KR20220118195A - Natural killer cells with activated Metabotropic glutamate receptor 5 and uses thereof - Google Patents
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Abstract
Description
본 발명은 대사성 글루타메이트 수용체 5(Metabotropic glutamate receptor 5, mGluR5)가 활성화된 자연 살해(Natural killer, NK) 세포의 간질환 치료 용도에 관한 것이다. The present invention relates to the use of natural killer (NK) cells activated by metabolic glutamate receptor 5 (Metabotropic glutamate receptor 5, mGluR5) for the treatment of liver disease.
만성 간질환은 하나의 독립적인 질환이기보다는 만성 간염에서부터 섬유화를 거쳐 간경변증으로 진행하는 연속적인 질환이다. 만성 간질환은 국내에서 성인병 및 사망의 중요한 원인이므로 이 질환의 병인 규명, 예방과 치료 등에 대한 연구가 매우 중요하다. Chronic liver disease is not an independent disease, but a continuous disease that progresses from chronic hepatitis through fibrosis to cirrhosis. Chronic liver disease is an important cause of adult disease and death in Korea, so research on the etiology, prevention and treatment of this disease is very important.
만성 간질환의 진행 단계 중에서, 간섬유화는 만성 간내 염증으로 인한 세포외 기질(Extracellular matrix, ECM)의 과다한 침착으로 정의될 수 있으며 이러한 세포외 기질의 과다한 침착으로 만성 간질환이 지속되는 경우 결국은 간내 구조의 변형과 간세포수의 감소로 간경변으로 진행되게 된다. 구체적으로, 여러 원인으로 간세포가 손상되면 간성상세포(Hepatic stellate cell)와 쿠퍼세포(Kupffer cell) 등의 상호작용에 의해 각종 사이토카인과 활성 산소 등이 생성되고, 이로 인해 활성화된 간성상세포는 ECM을 생산한다. 이러한 섬유생성(Fibrogenesis) 과정으로 생산된 ECM은 간세포가 파괴되어 생긴 빈 공간을 채워서 간 형태를 유지하는 역할을 한다. 급성 간질환에서는 파괴된 공간을 간세포가 다시 재생하여 채워감에 따라 더 이상의 ECM 역할이 필요치 않아서 ECM을 파괴하는 섬유용해(Fibrolysis) 과정이 활발하게 일어나 정상 간으로 회복한다. 그러나 만성 간질환에서는 간세포가 지속적으로 파괴되어, 섬유용해 과정에 비해 상대적으로 섬유생성 과정이 과도하게 지속됨에 따라 ECM이 이상 증식되어 간섬유증으로 진행한다.Among the progressive stages of chronic liver disease, hepatic fibrosis can be defined as excessive deposition of extracellular matrix (ECM) due to chronic intrahepatic inflammation. It progresses to cirrhosis due to a change in the structure of the liver and a decrease in the number of hepatocytes. Specifically, when hepatocytes are damaged due to various causes, various cytokines and reactive oxygen species are generated by the interaction between hepatic stellate cells and Kupffer cells, and thus the activated hepatocytes are ECM is produced. The ECM produced by this Fibrogenesis process fills the empty space created by the destruction of hepatocytes and plays a role in maintaining the liver shape. In acute liver disease, as hepatocytes regenerate and fill the destroyed space, the role of the ECM is no longer needed, so the fibrolysis process that destroys the ECM actively occurs to restore a normal liver. However, in chronic liver disease, hepatocytes are continuously destroyed, and as the fibrosis process continues excessively compared to the fibrolytic process, the ECM proliferates abnormally and progresses to liver fibrosis.
일반적으로 간섬유증은 가역적이고, thin fibril로 구성되며 결절 형성이 없고 간손상 원인이 소실되면 정상 회복이 가능하나, 간섬유증 과정이 반복적으로 지속되면 ECM 간의 교환결합(Crosslinking)이 증가하여 thick fibril을 형성하고 결절이 있는 간경변으로 진행한다.In general, hepatic fibrosis is reversible, composed of thin fibril, no nodule formation, and normal recovery is possible when the cause of liver damage is eliminated. It forms and progresses to nodular cirrhosis.
ECM은 교원질(Collagen), 당단백질(Glycoprotein), 프로테오 글라이칸(Proteoglycan) 등으로 구성된다. 이 중 특히 교원질 증가는 간섬유증의 중요한 원인이 되고 교원질은 여러 원인에 의해 활성화된 간성상세포가 주로 생산한다.ECM is composed of collagen, glycoprotein, proteoglycan, and the like. Among them, an increase in collagen is an important cause of liver fibrosis, and collagen is mainly produced by hepatocytes activated by various causes.
전술한 바와 같이 간섬유화는 간질환의 전단계로써 그 원인도 어느 정도 알려져 있으나, 그럼에도 불구하고 아직까지 간섬유화에 대한 구체적인 치료 방법이 존재하지 않는 실정이다.As described above, the cause of liver fibrosis as a pre-stage of liver disease is also known to some extent. Nevertheless, there is no specific treatment method for liver fibrosis yet.
이러한 배경 하에서, 본 발명자들은 NK 세포 내에 mGluR5가 존재하고, mGluR5가 활성화되면 간섬유화를 유발할 수 있는 활성화된 간성상세포에 대한 NK 세포의 세포독성이 증대하고 그 결과 간섬유화가 완화되는 것을 확인함으로써, NK 세포 특이적 mGluR5 활성이 간섬유화의 억제에 기여함을 확인하여 본 발명을 완성하였다.Under this background, the present inventors confirmed that mGluR5 exists in NK cells, and when mGluR5 is activated, the cytotoxicity of NK cells to activated hepatic stellate cells, which can cause liver fibrosis, is increased, and as a result, liver fibrosis is alleviated. , The present invention was completed by confirming that NK cell-specific mGluR5 activity contributes to the inhibition of liver fibrosis.
본 발명의 하나의 목적은 대사성 글루타메이트 수용체 5(metabotropic glutamate receptor 5, mGluR5)의 작용제(agonist)를 유효성분으로 포함하는, 간질환의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.One object of the present invention is to provide a pharmaceutical composition for preventing or treating liver disease, comprising an agonist of metabolic glutamate receptor 5 (mGluR5) as an active ingredient.
본 발명의 다른 하나의 목적은 mGluR5가 활성화된 NK 세포를 유효성분으로 포함하는, 간질환의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating liver disease, comprising mGluR5 activated NK cells as an active ingredient.
본 발명의 또 다른 하나의 목적은 상기 약학적 조성물을 개체에 투여하는 단계를 포함하는, 간질환의 치료방법을 제공하는 것이다.Another object of the present invention is to provide a method for treating liver disease, comprising administering the pharmaceutical composition to a subject.
이를 구체적으로 설명하면 다음과 같다. 한편, 본 발명에서 개시된 각각의 설명, 및 실시형태는 각각의 다른 설명, 및 실시형태에도 적용될 수 있다. 즉, 본 발명에서 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 볼 수 없다.This will be described in detail as follows. On the other hand, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be considered that the scope of the present invention is limited by the specific descriptions described below.
또한, 당해 기술분야의 통상의 지식을 가진 자는 통상의 실험만을 사용하여 본 발명에 기재된 본 발명의 특정 양태에 대한 다수의 등가물을 인지하거나 확인할 수 있다. 또한, 이러한 등가물은 본 발명에 포함되는 것으로 의도된다.In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Also, such equivalents are intended to be encompassed by the present invention.
상기 목적을 달성하기 위하여, 본 발명의 하나의 양태는 대사성 글루타메이트 수용체 5(metabotropic glutamate receptor 5, mGluR5)의 작용제(agonist)를 유효성분으로 포함하는, 간질환의 예방 또는 치료용 약학적 조성물을 제공한다.In order to achieve the above object, one aspect of the present invention provides a pharmaceutical composition for preventing or treating liver disease, comprising an agonist of metabolic glutamate receptor 5 (mGluR5) as an active ingredient do.
본 발명에서 용어, "간질환(Hepatic disease, Liver disease)" 만성 간염에서부터 섬유화를 거쳐 간경변증으로 진행하는 연속적인 질환이다. 본 발명에 있어서, 상기 간질환은 간섬유화, 및 이로부터 비롯되는 간경변, 비알코올성 간질환 및 알코올성 간질환을 포함할 수 있으며, 전술한 질환으로부터 진행되는 간질환을 제한 없이 포함할 수 있으나, 구체적으로 간섬유화일 수 있다.As used herein, the term "hepatic disease (Liver disease)" is a continuous disease that progresses from chronic hepatitis to cirrhosis through fibrosis. In the present invention, the liver disease may include liver fibrosis and cirrhosis resulting therefrom, non-alcoholic liver disease and alcoholic liver disease, and may include, without limitation, liver disease proceeding from the above-described disease, but specifically may be liver fibrosis.
본 발명에서 용어, "간섬유화(Liver fibrosis)"는 만성 간내 염증으로 인한 세포외 기질(Extracellular matrix, ECM)이 과다하게 침착된 것으로, 이러한 세포외 기질의 과다한 침착으로 만성 간질환이 지속되는 경우 결국은 간내 구조의 변형과 간세포수의 감소로 간경변으로 진행되게 된다. ECM은 교원질(Collagen), 당단백질(Glycoprotein), 프로테오 글라이칸(Proteoglycan) 등으로 구성된다. 이 중 특히 교원질 증가는 간섬유증의 중요한 원인이 되고 교원질은 여러 원인에 의해 활성화된 간성상세포가 주로 생산하는 것으로 알려져 있다.As used herein, the term "liver fibrosis" refers to excessive deposition of extracellular matrix (ECM) due to chronic intrahepatic inflammation, and when chronic liver disease persists due to excessive deposition of the extracellular matrix Eventually, liver cirrhosis progresses due to a change in the structure of the liver and a decrease in the number of hepatocytes. ECM is composed of collagen, glycoprotein, proteoglycan, and the like. In particular, it is known that the increase in collagen is an important cause of liver fibrosis, and collagen is mainly produced by hepatocytes activated by various causes.
본 발명에서 용어, "대사성 글루타메이트 수용체(metabotropic glutamate receptor, mGluR, GRM)"는 간접 대사성 과정을 통해 활성화되는 글루타메이트 수용체의 한 유형으로, G- 단백질 결합 수용체(G-protein-coupled receptors, GPCRs)의 그룹 C 패밀리의 구성원이며, 흥분성 신경전달물질로 기능하는 아미노산 인 글루타메이트와 결합한다. 상기 mGluR은 mGluR1부터 mGluR8의 유형이 존재하며, 수용체 구조와 생리적 활동에 따라 그룹 I, II 및 III으로 나뉘고, mGluR7a 및 mGluR7b와 같은 하위 유형으로 더 분류할 수 있다.As used herein, the term "metabotropic glutamate receptor (mGluR, GRM)" is a type of glutamate receptor that is activated through an indirect metabolic process, and of G-protein-coupled receptors (GPCRs) It is a member of the Group C family and binds to glutamate, an amino acid that functions as an excitatory neurotransmitter. The mGluR types are mGluR1 to mGluR8, and are divided into groups I, II and III according to receptor structures and physiological activities, and can be further classified into subtypes such as mGluR7a and mGluR7b.
mGluR1 및 mGluR5를 포함한 그룹 I의 mGluR은 흥분성 아미노산 유사체 L- 퀴 스쿠알산(L-quisqualic acid)에 의해 가장 강하게 자극된다. 수용체를 자극하면 관련 효소인 포스포리파아제 C(phospholipase C)가 세포의 원형질막에서 포스포이노시티드(phosphoinositide) 인지질을 가수 분해하고, 이는 이노시톨 1,4,5-트리스포스페이트(inositol 1,4,5-trisphosphate, IP3) 및 디아실 글리세롤(diacyl glycerol)의 형성을 유도한다. 친수성인 IP3는 소포체로 이동하여 수용체에 고정을 통해 칼슘 채널의 개방을 유도하여 세포질 칼슘 농도를 증가시키고, 친유성인 디아실 글리세롤은 막에 남아있어 단백질 키나아제 C의 활성화를 위한 보조 인자로 작용한다. Group I mGluRs, including mGluR1 and mGluR5, are most strongly stimulated by the excitatory amino acid analog L-quisqualic acid. Upon stimulation of the receptor, a related enzyme, phospholipase C, hydrolyzes phosphoinositide phospholipids in the cell's plasma membrane, which in turn hydrolyzes inositol 1,4,5-triphosphate. -trisphosphate, IP3) and induces the formation of diacyl glycerol. The hydrophilic IP3 moves to the endoplasmic reticulum and induces the opening of calcium channels through fixation to the receptor, thereby increasing the cytoplasmic calcium concentration, while the lipophilic diacylglycerol remains in the membrane and acts as a cofactor for the activation of protein kinase C .
상기 그룹 I의 mGluR의 작용은 흥분성이며 전도도를 증가시켜 시냅스 전 세포에서 더 많은 글루타메이트가 방출되도록 할 수 있지만, 억제성 시냅스 후 전위 또는 억제성 연접후 전위를 증가시키기도 하며, 글루타메이트 방출을 억제하고 전압 의존적 칼슘 채널을 조절할 수 있다.The action of group I mGluRs is excitatory and can increase the conductance to release more glutamate from presynaptic cells, but also increase the inhibitory post-synaptic potential or the inhibitory post-synaptic potential, inhibiting glutamate release and increasing the voltage It can regulate calcium-dependent calcium channels.
본 발명은 신경세포 외에서 mGluR5의 발현, 특히 자연 살해(Natural killer, NK) 세포에서 mGluR5의 발현을 최초로 확인한 것에 의의가 있다.The present invention is significant in confirming for the first time the expression of mGluR5 in extraneuronal cells, in particular, in natural killer (NK) cells.
본 발명에 있어서, 상기 mGluR5 작용제(agonist)는 자연 살해(Natural killer, NK) 세포의 mGluR5을 활성화시키는 것일 수 있다.In the present invention, the mGluR5 agonist may be one that activates mGluR5 of natural killer (NK) cells.
본 발명의 목적상, 상기 NK 세포는 구체적으로 간 및 혈액 내 NK 세포일 수 있다.For the purposes of the present invention, the NK cells may specifically be NK cells in the liver and blood.
본 발명의 일 실시예에서, 마우스에서 분리된 간 및 혈액 내 NK 세포에서 세포의 흥분 독성(excitotoxicity)을 증가시키는 그룹 I mGluR과 감소시키는 그룹 II 및 그룹 III mGluR의 유전자 발현 정도를 비교한 결과(도 1A), 흥분 독성을 증가시키는 Group I mGluR을 구성하는 mGluR1의 유전자 Grm1과 mGluR5의 유전자 Grm5 중 Grm5의 발현이 유의하게 더 높은 것을 확인하였다.In one embodiment of the present invention, a result of comparing the gene expression levels of group I mGluR increasing excitotoxicity and decreasing group II and group III mGluR in NK cells in liver and blood isolated from mice ( 1A), it was confirmed that the expression of Grm5 among the mGluR1 gene Grm1 and mGluR5 gene Grm5 constituting the Group I mGluR that increases excitotoxicity was significantly higher.
기존에 글루타메이트 수용체가 잘 발달되어 있다고 알려진 뇌 조직과, 간 내 실질세포인 마우스 간세포, 그리고 마우스 NK 세포에서 mGluR1의 유전자 Grm1과 mGluR5의 유전자 Grm5 발현 정도를 RT-PCR로 비교한 결과(도 1B), 뇌 조직에서는 알려진 대로 Grm1 및 Grm5의 밴드가 짙게 형성되었고, 간세포에서는 Grm1, Grm5 모두 밴드가 형성되지 않았다. 반면, NK 세포에서는 Grm1, Grm5 모두 밴드가 형성되었고, 그 중에서도 Grm5 가 더 짙게 형성된 것을 확인하였다.The results of comparing the expression levels of mGluR1 genes Grm1 and mGluR5 genes Grm5 in brain tissue, which are known to have well-developed glutamate receptors, mouse hepatocytes, which are parenchymal cells in the liver, and mouse NK cells, by RT-PCR (Fig. 1B) , Grm1 and Grm5 bands were densely formed in brain tissue, and neither Grm1 nor Grm5 bands were formed in hepatocytes. On the other hand, in NK cells, both Grm1 and Grm5 bands were formed, and it was confirmed that Grm5 was formed more densely among them.
마우스 NK 세포에서 mGluR5가 유전자 수준이 아닌 단백질 수준에서도 발현하는지 웨스턴 블랏으로 확인한 결과(도 1C), 유전자 수준에서와 마찬가지로, 뇌 조직 및 NK 세포에서 mGluR5 밴드가 확인되었다.As a result of confirming by Western blot whether mGluR5 was expressed at the protein level rather than the gene level in mouse NK cells ( FIG. 1C ), mGluR5 bands were confirmed in brain tissue and NK cells as in the gene level.
또한, 마우스 NK 세포 내의 mGluR5을 단백질 수준에서 보여주는 유세포 분석 결과를 히스토그램 도표로 나타낸 결과(도 1C), mGluR5 항체로 염색한 NK 세포에서 비염색(Unstained) 대조군 및 이소타입 대조군(isotype)에 비해 mGluR5가 월등하게 발현하는 것을 확인하였다. In addition, the results of flow cytometry showing mGluR5 in mouse NK cells at the protein level as a histogram chart (Fig. 1C), mGluR5 in NK cells stained with mGluR5 antibody compared to unstained control and isotype control (isotype) was found to be superiorly expressed.
이에 따라, 간 내 NK 세포에서 mGluR5가 존재함을 확인하였다.Accordingly, it was confirmed that mGluR5 was present in NK cells in the liver.
즉, 상기 mGluR5 작용제는 간 내 NK 세포에 존재하는 mGluR5를 활성화시키는 것일 수 있다.That is, the mGluR5 agonist may activate mGluR5 present in NK cells in the liver.
상기 작용제는 CHPG[(RS)-2-Chloro-5-hydroxyphenylglycine] 등일 수 있으나, 이에 제한되지 않는다. The agent may be CHPG[(RS)-2-Chloro-5-hydroxyphenylglycine], but is not limited thereto.
본 발명에 있어서, 상기 mGluR5가 활성화된 NK 세포는 활성화된 간성상세포에 대한 세포독성이 증가될 수 있다.In the present invention, the mGluR5 activated NK cells may have increased cytotoxicity to activated hepatic stellate cells.
본 발명의 일 실시예에서, 마우스 NK 세포에 mGluR5 작용제인 CHPG 100 μM을 처리시 Grm5의 발현 변화를 확인한 결과(도 2A), CHPG를 처리한 NK 세포는 처리하지 않은 NK 세포보다 Grm5의 발현이 상승하였다.In one embodiment of the present invention, as a result of confirming the change in the expression of Grm5 when the mouse NK cells were treated with 100 μM of the mGluR5 agonist CHPG ( FIG. 2A ), the expression of Grm5 was higher in the NK cells treated with CHPG than the NK cells that were not treated. rose.
타겟 세포(Day 1 간성상세포(비활성 간성상세포)와 Day 4 간성상세포(활성화된 간성상세포)) 및 이펙터 세포(CHPG 100 μM을 처리 또는 처리하지 않은 간 내 NK 세포)를 공동배양한 뒤 발생하는 Calcein-AM 형광 강도으로 세포독성을 평가한 결과(도 2B), 공동배양된 NK 세포가 간성상세포를 사멸시키는 정도는 CHPG 처리군이 CHPG를 처리하지 않은 군보다 유의하게 높았으며, 모든 타겟 세포에서 동일하였다.Target cells (Day 1 hepatocytes (inactive hepatocytes) and Day 4 hepatocytes (activated hepatocytes)) and effector cells (NK cells in the liver with or without
또한, 타겟 세포(LX-2 세포주(활성화된 사람 간성상세포주)) 및 이펙터 세포(CHPG 100 μM을 처리 또는 처리하지 않은 사람 간 유래 NK 세포)를 공동배양한 뒤 발생하는 Calcein-AM 형광 강도로 세포독성을 평가한 결과(도 2C), 도 2B의 결과와 마찬가지로 CHPG를 처리한 NK 세포의 세포독성이 더 높았다.In addition, the Calcein-AM fluorescence intensity generated after co-culture of target cells (LX-2 cell line (activated human hepatic stellate cell line)) and effector cells (human liver-derived NK cells treated with or without
상기 결과에 따라, NK 세포의 mGluR5가 활성화 되면 간 섬유화를 유발할 수 있는 활성화된 간성상세포를 사멸시킬 수 있는 능력이 증대됨을 확인하였다.According to the above results, it was confirmed that when mGluR5 of NK cells is activated, the ability to kill activated hepatic stellate cells that can cause liver fibrosis is increased.
또한, 본 발명의 다른 일 실시예에서, CHPG의 생체 내 투여가 실제로 간섬유화를 완화시킬 수 있음을 확인하였다(도 3).In addition, in another embodiment of the present invention, it was confirmed that the in vivo administration of CHPG can actually alleviate liver fibrosis (FIG. 3).
즉, 본 발명의 mGluR5 작용제는 간 내 NK 세포에 존재하는 mGluR5를 활성화시키고 활성화된 간성상세포에 대한 세포독성을 증가시켜 이를 사멸시킴으로써 간섬유화 치료 효과를 발휘하는 것일 수 있다.That is, the mGluR5 agonist of the present invention may activate mGluR5 present in NK cells in the liver, increase cytotoxicity to the activated hepatic stellate cells, and kill them, thereby exerting a therapeutic effect on liver fibrosis.
본 발명에서 용어, "예방"은 상기 약학적 조성물의 투여로 대상 질환의 발생, 확산 및 재발을 억제시키거나 지연시키는 모든 행위를 의미하고, "치료"는 상기 약학적 조성물의 투여로 대상 질환의 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.As used herein, the term “prevention” refers to any action that inhibits or delays the occurrence, spread, and recurrence of a target disease by administration of the pharmaceutical composition, and “treatment” refers to any act of inhibiting or delaying the occurrence, spread and recurrence of a target disease by administration of the pharmaceutical composition. It refers to any action that improves or changes the condition for the better.
본 발명의 구체적인 일 구현예에서, 상기 치료는 간질환, 구체적으로는 간경변, 비알코올성 간질환 및 알코올성 간질환, 보다 구체적으로는 간섬유화의 징후의 감소 또는 완화, 상기 질병의 정도의 축소, 질병의 지연 또는 완행(slowing), 질병 상태의 일시적 완화(palliation) 또는 안정화, 및 그 외 이로운 결과일 수 있으나, 이에 제한되지 않는다.In a specific embodiment of the present invention, the treatment is a liver disease, specifically liver cirrhosis, non-alcoholic liver disease and alcoholic liver disease, more specifically reducing or alleviating the signs of liver fibrosis, reducing the severity of the disease, disease may be, but not limited to, delay or slowing of the disease state, palliation or stabilization of the disease state, and other beneficial outcomes.
본 발명의 약학적 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형체 또는 희석제를 추가로 포함할 수 있다. 이때, 상기 약학적 조성물에 포함되는 유효성분의 함량은 특별히 이에 제한되지 않으나, 조성물 총 중량에 대하여 0.0001 중량% 내지 10 중량%로, 바람직하게는 0.001 중량% 내지 1 중량%를 포함할 수 있다.The pharmaceutical composition of the present invention may further include an appropriate carrier, excipient or diluent commonly used in the preparation of pharmaceutical compositions. At this time, the content of the active ingredient included in the pharmaceutical composition is not particularly limited thereto, but may include 0.0001 wt% to 10 wt%, preferably 0.001 wt% to 1 wt%, based on the total weight of the composition.
상기 약학적 조성물은 정제, 환제, 산제, 과립제, 캡슐제, 현탁제, 내용액제, 유제, 시럽제, 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제 및 좌제으로 이루어진 군으로부터 선택되는 어느 하나의 제형을 가질 수 있으며, 경구 또는 비경구의 여러 가지 제형일 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구 투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 하나 이상의 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로오스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 스테아린산 마그네슘, 탈크 등과 같은 윤활제들도 사용된다. 경구 투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테로 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.The pharmaceutical composition is any selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, internal solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories It may have one dosage form, and may be oral or parenteral various dosage forms. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include one or more compounds and at least one excipient, for example, starch, calcium carbonate, sucrose or lactose ( lactose), gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.
본 발명의 약학적 조성물은 약학적으로 유효한 양으로 투여할 수 있다.The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount.
본 발명에서 용어, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 질병의 종류, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다. 본 발명의 조성물의 바람직한 투여량은 환자의 상태 및 체중, 질병의 정도, 약물 형태, 투여경로 및 기간에 따라 다르지만, 바람직한 효과를 위해서, 본 발명의 조성물은 1일 0.0001 내지 500mg/kg으로, 바람직하게는 0.001 내지 200mg/kg으로 투여하는 것이 바람직할 수 있다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 조성물은 쥐, 가축, 인간 등의 다양한 포유동물에 다양한 경로로 투여할 수 있으며, 투여의 방식은 당업계의 통상적인 방법이라면 제한없이 포함하며, 예를 들어, 경구, 직장 또는 정맥, 근육, 피하, 자궁내 경막 또는 뇌혈관 내 주사에 의해 투여될 수 있다. 구체적으로는 경구 투여일 수 있으나, 이에 제한되지 않는다.As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the subject's type and severity, age, sex, and disease. It may be determined according to the type, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. and may be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those skilled in the art. The preferred dosage of the composition of the present invention varies depending on the condition and weight of the patient, the degree of disease, the drug form, the route and period of administration, but for a desirable effect, the composition of the present invention is 0.0001 to 500 mg/kg per day, preferably It may be desirable to administer at a dose of 0.001 to 200 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The composition can be administered to various mammals such as mice, livestock, and humans by various routes, and the method of administration includes without limitation as long as it is a conventional method in the art, for example, oral, rectal or intravenous, muscle, It may be administered by subcutaneous, intrauterine dural or intracerebrovascular injection. Specifically, it may be oral administration, but is not limited thereto.
또한, 본 발명의 약학적 조성물은 인간에 적용되는 의약품뿐만 아니라, 동물 의약품의 형태로도 사용될 수 있다. 여기에서, 동물이란 가축 및 애완동물을 포함하는 개념이다.In addition, the pharmaceutical composition of the present invention can be used in the form of veterinary drugs as well as pharmaceuticals applied to humans. Here, the animal is a concept including livestock and pets.
본 발명의 다른 하나의 양태는 mGluR5가 활성화된 NK 세포를 유효성분으로 포함하는, 간질환의 예방 또는 치료용 약학적 조성물을 제공한다.Another aspect of the present invention provides a pharmaceutical composition for preventing or treating liver disease, comprising mGluR5 activated NK cells as an active ingredient.
여기에서 사용되는 용어는 전술한 바와 같다.The terms used herein are the same as described above.
본 발명에 있어서, 상기 간질환은 간섬유화, 간경변, 비알코올성 간질환 및 알코올성 간질환으로 이루어지는 군으로부터 선택되는 어느 하나 이상일 수 있고, 구체적으로는 간섬유화일 수 있으나, 이에 제한되지 않는다.In the present invention, the liver disease may be any one or more selected from the group consisting of liver fibrosis, liver cirrhosis, non-alcoholic liver disease and alcoholic liver disease, and specifically may be liver fibrosis, but is not limited thereto.
본 발명에 있어서, 상기 mGluR5의 활성화는 작용제의 처리에 의한 것일 수 있다. In the present invention, the activation of mGluR5 may be by treatment with an agonist.
상기 작용제는 CHPG[(RS)-2-Chloro-5-hydroxyphenylglycine] 등일 수 있으나, 이에 제한되지 않는다. The agent may be CHPG[(RS)-2-Chloro-5-hydroxyphenylglycine], but is not limited thereto.
본 발명의 mGluR5가 활성화된 NK 세포는 mGluR5 작용제의 처리에 의해 NK 세포에 존재하는 mGluR5가 활성화된 것일 수 있으며, 활성화된 간성상세포에 대한 세포독성이 증가된 것일 수 있다. 이에 따라, 상기 mGluR5가 활성화된 NK 세포는 활성화된 간성상세포를 사멸시킴으로써 간섬유화 치료 효과를 발휘할 수 있다. 이에 대해서는 전술한 바와 같다. The mGluR5 activated NK cells of the present invention may be those in which mGluR5 present in NK cells is activated by treatment with an mGluR5 agonist, and may have increased cytotoxicity to activated hepatocytes. Accordingly, the mGluR5-activated NK cells can exert a therapeutic effect on liver fibrosis by killing the activated hepatic stellate cells. This is the same as described above.
본 발명의 조성물은 약학적으로 유효한 양의 mGluR5가 활성화된 NK 세포를 포함하여 투여할 수 있으며, 구체적으로 1회 투여당 적어도 천만 개 이상, 또는 적어도 백만 개 이상의 NK 세포를 포함하여 투여할 수 있으나, 이에 특별히 제한되지 않는다.The composition of the present invention may be administered including a pharmaceutically effective amount of mGluR5 activated NK cells, and specifically, may be administered including at least 10 million or more, or at least 1 million or more NK cells per one administration. , but is not particularly limited thereto.
본 발명의 또 다른 하나의 양태는 상기 약학적 조성물을 개체에 투여하는 단계를 포함하는, 간질환의 예방 또는 치료방법을 제공한다.Another aspect of the present invention provides a method for preventing or treating liver disease, comprising administering the pharmaceutical composition to an individual.
여기에서 사용되는 용어는 전술한 바와 같다.The terms used herein are the same as described above.
본 발명에 있어서, 상기 간질환은 간섬유화, 간경변, 비알코올성 간질환 및 알코올성 간질환으로 이루어지는 군으로부터 선택되는 어느 하나 이상일 수 있고, 구체적으로는 간섬유화일 수 있으나, 이에 제한되지 않는다.In the present invention, the liver disease may be any one or more selected from the group consisting of liver fibrosis, liver cirrhosis, non-alcoholic liver disease and alcoholic liver disease, and specifically may be liver fibrosis, but is not limited thereto.
본 발명의 "개체"는 대상 질환이 발병하였거나 발병할 수 있는 모든 동물을 의미하며, 본 발명의 약학적 조성물을 간질환의 개체 또는 의심 개체에 투여함으로써, 개체를 효율적으로 치료 또는 예방할 수 있다. 본 발명의 약학적 조성물은 간질환을 예방 또는 치료 목적으로 하는 개체이면 특별히 한정되지 않고, 어떠한 개체에든 적용 가능하다. 예를 들면, 원숭이, 개, 고양이, 토끼, 모르모트, 랫트, 마우스, 소, 양, 돼지, 염소 등과 같은 동물, 조류 및 어류 등 어느 것이나 사용할 수 있다.The "subject" of the present invention means any animal that has or can develop the target disease, and by administering the pharmaceutical composition of the present invention to an individual or suspected individual having liver disease, the individual can be effectively treated or prevented. The pharmaceutical composition of the present invention is not particularly limited as long as it is an individual for the purpose of preventing or treating liver disease, and can be applied to any individual. For example, any animal such as monkey, dog, cat, rabbit, guinea pig, rat, mouse, cow, sheep, pig, goat, birds and fish may be used.
본 발명의 "투여"는, 어떠한 적절한 방법으로 환자에게 소정의 물질을 도입하는 것을 의미하며, 상기 결합체의 투여 경로는 약물이 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 투여될 수 있다. 복강내 투여, 정맥내 투여, 근육내 투여, 피하 투여, 피내 투여, 경구 투여, 국소 투여, 비내 투여, 폐내 투여, 직장 내 투여 등이 될 수 있으나, 이에 제한되지는 않는다. 그러나 경구 투여시, 펩타이드는 소화가 되기 때문에 경구용 조성물은 활성 약제를 코팅하거나 위에서의 분해로부터 보호되도록 제형화 하는 것이 바람직하다. 또한, 약제학적 조성물은 활성 물질이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다."Administration" of the present invention means introducing a predetermined substance into a patient by any suitable method, and the administration route of the conjugate can be administered through any general route as long as the drug can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, etc. may be, but are not limited thereto. However, when administered orally, since the peptide is digestible, it is preferred that the oral composition be formulated to coat the active agent or to protect it from degradation in the stomach. In addition, the pharmaceutical composition may be administered by any device capable of transporting the active agent to a target cell.
본 발명의 목적상, 특정 환자에 대한 구체적인 치료적 유효량은 달성하고자 하는 반응의 종류와 정도, 경우에 따라 다른 제제가 사용되는지의 여부를 비롯한 구체적 조성물, 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료기간, 구체적 조성물과 함께 사용되거나 동시 사용되는 약물을 비롯한 다양한 인자와 의약 분야에 잘 알려진 유사 인자에 따라 다르게 적용하는 것이 바람직하다.For the purposes of the present invention, a specific therapeutically effective amount for a particular patient will depend on the specific composition, age, weight, general health, and sex of the patient, including the type and extent of the response to be achieved and, if desired, whether other agents are used. It is preferable to apply differently depending on various factors including diet, administration time, administration route and secretion rate of the composition, treatment period, and drugs used together or concurrently with a specific composition and similar factors well known in the pharmaceutical field.
상기 치료적 유효량은 제공된 증상의 세트로 의약 분야의 통상적인 투여량 결정 테스트를 통해 당업자에 의해 확인될 수 있다. Said therapeutically effective amount can be ascertained by one of ordinary skill in the art through routine dosage determination tests in the art of medicine with a given set of conditions.
본 발명의 약학적 조성물의 투여빈도는 특별히 이에 제한되지 않으나, 1일 1회 투여하거나 또는 용량을 분할하여 수회 투여할 수 있다. 본 발명의 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와는 순차적 또는 동시에 투여할 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 유발을 최소화 하면서 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다.The frequency of administration of the pharmaceutical composition of the present invention is not particularly limited thereto, but may be administered once a day or administered several times by dividing the dose. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount while minimizing the occurrence of side effects, and can be easily determined by those skilled in the art.
본 발명의 약학적 조성물은 간질환의 예방 및 치료를 위하여 단독으로, 또는 수술, 호르몬 치료, 약물 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다. The pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, hormone therapy, drug therapy, and biological response modifiers for the prevention and treatment of liver disease.
전술한 바와 같이, 본 발명은 NK 세포 내에 mGluR5가 존재하고, mGluR5가 활성화되면 간섬유화를 유발할 수 있는 활성화된 간성상세포에 대한 NK 세포의 세포독성이 증대하고, mGluR5 작용제의 생체 내 처리 시 간섬유화가 완화되는 것을 확인함으로써, NK 세포 특이적 mGluR5 활성이 간섬유화의 억제에 기여함을 최초로 확인한 것에 의의가 있다.As described above, in the present invention, mGluR5 is present in NK cells, and when mGluR5 is activated, the cytotoxicity of NK cells to activated hepatic stellate cells, which can cause liver fibrosis, is increased, and the in vivo treatment time of mGluR5 agonists By confirming that fibrosis is alleviated, it is significant that it was first confirmed that NK cell-specific mGluR5 activity contributes to the inhibition of liver fibrosis.
본 발명은 NK 세포 내에 mGluR5가 존재하고, mGluR5가 활성화되면 간섬유화를 유발할 수 있는 활성화된 간성상세포에 대한 NK 세포의 세포독성이 증대하고, 그 결과 간섬유화가 완화되는 것을 확인하였는 바, NK 세포 특이적 mGluR5 활성을 간질환 치료에 적용할 수 있다.The present invention confirmed that mGluR5 is present in NK cells, and when mGluR5 is activated, the cytotoxicity of NK cells to activated hepatic stellate cells, which can cause liver fibrosis, is increased, and as a result, hepatic fibrosis is alleviated. Cell-specific mGluR5 activity can be applied to the treatment of liver disease.
도 1은 대사성 글루타메이트 수용체인(metabotropic glutamate receptors, mGluR)의 뇌 조직이나 간세포 이외에 자연 살해(Natural killer, NK) 세포 내에서의 존재를 확인한 결과이다. (A) qRT-PCR 분석은 그룹 I, 그룹 II 및 그룹 III mGluR에 대해 마우스의 간 NK 세포에서 수행되었다. (B) RT-PCR은 마우스의 뇌 조직, 간세포 및 NK 세포에서 Grm1 및 Grm5에 대해 수행되었다. (C) mGluR5의 제시는 마우스의 뇌 조직 및 분리된 간 NK 세포에서 웨스턴 블롯에 의해 입증되었다. (D) 마우스의 간 NK 세포에서 mGluR5를 확인하기 위해 유세포 분석을 수행하였다. 데이터는 평균 ± SEM으로 표시된다. * p <0.05, ** p <0.01, *** p <0.001.
도 2는 mGluR5의 활성화가 NK 세포의 정지 및 활성화 된 간 성상 세포에 대한 세포 독성을 증가시키는지의 여부를 확인한 결과이다. (A) Grm5 발현의 비교는 qRT-PCR에 의해 마우스의 비히클 처리 및 CHPG[(RS)-2-Chloro-5-hydroxyphenylglycine] 처리 간 NK 세포에서 수행되었다. (B) 간 NK 세포를 비히클 또는 CHPG로 처리된 마우스의 D1 및 D4 간 성상세포와 공동배양하여 간 NK 세포의 세포독성을 분석하였다. (C) CHPG를 처리하거나 처리하지 않은 LX-2 세포주와 공동배양된 인간 간 NK 세포에서 세포 독성을 평가하였다. 데이터는 평균 ± SEM으로 표시된다. * p <0.05, ** p <0.01, *** p <0.001. 축척 막대 = 50 μm.
도 3은 마우스에서 CHPG의 전신 처리에 의한 mGluR5 활성화가 간 섬유증을 완화시키는지의 여부를 확인한 결과이다. (A) 마우스는 2 주 동안 CHPG를 처리하거나 처리하지 않으면서 동시에 CCl4 처리된 후 1 주 뒤에 비히클 또는 CHPG로 처리되었다. (B) 간 중량 및 체중을 측정하였다. (C) AST 및 ALT는 혈청에서 측정되었다. (D) 조직학적 관찰을 위해 조직 염색 하였다. (E) 비히클 처리된 마우스와 CHPG 처리된 마우스의 간 조직에서 Col1a1의 발현을 qRT-PCR로 비교하였다. 데이터는 평균 ± SEM으로 표시된다. * p <0.05, ** p <0.01, *** p <0.001. 축척 막대 = 50 μm.1 is a result confirming the presence of metabolic glutamate receptors (metabotropic glutamate receptors, mGluR) in natural killer (NK) cells in addition to brain tissue or hepatocytes. (A) qRT-PCR analysis was performed on liver NK cells of mice for Group I, Group II and Group III mGluRs. (B) RT-PCR was performed for Grm1 and Grm5 in mouse brain tissue, hepatocytes and NK cells. (C) Presentation of mGluR5 was demonstrated by Western blot in mouse brain tissue and isolated liver NK cells. (D) Flow cytometry was performed to identify mGluR5 in mouse liver NK cells. Data are presented as mean ± SEM. *p <0.05, **p <0.01, ***p <0.001.
Figure 2 is the result of confirming whether the activation of mGluR5 increases the cytotoxicity to the NK cell arrest and activated hepatic stellate cells. (A) Comparison of Grm5 expression was performed in vehicle-treated and CHPG[(RS)-2-Chloro-5-hydroxyphenylglycine]-treated liver NK cells of mice by qRT-PCR. (B) Hepatic NK cells were co-cultured with D1 and D4 hepatic astrocytes of mice treated with vehicle or CHPG to analyze the cytotoxicity of liver NK cells. (C) Cytotoxicity was evaluated in human liver NK cells co-cultured with LX-2 cell line with or without CHPG treatment. Data are presented as mean ± SEM. *p <0.05, **p <0.01, ***p <0.001. Scale bar = 50 μm.
3 is a result confirming whether mGluR5 activation by systemic treatment of CHPG in mice alleviates liver fibrosis. (A) Mice were treated with vehicle or CHPG 1 week after CCl 4 treatment with or without CHPG for 2 weeks. (B) Liver weight and body weight were measured. (C) AST and ALT were measured in serum. (D) The tissue was stained for histological observation. (E) The expression of Col1a1 in liver tissues of vehicle-treated mice and CHPG-treated mice was compared by qRT-PCR. Data are presented as mean ± SEM. *p <0.05, **p <0.01, ***p <0.001. Scale bar = 50 μm.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명하고자 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. These Examples are for explaining the present invention in more detail, and the scope of the present invention is not limited by these Examples.
실시예 1. 대사성 글루타메이트 수용체(metabotropic glutamate receptors, mGluR)의 자연 살해(Natural killer, NK) 세포 내 존재 확인Example 1. Confirmation of the presence of natural killer (NK) cells of metabotropic glutamate receptors (mGluR)
mGluR이 뇌 조직이나 간세포 이외에 NK 세포에 존재하는지를 확인하였다.It was confirmed whether mGluR was present in NK cells other than brain tissue or hepatocytes.
먼저 간 내 NK 세포를 분리하기 위해, 간을 70μm 나일론 메쉬 필터를 이용하여 분쇄하였다. 이후 40g로 5분 동안 원심분리하여 상층액을 수득한 후 인산완충생리식염수(Phosphate buffered saline, PBS)로 세척한 뒤 펠릿(pellet)을 40% 펄콜(Percoll, GE Healthcare, Little Chalfont, UK)로 풀었다. 이를 1400g, 4℃에서 30분동안 원심분리한 후 펠릿을 적혈구 용혈 완충액으로 세척하여 적혈구를 용혈시켰다. 이를 통하여 얻은 간의 단핵세포들을 NK 세포 분리 키트 (마우스의 경우 #130-115-818, 사람의 경우 #130-092-657, Miltenyi Biotec, Auburn, CA)를 이용하여 분리한다.First, in order to isolate NK cells in the liver, the liver was pulverized using a 70 μm nylon mesh filter. Thereafter, the supernatant was obtained by centrifugation at 40 g for 5 minutes, washed with phosphate buffered saline (PBS), and the pellet was washed with 40% Percoll (Percoll, GE Healthcare, Little Chalfont, UK). loosened After centrifugation at 1400 g, 4°C for 30 minutes, the pellet was washed with red blood cell hemolysis buffer to hemolyze red blood cells. The liver mononuclear cells obtained through this are isolated using an NK cell isolation kit (#130-115-818 for mice, #130-092-657 for humans, Miltenyi Biotec, Auburn, CA).
간세포를 분리하기 위해, 마우스의 간문맥을 통하여 EGTA(Ethylene glycol tetraacetic acid) 용액(0.5 mM EGTA, 25 mM 트라이신(Tricine), pH 7.2, 0.44 mM KH2PO4, 5.4 mM KCl, 140 mM NaCl, 0.34 mM Na2HPO4)를 간으로 관류(perfusion)하였다. 이후 관류 완충액(HBSS(Hanks' balanced salt solution) 버퍼 내 0.075%(w/v) 콜라게나제 타입 I(collagenase type I) 및 0.02%(w/v) DNase I)과 소화(digestion) 완충액(HBSS 버퍼 내 0.009%(v/v) 콜라게나제 타입 I 및 0.2%(w/v) DNase I)을 간으로 관류한다. 관류가 완료된 간은 70μm 세포 여과기(cell strainer)를 통해 분쇄한 후 40g로 5분 동안 원심분리하여 간세포를 분리하였다. 해당 간세포는 50%v/v 펄콜 구배 용액으로 희석 후 4℃에서 1400g로 10분동안 원심분리하여 정제하였다.To isolate hepatocytes, ethylene glycol tetraacetic acid (EGTA) solution (0.5 mM EGTA, 25 mM Tricine, pH 7.2, 0.44 mM KH 2 PO 4 , 5.4 mM KCl, 140 mM NaCl, 0.34 mM Na 2 HPO 4 ) was perfused into the liver. Then, perfusion buffer (0.075% (w/v) collagenase type I and 0.02% (w/v) DNase I in Hanks' balanced salt solution (HBSS) buffer) and digestion buffer (HBSS) Perfuse the liver with 0.009% (v/v) collagenase type I and 0.2% (w/v) DNase I) in buffer. After the perfusion was completed, the liver was pulverized through a 70 μm cell strainer, and then centrifuged at 40 g for 5 minutes to separate the liver cells. The hepatocytes were purified by dilution with a 50% v/v Percol gradient solution and centrifugation at 1400 g at 4° C. for 10 minutes.
정량적 실시간 중합효소연쇄반응(quantitative real time polymerase chain reaction, qRT-PCR)은 그룹 I, 그룹 II 및 그룹 III mGluR에 대해 마우스의 간 NK 세포에서 수행되었다. qRT-PCR을 수행하기 위해, 세포 혹은 조직에서의 RNA를 TRIzol 시약(Thermo Fisher Scientific, Waltham, MA)을 이용하여 분리하고 ReverTra Aceⓒ qPCR RT Master Mix with gDNA Remover(Toyobo, Osaka, Japan)을 이용하여 cDNA로 역전사시켰다. 이후, SYBR green(Toyobo)과 CFX96 Real-Time system(Bio-Rad, Hercules, CA)을 이용하여 qRT-PCR을 진행하고 타겟 유전자의 발현은 18s 리보솜 RNA를 하우스키핑 유전자로 두어 폴드-변경(fold-change)으로 나타내었다.Quantitative real time polymerase chain reaction (qRT-PCR) was performed in mouse liver NK cells for group I, group II and group III mGluRs. To perform qRT-PCR, RNA from cells or tissues was isolated using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA) and ReverTra Ace© qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan) was used. and reverse transcribed into cDNA. Then, qRT-PCR was performed using SYBR green (Toyobo) and CFX96 Real-Time system (Bio-Rad, Hercules, CA), and the expression of the target gene was changed by placing 18s ribosomal RNA as a housekeeping gene. -change).
실시간 중합효소연쇄반응(real time polymerase chain reaction, RT-PCR)은 마우스의 뇌 조직, 간세포 및 NK 세포에서 Grm1 및 Grm5에 대해 수행되었다. RT-PCR 은 qRT-PCR 과 동일한 과정을 거친 후에 아가로스 젤 로딩 버퍼를 혼합하고 이를 2% 아가로스 젤에서 전기영동시켜 나타내었다.Real time polymerase chain reaction (RT-PCR) was performed for Grm1 and Grm5 in mouse brain tissue, hepatocytes and NK cells. RT-PCR was shown by mixing agarose gel loading buffer and electrophoresis on 2% agarose gel after going through the same process as qRT-PCR.
마우스의 뇌 조직 및 분리된 간 NK 세포에 대해 웨스턴 블랏을 수행하기 위해, RIPA 용해 버퍼(10% SDS(Sodium dodecyl sulfate), pH 7.5, 150 mM NaCl, 1 mM PMSF(Phenylmethylsulfonyl fluoride), 30 mM 트리스, 1 mM Na3VO4, 10% 글리세롤)를 사용하여 조직 혹은 세포에서 단백질을 분리하였다. 10% SDS 폴리아크릴아마니드 젤 전기영동을 통해 단백질을 분리하고, 니트로셀룰로스 막(nitrocellulose membrane, #88018, Thermo Fisher Scientific)으로 옮겼다. 이후, 막을 탈지 분유로 블로킹하고 1차 항체와 함께 4℃에서 약 15시간 동안 보관한 뒤, 2차 항체와 함께 상온에서 1시간 보관하였다. 면역 반응 밴드들은 ImageQuantTM LAS 4000(GE Healthcare, Little Chalfont, UK)을 이용하여 시각화하였다.For Western blotting of mouse brain tissue and isolated liver NK cells, RIPA lysis buffer (10% sodium dodecyl sulfate (SDS), pH 7.5, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 30 mM Tris) , 1 mM Na 3 VO 4 , 10% glycerol) was used to separate proteins from tissues or cells. Proteins were separated by 10% SDS polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (#88018, Thermo Fisher Scientific). Thereafter, the membrane was blocked with skim milk powder and stored at 4° C. together with the primary antibody for about 15 hours, and then stored with the secondary antibody at room temperature for 1 hour. Immune response bands were visualized using ImageQuant ™ LAS 4000 (GE Healthcare, Little Chalfont, UK).
다음으로, 마우스의 간 NK 세포에서 유세포분석을 수행하기 위해, Alexa 647-결합된 항-mGluR5 항체(abcam)을 사용하여 mGluR5을 세포내 염색하였다. 대조군으로 이소타입 대조군(isotype, abcam)과 비염색 대조군(unstanined)을 사용하였다. 염색된 세포들은 BD LSR Fortessa (BD bioscience, San Jose, CA)과 FlowJo software (Tree Star, Ashland, OR)을 사용하여 분석하였다.Next, to perform flow cytometry on mouse liver NK cells, mGluR5 was stained intracellularly using Alexa 647-conjugated anti-mGluR5 antibody (abcam). As controls, an isotype control group (isotype, abcam) and an unstained control group (unstained) were used. Stained cells were analyzed using BD LSR Fortessa (BD bioscience, San Jose, CA) and FlowJo software (Tree Star, Ashland, OR).
전술한 방법에 따라, 마우스에서 분리된 간 내 NK 세포에서 세포의 흥분 독성(excitotoxocity)을 증가시키는 그룹 I mGluR과 감소시키는 그룹 II 및 그룹 III mGluR의 유전자 발현 정도를 비교한 결과(도 1A), 흥분 독성을 증가시키는 Group I mGluR을 구성하는 mGluR1의 유전자 Grm1과 mGluR5의 유전자 Grm5 중 Grm5의 발현이 유의하게 더 높은 것을 확인하였다.As a result of comparing the gene expression levels of Group I mGluRs that increase excitotoxocity and Group II and Group III mGluRs that decrease cell excitotoxocity in NK cells in the liver isolated from mice according to the above-described method (FIG. 1A), It was confirmed that the expression of Grm5 among mGluR1 gene Grm1 and mGluR5 gene Grm5 constituting Group I mGluR that increases excitotoxicity was significantly higher.
기존에 글루타메이트 수용체가 잘 발달되어 있다고 알려진 뇌 조직과, 간 내 실질세포인 마우스 간세포, 그리고 마우스 NK 세포에서 mGluR1의 유전자 Grm1과 mGluR5의 유전자 Grm5 발현 정도를 RT-PCR로 비교한 결과(도 1B), 뇌 조직에서는 알려진 대로 Grm1 및 Grm5의 밴드가 짙게 형성되었고, 간세포에서는 Grm1, Grm5 모두 밴드가 형성되지 않았다. 반면, NK 세포에서는 Grm1, Grm5 모두 밴드가 형성되었고, 그 중에서도 Grm5 가 더 짙게 형성된 것을 확인하였다.The results of comparing the expression levels of mGluR1 genes Grm1 and mGluR5 genes Grm5 in brain tissue, which are known to have well-developed glutamate receptors, mouse hepatocytes, which are parenchymal cells in the liver, and mouse NK cells, by RT-PCR (Fig. 1B) , Grm1 and Grm5 bands were densely formed in brain tissue, and neither Grm1 nor Grm5 bands were formed in hepatocytes. On the other hand, in NK cells, both Grm1 and Grm5 bands were formed, and it was confirmed that Grm5 was formed more densely among them.
마우스 NK 세포에서 mGluR5가 유전자 수준이 아닌 단백질 수준에서도 발현하는지 웨스턴 블랏으로 확인한 결과(도 1C), 유전자 수준에서와 마찬가지로, 뇌 조직 및 NK 세포에서 mGluR5 밴드가 확인되었다.As a result of confirming by Western blot whether mGluR5 was expressed at the protein level rather than the gene level in mouse NK cells ( FIG. 1C ), mGluR5 bands were confirmed in brain tissue and NK cells as in the gene level.
또한, 마우스 NK 세포 내의 mGluR5을 단백질 수준에서 보여주는 유세포 분석 결과를 히스토그램 도표로 나타낸 결과(도 1C), mGluR5 항체로 염색한 NK 세포에서 비염색(Unstained) 대조군 및 이소타입 대조군(isotype)에 비해 mGluR5가 월등하게 발현하는 것을 확인하였다. In addition, the results of flow cytometry showing mGluR5 in mouse NK cells at the protein level as a histogram chart (Fig. 1C), mGluR5 in NK cells stained with mGluR5 antibody compared to unstained control and isotype control (isotype) was found to be superiorly expressed.
상기 결과에 따라, 간 내 NK 세포에서 mGluR5가 존재함을 확인하였다.According to the above results, it was confirmed that mGluR5 was present in NK cells in the liver.
실시예 2. mGluR5의 활성화에 따른 NK 세포의 정지 및 활성화된 간 성상 세포에 대한 세포독성 증가 여부 확인Example 2. Confirmation of increase in cytotoxicity to NK cell arrest and activated hepatic stellate cells according to activation of mGluR5
mGlu5 선택적 작용제(agonist)인 CHPG((RS)-2-Chloro-5-hydroxyphenylglycine, Tocris) 100μM을 NK 세포에 처리시 Grm5의 발현의 차이가 있는지를 실시예 1의 qRT-PCR 방법으로 확인하였다.When NK cells were treated with 100 μM of CHPG ((RS)-2-Chloro-5-hydroxyphenylglycine, Tocris), which is an mGlu5 selective agonist, it was confirmed by the qRT-PCR method of Example 1.
다음으로, CHPG 처리시 NK 세포가 간성상세포를 사멸시키는 정도가 강해지는 지를 확인하였다. Next, it was confirmed whether the degree of NK cells apoptosis of hepatic stellate cells becomes stronger during CHPG treatment.
간성상세포를 분리하기 위해, 실시예 1의 방법으로 간세포 분리 후, 간세포를 제외한 상층액을 4℃에서 510g로 10분 동안 원심분리하여 간내 비실질세포를 펠릿으로 수득하였다. 상기 펠릿을 11.5% Opti-Prep 구배(Sigma-Aldrich, St. Louis, MO)로 희석 후 4℃에서 1800g로 17분 동안 원심분리하여 간성상세포층을 수득하였다. 세포의 생존률은 Trypan blue 염색으로 90% 이상임을 확인한다.To isolate hepatic stellate cells, hepatocytes were separated by the method of Example 1, and the supernatant except for hepatocytes was centrifuged at 510 g at 4 °C for 10 minutes to obtain non-parenchymal cells in the liver as pellets. The pellet was diluted with a 11.5% Opti-Prep gradient (Sigma-Aldrich, St. Louis, MO) and centrifuged at 1800 g at 4° C. for 17 minutes to obtain a hepatic stellate cell layer. It is confirmed that the cell viability is more than 90% by Trypan blue staining.
상기 수득한 간성상세포에 대한 세포독성 평가를 수행하기 위해, 먼저 Calcein-AM(Invitrogen, Waltham, MA)를 1mg/dL가 되도록 다이메틸 설폭사이드(dimethyl sulfoxide)에서 희석하였다. Day 1(비활성 간성상세포) 및 Day 4(활성화된 간성상세포)로 배양된 간성상세포를 타겟(target) 세포로, CHPG 100 μM을 처리 또는 처리하지 않은 간 내 NK 세포를 이펙터(effector) 세포로 명명하고, 타겟 세포를 15μM의 Calcein-AM으로 37℃에서 30분 동안 라벨링하였다. 타겟 세포와 이펙터 세포를 1:10 (세포수/세포수)으로 혼합하고 5% 이산화탄소 인큐베이터에서 37℃, 2시간 동안 공동배양한 후 상층액을 수득하였다. Calcein-AM 형광 강도는 마이크로플레이트 스펙트로플루오로미터(microplate spectrofluorometer)로 측정하였다. 여기 필터(Excitation filter)는 485nm로 맞추고 브랜드-패스 필터(brand-pass filter)를 535nm로 맞추었다. 세포독성은 하기 수식에 따라 산출하였다.In order to perform cytotoxicity evaluation on the obtained hepatocytes, first, Calcein-AM (Invitrogen, Waltham, MA) was diluted in dimethyl sulfoxide to 1 mg/dL. Hepatocytes cultured on Day 1 (inactive hepatocytes) and Day 4 (activated hepatocytes) were used as target cells, and NK cells in the liver treated with or without
세포독성(cytotoxicity) = [(테스트 방출 값 - 자연 방출 값) / (최대 방출 값 - 자연 방출 값)] X100Cytotoxicity = [(Test Release Value - Natural Release Value) / (Maximum Release Value - Natural Release Value)] X100
다음으로, LX-2 세포주(활성화된 사람 간성상세포주)를 타겟 세포로, CHPG 100 μM을 처리 또는 처리하지 않은 사람 간 유래 NK 세포를 이펙터 세포로 명명하고, 상기 세포독성 평가 방법에 따라 LX-2 세포주에 대한 세포독성 평가를 수행하였다.Next, the LX-2 cell line (activated human hepatic stellate cell line) was designated as the target cell, and human liver-derived NK cells treated with or without
전술한 방법에 따라, 마우스 NK 세포에 mGluR5 작용제인 CHPG 100 μM을 처리시 Grm5의 발현 변화를 확인한 결과(도 2A), CHPG를 처리한 NK 세포는 처리하지 않은 NK 세포보다 Grm5의 발현이 상승하였다.As a result of confirming the change in the expression of Grm5 when 100 μM of CHPG, an mGluR5 agonist, was treated in mouse NK cells according to the above-described method ( FIG. 2A ), the expression of Grm5 was increased in NK cells treated with CHPG than in NK cells that were not treated. .
타겟 세포(Day 1 간성상세포(비활성 간성상세포)와 Day 4 간성상세포(활성화된 간성상세포)) 및 이펙터 세포(CHPG 100 μM을 처리 또는 처리하지 않은 간 내 NK 세포)를 공동배양한 뒤 발생하는 Calcein-AM 형광 강도으로 세포독성을 평가한 결과(도 2B), 공동배양된 NK 세포가 간성상세포를 사멸시키는 정도는 CHPG 처리군이 CHPG를 처리하지 않은 군보다 유의하게 높았으며, 모든 타겟 세포에서 동일하였다.Target cells (Day 1 hepatocytes (inactive hepatocytes) and Day 4 hepatocytes (activated hepatocytes)) and effector cells (NK cells in the liver with or without
또한, 타겟 세포(LX-2 세포주(활성화된 사람 간성상세포주)) 및 이펙터 세포(CHPG 100 μM을 처리 또는 처리하지 않은 사람 간 유래 NK 세포)를 공동배양한 뒤 발생하는 Calcein-AM 형광 강도로 세포독성을 평가한 결과(도 2C), 도 2B의 결과와 마찬가지로 CHPG를 처리한 NK 세포의 세포독성이 더 높았다.In addition, the Calcein-AM fluorescence intensity generated after co-culture of target cells (LX-2 cell line (activated human hepatic stellate cell line)) and effector cells (human liver-derived NK cells treated with or without
상기 결과에 따라, NK 세포의 mGluR5가 활성화 되면 간 섬유화를 유발할 수 있는 활성화된 간성상세포를 사멸시킬 수 있는 능력이 증대됨을 확인하였다.According to the above results, it was confirmed that when mGluR5 of NK cells is activated, the ability to kill activated hepatic stellate cells that can cause liver fibrosis is increased.
실시예 3. CHPG의 전신 투여에 의한 mGluR5 활성화의 간 섬유증 완화 여부 확인Example 3. Confirmation of Relief of Hepatic Fibrosis by mGluR5 Activation by Systemic Administration of CHPG
도 3A에 따라 3주간 마우스에 사염화탄소(CCl4)와 CHPG를 투여하였다. 마우스를 두 군으로 나누어 한 군은 마우스에 사염화탄소를 2주간 투여하여 간섬유화를 유발한 후 나머지 일주일 동안 아무 투여도 하지 않았고, 다른 한 군은 마우스에 사염화탄소 및 CHPG를 2주간 투여하고 나머지 일주일 동안 사염화탄소를 제외한 CHPG 만 투여하였다.According to FIG. 3A, carbon tetrachloride (CCl 4 ) and CHPG were administered to mice for 3 weeks. The mice were divided into two groups, and one group was administered carbon tetrachloride to the mice for 2 weeks to induce liver fibrosis, and then no administration was given for the remaining week. Except for CHPG, only CHPG was administered.
각 군의 마우스 체중 변화를 관찰하고, 최종 부검시 체중 대비 간 무게(Liver weight/Body weight)을 기록하였다. Changes in the weight of mice in each group were observed, and the liver weight (Liver weight/Body weight) compared to the body weight at the final autopsy was recorded.
각 군의 마우스 혈장에서 간세포의 파괴로 인한 염증 지표인 ALT(alanine aminotransferase), AST(aspartate aminotransferase) 농도를 측정하기 위해, 마우스로부터 채혈 후 원심분리로 혈장을 분리해 낸 뒤 키트(IDEXX Laboratories, Westbrook, ME)를 사용하여 측정하였다. In order to measure the concentration of ALT (alanine aminotransferase) and AST (aspartate aminotransferase), which are inflammatory markers due to hepatocyte destruction, in the plasma of each group of mice, after collecting blood from the mouse, plasma was separated by centrifugation, and the kit (IDEXX Laboratories, Westbrook) , ME) was used.
각 군의 간 조직을 염색하기 위해, 간 조직을 10% 포르말린으로 고정한 후 파라핀으로 포매하였다. 이를 4μm의 두께로 자른 후 슬라이드 글라스에 부착하고, H&E(hematoxylin&eosion) 시약 및 시리우스 레드(sirius red) 시약으로 염색하여 광학 현미경으로 관찰하였다.To stain the liver tissues of each group, the liver tissues were fixed with 10% formalin and then embedded with paraffin. After cutting it to a thickness of 4 μm, it was attached to a slide glass, stained with hematoxylin & eosion (H&E) reagent and Sirius red reagent, and observed with an optical microscope.
다음으로, 각 군의 간 조직에서 콜라겐 타입 I(collagen type I)의 유전자인 Col1a1 발현을 실시예 1의 qRT-PCR 방법으로 정량하였다.Next, the expression of Col1a1, a gene of collagen type I, in liver tissues of each group was quantified by the qRT-PCR method of Example 1.
전술한 방법에 따라, 3주 동안의 마우스 체중과 3주 후 부검 시 체중 대비 간 무게(Liver weight/Body weight)를 확인한 결과(도 3B), 각 군간 마우스의 체중과 간 무게는 유의한 차이가 없는 것을 확인하였으며, 각 군간 ALT와 AST 또한 유의한 차이가 없었다(도 3C).According to the method described above, as a result of checking the mouse weight for 3 weeks and the liver weight compared to the body weight at the time of autopsy after 3 weeks (Fig. 3B), there was no significant difference between the mouse weight and the liver weight between each group. There was no significant difference in ALT and AST between each group (FIG. 3C).
각 군의 간 조직 절편을 염색하여 광학 현미경으로 관찰한 결과, CHPG를 투여하지 않은 군보다 CHPG를 투여한 군에서 섬유성 격벽(fibrous septa) 등 간섬유화를 시사할 만한 소견이 현저하게 감소한 것이 확인되었다.As a result of staining the liver tissue sections of each group and observing them under an optical microscope, it was confirmed that the group treated with CHPG significantly reduced findings suggestive of liver fibrosis, such as fibrous septa, compared to the group not treated with CHPG. became
또한, 각 군의 간 조직에서 간섬유화 발병시 상승하는 Col1a1의 발현을 정량화한 결과(도 3E), CHPG를 투여하지 않은 군보다 CHPG를 투여한 군의 간 조직 내 Col1a1 발현이 유의하게 더 낮은 것을 확인하였다. In addition, as a result of quantifying the expression of Col1a1 that rises upon the onset of hepatic fibrosis in the liver tissues of each group ( FIG. 3E ), it was found that the expression of Col1a1 in the liver tissues of the group administered with CHPG was significantly lower than that of the group not administered with CHPG. Confirmed.
상기 결과에 따라, CHPG의 생체 내 투여가 실제로 간섬유화를 완화시킬 수 있음을 알 수 있다.According to the above results, it can be seen that the in vivo administration of CHPG can actually alleviate liver fibrosis.
상기 실시예의 결과로부터, NK 세포 내에 mGluR5가 존재하고, mGluR5가 활성화되면 간섬유화를 유발할 수 있는 활성화된 간성상세포에 대한 NK 세포의 세포독성이 증대하고, mGluR5 작용제의 생체 내 투여 시 간섬유화가 완화되는 것을 확인하였는 바, NK 세포 특이적 mGluR5 활성은 간섬유화의 억제에 기여하여 잠재적 치료 선택지가 될 수 있음을 알 수 있다.From the results of the above example, mGluR5 is present in NK cells, and when mGluR5 is activated, the cytotoxicity of NK cells to activated hepatic stellate cells that can cause liver fibrosis increases, and liver fibrosis occurs when mGluR5 agonists are administered in vivo. As it was confirmed that alleviation, NK cell-specific mGluR5 activity contributes to the inhibition of liver fibrosis, suggesting that it can be a potential treatment option.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 청구범위의 의미, 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.
Claims (10)
A pharmaceutical composition for preventing or treating liver disease, comprising an agonist of metabolic glutamate receptor 5 (mGluR5) as an active ingredient.
The composition of claim 1, wherein the liver disease is at least one selected from the group consisting of liver fibrosis, cirrhosis, non-alcoholic liver disease and alcoholic liver disease.
The composition of claim 1, wherein the agonist of mGluR5 activates mGluR5 of Natural killer (NK) cells.
The composition of claim 3, wherein the mGluR5 activated NK cells have increased cytotoxicity to activated hepatic stellate cells.
The composition of claim 1, wherein the agent is CHPG[(RS)-2-Chloro-5-hydroxyphenylglycine].
A pharmaceutical composition for preventing or treating liver disease, comprising mGluR5 activated NK cells as an active ingredient.
The composition of claim 6, wherein the liver disease is at least one selected from the group consisting of liver fibrosis, cirrhosis, non-alcoholic liver disease and alcoholic liver disease.
7. The composition of claim 6, wherein the activation of mGluR5 is by an agonist.
9. The composition of claim 8, wherein the agent is CHPG[(RS)-2-Chloro-5-hydroxyphenylglycine].
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| 1. 한광협 외, 간섬유화, 대한간학회, 2010 8차 PG, 49-58. |
| 2. 이관식, 간섬유화, 대한소화기학회지 2006;48:297-305. |
| Cell Metab. 30(5), 877-889, 2019. * |
| Neuroreport, 21(10< 704-708, 2010. * |
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