KR20230006413A - Composition for promoting skin regeneration, comprising human trophoblast cell derivatives as an active ingredient - Google Patents
Composition for promoting skin regeneration, comprising human trophoblast cell derivatives as an active ingredient Download PDFInfo
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- KR20230006413A KR20230006413A KR1020220107789A KR20220107789A KR20230006413A KR 20230006413 A KR20230006413 A KR 20230006413A KR 1020220107789 A KR1020220107789 A KR 1020220107789A KR 20220107789 A KR20220107789 A KR 20220107789A KR 20230006413 A KR20230006413 A KR 20230006413A
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- human trophoblast
- culture medium
- mesenchymal stem
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- stem cells
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Abstract
Description
본 발명은 인간영양막세포(Trophoblasts) 유래물에 관한 것으로, 보다 상세하게는, 태반 속 융모막 세포층 중 영양막세포로부터 제조한 인간영양막세포 배양액 및 유래물의 피부재생 및 피부상처 치유 촉진 효능 등에 관한 것이다.The present invention relates to human trophoblasts-derived products, and more particularly, to human trophoblast cultures prepared from trophoblasts in the chorion cell layer in the placenta and skin regeneration and skin wound healing promoting efficacy of the derivatives.
양막(amnion membrane)은 태반을 구성하고 있는 가장 안쪽에 있는 막으로, 양수와 태아를 둘러싸고 있어 태아를 외부의 병원균으로부터 보호하고 양수와 태아의 생체항상성을 유지하여 태아가 정상적으로 성장/발달할 수 있는 환경을 조성하는 역할을 한다. 1910년 Davis가 처음 양막을 생체 재료로 사용한 후(Davis JW et al., Johns Hopkins Hosp Rep, 15; 307, 1910) 화상, 창상 및 안과 수술 후 상처 치유의 목적으로 널리 사용되고 있다(Faulk WP et al., The Lancet, 315(8179):1156-1158, 1980, Mohammadi AA et al., Burns, 39(2):349-353, 2013, Dua HS et, al., Survey of Ophthalmology, 49(1):51-77, 2004). 양막은 표피세포 성장인자 (epidermal growth factor, EGF), 섬유아세포 성장인자 (fibroblast growth factor, FGF), 전환 성장인자 (transforming growth factor, TGF-b), 신경 성장인자 (nerve growth factor, NGF) 및 간장 성장인자 (hepatic growth factor, HGF)등을 포함하고 있는 것으로 알려져 있으며 그 자체로 콜라겐으로 구성된 세포외 기질과 상피세포로 구성되어 있다. 또한, 항염증 (anti-inflammation), 항균 (anti-bacterial) 성분을 포함하고 있으며 혈관생성 조절 성분 (angiogenic modulatory properties)들이 존재한다(Hao Y et al., Cornea, 19(3):348-352, 2000). The amnion membrane is the innermost membrane that makes up the placenta. It surrounds the amniotic fluid and the fetus to protect the fetus from external pathogens and maintains the homeostasis of the amniotic fluid and the fetus so that the fetus can grow/develop normally. It plays a role in shaping the environment. After Davis first used amnion as a biomaterial in 1910 (Davis JW et al., Johns Hopkins Hosp Rep, 15; 307, 1910), it has been widely used for the purpose of wound healing after burns, wounds, and ophthalmic surgeries (Faulk WP et al. ., The Lancet, 315(8179):1156-1158, 1980, Mohammadi AA et al., Burns, 39(2):349-353, 2013, Dua HS et, al., Survey of Ophthalmology, 49(1) :51-77, 2004). Amniotic membrane contains epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor (TGF-b), nerve growth factor (NGF) and It is known to contain hepatic growth factor (HGF), etc., and is itself composed of an extracellular matrix composed of collagen and epithelial cells. In addition, it contains anti-inflammation and anti-bacterial components and has angiogenic modulatory properties (Hao Y et al., Cornea, 19(3):348-352 , 2000).
엑소좀 (exosome)은 세포밖 소포체, 세포외 소포 (EVs, extracellular vesicles), 분비성 미세소포 등으로 알려졌으며 세포 간 정보교환을 위해 세포들이 외부 환경으로 분비하는 나노 크기의 소포체이다(Thery C et al., J Extracel Vesic. 2018;7(1):1535750.) 엑소좀은 단백질, 지질, 핵산, 대사물질 등 생물학적 활성을 보이는 다양한 물질들을 포함하여 이러한 기능적 수하물을 운반하는 매개체로 세포 간 소통의 역할을 수행하는 것으로 알려졌다. 최근에는 엑소좀 내 miRNA가 생체 내 중요한 기능을 수행하는 것으로 알려지기도 하였다(Yanez-Mo M et al., J Extracel Vesic. 2015;4:27066, Colombo M et al., Ann Rev Cell Dev Biol. 2014;30:255-89, Valadi H et al., Nat Cell Biol. 2007;9(6):654-9). 엑소좀은 다양한 체액에서도 발견되어 질병의 진단에도 사용되고 있으며, 비교적 안정적으로 제조와 보관이 가능하여 진단 및 치료제로 개발되고 있다.Exosomes are known as extracellular vesicles, extracellular vesicles (EVs), secretory microvesicles, etc., and are nano-sized vesicles secreted by cells into the external environment to exchange information between cells (Thery C et al. al., J Extracel Vesic. 2018;7(1):1535750.) Exosomes are carriers of functional baggage, including various biologically active substances such as proteins, lipids, nucleic acids, and metabolites, and are involved in cell-to-cell communication. known to play a role. Recently, miRNAs in exosomes have been known to perform important functions in vivo (Yanez-Mo M et al., J Extracel Vesic. 2015;4:27066, Colombo M et al., Ann Rev Cell Dev Biol. 2014 30:255-89, Valadi H et al., Nat Cell Biol. 2007;9(6):654-9). Exosomes are found in various bodily fluids and are used for diagnosis of diseases, and are being developed as diagnostic and therapeutic agents because they can be manufactured and stored relatively stably.
중간엽줄기세포 (Mesenchymal stem cells, MSCs)는 성체줄기세포로 수정란이나 난자를 이용하지 않기 때문에 윤리적 논란에서 자유로워 활용도가 높고, 자가 중간엽줄기세포를 세포치료제 및 이식에 사용할 경우 면역거부반응을 줄일 수 있다. 줄기세포의 종류를 중간엽줄기세포(MSC), 조혈줄기세포(HSC), 신경줄기세포(NSC), 배아유래줄기세포(ES-derived cells) 등으로 구분하여 임상연구 비율을 분석하였을 때, 중간엽줄기세포가 69%로 전체 임상연구의 최대 다수를 차지하고 있었고, 조혈줄기세포가 13%, 신경줄기세포가 4% 이었다. 이렇듯 배아줄기세포 연구와는 다르게 중간엽줄기세포 치료법은 상당수가 이미 임상 활용 단계에 와 있으며, 해외를 비롯한 많은 연구소들의 성체줄기세포 연구는 이미 파킨슨병, 연골손상, 소경, 암, 척수손상 등 60종 이상의 병을 치료한 결과가 발표되었고 현재 세계적으로 약 300종의 병에 대해서도 임상시험이 진행 중이다. 우리나라도 중간엽줄기세포를 이용하여 뇌졸중치료제, 파킨슨병 진행 억제, 퇴행성 관절염 치료, 간경변 줄기세포 신약 개발, 뼈, 연골 관련 질환 치료, 혈액 관련 질환, 심혈관계 질환 등 여러 임상 분야에 적용되어 활발하게 진행되고 있다. 하지만, 중간엽줄기세포는 여러 조직으로부터 획득은 가능하나, 얻을 수 있는 세포의 양이 매우 적을 뿐만 아니라 수명이 짧아 미분화 능력을 잃어버리며 퇴화되어 버리는 치명적인 단점이 있다. 또한, 체외 상태에서 배양 시 증식이 매우 느려 임상에 이용할 만큼의 양을 확보하기가 어렵고, 체외 배양 시 사용되는 동물유래 혈청 (Fetal Bovine Serum, FBS)으로 임상 적용에 한계점이 있는 실정이다. Mesenchymal stem cells (MSCs) are adult stem cells that are free from ethical controversies and highly usable because they do not use fertilized eggs or eggs. can be reduced When the clinical research ratio was analyzed by dividing the types of stem cells into mesenchymal stem cells (MSC), hematopoietic stem cells (HSC), neural stem cells (NSC), and embryo-derived stem cells (ES-derived cells), mesenchymal Stem cells accounted for the largest majority of all clinical studies at 69%, followed by hematopoietic stem cells at 13% and neural stem cells at 4%. Unlike embryonic stem cell research, many of the mesenchymal stem cell therapies are already in the clinical application stage, and adult stem cell research by many research institutes, including overseas, has already been carried out for Parkinson's disease, cartilage damage, blindness, cancer, spinal cord injury, etc. 60 The results of treating more than three types of diseases have been published, and clinical trials are currently underway for about 300 types of diseases worldwide. In Korea, mesenchymal stem cells are also actively applied to various clinical fields such as stroke treatment, Parkinson's disease progression inhibition, degenerative arthritis treatment, liver cirrhosis stem cell drug development, bone and cartilage related disease treatment, blood related disease, cardiovascular disease, etc. It's going on. However, although mesenchymal stem cells can be obtained from various tissues, the amount of cells that can be obtained is very small and have a fatal disadvantage in that they lose their undifferentiated ability and degenerate due to their short lifespan. In addition, proliferation is very slow during in vitro culture, and it is difficult to secure an amount sufficient for clinical use, and there are limitations in clinical application as animal-derived serum (Fetal Bovine Serum, FBS) used for in vitro culture.
인체의 연조직과 체중을 지탱해주고 내부기관을 둘러싸서 내부 장기를 외부의 충격으로부터 보호해 주는 뼈는 근육이나 장기를 구조적으로 지탱할 뿐만 아니라 체내의 칼슘이나 다른 필수 무기질 즉 인이나 마그네슘과 같은 물질을 저장하는 인체의 중요한 부분 중 하나이다. 성장이 끝난 성인의 뼈는 오래된 뼈는 제거하고 새로운 뼈로 대체하는 생성과 흡수 과정을 지속적으로 반복 재생하면서 균형을 유지하게 되는데, 이를 골재형성(bone remodeling)이라고 한다(Yamaguchi A. et al., Tanpakushitsu Kakusan Koso., 50(6Suppl);664-669, 2005). 이러한 뼈의 순환은 성장과 스트레스에 의해 일어나는 뼈의 미세한 손상을 회복시키고 그 기능을 유지하는데 필수적이다. 성인에서는 매년 골격의 약 10-30%가 골흡수-골형성의 리모델링을 통하여 재형성된다.Bone, which supports the body's soft tissues and weight and protects internal organs from external shocks by surrounding internal organs, not only structurally supports muscles and organs, but also stores calcium and other essential minerals in the body, such as phosphorus and magnesium. is one of the most important parts of the human body. The bone of an adult after growth is maintained in balance by continuously repeating the process of generation and absorption of removing old bone and replacing it with new bone, which is called bone remodeling (Yamaguchi A. et al., Tanpakushitsu). Kakusan Koso., 50(6Suppl);664-669, 2005). This bone circulation is essential for restoring fine damage to bone caused by growth and stress and maintaining its function. In adults, approximately 10-30% of the skeleton is remodeled each year through bone resorption-osteogenesis remodeling.
골재형성은 뼈를 생성하는 조골세포(osteoblast) 및 뼈를 파괴하는 파골세포(osteoclast)가 관여하며 서로 밀접한 연관성으로 골의 항상성이 유지된다. 예를 들어, 조골세포는 RANKL(receptor activator of nuclear factor-_ligand), 및 그의 유도 수용체인 OPG(osteoprotegerin)과 같은 물질의 분비를 통해 골흡수를 담당하는 파골세포의 분화를 조절함으로써 체내의 골 항상성을 유지한다. 이러한 골의 항상성이 물리적 영향, 호르몬 체계 등에 의해 유지되지 않은 경우 및 골조직 손상시 동반되는 골질환의 치료와 관련하여 과거에는 주로 골 무기질, 즉 칼슘과 인의 대사 이상만을 중심으로 연구가 진행되어 왔으며, 그 기전 규명에 있어서 진전을 보지 못하였다. 일반적으로, 골다공증의 치료 및 예방을 위하여 칼슘이 함유된 식이요법이 추천되며, 폐경기의 여성들에게는 에스트로겐 또는 비타민 D 투여가 추천되고 있다. 그 외에, 비스포스포네이트(bisphosphonate) 계열 약물이 파골세포를 억제하고 사멸을 유도하는 골흡수 억제제로서 새로운 대체 치료제로 주목받고 있다. 그러나, 골질환 치료제로 널리 사용되는 칼슘보강제는 부갑상선 호르몬의 분비를 억제하며 골흡수에 의한 골량 감소를 방지하나, 골량 유지의 개인 차이가 심한 것으로 알려져 있다(Heandy R.P. principles of bone biology, Academic press, 1007-1017, 1996). 또한, 에스트로겐이나 칼시토닌을 이용한 호르몬 요법의 경우 골밀도를 증가시키고 직장암의 발생을 감소시킨다는 보고도 있으나, 유방암, 심근경색, 정맥 혈전증 등의 부작용이 보고된 바 있다(Nelson, H.D et al., JAMA, 288:872-881, 2002; Lemay, A., J.Obstet. Bynaecol. Can., 24:711-7152-3). 또한, 비스포스포네이트 제제의 경우, 최근 복용하는 환자에서 턱뼈의 괴사, 중증 심방세동, 뼈나 관절의 무력화 또는 근골격의 통증이 발생하는 사례가 해마다 증가되고 있다(Coleman RE., Br J Cancer, 98:1736-1740(2008). 따라서 부작용 발현이 적으면서도 효과적으로 골흡수를 억제할 수 있는 새로운 골질환 치료제의 개발이 요구되고 있는 실정이다.Osteoblasts that produce bone and osteoclasts that destroy bone are involved in bone remodeling, and bone homeostasis is maintained in close association with each other. For example, osteoblasts maintain bone homeostasis in the body by regulating the differentiation of osteoclasts responsible for bone resorption through the secretion of substances such as RANKL (receptor activator of nuclear factor-_ligand) and its induced receptor, OPG (osteoprotegerin). keep In the past, in relation to the treatment of bone diseases that accompany bone tissue damage and when bone homeostasis is not maintained by physical influences or hormonal systems, studies have been conducted mainly focusing only on abnormalities in bone mineral metabolism, that is, calcium and phosphorus. No progress has been made in identifying the mechanism. In general, a diet containing calcium is recommended for the treatment and prevention of osteoporosis, and estrogen or vitamin D administration is recommended for postmenopausal women. In addition, bisphosphonate-based drugs are attracting attention as new alternative treatments as bone resorption inhibitors that inhibit osteoclasts and induce apoptosis. However, calcium supplements, which are widely used as a treatment for bone diseases, suppress the secretion of parathyroid hormone and prevent bone mass loss due to bone resorption, but it is known that individual differences in bone mass maintenance are severe (Heandy R.P. principles of bone biology, Academic press, 1007-1017, 1996). In addition, there are reports that hormone therapy using estrogen or calcitonin increases bone density and reduces the incidence of rectal cancer, but side effects such as breast cancer, myocardial infarction, and venous thrombosis have been reported (Nelson, H.D et al., JAMA, 288:872-881, 2002; Lemay, A., J. Obstet. Bynaecol. Can., 24:711-7152-3). In addition, in the case of bisphosphonate preparations, cases of jawbone necrosis, severe atrial fibrillation, bone or joint incapacity, or musculoskeletal pain are increasing year by year in patients who have recently taken them (Coleman RE., Br J Cancer, 98:1736- 1740 (2008) Therefore, there is a demand for the development of new therapeutic agents for bone diseases that can effectively inhibit bone resorption while having fewer side effects.
본 발명은 전술한 종래기술의 문제점을 해결하기 위한 것으로, 중간엽줄기세포의 수급과 생체 외 배양 조건은 대량 생산을 위한 제품화 과정과 임상 적용에서의 문제를 가지고 있었다. 이에, 본 발명자들은 태반 속 융모막 세포층 중 하나인 영양막으로부터 획득되는 세포의 배양액을 이용하여 중간엽줄기세포의 증식 및 FBS와 같은 동물유래물질 배재 조건 하의 체외배양 조건을 확립하였다. The present invention is to solve the above-mentioned problems of the prior art, and the supply and demand of mesenchymal stem cells and in vitro culture conditions had problems in the commercialization process for mass production and clinical application. Accordingly, the present inventors established in vitro culture conditions under conditions for proliferation of mesenchymal stem cells and exclusion of animal-derived substances such as FBS using a culture medium of cells obtained from the trophoblast, one of the chorionic cell layers in the placenta.
또한, 인간영양막세포의 배양액 및 이의 유래물에서 피부 및 골재생 촉진효과를 확인하여 이를 유효성분으로 하는 조성물을 제공한다. In addition, by confirming the effect of promoting skin and bone regeneration in the culture medium of human trophoblast cells and its derivatives, a composition containing it as an active ingredient is provided.
그러나, 본 발명이 해결하고자 하는 과제는 이상에서 언급한 과제로 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 해당 기술분야의 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the problem to be solved by the present invention is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.
본 발명의 일 구체예에 따르면, 인간 영양막 세포 배양액 및 이의 유래물로 이루어진 군으로부터 선택되는 어느 하나 이상을 유효성분으로 포함하는 피부재생 촉진용 조성물이 제공된다. According to one embodiment of the present invention, a composition for promoting skin regeneration comprising at least one selected from the group consisting of human trophoblast cell culture medium and derivatives thereof as an active ingredient is provided.
일 측에 따르면, 상기 피부재생 촉진용 조성물은 인간중간엽줄기세포를 더 포함할 수 있다. According to one side, the composition for promoting skin regeneration may further include human mesenchymal stem cells.
일 측에 따르면, 상기 피부재생 촉진은 피부 결손부위 또는 피부 상처의 회복을 촉진하는 것일 수 있다. According to one side, the promotion of skin regeneration may be to promote recovery of skin defects or skin wounds.
본 발명의 또 다른 실시예에 따르면, 상기 피부재생 촉진용 조성물 중 어느 하나를 포함하는 화장료 조성물이 제공된다. According to another embodiment of the present invention, a cosmetic composition comprising any one of the compositions for promoting skin regeneration is provided.
일 측에 따르면 상기 화장료 조성물은 스킨, 스킨 소프트너, 스킨토너, 아스트린젠트, 로션, 밀크로션, 모이스쳐로션, 영양로션, 마사지크림, 영양크림, 아이 크림, 모이스쳐 크림, 핸드크림, 에센스, 영양에센스, 팩, 클렌징폼, 클렌징 워터, 클렌징 로션, 클렌징 크림, 바디로션, 바디클렌져, 비누 또는 파우더로 제형화되는 것을 특징으로 하는 것일 수 있다. According to one side, the cosmetic composition is skin, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrient lotion, massage cream, nutrient cream, eye cream, moisture cream, hand cream, essence, nutrient essence, It may be formulated into a pack, cleansing foam, cleansing water, cleansing lotion, cleansing cream, body lotion, body cleanser, soap or powder.
본 발명의 또 다른 구체예에 따르면, According to another embodiment of the present invention,
(a)인간영양막세포를 체외배양하는 단계;(a) culturing human trophoblast cells in vitro;
(b)상기 배양된 인간영양막세포를 포함하는 용액을 원심분리하여 상층액을 수거하는 단계; 및(b) collecting the supernatant by centrifuging the solution containing the cultured human trophoblast cells; and
(c)상기 수거된 상층액을 다공성 필터를 이용하여 멸균 정제하여 배양액을 수득하는 단계;(c) obtaining a culture solution by sterilizing and purifying the collected supernatant using a porous filter;
를 포함하는, 피부재생 촉진용 조성물의 제조방법이 제공된다. Containing, there is provided a method for producing a composition for promoting skin regeneration.
일 측에 따르면, 상기 (a)단계의 상기 체외배양은 50 내지 100%의 세포밀도(confluency)로 48 내지 72시간 배양 후 배양액을 교체하는 것일 수 있다. According to one side, the in vitro culture in step (a) may be to replace the culture medium after culturing for 48 to 72 hours at a cell density (confluency) of 50 to 100%.
일 측에 따르면, 상기 (c)단계의 다공성 필터의 공극지름은 0.2μm이하일 수 있다. According to one side, the pore diameter of the porous filter of step (c) may be 0.2 μm or less.
일 측에 따르면, According to one side,
상기 단계 이후에, After the above step,
(d) 300G에서 10분, 2,000G에서 10분, 10,000G에서 30분, 100,000G에서 70분동안 원심분리하는 단계; 및(d) centrifugation at 300G for 10 minutes, 2,000G for 10 minutes, 10,000G for 30 minutes, and 100,000G for 70 minutes; and
(e) 상기 원심분리를 동일하게 1회 더반복하는 단계;(e) repeating the same centrifugation one more time;
를 더 포함할 수 있다. may further include.
본 발명의 인간영양막세포 배양액 및 유래물은 줄기세포 증식 촉진효과를 가져 동물유래물질인 FBS가 없는 조건에서의 인간 중간엽줄기세포의 체외배양을 가능하게 한다. The human trophoblast cell culture medium and derivatives of the present invention have a stem cell proliferation promoting effect, enabling in vitro culture of human mesenchymal stem cells in the absence of animal-derived FBS.
또한, 본 발명의 인간영양막세포 유래물은 조골세포 분화와 골재생 촉진효과를 가져 골질환의 예방 또는 치료용 조성물로 이용될 수 있고, 피부재생 촉진효과를 가져 피부재생 촉진 또는 피부상처 치료용 조성물로 이용될 수 있다. In addition, the human trophoblastic cell-derived product of the present invention has an effect of promoting osteoblast differentiation and bone regeneration, so that it can be used as a composition for preventing or treating bone diseases, and has an effect of promoting skin regeneration, so that it can be used as a composition for promoting skin regeneration or treating skin wounds. can be used as
본 발명의 효과는 상기한 효과로 한정되는 것은 아니며, 본 발명의 상세한 설명 또는 청구범위에 기재된 발명의 구성으로부터 추론 가능한 모든 효과를 포함하는 것으로 이해되어야 한다.The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the detailed description of the present invention or the configuration of the invention described in the claims.
도 1은 제조 프로토콜에 따른 NTA(Nanoparticle Tracking Analysis)분석결과와 골재생 효능에 대한 평가이다.
도 2는 FBS 존재하에 인간영양막세포 배양액을 중간엽줄기세포 증식단계에 처리한 결과이다.
도 3은 FBS 부재하에 인간영양막세포 배양액을 중간엽줄기세포 증식단계에 처리한 결과이다.
도 4는 FBS 부재하에 인간영양막세포 배양액 처리군과 중간엽줄기세포 배양액 처리군의 세포분포도 및 모양을 확인한 것이다.
도 5는 인간영양막세포의 줄기세포 유지 관련 유전자의 발현을 나타낸 것이다.
도 6은 인간영양막세포와 중간엽줄기세포의 체외배양한 모습과 나노입자 분석결과를 나타낸 것이다.
도 7은 FBS 부재하에 인간영양막세포 배양액과 중간엽줄기세포 배양액의 중간엽줄기세포 증식효능을 비교한 것이다.
도 8은 중간엽줄기세포 배양액, 인간영양막세포 배양액 및 인간영양막세포 유래물의 항노화 효능을 SA-β-GAL염색법을 적용하여 나타낸 것이다.
도 9는 FBS 부재하에 도 8과 동일한 조건에서의 항노화 효능을 SA-
도 10은 마우스 창상부위에 인간영양막세포 배양액을 처리하여 일자에 따라 창상회복 정도를 나타낸 것이다.
도 11은 마우스 창상치유 실험에서 조직염색법을 통해 상피세포층과 진피층의 두께가 증가함을 확인한 것이다.
도 12는 인간영양막세포 배양액의 골분화 촉진 효과를 ALP활성도 및 칼슘침착도로 나타낸 것이다.
도 13은 인간영양막세포 배양액을 처리한 경우의 미네랄 침착도 평가를 알리자린 레드 에스 염색 결과로 나타낸 것이다.
도 14는 인간영양막세포 배양액을 처리함에 따라 골재생 관련 유전자의 발현 변화를 확인한 것이다.
도 15는 인간영양막세포 배양액을 처리하여 중간엽줄기세포를 골분화 유도배양하고 RNA 시퀀싱 분석한 결과를 나타낸 것이다.
도 16은 인간영양막세포 배양액 처리에 따른 세포신호전달계(KEGG pathway) 분석결과를 나타낸 것이다.
도 17은 증가된 MAPK 신호전달계를 웨스턴블롯으로 확인한 것이다.
도 18은 신호전달계 중 BMP2의 발현은 증가하고 GREM1의 발현이 감소됨을 확인한 것이다.
도 19는 인간영양막세포 배양액 처리에 따라 사이토카인 연계 신호전달계의 활성 변화를 비교한 것이다.
도 20은 인간영양막세포 배양액의 지방분화 억제효과를 확인한 사진 및 지방분화 인자 발현정도를 표시한 것이다.
도 21은 인간영양막세포 유래물의 골분화 촉진 효과를 ALP활성도 및 칼슘침착도로 확인한 것이다.
도 22는 인간영양막세포 유래물과 배양액을 함께 사용하는 경우의 골분화 촉진효과가 상승됨을 ALP 활성도를 통해 확인한 것이다.
도 23은 인간영양막세포 유래물의 미네랄 침착도 평가를 알리자린 레드 에스 염색을 통해 나타낸 것이다.
도 24는 인간영양막세포 유래물로 골분화 촉진시 골재생 관련 전사인자 유전자의 발현을 측정한 것이다.
도 25는 인간영양막세포 유래물의 골재생 효능을 동물실험으로 검증하는 microCT 결과이다.
도 26은 상기 동물실험에서 H&E 및 MT 염색방법을 이용한 조직학적 분석결과이다.
도 27은 상기 동물실험에서 골재생 마커 인자 발현을 형광분석법으로 평가한 결과이다.
도 28은 인간영양막세포 유래물(엑소좀)의 miRNA를 분석한 결과이다.
도 29는 인간영양막세포 유래물(엑소좀)에서 더 높게 발현되는 miRNA의 표적유전자들을 나타낸 것이다.
도 30은 ClueGO bioinformatic tool로 분석한 신호전달계를 나타낸 것이다.
도 31은 DAVID bioinformatic tool로 인간영양막세포 유래물(엑소좀)에서 발현되는상위 1%의 miRNA를 분석한 결과이다.
도 32는 상기 도 31과 같은 분석을 string과 ClueGO bioinformatic tool로 수행한 결과이다.
도 33은 인간영양막세포 유래물(엑소좀)과 중간엽줄기세포의 엑소좀 내의 miRNA의 역할을 도식화한 것이다.
도 34는 인간 산모 태반으로부터 영양막세포를 분리하는 과정이다.
도 35는 분리된 세포를 체외배양한 결과를 나타낸다.
도 36은 배양 72시간 후의 다핵세포화와 인간영양막세포 특이적 마커 단백질인 시토케라틴7의 발현을 보여준다.
도 37은 일차 인간영양막세포 배양액을 중간엽줄기세포 배양시 처리한 결과를 나타낸다.
도 38은 일차 인간영양막세포 배양액을 중간엽줄기세포 배양액 및 인간영양막세포 배양액과 줄기세포 증식효능을 비교실험한 것이다.
도 39는 FBS 부재하에 일차 인간영양막세포 배양액의 중간엽줄기세포의 증식 촉진 효능을 확인한 것이다.
도 40은 일차 인간영양막세포 배양액의 골재생 효능을 ALP활성화, 칼슘침착도 및 RUNX2 유전자 발현 변화로 확인한 결과이다. Figure 1 is an evaluation of the NTA (Nanoparticle Tracking Analysis) analysis results and bone regeneration efficacy according to the manufacturing protocol.
Figure 2 shows the result of treating the human trophoblast cell culture medium in the mesenchymal stem cell proliferation step in the presence of FBS.
Figure 3 shows the result of treating the human trophoblast cell culture medium in the mesenchymal stem cell proliferation step in the absence of FBS.
Figure 4 confirms the cell distribution and shape of the human trophoblast cell culture medium-treated group and the mesenchymal stem cell culture medium-treated group in the absence of FBS.
5 shows the expression of stem cell maintenance-related genes in human trophoblast cells.
6 shows the in vitro culture of human trophoblast cells and mesenchymal stem cells and the results of nanoparticle analysis.
Figure 7 compares the mesenchymal stem cell proliferation efficacy of human trophoblast cell culture medium and mesenchymal stem cell culture medium in the absence of FBS.
8 shows the anti-aging efficacy of the mesenchymal stem cell culture medium, the human trophoblast cell culture medium, and the human trophoblast cell-derived product by applying the SA-β-GAL staining method.
Figure 9 shows the anti-aging efficacy in the same conditions as in Figure 8 in the absence of FBS SA-
Figure 10 shows the degree of wound recovery according to the date of treatment of human trophoblast cell culture medium on mouse wounds.
11 confirms that the thickness of the epithelial cell layer and the dermal layer increases through tissue staining in mouse wound healing experiments.
12 shows the bone differentiation promoting effect of the human trophoblast cell culture medium in terms of ALP activity and calcium deposition.
13 shows the results of alizarin red S staining of mineral deposition in the case of treatment of human trophoblast cell culture medium.
14 confirms changes in the expression of bone regeneration-related genes as the human trophoblast cell culture medium is treated.
15 shows the results of RNA sequencing analysis after culturing osteogenic differentiation of mesenchymal stem cells by treating human trophoblast cell culture medium.
16 shows the results of KEGG pathway analysis according to the treatment of human trophoblast cell culture medium.
Figure 17 confirms the increased MAPK signaling pathway by Western blot.
18 confirms that the expression of BMP2 increases and the expression of GREM1 decreases in the signal transduction system.
Figure 19 compares changes in the activity of the cytokine-linked signaling pathway according to the treatment of human trophoblast cell culture medium.
20 is a photograph confirming the adipogenesis inhibitory effect of the human trophoblast cell culture medium and displays the expression level of adipogenic factors.
21 confirms the bone differentiation promoting effect of human trophoblast cell-derived products by ALP activity and calcium deposition.
22 confirms through ALP activity that the bone differentiation promoting effect is increased when the human trophoblast cell-derived product and the culture medium are used together.
23 shows the evaluation of mineral deposition in human trophoblast cell-derived products through alizarin red S staining.
24 is a measurement of the expression of transcription factor genes related to bone regeneration when bone differentiation is promoted with human trophoblast cell-derived products.
25 shows microCT results verifying the bone regeneration efficacy of human trophoblast cell-derived products through animal experiments.
26 is a histological analysis result using H&E and MT staining methods in the animal experiment.
27 is a result of evaluating bone regeneration marker expression in the animal experiment by fluorescence analysis.
28 is a result of analyzing miRNAs derived from human trophoblast cells (exosomes).
29 shows target genes of miRNAs that are more highly expressed in human trophoblast cell-derived material (exosomes).
30 shows the signal transduction system analyzed by the ClueGO bioinformatic tool.
Figure 31 is the result of analyzing the top 1% of miRNAs expressed in human trophoblast cell-derived material (exosomes) with DAVID bioinformatic tool.
32 is a result of performing the same analysis as in FIG. 31 with string and ClueGO bioinformatic tool.
33 is a schematic diagram of the roles of miRNAs in human trophoblast cell-derived (exosomes) and exosomes of mesenchymal stem cells.
34 is a process of separating trophoblast cells from human maternal placenta.
35 shows the results of in vitro culture of the isolated cells.
36 shows multinucleated cells after 72 hours of culture and expression of
37 shows the results of treatment of primary human trophoblast cell culture medium during mesenchymal stem cell culture.
38 is a comparison experiment of primary human trophoblast cell culture medium with mesenchymal stem cell culture medium and human trophoblast cell culture medium and stem cell proliferation efficacy.
39 confirms the proliferation promoting effect of mesenchymal stem cells in the primary human trophoblast cell culture medium in the absence of FBS.
40 shows the results of confirming the bone regeneration efficacy of the primary human trophoblast cell culture medium through changes in ALP activation, calcium deposition, and RUNX2 gene expression.
이하에서, 첨부된 도면을 참조하여 실시예들을 상세하게 설명한다. 각 도면에 제시된 동일한 참조 부호는 동일한 부재를 나타낸다.Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. Like reference numerals in each figure indicate like elements.
아래 설명하는 실시예들에는 다양한 변경이 가해질 수 있다. 아래 설명하는 실시예들은 실시 형태에 대해 한정하려는 것이 아니며, 이들에 대한 모든 변경, 균등물 내지 대체물을 포함하는 것으로 이해되어야 한다.Various changes may be made to the embodiments described below. The embodiments described below are not intended to be limiting on the embodiments, and should be understood to include all modifications, equivalents or substitutes thereto.
실시예에서 사용한 용어는 단지 특정한 실시예를 설명하기 위해 사용된 것으로, 실시예를 한정하려는 의도가 아니다. 단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한, 복수의 표현을 포함한다. 본 명세서에서, "포함하다" 또는 "가지다" 등의 용어는 명세서 상에 기재된 특징, 숫자, 단계, 동작, 구성 요소, 부품 또는 이들을 조합한 것이 존재함을 지정하려는 것이지, 하나 또는 그 이상의 다른 특징들이나 숫자, 단계, 동작, 구성 요소, 부품 또는 이들을 조합한 것들의 존재 또는 부가 가능성을 미리 배제하지 않는 것으로 이해되어야 한다.Terms used in the examples are used only to describe specific examples, and are not intended to limit the examples. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "include" or "have" are intended to designate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features It should be understood that the presence or addition of numbers, steps, operations, components, parts, or combinations thereof is not precluded.
다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 실시예가 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥 상 가지는 의미와 일치하는 의미를 가지는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다.Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiment belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in the present application, they should not be interpreted in an ideal or excessively formal meaning. don't
또한, 첨부 도면을 참조하여 설명함에 있어, 도면 부호에 관계없이 동일한 구성 요소는 동일한 참조 부호를 부여하고 이에 대한 중복되는 설명은 생략하기로 한다. 실시예를 설명함에 있어서 관련된 공지 기술에 대한 구체적인 설명이 실시예의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다.In addition, in the description with reference to the accompanying drawings, the same reference numerals are given to the same components regardless of reference numerals, and overlapping descriptions thereof will be omitted. In describing the embodiment, if it is determined that a detailed description of a related known technology may unnecessarily obscure the gist of the embodiment, the detailed description will be omitted.
본 발명의 일 실시예에 따르면, 인간 영양막 세포 배양액 및 이의 유래물로 이루어진 군으로부터 선택되는 어느 하나 이상을 포함하는, 중간엽 줄기세포 증식 촉진용 조성물이 제공된다. According to one embodiment of the present invention, a composition for promoting mesenchymal stem cell proliferation is provided, comprising at least one selected from the group consisting of human trophoblast cell culture medium and derivatives thereof.
본 명세서에서 사용되는 용어 "인간영양막세포"는 인간 태반의 일 구성부분으로서, 보다 구체적으로는 배포 외부에 위치한 배자의 외배엽층으로, 난자를 자궁벽에 부착시키며 배자에 영양분을 공급하는 조직을 의미한다. 이것에서 융모막 및 양막이 유래하며, 영양막의 내세포층은 융모를 덮으며, 이를 세포영양막이라 부른다. 상기 인간영양막세포는 시판되는 세포를 이용할 수 있고 또는 인간 산모의 출산 후 분리된 태반으로부터 추출되어 제공될 수 있다. As used herein, the term "human trophoblast" refers to a component of the human placenta, more specifically, the ectodermal layer of the embryo located outside the distribution, which attaches the egg to the uterine wall and supplies nutrients to the embryo. . From this, the chorion and amnion are derived, and the inner cell layer of the trophoblast covers the villi, which is called the cytotrophoblast. The human trophoblast cells may be commercially available cells or may be provided by being extracted from the placenta separated after childbirth of a human mother.
본 명세서에서 사용되는 용어 “인간영양양막세포 배양액”은 상기 인간영양막세포를 체외배양한 배양액을 의미한다. 바람직하게는 항생제 및 FBS의 존재 하에 세포성장배지에서 체외배양될 수 있으며 구체적인 제조방법은 후술하는 바와 같다. 상기 인간영양막세포 배양액은 하기 인간영양막세포 유래물을 포함할 수 있다. As used herein, the term “human trophoblast culture medium” refers to a culture medium in which the human trophoblast cells are cultured in vitro. Preferably, it can be cultured in vitro in a cell growth medium in the presence of antibiotics and FBS, and a specific manufacturing method is as described below. The human trophoblast cell culture medium may include the following human trophoblast cell-derived products.
일 측에 따르면, 상기 중간엽 줄기세포 증식 촉진용 조성물은, 동물혈청을 포함하지 않을 수 있다. 상기 동물혈청을 포함하지 않는 것이란 바람직하게는 FBS를 포함하지 않는 것을 의미하며, FBS의 부재 하에서도 인간중간엽줄기세포의 증식을 촉진할 수 있다. 본 명세서에서 사용된 용어 "FBS"는 소태아 혈청을 말하며, FBS는 세포생장에 필수적인 성장인자, 시토카인, 단백질 등이 다양하게 포함되어 줄기세포용 배지 첨가제로 널리 활용되고 있다. 하기 실시예 2, 실시예 3-2 및 실시예 6-2에서 검증하는 바와 같이, FBS가 부존재하는 조건에서도 상기 조성물은 중간엽 줄기세포의 증식을 촉진하는 효과를 가진다. According to one side, the composition for promoting mesenchymal stem cell proliferation may not contain animal serum. Not including the animal serum preferably means not including FBS, and the proliferation of human mesenchymal stem cells can be promoted even in the absence of FBS. As used herein, the term "FBS" refers to fetal bovine serum, and FBS contains various growth factors, cytokines, and proteins essential for cell growth, and is widely used as a medium additive for stem cells. As verified in Examples 2, 3-2 and 6-2 below, the composition has an effect of promoting the proliferation of mesenchymal stem cells even in the absence of FBS.
일 측에 따르면, 상기 유래물은 엑소좀을 포함할 수 있다. 본 명세서에서 사용된 용어 "인간영양막세포 유래물"은 인간 영양막 세포가 포함하거나 외부로 분비하는 막성 소포체로서 내부에 지질, 핵산, 단백질, 대사물질 등 생물학적 활성을 보이는 다양한 물질을 포함하는 것을 의미한다. 상기 유래물은 엑소좀, 미세소포체, 세포외소포체, 마이크로베지클 또는 마이크로파티클 중 선택되는 어느 하나 이상일 수 있으나 이에 제한되지 않으며, 본 명세서에서는 “유래물”과 “엑소좀”을 서로 같은 의미로써 교환적으로 사용될 수 있다. 상기 유래물은 바람직하게는 중간엽줄기세포의 증식을 촉진하는 성분 및 골분화 유도된 중간엽 줄기세포가 골세포로 분화하는 것을 촉진하는 성분 중 어느 하나 이상을 포함할 수 있으며, 더욱 바람직하게는 has-miR-1246, has-miR-1290, has-miR-423-5p 및 has-let-7b-5p로 이루어진 군에서 선택되는 어느 하나 이상의 miRNA를 포함할 수 있다. According to one side, the derivative may include exosomes. As used herein, the term "human trophoblast-derived product" refers to membranous vesicles that human trophoblast cells contain or secrete to the outside, and contain various substances exhibiting biological activity such as lipids, nucleic acids, proteins, and metabolites therein. . The derivative may be any one or more selected from exosomes, microvesicles, extracellular vesicles, microvesicles, or microparticles, but is not limited thereto. can be used interchangeably. The derivative may preferably include any one or more of a component that promotes the proliferation of mesenchymal stem cells and a component that promotes the differentiation of mesenchymal stem cells induced to osteodifferentiation into bone cells, and more preferably It may include one or more miRNAs selected from the group consisting of has-miR-1246, has-miR-1290, has-miR-423-5p, and has-let-7b-5p.
일 측에 따르면, 상기 중간엽 줄기세포는 골수, 탯줄 및 지방으로 이루어지는 군에서 선택되는 어느 하나에서 유래한 것일 수 있으며, 전분화능 줄기세포로 부터 분화된 것일 수 있다. 본 명세서에서 사용된 용어 "인간 중간엽줄기세포"는 인간의 다분화능을 가진 기질세포로, 제한적이지만 다양한 분화능을 가지는 줄기세포를 말하며, 유래 조직에 의해 한정되지 않으나, 바람직하게는 골수, 탯줄 및 지방으로 이루어지는 군에서 선택되는 어느 하나에서 유래한 인간중간엽줄기세포일 수 있다. 또한, 본 발명의 중간엽 줄기세포가 전분화능 줄기세포로 부터 분화된 것인 경우 상기 전분화능 줄기세포는 배아줄기세포(ESC)일 수 있으며, 인간 유도만능 줄기세포(iPSC)일 수 있다. 하기 실시예 3-2에서 검증하는 바와 같이, 본 발명의 상기 중간엽 줄기세포의 증식 촉진용 조성물은 골수, 탯줄 및 지방 유래의 중간엽 줄기세포 모두에 대하여 증식을 촉진하는 효과가 존재하였다. According to one side, the mesenchymal stem cells may be derived from any one selected from the group consisting of bone marrow, umbilical cord and fat, and may be differentiated from pluripotent stem cells. As used herein, the term "human mesenchymal stem cell" refers to stem cells having a limited but diverse differentiation potential as human stromal cells with pluripotency, and is not limited by the tissue of origin, but is preferably bone marrow, umbilical cord and It may be a human mesenchymal stem cell derived from any one selected from the group consisting of fat. In addition, when the mesenchymal stem cells of the present invention are differentiated from pluripotent stem cells, the pluripotent stem cells may be embryonic stem cells (ESC) or human induced pluripotent stem cells (iPSC). As verified in Example 3-2 below, the composition for promoting proliferation of mesenchymal stem cells of the present invention had an effect of promoting proliferation of all mesenchymal stem cells derived from bone marrow, umbilical cord and adipose tissue.
일 측에 따르면, 상기 인간 영양막 세포 배양액은 전체 조성물의 20 내지 60 부피 %로 포함될 수 있다. 실시예 3-2에서 검증하듯 상기 조성에서 본 발명의 인간영양막세포 배양액은 중간엽줄기세포 배양액보다 중간엽줄기세포의 증식을 촉진하는 효과가 우수하였다. According to one side, the human trophoblast cell culture medium may be included in 20 to 60% by volume of the total composition. As verified in Example 3-2, the human trophoblast cell culture medium of the present invention was more effective in promoting the proliferation of mesenchymal stem cells than the mesenchymal stem cell culture medium in the above composition.
본 발명의 다른 실시예에 따르면, According to another embodiment of the present invention,
(a) 세포배양배지에 중간엽 줄기세포를 배양하는 단계;(a) culturing mesenchymal stem cells in a cell culture medium;
(b) 중간엽 줄기세포에 인간 영양막 세포 배양액 및 이의 유래물로 이루어진 군으로부터 선택되는 어느 하나 이상을 포함하는 조성물을 처리하는 단계; 및(b) treating mesenchymal stem cells with a composition comprising at least one selected from the group consisting of human trophoblast cell culture medium and derivatives thereof; and
(c) 적어도 24시간 이상 연속하여 배양하는 단계;를 포함하는, 중간엽 줄기세포의 증식을 촉진하는 방법이 제공된다. (c) continuously culturing for at least 24 hours or more; including, a method for promoting the proliferation of mesenchymal stem cells is provided.
일 측에 따르면, 상기 (a)단계 및 (c) 단계에서의 배양은 동물혈청을 포함하지 않는 배지에서 이루어질 수 있다.According to one side, the culture in steps (a) and (c) may be performed in a medium that does not contain animal serum.
본 발명에 따른 인간영양막세포 배양액 또는 이의 유래물을 함유하는 조성물이 동물혈청이 부존재하는 환경에서도 중간엽줄기세포의 증식을 촉진할 수 있음은 상술한 바와 같다. As described above, the composition containing the human trophoblast cell culture medium or a derivative thereof according to the present invention can promote the proliferation of mesenchymal stem cells even in the absence of animal serum.
본 발명의 또 다른 실시예에 따르면, According to another embodiment of the present invention,
(a)인간영양막세포를 체외배양하는 단계;(a) culturing human trophoblast cells in vitro;
(b)상기 배양된 인간영양막세포를 포함하는 용액을 원심분리하여 상층액을 수거하는 단계; 및(b) collecting the supernatant by centrifuging the solution containing the cultured human trophoblast cells; and
(c)상기 수거된 상층액을 다공성 필터를 이용하여 멸균 정제하여 배양액을 수득하는 단계; 를 포함하는, 중간엽 줄기세포 증식 촉진용 조성물의 제조방법이 제공된다. 본 명세서에서 사용된 용어 "체외배양"은 인체 및 여하의 동물의 체외에서 배양되는 것을 의미하며, 특정한 배양 조건으로 한정되지 않으나 바람직하게는 항생제와 배지첨가제 하에 진행될 수 있다. 더욱 바람직하게는 1% 항생제의 존재 하에 배양될 수 있다. (c) obtaining a culture solution by sterilizing and purifying the collected supernatant using a porous filter; There is provided a method for producing a composition for promoting mesenchymal stem cell proliferation comprising a. As used herein, the term "in vitro culture" refers to culture outside the body of a human body or any animal, and is not limited to specific culture conditions, but may preferably be carried out under antibiotics and medium additives. More preferably, it may be cultured in the presence of 1% antibiotics.
일 측에 따르면, 상기 (a)단계의 체외배양은 바람직하게는 50 내지 100%의 세포밀도(confluency)로 48 내지 72시간 배양 후 배양액을 교체하는 것일 수 있다. 바람직하게는 50%의 세포밀도로 72시간 배양 후 배양액을 교체하고 48시간 후에 배양액을 수거하여 이루어질 수 있다. According to one side, the in vitro culture of step (a) may be to replace the culture medium after culturing for 48 to 72 hours, preferably at a cell density (confluency) of 50 to 100%. Preferably, it can be achieved by replacing the culture medium after culturing for 72 hours at a cell density of 50% and collecting the culture medium after 48 hours.
일 측에 따르면, 상기 (c)단계의 다공성 필터의 공극지름은 0.2μm이하일 수 있다.According to one side, the pore diameter of the porous filter of step (c) may be 0.2 μm or less.
일 측에 따르면, 상기 단계 이후에, According to one side, after the above step,
(d) 300G에서 10분, 2,000G에서 10분, 10,000G에서 30분, 100,000G에서 70분동안 원심분리하는 단계; 및(d) centrifugation at 300G for 10 minutes, 2,000G for 10 minutes, 10,000G for 30 minutes, and 100,000G for 70 minutes; and
(e) 상기 원심분리를 동일하게 1회 더 반복하는 단계;(e) repeating the same centrifugation one more time;
를 더 포함할 수 있다.may further include.
바람직하게는, 상기의 원심분리를 통해 하부에 침전된 침전물(pellet)을 수득할 수 있다. Preferably, it is possible to obtain a precipitate (pellet) precipitated at the bottom through the above centrifugation.
본 발명의 또 다른 실시예에 따르면, 상기 제조방법 중 어느 하나의 제조방법으로 제조된, 중간엽 줄기세포 증식 촉진용 조성물이 제공된다. According to another embodiment of the present invention, a composition for promoting mesenchymal stem cell proliferation, prepared by any one of the above manufacturing methods, is provided.
본 발명의 일 실시예에 따르면, 인간 영양막 세포 배양액 및 이의 유래물로 이루어진 군으로부터 선택되는 어느 하나 이상을 포함하는 중간엽 줄기세포의 골분화 촉진용 조성물이 제공된다. 본 발명에서 개시하는 인간영양막세포 배양액과 그 유래물은 단독으로 사용되는 경우에도 중간엽 줄기세포의 골분화를 촉진하는 효과를 가지며, 하기 실시예 5-1에서 검증하는 바와 같이 배양액과 유래물을 함께 처리하는 경우 상승효과를 가질 수 있다.According to one embodiment of the present invention, there is provided a composition for promoting bone differentiation of mesenchymal stem cells comprising at least one selected from the group consisting of human trophoblast cell culture medium and derivatives thereof. The human trophoblast cell culture medium and its derivatives disclosed in the present invention have an effect of promoting the osteogenic differentiation of mesenchymal stem cells even when used alone, and as verified in Example 5-1 below, the culture medium and derivatives When treated together, it can have a synergistic effect.
본 명세서에서 사용된 용어 “골분화 촉진”이란 골분화가 유도된 중간엽 줄기세포가 골세포로 분화하는 것을 촉진하는 것을 말한다. 상기 골분화의 유도는 바람직하게는 중간엽줄기세포를 골분화 유도 배지(OIM)에 처리하여 이루어질 수 있다. 인간영양막세포 배양액 또는 유래물을 포함하는 조성물은 중간엽줄기세포의 골분화를 촉진하여 높은 ALP활성도 및 미네랄 침착도를 보여줄 수 있으며, 우수한 조골세포 분화 촉진 및 골형성 촉진 효능을 가질 수 있다. As used herein, the term “stimulation of bone differentiation” refers to promoting the differentiation of mesenchymal stem cells in which bone differentiation is induced into osteocytes. The induction of osteogenic differentiation may preferably be performed by treating the mesenchymal stem cells in an osteogenic differentiation induction medium (OIM). A composition containing human trophoblast cell culture medium or derivatives can promote bone differentiation of mesenchymal stem cells to show high ALP activity and mineral deposition, and can have excellent osteoblast differentiation promotion and osteogenesis promoting effects.
상기 ALP활성도는 조골세포의 초기 분화 및 골형성 지표로서, ALP(Alkaline Phosphatase)효소가 활성화된 정도를 나타낸다. 바람직하게는 아나스펙, Inc, Fermont, Canada의 ALP 키트를 사용하여 405 ㎚에서 측정될 수 있다. The ALP activity is an indicator of early differentiation and bone formation of osteoblasts, and indicates the degree to which ALP (Alkaline Phosphatase) enzyme is activated. Preferably at 405 nm using an ALP kit from Anaspec, Inc, Fermont, Canada.
상기 미네랄 침착도는 조골세포의 중후반 분화의 마커로 널리 이용되는 알리자린 레드 에스(Alizarin Red S) 염색으로 평가될 수 있다. 바람직하게는 Millipore 사의 염색액을 제조사의 지침에 따라 사용하여 측정할 수 있다. The degree of mineral deposition can be evaluated by Alizarin Red S staining, which is widely used as a marker for mid-late differentiation of osteoblasts. Preferably, it can be measured using Millipore's staining solution according to the manufacturer's instructions.
일 측에 따르면, 상기 인간 영양막 세포 배양액은 전체 조성물의 10 내지 40부피 %로 포함될 수 있다. 하기 실시예 4에서 검증하듯이, 인간영양막세포 배양액의 상기 조성에서 골분화 유도된 중간엽줄기세포의 골세포로의 분화를 촉진하는 효과를 가졌다. According to one side, the human trophoblast cell culture medium may be included in 10 to 40% by volume of the total composition. As verified in Example 4 below, the composition of the human trophoblast cell culture medium had an effect of promoting the differentiation of osteogenic induced mesenchymal stem cells into osteocytes.
일 측에 따르면, 상기 유래물은 엑소좀을 포함할 수 있고, 상기 유래물은 1Ⅹ108 내지 1Ⅹ109 입자/mL로 포함될 수 있다. 상기 “인간영양막세포 유래물” 및 “엑소좀”에 관한 설명은 앞서 설명한 바와 같으므로 생략한다. 실시예 5에서 검증하듯이, 인간영양막세포 유래물이 1Ⅹ108 내지 1Ⅹ109 입자/mL로 포함되는 경우 골분화 촉진 효과가 우수하였다. According to one side, the derivative may include an exosome, and the derivative may be included in 1Ⅹ10 8 to 1Ⅹ10 9 particles/mL. Descriptions of the “human trophoblast cell-derived product” and “exosome” are omitted as they have been described above. As verified in Example 5, the bone differentiation promoting effect was excellent when the human trophoblast cell-derived product was included in an amount of 1Ⅹ10 8 to 1Ⅹ10 9 particles/mL.
본 발명의 또 다른 실시예에 따르면, According to another embodiment of the present invention,
(a)인간영양막세포를 체외배양하는 단계;(a) culturing human trophoblast cells in vitro;
(b)상기 배양된 인간영양막세포를 포함하는 용액을 원심분리하여 상층액을 수거하는 단계; 및(b) collecting the supernatant by centrifuging the solution containing the cultured human trophoblast cells; and
(c)상기 수거된 상층액을 다공성 필터를 이용하여 멸균 정제하여 배양액을 수득하는 단계; 를 포함하는, 중간엽 줄기세포의 골분화 촉진용 조성물의 제조방법이 제공된다. (c) obtaining a culture solution by sterilizing and purifying the collected supernatant using a porous filter; There is provided a method for producing a composition for promoting bone differentiation of mesenchymal stem cells, comprising a.
일 측에 따르면, 상기 단계 이후에, According to one side, after the above step,
(d) 상기 배양액에 엑소좀 단리 용액을 처리하는 단계; 및(d) treating the culture medium with an exosome isolation solution; and
(e) 상기 처리된 용액을 원심분리하여 엑소좀을 수득하는 단계;를 더 포함할 수 있다.(e) obtaining exosomes by centrifuging the treated solution; may further include.
상기 엑소좀 단리 용액은 당 업계에 공지된 모든 종류의 단리방법에 사용되는 것일 수 있으나 바람직하게는 엑소좀을 석출하기 위한 용액일 수 있으며, 시판되는 엑소좀 단리 용액을 이용할 수 있다. The exosome isolation solution may be used in all kinds of isolation methods known in the art, but preferably may be a solution for precipitating exosomes, and a commercially available exosome isolation solution may be used.
본 발명의 또 다른 구체예에 따르면, 상기 방법 중 어느 하나의 방법으로 제조된, 중간엽 줄기세포의 골분화 촉진용 조성물이 제공된다. According to another embodiment of the present invention, a composition for promoting bone differentiation of mesenchymal stem cells prepared by any one of the above methods is provided.
본 발명의 일 구체예에 따르면, 인간 영양막 세포 배양액 및 이의 유래물로 이루어진 군으로부터 선택되는 어느 하나 이상을 포함하는 골질환의 예방 또는 치료용 약학적 조성물이 제공된다. According to one embodiment of the present invention, a pharmaceutical composition for preventing or treating bone disease comprising at least one selected from the group consisting of human trophoblast cell culture medium and derivatives thereof is provided.
본 발명에서 사용되는 용어 “예방”이란 본 발명에 따른 약학적 조성물의 투여에 의해 골질환 또는 피부질환을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.The term "prevention" used in the present invention refers to any action that suppresses or delays the onset of a bone disease or skin disease by administration of the pharmaceutical composition according to the present invention.
본 발명에서 사용되는 용어 “치료”란 본 발명에 따른 약학적 조성물의 투여에 의해 골질환,피부질환 또는 피부상처부위의 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.As used herein, the term “treatment” refers to any activity that improves or beneficially changes symptoms of bone disease, skin disease or skin wound by administration of the pharmaceutical composition according to the present invention.
본 발명에서 사용되는 용어 “개체” 란 골 질환의 발생 위험도가 높거나 골 질환 또는 피부질환이 발생한 포유동물로서 야생동물과 가축을 가리지 않으며, 바람직하게는 인간일 수 있다.The term "individual" used in the present invention is a mammal with a high risk of bone disease or having a bone disease or skin disease, regardless of wild or domestic animals, preferably humans.
일 측에 따르면, 상기 조성물은 1Ⅹ108 내지 1Ⅹ109 입자/mL로 상기 유래물을 포함할 수 있다.According to one side, the composition may include the derivative in an amount of 1Ⅹ10 8 to 1Ⅹ10 9 particles/mL.
일 측에 따르면, 상기 골질환은 골결손, 골다공증, 골다공증성 골절, 당뇨성 골절, 불유합골절, 골형성 부전증, 골연화증 및 이로 인한 골절, 골형성장애, 퇴행성 골질환 및 부정교합으로 이루어진 군으로부터 선택되는 어느 하나일 수 있다. According to one side, the bone disease is selected from the group consisting of bone defect, osteoporosis, osteoporotic fracture, diabetic fracture, nonunion fracture, osteogenesis imperfecta, osteomalacia and resulting fracture, osteogenesis disorder, degenerative bone disease and malocclusion can be any one of them.
일 측에 따르면, 상기 조성물은 골분화 유도된 중간엽줄기세포의 골세포로의 분화를 촉진시키는 것일 수 있다. 따라서, 상기 조성물은 골분화가 유도된 중간엽줄기세포가 이용되는 줄기세포치료방법에 있어서, 중간엽줄기세포의 골분화를 촉진하여 치료효과를 개선하기 위한 용도로 사용될 수 있다. According to one aspect, the composition may promote the differentiation of osteogenic mesenchymal stem cells into osteocytes. Therefore, the composition can be used for improving the therapeutic effect by promoting bone differentiation of mesenchymal stem cells in a stem cell treatment method using mesenchymal stem cells in which bone differentiation is induced.
일 측에 따르면, 상기 조성물은 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸, 외용제, 좌제 또는 멸균 주사용 액으로 제형화될 수 있다. According to one side, the composition may be formulated as a powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions.
상기 약학적 조성물은 약학적으로 허용되는 담체를 더 포함할 수 있다. 이러한 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 상기 약학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다.The pharmaceutical composition may further include a pharmaceutically acceptable carrier. Such carriers are commonly used in formulations, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, and polyvinylpyrrolidone. , cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, but are not limited thereto. The pharmaceutical composition may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components.
상기 약학적 조성물은 비경구 투여가 바람직하고, 예컨대 정맥내 투여, 복강내 투여, 종양내 투여, 근육내 투여, 피하 투여, 또는 국부 투여를 이용하여 투여할 수 있다.The pharmaceutical composition is preferably administered parenterally, and may be administered using, for example, intravenous administration, intraperitoneal administration, intratumoral administration, intramuscular administration, subcutaneous administration, or local administration.
상기 약학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 질병 증상의 정도, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 보통으로 숙련된 의사는 목적하는 치료에 효과적인 투여량을 용이하게 결정 및 처방할 수 있다.The suitable dosage of the pharmaceutical composition varies depending on factors such as formulation method, administration method, patient's age, weight, sex, severity of disease symptoms, food, administration time, administration route, excretion rate and reaction sensitivity, A ordinarily skilled physician can readily determine and prescribe effective dosages for the desired treatment.
상기 약학적 조성물은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화됨으로써 단위 용량 형태로 제조되거나 또는 다용량 용기내에 내입시켜 제조될 수 있다. 이 때, 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical composition is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art, or It can be prepared by placing it in a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet, or capsule, and may additionally contain a dispersing agent or a stabilizer.
본 발명의 일 실시예에 따르면, 인간 영양막 세포 배양액 및/또는 이의 유래물을 개체에 투여하는 단계를 포함하는 골질환 치료방법으로서, 상기 개체는 골 질환이 발생한 인간을 제외한 포유동물인 골질환의 치료방법이 제공된다. According to one embodiment of the present invention, a method for treating a bone disease comprising administering a human trophoblast cell culture medium and/or a derivative thereof to a subject, wherein the subject is a mammal other than a human having a bone disease, and the bone disease is treated. Treatment is provided.
일 측에 따르면, 상기 치료방법은 중간엽줄기세포를 개체에 투여하는 단계를 추가로 포함하고, 상기 중간엽줄기세포는 골분화가 유도된 중간엽줄기세포이거나 상기 치료방법은 중간엽줄기세포와 함께 중간엽줄기세포의 골분화 유도 물질을 개체에 투여하는 것일 수 있다. According to one side, the treatment method further comprises the step of administering mesenchymal stem cells to a subject, wherein the mesenchymal stem cells are mesenchymal stem cells in which bone differentiation is induced, or the treatment method is performed together with mesenchymal stem cells. It may be to administer an osteogenic differentiation-inducing substance of mesenchymal stem cells to a subject.
본 발명의 상기 조성물이 골분화 유도된 중간엽줄기세포가 골세포로 분화되는 것을 촉진하는 효과를 가지고 있음은 상술한 바와 같다. 바람직하게는 줄기세포치료에 있어서, 골분화 유도된 중간엽 줄기세포를 투여하거나 또는 중간엽 줄기세포와 골분화 유도물질을 함께 투여하는 경우 이와 동시에 또는 순차로 본 발명의 상기 조성물을 투여하여 치료효과를 개선할 수 있다. As described above, the composition of the present invention has an effect of promoting the differentiation of mesenchymal stem cells induced by osteogenic differentiation into osteocytes. Preferably, in stem cell therapy, when the osteogenic differentiation-induced mesenchymal stem cells are administered or the mesenchymal stem cells and the osteogenic differentiation-inducing substance are administered together, the composition of the present invention is administered simultaneously or sequentially to achieve a therapeutic effect. can improve
일 측에 따르면, 상기 치료방법은 골분화 유도된 중간엽줄기세포를 개체에 투여하는 단계를 추가로 포함하고, 상기 중간엽줄기세포의 투여는 상기 배양액 및/또는 유래물의 투여와 동시에 또는 순차로 이루어지는 것이고, 순차로 투여되는 경우 그 선/후는 무방하나 바람직하게는 중간엽줄기세포의 투여 후 인간 영양막 세포 배양액 및/또는 이의 유래물을 투여하는 것일 수 있다. According to one aspect, the treatment method further comprises the step of administering osteogenic differentiation-induced mesenchymal stem cells to a subject, and the administration of the mesenchymal stem cells is simultaneously or sequentially with the administration of the culture medium and / or derivatives. When administered sequentially, it may be administered before/after, but preferably, human trophoblast cell culture medium and/or derivatives thereof may be administered after administration of mesenchymal stem cells.
일 측에 따르면, 상기 치료방법에서 개체는 CT 촬영으로 골질환 발생이 확인된 개체일 수 있으며, 상기 치료방법은 상기 투여 단계 이후에 상기 골질환이 발생한 환부의 CT 촬영을 수행하는 단계를 추가로 포함할 수 있다.According to one side, in the treatment method, the subject may be an individual whose occurrence of bone disease has been confirmed by CT imaging, and the treatment method further includes the step of performing CT imaging of the affected area where the bone disease occurs after the administration step. can include
본 발명의 상기 조성물에 의한 치료효과는 조골세포의 골형성과 관련되어 확인될 수 있다. 하기 실시예 5-4의 동물실험에서는 인위적으로 골결손을 유발한 후 본 발명의 조성물에 의해 결손부위가 회복되는 것으로 검증하였다. 본 발명의 조성물 처리에 따른 치료효과는 처리 전후 CT촬영을 통한 육안비교, 조직염색법을 통한 육안비교 및 골재생 마커인 OCN(osteocalcin) 발현량 비교를 통해 확인될 수 있으나 이에 제한되는 것은 아니다. The therapeutic effect of the composition of the present invention can be confirmed in relation to bone formation of osteoblasts. In the animal experiments of Examples 5-4 below, it was verified that the bone defect was artificially induced and then the defect was recovered by the composition of the present invention. The therapeutic effect of treatment with the composition of the present invention can be confirmed through visual comparison through CT imaging before and after treatment, visual comparison through tissue staining, and OCN (osteocalcin) expression level comparison, which is a bone regeneration marker, but is not limited thereto.
본 발명의 일 측에 따르면, 인간 영양막 세포 배양액 및 이의 유래물로 이루어진 군으로부터 선택되는 어느 하나 이상을 포함하는 피부재생 촉진용 조성물이 제공된다. According to one aspect of the present invention, a composition for promoting skin regeneration comprising at least one selected from the group consisting of human trophoblast cell culture medium and derivatives thereof is provided.
상기 “피부재생” 이란 상처부위의 회복, 노화된 진피/표피 세포의 대체, 피부질환의 치료 및 탈락된 부위의 재생 등을 포함할 수 있으며, 바람직하게는 창상과 같이 외부의 물리적 자극으로 인해 소실된 피부층의 수복을 의미한다. 하기 실시예 3-4에서 검증하는 바와 같이, 본 발명의 조성물은 창상부위의 피부를 재생하는 효과가 존재하였다. The term "skin regeneration" may include recovery of a wound, replacement of aged dermal/epidermal cells, treatment of skin disease, and regeneration of an exfoliated area, and is preferably lost due to an external physical stimulus such as a wound. means the restoration of the damaged skin layer. As verified in Examples 3-4 below, the composition of the present invention had an effect of regenerating the skin of the wound site.
일 실시예에 따르면, 상기 조성물은 인간중간엽줄기세포를 더 포함할 수 있다. 인간중간엽줄기세포를 피부 상처 부위에 처리하는 경우에도 피부재생 촉진 효과를 보이나, 본 발명의 인간영양막세포 배양액 및 유래물로 이루어진 군으로부터 선택되는 어느 하나 이상을 포함하는 조성물과 함께 사용되는 경우 더 우수한 피부재생 촉진 효과가 있음을 실시예 3-4에서 검증하였다. According to one embodiment, the composition may further include human mesenchymal stem cells. Even when human mesenchymal stem cells are treated on skin wounds, skin regeneration promoting effects are shown, but when used with a composition containing at least one selected from the group consisting of human trophoblast cell culture medium and derivatives of the present invention, it is more It was verified in Examples 3-4 that there was an excellent skin regeneration promoting effect.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명하기로 한다. 하기 실시예는 본 발명을 예시하기 위한 목적으로 기술된 것으로서, 본 발명의 범위가 이에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. The following examples are described for the purpose of illustrating the present invention, but the scope of the present invention is not limited thereto.
실시예 1: 인간영양막세포 배양액(Conditioned Medium, CM) 및 유래물(Extracellular Vesicles, EV)제조 프로토콜 확립Example 1: Human Trophoblast Cell Culture Media (Conditioned Medium, CM) and Derivatives (Extracellular Vesicles, EV) Manufacturing Protocol Establishment
인간영양막세포는 ATCC로부터 구매하여 RPMI1640 배양액에 1% 항생제, 5% 엑소좀 디플리티드 FBS(exosome depleted FBS)를 넣어 체외 배양하였다. 인간영양막세포는 태반 속 융모막으로부터 분리하여 사용할 수 있으며, 이에 대한 방법은 이미 알려져 있다 (Lucas Sagrillo-Fagundes et al., Journal of Visualized Experiments 2016). Human trophoblasts were purchased from ATCC and cultured in vitro by adding 1% antibiotics and 5% exosome depleted FBS to RPMI1640 culture medium. Human trophoblast cells can be isolated and used from the chorion in the placenta, and methods for this are already known (Lucas Sagrillo-Fagundes et al., Journal of Visualized Experiments 2016).
실시예 1-1: 골재생 효과 확인용 인간영양막세포 배양액 및 유래물의 제조조건Example 1-1: Production conditions of human trophoblast cell culture medium and derivatives for confirming the bone regeneration effect
골재생 효과 확인용 인간영양막세포 배양액 및 유래물의 제조 조건은 다음과 같다. 1.5 x 106 세포로 75cm2 배양 컨테이너에서 72시간 배양 후 (50%세포밀도) 엑소좀 제거된 FBS(exosome depleted FBS)를 포함하는 배양액으로 교체한다. 이후 48시간 경과 후 상기 배양액을 수거하여 이를 인간영양막세포 배양액으로 사용하였다. 인간영양막세포 유래물은 상기 배양액으로부터 제조하였으며, 엑소좀 분리 용액을 사용하거나 초원심분리 방법으로 제조하엿다. 엑소좀 분리 용액을 사용하는 경우, 인간영양막세포 배양액 10mL에 2mL의 엑소퀵(Exoquick) 용액 (SBI 제조)을 섞어서 (5:1) 하룻밤 동안 4°C에서 엑소좀이 분리되도록 한 후 원심분리기를 이용하여 엑소좀을 수집하였다. 초원심분리 방법으로 제조하는 경우, 1) 300G에서 10분, 2) 2,000G에서 10분, 3) 10,000G에서 30분, 4) 100,000G에서 70분의 과정을 두 번 반복하여 제조하였다. 분리된 엑소좀은 1/10 또는 1/20으로 희석하여 NTA 장비를 이용하여 사이즈와 농도를 분석하였다. Conditions for preparing the human trophoblast cell culture medium and derivatives for confirming the bone regeneration effect are as follows. After culturing for 72 hours (50% cell density) in a 75 cm 2 culture container with 1.5 x 10 6 cells, the culture medium containing exosome depleted FBS is replaced. After 48 hours, the culture medium was collected and used as a human trophoblast cell culture medium. The human trophoblast cell-derived product was prepared from the above culture medium, using an exosome separation solution or by ultracentrifugation. When using the exosome isolation solution, mix 2mL of Exoquick solution (manufactured by SBI) with 10mL of human trophoblast cell culture medium (5:1), allow exosomes to separate overnight at 4°C, and then centrifuge. Exosomes were collected using When prepared by the ultracentrifugation method, 1) 300G for 10 minutes, 2) 2,000G for 10 minutes, 3) 10,000G for 30 minutes, 4) 100,000G for 70 minutes, and the process was repeated twice. The separated exosomes were diluted 1/10 or 1/20 and analyzed for size and concentration using NTA equipment.
실시예 1-2: 줄기세포증식 및 피부재생 효과 확인용 인간영양막세포 배양액 및 유래물의 제조 방법Example 1-2: Preparation method of human trophoblast cell culture medium and derivatives for confirming stem cell proliferation and skin regeneration effects
중간엽 줄기세포 증식 및 피부재생 효과 확인용 인간영양막세포 배양액 및 유래물의 제조 조건은 다음과 같다. 75cm2 배양 컨테이너에서 1.5 x 106 세포를 배양하여 90% 세포밀도(confluency)가 되도록 한 후, PBS로 2회 세척하였다. FBS가 존재하지 않는 DMEM F12 또는 MEM에 1% 페니실린/스트렙토마이신을 첨가한 배양액에서 24시간 배양 후 수거하여 이를 인간영양막세포 배양액으로 사용하였다. 인간영양막세포 유래물은 초원심분리방법으로 엑소좀을 수집하였으며, 1) 300G에서 10분, 2) 2,000G에서 10분, 3) 10,000G에서 30분, 4) 100,000G에서 70분의 과정을 두 번 반복하여 제조하였다. 분리된 엑소좀은 1/10 또는 1/20으로 희석하여 NTA 장비를 이용하여 사이즈와 농도를 분석하였다. Conditions for preparing the human trophoblast cell culture medium and derivatives for confirming mesenchymal stem cell proliferation and skin regeneration effects are as follows. After culturing 1.5 x 10 6 cells in a 75 cm 2 culture container to reach 90% confluency, they were washed twice with PBS. After culturing for 24 hours in a culture medium in which 1% penicillin/streptomycin was added to DMEM F12 or MEM without FBS, it was collected and used as a human trophoblast cell culture medium. Human trophoblast cell-derived exosomes were collected by ultracentrifugation, 1) 300G for 10 minutes, 2) 2,000G for 10 minutes, 3) 10,000G for 30 minutes, 4) 100,000G for 70 minutes. It was prepared in duplicate. The separated exosomes were diluted 1/10 or 1/20 and analyzed for size and concentration using NTA equipment.
실시예 1-3: 최적프로토콜 확립Example 1-3: Establishment of optimal protocol
세포 유래물 수집에 있어 최적의 세포밀도 및 배양시간을 파악하기 위하여 다음과 같이 실험하였다. 50% 이상, 100% 이하의 세포밀도(confluency)에서 48시간 배양하여 수급된 세포유래물(엑소좀 포함)을 프로토콜 1로, 100%초과의 세포밀도에서 72시간 배양하여 수급된 세포유래물을 프로토콜 2로 나누었다. NTA 분석 후 중간엽줄기세포의 골분화 유도 시 처리하여 초기 골분화 마커인 ALP활성도를 평가하였다. 프로토콜 1과 2에서 비슷한 세포유래물 입자(particle)의 분포도가 나타났으나 차이도 확인되었다. 프로토콜 1의 세포유래물 입자 분포도가 넓게 나타나며, 프로토콜 2는 대부분 50~350nm의 입자가 분석되었다. ALP 활성도 평가에서는 프로토콜 1에서 유의적 결과를 얻을 수 있었다. 프로토콜에 따른 NTA 분석 결과와 골재생 효능에 대한 평가 결과는 도 1에 나타내었다.In order to determine the optimal cell density and incubation time for cell-derived collection, the following experiments were conducted. Cell-derived products (including exosomes) obtained by culturing for 48 hours at a cell density of 50% or more and 100% or less were cultured for 72 hours at a cell density of more than 100
실시예 2: 인간영양막세포 배양액의 중간엽줄기세포의 증식 촉진 효능 확인Example 2: Confirmation of the proliferation promoting effect of mesenchymal stem cells in human trophoblast cell culture medium
본 발명에서 사용한 인간 중간엽줄기세포는 골수유래 중간엽줄기세포로 promocell로부터 구입하였으며, 5%(v/v) FBS(Fetal bonine serum) 및 1%(w/v) 페니실린/스트렙토마이신을 포함하는 MEM-alpha에서 37 ℃, 5% CO2 습한 환경의 배양조건에서 세포를 배양하였다. Human mesenchymal stem cells used in the present invention were bone marrow-derived mesenchymal stem cells purchased from promocell, containing 5% (v/v) Fetal bonine serum (FBS) and 1% (w/v) penicillin/streptomycin. Cells were cultured in MEM-alpha at 37 °C and 5% CO 2 in a humid environment.
중간엽줄기세포를 동일 세포수로 부착시킬 때 인간영양막세포 배양액 (Trophoblast CM 또는 TB CM)을 5%(100uL), 10%(200uL) 및 30%(v/v, 600uL) 로 처리하고 24, 48, 72시간 후 세포의 증식을 이미지 또는 정량화하여 평가하였다. 인간영양막세포 배양액 처리량이 많을수록 중간엽줄기세포의 증식이 촉진되는 것으로 확인할 수 있었으며, 배양 시간이 증가할수록 또한 증식이 촉진되었다. 평가 결과는 도 2에 나타내었다.When attaching mesenchymal stem cells with the same number of cells, human trophoblast CM or TB CM was treated with 5% (100uL), 10% (200uL), and 30% (v/v, 600uL) and 24, After 48 and 72 hours, cell proliferation was evaluated by imaging or quantification. It was confirmed that the proliferation of mesenchymal stem cells was promoted as the treatment amount of the human trophoblast cell culture medium increased, and the proliferation was also promoted as the culture time increased. The evaluation results are shown in FIG. 2 .
중간엽줄기세포 배양 시 동물혈청인 FBS를 제외하고 인간영양막세포 배양액을 처리하여도 중간엽줄기세포의 증식이 유지 또는 촉진되는 지를 확인하였다. 중간엽줄기세포를 동일 세포수로 처리 후 부착시킬 때 FBS를 제외하고 인간영양막세포 배양액 (Trophoblast CM) 또는 중간엽줄기세포 배양액(MSC CM)을 각각 5%(100 uL), 10%(200 uL), 20%(400 uL) 및 30%(v/v, 600 uL)로 처리하고 24시간이 경과한 후의 현미경 사진을 도 3에 나타내었다. FBS가 처리되지 않은 그룹과 비교하여 인간영양막세포배양액이 처리된 군은 배양액 처리량이 증가할수록 중간엽줄기세포의 증식을 촉진함을 확인할 수 있었으며 FBS만 처리된 군과 비교하여도 인간영양막세포 배양액 처리군에서 중간엽줄기세포의 증식이 촉진됨을 확인할 수 있었다. During the culture of mesenchymal stem cells, it was confirmed whether the proliferation of mesenchymal stem cells was maintained or promoted even when the human trophoblast cell culture medium was treated with the exception of animal serum FBS. When attaching mesenchymal stem cells after treatment with the same number of cells, 5% (100 uL) and 10% (200 uL) of human trophoblast cell culture medium (Trophoblast CM) or mesenchymal stem cell culture medium (MSC CM), respectively, except for FBS ), 20% (400 uL) and 30% (v / v, 600 uL), and micrographs after 24 hours have elapsed are shown in FIG. 3. Compared to the group not treated with FBS, it was confirmed that the group treated with human trophoblast cell culture medium promoted the proliferation of mesenchymal stem cells as the amount of culture medium treatment increased. In the group, it was confirmed that the proliferation of mesenchymal stem cells was promoted.
FBS 처리 없이 중간엽줄기세포 배양액과 인간영양막세포 배양액 처리 시 중간엽줄기세포 배양과정에서 세포 분포도 및 모양을 확인하였다. FBS가 처리되지 않은 중간엽줄기세포는 24시간 후 잘 부착되지 않은 모습이었으나 FBS와 인간영양막세포배양액이 처리된 군에서 안정적으로 배양이 유지되는 모습을 확인할 수 있었다. 중간엽줄기세포 배양액 5% 처리군은 같은 양의 인간영양막세포 배양액 처리군과 비교하여 중간엽줄기세포의 부착 및 배양이 잘 이루어지지 않았다. 30% 배양액 처리군에서는 모두 중간엽줄기세포의 배양이 안정적으로 관찰되었으나 세포 하나 하나의 모습이 선명하게 관찰되는 인간영양막세포 배양액 처리군과 비교하여 중간엽줄기세포 배양액 처리군은 배양된 세포의 수도 적고 세포의 모습이 불분명하게 관찰되었다. 평가 결과는 도 4에 나타내었다. When treating mesenchymal stem cell culture medium and human trophoblast cell culture medium without FBS treatment, the cell distribution and shape were confirmed during the culture process of mesenchymal stem cells. Mesenchymal stem cells not treated with FBS did not adhere well after 24 hours, but it was confirmed that the culture was stably maintained in the group treated with FBS and human trophoblast cell culture medium. The mesenchymal stem
실시예 3: 인간영양막세포와 중간엽 줄기세포와의 비교실험Example 3: Comparative experiment between human trophoblast cells and mesenchymal stem cells
실시예 3-1: 인간영양막세포 및 중간엽 줄기세포의 특성 확인Example 3-1: Confirmation of characteristics of human trophoblast cells and mesenchymal stem cells
먼저, 인간영양막세포의 줄기세포 유지능력(stemness potential)을 중간엽줄기세포와 비교하기 위해 줄기세포 유지 관련 유전자인 OCT4, NANOG, SOX2 및 KLF4의 발현을 PCR로 확인하여 도 5에 나타내었다. 인간영양막세포는 ATCC로부터 구입하여 RPMI 1640 배양액에 5%(v/v) FBS, 1%(v/v) 페니실린/스트렙토마이신을 첨가하여 배양하였다. 인간영양막세포(TSC)가 중간엽줄기세포(MSC)보다 상기 OCT4, NANOG 및 SOX2의 유전자 발현률이 높게 나타났다. 세포 유래물은 세포가 지닌 특이적 능력을 포함하므로, 본 실험을 통해 인간영양막세포의 유래물이 중간엽줄기세포보다 우수한 재생 능력을 나타낼 수 있음을 확인할 수 있다. First, in order to compare the stem cell maintenance potential of human trophoblast cells with mesenchymal stem cells, the expression of OCT4, NANOG, SOX2, and KLF4, which are stem cell maintenance related genes, was confirmed by PCR and shown in FIG. 5 . Human trophoblasts were purchased from ATCC and cultured by adding 5% (v/v) FBS and 1% (v/v) penicillin/streptomycin to RPMI 1640 medium. Human trophoblast cells (TSC) showed higher gene expression rates of OCT4, NANOG and SOX2 than mesenchymal stem cells (MSC). Since the cell-derived product includes the specific ability of the cell, through this experiment, it can be confirmed that the human trophoblast cell-derived product can exhibit superior regenerative ability than the mesenchymal stem cell.
두 번째로, 인간영양막세포와 중간엽줄기세포 체외 배양 시 모습과 세포 유래물 속 나노입자 분석 결과를 도 6에 나타내었다. 두 줄기세포 모두 2.0 × 1010 내지 2.5 × 1010 입자/mL농도와 직경 150nm 이하의 입자 크기를 갖는 것으로 확인되었으며, 서로 비슷한 분포도 양상을 보여 유래물 회수율의 차이는 없는 것으로 확인되었다.Second, the appearance of human trophoblast cells and mesenchymal stem cells during in vitro culture and the results of nanoparticle analysis in cell-derived products are shown in FIG. 6 . Both stem cells were confirmed to have a concentration of 2.0 × 10 10 to 2.5 × 10 10 particles/mL and a particle size of 150 nm or less in diameter, and it was confirmed that there was no difference in the recovery rate of the derivatives, showing similar distribution patterns.
실시예 3-2: FBS-free 조건에서 중간엽줄기세포 증식 평가Example 3-2: Evaluation of mesenchymal stem cell proliferation in FBS-free conditions
골수유래(BM), 탯줄유래(UC) 및 지방유래 (AD) 중간엽줄기세포의 배양 시 동물혈청인 FBS를 제외하고 인간영양막세포 배양액을 처리하여 중간엽줄기세포의 증식이 유지 또는 촉진되는지를 확인하였다. 중간엽줄기세포를 동일 세포수로 처리 후 부착시킬 때 FBS를 제외하고 인간영양막세포 배양액 (TSC-CM) 또는 중간엽줄기세포 배양액(MSC-CM)을 각각 20%, 40% 및 60 %(v/v)로 처리하고 24시간, 48시간 후에 CCK8으로 정량분석 평가하여 도 7에 나타내었다. FBS가 처리되지 않은 그룹(serum free medium, SFM)과 비교하여 인간영양막세포 배양액이 처리된 군은 배양액 처리량이 증가할수록 중간엽줄기세포의 증식 유지 및 촉진능력이 향상되었다(TSC-CM은 적색 *로 통계적 유의성을 표시함). 중간엽줄기세포 배양액 처리군도 FBS 없이 중간엽줄기세포 자신의 증식 유지 및 촉진 효능이 나타났다(MSC-CM은 흑색 *로 통계적 유의성을 표시함). 하지만, 인간영양막세포 배양액 처리 군에서는 40% 이하의 적은 양의 배양액 처리 시에도 증식 촉진 효능이 나타나는 것으로 확인되었다. During the culture of bone marrow-derived (BM), umbilical cord-derived (UC), and adipose-derived (AD) mesenchymal stem cells, treatment with human trophoblast cell culture medium excluding animal serum FBS was performed to determine whether the proliferation of mesenchymal stem cells was maintained or promoted. Confirmed. When attaching mesenchymal stem cells after treatment with the same number of cells, 20%, 40%, and 60% (v /v), and after 24 hours and 48 hours, quantitative analysis and evaluation with CCK8 are shown in FIG. 7 . Compared to the group not treated with FBS (serum free medium, SFM), the group treated with human trophoblast culture medium improved the ability to maintain and promote the proliferation of mesenchymal stem cells as the amount of culture medium increased (TSC-CM is red * indicates statistical significance). The mesenchymal stem cell culture medium-treated group also showed an effect of maintaining and promoting the proliferation of mesenchymal stem cells themselves without FBS (statistical significance is indicated by black * for MSC-CM). However, in the human trophoblast cell culture medium-treated group, it was confirmed that the growth promoting effect was shown even when the culture medium was treated with a small amount of 40% or less.
실시예 3-3: 노화세포에서의 항노화 효능 확인Example 3-3: Confirmation of anti-aging efficacy in senescent cells
계대수 9 이상의 노화된 골수유래 중간엽 줄기세포를 이용하여 중간엽줄기세포 배양액(MSC-CM), 인간영양막세포 배양액(TSC-CM) 및 인간영양막세포 유래물(엑소좀)(TSC-Exos)의 항노화 효능을 확인하였다. 모두 30%로 처리되었으며, 세포노화의 지표인 SA-β-GAL(Senescence-associated beta-galactosidase) 염색법을 적용하여 항노화 효능을 검증하였고 그 결과를 도 8에 나타내었다. 인간영양막세포 배양액과 유래물 처리 군에서 유의적으로 염색 세포의 수가 감소함을 확인하였다. 또한, 동일한 조건에서 FBS 처리된 군(GM), FBS 처리되지 않은 SFM(serum free media)군 및 MSC-CM 처리군과 인간영양막세포 배양액 처리군을 비교실험하여 결과를 도 9에 나타내었다. 인간영양막세포 배양액(TB-CM) 처리군에서 FBS 처리군과 비슷한 세포수가 유지됨 및 항노화 효능이 나타남을 확인할 수 있었고, 특히 중간엽 줄기세포 배양액(MSC-CM) 처리군과 비교하면 2배 이상의 항노화 효능이 확인되었다. Mesenchymal stem cell culture medium (MSC-CM), human trophoblast cell culture medium (TSC-CM) and human trophoblast cell-derived product (exosomes) (TSC-Exos) using aged bone marrow-derived mesenchymal stem cells with
실시예 3-4: 피부상처 치유 촉진 효능Example 3-4: Efficacy of promoting skin wound healing
중간엽줄기세포 배양액(MSC-CM)과 인간영양막세포 배양액(TSC-CM)의 상처치유 촉진 효능을 비교 분석하기 위해 면역결핍마우스 피부에 지름 10mm의 동일한 원형의 상처를 유도한 후 5×105cell의 중간엽줄기세포를 100 uL PBS에 섞어서 도입하였다. 그리고, 상처부위에 3일마다 100 uL의 중간엽줄기세포 및 인간영양막세포 배양액을 처리하여 15일 동안 관찰하여 이를 도 10에 나타내었다. 5일 이후부터 중간엽줄기세포 배양액보다 우수한 상처치유능력을 확인할 수 있었으며, 15일에 인간영양막세포 배양액 처리군에서 상처가 모두 치유된 것을 확인하였다. 중간엽줄기세포 배양액 처리군도 상처치유 효능을 보였으나 인간영양막세포 배양액 처리군보다 우수하지는 않았다. 상처치유 효능을 H&E 염색법과 MT 염색법을 통해 조직학적 분석으로 확인하여 도 11에 나타내었으며, 인간영양막세포 배양액 처리군에서 피부 상피세포층과 진피층의 두께가 증가함을 확인할 수 있었다. 따라서, 본원발명의 인간영양막세포 배양액은 상처치유 촉진효능 및 피부재생 촉진효능을 가짐을 확인할 수 있었다. To compare and analyze the efficacy of mesenchymal stem cell culture medium (MSC-CM) and human trophoblast cell culture medium (TSC-CM) in promoting wound healing, 5 × 10 5 identical circular wounds with a diameter of 10 mm were induced in the skin of immunodeficient mice. The mesenchymal stem cells of the cells were introduced by mixing in 100 uL PBS. In addition, the wound site was treated with 100 uL of mesenchymal stem cell and human trophoblast cell culture medium every 3 days and observed for 15 days, as shown in FIG. 10 . From the 5th day onward, it was confirmed that the wound healing ability was superior to that of the mesenchymal stem cell culture medium, and it was confirmed that all wounds were healed in the human trophoblast cell culture medium treatment group on the 15th day. The mesenchymal stem cell culture medium treatment group also showed wound healing efficacy, but it was not superior to the human trophoblast cell culture medium treatment group. The wound healing efficacy was confirmed by histological analysis through the H&E staining method and the MT staining method, and is shown in FIG. Therefore, it was confirmed that the human trophoblast cell culture medium of the present invention has a wound healing promoting effect and skin regeneration promoting effect.
실시예 4: 인간영양막세포 배양액의 골재생 촉진효능 평가Example 4: Evaluation of bone regeneration promoting effect of human trophoblast cell culture medium
실시예 4-1: ALP 활성도 평가Example 4-1: ALP activity evaluation
인간중간엽줄기세포의 골분화 초기단계에서 ALP활성화도를 측정하였다. ALP 활성도는 중간엽줄기세포의 골형성 유도 배지(osteogenesis induction medium, OIM)에 인간영양막세포 배양액(TB-CM) 10%, 20%, 30% 및 40%(v/v)를 각각 처리한 후 3일차 및 7일차에 ALP활성도를 평가하였다. 차가운 PBS버퍼로 2회 세척한 다음, 0.1% 트리톤(Triton) X-10이 첨가된 세포막 분해 용액(cell lysis buffer)로 용해(lysis) 시켰다. 용해된 세포를 섭씨 4도, 13000 rpm에서 30분간 원심분리한 후 상층액을 회수하여 브래드포드법으로 정량하고, 동량의 단백질을 ALP kit(아나스펙, Inc, Fermont, Canada)로 제조사의 지침에 따라 405 ㎚에서 ALP 활성도를 측정하였으며, 그 결과를 도 12의 좌측에 나타내었다. 인간영양막세포 배양액 처리군에서는 처리하지 않은 경우에 비하여 최대 1.5배까지 ALP 활성도가 증가된 것으로 확인되었다. 인간영양막세포 유래물 (TB EV, protocol 1 EV)의 골재생 촉진 효능 평가는 ALP 활성도로 분석되었으며 이에 대한 결과는 도 1에 나타낸 바와 같다. ALP activation was measured in the early stage of bone differentiation of human mesenchymal stem cells. ALP activity was measured after treatment with 10%, 20%, 30%, and 40% (v/v) of human trophoblast cell culture medium (TB-CM) in osteoogenesis induction medium (OIM) of mesenchymal stem cells, respectively. ALP activity was evaluated on the 3rd and 7th days. After washing twice with cold PBS buffer, lysis was performed with a cell lysis buffer containing 0.1% Triton X-10. The lysed cells were centrifuged at 4 degrees Celsius and 13000 rpm for 30 minutes, and the supernatant was recovered and quantified by the Bradford method. ALP activity was measured at 405 nm according to the method, and the results are shown on the left side of FIG. 12 . In the human trophoblast cell culture solution-treated group, it was confirmed that the ALP activity was increased by up to 1.5 times compared to the untreated group. The bone regeneration promoting efficacy of human trophoblast cell-derived (TB EV,
실시예 4-2: 칼슘 침착도 평가Example 4-2: Evaluation of calcium deposition
골 형성 후기 단계에 나타나는 칼슘 침착은 후기 골 형성 지표로 사용되어 이에 대한 실험을 실시하였다. 동일 실험에서 골분화 14일째 세포에 침착된 칼슘을 정량적으로 분석하였으며(DICA-500, Bioassay System) 이를 도 12의 우측에 나타내었다. 칼슘침착도는 인간영양막세포 배양액 처리군이 처리되지 않은 군과 비교하여 3배가량 증가한 것으로 분석되었다. Calcium deposition in the late stage of bone formation was used as an indicator of late bone formation, and an experiment was conducted on this. In the same experiment, calcium deposited in cells on
실시예 4-3: 알리자린 레드 에스 염색 평가Example 4-3: Alizarin Red S staining evaluation
알리자린 레드 에스(Alizarin Red S) 염색 평가는 조골세포의 분화 중 중후반 지표로 널리 사용되어 이에 관한 실험을 진행하였다. 인간영양막세포 배양액의 미네랄 침착 촉진 효능을 평가하기 위해서 골형성 유도 배지(osteogenesis induction medium, OIM)에서 중간엽줄기세포를 분화시킬 때 인간영양막세포 배양액 10%, 20% 및 30%(v/v)를 각각 처리하고, 처리하지 않은 대조군과 함께 미네랄의 함량을 분화 14일 후에 측정하였다. 미네랄의 함량은 Alizarin Red S 염색액 (Millipore)을 사용하여 제조사의 지침에 따라 측정하였으며, 그 결과를 도 13에 나타내었다. 이를 통해서 인간영양막세포 배양액을 처리한 군이 처리하지 않은 군과 비교하여 미네랄 침착 유도 효과가 높음을 확인할 수 있었다(*P<0.05).Alizarin Red S staining evaluation was widely used as a mid-to-late indicator during differentiation of osteoblasts, and experiments were conducted on this. To evaluate the mineral deposition promoting effect of human trophoblast cell culture medium, 10%, 20% and 30% (v/v) of human trophoblast cell culture medium when differentiating mesenchymal stem cells in osteoogenesis induction medium (OIM) were treated, respectively, and the mineral content was measured 14 days after differentiation together with the untreated control group. The mineral content was measured using Alizarin Red S staining solution (Millipore) according to the manufacturer's instructions, and the results are shown in FIG. 13 . Through this, it was confirmed that the group treated with the human trophoblast cell culture medium had a higher mineral deposition induction effect than the untreated group (*P<0.05).
실시예 4-4: 골재생 관련 전사인자 유전자 발현정도 평가Example 4-4: Evaluation of transcription factor gene expression level related to bone regeneration
골재생 관련 중요 전사인자 유전자인 초기의 IBSP 및 ALP, 중기의 RUNX2 및 Osterix 그리고 후기의 OCN 및 OPN 유전자의 발현을 측정하였다. 인간영양막세포 배양액을 중간엽줄기세포 골분화 유도 시 0%, 10%, 20%, 30% 및 40%로 각각 처리 후 PCR을 통하여 확인하였으며 그 결과를 도 14에 나타내었다. TB-CM 처리량이 증가할수록 해당 유전자 발현이 모두 증가하는 것을 확인하였으며, 이를 통해 인간영양막세포 배양액이 중간엽줄기세포의 골분화를 촉진할 수 있음을 확인하였다. The expression of IBSP and ALP in the early stage, RUNX2 and Osterix in the middle stage, and OCN and OPN genes in the late stage, which are important transcription factor genes related to bone regeneration, were measured. The human trophoblast cell culture medium was treated with 0%, 10%, 20%, 30%, and 40% when inducing bone differentiation of mesenchymal stem cells, respectively, and then confirmed through PCR, and the results are shown in FIG. 14 . It was confirmed that the corresponding gene expression increased as the amount of TB-CM treatment increased, and through this, it was confirmed that the human trophoblast cell culture medium could promote the osteogenic differentiation of mesenchymal stem cells.
실시예 4-5: RNA 시퀀싱 분석Example 4-5: RNA sequencing analysis
인간영양막세포 배양액이 중간엽줄기세포의 골분화 촉진 효능을 가짐은 상기 실시예 4-1 내지 4-3에서 확인한 바와 같다. 이의 기전을 확인하기 위해 RNA 시퀀싱 분석을 실시하였다. 중간엽줄기세포를 골형성유도배지(OIM)에서 배양하여 골분화 유도 시, 인간영양막세포 배양액을 20% 처리하여 7일간 골분화 유도배양하고 RNA 시퀀싱 분석하여 그 결과를 도 15에 나타내었다. 분석 결과 인간영양막세포 배양액 처리에 따라 세포 내 골재생 촉진과 관련된 사이토카인 및 케모카인 반응, 염증, 성장인자 활성화 관련 세포 반응이 분석되었으며 골재생에 직접적 영향을 주는 BMP 바인딩(BMP binding)도 확인되었다.It is as confirmed in Examples 4-1 to 4-3 that the human trophoblast cell culture medium has an effect of promoting bone differentiation of mesenchymal stem cells. RNA sequencing analysis was performed to confirm this mechanism. When osteogenic differentiation was induced by culturing mesenchymal stem cells in an osteoinduction medium (OIM), human trophoblast cell culture medium was treated with 20%, bone differentiation induction cultured for 7 days, and RNA sequencing analysis was performed, and the results are shown in FIG. 15 . As a result of the analysis, cytokine and chemokine reactions related to the promotion of intracellular bone regeneration, inflammation, and cellular responses related to the activation of growth factors were analyzed according to the treatment of human trophoblast cell culture medium, and BMP binding, which directly affects bone regeneration, was also confirmed.
실시예 4-6: 신호전달계 분석Example 4-6: Signal Transduction System Analysis
인간영양막세포 배양액 처리에 따른 세포신호전달계 분석결과를 도 16에 나타내었으며, 사이토카인 연계 신호전달계와 MAPK 신호전달계가 유의적으로 증가되는 것으로 확인되었다. 인간영양막세포 배양액에 의한 중간엽줄기세포 골분화 촉진 시 MAPK 신호전달계가 증가됨을 웨스턴 블롯(western blot)으로 확인하여 이를 도 17에 나타내었다. 또한, BMP2의 발현은 증가하고 BMP2 신호전달계 저해인자(antagonist)인 GREM1의 발현은 감소됨을 PCR로 확인하여 도 18에 나타내었다. 따라서, 인간영양막세포 배양액 처리에 따라 BMP2 바인딩이 증가하여 골재생을 촉진하는 기능이 있음을 확인하였다. The results of analysis of the cell signaling pathway according to the treatment of the human trophoblast cell culture medium are shown in FIG. 16, and it was confirmed that the cytokine-linked signaling pathway and the MAPK signaling pathway were significantly increased. It was confirmed by western blot that the MAPK signaling pathway was increased when the human trophoblast cell culture medium promoted bone differentiation of mesenchymal stem cells, and this is shown in FIG. 17 . In addition, it was confirmed by PCR that the expression of BMP2 increased and the expression of GREM1, an antagonist of the BMP2 signaling system, decreased, as shown in FIG. 18 . Therefore, it was confirmed that the binding of BMP2 increased according to the treatment of the human trophoblast cell culture medium, thereby promoting bone regeneration.
또한, 골분화 유도배지(OIM)에서 인간영양막세포 배양액 처리 여부에 따라 CXCL1, CXCL3, CXCL6, CXCL8, CCL7, CCL11, CCL13, IL-6, MMP-1, MMP-3, MMP-8 및 MMP-13 유전자의 발현율의 변화를 PCR로 확인하여 도 19에 나타내었다. 대조군과 비교하여 사이토카인 연계 신호전달계 활성이 증가하여 중간엽 줄기세포의 골분화를 촉진함을 확인할 수 있었다. In addition, CXCL1, CXCL3, CXCL6, CXCL8, CCL7, CCL11, CCL13, IL-6, MMP-1, MMP-3, MMP-8 and MMP- Changes in expression rates of 13 genes were confirmed by PCR and are shown in FIG. 19 . Compared to the control group, it was confirmed that the activity of the cytokine-linked signaling pathway was increased to promote bone differentiation of mesenchymal stem cells.
실시예 4-7: 지방분화 억제 효능Example 4-7: Adipogenesis inhibitory effect
중간엽줄기세포는 지방세포와 골세포로 모두 분화가 가능하며, 지방분화가 증가하는 경우 골세포량이 줄어 골다공증 등 골재생 관련 질환에 노출된다. 따라서, 지방분화 억제 효능은 골분화 촉진 효능과 밀접한 관련이 있으며, 이를 검증하고자 하기와 같이 실험하였다. Mesenchymal stem cells can differentiate into both fat cells and bone cells, and when fat differentiation increases, the amount of bone cells decreases and is exposed to diseases related to bone regeneration, such as osteoporosis. Therefore, the effect of inhibiting fat differentiation is closely related to the effect of promoting bone differentiation, and the experiment was conducted as follows to verify this.
5×104입자/mL의 중간엽줄기세포를 24 웰 플레이트(well plate)에 도입 후 90%이상 세포밀도(confluency)일 때 지방유도 배양액 (adipogenesis induced medium, AIM)을 처리하여 14일동안 배양하였다. 이 때 인간영양막세포 배양액(TB-CM)을 10%, 20% 및 50%(v/v)로 처리하여 2~3일에 한 번씩 배양액을 교체하여 주었다. 14일 후 Oil red O 염색법으로 중간엽줄기세포의 지방분화도를 측정하였으며 지방재생 관련 유전자인 아디포넥틴(adiponectin), 렙틴(leptin) 및 퍼옥시좀 증식체 활성화 수용체 감마(PPAR-γ)의 상대적 발현정도를 측정하여 도 20에 나타내었다. 붉은색 염색이 많이 나타날수록 지방 분화가 증가된 것으로 평가되며, 인간영양막세포 배양액 처리군에서 유의적으로 지방분화 감소가 관찰되었다. 또한 지방재생 관련 유전자인 아디포넥틴, 렙틴 및 퍼옥시좀 증식체 활성화 수용체 감마의 상대적 발현량이 인간영양막세포 배양액 처리 농도 증가에 따라 현저히 감소함을 확인할 수 있었다. 5×10 4 Particles/mL of mesenchymal stem cells were introduced into a 24-well plate, and then cultured for 14 days by treating with adipogenesis induced medium (AIM) at a cell density of 90% or higher. did At this time, human trophoblast cell culture medium (TB-CM) was treated with 10%, 20%, and 50% (v/v), and the culture medium was replaced once every 2 to 3 days. After 14 days, the degree of fat differentiation of mesenchymal stem cells was measured by Oil red O staining, and the relative expression levels of fat regeneration-related genes, adiponectin, leptin, and peroxisome proliferator-activated receptor gamma (PPAR-γ) was measured and shown in FIG. 20 . The higher the amount of red staining, the more fat differentiation was evaluated, and a significant decrease in fat differentiation was observed in the human trophoblast cell culture solution treatment group. In addition, it was confirmed that the relative expression levels of adiponectin, leptin, and peroxisome proliferator-activated receptor gamma, which are genes related to fat regeneration, significantly decreased as the treatment concentration of the human trophoblast cell culture medium increased.
따라서, 본원발명의 인간영양막세포 배양액이 지방분화 유도된 중간엽줄기세포의 분화 및 증식을 억제함을 확인할 수 있었다. Accordingly, it was confirmed that the human trophoblast cell culture medium of the present invention inhibits the differentiation and proliferation of mesenchymal stem cells induced by adipocyte differentiation.
실시예 5: 인간영양막세포 유래물(엑소좀)의 골재생 촉진효능 평가Example 5: Evaluation of the bone regeneration promoting effect of human trophoblast cell-derived products (exosomes)
실시예 5-1: ALP활성도 및 칼슘침착도 측정Example 5-1: Measurement of ALP activity and calcium deposition
본 실시예에서의 인간영양막세포 유래물(엑소좀, TSC-EV)은 상기 실시예 1-1에 따라 제조되었다. 골분화 유도배지에서 골분화를 유도할 때, 인간영양막세포 유래물(엑소좀)을 1Ⅹ108 및 1Ⅹ109 입자/mL로 처리한 후 평가하였다. ALP활성도는 3일 및 7일에서 측정되었고, 칼슘침착도는 14일에서 측정되었으며 그 결과를 도 21에 나타내었다. 인간영양막세포 유래물(엑소좀)을 처리한 군에서 ALP 활성도는 농도에 따라 1.3배 내지 2배가 증가하였으며, 칼슘침착도는 3배 증가한 것으로 분석되어 골재생효능이 있음을 확인하였다.The human trophoblast cell-derived product (exosome, TSC-EV) in this Example was prepared according to Example 1-1. When osteogenic differentiation was induced in the osteogenic differentiation induction medium, human trophoblast cell-derived products (exosomes) were treated with 1Ⅹ10 8 and 1Ⅹ10 9 particles/mL and then evaluated. ALP activity was measured on the 3rd and 7th days, and calcium deposition was measured on the 14th day, and the results are shown in FIG. 21 . In the group treated with human trophoblast cell derivatives (exosomes), the ALP activity increased 1.3 to 2 times depending on the concentration, and the calcium deposition was analyzed to increase 3 times, confirming the bone regeneration effect.
인간영양막세포 유래물(TSC-EV)과 인간영양막세포 배양액(TSC-CM)을 함께 처리하는 경우의 ALP 활성도를 단독 처리하는 경우와 비교하여 도 22에 나타내었다. 상기 유래물과 상기 배양액을 함께 처리하는 경우, 단독으로 처리하는 경우보다 ALP 활성도가 3일차 및 7일차에서 모두 유의적으로 증가하였다.Figure 22 shows the ALP activity when both human trophoblast cell-derived material (TSC-EV) and human trophoblast cell culture medium (TSC-CM) were treated together and compared with when treated alone. When the derivative and the culture medium were treated together, the ALP activity was significantly increased on both the 3rd and 7th days, compared to the case of treatment alone.
실시예 5-2: 알리자린 레드 에스 염색 평가Example 5-2: Alizarin Red S staining evaluation
알리자린 레드 에스(Alizarin Red S) 염색 평가로 인간영양막세포 유래물(엑소좀, TSC-EV)의 미네랄 침착 촉진 효능을 평가하였다. 골형성 유도배지에서 인간영양막세포 유래물을 각각 1Ⅹ108 및 1Ⅹ109입자/mL로 처리 후 결과를 도 23에 나타내었다. 분화 14일차에서 인간영양막세포 유래물을 처리한 군이 대조군에 비해 미네랄 침착도가 높음을 확인할 수 있었다. The mineral deposition promoting effect of the human trophoblast cell-derived material (exosome, TSC-EV) was evaluated by Alizarin Red S staining evaluation. 23 shows the results after treating the human trophoblast-derived material with 1Ⅹ10 8 and 1Ⅹ10 9 particles/mL, respectively, in the osteogenic induction medium. On the 14th day of differentiation, it was confirmed that the group treated with the human trophoblast-derived material had a higher degree of mineral deposition than the control group.
실시예 5-3: 골재생 관련 전사인자 유전자 발현정도 평가Example 5-3: Evaluation of transcription factor gene expression level related to bone regeneration
골재생 관련 중요 전사인자 유전자인 초기의 IBSP 및 ALP, 중기의 RUNX2 및 Osterix 그리고 후기의 OCN 및 OPN 유전자의 발현을 측정하였다. 인간영양막세포 유래물을 중간엽줄기세포 골분화 유도 시 1Ⅹ108 및 1Ⅹ109입자/mL 로 각각 처리 후 PCR을 통하여 확인하였으며 그 결과를 도 24에 나타내었다. 인간영양막세포 유래물의 처리량이 증가할수록 해당 유전자 발현이 모두 증가하는 것을 확인하였으며, 이를 통해 인간영양막세포 유래물(엑소좀)이 중간엽줄기세포의 골분화를 촉진할 수 있음을 확인할 수 있다. The expression of IBSP and ALP in the early stage, RUNX2 and Osterix in the middle stage, and OCN and OPN genes in the late stage, which are important transcription factor genes related to bone regeneration, were measured. The human trophoblast cell-derived product was treated with 1Ⅹ10 8 and 1Ⅹ10 9 particles/mL, respectively, when inducing bone differentiation of mesenchymal stem cells, and then confirmed through PCR, and the results are shown in FIG. 24 . It was confirmed that the corresponding gene expression increased as the amount of treatment of human trophoblast cell-derived products increased, and through this, it was confirmed that human trophoblast cell-derived products (exosomes) can promote bone differentiation of mesenchymal stem cells.
실시예 5-4: 동물실험Example 5-4: Animal testing
인간영양막세포 유래물의 골재생 효능을 검증하기 위해 전임상 실험(동물실험)을 진행하였다. 랫트의 두개골에 직경 6mm의 원형으로 골결손을 유도하였다. 결손부위를 그대로 둔 경우, 지지체만 처리한 경우, 지지체 처리에 더하여 체외에서 5일간 골분화 유도된 중간엽줄기세포(5Ⅹ105세포)를 처리한 경우, 상기 중간엽 줄기세포에 인간영양막세포 유래물을 1Ⅹ109입자/mL 농도로 처리한 경우 및 상기 중간엽 줄기세포에 BMP2를 500ng/mL로 처리한 경우에 대하여 평가하였다. 8주 후 조직을 적출하여 골재생 정도를 microCT로 확인하여 그 결과를 도 25에 나타내었다. microCT 결과 인간영양막세포 유래물 (TSC-EV) 처리군에서 유의적으로 골결손 부위가 채워져 골형성을 촉진함을 확인하였으며, 결손부위의 직경 및 면적 모두 MSC만 처리한 군 및 MSC+BMP2 처리군 보다 유의하게 감소함을 확인하였다. Preclinical experiments (animal experiments) were conducted to verify the bone regeneration efficacy of the human trophoblast cell-derived product. A bone defect was induced in a circular shape with a diameter of 6 mm on the skull of the rat. When the defective part is left as it is, when only the scaffold is treated, when the bone differentiation-induced mesenchymal stem cells (5Ⅹ10 5 cells) are treated in vitro for 5 days in addition to the scaffold treatment, the mesenchymal stem cells are treated with human trophoblast cell-derived was evaluated at a concentration of 1Ⅹ10 9 particles/mL and when the mesenchymal stem cells were treated with BMP2 at 500 ng/mL. After 8 weeks, tissues were extracted and the degree of bone regeneration was confirmed by microCT, and the results are shown in FIG. 25 . As a result of microCT, it was confirmed that in the human trophoblast cell-derived (TSC-EV) treated group, the bone defect was significantly filled and bone formation was promoted, and the diameter and area of the defect were both MSC-only and MSC+BMP2-treated groups. A more significant decrease was confirmed.
같은 실험에서, H&E(hematoxylin & eosin)와 MT(Masson's trichrome)염색방법을 이용하여 조직학적 분석을 하였다. 대조군과 실험군은 상기 microCT 실시예에서와 같으며 결과를 도 26에 나타내었다. 중간엽줄기세포에 인간영양막세포 유래물을 처리한 군(MSC+TSC-EV)에서 다른 대조군에 비해 유의하게 골결손 부위(defect region) 내 새로운 골형성을 촉진함을 확인할 수 있었다. In the same experiment, histological analysis was performed using H&E (hematoxylin & eosin) and MT (Masson's trichrome) staining methods. The control group and the experimental group were the same as in the microCT example, and the results are shown in FIG. 26 . In the group (MSC+TSC-EV) treated with human trophoblastic cell-derived mesenchymal stem cells, it was confirmed that new bone formation in the bone defect region was significantly promoted compared to other control groups.
또한 같은 실험에서, 새로이 골형성이 이루어지는 부위에서의 골재생 마커 인자인 OCN(osteocalcin) 발현을 해당 항체를 이용하여 형광분석법으로 평가하였다. 대조군과 실험군은 상기 microCT에서와 같으며 결과를 도 27에 나타내었다. 인간영양막세포 유래물 처리군 (MSC+TSC-EV)에서 유의적으로 높게 OCN이 발현됨을 형광 현미경 사진 및 형광 세기를 통해 확인할 수 있었고, 이는 인간영양막세포 유래물이 골결손 부위에서 새로운 골형성을 촉진하는 효과를 가짐을 뒷받침한다. Also, in the same experiment, the expression of OCN (osteocalcin), a bone regeneration marker factor, at the site where new bone formation takes place was evaluated by fluorescence analysis using the corresponding antibody. The control group and the experimental group were the same as in the microCT, and the results are shown in FIG. 27 . It was confirmed through fluorescence micrographs and fluorescence intensity that OCN was significantly higher in the human trophoblast cell-derived treatment group (MSC+TSC-EV), indicating that the human trophoblast cell-derived protein inhibits new bone formation at the bone defect site. It supports that it has a stimulating effect.
실시예 5-5: miRNA 분석Example 5-5: miRNA analysis
상기 실시예 5-1 내지 5-4에서 확인한 바와 같이, 인간영양막세포 유래물(엑소좀)은 우수한 골재생효과를 보여주었다. 인간영양막세포 엑소좀의 재생 효능 기전을 분석하기 위해 엑소좀 내의 miRNA를 분석하고, 현재 엑소좀 바이오시장에서 사용 중인 중간엽줄기세포 엑소좀과 비교분석하여 그 결과를 도 28 및 표 1에 나타내었다. As confirmed in Examples 5-1 to 5-4, the human trophoblast cell-derived product (exosome) showed an excellent bone regeneration effect. In order to analyze the mechanism of regeneration efficacy of human trophoblast cell exosomes, miRNAs in exosomes were analyzed, and mesenchymal stem cell exosomes currently used in the exosome bio market were compared and analyzed, and the results are shown in FIG. 28 and Table 1. .
분석 결과 인간영양막세포 엑소좀 (TSC-Exos)에서는 전체 RNA 조성 중 세포 내 전사 유전자 조절을 할 수 있는 snRNA(small nuclear RNA, miRNA를 포함함)가 70% 이상을 차지하였고, 반면 중간엽줄기세포 엑소좀(MSC-Exos) 속에는 단백질 번역 기능의 rRNA(ribosomal RNA)가 77% 분포되어 있었다. 또한, total reads counts의 비는 500,223/24,359로 약 20.54를 나타내었으며, 인간영양막세포 엑소좀의 총 miRNA 수가 중간엽줄기세포 엑소좀 보다 약 20배 많이 분포된 것으로 확인되었다. 또한 miRNA 분포 양상도 두 세포에서 다르게 나타났다. 상기 표 1의 인간영양막세포 엑소좀에서는 has-miR-1246, has-miR-1290, has-miR-423-5p 및 has-let-7b-5p가 98%를 차지하고 있었으며, 중간엽줄기세포 엑소좀에서의 miRNA 구성과 차이를 보였다. As a result of the analysis, in human trophoblast cell exosomes (TSC-Exos), snRNA (including small nuclear RNA, miRNA) capable of regulating intracellular transcriptional genes among the total RNA composition accounted for more than 70%, whereas mesenchymal stem cells In exosomes (MSC-Exos), 77% of rRNA (ribosomal RNA) for protein translation was distributed. In addition, the ratio of total reads counts was 500,223/24,359, which was about 20.54, and it was confirmed that the total number of miRNAs in human trophoblast cell exosomes was distributed about 20 times more than that in mesenchymal stem cell exosomes. In addition, miRNA distribution patterns were also different in the two cells. In the human trophoblast cell exosomes in Table 1, has-miR-1246, has-miR-1290, has-miR-423-5p, and has-let-7b-5p accounted for 98%, and mesenchymal stem cell exosomes showed differences from miRNA composition in .
중간엽줄기세포 엑소좀 속 miRNA와 비교하여 인간영양막세포 엑소좀에서 100 fold 이상 높게 발현되는 miRNA는 23개로 나타났으며 이들의 표적 유전자들을 도 29에 나타내었고, 이를 기반으로 세포 내 영향을 주는 신호전달계를 ClueGO bioinformatic tool을 이용하여 분석하여 이를 도 30에 나타내었다. 골재생과 관련된 병소 유착(Focal adhesion), AMPK 및 MAPK 신호전달계가 유의적으로 분석되었고, 인간영양막세포 엑소좀 속의 miRNA가 골재생 효능에 영향을 미칠 수 있음을 보여준다. Compared to miRNAs in mesenchymal stem cell exosomes, 23 miRNAs were expressed more than 100 fold higher in human trophoblast cell exosomes, and their target genes are shown in FIG. 29, based on which signals affecting the cell The delivery system was analyzed using the ClueGO bioinformatic tool and is shown in FIG. 30 . Focal adhesion, AMPK and MAPK signaling pathways related to bone regeneration were significantly analyzed, and miRNAs in human trophoblast exosomes can affect bone regeneration efficacy.
다음으로, 인간영양막세포 엑소좀의 98% 이상을 점유하고 있는 상위 1%의 4개 miRNA(has-miR-1246, has-miR-1290, has-miR-423-5p 및 has-let-7b-5p)의 표적 유전자들을 기반으로 DAVID bioinformatic tool을 이용하여 세포 내에서 변화 가능한 생물학적 과정(biological processes)와 신호전달계를 분석하여 도 31에 나타내었다. 그 중 재생에 관련된 세포 증식 (cell proliferation), 신경계 발달(nerve system development), 상처치유 (wound healing), 그리고 줄기세포 조절 (regulation of stem cell)등이 분석되었다. 이 결과는 string과 ClueGO bioinformatic tool에 의해서도 동일하게 분석되었으며 이를 도 32에 나타내었다. 통합적으로 인간영양막세포과 중간엽줄기세포 엑소좀 속 miRNA에 의해 조절 가능한 세포 기능들 및 두 세포 유래 엑소좀의 miRNA에 의해 동일하게 조절 가능한 세포기능을 모식화 하여 도 33에 표시하였다. Next, the top 1% of the four miRNAs (has-miR-1246, has-miR-1290, has-miR-423-5p and has-let-7b- Based on the target genes of 5p), the biological processes and signal transduction system that can be changed in the cell were analyzed using the DAVID bioinformatic tool, and are shown in FIG. 31 . Among them, cell proliferation related to regeneration, nerve system development, wound healing, and regulation of stem cells were analyzed. This result was analyzed in the same way by string and ClueGO bioinformatic tool, and it is shown in FIG. 32. Integratively, the cellular functions regulatable by miRNA in exosomes of human trophoblast cells and mesenchymal stem cells and the cellular functions equally regulatable by miRNAs in exosomes derived from both cells are modeled and shown in FIG. 33 .
실시예 6: 산모 태반으로부터 분리된 인간영양막세포 배양액의 효능 검증Example 6: Efficacy verification of human trophoblast cell culture isolated from maternal placenta
상시 실시예 1 내지 5에서는 ATCC사에서 구입한 인간영양막세포를 이용하였다. 인간 산모 태반으로부터 얻은 영양막세포에 대해서도 동일한 재생효능이 나타나는지를 검증하기 위해 다음과 같이 실험하였다. 이하에서 ATCC사에서 구입한 인간영양막세포와 구분하기 위하여 산모 태반으로부터 분리된 인간영양막세포를 일차(primary) 인간영양막세포로 지칭한다. In Examples 1 to 5, human trophoblast cells purchased from ATCC were used. The following experiment was conducted to verify whether the same regenerative effect was exhibited on trophoblast cells obtained from the placenta of a human mother. Hereinafter, in order to differentiate from human trophoblast cells purchased from ATCC, human trophoblast cells isolated from the mother's placenta are referred to as primary human trophoblast cells.
실시예 6-1: 일차 인간영양막세포의 분리Example 6-1: Isolation of primary human trophoblast cells
분만한 산모의 태반으로부터 일차(primary) 인간영양막세포를 분리하여 15일간 체외 배양하였으며, 매일 배양액을 수거하여 중간엽줄기세포의 증식과 골재생 효능을 확인하고자 하였다. 세포 분리과정은 선행연구 (B. Simon, M. Bucher, A. Maloyan, A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta. J Vis Exp,(2017))의 방법을 따랐으며, 이는 다음과 같다. 분만 후 30분안에 가로 및 세로 2 내지 3 cm로 태반 조직을 자른 후 유리판으로 밀어 융모조직(villous tissue) 주변 혈관을 제거한 후 PBS로 세척하였다. 가위로 조직을 보다 세밀히 자른 후 DNase와 트립신(trypsin)을 이용한 다이제스천(digestion)과정을 3번 진행하여 부유층을 수거하였다. 수거한 부유층은 FBS와 함께 원심분리를 실시하고 적혈구층과 FBS층 사이 흰색의 영양막(trophoblast)세포 층을 확인하고 수집하였다. 수집된 영양막 세포들은 밀도 차등(density gradient) 원심분리법으로 다시 한번 분리과정을 거쳐 최종 영양막 세포를 태반조직으로부터 분리해냈다. 상기 분리과정은 도 34에 나타내었다. 본 분리과정에서 융모 조직 100g당 3.5×107세포의 일차 영양막세포를 얻을 수 있었다. 분리된 세포는 IMDM 배양액에 10%(v/v)FBS, 1%(v/v)페니실린/스트렙토마이신 항생제를 포함하는 배양액에서 체외배양을 진행하였으며, 현미경 사진 및 나노입자 분석결과를 도 35에 나타내었다. 배양 4일을 전후로 나노입자의 농도가 감소하고 입자의 크기가 커지는 것으로 확인되었다. 일차 인간영양막세포는 증식과 계대를 하지 않는 특징을 가졌으며, 도 36 좌측의 현미경사진에서 확인할 수 있듯이 체외배양 72시간 후에는 다핵세포(multinucelated cells)의 모습을 보였다. 또한, 도 36의 우측에서 특이적 마커 단백질인 시토케라틴 7(cytokeratin 7)을 발현함을 확인하여, 본 세포가 일차 인간영양막세포임을 확인하였다. Primary human trophoblast cells were isolated from the placenta of a pregnant woman and cultured in vitro for 15 days, and the culture medium was collected every day to confirm the proliferation of mesenchymal stem cells and the efficacy of bone regeneration. The cell isolation process is a preceding study (B. Simon, M. Bucher, A. Maloyan, A Primary Human Trophoblast Model to Study the Effect of Inflammation Associated with Maternal Obesity on Regulation of Autophagy in the Placenta. J Vis Exp, (2017)) was followed, which is as follows. Within 30 minutes after delivery, the placental tissue was cut into 2 to 3 cm horizontally and vertically, pushed to a glass plate to remove blood vessels around the villous tissue, and then washed with PBS. After more finely cutting the tissue with scissors, a digestion process using DNase and trypsin was performed three times to collect the floating layer. The collected floating layer was subjected to centrifugation together with FBS, and a white trophoblast cell layer between the red blood cell layer and the FBS layer was identified and collected. The collected trophoblast cells were separated again by density gradient centrifugation to separate the final trophoblast cells from the placental tissue. The separation process is shown in FIG. 34 . In this isolation process, 3.5×10 7 cells of primary trophoblast cells were obtained per 100 g of villous tissue. The isolated cells were cultured in vitro in a culture medium containing 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin antibiotics in the IMDM culture medium, and the micrograph and nanoparticle analysis results are shown in FIG. showed up It was confirmed that the concentration of nanoparticles decreased and the size of the particles increased around 4 days of culture. Primary human trophoblast cells were characterized by non-proliferation and passage, and as shown in the photomicrograph on the left of FIG. 36, multinucleated cells appeared after 72 hours of in vitro culture. In addition, it was confirmed from the right side of FIG. 36 that the specific marker protein,
실시예 6-2: 일차 인간영양막세포 배양액의 중간엽줄기세포 증식 촉진 효능 확인Example 6-2: Confirmation of mesenchymal stem cell proliferation promotion effect of primary human trophoblast cell culture medium
일차 인간영양막세포 배양액 (PTB-CM)을 중간엽줄기세포 배양 시 각각 0%, 15%, 30% 및 60%(v/v)로 처리하고 24일간 배양하여 현미경 사진 및 세포 수를 도 37에 나타내었다. 일차 인간영양막세포 배양액의 처리 농도가 증가함에 따라 세포 증식이 촉진됨을 확인하여 일차 인간영양막세포 배양액의 중간엽줄기세포 증식 효능을 확인하였다.Primary human trophoblast cell culture medium (PTB-CM) was treated with 0%, 15%, 30%, and 60% (v/v), respectively, when culturing mesenchymal stem cells, and cultured for 24 days. Micrographs and cell numbers are shown in FIG. 37 showed up It was confirmed that cell proliferation was promoted as the treatment concentration of the primary human trophoblast cell culture medium increased, confirming the mesenchymal stem cell proliferation effect of the primary human trophoblast cell culture medium.
또한, 산모태반으로부터 얻은 일차 인간영양막세포 배양액(PTB-CM)을 중간엽줄기세포 배양액 (MSC-CM) 및 실시예 1 내지 4의 인간영양막세포 배양액(TSC-CM)과 중간엽줄기세포 증식효능을 비교실험하여 그 결과를 도 38에 나타내었다. 산모태반에서 얻어진 일차 인간영양막세포 배양액(PTB-CM)은 인간영양막세포 배양액(TSC-CM)보다는 낮으나, 중간엽줄기세포 배양액(MSC-CM)보다 우수한 세포 증식 효능을 보였다. In addition, primary human trophoblast cell culture medium (PTB-CM) obtained from maternal placenta was used as mesenchymal stem cell culture medium (MSC-CM) and human trophoblast cell culture medium (TSC-CM) of Examples 1 to 4 and mesenchymal stem cell proliferation efficacy A comparison experiment was performed and the results are shown in FIG. 38 . Primary human trophoblast cell culture medium (PTB-CM) obtained from maternal placenta showed lower cell growth efficacy than human trophoblast culture medium (TSC-CM) but superior to mesenchymal stem cell culture medium (MSC-CM).
FBS 없이 일차 인간영양막세포 배양액(PTB-CM)만을 처리하는 경우에도 중간엽줄기세포의 증식 촉진 효능이 나타나는지 확인하였다. FBS를 처리하지 않은 군과 5% FBS를 처리한 군, FBS 없이 1 내지 7일에 수거된 일차 인간영양막세포 배양액을 처리한 군(Early PTB-CM) 및 FBS 없이 8 내지 14일에 수거된 일차 인간영양막세포 배양액을 처리한 군(Late PTB-CM)을 비교실험하였으며 그 결과를 도 39에 나타내었다. PTB-CM이 처리된 군들은 각각 20%, 40% 및 60%(v/v)로 처리되었다. 일차 인간영양막세포 배양액이 FBS 부존재 하에서도 중간엽줄기세포 증식을 촉진할 수 있음을 확인할 수 있었으며, 특히 초기에 수거된 PTB-CM(Early PTB-CM)의 60% 처리군에서는 FBS 미처리군에 비해 2배 이상의 증식 촉진 효능이 확인되었다. 전체적으로 1 내지 7일에 수거된 초기 배양액(Early PTB-CM) 처리군에서 8 내지 14일에 수거된 후기 배양액(Late PTB-CM) 처리군보다 증식 효능이 높게 나타났다. It was also confirmed that the proliferation promoting effect of mesenchymal stem cells appeared even when only the primary human trophoblast cell culture medium (PTB-CM) was treated without FBS. The group not treated with FBS and the group treated with 5% FBS, the group treated with primary human trophoblast cell culture medium harvested on
실시예 6-3: 일차 인간영양막세포 배양액의 골재생 효능 확인Example 6-3: Confirmation of bone regeneration efficacy of primary human trophoblast cell culture medium
일차 인간영양막세포 배양액의 골재생 효능을 ALP활성화, 칼슘침착도 및 RUNX2 유전자 발현 변화로 확인하였다. The bone regeneration efficacy of the primary human trophoblast cell culture medium was confirmed by changes in ALP activation, calcium deposition and RUNX2 gene expression.
조골세포에 골형성유도배지(OIM) 처리 시 일차 인간영양막세포 배양액(PTB-CM)을 10% 및 30%로 처리한 후 ALP 활성도, 칼슘침착도 및 RUNX2 발현량을 확인하여 도 40에 나타내었다. ALP 활성도 및 RUNX2 발현량은 7일차에 측정되었으며, 칼슘침착도는 14일차에 측정되었다. 일차 인간영양막세포 배양액 처리군에서 미처리군 및 대조군과 비교하여 ALP 활성도, 칼슘침착도 및 RUNX2 발현량이 유의하게 증가된 것으로 확인되어 일차 인간영양막세포 배양액도 골재생 촉진 효능을 가짐을 확인하였다. When osteoblasts were treated with bone formation induction medium (OIM), primary human trophoblast culture medium (PTB-CM) was treated with 10% and 30%, and then ALP activity, calcium deposition, and RUNX2 expression levels were confirmed and shown in FIG. 40 . ALP activity and RUNX2 expression levels were measured on the 7th day, and calcium deposition was measured on the 14th day. It was confirmed that ALP activity, calcium deposition, and RUNX2 expression levels were significantly increased in the primary human trophoblast culture medium-treated group compared to the untreated group and the control group, confirming that the primary human trophoblast cell culture medium also had bone regeneration promoting efficacy.
이상과 같이 실시예들이 비록 한정된 실시예와 도면에 의해 설명되었으나, 해당 기술분야에서 통상의 지식을 가진 자라면 상기의 기재로부터 다양한 수정 및 변형이 가능하다. 예를 들어, 설명된 기술들이 설명된 방법과 다른 순서로 수행되거나, 및/또는 설명된 구성요소들이 설명된 방법과 다른 형태로 결합 또는 조합되거나, 다른 구성요소 또는 균등물에 의하여 대치되거나 치환되더라도 적절한 결과가 달성될 수 있다.As described above, although the embodiments have been described with limited examples and drawings, those skilled in the art can make various modifications and variations from the above description. For example, even if the described techniques are performed in a different order from the described method, and/or the described components are combined or combined in a different form than the described method, or substituted or replaced by other components or equivalents. Appropriate results can be achieved.
그러므로, 다른 구현들, 다른 실시예들 및 청구범위와 균등한 것들도 후술하는 청구범위의 범위에 속한다.Therefore, other implementations, other embodiments, and equivalents of the claims are within the scope of the following claims.
Claims (9)
A composition for promoting skin regeneration comprising at least one selected from the group consisting of human trophoblast cell culture medium and derivatives thereof as an active ingredient.
The composition for promoting skin regeneration according to claim 1, wherein the composition for promoting skin regeneration further comprises human mesenchymal stem cells.
The composition for promoting skin regeneration according to claim 1 or 2, wherein the skin regeneration promotion promotes recovery of skin defects or skin wounds.
A cosmetic composition comprising the composition for accelerating skin regeneration according to any one of claims 1 or 2.
The cosmetic composition of claim 4, skin, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrient lotion, massage cream, nutrient cream, eye cream, moisture cream, hand cream, essence, nutrient essence, pack , A cosmetic composition characterized in that it is formulated into a cleansing foam, cleansing water, cleansing lotion, cleansing cream, body lotion, body cleanser, soap or powder.
(b)상기 배양된 인간영양막세포를 포함하는 용액을 원심분리하여 상층액을 수거하는 단계; 및
(c)상기 수거된 상층액을 다공성 필터를 이용하여 멸균 정제하여 배양액을 수득하는 단계;
를 포함하는, 피부재생 촉진용 조성물의 제조방법.
(a) culturing human trophoblast cells in vitro;
(b) collecting the supernatant by centrifuging the solution containing the cultured human trophoblast cells; and
(c) obtaining a culture solution by sterilizing and purifying the collected supernatant using a porous filter;
Containing, a method for producing a composition for promoting skin regeneration.
상기 (a)단계의 상기 체외배양은 50 내지 100%의 세포밀도(confluency)로 48 내지 72시간 배양 후 배양액을 교체하는 것인, 피부재생 촉진용 조성물의 제조방법.
According to claim 6,
The in vitro culture of step (a) is to replace the culture medium after culturing for 48 to 72 hours at a cell density (confluency) of 50 to 100%, a method for producing a composition for promoting skin regeneration.
상기 (c)단계의 다공성 필터의 공극지름은 0.2μm이하인, 피부재생 촉진용 조성물의 제조방법.
According to claim 6,
The pore diameter of the porous filter in step (c) is 0.2 μm or less, a method for producing a composition for promoting skin regeneration.
상기 단계 이후에,
(d) 300G에서 10분, 2,000G에서 10분, 10,000G에서 30분, 100,000G에서 70분동안 원심분리하는 단계; 및
(e) 상기 원심분리를 동일하게 1회 더반복하는 단계;
를 더 포함하는, 피부재생 촉진용 조성물의 제조방법.
According to claim 6,
After the above steps,
(d) centrifugation at 300G for 10 minutes, 2,000G for 10 minutes, 10,000G for 30 minutes, and 100,000G for 70 minutes; and
(e) repeating the same centrifugation one more time;
Further comprising a method for producing a composition for promoting skin regeneration.
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| CA3140452A1 (en) * | 2018-06-11 | 2019-12-19 | Angelita De Francisco | Extracellular vesicles derived from mesenchymal stem cells |
| KR102049505B1 (en) * | 2019-05-14 | 2019-11-29 | 이장호 | Production Methods of Immune-tolerant Extracellular Vesicle containing Lactate Dehydrogenase B and Peroxisome proliferator-activated receptor gamma coactivator 1-alpha and Therapeutic Composition for Anti-Cancer thereof |
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2022
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR20150083440A (en) * | 2014-01-08 | 2015-07-17 | 사회복지법인 삼성생명공익재단 | Stem cells derived from chorionic trophoblast layer without villi, and cellular therapeutic agents comprising the same |
| KR20210143721A (en) * | 2019-03-27 | 2021-11-29 | 플루리스템 리미티드 | Methods and compositions for aesthetic and cosmetic treatment and stimulation of hair growth |
| KR20220107789A (en) * | 2021-01-26 | 2022-08-02 | 현대모비스 주식회사 | Rctb system and control method thereof |
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