KR102572595B1 - Composition for preventing or treating anti-inflammatory diseases comprising immortalized feline mesenchymal stem cells and their exosome-rich conditoned medium - Google Patents

Composition for preventing or treating anti-inflammatory diseases comprising immortalized feline mesenchymal stem cells and their exosome-rich conditoned medium Download PDF

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KR102572595B1
KR102572595B1 KR1020200166709A KR20200166709A KR102572595B1 KR 102572595 B1 KR102572595 B1 KR 102572595B1 KR 1020200166709 A KR1020200166709 A KR 1020200166709A KR 20200166709 A KR20200166709 A KR 20200166709A KR 102572595 B1 KR102572595 B1 KR 102572595B1
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김윤배
최은경
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충북대학교 산학협력단
주식회사 디자인셀
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Abstract

본 발명은 불멸화 고양이줄기세포 또는 이의 배양액을 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 조성물에 관한 것이다. 보다 구체적으로, 본 발명은 고양이지방줄기세포(feline adipose-derived mesenchymal stem cells, fADMSC)를 HPV16의 E6 및 E7 유전자를 포함하는 렌티바이러스 벡터(lentiviral vector)로 처리하여 분화유도하여 얻은 불멸화 고양이줄기세포(기탁번호: KCTC 14390BP), 상기 불멸화 고양이줄기세포의 배양액을 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 약제학적 조성물에 관한 것이다. The present invention relates to a composition for preventing or treating inflammatory diseases comprising immortalized feline stem cells or a culture medium thereof as an active ingredient. More specifically, the present invention provides immortalized feline stem cells obtained by inducing differentiation by treating feline adipose-derived mesenchymal stem cells (fADMSC) with a lentiviral vector containing the E6 and E7 genes of HPV16. (Accession number: KCTC 14390BP), and relates to a pharmaceutical composition for preventing or treating inflammatory diseases comprising the culture medium of the immortalized feline stem cells as an active ingredient.

Description

불멸화 고양이줄기세포 또는 이의 엑소좀풍부배양액을 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 조성물{Composition for preventing or treating anti-inflammatory diseases comprising immortalized feline mesenchymal stem cells and their exosome-rich conditoned medium}Composition for preventing or treating anti-inflammatory diseases comprising immortalized feline mesenchymal stem cells and their exosome-rich conditoned medium}

본 발명은 불멸화 고양이줄기세포 또는 이의 배양액을 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 조성물에 관한 것이다. 보다 구체적으로, 본 발명은 고양이지방줄기세포(feline adipose-derived mesenchymal stem cells, fADMSC)를 HPV16의 E6 및 E7 유전자를 포함하는 렌티바이러스 벡터(lentiviral vector)로 처리하여 분화유도하여 얻은 불멸화 고양이줄기세포(immortalized feline adipose-derived mesenchymal stem cells: im-fADMSC, 기탁번호: KCTC 14390BP), 상기 불멸화 고양이줄기세포의 엑소좀풍부배양액(exosome-rich conditioned medium, ERCM)을 유효성분으로 포함하는 염증성 질환, 예를 들면 피부염 또는 관절염의 예방 또는 치료용 약제학적 조성물에 관한 것이다. The present invention relates to a composition for preventing or treating inflammatory diseases comprising immortalized feline stem cells or a culture medium thereof as an active ingredient. More specifically, the present invention provides immortalized feline stem cells obtained by inducing differentiation by treating feline adipose-derived mesenchymal stem cells (fADMSC) with a lentiviral vector containing the E6 and E7 genes of HPV16. (immortalized feline adipose-derived mesenchymal stem cells: im-fADMSC, accession number: KCTC 14390BP), inflammatory diseases containing exosome-rich conditioned medium (ERCM) of the immortalized feline stem cells as an active ingredient, e.g. For example, it relates to a pharmaceutical composition for preventing or treating dermatitis or arthritis.

최근 피부질환이 도시인과 반려동물에서 크게 증가하고 있다. 즉, 감염성 피부질환은 물론, 미세먼지 등의 환경오염과 건조한 기후, 꽃가루, 집안의 카펫에 서식하는 진드기 등등 직접적으로 접촉성 피부질환을 유발하는 원인 뿐만 아니라 면역과민반응을 유발하는 다양한 알러젠에 노출되는 기회가 점차 커지고 있다. 특히 반려동물은 외부에서 흙이나 잔디밭에서 뒹굴기도 하고, 서로 싸우기도 하며, 쓰레기통 등 환경오염물질에 노출될 기회가 많아 피부염 발생이 빈번하다. 더욱이 반려동물 중 개는 수명이 10~15년으로 10세 이상에서는 감염병, 피부염, 관절염, 호흡기질병 등 염증성 질환으로 고통받게 된다. Recently, skin diseases are greatly increasing in urban residents and companion animals. In other words, exposure to various allergens that cause immune hypersensitivity reactions as well as direct causes of contact skin diseases such as infectious skin diseases, environmental pollution such as fine dust, dry climate, pollen, mites living in carpets in the house, etc. Opportunities are gradually increasing. In particular, companion animals often roll around in the soil or lawn outside, fight with each other, and have many opportunities to be exposed to environmental pollutants such as trash cans. Moreover, among companion animals, dogs have a lifespan of 10 to 15 years, and those over 10 years old suffer from inflammatory diseases such as infectious diseases, dermatitis, arthritis, and respiratory diseases.

관절염은 퇴행성 골관절염(osteoarthritis, OA)과 염증성 류마티스 관절염(rheumatoid arthritis, RA)으로 구별된다. 골관절염은 나이가 들어감에 따라 하중에 의해 관절연골이 퇴행성으로 마모되면서 ebernated bone surfaces가 부딪히면서 통증을 유발하고, 골두가 옆으로 자라 osteophytes가 형성되어 운동장애를 유발한다. 이에 비해 류마티스 관절염은 관절강 내로 neutrophils, macrophages, lymphocytes, dendritic cells, plasma cells 등의 염증세포가 침윤되면서 염증성 synovial membranes이 증식하여 pannus를 형성함으로써 심하게 붓고 통증이 유발되며, 관절골두의 변형 및 유합으로 운동장애를 유발한다. 이러한 염증반응은 관절 이외에도 여러 장기를 침범하는 전신성 만성 염증성 질환으로 확대되기도 한다. 류마티스 관절염의 원인은 아직 명확히 밝혀지지는 않았으나 현재는 자가면역 질환의 일종으로 유전적 및 환경적 요인에 의한 면역세포의 편향된 분화가 그 원인인 것으로 보고되고 있다. 즉, CD4+ T helper cells (Th1) 등의 활성화로 collagen과 같은 관절강 내의 proteins에 대한 자가항체가 형성되면 interferon-γ (IFN-γ), interleukin-2 (IL-2), complements 등의 chamoattraction에 의해 macrophages가 동원되며, macrophages에서 분비되는 tumor-necrosis factor-α (TNF-α)에 의해 다양한 세포로부터 cytokines이 분비됨에 따라 nitric oxide synthase (NOS)와 cyclooxygenase-II (COX-II)가 활성화되어 염증반응이 극대화된다.Arthritis is divided into degenerative osteoarthritis (OA) and inflammatory rheumatoid arthritis (RA). Osteoarthritis causes degenerative wear of articular cartilage due to load with age, causing pain as ebernated bone surfaces collide, and bone heads grow sideways to form osteophytes, resulting in movement disorders. In contrast, in rheumatoid arthritis, inflammatory cells such as neutrophils, macrophages, lymphocytes, dendritic cells, and plasma cells infiltrate into the joint cavity, and inflammatory synovial membranes proliferate to form pannus, causing severe swelling and pain. cause disability This inflammatory response may be extended to a systemic chronic inflammatory disease that invades various organs in addition to the joints. Although the cause of rheumatoid arthritis has not yet been clarified yet, it is now reported that biased differentiation of immune cells by genetic and environmental factors as a kind of autoimmune disease is the cause. In other words, when autoantibodies against proteins in the articular cavity such as collagen are formed by activation of CD4+ T helper cells (Th1), interferon-γ (IFN-γ), interleukin-2 (IL-2), and complements, etc. Macrophages are mobilized, and as cytokines are secreted from various cells by tumor-necrosis factor-α (TNF-α) secreted from macrophages, nitric oxide synthase (NOS) and cyclooxygenase-II (COX-II) are activated to initiate an inflammatory response this is maximized

따라서 아토피 피부염이나 류마티스 관절염 치료를 위해 NOS 억제제인 스테로이드(corticosteroids)와 COX-II 억제제인 비스테로이드성 소염제(non-steroidal anti-inflammatory drugs, NSAIDs)가 널리 사용되고 있다. 하지만 스테로이드는 장기간 사용 시 효과가 떨어지고 면역기능을 억제하는 심각한 부작용을 유발할 수 있으며, 비스테로이드성 소염제는 일시적인 통증과 염증을 완화시켜 주지만 근본적인 치료에는 한계가 있고 위궤양 등 2차적인 부작용을 야기한다.Therefore, NOS inhibitors corticosteroids and COX-II inhibitors non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of atopic dermatitis or rheumatoid arthritis. However, steroids are ineffective when used for a long period of time and can cause serious side effects that suppress immune function. Nonsteroidal anti-inflammatory drugs temporarily relieve pain and inflammation, but have limitations in fundamental treatment and cause secondary side effects such as stomach ulcers.

한편, 최근에는 줄기세포(stem cells)가 면역조절 기전을 통해 다양한 피부염 및 관절염 등을 효과적으로 개선시켜 주는 것으로 밝혀졌다. 예를 들어, 제대혈줄기세포(umbilical cord blood stem cells, UCBSC)를 피하로 주사하면 장기간 아토피 피부염의 재발을 방지할 수 있고, 지방줄기세포(adipose-derived mesenchymal stem cells, ADMSC)를 관절강이나 혈관 내로 주사하면 관절염을 효과적으로 치료할 수 있고, 신경줄기세포(neural stem cells, NSC)를 혈관 내로 주사했을 때 말초 면역계를 조절함으로써 다발성경화증을 회복시켜 주는 것으로 밝혀졌다. 하지만 줄기세포를 효율적으로 사용하려면, 환자로부터 반복적으로 채취하여 충분한숫자로 증식배양해야 하는 어려움이 있다. 따라서 한 번 채취한 줄기세포를 불멸화하여 계속 사용할 수 있는 줄기세포주(stem cell line)를 확립할 경우, ① 반복적으로 채취할 때의 환자의 고통을 줄여 줄 수 있고, ② 줄기세포 시술비용이 크게 감소하며, ③ 줄기세포의 특성이 지속적으로 유지되어 동일한 효과를 얻을 수 있을 뿐만 아니라, ④ 나이 많은 환자로부터 얻은 줄기세포의 활력/증식능 저하 문제를 해결할 수 있다.Meanwhile, it has recently been found that stem cells effectively improve various dermatitis and arthritis through an immunoregulatory mechanism. For example, subcutaneous injection of umbilical cord blood stem cells (UCBSC) can prevent long-term recurrence of atopic dermatitis, and adipose-derived mesenchymal stem cells (ADMSC) can be injected into the joint cavity or blood vessels. Injections can effectively treat arthritis, and intravascular injections of neural stem cells (NSCs) have been shown to reverse multiple sclerosis by regulating the peripheral immune system. However, in order to efficiently use stem cells, there is a difficulty in that they must be repeatedly collected from a patient and grown and cultured in sufficient numbers. Therefore, if a stem cell line that can be used continuously is established by immortalizing stem cells once harvested, the patient's pain during repeated collection can be reduced, and the cost of stem cell treatment can be greatly reduced. ③ The characteristics of stem cells are continuously maintained to obtain the same effect, and ④ the problem of reduced vitality/proliferative ability of stem cells obtained from elderly patients can be solved.

세포 이식에 사용되는 줄기세포는 크게 배아줄기세포와 성체줄기세포로 구분된다. 배아줄기세포는 inner cell mass에서 기원하며 체내의 모든 세포로 분화할 수 있지만 종양으로 발전할 수 있어서 아직 치료제로 사용하기에는 기술적으로 풀어야 할 문제가 많이 남아 있다. 반면, 성체줄기세포는 성숙한 개체에서 얻을 수 있으며, 종양발생 위험이 없이 활용할 수 있는 장점이 있다. 성체줄기세포는 골수, 제대혈, 지방조직 등에서 얻을 수 있는데, 골수줄기세포는 채취할 때 고통이 따르며, 제대혈줄기세포는 출산 시에만 채취할 수 있는데 동물이 가정에서 출산할 때는 미생물 오염이 심하여 동물병원에서 출산할 때 무균 채취해야 한다. 이에 비해 지방줄기세포는 양이 많고, 동물병원에서 쉽게 무균 채취할 수 있다.Stem cells used for cell transplantation are largely classified into embryonic stem cells and adult stem cells. Embryonic stem cells originate from the inner cell mass and can differentiate into all cells in the body, but can develop into tumors, so many technical problems remain to be solved before they can be used as therapeutic agents. On the other hand, adult stem cells can be obtained from mature individuals and have the advantage of being utilized without the risk of tumor development. Adult stem cells can be obtained from bone marrow, cord blood, adipose tissue, etc. Bone marrow stem cells are painful to collect, and cord blood stem cells can only be collected at birth. Aseptically harvested at birth. In contrast, adipose stem cells are large in quantity and can be easily collected aseptically at veterinary hospitals.

인간 또는 동물로부터 분리한 1차세포(primary cells)는 연속 배양할 때 계속 자라지 못하고 제한된 수의 분열을 한 후 성장이 정지하는데, 이를 in vitro 세포복제의 노화(replicative senescence)라 하며, 이 세포가 노화되지 않고 정상세포로 무한 증식할 수 있는 능력을 지니는 것을 불멸화세포(immortalized cells)라 한다. 따라서 본 발명은 한 번 채취로 계속 증식 배양하여 염증성 질환을 치료할 수 있는 불멸화 고양이줄기세포를 확립하고, 더 나아가 줄기세포로부터 유효성분인 기능물질을 다량으로 함유한 엑소좀 풍부 배양액을 수득함으로써 대표적인 염증성 질환인 아토피 피부염과 류마티스 관절염 모델에서 그 효능을 확인하였다. Primary cells isolated from humans or animals do not continue to grow when continuously cultured, but after a limited number of divisions, growth stops. This is called in vitro cell replication senescence (replicative senescence). Cells that have the ability to proliferate indefinitely as normal cells without aging are called immortalized cells. Therefore, the present invention establishes immortalized feline stem cells that can treat inflammatory diseases by continuously growing and culturing once collected, and furthermore, by obtaining a culture medium rich in exosomes containing a large amount of functional substances as active ingredients from stem cells, representative inflammatory Its efficacy was confirmed in the disease models of atopic dermatitis and rheumatoid arthritis.

대한민국 등록특허 제10-1740357호Republic of Korea Patent No. 10-1740357

세포의 노화는 체내에서 일생 동안 진행되는 현상으로, 세포에 내재하는 노화프로그램에 의한 세포생리활성의 변화에 기인한다. 세포 불멸화의 방법으로는 크게 SV40, hTERT, HPV E6/E7, EBV, MycT58A, RasV12 및 p53 등의 유전자를 탑재하는 방법이 있으며, 이 방법들은 복제노화(replicative senescence)를 극복하고 성공적으로 세포분화를 무한히 영속하도록 유도한다. 세포 불멸화를 위한 일반적인 전략으로 simian virus 40 (SV40) T 항원을 이용하는 것인데, 최근 연구에서 SV40T 항원이 telomerase 활성을 유도할 수 있음이 보고되어 다양한 분야에서 손쉽게 불멸화를 유도할 수 있는 방법으로 인정받고 있다. 가장 최근에는 chromatin 말단소체(telomere)의 길이를 유지하도록 돕는 telomerase reverse transcription protein (TERT)이 정상세포의 불멸화 시 안정한 유전형질과 특정 표현형질 마커들을 유지하는 특성을 나타내는 등 방법론적으로 우수한 불멸화 전략으로 보고되었다. 하지만 상피세포와 같은 특정 세포에서 TERT의 과발현이 세포 불멸화를 유도하지 못하거나 세포사멸을 유도하는 등의 문제를 내포하고도 있어 일반적인 불멸화 방법으로는 제한성이 있다. 그럼에도 불구하고 TERT 과발현이 줄기세포(특히, mesenchymal stem cells, MSC)에서 그 줄기세포의 특성을 증가시킨다는 보고가 있어 전략적인 불멸화 방법으로 고려할 수 있다. Cellular aging is a phenomenon that progresses throughout life in the body, and is caused by changes in cell physiological activity by aging programs inherent in cells. Cell immortalization methods include methods of loading genes such as SV40, hTERT, HPV E6/E7, EBV, MycT58A, RasV12 and p53, and these methods overcome replicative senescence and successfully differentiate cells. induces to perpetuate indefinitely. A general strategy for cell immortalization is to use the simian virus 40 (SV40) T antigen. A recent study reported that the SV40T antigen can induce telomerase activity, and it is recognized as a method that can easily induce immortalization in various fields. . Most recently, telomerase reverse transcription protein (TERT), which helps to maintain the length of chromatin telomeres, is a methodologically superior immortalization strategy that shows the characteristics of maintaining stable genetic traits and specific phenotypic markers during immortalization of normal cells. reported However, overexpression of TERT in specific cells such as epithelial cells has problems such as failing to induce cell immortalization or inducing apoptosis, so there are limitations as a general immortalization method. Nevertheless, there is a report that overexpression of TERT increases the characteristics of stem cells (especially mesenchymal stem cells, MSC), so it can be considered as a strategic immortalization method.

본 발명자들은 이와 같이 전반적인 기술고찰을 통해 human papillomavirus (HPV)의 E6/E7이 각각 hTERT의 프로모터 활성화와 telomerase 활성화를 도와 안정적인 세포의 불멸화를 성공적으로 유도할 전략으로 판단되었다. 아울러 본 발명에서는 형질전환 벡터(transfection vector)에 저항성을 띠는 특징을 감안하여 불멸화 성공률을 높이는 방법으로 viral vector (retroviral 및 lentiviral) systems을 도입하였다. Retroviral vector는 분열하는 세포에서 효과적으로 불멸화 유도가 가능하며, 이는 retroviral vector가 능동적으로 핵막을 통과할 수는 없지만, 세포가 분열하는 동안 나타나는 핵막의 disintegration 과정에서 viral DNA가 유전체로 들어가 integration됨으로써 가능한 방법으로 안정적인 불멸화가 가능하다. 반면, lentiviral vector는 HIV genome인 복합구조로 구성되어 있는데, retrovirus가 가지는 기본유전자 (env, gag, pol) 이외에 조절유전자 (tat, rev)와 보조유전자 (vpr, vif, vpu, nef)를 더 가지고 있어 핵막을 능동적으로 통과하여 분열세포 및 비분열세포의 불멸화에 사용될 수 있다. 이는 HIV 유전자 중 vpr 유전자, gag 유전자와 integrase 효소에 의해 만들어지는 이입 전 복합물질이 대상 세포의 핵막을 직접 통과할 수 있는 장점이 중요한 전략이 될 수 있으며, 이런 이유로 유전자 도입의 방법으로 in vitro 및 in vivo 모두에서 각광받는 viral vector에 속한다. 숙주세포로의 유전자 전달을 위해 필요한 바이러스 입자들은 3가지 plasmids 즉, 전달하고자 하는 유전자가 재조합된 바이러스 vector, 재조합 바이러스 생산에 필요한 단백질 plasmids (예, gag, pol, tat, rev, nef, vpr, vpu and vif)를 포함하는 packaging plasmid, 그리고 피막(vesicular stomatitis virus G: VSV-G) 단백질 plasmid에 의한 일시적 transduction에 의해 보통 3×106 IU/mL 정도의 재조합 바이러스 입자를 얻을 수 있다.Through this general technical review, the present inventors determined that E6/E7 of human papillomavirus (HPV) would be a strategy to successfully induce immortalization of stable cells by assisting promoter activation and telomerase activation of hTERT, respectively. In addition, in the present invention, viral vector (retroviral and lentiviral) systems were introduced as a method of increasing the success rate of immortalization in consideration of the resistance to transfection vectors. Retroviral vectors can effectively induce immortalization in dividing cells, and although retroviral vectors cannot actively pass through the nuclear membrane, viral DNA enters and integrates into the genome during the nuclear membrane disintegration process that occurs during cell division. Stable immortality is possible. On the other hand, the lentiviral vector is composed of the complex structure of the HIV genome, and has additional regulatory genes (tat, rev) and auxiliary genes (vpr, vif, vpu, nef) in addition to the basic genes (env, gag, pol) of the retrovirus. It can actively pass through the nuclear membrane and be used for immortalization of dividing and non-dividing cells. For this reason, the advantage of being able to directly pass through the nuclear membrane of the target cell can be an important strategy for complex substances made by the vpr gene, gag gene and integrase enzyme among the HIV genes, and for this reason, in vitro and It belongs to a viral vector that is in the spotlight both in vivo. Viral particles required for gene transfer to host cells include three plasmids: a viral vector in which the gene to be transferred is recombined, and protein plasmids required for recombinant virus production (e.g., gag, pol, tat, rev, nef, vpr, vpu and vif), and transient transduction by vesicular stomatitis virus G (VSV-G) protein plasmid, usually about 3×10 6 IU/mL of recombinant virus particles can be obtained.

따라서 본 발명에서는 줄기세포의 불멸화 전략으로 분열세포와 비분열세포 모두에 감염시킬 수 있는 능률을 가진 lentiviral vector를 활용하여 세포복제노화(replicative senescence)를 억제함으로써 불멸화세포시스템을 구축하고자 하였다. HPV의 E7 단백질은 기능·구조적으로 adenoviurs의 E1A 단백질과 유사하며 일부 유형의 세포를 불멸화하는 데에도 사용된다. E7 단백질은 그 자체로 epithelial cells을 불멸화시킬 수 있지만, E6 및 E7의 발현은 형질전환효율을 증가시킨다. 불멸화 유도를 위해 Accession No. NC_001526.2 정보를 탑재한 Lenti-HPV-16 E6/E7 particles (도 1)을 적용하였으며, 각 과정은 각 시약의 제조사 지침에 따라 수행하였다. 상기 과정에 따라 제조된 불멸화 고양이줄기세포를 기탁번호: KCTC 14390BP 하에 기탁하였다. Therefore, in the present invention, as a stem cell immortalization strategy, an immortalized cell system was constructed by using a lentiviral vector capable of infecting both dividing and non-dividing cells and suppressing cell replicative senescence. The HPV E7 protein is functionally and structurally similar to the E1A protein of adenoviurs and is also used to immortalize some types of cells. E7 protein itself can immortalize epithelial cells, but expression of E6 and E7 increases transformation efficiency. To induce immortalization, Accession No. Lenti-HPV-16 E6/E7 particles (Fig. 1) loaded with NC_001526.2 information were applied, and each process was performed according to the manufacturer's instructions for each reagent. Immortalized feline stem cells prepared according to the above procedure were deposited under Accession Number: KCTC 14390BP.

상기 준비한 불멸화 고양이줄기세포(1x106 cells/mL)를 Dulbecco's modified Eagle's medium (DMEM) 배지에 접종하고 5% 이산화탄소(CO2) 공급 배양기에서 37℃의 온도로 24시간 동안 배양하였다. 이후 줄기세포로부터 엑소좀이 풍부하게 함유된 배양액(ERCM)을 수득하기 위해 상기 배양 배지에 TNF-α를 50 ng/mL의 농도로 첨가하고 산소(O2) 농도를 3%로 낮춘 후, 24시간 동안 배양한 다음, 배양액을 채취하여 필터로 거른 후 저온에서 10배 농축하여 본 발명에 따른 줄기세포 유래 엑소좀 풍부 배양액을 수득하였다. 한편, 비교 예로써, 불멸화 줄기세포로부터 엑소좀이 함유된 배양액을 수득하는 과정에서, TNF-α를 처리하지 않고 산소 농도를 21%로 유지한 채 정상상태로 배양한 줄기세포 정상배양액(CM)을 수득하였다.The prepared immortalized cat stem cells (1x10 6 cells/mL) were inoculated in Dulbecco's modified Eagle's medium (DMEM) medium and cultured for 24 hours at 37°C in an incubator supplied with 5% carbon dioxide (CO 2 ). Thereafter, in order to obtain a culture medium (ERCM) rich in exosomes from stem cells, TNF-α was added to the culture medium at a concentration of 50 ng/mL and the oxygen (O 2 ) concentration was lowered to 3%, and then 24 After culturing for a period of time, the culture solution was collected, filtered through a filter, and concentrated 10 times at low temperature to obtain a stem cell-derived exosome-rich culture solution according to the present invention. On the other hand, as a comparative example, in the process of obtaining a culture solution containing exosomes from immortalized stem cells, stem cell normal culture medium (CM) cultured in a normal state while maintaining the oxygen concentration at 21% without treatment with TNF-α was obtained.

그 결과, 본 발명에 따른 불멸화 고양이줄기세포 및 이의 배양액의 경우 Carrageenan로 인한 피부 삼출물 및 염증지표, 염증세포의 침윤 및 피부염 억제 효능을 나타낼 뿐만 아니라, 수술적 퇴행성 관절염(연골결손) 치료 효능이 우수함을 확인하였으며, 특히 불멸화 고양이줄기세포를 함유한 배양액에 TNF-α를 처리하고, 1~5%의 저산소농도 조건 하에서 12~72시간 배양하여 수득한 불멸화 고양이줄기세포 엑소좀 풍부 배양액의 경우, TNF-α의 처리 없이 정상상태로 배양한 배양액에 비해서 더욱 우수한 피부염 및 관절염 치료 효과가 있는 것으로 나타났다. As a result, in the case of the immortalized feline stem cell and its culture medium according to the present invention, it not only shows the effect of inhibiting skin exudate and inflammatory markers, inflammatory cell infiltration and dermatitis due to Carrageenan, but also has excellent treatment efficacy for surgical degenerative arthritis (cartilage defect) was confirmed, in particular, in the case of immortalized feline stem cell exosome-rich culture obtained by treating the culture medium containing immortalized feline stem cells with TNF-α and culturing for 12 to 72 hours under 1-5% hypoxic conditions, TNF It was found that there was a more excellent treatment effect for dermatitis and arthritis compared to the culture medium cultured in a normal state without treatment with -α.

본 발명은 항염증 활성을 갖는 고양이지방줄기세포(feline adipose-derived mesenchymal stem cells, fADMSC)를 HPV16의 E6 및 E7 유전자를 포함하는 렌티바이러스 벡터(lentiviral vector)로 처리하여 분화유도하여 얻은 불멸화 고양이줄기세포(기탁번호: KCTC 14390BP)를 제공하는 것을 목적으로 한다. The present invention is an immortalized feline stem cell obtained by inducing differentiation by treating feline adipose-derived mesenchymal stem cells (fADMSC) having anti-inflammatory activity with a lentiviral vector containing the E6 and E7 genes of HPV16. The purpose is to provide cells (accession number: KCTC 14390BP).

본 발명은 또한 불멸화 고양이줄기세포(기탁번호: KCTC 14389BP) 또는 이의 배양액을 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 약제학적 조성물을 제공하는 것을 목적으로 한다. Another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases comprising immortalized feline stem cells (accession number: KCTC 14389BP) or a culture medium thereof as an active ingredient.

본 발명은 또한 불멸화 고양이줄기세포 엑소좀 풍부 배양액의 제조 방법을 제공하는 것을 목적으로 한다. Another object of the present invention is to provide a method for preparing an exosome-rich culture medium of immortalized feline stem cells.

본 명세서에서 사용된 용어 “줄기세포”는 특정 세포로 분화가 진행되지 않은 세포로서, 필요한 경우, 신경, 피부, 혈액, 근육, 골, 연골 등 신체를 구성하는 모든 종류의 세포로 분화할 능력을 가진 세포를 의미한다. As used herein, the term "stem cell" is a cell that has not undergone differentiation into a specific cell, and, if necessary, has the ability to differentiate into all types of cells constituting the body, such as nerves, skin, blood, muscle, bone, and cartilage. means cells with

본 명세서에서 사용된 용어 “엑소좀(exosomes)”은 정교한 RNA 및 단백질 운반물질이 들어있는 작은 크기(30~150 nm)의 소포로, 여러 종류의 세포들로부터 분비되는 막 구조의 소낭체를 의미한다. As used herein, the term “exosomes” refers to membrane-structured vesicles secreted from various types of cells as small-sized (30-150 nm) vesicles containing sophisticated RNA and protein carriers. do.

본 명세서에서 사용된 용어 "염증성 질환(Inflammatory diseases)"은 염증유발인자 또는 방사선조사 등 유해한 자극으로 인해 인체 면역체계를 과도하게 항진시켜 대식세포와 같은 면역세포에서 분비되는 TNF-α (tumor necrosis factor-α), IL-1 (interleukin-1), IL-6, 프로스타글란딘 (prostagladins), 루코트리엔(leucotrienes) 또는 산화질소(nitric oxide, NO)와 같은 염증유발물질 (염증성 사이토카인)에 의해 유발되는 질환을 의미한다.As used herein, the term "inflammatory diseases" refers to TNF-α (tumor necrosis factor) secreted from immune cells such as macrophages by excessively enhancing the human immune system due to harmful stimuli such as inflammation-inducing factors or irradiation. -α), IL-1 (interleukin-1), IL-6, prostaglandins, leucotrienes or nitric oxide (NO) by pro-inflammatory substances (inflammatory cytokines) disease that causes it.

본 명세서에서 사용된 용어 "예방"은 본 발명에 따른 조성물의 투여로 염증성 질환을 억제 또는 지연시키는 모든 행위를 의미한다.As used herein, the term "prevention" refers to any action that suppresses or delays an inflammatory disease by administering the composition according to the present invention.

본 명세서에서 사용된 용어 "치료“는 치료하고자 하는 개개인 또는 세포의 천연 과정을 변경시키기 위해 임상적으로 개입하는 모든 행위를 의미하는데, 임상 병리 상태가 진행되는 동안 또는 이를 예방하기 위해 수행할 수 있다. 목적하는 치료 효과에는 질병의 발생 또는 재발을 예방하고, 증상을 완화시키며, 질병에 따른 모든 직접 또는 간접적인 병리학적 결과를 저하시키며, 전이를 예방하고, 질병 진행 속도를 감소시키며, 질병 상태를 경감 또는 일시적으로 완화시키며, 차도시키거나 예후를 개선시키는 것이 포함된다. 본 발명의 목적상 상기 치료는 본 발명에 따른 조성물의 투여로 염증성 질환의 증세가 호전되거나 완치되는 모든 행위를 포함하는 것으로 해석될 수 있으나, 특별히 이에 제한되지는 않는다.As used herein, the term "treatment" refers to any action that clinically intervenes to alter the natural process of an individual or cell to be treated, which can be performed during the course of a clinical pathological condition or to prevent it. The desired therapeutic effect includes preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, and improving the disease state. For the purpose of the present invention, the treatment is interpreted to include all actions in which the symptoms of an inflammatory disease are improved or cured by administration of the composition according to the present invention. It may be, but is not particularly limited thereto.

본 명세서에서 사용된 용어 "완화"는 본 발명에 따른 조성물의 투여로 염증성 질환이 호전되거나 이롭게 되는 모든 행위를 말한다.As used herein, the term "alleviation" refers to any action that improves or benefits an inflammatory disease by administration of a composition according to the present invention.

본 발명은 고양이지방줄기세포(feline adipose-derived mesenchymal stem cells, fADMSC)를 Lenti-HPV-16 E6/E7 플랫폼을 이용하여 불멸화함으로써 지속적인 증식배양을 통해 반복적인 줄기세포 채취 없이 동일 특성 및 효능을 가진 불멸화 고양이줄기세포주(DC-immortalized feline adipose-derived mesenchymal stem cells, DC-im-fADMSC)를 확립하였다. 본 발명은 또한 상기 불멸화 고양이줄기세포, 상기 불멸화 고양이줄기세포의 배양액, 특히 상기 불멸화 고양이줄기세포로부터 수득한 다량의 엑소좀이 함유된 엑소좀 풍부 배양액을 포함하는 염증성 질환의 예방 또는 치료용 조성물이 피부염 및 관절염에 우수한 효과가 있음을 확인하였다. 따라서, 본 발명에 따른 조성물은 염증 치료제로서 유용하게 사용될 수 있을 것으로 기대된다. The present invention immortalizes feline adipose-derived mesenchymal stem cells (fADMSC) using the Lenti-HPV-16 E6/E7 platform to achieve the same characteristics and efficacy without repeated stem cell collection through continuous proliferation culture. An immortalized feline stem cell line (DC-immortalized feline adipose-derived mesenchymal stem cells, DC-im-fADMSC) was established. The present invention also relates to a composition for preventing or treating inflammatory diseases comprising the immortalized feline stem cells, a culture medium of the immortalized feline stem cells, and particularly an exosome-rich culture medium containing a large amount of exosomes obtained from the immortalized feline stem cells. It was confirmed that there is an excellent effect on dermatitis and arthritis. Therefore, the composition according to the present invention is expected to be useful as an anti-inflammatory agent.

도 1은 줄기세포 불멸화 플랫폼 Lenti-HPV-16 E6/E7 system의 모식도를 나타낸다.
도 2는 줄기세포를 불멸화하는 과정을 나타낸다.
도 3은 줄기세포가 불멸화되었음을 RT-PCR 유전자 분석을 통해 확인한 결과를 나타낸다.
도 4는 정상산소농도와 저산소농도에서 배양한 후의 정상(비불멸화) 줄기세포와 불멸화 줄기세포의 생존율 변화를 확인한 결과른 나타낸다.
도 5는 정상배양액(CM)과 엑소좀풍부배양액(ERCM) 내 엑소좀 함량을 CD9 지표에 대한 RT-PCR 분석 결과를 나타낸다.
도 6은 고양이줄기세포와 줄기세포 배양액이 Carrageenan으로 인한 피부염을 억제효능에 대한 현미경소견을 나타낸다.
도 7은 고양이줄기세포와 줄기세포 배양액의 수술적 퇴행성 관절염(연골결손) 치료효능에 대한 육안소견을 나타낸다.
도 8은 고양이줄기세포와 줄기세포 배양액의 수술적 퇴행성 관절염(연골결손) 치료효능에 대한 ICRS score를 나타낸다.
도 9는 고양이줄기세포와 줄기세포 배양액의 수술적 퇴행성 관절염(연골결손) 치료효능에 대한 현미경소견을 나타낸다.
도 10은 고양이줄기세포와 줄기세포 배양액의 수술적 퇴행성 관절염(연골결손) 치료효능에 대한 연골재생 현미경소견을 나타낸다. Figure 1 shows a schematic diagram of the stem cell immortalization platform Lenti-HPV-16 E6 / E7 system.
Figure 2 shows the process of immortalizing stem cells.
Figure 3 shows the result of confirming through RT-PCR gene analysis that the stem cells are immortalized.
Figure 4 shows the result of confirming the change in viability of normal (non-immortalized) stem cells and immortalized stem cells after culturing in normal oxygen concentration and hypoxic concentration.
Figure 5 shows the results of RT-PCR analysis for the CD9 index of the exosome content in normal culture medium (CM) and exosome-rich culture medium (ERCM).
Figure 6 shows the microscopic findings of the inhibitory effect of feline stem cells and stem cell cultures on dermatitis caused by Carrageenan.
Figure 7 shows the visual findings of the treatment efficacy of surgical degenerative arthritis (cartilage defect) of feline stem cells and stem cell culture medium.
Figure 8 shows the ICRS score for the treatment efficacy of surgical degenerative arthritis (cartilage defect) of feline stem cells and stem cell cultures.
Figure 9 shows the microscopic findings of the therapeutic efficacy of surgical degenerative arthritis (cartilage defect) of feline stem cells and stem cell cultures.
Figure 10 shows cartilage regeneration microscopic findings on the therapeutic efficacy of surgical degenerative arthritis (cartilage defect) of feline stem cells and stem cell cultures.

이하, 발명의 구체적인 구현예에 따른 불멸화 고양이줄기세포 또는 이의 배양액을 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 조성물에 대하여 상세하게 설명하기로 한다. 다만, 이는 발명의 하나의 예시로써 제시되는 것으로, 이에 의해 발명의 권리범위가 한정되는 것은 아니며, 발명의 권리범위 내에서 구현예에 대한 다양한 변형이 가능함은 당업자에게 자명하다.Hereinafter, a composition for preventing or treating inflammatory diseases comprising immortalized feline stem cells or a culture solution thereof according to a specific embodiment of the present invention as an active ingredient will be described in detail. However, this is presented as an example of the invention, whereby the scope of the invention is not limited, and it is obvious to those skilled in the art that various modifications to the embodiments are possible within the scope of the invention.

본 명세서 전체에서 특별한 언급이 없는 한 "포함" 또는 "함유"라 함은 어떤 구성 요소(또는 구성 성분)를 별다른 제한 없이 포함함을 지칭하며, 다른 구성 요소(또는 구성 성분)의 부가를 제외하는 것으로 해석될 수 없다.Throughout this specification, unless otherwise specified, "include" or "include" refers to including a certain component (or component) without particular limitation, and excludes the addition of other components (or components). cannot be interpreted as

제1구현예에 따르면, According to the first embodiment,

본 발명은 항염증 활성을 갖는 고양이지방줄기세포(feline adipose-derived mesenchymal stem cells, fADMSC)를 HPV16의 E6 및 E7 유전자를 포함하는 렌티바이러스 벡터(lentiviral vector)로 처리하여 분화유도하여 얻은 불멸화 고양이줄기세포(기탁번호: KCTC 14390BP)를 제공하고자 한다. The present invention is an immortalized feline stem cell obtained by inducing differentiation by treating feline adipose-derived mesenchymal stem cells (fADMSC) having anti-inflammatory activity with a lentiviral vector containing the E6 and E7 genes of HPV16. Cells (accession number: KCTC 14390BP) are intended to be provided.

본 발명에 따른 불멸화 고양이줄기세포(기탁번호: KCTC 14390BP)에 있어서, 상기 불멸화 고양이줄기세포는 피부염 또는 류마티스 관절염 억제 효과를 갖는 것을 특징으로 한다. 예를 들면, 상기 불멸화 고양이줄기세포는 Carrageenan로 인한 피부 삼출물 및 염증지표, 염증세포의 침윤 및 피부염 억제 효능을 나타낼 뿐만 아니라, 수술적 퇴행성 관절염(연골결손)에 대한 치료 효과를 가질 수 있다. In the immortalized feline stem cells (accession number: KCTC 14390BP) according to the present invention, the immortalized feline stem cells are characterized in that they have an inhibitory effect on dermatitis or rheumatoid arthritis. For example, the immortalized feline stem cells not only show effects on carrageenan-induced skin exudation and inflammation, inflammatory cell infiltration and dermatitis inhibition, but also have a therapeutic effect on surgically degenerative arthritis (cartilage defect).

제2구현예에 따르면,According to the second embodiment,

본 발명은 불멸화 고양이줄기세포(기탁번호: KCTC 14390BP) 또는 이의 배양액을 유효성분으로 포함하는 염증성 질환의 예방 또는 치료용 약제학적 조성물을 제공하고자 한다. The present invention is intended to provide a pharmaceutical composition for preventing or treating inflammatory diseases comprising immortalized feline stem cells (accession number: KCTC 14390BP) or a culture solution thereof as an active ingredient.

본 발명에 따른 염증성 질환의 예방 또는 치료용 약제학적 조성물에 있어서, 상기 배양액은 불멸화 줄기세포를 함유한 배양액에 TNF-α를 처리하고, 1~5%의 저산소농도 조건 하에서 12~72시간 배양하여 수득하여 엑소좀이 풍부한 것을 특징으로 한다. 바람직하기는, 상기 배양액은 불멸화 줄기세포를 함유한 배양액에 TNF-α를 처리하고, 3%의 저산소농도 조건 하에서 24시간 배양하여 수득하여 엑소좀이 풍부할 수 있다. 상기 TNF-α는 3 내지 100 ng/mL의 농도로 처리될 수 있다. In the pharmaceutical composition for preventing or treating inflammatory diseases according to the present invention, the culture medium is treated with TNF-α in the culture medium containing immortalized stem cells, and cultured for 12 to 72 hours under a hypoxic condition of 1 to 5%. obtained and characterized in that it is rich in exosomes. Preferably, the culture medium may be obtained by treating the culture medium containing immortalized stem cells with TNF-α and culturing it for 24 hours under a hypoxic condition of 3% to be rich in exosomes. The TNF-α may be treated at a concentration of 3 to 100 ng/mL.

본 발명에 따른 염증성 질환의 예방 또는 치료용 약제학적 조성물에 있어서, 상기 염증성 질환은 아토피성 피부염, 뇌염, 염증성 장염, 만성 폐쇄성 폐질환, 폐혈병성 쇼크증, 폐섬유증, 미분화 척추관절증, 미분화 관절병증, 관절염, 염증성 골용해, 만성 바이러스 또는 박테리아 감염에 의한 만성 염증질환, 대장염, 염증성 장질환, 1형 당뇨병, 류마티스 관절염, 반응성 관절염(Reactive Arthritis), 골관절염, 건선, 공피증, 골다공증, 아테롬성 동맥경화증, 심근염, 심내막염, 심낭염, 낭성 섬유증, 하시모토 갑상선염, 그레이브스병, 나병, 매독, 라임 질환(Lyme), 보렐리아증(Borreliosis), 신경성-보렐리아증, 결핵, 사르코이드증(Sarcoidosis), 낭창, 원판성 낭창, 동창성 루프스, 루프스 신염, 전신성 홍반성 루프스, 황반변성, 포도막염, 과민대장 증후군, 크로씨병, 쇼그랜 증후군, 섬유근통, 만성피로 증후군, 만성피로 면역부전 증후군, 근육통성 뇌척수염, 근위축성 측삭경화증, 파킨스병 및 다발경화증로 이루어진 군으로부터 하나 또는 그 이상 선택되는 것을 특징으로 한다. 바람직하기는, 상기 염증성 질환은 아토피성 피부염 또는 류마티스 관절염일 수 있다. In the pharmaceutical composition for preventing or treating inflammatory diseases according to the present invention, the inflammatory diseases include atopic dermatitis, encephalitis, inflammatory enteritis, chronic obstructive pulmonary disease, septic shock, pulmonary fibrosis, undifferentiated spondyloarthrosis, and undifferentiated joint Conditions, arthritis, inflammatory osteolysis, chronic inflammatory diseases caused by chronic viral or bacterial infection, colitis, inflammatory bowel disease, type 1 diabetes, rheumatoid arthritis, reactive arthritis, osteoarthritis, psoriasis, scleroderma, osteoporosis, atherosclerosis Sclerosis, myocarditis, endocarditis, pericarditis, cystic fibrosis, Hashimoto's thyroiditis, Graves' disease, leprosy, syphilis, Lyme disease, Borreliosis, neurogenic-borreliosis, tuberculosis, sarcoidosis, lupus, disc Lupus lupus, lupus lupus, lupus nephritis, systemic lupus erythematosus, macular degeneration, uveitis, irritable bowel syndrome, Croissie's disease, Sjogren's syndrome, fibromyalgia, chronic fatigue syndrome, chronic fatigue immunodeficiency syndrome, myalgic encephalomyelitis, amyotrophic lateral Characterized in that one or more selected from the group consisting of sclerosis, Parkinson's disease and multiple sclerosis. Preferably, the inflammatory disease may be atopic dermatitis or rheumatoid arthritis.

본 발명에 따른 염증성 질환의 예방 또는 치료용 약제학적 조성물에 있어서, 상기 약제학적 조성물은 Carrageenan로 인한 피부 삼출물 및 염증지표, 염증세포의 침윤 및 피부염 억제 효능을 나타낼 뿐만 아니라, 수술적 퇴행성 관절염(연골결손) 억제 효과를 가지는 것을 특징으로 한다. In the pharmaceutical composition for preventing or treating inflammatory diseases according to the present invention, the pharmaceutical composition not only exhibits skin exudate and inflammatory markers due to Carrageenan, inflammatory cell infiltration and dermatitis inhibitory effect, but also treats surgical degenerative arthritis (cartilage deficiency) characterized in that it has an inhibitory effect.

본 발명에 따른 염증성 질환의 예방 또는 치료용 약제학적 조성물은 각각의 사용 목적에 맞게 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁제, 에멀젼, 시럽, 에어로졸 등의 경구 제형, 멸균 주사 용액의 형태 등 다양한 형태로 제형화하여 사용할 수 있으며, 경구 투여하거나 정맥 내, 복강 내, 피하, 직장, 국소 투여 등을 포함한 다양한 경로를 통해 투여될 수 있다.The pharmaceutical composition for preventing or treating inflammatory diseases according to the present invention is formulated according to conventional methods according to the purpose of use, such as oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and sterile injections. It can be formulated and used in various forms such as the form of a solution, and can be administered through various routes including oral administration or intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like.

본 발명에 따른 염증성 질환의 예방 또는 치료용 약제학적 조성물에 포함될 수 있는 적합한 담체, 부형제 및 희석제의 예로는 락토오스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 비정질 셀룰로즈, 폴리비닐 피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등을 들 수 있다.Examples of suitable carriers, excipients and diluents that may be included in the pharmaceutical composition for preventing or treating inflammatory diseases according to the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil; and the like. .

본 발명에 따른 염증성 질환의 예방 또는 치료용 약제학적 조성물은 충전제, 항응집제, 윤활제, 습윤제, 향료, 유화제, 방부제 등을 추가로 포함할 수 있다.The pharmaceutical composition for preventing or treating inflammatory diseases according to the present invention may further include fillers, anti-aggregating agents, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, and the like.

본 발명에 따른 염증성 질환의 예방 또는 치료용 약제학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다. 구체적으로, 본 발명에 따른 염증성 질환의 예방 또는 치료용 약제학적 조성물의 약제학적으로 유효한 양은 환자의 나이, 성별, 체중에 따라 달라질 수 있으며, 일반적으로 줄기세포는 개체당 1천~2억 세포, 엑소좀풍부배양액은 체중 ㎏당 1 내지 50 mg을 매일 또는 격일로 투여하거나 1일 1 내지 3회로 나누어 투여할 수 있다. 그러나, 투여 경로, 질병의 중증도, 성별, 체중, 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.The pharmaceutical composition for preventing or treating inflammatory diseases according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art. Specifically, the pharmaceutically effective amount of the pharmaceutical composition for preventing or treating inflammatory diseases according to the present invention may vary depending on the age, sex, and weight of the patient, and in general, stem cells are 100 to 200 million cells per subject, The exosome-rich culture medium may be administered in an amount of 1 to 50 mg per kg of body weight every day or every other day or divided into 1 to 3 times a day. However, since it may increase or decrease according to the route of administration, severity of disease, sex, weight, age, etc., the dosage is not limited to the scope of the present invention in any way.

제3구현예에 따르면,According to the third embodiment,

본 발명은 불멸화 고양이줄기세포(기탁번호: KCTC 14390BP) 또는 이의 배양액을 유효성분으로 포함하는 염증성 질환의 예방 또는 완화용 식품 조성물을 제공하고자 한다. The present invention is intended to provide a food composition for preventing or alleviating inflammatory diseases comprising immortalized feline stem cells (accession number: KCTC 14390BP) or a culture solution thereof as an active ingredient.

본 발명에 따른 염증성 질환의 예방 또는 완화용 식품 조성물에 있어서, 상기 배양액은 불멸화 줄기세포를 함유한 배양액에 TNF-α를 처리하고, 1~5%의 저산소농도 조건 하에서 12~72시간 배양하여 수득하여 엑소좀이 풍부한 것을 특징으로 한다. 바람직하기는, 상기 배양액은 불멸화 줄기세포를 함유한 배양액에 TNF-α를 처리하고, 3%의 저산소농도 조건 하에서 24시간 배양하여 수득하여 엑소좀이 풍부할 수 있다. 상기 TNF-α는 3 내지 100 ng/mL의 농도로 처리될 수 있다. In the food composition for preventing or alleviating inflammatory diseases according to the present invention, the culture medium is obtained by treating the culture medium containing immortalized stem cells with TNF-α and culturing for 12 to 72 hours under conditions of 1 to 5% hypoxia. Therefore, it is characterized in that it is rich in exosomes. Preferably, the culture medium may be obtained by treating the culture medium containing immortalized stem cells with TNF-α and culturing it for 24 hours under a hypoxic condition of 3% to be rich in exosomes. The TNF-α may be treated at a concentration of 3 to 100 ng/mL.

본 발명에 따른 염증성 질환의 예방 또는 치료용 약제학적 조성물에 있어서, 상기 식품 조성물은 Carrageenan로 인한 피부 삼출물 및 염증지표, 염증세포의 침윤 및 피부염 억제 효능을 나타낼 뿐만 아니라, 수술적 퇴행성 관절염(연골결손) 억제, 개선 또는 완화 효과를 가지는 것을 특징으로 한다. In the pharmaceutical composition for preventing or treating inflammatory diseases according to the present invention, the food composition not only exhibits skin exudate and inflammatory markers due to Carrageenan, inflammatory cell infiltration and dermatitis inhibitory effect, but also treats surgical degenerative arthritis (chondral defect). ) characterized in that it has an inhibitory, ameliorating or alleviating effect.

본 발명에 따른 염증성 질환의 예방 또는 완화용 식품 조성물은 상기 유효 성분 이외에 감미제, 풍미제, 생리활성 성분, 미네랄 등을 추가로 포함할 수 있다. The food composition for preventing or alleviating inflammatory diseases according to the present invention may further include sweeteners, flavors, physiologically active ingredients, minerals, and the like in addition to the active ingredients.

본 발명에 따른 염증성 질환의 예방 또는 완화용 식품 조성물은 필요에 따라 보존제, 유화제, 산미료, 점증제 등을 포함할 수 있다. 이러한 보존제, 유화제 등은 그것이 첨가되는 용도를 달성할 수 있는 한 극미량으로 첨가되어 사용되는 것이 바람직하다. 극미량이란 수치적으로 표현할 때 식품 조성물 전체 중량을 기준으로 할 때 0.0005중량％ 내지 약 0.5중량％ 범위를 의미한다.The food composition for preventing or alleviating inflammatory diseases according to the present invention may include a preservative, an emulsifier, an acidulant, a thickening agent, and the like, if necessary. These preservatives, emulsifiers, etc. are preferably added and used in very small amounts as long as the purpose to which they are added can be achieved. A trace amount means a range of 0.0005% by weight to about 0.5% by weight based on the total weight of the food composition when expressed numerically.

제4구현예에 따르면, According to the fourth embodiment,

A) 고양이지방줄기세포(feline adipose-derived mesenchymal stem cells, fADMSC)를 HPV16의 E6 및 E7 유전자를 포함하는 렌티바이러스 벡터(lentiviral vector)로 처리하여 분화유도하여 불멸화 고양이줄기세포를 얻는 단계; A) obtaining immortalized feline stem cells by treating feline adipose-derived mesenchymal stem cells (fADMSC) with a lentiviral vector containing HPV16 E6 and E7 genes to induce differentiation;

B) 상기 불멸화 고양이줄기세포를 DMEM (Dulbecco's modified Eagle's medium) 배지에 접종하여 5%의 이산화탄소 공급 배양기에서 배양하는 단계; 및B) inoculating the immortalized feline stem cells in DMEM ( Dulbecco's modified Eagle's medium ) medium and culturing them in an incubator supplying 5% carbon dioxide; and

C) 상기 배양 배지에 TNF-α를 첨가하고 TNF-α를 처리하고 1~5%의 저산소농도 조건 하에서 12~72시간 배양하는 단계를 포함하는 불멸화 고양이줄기세포 엑소좀 풍부 배양액의 제조 방법을 제공하고자 한다. C) adding TNF-α to the culture medium, treating TNF-α, and culturing for 12-72 hours under 1-5% hypoxic conditions Provide a method for producing an exosome-rich culture solution of immortalized feline stem cells want to do

본 발명에 따른 불멸화 고양이줄기세포 엑소좀 풍부 배양액의 제조 방법에 있어서, 상기 불멸화 고양이줄기세포는 기탁번호: KCTC 14390BP인 것을 특징으로 한다. In the method for preparing an exosome-rich culture solution of immortalized feline stem cells according to the present invention, the immortalized feline stem cells are characterized by accession number: KCTC 14390BP.

본 발명에 따른 불멸화 고양이줄기세포 엑소좀 풍부 배양액의 제조 방법에 있어서, 상기 TNF-α는 3 내지 100 ng/mL의 농도로 첨가되는 것을 특징으로 한다. In the method for preparing an exosome-rich culture solution of immortalized feline stem cells according to the present invention, the TNF-α is added at a concentration of 3 to 100 ng/mL.

본 발명에 따른 불멸화 고양이줄기세포 엑소좀 풍부 배양액의 제조 방법에 있어서, 상기 불멸화 고양이줄기세포 엑소좀 풍부 배양액은 피부염 또는 류마티스 관절염 억제 효과를 갖는 것을 특징으로 한다. 예를 들면, 상기 불멸화 고양이줄기세포 엑소좀 풍부 배양액은 Carrageenan로 인한 피부 삼출물 및 염증지표, 염증세포의 침윤 및 피부염 억제 효능을 나타낼 뿐만 아니라, 수술적 퇴행성 관절염(연골결손) 억제 효과를 가질 수 있다. In the preparation method of the exosome-rich culture solution of immortalized feline stem cells according to the present invention, the culture solution rich in exosomes of immortalized feline stem cells is characterized in that it has an inhibitory effect on dermatitis or rheumatoid arthritis. For example, the immortalized feline stem cell exosome-rich culture medium not only exhibits skin exudate and inflammatory markers caused by Carrageenan, infiltration of inflammatory cells and dermatitis inhibitory effects, but also has an inhibitory effect on surgical degenerative arthritis (cartilage defect). .

이하, 구체적인 실시예를 통해 본 발명을 보다 구체적으로 설명한다. 하기 실시예는 본 발명의 이해를 돕기 위한 예시에 불과하며, 본 발명의 범위가 이에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through specific examples. The following examples are merely examples to aid understanding of the present invention, and the scope of the present invention is not limited thereto.

<실시예> <Example>

실시예 1. 줄기세포 불멸화Example 1. Stem cell immortalization

(1) 줄기세포 불멸화 과정(1) Stem cell immortalization process

나이 2살의 건강한 수컷 코리안숏헤어(Korean short hair)종 고양이로부터 지방조직을 채취하고, 이미 참고문헌에 제시된 방법에 따라 지방줄기세포를 분리, 배양하였다. 불멸화하기 위한 세포는 60 mm2 culture dish에 70%가 되도록 준비한 후 1 mL culture medium, 2 mL pLenti-III-HPV-16 E6/E7 particle (Abm), 그리고 5 μg/mL polybrene (Sigma-Aldrich)을 혼합하여 8시간 배양한 후 같은 medium 혼합액으로 교체 후 3일 동안 배양하였다(도 2). 그 다음, culture medium으로 교체하여 불멸화된 세포를 관찰하면서 불멸화 특징 시 나타나는 유전자 발현을 RT-PCR (표 1)로 확인하였다.Adipose tissue was collected from a 2-year-old healthy male Korean short hair cat, and adipose stem cells were isolated and cultured according to the method already suggested in the references. Cells for immortalization were prepared to 70% confluency in a 60 mm 2 culture dish, 1 mL culture medium, 2 mL pLenti-III-HPV-16 E6/E7 particle (Abm), and 5 μg/mL polybrene (Sigma-Aldrich) were cultured for 8 hours by mixing, and then cultured for 3 days after replacing with the same medium mixture (FIG. 2). Then, the culture medium was replaced and the immortalized cells were observed, and gene expression during immortalization was confirmed by RT-PCR (Table 1).

표 1: 줄기세포 불멸화를 확인하기 위한 유전자 분석 reverse transcriptase-polymerase chain reaction (RT-PCR) primers 목록Table 1: List of reverse transcriptase-polymerase chain reaction (RT-PCR) primers for genetic analysis to confirm stem cell immortalization

GeneGene PrimerPrimer SequenceSequence Accession No.Accession no. HPV-16 E6/E7HPV-16 E6/E7 5’ primer5’ primer 5’-CGCAAATGGGCGGTAGGCGTG5'-CGCAAATGGGCGGTAGGCGTG NC_001526.2NC_001526.2 3’ primer3’ primer 5’-TAGTCAGCCATGGGGCGGAGA5'-TAGTCAGCCATGGGGCGGAGA HPV-16 E6/E7
(NO selection)HPV-16 E6/E7
(NO selection) 5’ primer5’ primer 5’-CGCAAATGGGCGGTAGGCGTG5'-CGCAAATGGGCGGTAGGCGTG 3’ primer3’ primer 5’-GAAAGCCATACGGGAAGCAATAG5'-GAAAGCCATACGGGAAGCAATAG Feline β-actinFeline β-actin 5’ primer5’ primer 5’-GAACGGGAAACTTGTCATCA5′-GAACGGGAAACTTGTCATCA NM_001009307.1NM_001009307.1 3’ primer3’ primer 5’-AGAGATGATGACCCTCTTGG5'-AGAGATGATGACCCTCTTGG

(2) RT-PCR을 이용한 줄기세포 불멸화 확인(2) Confirmation of immortalization of stem cells using RT-PCR

불멸화 후 최소 일주일 정도 배양하여 일정 부분의 세포를 분리하여 Trizol (Thermo Fisher Scientific, MA, USA)로 total RNA를 추출하였다. 2 μg의 total RNA를 대상으로 Oligo dT primer와 TOP scriptTM Reverse Transcriptase (Enzynomics, Daejeon, Korea)를 사용한 역전사반응을 통해 PCR의 주형으로 사용할 cDNA를 합성하였다. 합성한 cDNA를 이용하여 RT-PCR을 진행하였으며, RT-PCR master mix는 10 μL 2X enzyme Mastermix, 7 μL RNase-free water, 각각 1 μL primer (각 10 pmole), 그리고 1 μL cDNA로 조성하였다. PCR 프로그램은 95℃에서 10분, denature 95℃에서 15초, annealing 67.4℃에서 15초, 그리고 extension 72℃에서 10초이며 35 cycles로 진행하였다. 유전자의 발현 정도는 β-actin primer를 standard로 하여 유전자의 발현량을 확인하였으며, primers는 Gene bank와 시약에 기재되어 있는 정보를 바탕으로 제작되었다.After immortalization, the cells were cultured for at least one week, and a certain portion of the cells was isolated, and total RNA was extracted with Trizol (Thermo Fisher Scientific, MA, USA). For 2 μg of total RNA, cDNA to be used as a PCR template was synthesized through reverse transcription using Oligo dT primer and TOP scriptTM Reverse Transcriptase (Enzynomics, Daejeon, Korea). RT-PCR was performed using the synthesized cDNA, and the RT-PCR master mix was composed of 10 μL 2X enzyme Mastermix, 7 μL RNase-free water, 1 μL primer each (10 pmole each), and 1 μL cDNA. The PCR program was 35 cycles at 95°C for 10 minutes, denature at 95°C for 15 seconds, annealing at 67.4°C for 15 seconds, and extension at 72°C for 10 seconds. The expression level of the gene was checked using the β-actin primer as a standard, and the primers were prepared based on the information described in the Gene bank and reagents.

(3) 줄기세포 불멸화 확인(3) Confirmation of immortalization of stem cells

줄기세포 불멸화 여부를 확인하고자, primary cells (대조군)과 함께 세포의 모양(morphology)를 비교하고, pLenti-III-HPV-16 E6/E7 particles 삽입으로 인한 HPV 유전자 발현을 RT-PCR로 분석하여 확인하였다. 불멸화된 고양이지방줄기세포는 배양과정에서 약간의 morphology 변화가 관찰되었다. 즉, 불멸화 세포에서 primary cells에 비해 세포의 길이가 약간 짧아졌으며(도 3A), doubling time 주기가 감소하여 세포의 성장속도가 빨라진 것으로 확인되었다. 유전자 발현은 불멸화된 세포에서 HPV 유전자가 과발현된 것이 확인되었다(도 3B).To confirm stem cell immortality, the morphology of the cells was compared with primary cells (control), and HPV gene expression due to the insertion of pLenti-III-HPV-16 E6/E7 particles was analyzed by RT-PCR and confirmed. did Immortalized feline adipose stem cells showed slight morphology changes during the culture process. That is, it was confirmed that the length of the immortalized cells was slightly shorter than that of the primary cells (FIG. 3A), and the doubling time period was reduced, resulting in increased cell growth rate. Gene expression confirmed that the HPV gene was overexpressed in the immortalized cells (FIG. 3B).

또한, 정상(비불멸화) 고양이줄기세포(fADMSC)와 불멸화 고양이줄기세포 (im-fADMSC)를 정상의 산소농도(20%)와 저산소농도(3%)에서 2일 동안 배양하면서 생존율 변화를 측정한 결과, 비불멸화 줄기세포는 정상 산소농도에서 2일만에 2배 가까이(168%) 증식했지만 저산소농도에서는 대부분 사멸하여(5%) 저산소 환경에서 생존하지 못하는 것으로 확인되었다(도 4). 이에 비해 불멸화 줄기세포는 정상 산소농도에서 2일만에 2배 이상(215%) 증식하여 정상세포보다 빠른 증식율을 나타냈으며, 저산소 농도에서도 147%의 증식능을 나타내 저산소 환경에 저항성을 보이는 것으로 확인되었다.In addition, normal (non-immortalized) feline stem cells (fADMSC) and immortalized feline stem cells (im-fADMSC) were cultured for 2 days in normal oxygen concentration (20%) and hypoxia (3%), and the change in viability was measured. As a result, it was confirmed that the non-immortalized stem cells proliferated nearly twice (168%) in 2 days in normal oxygen concentration, but were mostly killed (5%) in hypoxic concentration and did not survive in hypoxic environment (FIG. 4). In contrast, immortalized stem cells proliferated more than twice (215%) in 2 days in normal oxygen concentration, showing a faster proliferation rate than normal cells, and showed resistance to hypoxic environment by showing 147% proliferation ability even in low oxygen concentration.

(5) 줄기세포 엑소좀 함량 분석 결과(5) Stem cell exosome content analysis result

불멸화 줄기세포를 산소농도 21%에서 배양한 정상배양액(CM)과 TNF-α 처리 후 산소농도 3%에서 저산소배양한 엑소좀풍부배양액(ERCM)의 엑소좀 함량을 CD9에 대한 RT-PCR로 분석한 결과, ERCM에서 CM에 비해 50배 이상의 CD9 지표가 검출되어 ERCM 내 고농도 엑소좀 함유가 확인되었다(도 5).Analysis of exosome content in normal culture medium (CM) of immortalized stem cells cultured at 21% oxygen concentration and exosome-rich culture medium (ERCM) cultured in hypoxia at 3% oxygen concentration after TNF-α treatment by RT-PCR for CD9 As a result, 50 times more CD9 index was detected in ERCM than in CM, confirming the high concentration of exosomes contained in ERCM (FIG. 5).

실시예 2. 줄기세포의 피부염 치료효능Example 2. Dermatitis treatment efficacy of stem cells

(1) 줄기세포와 줄기세포 배양액의 Carrageenan 유도 피부염 억제효과(삼출액 및 염증지표물질)(1) Carrageenan-induced dermatitis inhibitory effect of stem cells and stem cell culture media (exudate and inflammatory markers)

Carrageenan으로 유도한 피부염으로 인한 피부 삼출액량 및 삼출액 내 염증지표물질에 대한 고양이줄기세포와 줄기세포 배양액의 억제효과를 평가하였다. 실험 첫 날 마우스 등의 털을 제거하고, 10 mL의 멸균공기를 피하로 주사하고, 2일 및 5일 후 5 mL의 공기를 추가 주사해 공기주머니(air-pouch)를 유지하였다. 공기주머니 내로 생리식염수에 1%로 희석한 1 mL carrageenan에 3x104 cells의 정상(비불멸화) 고양이줄기세포(fADMSC), 불멸화 고양이줄기세포(im-fADMSC), 100 μL의 정상배양액(CM) 또는 엑소좀풍부배양액(ERCM)을 혼합하여 주사하고, 6시간 후 1 mL의 생리식염수로 공기주머니를 세척해 내었다. 회수한 주입한 1 mL를 뺀 세척액량, 즉 삼출액량을 측정하고, 삼출액 내의 알부민 단백질과 염증지표물질[tumor-necrosis factor-α (TNF-α), interleukin-6 (IL-6), nitric oxide (NO) 및 prostaglandin E2 (PGE2)]을 혈액생화학분석기, ELISA 키트 및 Griess 시약으로 분석하였다.The inhibitory effects of feline stem cells and stem cell cultures on the amount of skin exudate and inflammatory markers in the exudate caused by carrageenan-induced dermatitis were evaluated. On the first day of the experiment, the hair on the back of the mouse was removed, 10 mL of sterile air was subcutaneously injected, and after 2 and 5 days, 5 mL of air was additionally injected to maintain an air-pouch. 3x10 4 cells of normal (non-immortalized) feline stem cells (fADMSC), immortalized feline stem cells (im-fADMSC), 100 μL of normal culture medium (CM) or Exosome-rich culture medium (ERCM) was mixed and injected, and after 6 hours, the air bag was washed out with 1 mL of physiological saline. The amount of washing liquid minus 1 mL of the recovered injection, that is, the amount of exudate, was measured, and albumin protein and inflammatory markers [tumor-necrosis factor-α (TNF-α), interleukin-6 (IL-6), nitric oxide in the exudate were measured. (NO) and prostaglandin E 2 (PGE 2 )] were analyzed with a blood biochemistry analyzer, ELISA kit and Griess reagent.

시험 결과, Carrageenan 투여로 인한 피부염으로 삼출액량이 2배 가까이 증가하고, 삼출액 내 알부민 농도가 2.5배 상승하였는 바, 이러한 혈액성분 누출은 3x104 cells의 정상(비불멸화) 줄기세포(fADMSC)와 불멸화 줄기세포(im-fADMSC) 투여로 완화되었으며, 특히 불멸화 줄기세포가 비불멸화 줄기세포에 비해 우수하였다 (표 2). 또한 Carrageenan에 의해 크게 증가했던 삼출액 내 염증성 사이토카인(TNF-α 및 IL-6)과 염증매개물질(NO 및 PGE2) 역시 줄기세포 복합투여로 억제되었으며, 이러한 효과 또한 비불멸화 줄기세포보다 불멸화 줄기세포가 우수하였다. 더 나아가 줄기세포 대신 100 μL의 줄기세포 배양액을 투여했을 때에도 삼출액 및 염증지표물질이 감소하였는 바, 엑소좀풍부배양액(ERCM)이 정상배양액(CM)보다 우수하였다.As a result of the test, the amount of exudate increased nearly 2 times due to dermatitis caused by administration of Carrageenan, and the concentration of albumin in the exudate increased 2.5 times. It was relieved by cell (im-fADMSC) administration, and in particular, immortalized stem cells were superior to non-immortalized stem cells (Table 2). In addition, inflammatory cytokines (TNF-α and IL-6) and inflammatory mediators (NO and PGE 2 ) in the exudate, which were significantly increased by Carrageenan, were also suppressed by the combined administration of stem cells, and these effects were also reduced by immortalized stem cells than non-immortalized stem cells. Cells were excellent. Furthermore, even when 100 μL of stem cell culture medium was administered instead of stem cells, exudate and inflammatory markers were reduced, and exosome-rich culture medium (ERCM) was superior to normal culture medium (CM).

표 2: Carrageenan으로 유도된 피부 삼출액 및 염증지표물질에 대한 고양이줄기세포와 줄기세포 배양액의 억제효과Table 2: Inhibitory effects of feline stem cells and stem cell cultures on skin exudates and inflammatory markers induced by carrageenan

투여administration 생리식염수saline solution Carrageenan 단독Carrageenan alone +fADMSC
(3x104 cells)+fADMSC
(3x10 4 cells) +im-fADMSC
(3x104 cells)+im-fADMSC
(3x10 4 cells) +CM
(100 μL)+CM
(100 µL) +ERCM
(100 μL)+ERCM
(100 µL) 삼출액
(mL)exudate
(mL) 0.29±0.030.29±0.03 0.57±0.02*0.57±0.02* 0.45±0.080.45±0.08 0.34±0.02# 0.34±0.02 # 0.38±0.120.38±0.12 0.36±0.05# 0.36±0.05 # 일부민
(g/dL)some people
(g/dL) 0.26±0.020.26±0.02 0.69±0.04*0.69±0.04* 0.41±0.04# 0.41±0.04 # 0.32±0.03# 0.32±0.03 # 0.43±0.02# 0.43±0.02 # 0.36±0.04# 0.36±0.04 # TNF-α
(pg/dL)TNF-α
(pg/dL) 7.7±0.47.7±0.4 166.1±31.0*166.1±31.0* 121.9±23.2# 121.9±23.2 # 88.2±15.0# 88.2±15.0 # 131.0±11.2# 131.0±11.2 # 98.3±12.8# 98.3±12.8 # IL-6
(pg/dL)IL-6
(pg/dL) 17.9±16.517.9±16.5 443.4±43.3*443.4±43.3* 402.0±27.6402.0±27.6 301.8±23.4# 301.8±23.4 # 388.0±17.7388.0±17.7 313.8±20.4# 313.8±20.4 # NO
(μM)NO
(μM) 1.32±0.361.32±0.36 2.96±0.43*2.96±0.43* 1.73±0.12# 1.73±0.12 # 1.54±0.24# 1.54±0.24 # 1.99±0.02# 1.99±0.02 # 1.59±0.14# 1.59±0.14 # PGE2
(ng/dL)PGE 2
(ng/dL) 2.84±0.192.84±0.19 3.42±0.133.42±0.13 1.94±0.11# 1.94±0.11 # 1.66±0.04# 1.66±0.04 # 2.11±0.21# 2.11±0.21 # 1.73±0.03# 1.73±0.03 #

TNF-α, tumor-necrosis factor-α; IL-6, interleukin-6; NO, nitric oxide; PGE2, prostaglandin E2.*용매(생리식염수) 대조군에 비해 통계적으로 유의함(P<0.05).TNF-α, tumor-necrosis factor-α; IL-6, interleukin-6; NO, nitric oxide; PGE 2 , prostaglandin E 2 .*solvent (physiological saline) statistically significant compared to the control group (P<0.05).

#Carrageenan 단독투여군에 비해 통계적으로 유의함(P<0.05). # Statistically significant compared to the carrageenan alone group (P<0.05).

(2) 줄기세포의 Carrageenan 유도 피부염 억제효과(염증세포 침윤)(2) Carrageenan-induced dermatitis inhibitory effect of stem cells (inflammatory cell infiltration)

앞의 공기주머니 세척액 내 염증세포, 즉 총백혈구(white blood cells, WBC), 중호성백혈구(neutrophils), 산호성백혈구(eosinophils), 염기호성백혈구(basophils), 단핵구(monocytes) 및 림프구(lymphocytes)를 세포측정기(coulter counter)로 분석하였다.Inflammatory cells in the preceding air bag lavage fluid, i.e. white blood cells (WBC), neutrophils, eosinophils, basophils, monocytes and lymphocytes was analyzed with a coulter counter.

시험 결과, Carrageenan 투여로 인한 피부염으로 총백혈구수, 특히 중호성백혈구수가 10배 가까이 증가한 반면, 이러한 백혈구 침윤은 정상(비불멸화) 줄기세포(fADMSC)와 불멸화 줄기세포(im-fADMSC) 투여로 완화되었는데, 특히 불멸화 줄기세포가 비불멸화 줄기세포에 비해 우수하였다(표 3). 또한 carrageenan에 의해 크게 증가했던 단핵구 및 림프구 침윤 역시 줄기세포 복합투여로 억제되었으며, 이러한 효과 또한 비불멸화 줄기세포보다 불멸화 줄기세포가 우수하였다. 더 나아가 줄기세포 대신 100 μL의 줄기세포 배양액을 투여했을 때에도 염증세포 침윤이 억제되었는 바, 엑소좀풍부배양액(ERCM)이 정상배양액(CM)보다 우수하였다.As a result of the test, the total white blood cell count, especially the mesophil count, increased nearly 10-fold due to dermatitis caused by Carrageenan administration, while this leukocyte infiltration was alleviated by the administration of normal (non-immortalized) stem cells (fADMSC) and immortalized stem cells (im-fADMSC). In particular, immortalized stem cells were superior to non-immortalized stem cells (Table 3). In addition, monocyte and lymphocyte infiltration, which had been significantly increased by carrageenan, were also inhibited by the combined administration of stem cells, and these effects were also superior to immortalized stem cells than non-immortalized stem cells. Furthermore, even when 100 μL of stem cell culture medium was administered instead of stem cells, inflammatory cell infiltration was suppressed, and exosome-rich culture medium (ERCM) was superior to normal culture medium (CM).

표 3: Carrageenan으로 유도된 염증세포 침윤에 대한 고양이줄기세포와 줄기세포 배양액의 억제효과Table 3: Inhibitory effect of feline stem cells and stem cell culture medium on carrageenan-induced inflammatory cell infiltration

투여administration 생리식염수saline solution Carrageenan 단독Carrageenan alone +fADMSC
(3x104 cells)+fADMSC
(3x10 4 cells) +im-fADMSC
(3x104 cells)+im-fADMSC
(3x10 4 cells) +CM
(100 μL)+CM
(100 µL) +ERCM
(100 μL)+ERCM
(100 µL) 총백혈구total white blood cells 0.96±0.150.96±0.15 9.44±3.02*9.44±3.02* 5.31±0.63# 5.31±0.63 # 3.39±0.57# 3.39±0.57 # 5.01±0.33# 5.01±0.33 # 3.13±0.50# 3.13±0.50 # 중호성백혈구Neutrophils 0.16±0.040.16±0.04 0.94±0.41*0.94±0.41* 0.63±0.23# 0.63±0.23 # 0.40±0.16# 0.40±0.16 # 0.86±0.530.86±0.53 0.43±0.23# 0.43±0.23 # 산호성백혈구coral leukocyte 0.04±0.020.04±0.02 0.06±0.010.06±0.01 0.07±0.020.07±0.02 0.05±0.060.05±0.06 0.08±0.020.08±0.02 0.05±0.020.05±0.02 염기호성백혈구basophilic leukocyte 0.06±0.030.06±0.03 0.09±0.300.09±0.30 0.18±0.060.18±0.06 0.11±0.120.11±0.12 0.08±0.060.08±0.06 0.10±0.100.10±0.10 단핵구monocyte 0.07±0.030.07±0.03 0.43±0.13*0.43±0.13* 0.18±0.06# 0.18±0.06 # 0.14±0.04# 0.14±0.04 # 0.20±0.05# 0.20±0.05 # 0.16±0.04# 0.16±0.04 # 림프구lymphocyte 0.67±0.190.67±0.19 7.93±2.85*7.93±2.85* 3.44±0.52# 3.44±0.52 # 2.05±0.88# 2.05±0.88 # 3.99±0.82# 3.99±0.82 # 2.22±0.56# 2.22±0.56 #

*용매 대조군에 비해 통계적으로 유의함(P<0.05).*Statistically significant compared to solvent control (P<0.05).

#Carrageenan 단독투여군에 비해 통계적으로 유의함(P<0.05). # Statistically significant compared to the carrageenan alone group (P<0.05).

(3) 줄기세포의 Carrageenan 유도 피부염 억제효과(현미경소견)(3) Carrageenan-induced dermatitis inhibitory effect of stem cells (microscopic findings)

앞의 공기주머니 내막을 포함한 피부조직을 채취하여 4% 중성포르말린 용액으로 고정한 후, 파라핀 절편을 만들어 hematoxylin & eosin (H&E)으로 염색하고, 광학현미경 하에서 피부염증반응을 관찰하였다.Skin tissue including the inner membrane of the air sac was collected, fixed in 4% neutral formalin solution, paraffin sections were made, stained with hematoxylin & eosin (H&E), and skin inflammatory reactions were observed under an optical microscope.

그 결과, Carrageenan 투여로 인한 피부염 소견으로 피하조직에 다량의 염증세포가 침윤되어 두꺼운 층을 형성하고 있었지만, 이러한 염증소견은 정상(비불멸화) 줄기세포(fADMSC)와 불멸화 줄기세포(im-fADMSC) 투여로 확연히 완화되었는데, 특히 불멸화 줄기세포가 비불멸화 줄기세포에 비해 훨씬 우수하였다(도 6). 더 나아가 줄기세포 대신 100 μL의 줄기세포 배양액을 투여했을 때에도 염증반응이 상당히 완화되었는 바, 엑소좀풍부배양액(ERCM)이 정상배양액(CM)보다 우수하였다.As a result, a large amount of inflammatory cells infiltrated into the subcutaneous tissue and formed a thick layer due to dermatitis due to Carrageenan administration. It was significantly alleviated by administration, and in particular, immortalized stem cells were much better than non-immortalized stem cells (FIG. 6). Furthermore, even when 100 μL of the stem cell culture medium was administered instead of the stem cells, the inflammatory reaction was significantly alleviated, and the exosome-rich culture medium (ERCM) was superior to the normal culture medium (CM).

실시예 3. 줄기세포의 류마티스 관절염 치료효능Example 3. Therapeutic efficacy of stem cells for rheumatoid arthritis

(1) 퇴행성 관절염(연골결손) 유발 및 줄기세포 투여(1) Degenerative arthritis (cartilage defect) induction and stem cell administration

연골결손(articular cartilage defect, ACD) 모델 제작에는 성장판 생장이 멈춘 생후 12주 이상의 2.8-3.2 kg의 수컷 New Zealand White (NZW) 토끼를 사용하였다. 토끼에 Zoletil 50 (15 mg/kg)과 Rompum (5 mg/kg)을 근육 내로 주사하여 전신마취하였다. 우측 무릎 주변의 털을 제거하고 Povidone과 70% ethanol로 피부를 소독하고, 수술 부위를 무균 조작하여 수술을 개시하였다. 무릎 관절의 내측 피부를 절개하고 근막과 관절낭을 절개한 후 무릎뼈를 외측으로 젖혀 관절낭을 노출시켰다. 대퇴골의 내과부(무릎관절고랑 정중앙)에 직경 1 mm 드릴을 이용하여 직경 5 mm, 깊이 2 mm의 연골결손 부위를 만들고, 소파기로 결손부위 내의 연골을 모두 제거한 후 관절낭을 봉합하였다. 이어 피하조직과 피부를 봉합한 후 povidone으로 소독하였다. 연골이 제거된 시점에서 30분 후, 1x106 cells의 정상(비불멸화) 줄기세포(fADMSC), 불멸화 줄기세포(im-fADMSC), 100 μL의 정상배양액(CM) 또는 엑소좀풍부배양액(ERCM)을 관절강 내로 주사하였다. 수술 후 3일간 항생제 Foxolin (10 mg/kg)과 진통제 Maritrol (3 mg/kg)을 매일 2회 근육 내로 주사하였고, 이후 3일간 Foxolin을 매일 1회 추가로 처치하였다.To construct the articular cartilage defect (ACD) model, male New Zealand White (NZW) rabbits aged 12 weeks or older and weighing 2.8-3.2 kg with growth plate growth stopped were used. Rabbits were anesthetized by intramuscular injection of Zoletil 50 (15 mg/kg) and Rompum (5 mg/kg). The hair around the right knee was removed, the skin was disinfected with Povidone and 70% ethanol, and the surgical site was operated aseptically to initiate the operation. The inner skin of the knee joint was incised, and the fascia and joint capsule were incised, and then the kneecap was bent outward to expose the joint capsule. A cartilage defect with a diameter of 5 mm and a depth of 2 mm was created using a drill with a diameter of 1 mm in the medial part of the femur (center of the knee joint sulcus). After removing all the cartilage in the defect with a sofa, the joint capsule was sutured. Then, the subcutaneous tissue and skin were sutured and disinfected with povidone. 30 minutes after the cartilage was removed, 1x10 6 cells of normal (non-immortalized) stem cells (fADMSC), immortalized stem cells (im-fADMSC), 100 μL of normal culture medium (CM) or exosome-rich culture medium (ERCM) was injected into the joint cavity. After surgery, the antibiotic Foxolin (10 mg/kg) and the analgesic Maritrol (3 mg/kg) were intramuscularly injected twice daily for 3 days, and Foxolin was additionally treated once daily for 3 days.

(2) 퇴행성 관절염(연골결손) 치료효과(육안소견) 확인(2) Degenerative arthritis (cartilage defect) treatment effect (visual observation) confirmation

줄기세포 이식 4주 후에 부검하여 관절의 외측 femoral condyle의 손상 부위가 포함되도록 하여 관절면을 절단한 후 연골결손 부위의 변화, 연골결손 부위 표면상태, 결손 경계부위와 새롭게 생긴 조직과의 경계부의 연속성에 대한 육안관찰, 그리고 사진 촬영을 통한 연골재생 정도를 International Cartilage Repair Society (ICRS) scoring system에 따라 점수화하여 분석하였다(표 4).After 4 weeks of stem cell transplantation, an autopsy is performed to include the damaged part of the femoral condyle on the outside of the joint, and the joint surface is cut. The degree of cartilage regeneration through visual observation and photography was scored and analyzed according to the International Cartilage Repair Society (ICRS) scoring system (Table 4).

표 4: 연골조직 점수표[International Cartilage Repair Society (ICRS) scoring system]Table 4: Cartilage tissue scoring system [International Cartilage Repair Society (ICRS) scoring system]

시험 결과, 육안적인 결손연골 소견에서 생리식염수만을 투여한 군에서는 결손부위가 약간 차올라온 데 비해, 비불멸화 줄기세포 투여 동물에서는 상당히 복구되었으며, 특히 불멸화 줄기세포 이식한 동물에서는 결손연골이 온전하게 복구되었다(도 7). 더 나아가 줄기세포 대신 100 μL의 줄기세포 배양액을 투여했을 때에도 결손연골이 상당히 복구되었는 바, 엑소좀풍부배양액(ERCM)이 정상배양액(CM)보다 우수하였다. 이를 ICRS 점수로 정량화했을 때, 생리식염수 투여군에 비해 비불멸화 줄기세포는 약 2.2배, 불멸화 줄기세포는 약 3.1배, 정상배양액은 약 2.0배, 그리고 엑소좀풍부배양액은 약 2.9배 연골재생을 촉진시키는 것으로 확인되었다(도 8). As a result of the test, in the macroscopic findings of the defective cartilage, in the group administered only with physiological saline, the defect area was slightly filled up, whereas in the non-immortalized stem cell-treated animals, it was significantly restored. became (FIG. 7). Furthermore, even when 100 μL of stem cell culture medium was administered instead of stem cells, the defect cartilage was significantly restored, and the exosome-rich culture medium (ERCM) was superior to the normal culture medium (CM). When this was quantified by ICRS score, non-immortalized stem cells increased by about 2.2 times, immortalized stem cells by about 3.1 times, normal culture medium by about 2.0 times, and exosome-enriched culture medium by about 2.9 times compared to the saline-treated group. Cartilage regeneration was promoted. It was confirmed to do (FIG. 8).

(3) 퇴행성 관절염(연골결손) 치료효과(현미경소견) 확인(3) Degenerative arthritis (cartilage defect) treatment effect (microscopic findings) confirmed

관절조직은 10% 중성포르말린 용액으로 고정한 후 10% EDTA (pH 8.0) 용액으로 충분히 탈회시킨 다음 파라핀 절편을 제작하였다. 조직절편은 hematoxylin & eosin (H&E)으로 염색하여 연골구조를 확인하고, 현미경 영상분석 프로그램인 Image J 4.8v [연골재생부위/절단면 넓이(Regeneration tissue ratio)]의 비율 분석을 통해 연골재생 면적을 정량 분석하였다. 또한 Safranin O 염색을 통해 연골재생지표인 proteoglycans의 함량을 확인하였으며, 관절연골의 세포외기질(extracellular matrix, ECM) 중 50%를 차지하는 Collagen II에 대한 면역염색을 통해 조직학적으로 연골재생 정도를 확인하였다.The joint tissue was fixed with 10% neutral formalin solution, sufficiently demineralized with 10% EDTA (pH 8.0) solution, and then paraffin sections were prepared. Tissue sections were stained with hematoxylin & eosin (H&E) to confirm cartilage structure, and the area of cartilage regeneration was quantified by analyzing the ratio of [Regeneration tissue ratio] in Image J 4.8v, a microscope image analysis program. analyzed. In addition, the content of proteoglycans, which is an index of cartilage regeneration, was confirmed through Safranin O staining, and the degree of cartilage regeneration was confirmed histologically through immunostaining for Collagen II, which accounts for 50% of the extracellular matrix (ECM) of articular cartilage. did

시험 결과, 생리식염수만을 투여한 군에서는 결손부위가 확연히 관찰된 데 비해, 비불멸화 줄기세포 투여 동물에서는 연골구조(H&E), 연골재생(Safranin O) 및 세포외기질(Collagen II)에서 모두 상당한 회복효과를 보여 주었으며, 특히 불멸화 줄기세포 이식한 동물에서는 연골 및 기질의 재생으로 결손연골이 온전하게 복구되었다(도 9). 더 나아가 줄기세포 대신 100 μL의 줄기세포 배양액을 투여했을 때에도 상당한 연골재생이 확인되었는 바, 엑소좀풍부배양액(ERCM)이 정상배양액(CM)보다 우수하였다. 이를 Regeneration tissue ratio로 정량화했을 때, 생리식염수, 비불멸화 줄기세포, 불멸화 줄기세포, 정상배양액 및 엑소좀풍부배양액 투여군에서 각각 45%, 76%, 91%, 63% 및 88%로 불멸화 줄기세포 > 엑소좀풍부배양액 > 비불멸화 줄기세포 > 정상배양액의 순으로 우수한 연골재생 촉진효능을 보여 주었다(도 10).As a result of the test, the defect area was clearly observed in the group administered only with physiological saline, whereas significant recovery was observed in cartilage structure (H&E), cartilage regeneration (Safranin O), and extracellular matrix (Collagen II) in the non-immortalized stem cell-administered animals. In particular, in animals implanted with immortalized stem cells, defective cartilage was completely restored through regeneration of cartilage and matrix (FIG. 9). Furthermore, significant cartilage regeneration was confirmed even when 100 μL of the stem cell culture medium was administered instead of the stem cells, and the exosome-rich culture medium (ERCM) was superior to the normal culture medium (CM). When this was quantified by Regeneration tissue ratio, 45%, 76%, 91%, 63%, and 88% in physiological saline, non-immortalized stem cells, immortalized stem cells, normal culture medium, and exosome-rich culture medium administration group, respectively. Exosome-rich culture medium> non-immortalized stem cells> normal culture medium showed excellent cartilage regeneration promoting effect (FIG. 10).

실시예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.It was looked at based on the embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.

기탁기관명 : 한국생명공학연구원Name of Depositary Institution: Korea Research Institute of Bioscience and Biotechnology

수탁번호 : KCTC14390BPAccession number: KCTC14390BP

수탁일자 : 20201126Entrusted date: 20201126

Claims (10)

고양이지방줄기세포(feline adipose-derived mesenchymal stem cells, fADMSC)를 HPV16의 E6 및 E7 유전자로 구성되는 렌티바이러스 벡터(lentiviral vector)로 처리하여 얻은 불멸화 고양이줄기세포 또는 이의 배양액을 유효성분으로 포함하는 염증성 질환 치료용 약제학적 조성물로서,
상기 염증성 질환은 아토피 피부염 또는 류마티스 관절염인 약제학적 조성물.
Immortalized feline stem cells obtained by treating feline adipose-derived mesenchymal stem cells (fADMSC) with a lentiviral vector consisting of the E6 and E7 genes of HPV16 or an inflammatory agent containing the culture medium as an active ingredient As a pharmaceutical composition for the treatment of diseases,
The inflammatory disease is atopic dermatitis or rheumatoid arthritis pharmaceutical composition.
 제1항에 있어서,
상기 불멸화 고양이줄기세포는 기탁번호 KCTC 14390BP로 수탁된 것을 특징으로 하는, 약제학적 조성물.
According to claim 1,
The immortalized feline stem cells are characterized in that deposited under accession number KCTC 14390BP, pharmaceutical composition.
 제1항에 있어서,
상기 배양액은 불멸화 줄기세포를 함유한 배양액에 TNF-α를 처리하고, 1~5%의 저산소농도 조건 하에서 12~72시간 배양하여 수득하여 엑소좀이 풍부한 것을 특징으로 하는 것인, 약제학적 조성물.
According to claim 1,
The culture solution is obtained by treating TNF-α in the culture medium containing immortalized stem cells and culturing for 12 to 72 hours under conditions of 1-5% hypoxia, characterized in that the exosomes are rich. Pharmaceutical composition.
 제3항에 있어서,
상기 TNF-α는 3 내지 100 ng/mL의 농도로 처리되는 것을 특징으로 하는 것인, 약제학적 조성물.
According to claim 3,
The pharmaceutical composition, characterized in that the TNF-α is treated at a concentration of 3 to 100 ng / mL.
 삭제delete 삭제delete A) 고양이지방줄기세포(feline adipose-derived mesenchymal stem cells, fADMSC)를 HPV16의 E6 및 E7 유전자로 구성되는 렌티바이러스 벡터(lentiviral vector)로 처리하여 불멸화 고양이줄기세포를 얻는 단계;
B) 상기 불멸화 고양이줄기세포를 DMEM (Dulbecco's modified Eagle's medium) 배지에 접종하여 5%의 이산화탄소 공급 배양기에서 배양하는 단계; 및
C) 상기 배양 배지에 TNF-α를 첨가하고 1~5%의 저산소농도 조건 하에서 12~72시간 배양하는 단계를 포함하는 불멸화 고양이줄기세포 엑소좀 풍부 배양액의 제조 방법.
A) obtaining immortalized feline stem cells by treating feline adipose-derived mesenchymal stem cells (fADMSC) with a lentiviral vector composed of HPV16 E6 and E7 genes;
B) inoculating the immortalized feline stem cells in DMEM ( Dulbecco's modified Eagle's medium ) medium and culturing them in an incubator supplying 5% carbon dioxide; and
C) A method for producing an exosome-rich culture solution of immortalized feline stem cells comprising the step of adding TNF-α to the culture medium and culturing for 12 to 72 hours under a hypoxic condition of 1 to 5%.
 제7항에 있어서.
상기 불멸화 고양이줄기세포는 기탁번호: KCTC 14390BP로 수탁된 것을 특징으로 하는 것인, 방법.
According to claim 7.
The immortalized feline stem cells are characterized in that deposited under the accession number: KCTC 14390BP, the method.
 제7항에 있어서,
상기 TNF-α는 3 내지 100 ng/mL의 농도로 첨가되는 것을 특징으로 하는 것인, 방법.
According to claim 7,
The method, characterized in that the TNF-α is added at a concentration of 3 to 100 ng / mL.
 제7항에 있어서,
상기 불멸화 고양이줄기세포 엑소좀 풍부 배양액은 아토피성 피부염 및 류마티스 관절염 억제 효과가 우수한 것을 특징으로 하는 것인, 방법.
According to claim 7,
The method, characterized in that the immortalized feline stem cell exosome-rich culture medium has an excellent inhibitory effect on atopic dermatitis and rheumatoid arthritis.
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