sodium butyrate, a metabolite of the strain, was treated with liver transplant patient-derived PBMC, the expression of Th17 and Treg, which are immune cells in the liver transplant patient group, was regulated. In addition, it was confirmed that the expression of inflammatory cytokines or chemokines IL-17, GM-CSF, IL-6, IL-15, IFN-α, IL-2, IL-1β, TNF-α and IL-8 was inhibited, and the expression of anti-inflammatory cytokine IL-10 and T cell regulatory cytokine TGF-β was increased. Therefore, it is possible to realize personalized medicine by diagnosing the intestinal flora composition ratio and abnormality of the liver transplant patient group, and confirming the increase in useful intestinal flora according to the treatment of the active substance of the present invention. It can be usefully used in related industries.
도 1은 간이식 환자군과 건강한 성인의 장내 균총의 다양성을 α-diversity 및 β-diversity로 분석한 도이다(A: Observed OTU 및 Shannon 분석 결과, B: PCoA plot 및 bray curstis distance 분석 결과).
도 2는 간이식 환자군과 건강한 성인의 장내 균총의 분류군을 linear discriminant analysis (LDA) effect size (LEfSe)이용하여 분석한 도이다.
도 3은 간이식 환자군과 건강한 성인군에서 각 군의 개인별 장내 균총을 LEfSe으로 분석하여, 비교한 도이다(A: 개인별 균총 분석 결과, B: 각 군별 증가 균총 비교).
도 4는 간이식 환자군과 건강한 성인군에서 증가 또는 감소하는 균주를 비교한 도이다.
도 5는 간이식 환자군과 건강한 성인군의 면역 세포의 발현을 유세포 분석으로 확인한 도이다.
도 6은 간이식 환자군에서 체내 면역억제제 양에 따른 장내 균총의 다양성을 α-diversity 및 β-diversity로 분석한 도이다(A: Observed OTU 및 Shannon 분석 결과, B: β-diversity 분석 결과).
도 7은 간이식 환자군에서 체내 면역억제제 양에 따른 장내 균총의 분류군을 linear discriminant analysis (LDA) effect size (LEfSe)이용하여 분석한 도이다.
도 8은 간이식 환자군에서 체내 면역억제제 양에 따른 증가 또는 감소하는 균주를 비교한 도이다.
도 9는 간이식 환자군과 면역 관용상태 환자군의 장내 균총의 다양성을 α-diversity 및 β-diversity로 분석한 도이다(A: Observed OTU 및 Shannon 분석 결과, B: PCoA plot 및 bray curstis distance 분석 결과)
도 10은 간이식 환자군과 면역 관용상태 환자군의 장내 균총의 분류군을 linear discriminant analysis (LDA) effect size (LEfSe)이용하여 분석한 도이다.
도 11은 간이식 환자군과 면역 관용상태 환자군에서 각 군의 개인별 장내 균총을 LEfSe으로 분석하여, 비교한 도이다.
도 12는 간이식 환자군과 면역 관용상태 환자군에서의 증가 또는 감소하는 균주를 비교한 도이다.
도 13은 간이식 환자군과 면역 관용상태 환자군에서의 면역 세포의 발현을 유세포 분석으로 확인한 도이다.
도 14는 간이식 환자군과 면역 관용상태 환자군에서의 사이토카인 및 케모카인의 발현차이를 비교한 도이다.
도 15는 간이식 환자군과 면역 관용상태 환자군에서의 사이토카인 및 케모카인의 발현차이를 비교한 도이다.
도 16은 간이식 환자군의 PBMC에, 감소되어 있는 마이크로바이옴의 처리에 따른 Treg의 발현을 유세포 분석으로 분석한 도이다(A: 유세포분석 결과, B: Treg 발현 정량화).
도 17은 간이식 환자군의 PBMC에, 감소되어 있는 마이크로바이옴의 처리에 따른 Th17의 발현을 유세포 분석으로 분석한 도이다(A: 유세포분석 결과, B: Th17 발현 정량화).
도 18은 간이식 환자군의 PBMC에, 감소되어 있는 마이크로바이옴의 처리에 따른 사이토카인의 발현을 ELISA로 분석한 도이다(A: IL-17 발현 정량화, B: IL-10 발현 정량화, C: TGF-β 발현 정량화).
도 19는 간이식 환자군의 PBMC에 바이크로바이옴의 대사체인 소듐 부티레이트의 처리에 따른 IL-17의 발현을 ELISA로 분석한 도이다.1 is a diagram illustrating the diversity of intestinal flora in liver transplant patients and healthy adults by α-diversity and β-diversity (A: Observed OTU and Shannon analysis results, B: PCoA plot and bray curstis distance analysis results).
2 is a diagram illustrating the analysis of taxa of the intestinal flora of a liver transplant patient group and healthy adults using linear discriminant analysis (LDA) effect size (LEfSe).
Figure 3 is a diagram comparing the intestinal flora of each group in a liver transplant patient group and a healthy adult group by analyzing with LEfSe (A: result of individual flora analysis, B: comparison of increased flora in each group).
Figure 4 is a comparison of strains increased or decreased in the liver transplant patient group and healthy adult group.
5 is a diagram confirming the expression of immune cells in a liver transplant patient group and a healthy adult group by flow cytometry.
6 is a diagram illustrating the diversity of intestinal flora according to the amount of immunosuppressant in the body in the liver transplant patient group by α-diversity and β-diversity (A: Observed OTU and Shannon analysis results, B: β-diversity analysis results).
7 is a diagram illustrating the analysis of taxa of intestinal flora according to the amount of immunosuppressant in the body in a liver transplant patient group using linear discriminant analysis (LDA) effect size (LEfSe).
8 is a diagram comparing strains that increase or decrease according to the amount of immunosuppressant in the body in a liver transplant patient group.
9 is a diagram illustrating the diversity of intestinal flora in the liver transplant patient group and the immune tolerant patient group by α-diversity and β-diversity analysis (A: Observed OTU and Shannon analysis results, B: PCoA plot and bray curstis distance analysis results)
10 is a diagram illustrating the analysis of taxa of intestinal flora in a liver transplant patient group and an immune tolerant patient group using linear discriminant analysis (LDA) effect size (LEfSe).
11 is a diagram comparing the intestinal microflora of individuals in each group in a liver transplant patient group and an immune tolerant patient group by LEfSe analysis.
12 is a diagram comparing increased or decreased strains in a liver transplant patient group and an immune tolerant patient group.
13 is a diagram confirming the expression of immune cells in a liver transplant patient group and an immune tolerant patient group by flow cytometry.
14 is a diagram comparing differences in cytokine and chemokine expression between a liver transplant patient group and an immune tolerant patient group.
15 is a diagram comparing differences in cytokine and chemokine expression between a liver transplant patient group and an immune tolerant patient group.
FIG. 16 is a flow cytometry analysis of Treg expression in PBMCs of a liver transplant patient group according to microbiome treatment (A: result of flow cytometry, B: quantification of Treg expression).
FIG. 17 is a flow cytometric analysis of Th17 expression in PBMC of a liver transplant patient group following microbiome treatment (A: result of flow cytometry, B: quantification of Th17 expression).
Figure 18 is a diagram showing the expression of cytokines according to the treatment of the microbiome, which is reduced in PBMC of the liver transplant patient group, analyzed by ELISA (A: quantification of IL-17 expression, B: quantification of IL-10 expression, C: TGF-β expression quantification).
19 is an ELISA analysis of the expression of IL-17 according to the treatment of sodium butyrate, a metabolite of the biobiome, in PBMCs of the liver transplant patient group.
본 발명은 소듐 부티레이트(Sodium Butyrate) 또는 마이크로바이옴을 유효성분으로 포함하는, 이식거부반응의 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating transplant rejection, comprising sodium butyrate or microbiome as an active ingredient.
본 발명에서 사용되는 용어 "예방"은 본 발명의 조성물의 투여로 특정 질환의 증상을 억제하거나 진행을 지연시키는 모든 행위를 의미한다.As used herein, the term "prevention" refers to any action that suppresses symptoms or delays the progression of a specific disease by administering the composition of the present invention.
본 발명에서 사용되는 용어 "치료"는 본 발명의 조성물의 투여로 특정 질환의 증상을 호전 또는 이롭게 변경시키는 모든 행위를 의미한다.The term "treatment" used in the present invention refers to all activities that improve or beneficially change the symptoms of a specific disease by administration of the composition of the present invention.
본 발명의 약학 조성물에는 유효성분 이외에 보조제(adjuvant)를 추가로 포함할 수 있다. 상기 보조제는 당해 기술분야에 알려진 것이라면 어느 것이나 제한 없이 사용할 수 있으나, 예를 들어 프로인트(Freund)의 완전 보조제 또는 불완전 보조제를 더 포함하여 그 효과를 증가시킬 수 있다.The pharmaceutical composition of the present invention may further include an adjuvant in addition to the active ingredient. As long as the adjuvant is known in the art, any one may be used without limitation, but, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase the effect.
본 발명에 따른 약학 조성물은 유효성분을 약학적으로 허용된 담체에 혼입시킨 형태로 제조될 수 있다. 여기서, 약학적으로 허용된 담체는 제약 분야에서 통상 사용되는 담체, 부형제 및 희석제를 포함한다. 본 발명의 약학 조성물에 이용할 수 있는 약학적으로 허용된 담체는 이들로 제한되는 것은 아니지만, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로스, 메틸 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다.The pharmaceutical composition according to the present invention may be prepared in the form of incorporating the active ingredient into a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers usable in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
본 발명의 약학 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀전, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 또는 멸균 주사용액의 형태로 제형화하여 사용될 수 있다.The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions according to conventional methods.
제제화할 경우에는 통상 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제될 수 있다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 그러한 고형 제제는 유효성분에 적어도 하나 이상의 부형제, 예를 들면 전분, 칼슘 카르보네이트, 수크로스, 락토오스, 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용될 수 있다. 경구투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데, 일반적으로 사용되는 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수용성용제, 현탁제, 유제, 동결건조 제제 및 좌제가 포함된다. 비수용성용제, 현탁제로는 프로필렌 글리콜, 폴리에틸렌 글리콜, 올리브유와 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.When formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations may be prepared by mixing active ingredients with at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. may be included in addition to commonly used diluents such as water and liquid paraffin. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for suppositories, witepsol, tween 61, cacao paper, laurin paper, glycerogelatin, and the like may be used.
본 발명에 따른 약학 조성물은 개체에 다양한 경로로 투여될 수 있다. 투여의 모든 방식이 예상될 수 있는데, 예를 들면 경구, 정맥, 근육, 피하, 복강내 주사에 의해 투여될 수 있다.The pharmaceutical composition according to the present invention can be administered to a subject by various routes. All modes of administration are contemplated, eg oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.
본 발명에 따른 약학 조성물의 투여량은 개체의 연령, 체중, 성별, 신체 상태 등을 고려하여 선택된다. 상기 약학 조성물 중 포함되는 유효성분의 농도는 대상에 따라 다양하게 선택할 수 있음은 자명하며, 바람직하게는 약학 조성물에0.01 ~ 5,000 ㎍/ml의 농도로 포함되는 것이다. 그 농도가 0.01 ㎍/ml 미만일 경우에는 약학 활성이 나타나지 않을 수 있고, 5,000 ㎍/ml를 초과할 경우에는 인체에 독성을 나타낼 수 있다.The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, and physical condition of the subject. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be variously selected according to the subject, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. If the concentration is less than 0.01 μg/ml, pharmacological activity may not appear, and if the concentration exceeds 5,000 μg/ml, toxicity to the human body may be exhibited.
본 발명의 일실시예에 따르면, 상기 마이크로바이옴은, 페칼리박테리움 프로스니치(Faecalibacterium prausnitzii), 비피도박테리움 비피듐(Bifidobacterium bifidum), 비피도박테리움 롱검(Bifidobacterium longum) 및 아커멘시아 뮤시니필리아(Akkermansia muciniphilla)로 이루어진 군에서 선택된 균주인 것일 수 있으나, 이에 제한되지는 않는다.According to an embodiment of the present invention, the microbiom is the FAECALIBACTERIUM Prausnitzii, Bifidobacterium Bipidum , Bifido Bacterium Long Sword ONGUM) and Akkermansia Muciniphilla may be a selected strain, but it is not limited thereto.
본 발명에서 용어 "마이크로바이옴"은 인체에 서식하는 미생물의 유전정보 전체나, 미생물 자체를 일컫는 것으로, 인간의 몸에 서식하며 공생하는 미생물인 마이크로바이오타(microbiota)와 게놈(genome)의 합성어이며, 인체 마이크로바이온의 수는 순수한 인체의 세포수보다 두 배 이상 많고 유전자 수는 100배이상 많은 것으로 알려져 있으며, 마이크로 바이옴은 유익균과 유해균이 생성되는 원리와 질병간의 연관성 등을 분석할 수 있어, 신약 개발 및 불치병 치료와 같은 인체내 미생물 환경의 연구에 폭넓게 활용될 수 있는 분야를 뜻한다.In the present invention, the term "microbiome" refers to the entire genetic information of microorganisms living in the human body or the microorganisms themselves. It is a compound word of microbiota and genome, which are microorganisms that live and coexist in the human body. It means a field that can be widely used in the study of the microbial environment in the human body, such as
본 발명의 일실시예에 따르면, 상기 조성물은, 면역 세포를 조절하는 것일 수 있고, 상기 면역세포는 Th17 또는 Treg인 것일 수 있다.According to one embodiment of the present invention, the composition may regulate immune cells, and the immune cells may be Th17 or Treg.
본 발명의 일실시예에 따르면, 상기 면역세포를 조절하는 것은 Th17의 발현을 감소시키는 것일 수 있고, Treg의 발현을 증가시키는 것일 수 있다.According to one embodiment of the present invention, regulating the immune cells may decrease Th17 expression or increase Treg expression.
본 발명의 일실시예에 따르면, 상기 조성물은, IL-17, GM-CSF, IL-6, IL-15, IFN-α, IL-2, IL-1β TNF-α 및 IL-8으로 이루어진 군에서 선택된 염증성 사이토카인 또는 케모카인의 발현을 감소시키는 것일 수 있다.According to one embodiment of the present invention, the composition may reduce the expression of an inflammatory cytokine or chemokine selected from the group consisting of IL-17, GM-CSF, IL-6, IL-15, IFN-α, IL-2, IL-1β, TNF-α, and IL-8.
본 발명의 일실시예에 따르면, 상기 조성물은, 항염증성 사이토카인인 IL-10 또는 T 세포 조절 사이토카인인 TGF-β의 발현을 증가시키는 것일 수 있다.According to one embodiment of the present invention, the composition may increase the expression of anti-inflammatory cytokine IL-10 or T cell regulatory cytokine TGF-β.
본 발명의 일실시예에 따르면, 상기 이식거부반응은 세포, 혈액, 조직 및 장기로 이루어진 군에서 선택된 1종 이상의 이식거부반응인 것일 수 있다.According to one embodiment of the present invention, the transplant rejection may be one or more types of transplant rejection selected from the group consisting of cells, blood, tissues and organs.
본 발명의 일실시예에 따르면, 상기 이식거부반응은 골수 이식, 심장 이식, 각막 이식, 장 이식, 간 이식, 폐 이식, 췌장 이식, 신장 이식 및 피부 이식의 거부 반응으로 이루어진 군에서 선택된 1종 이상인 것일 수 있다.According to one embodiment of the present invention, the transplant rejection may be at least one selected from the group consisting of bone marrow transplantation, heart transplantation, corneal transplantation, intestinal transplantation, liver transplantation, lung transplantation, pancreas transplantation, kidney transplantation, and skin transplantation rejection.
또한, 본 발명은 소듐 부티레이트(Sodium Butyrate) 또는 마이크로바이옴을 유효성분으로 포함하는, 이식거부반응의 예방 또는 개선용 식품조성물을 제공한다.In addition, the present invention provides a food composition for preventing or improving transplant rejection, containing sodium butyrate or microbiome as an active ingredient.
본 발명에서 사용되는 용어 "개선"은 치료되는 상태와 관련된 파라미터, 예를 들면 증상의 정도를 적어도 감소시키는 모든 행위를 의미한다.As used herein, the term "improvement" refers to any action that at least reduces a parameter associated with the condition being treated, eg, the severity of a symptom.
본 발명의 식품 조성물은 본 발명의 유효성분을 함유하는 것 외에 통상의 식품 조성물과 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다.In addition to containing the active ingredient of the present invention, the food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients like conventional food compositions.
상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 향미제는 천연 향미제 (타우마틴), 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등) 및 합성 향미제 (사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 본 발명의 식품 조성물은 상기 약학적 조성물과 동일한 방식으로 제제화되어 기능성 식품으로 이용하거나, 각종 식품에 첨가할 수 있다. 본 발명의 조성물을 첨가할 수 있는 식품으로는 예를 들어, 음료류, 육류, 초코렛, 식품류, 과자류, 피자, 라면, 기타 면류, 껌류, 사탕류, 아이스크림류, 알코올 음료류, 비타민 복합제 및 건강보조식품류 등이 있다.Examples of the aforementioned natural carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agents described above, natural flavoring agents (thaumatin), stevia extracts (eg rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can advantageously be used. The food composition of the present invention can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.
또한 상기 식품 조성물은 유효성분인 추출물 외에 여러 가지 영양제, 비타민, 광물 (전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제 (치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그밖에 본 발명의 식품 조성물은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다.In addition to the active ingredient extract, the food composition may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like. In addition, the food composition of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages, and vegetable beverages.
본 발명의 기능성 식품 조성물은 이식거부반응의 예방 또는 치료 목적으로, 정제, 캅셀, 분말, 과립, 액상, 환 등의 형태로 제조 및 가공될 수 있다. 본 발명에서 '건강기능성 식품 조성물'이라 함은 건강기능식품에 관한 법률 제6727호에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 말하며, 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미한다. 본 발명의 건강기능식품은 통상의 식품 첨가물을 포함할 수 있으며, 식품 첨가물로서의 적합 여부는 다른 규정이 없는 한, 식품의약품안전청에 승인된 식품 첨가물 공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 판정한다. 상기 '식품 첨가물 공전'에 수재된 품목으로는 예를 들어, 케톤류, 글리신, 구연산칼슘, 니코틴산, 계피산 등의 화학적 합성물; 감색소, 감초추출물, 결정셀룰로오스, 고량색소, 구아검 등의 천연첨가물; L-글루타민산나트륨 제제, 면류첨가알칼리제, 보존료 제제, 타르색소제제 등의 혼합제제류 등을 들 수 있다. 예를 들어, 정제 형태의 건강기능식품은 본 발명의 유효성분을 부형제, 결합제, 붕해제 및 다른 첨가제와 혼합한 혼합물을 통상의 방법으로 과립화한 다음, 활택제 등을 넣어 압축성형하거나, 상기 혼합물을 직접 압축 성형할 수 있다. 또한 상기 정제 형태의 건강기능식품은 필요에 따라 교미제 등을 함유할 수도 있다. 캅셀 형태의 건강기능식품 중 경질 캅셀제는 통상의 경질 캅셀에 본 발명의 유효성분을 부형제 등의 첨가제와 혼합한 혼합물을 충진하여 제조할 수 있으며, 연질 캅셀제는 본 발명의 유효성분을 부형제 등의 첨가제와 혼합한 혼합물을 젤라틴과 같은 캅셀기제에 충진하여 제조할 수 있다. 상기 연질 캅셀제는 필요에 따라 글리세린 또는 소르비톨 등의 가소제, 착색제, 보존제 등을 함유할 수 있다. 환 형태의 건강기능식품은 본 발명의 유효성분과 부형제, 결합제, 붕해제 등을 혼합한 혼합물을 기존에 공지된 방법으로 성형하여 조제할 수 있으며, 필요에 따라 백당이나 다른 제피제로 제피할 수 있으며, 또는 전분, 탈크와 같은 물질로 표면을 코팅할 수도 있다. 과립 형태의 건강기능식품은 본 발명의 유효성분의 부형제, 결합제, 붕해제 등을 혼합한 혼합물을 기존에 공지된 방법으로 입상으로 제조할 수 있으며, 필요에 따라 착향제, 교미제 등을 함유할 수 있다.The functional food composition of the present invention may be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or treating transplant rejection. In the present invention, 'health functional food composition' refers to a food manufactured and processed using raw materials or ingredients having useful functionalities for the human body in accordance with Act No. 6727 on Health Functional Foods, and nutrients for the structure and function of the human body. The health functional food of the present invention may contain ordinary food additives, and its suitability as a food additive is determined by the standards and standards for the item in accordance with the General Rules and General Test Methods of Food Additives approved by the Korea Food and Drug Administration, unless otherwise specified. Examples of the items listed in the 'Food Additive Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations. For example, a health functional food in the form of a tablet is a mixture obtained by mixing the active ingredient of the present invention with excipients, binders, disintegrants, and other additives, granulated in a conventional manner, and then compression-molded by adding a lubricant or the like, or the mixture can be directly compressed. In addition, the health functional food in the form of a tablet may contain a flavoring agent and the like as needed. Among health functional foods in the form of capsules, hard capsules can be prepared by filling ordinary hard capsules with a mixture of the active ingredient of the present invention mixed with additives such as excipients, and soft capsules can be prepared by filling a mixture of the active ingredient of the present invention mixed with additives such as excipients into a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary. The health functional food in the form of a pill can be prepared by molding a mixture of the active ingredient of the present invention mixed with an excipient, a binder, a disintegrant, etc. by a conventionally known method, and, if necessary, can be coated with sucrose or other coating agent, or the surface can be coated with a material such as starch or talc. A health functional food in granular form may be prepared by a conventionally known method from a mixture of excipients, binders, disintegrants, etc. of the active ingredients of the present invention, and may contain flavoring agents, flavoring agents, etc., if necessary.
또한, 본 발명은 소듐 부티레이트(Sodium Butyrate) 또는 마이크로바이옴을 유효성분으로 포함하는 조성물을 개체에 투여하는 단계;를 포함하는, 간이식 환자군의 페칼리박테리움 속(Faecalibacterium sp.) 균주를 증가시키고, 박테로이데스 속(Bacteroides sp.) 균주를 감소시키는 방법을 제공한다.In addition, the present invention includes the step of administering a composition containing sodium butyrate or a microbiome as an active ingredient to an individual; Faecalibacterium sp. Increases the strain of liver transplant patients, and provides a method for reducing the strain of Bacteroides sp .
또한, 본 발명은 소듐 부티레이트(Sodium Butyrate) 또는 마이크로바이옴을 유효성분으로 포함하는, 이식거부반응의 예방 또는 치료용 조성물로서,In addition, the present invention is a composition for preventing or treating transplant rejection, comprising sodium butyrate or microbiome as an active ingredient,
상기 조성물은The composition
1) Treg의 발현을 증가시키고,1) increase Treg expression,
2) Th17의 발현을 감소시키며,2) reducing the expression of Th17;
3) IL-17, GM-CSF, IL-6, IL-15, IFN-α, IL-2, IL-1β TNF-α 및 IL-8으로 이루어진 군에서 선택된 염증성 사이토카인 또는 케모카인의 발현 억제시키고, 3) inhibiting the expression of an inflammatory cytokine or chemokine selected from the group consisting of IL-17, GM-CSF, IL-6, IL-15, IFN-α, IL-2, IL-1β, TNF-α and IL-8;
4) 항염증성 사이토카인인 IL-10 또는 T 세포 조절 사이토카인인 TGF-β의 발현을 증가시키는 것인, 조성물을 제공한다.4) to increase the expression of anti-inflammatory cytokine IL-10 or T cell regulatory cytokine TGF-β, provides a composition.
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 examples. These examples are merely for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.
<실시예 1> 간이식 환자 및 정상인의 장내균총 비교<Example 1> Comparison of intestinal flora between liver transplant patients and normal persons
<1-1> 장내균총 다양성 비교<1-1> Comparison of intestinal flora diversity
간이식(Liver transplantation, LT) 환자군과, 건강한 성인(healthy control, H)군을 비교하여, 장내균총의 다양성에 변화가 있는지 확인하고자 하였다. 구체적으로 건강한 성인과 간이식 환자의 fecal을 수집후 -70도에 분석 전까지 보관하였다. 메타지놈 분석을 위해 박테리아 genomic DNA 분리, 미생물 특이적인 16S rRNA 유전자 증폭, 차세대 염기서열 분석 (Next Generation Sequencing, NGS), 장내균총 분석 및 프로파일링으로 분석 하였다. 분리한 gDNA는 박테리아 특이적인 16S 유전자 서열 중 variant region 의 V3와 V4 부분을 포함할 수 있는 부위에서 제작된 프라이머로 유전자를 증폭시키고, 샘플을 구별할 수 있는 인덱스 서열을 부착하여 라이브러리를 제작하기 위해 2번의 PCR을 수행 한후 Illumina社의 Miseq을 이용하여 염기서열 해독을 진행하였다. 해독된 서열은 생물정보학적 분석 tool인 Quantitative Insights into Microbioal Ecology 2 (QIIME 2)과 Lefse, 통계처리는 Prism과 R을 이용하여 장내균총 프로파일링을 수행하였다. 그 후 각 군의 균총의 풍부도를 확인하기 위하여, α-diversity 분석 및 각 군간의 유사도 분석을 위하여 β-diversity 분석을 시행하였다. α-diversity 분석으로는 Observed OTUs 및 Shannon 분석을 시행하였으며, β-diversity 분석으로는 bray curstis distance에 기반하여 PCoA plot분석을 시행하였다.The liver transplantation (Liver transplantation, LT) patient group and healthy adult (healthy control, H) group were compared to determine whether there were any changes in the diversity of the intestinal flora. Specifically, fecal samples from healthy adults and liver transplant patients were collected and stored at -70°C until analysis. For metagenome analysis, bacterial genomic DNA isolation, microbial-specific 16S rRNA gene amplification, next-generation sequencing (NGS) analysis, and gut flora analysis and profiling were analyzed. For the isolated gDNA, the gene was amplified with primers prepared at a site capable of containing the V3 and V4 parts of the variant region among the bacterial-specific 16S gene sequences, and an index sequence capable of distinguishing samples was attached. PCR was performed twice to construct a library, and sequencing was performed using Illumina's Miseq. Intestinal flora profiling was performed using Quantitative Insights into Microbiological Ecology 2 (QIIME 2) and Lefse, which are bioinformatic analysis tools, and Prism and R for statistical processing. Then, in order to confirm the abundance of the flora of each group, α-diversity analysis and β-diversity analysis were performed to analyze the similarity between each group. Observed OTUs and Shannon analysis were performed for α-diversity analysis, and PCoA plot analysis was performed based on bray curstis distance for β-diversity analysis.
그 결과, 도 1에 나타낸 바와 같이, α-diversity 분석에서 Observed OTUs 레벨이 LT 군과 H 군에서 유의적으로 차이나는 것을 확인하였다. 또한, Shannon 분석에서 LT 군의 Shannon 레벨이 감소한 경향을 확인하였다(도 1A).As a result, as shown in FIG. 1, it was confirmed that the levels of Observed OTUs in the α-diversity analysis were significantly different between the LT and H groups. In addition, the Shannon analysis showed a tendency for the Shannon level of the LT group to decrease (FIG. 1A).
또한, β-diversity 분석에서, H 군과 LT 군은 permutational multivariate analysis of variance(PERMANOVA) 에서 유의적으로 차이가 나는 것을 확인하였으며, bray curstis distance 분포시 H와 LT의 각 군 내의 차이보다 H-LT 간의 차이가 유의적으로 증가한 것을 확인하였다(도 1B).In addition, in the β-diversity analysis, it was confirmed that the H group and the LT group were significantly different in the permutational multivariate analysis of variance (PERMANOVA), and the difference between H-LT was significantly higher than the difference between H and LT within each group at the time of bray curstis distance distribution (Fig. 1B).
<1-2> 장내균총 조성 비교<1-2> Comparison of intestinal flora composition
간이식 환자에서, 정상인과 비교하여, 장내균총의 조성에 차이가 있는지 확인하고자 하였으며, 문(Phylum), 과(Family), 속(Genus)의 조성을 확인하였다. 구체적으로 HC와 LT 군간에서 각 군을 대표할 수 있는 분류군(taxa)을 linear discriminant analysis(LDA) effect size(LEfSe)을 이용하여 분석하였다. LDA score가 3.0 이상이고, p-value가 0.05이하인 분류군이 유의성이 있는 것으로 간주하여 선별하였다. In liver transplant patients, compared to normal subjects, we tried to determine whether there was a difference in the composition of the intestinal flora, and the composition of Phylum, Family, and Genus was confirmed. Specifically, between the HC and LT groups, taxa that can represent each group were analyzed using linear discriminant analysis (LDA) effect size (LEfSe). Taxa with an LDA score of 3.0 or more and a p-value of 0.05 or less were considered significant and selected.
또한 각 군의 환자 및 건강한 성인의 개인별 균총 분석을 이용하여, 각 군간 유의적으로 증가되어 있는 균총을 비교하였다.In addition, by using individual flora analysis of patients and healthy adults in each group, the significantly increased flora of each group was compared.
그 결과, 도 2에 나타낸 바와 같이, LT 군에서는 H 군과 비교하여, Proteobacteria 문 및 Bacteroidetes 문이 유의하게 증가하였으며, Bacteroidaceae 과 및 Lachnospiraceae 과가 유의미하게 증가하였다. 또한, 박테로이데테스(Bacteroidetes) 속이 증가한 것을 확인하였다. LT 군에서 H 군과 비교하여 감소한 분류군으로는, Firmicutes 문이 유의하게 감소하였으며, Ruminococcaceae 과가 감소하였고, 페칼리박테리움(Faecalibacterium) 속 및 라크노스피라(Lachnospira) 속이 유의적으로 감소한 것을 확인하였다.As a result, as shown in FIG. 2, in the LT group, compared to the H group, the phylum Proteobacteria and Bacteroidetes increased significantly, and the Bacteroidaceae and Lachnospiraceae families significantly increased. In addition, it was confirmed that the genus Bacteroidetes increased. In the LT group, as the decreased taxa compared to the H group, it was confirmed that the Firmicutes phylum significantly decreased, the Ruminococcaceae family decreased, and the Faecalibacterium and Lachnospira genera significantly decreased.
또한, 각 군별 개인의 균총분석을 확인하였으며(도 3A), H 군에서 증가한 균총(도 3B, 초록색)과 LT 군에서 증가한 균총(도 3B, 빨간색)을 확인하였다.In addition, the colony analysis of individuals in each group was confirmed (Fig. 3A), and the increased flora in the H group (Fig. 3B, green) and the increased flora in the LT group (Fig. 3B, red) were confirmed.
상기의 결과를 바탕으로, H 군과 LT 군에서 증가 또는 감소한 마이크로바이옴을 확인하여 본 결과, LT 군에서는 장내 유익균을 포함하는 것으로 알려진 페칼리박테리움(Faecalibacterium) 속 과 페칼리박테리움 속 균주인 페칼리박테리움 프로스니치(Faecalibacterium Prausnitzi)가 모두 유의적으로 감소된 것을 확인하였으며, 비피도박테리움(Bifidobacterium) 속 및 아커멘시아(Akkermansia) 속이 감소하는 경향을 확인하였고, 장내 유해균을 포함하는 것으로 알려진 박테로이데스(Bacteroides) 속이 증가된 것을 확인하였다(도 4).Based on the above results, as a result of confirming the increased or decreased microbiome in the H and LT groups, it was confirmed that both the Faecalibacterium genus known to contain intestinal beneficial bacteria and Faecalibacterium Prausnitzi, a strain of the Faecalibacterium genus, were significantly reduced in the LT group, Bifidobacterium genus and Akkermansia It was confirmed that the genus tended to decrease, and it was confirmed that the genus Bacteroides, which is known to contain intestinal harmful bacteria, increased (FIG. 4).
<1-3> 면역세포 발현 차이 확인<1-3> Confirmation of differences in immune cell expression
간이식 환자에서, 정상인과 비교하여 면역세포의 발현에 차이가 있는지 확인하고자 하였다. 구체적으로, 자가 면역에 병인 세포로 알려진 Th17 세포와, 다양한 병인 T 세포를 조절하는 것으로 알려진 Treg 세포의 발현을 확인하였다.In liver transplant patients, compared to normal people, we wanted to confirm whether there was a difference in the expression of immune cells. Specifically, the expression of Th17 cells, which are known to be pathological cells in autoimmunity, and Treg cells, which are known to regulate various pathological T cells, were confirmed.
그 결과, 도 5에 나타낸 바와 같이 LT 군에서는 H 군과 비교하여, Th17의 발현이 유의적으로 증가하였으며, Treg의 발현이 유의적으로 감소한 것을 확인하여, LT 군에서는 면역세포의 불균형이 확인되었다.As a result, as shown in FIG. 5 , in the LT group, compared to the H group, it was confirmed that the expression of Th17 was significantly increased and the expression of Treg was significantly decreased, indicating an imbalance of immune cells in the LT group.
<실시예 2> 면역억제제 처리에 따른 장내균총 분석<Example 2> Intestinal flora analysis according to immunosuppressant treatment
간이식 환자에서 면역억제제의 투여(immunosuppressant, IS)에 따른 장내균총 변화를 확인하고자 하였다. 구체적으로, 상기 실시예 1-1 내지 1-2와 동일한 분석을 이용하였으며, 구체적으로 간이식 환자군에서 면역 억제제 용량대비 체내 농도비를 계산하여 그 중간값인 DC(Drug Concentrations)값을 기준으로, 1.6 이하인 군과, DC 1.6 초과인 군으로 분류하여, 면역억제제의 투여량에 따른 차이를 확인하였다. 각 군의 임상적 특성은 하기 표 1에 타나내었다.The purpose of this study was to examine changes in the intestinal flora according to immunosuppressant (IS) administration in liver transplant patients. Specifically, the same analysis as in Examples 1-1 to 1-2 was used. Specifically, the ratio of concentration in the body to immunosuppressant dose was calculated in the liver transplant patient group, and based on the median value of DC (Drug Concentrations), the group was classified into a group with a value of 1.6 or less and a group with a DC value exceeding 1.6, and differences according to the dosage of the immunosuppressant were confirmed. The clinical characteristics of each group are shown in Table 1 below.
그 결과, 도 6에 나타낸 바와 같이, Obseved OTUs, Shannon 및 β-diversity에서는 유의적인 차이는 없었으나, DC가 증가할수록 감소하는 경향을 확인하였다.As a result, as shown in FIG. 6, there was no significant difference in Obseved OTUs, Shannon, and β-diversity, but a tendency to decrease as DC increased.
또한, DC 1.6 초과 군에서는 분류군 중 Proteobacteria 문(phylum)이 현저히 증가하는 것을 확인하였으며, 페칼리박테리움(Faecalibacterium) 속이 감소하는 경향을 확인하였다(도 7).In addition, in the DC group exceeding 1.6, it was confirmed that the Proteobacteria phylum significantly increased among taxa, and the Faecalibacterium genus tended to decrease (FIG. 7).
또한, 구체적으로 증감하는 균총을 확인한 결과, 면역억제제의 양이 증가할 수록 페칼리박테리움(Faecalibacterium) 속 과 페칼리박테리움 속 균주인 페칼리박테리움 프로스니치(Faecalibacterium Prausnitzi), 비피도박테리움(Bifidobacterium) 속 및 아커멘시아(Akkermansia) 속이 감소하는 경향을 확인하였고, 박테로이데스(Bacteroides) 속이 면역억제제의 처리 양과 무관하게 증가되어 있는 것을 확인하였다(도 8).In addition, as a result of confirming the specific flora that increases and decreases, as the amount of immunosuppressive agent increases, it was confirmed that the genus Faecalibacterium, the genus Faecalibacterium Prausnitzi, the genus Bifidobacterium, and the genus Akkermansia decreased as the amount of immunosuppressant increased. It was confirmed that it increased regardless of the amount of treatment (FIG. 8).
<실시예 3> 면역 관용에 따른 장내 균총 비교<Example 3> Comparison of intestinal flora according to immune tolerance
<3-1>면역 관용에 따른 장내 균총 비교 및 면역 세포 발현 비교<3-1> Comparison of intestinal flora and immune cell expression according to immune tolerance
간이식 환자와, 간이식 후 면역억제제의 복용 없이도 면역 관용상태를 유지하는 환자군의 장내 균총이 차이를 나타내는지 확인하고자 하였다. 구체적으로 상기 실시예 1-1 및 1-2와 동일한 방법으로, 간이식 환자군(LT) 및 면역 관용군(Tolerance, T)의 장내 균총을 비교하였다. 또한, 각 군별 면역세포 분석을 실시예 1-3과 동일한 방법으로 확인하였다. 각 군의 환자의 임상 및 인구통계학적 매개변수는 하기 표 2에 나타내었다.The purpose of this study was to determine whether there was a difference in the intestinal microbiota between liver transplant patients and patients who maintained immune tolerance without taking immunosuppressive drugs after liver transplantation. Specifically, in the same manner as in Examples 1-1 and 1-2, the intestinal flora of the liver transplant patient group (LT) and the immune tolerance group (Tolerance, T) were compared. In addition, the analysis of immune cells for each group was confirmed in the same manner as in Examples 1-3. The clinical and demographic parameters of patients in each group are shown in Table 2 below.
그 결과, observed-OUTs와 Shannon 및 β-diversity에서 유의미한 차이가 확인되지 않았으나(도 9), LT 군에서 actinobacteria 문(phylum)이 증가하는 경향을 확인하였으며, Tolerance 군에서 Ruminococcus 과(Family)가 증가하는 것을 확인하였다. 또한, Tolerance 군에서 페칼리박테리움(Faecalibacterium) 속이 증가하는 것을 확인하였다(도 10).As a result, there was no significant difference between observed-OUTs, Shannon and β-diversity (FIG. 9), but the actinobacteria phylum increased in the LT group, and the Ruminococcus family increased in the Tolerance group. In addition, it was confirmed that Faecalibacterium genus increased in the Tolerance group (FIG. 10).
또한, 각 군별 환자들 간의 균총 변화를 속(genus) 수준에서 확인한 결과, Tolerance 군에서는 페칼리박테리움(Faecalibacterium) 속이 유의적으로 증가되어 있었으며(도 11), 페칼리박테리움이 속(genus) 및 페칼리박테리움 프로스니치 (Faecalibacterium Prausnitzi)가 증가된 경향을 확인하였다(도 12).In addition, as a result of confirming the change in flora between patients in each group at the genus level, in the Tolerance group, the Faecalibacterium genus was significantly increased (FIG. 11), and the Faecalibacterium genus and Faecalibacterium Prausnitzi were confirmed to increase (FIG. 12).
또한, 각 군별 면역세포 발현을 확인한 결과, Th17은 각 군별 유의미한 차이를 나타내지는 않았으나, Tolerance 군에서는 LT군과 비교하여 Treg의 발현이 유의적으로 증가되어 있는 것을 확인하였다(도 13).In addition, as a result of confirming the expression of immune cells in each group, there was no significant difference in Th17 in each group, but it was confirmed that Treg expression was significantly increased in the Tolerance group compared to the LT group (FIG. 13).
<3-2>면역 관용에 따른 사이토카인 및 케모카인 발현 비교<3-2> Comparison of cytokine and chemokine expression according to immune tolerance
간이식 환자와, 간이식 후 면역억제제의 복용 없이도 면역 관용상태를 유지하는 환자군의 면역세포에서, 사이토카인 및 케모카인의 발현을 비교하였다. 구체적으로, 분석 사이토카인 및 케모카인은 GM-CSF(granulocyte-macrophage colony-stimulating factor), CCL(Chemokine ligand), CXCL(C-X-C Motif Chemokine Ligand), IFN-α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-12p70, IL-15, IL-23, IP-10(interferon γ-induced protein), MCP-1(monocyte chemoattractant protein-1), MIP-1α(macrophage inflammatory protein-1α) 및 TNF-α(tumor necrosis factor-α)를 분석하였으며, 간이식 환자 및 면역 관용 환자군으로부터 분리된 혈장 샘플을 수집한 후, 분석 전까지 -80℃에서 보관하였으며, ProcartaPlex Human 14-Plex(PPX-14-MXPRMK7) 패널(InvitrogenTM, Thermo Fisher Scientific, Massachusetts, USA) 다중 사이토카인 분석을, Luminex® xMAP™ fluorescent bead- based technology(Luminex Corporation, 12,212 Technology Blvd, Austin, TX, 78727, USA)을 이용하여 분석하였다.Expressions of cytokines and chemokines were compared between liver transplant patients and immune cells of patients who maintained immune tolerance without taking immunosuppressive drugs after liver transplantation. Specifically, the analyzed cytokines and chemokines include granulocyte-macrophage colony-stimulating factor (GM-CSF), chemokine ligand (CCL), C-X-C motif chemokine ligand (CXCL), IFN-α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-12p70, IL-15, IL-23, IP-10 (interferon γ- induced protein), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1α (MIP-1α), and tumor necrosis factor-α (TNF-α) were analyzed. Plasma samples isolated from liver transplant patients and immune tolerant patients were collected and stored at -80 ° C until analysis. ProcartaPlex Human 14-Plex (PPX-14-MXPRMK7) panel (InvitrogenTM, Thermo Fisher Scientific, Massachusetts, USA) multiple cytokine assays were analyzed using Luminex® xMAP™ fluorescent bead-based technology (Luminex Corporation, 12,212 Technology Blvd, Austin, TX, 78727, USA).
그 결과, 도 14 및 도 15에 나타낸 바와 같이, Tolerance 군에서는 LT군과 비교하여, 염증과 관련된 사이토카인 및 케모카인인 GM-CSF, IL-6, IL-15, IFN-α, IL-2, IL-1β TNF-α 및 IL-8의 발현이 유의적으로 감소한 것을 확인하였다.As a result, as shown in FIGS. 14 and 15, in the Tolerance group, compared to the LT group, it was confirmed that the expression of cytokines and chemokines related to inflammation, GM-CSF, IL-6, IL-15, IFN-α, IL-2, IL-1β, TNF-α, and IL-8 decreased significantly.
<실시예 4> 마이크로바이옴의 면역세포 조절 효과 확인<Example 4> Confirmation of immune cell modulating effect of microbiome
상기 실시예 1 내지 실시예 3의 결과를 토대로, 상기 LT 군에서 감소하는 경향을 확인한 마이크로바이옴 균주들이, 면역세포를 조절할 수 있는지 확인하였다. 구체적으로, 간이식 환자로부터, 혈액을 수득하고, 말초혈액 단핵구(peripheral blood mononuclear cell, PBMC)를 분리하였다. 분리된 PBMC에 T 세포 활성조건인 anti-CD3 0.5ug/ml로 자극 한 조건에, 비피도박테리움 속 마이크로바이옴인 B. bifidum 및 B. longum, 페칼리박테리움 속 마이크로바이옴인 F. prausnitzii, 아커멘시아 속 균주인 A. muciniphilla를 각각 처리하고 3일간 배양하여, 유세포분석을 이용하여, 이식거부와 관련된 Th17 및 면역 조절 세포인 Treg 세포의 발현을 확인하였으며, ELISA를 이용하여, 염증성 사이토카인인 IL-17 및 항염증성 사이토카인인 IL-10, T 세포 조절 사이토카인인 TGF-β(Transforming growth factor beta)의 발현을 확인하였다. 대조군으로는 정상인의 PBMC(Nil) 및 환자 유래의 PBMC에 PBS를 처리하여 배양한 Vehicle 군을 이용하였다.Based on the results of Examples 1 to 3, it was confirmed whether the microbiome strains, which showed a decreasing trend in the LT group, could regulate immune cells. Specifically, blood was obtained from a liver transplant patient, and peripheral blood mononuclear cells (PBMCs) were isolated. Separated PBMCs were stimulated with 0.5ug/ml of anti-CD3, a condition for T cell activation, and treated with B. bifidum and B. longum , a microbiome of the genus Bifidobacterium, F. prausnitzii , a microbiome of the genus Faecalibacterium, and A. mucinipilla , a strain of the genus Acamencia, respectively, and cultured for 3 days, using flow cytometry to detect Th17 and immune regulatory cells, Treg cells. The expression of was confirmed, and the expression of the inflammatory cytokine IL-17, the anti-inflammatory cytokine IL-10, and the T cell regulatory cytokine TGF-β (Transforming growth factor beta) was confirmed using ELISA. As a control group, a vehicle group cultured by treating normal PBMC (Nil) and patient-derived PBMC with PBS was used.
또한, 페칼리박테리움 속 균주의 대사체인, 소듐 부티레이트(Sodium Butyrate)가 간이식 환자에서 항염증 효과가 있는지 확인하고자, 상기의 ELISA와 동일한 방법으로, 소듐 부티레이트(Sodium butyrate)를 0.5 mM 및 1 mM로 각각 처리하여, IL-17의 발현을 확인하였다.In addition, in order to confirm whether sodium butyrate, a metabolite of a strain of the genus Faecalibacterium, has an anti-inflammatory effect in liver transplant patients, sodium butyrate was treated with 0.5 mM and 1 mM, respectively, in the same manner as in the ELISA described above, and the expression of IL-17 was confirmed.
그 결과, 도 16에 나타낸 바와 같이, Vehicle 군과 비교하여 B. bifidum, B. longum, F. prausnitzii 및 A. muciniphilla를 각각 처리한 군에서는 Treg의 발현이 유의적으로 증가한 것을 확인하였다. 또한, Vehicle 군과 비교하여 B. bifidum, B. longum, F. prausnitzii 및 A. muciniphilla를 각각 처리한 군에서는 Th17의 발현이 유의적으로 감소하는 것을 확인하여(도 17), 본 발명의 B. bifidum, B. longum, F. prausnitzii 및 A. muciniphilla이 면역 세포를 조절할 수 있는 것을 확인하였다.As a result, as shown in FIG. 16, it was confirmed that Treg expression significantly increased in the group treated with B. bifidum , B. longum , F. prausnitzii , and A. mucinipilla , respectively, compared to the vehicle group. In addition, it was confirmed that the expression of Th17 was significantly decreased in the group treated with B. bifidum , B. longum , F. prausnitzii, and A. mucinipilla compared to the vehicle group (FIG. 17), confirming that B. bifidum , B. longum , F. prausnitzii , and A. mucinipilla of the present invention can regulate immune cells.
또한, 마이크로바이옴을 처리한 각 군에서는 항염증성 사이토카인인 IL-17의 발현이 유의적으로 감소하였으며(도 18A), 항염증성 사이토카인인 IL-10의 발현이 유의적으로 증가하고(도 18B), T 세포 조절 사이토카인인 TGF-β의 발현 또한 유의적으로 증가하는 것을 확인하였다(도 18C).In addition, in each group treated with the microbiome, the expression of the anti-inflammatory cytokine IL-17 was significantly decreased (FIG. 18A), the expression of the anti-inflammatory cytokine IL-10 was significantly increased (FIG. 18B), and the expression of TGF-β, a T cell regulatory cytokine, was also confirmed to be significantly increased (FIG. 18C).
또한, 페칼리박테리움속 균주의 대사체인 소듐 부티레이트(Sodium Butyrate)를 처리하면, IL-17의 발현이 유의적으로 감소하는 것을 확인하였다(도 19).In addition, it was confirmed that the expression of IL-17 was significantly reduced when sodium butyrate, a metabolite of a strain of the genus Faecalibacterium, was treated (FIG. 19).
따라서, 본 발명은, 간이식 환자군에서, 장내 균총의 다양성이 감소된 것을 확인하였으며, 특히 간이식 환자군에서, 장내 유익균으로 알려진 비피도박테리움 속 마이크로바이옴인 B. bifidum 및 B. longum, 페칼리박테리움 속 마이크로바이옴인 F. prausnitzii, 아커멘시아 속 균주인 A. muciniphilla가 유의적으로 감소한 것을 확인하였다. 또한, 상기의 마이크로바이옴 균주 또는 균주의 대사체인 소듐 부티레이트를 간이식 환자 유래의 PBMC에 처리하면, 간이식 환자군의 면역세포인 Th17 및 Treg의 발현을 조절하는 것을 확인하였다. 또한, 염증성 사이토카인 또는 케모카인인 IL-17, GM-CSF, IL-6, IL-15, IFN-α, IL-2, IL-1β TNF-α 및 IL-8의 발현을 억제하고, 항염증성 사이토카인인 IL-10 및 T 세포 조절 사이토카인인 TGF-β의 발현을 증가시키는 것을 확인하였다. 따라서, 간이식 환자군의 장내 균총 조성비 및 이상을 진단하고, 이에 따라 본 발명의 유효물질 처리에 따른 유용한 장내 균총의 증가를 확인하여, 개인별 맞춤의학 실현이 가능한 것을 확인하였다.Therefore, the present invention confirmed that the diversity of intestinal microbiota was reduced in the liver transplant patient group, and in particular, in the liver transplant patient group, it was confirmed that B. bifidum and B. longum , which are known as beneficial bacteria in the intestine, and B. In addition, it was confirmed that when the above microbiome strain or sodium butyrate, a metabolite of the strain, was treated with liver transplant patient-derived PBMC, the expression of Th17 and Treg, which are immune cells in the liver transplant patient group, was regulated. In addition, it was confirmed that the expression of inflammatory cytokines or chemokines IL-17, GM-CSF, IL-6, IL-15, IFN-α, IL-2, IL-1β, TNF-α and IL-8 was inhibited, and the expression of anti-inflammatory cytokine IL-10 and T cell regulatory cytokine TGF-β was increased. Therefore, it was confirmed that personalized medicine can be realized by diagnosing the intestinal flora composition ratio and abnormality of the liver transplant patient group and confirming the increase in useful intestinal flora according to the treatment of the active substance of the present invention.
Claims (14)
상기 마이크로바이옴은, 페칼리박테리움 프로스니치(Faecalibacterium prausnitzii), 비피도박테리움 비피듐(Bifidobacterium bifidum), 비피도박테리움 롱검(Bifidobacterium longum) 및 아커멘시아 뮤시니필리아(Akkermansia muciniphilla)로 이루어진 군에서 선택된 균주인 것인, 조성물.According to claim 1,
The microbiome is a strain selected from the group consisting of Faecalibacterium prausnitzii , Bifidobacterium bifidum , Bifidobacterium longum , and Akkermansia muciniphila . Composition.
상기 조성물은, 면역 세포를 조절하는 것인, 조성물.According to claim 1,
Wherein the composition modulates immune cells.
상기 면역세포는 Th17 또는 Treg인 것인, 조성물.According to claim 3,
Wherein the immune cells are Th17 or Treg.
상기 면역세포를 조절하는 것은 Th17의 발현을 감소시키는 것인, 조성물.According to claim 3,
Regulating the immune cells is to reduce the expression of Th17, the composition.
상기 면역세포를 조절하는 것은 Treg의 발현을 증가시키는 것인, 조성물.According to claim 3,
Regulating the immune cells is to increase the expression of Treg, the composition.
상기 조성물은, IL-17, GM-CSF, IL-6, IL-15, IFN-α, IL-2, IL-1β TNF-α 및 IL-8으로 이루어진 군에서 선택된 염증성 사이토카인 또는 케모카인의 발현을 감소시키는 것인, 조성물.According to claim 1,
The composition reduces the expression of an inflammatory cytokine or chemokine selected from the group consisting of IL-17, GM-CSF, IL-6, IL-15, IFN-α, IL-2, IL-1β TNF-α and IL-8 Composition.
상기 조성물은, 항염증성 사이토카인인 IL-10 또는 T 세포 조절 사이토카인인 TGF-β의 발현을 증가시키는 것인, 조성물.According to claim 1,
Wherein the composition increases the expression of the anti-inflammatory cytokine IL-10 or the T cell regulatory cytokine TGF-β.
상기 이식거부반응은 세포, 혈액, 조직 및 장기로 이루어진 군에서 선택된 1종 이상의 이식거부반응인 것인, 조성물.According to claim 1,
The transplant rejection is one or more types of transplant rejection selected from the group consisting of cells, blood, tissues and organs, the composition.
상기 이식거부반응은 골수 이식, 심장 이식, 각막 이식, 장 이식, 간 이식, 폐 이식, 췌장 이식, 신장 이식 및 피부 이식의 거부 반응으로 이루어진 군에서 선택된 1종 이상인 것인, 조성물.According to claim 9,
The transplant rejection reaction is at least one selected from the group consisting of bone marrow transplantation, heart transplantation, corneal transplantation, intestinal transplantation, liver transplantation, lung transplantation, pancreas transplantation, kidney transplantation and skin transplantation rejection reaction, composition.
상기 마이크로바이옴은, 페칼리박테리움 프로스니치(Faecalibacterium prausnitzii), 비피도박테리움 비피듐(Bifidobacterium bifidum), 비피도박테리움 롱검(Bifidobacterium longum) 및 아커멘시아 뮤시니필리아(Akkermansia muciniphilla)로 이루어진 군에서 선택된 균주인 것인, 조성물.According to claim 11,
The microbiome is a strain selected from the group consisting of Faecalibacterium prausnitzii , Bifidobacterium bifidum , Bifidobacterium longum , and Akkermansia muciniphila . Composition.
상기 조성물은
1) Treg의 발현을 증가시키고,
2) Th17의 발현을 감소시키며,
3) IL-17, GM-CSF, IL-6, IL-15, IFN-α, IL-2, IL-1β TNF-α 및 IL-8으로 이루어진 군에서 선택된 염증성 사이토카인 또는 케모카인의 발현 억제시키고,
4) 항염증성 사이토카인인 IL-10 또는 T 세포 조절 사이토카인인 TGF-β의 발현을 증가시키는 것인, 조성물.A composition for preventing or treating transplant rejection, comprising sodium butyrate or microbiome as an active ingredient,
The composition
1) increase Treg expression,
2) reducing the expression of Th17;
3) inhibiting the expression of an inflammatory cytokine or chemokine selected from the group consisting of IL-17, GM-CSF, IL-6, IL-15, IFN-α, IL-2, IL-1β, TNF-α and IL-8;
4) to increase the expression of the anti-inflammatory cytokine IL-10 or the T cell regulatory cytokine TGF-β, the composition.
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