KR20230137808A - Composition for promoting angiogenesis comprising adipose tissue-derived mesenchymal stem cells and human stromal vascular fraction - Google Patents
Composition for promoting angiogenesis comprising adipose tissue-derived mesenchymal stem cells and human stromal vascular fraction Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
Abstract
본 발명은 지방 조직 유래 중간엽 줄기세포 및 인간 기질 혈관 분획을 포함하는 혈관신생 촉진용 조성물에 관한 것으로, 본 발명의 혈관신생 촉진용 조성물은 혼합하여 하지에 이식할 때 상승적인 혈관 신생에 우수한 효과를 나타내므로 혈관신생 촉진 용도로 유용하게 사용될 수 있다.The present invention relates to a composition for promoting angiogenesis comprising adipose tissue-derived mesenchymal stem cells and human stromal vascular fraction. The composition for promoting angiogenesis of the present invention has an excellent effect on synergistic angiogenesis when mixed and transplanted to the lower extremity. Therefore, it can be usefully used to promote angiogenesis.
Description
본 발명은 지방 조직 유래 중간엽 줄기세포 및 인간 기질 혈관 분획을 포함하는 혈관신생 촉진용 의약 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for promoting angiogenesis comprising adipose tissue-derived mesenchymal stem cells and human stromal vascular fraction.
줄기세포 치료는 도입 초기부터 손상된 장기의 치료를 위해 매력적인 치료 옵션으로 떠올랐다. 줄기세포는 다분화능 (multipotency), 용이한 접근, 다양한 조직으로부터의 용이한 분리 및 배양, 높은 수율로 인해 세포 치료에 유리하다. 중간엽 줄기세포 (Mesenchymal stem cell)는 혈관과 세포주위에 이식되어 혈관 안정화에 중요한 역할을 할 수 있다.Stem cell therapy has emerged as an attractive treatment option for the treatment of damaged organs since its inception. Stem cells are advantageous for cell therapy due to their multipotency, easy access, easy isolation and culture from various tissues, and high yield. Mesenchymal stem cells can be transplanted into blood vessels and surrounding cells and play an important role in stabilizing blood vessels.
지방조직 유래 기질 혈관분획 (Adipose-derived stromal vascular fraction (SVF))은 중간엽 전구세포/줄기세포, 지방전구세포, 내피세포, pericytes, T 세포 및 M2 대식세포를 포함하는 이질적인 세포 집단이다. 갓 분리된 SVF 세포는 성형 수술 및 기타 임상 응용 분야에서 널리 사용된다.Adipose-derived stromal vascular fraction (SVF) is a heterogeneous cell population that includes mesenchymal progenitors/stem cells, preadipocytes, endothelial cells, pericytes, T cells, and M2 macrophages. Freshly isolated SVF cells are widely used in plastic surgery and other clinical applications.
지방유래 줄기세포 (adipose-derived stem cell, ASCs)는 지방세포, 골모세포, 연골모세포, 근섬유모세포 등으로 분화될 수 있는 다분화능을 가지고 있다. 또한, 지방유래 줄기세포는 중배엽 조직 세포 뿐만 아니라 외배엽 조직인 신경세포로도 분화될 수 있다는 것이 확인되었다.Adipose-derived stem cells (ASCs) have multipotent ability to differentiate into adipocytes, osteoblasts, chondroblasts, and myofibroblasts. In addition, it was confirmed that adipose-derived stem cells can be differentiated into not only mesodermal tissue cells but also ectodermal tissue nerve cells.
한편, VEGF-A/PDGF-b 및 FGF-2와 같은 혈관신생 인자는 혈관신생 촉진 효과에 강력한 시너지를 발휘한다. 유도된 만능 줄기 세포로부터 유래된 초기/후기 내피 전구 세포 또는 심근 세포와 같은, 이중줄기세포치료제는 또한 줄기 세포 호밍 (homing) 및 측분비 인자의 장기간 분비에 의한 허혈성 심장의 심장 기능 및 혈관 재생의 시너지 효과를 보여준다.Meanwhile, angiogenic factors such as VEGF-A/PDGF-b and FGF-2 exert strong synergy in promoting angiogenesis. Dual stem cell therapies, such as early/late endothelial progenitor cells or cardiomyocytes derived from induced pluripotent stem cells, can also improve cardiac function and vascular regeneration in ischemic hearts by stem cell homing and prolonged secretion of paracrine factors. It shows a synergistic effect.
다만, 이러한 줄기세포치료는 허혈성 심장질환에만 적용된다는 한계를 나타내었으며, 심장에만 국한 된 것이 아닌 다른 신체 부위에서의 VEGF-A/PDGF-b 및 FGF-2와 같은 혈관신생 인자를 모두 발현하고, 혈관 재생을 나타낼 수 있는 조성물의 개발이 절실히 요구되는 실정이다.However, this stem cell treatment has the limitation of being applied only to ischemic heart disease, and is not limited to the heart, expressing angiogenic factors such as VEGF-A/PDGF-b and FGF-2 in other body parts, There is an urgent need for the development of a composition that can demonstrate blood vessel regeneration.
본 발명자들은 높은 수준의 PDGF-b 및 VEGF-A를 발현하는 지방조직 유래 기질 혈관분획 (Adipose-derived stromal vascular fraction (SVF))과 높은 수준의 FGF-2를 발현하는 지방유래 줄기세포 (adipose-derived stem cell, ASC)를 혼합하여 하지에 이식할 때 상승적인 혈관 신생에 우수한 효과를 나타냄을 확인하여 본 발명을 완성하였다.The present inventors used adipose-derived stromal vascular fraction (SVF), which expresses high levels of PDGF-b and VEGF-A, and adipose-derived stem cells, which express high levels of FGF-2. The present invention was completed by confirming that a mixture of derived stem cells (ASC) exhibits an excellent effect on synergistic angiogenesis when transplanted to the lower extremities.
이에, 본 발명의 목적은 지방 조직 유래 중간엽 줄기세포 및 인간 기질혈관분획을 포함하는 혈관신생 촉진용 의약 조성물을 제공하는 것이다.Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for promoting angiogenesis comprising adipose tissue-derived mesenchymal stem cells and human stromal vascular fraction.
본 발명의 다른 목적은 지방 조직 유래 중간엽 줄기세포 및 인간 기질혈관분획을 포함하는 중증하지 허혈 예방 또는 치료용 의약 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating severe lower extremity ischemia comprising adipose tissue-derived mesenchymal stem cells and human stromal vascular fraction.
이하, 본 발명을 더욱 자세히 설명하고자 한다.Hereinafter, the present invention will be described in more detail.
본 발명의 일 양태는 지방 조직 유래 중간엽 줄기세포 (Adipose tissue-derived mesenchymal stem cells; ASC) 및 인간 기질혈관분획 (Human Stromal vascular Fraction; SVF)을 포함하는 혈관신생 촉진용 의약 조성물에 관한 것이다.One aspect of the present invention relates to a pharmaceutical composition for promoting angiogenesis containing adipose tissue-derived mesenchymal stem cells (ASC) and human stromal vascular fraction (SVF).
본 발명에 있어서 상기 지방 조직 유래 중간엽 줄기세포 및 인간 기질혈관분획은 1:9 내지 9:1 비율, 2:8 내지 8:2 비율, 3:7 내지 7:3 비율, 예를 들어, 2:8의 비율로 혼합된 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the adipose tissue-derived mesenchymal stem cells and human stromal vascular fraction are used in a ratio of 1:9 to 9:1, 2:8 to 8:2, and 3:7 to 7:3, for example, 2 It may be mixed at a ratio of :8, but is not limited to this.
본 명세서 상 용어 "지방 조직"은 백색지방, 황색지방 또는 갈색지방으로 구성된 조직을 의미한다.As used herein, the term “adipose tissue” refers to tissue composed of white fat, yellow fat, or brown fat.
본 발명에 있어서 지방조직은 살아있는 인간 또는 동물에서 얻게되며, 예를 들어, 하복부, 종아리, 허벅지, 팔뚝, 유방 또는 슬관절 지방조직으로부터 유래된 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, adipose tissue is obtained from a living human or animal, and may, for example, be derived from lower abdomen, calf, thigh, forearm, breast, or knee joint adipose tissue, but is not limited thereto.
본 명세서 상 용어 "줄기세포"란 자기 복제 능력을 가지면서 두 개 이상의 서로 다른 종류의 세포로 분화하는 능력을 가진 세포를 의미한다.As used herein, the term “stem cell” refers to a cell that has the ability to self-replicate and differentiate into two or more different types of cells.
본 명세서 상 용어 "중간엽 줄기세포"란 골, 연골, 지방, 골수간질, 근육, 신경 등으로 분화될 수 있는 미분화 된 줄기세포를 의미하는데, 성인에서는 일반적으로 골수에 머물러 있지만 제대혈, 말초혈액, 기타 조직 등에서도 존재하는 것을 의미한다.As used herein, the term "mesenchymal stem cells" refers to undifferentiated stem cells that can be differentiated into bone, cartilage, fat, bone marrow stroma, muscles, nerves, etc. In adults, they generally remain in the bone marrow, but are stored in umbilical cord blood, peripheral blood, This means that it also exists in other organizations.
본 발명에 있어서 인간 기질혈관분획은 지방 전구 세포 (Preadipocytes), 내피세포 (EC), 혈관내피전구 세포 (EPC), 혈관 평활근 세포 (SMC), 혈관 주위 세포 (Pericytes), 벽세포, 대식세포 (Macrophages) 및 섬유 모세포로 이루어진 군에서 선택된 1종 이상의 미분화 세포로 혼합된 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the human stromal vascular fraction includes preadipocytes (Preadipocytes), endothelial cells (EC), vascular endothelial progenitor cells (EPC), vascular smooth muscle cells (SMC), pericytes, mural cells, and macrophages ( It may be a mixture of one or more types of undifferentiated cells selected from the group consisting of macrophages and fibroblasts, but is not limited thereto.
본 발명에 있어서 지방 조직 유래 중간엽 줄기세포는 섬유아세포증식인자 (FGF-2), 혈소판유래성장인자 서브유닛 b (PDGF-b) 및 혈관내피성장인자 A (VEGF-A)로 이루어진 군에서 선택된 1종 이상의 인자의 발현 특성을 갖는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, adipose tissue-derived mesenchymal stem cells are selected from the group consisting of fibroblast growth factor (FGF-2), platelet-derived growth factor subunit b (PDGF-b), and vascular endothelial growth factor A (VEGF-A). It may have expression characteristics of one or more types of factors, but is not limited thereto.
본 발명에 있어서 인간 지질혈관분획은 섬유아세포증식인자-2 (FGF-2), 혈소판유래성장인자 서브유닛 b (PDGF-b) 및 혈관내피성장인자 A (VEGF-A)로 이루어진 군에서 선택된 1종 이상의 인자의 발현 특성을 갖는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the human lipid vascular fraction is one selected from the group consisting of fibroblast growth factor-2 (FGF-2), platelet-derived growth factor subunit b (PDGF-b), and vascular endothelial growth factor A (VEGF-A). It may have expression characteristics of more than one type of factor, but is not limited thereto.
본 발명에 있어서 혈관신생 (Angiogenesis)은 기존 혈관의 내피세포가 세포외 기질을 분해하고, 이동, 분열 및 분화하여 새로운 모세혈관을 형성하는 현상으로, 성장과 생식, 상처 치유 등의 생리적 과정에서 나타난다. 또한, 혈관신생은 혈관내피세포의 성장, 관절염, 당뇨병 등의 병리적 상태와도 연관이 있다. 혈관신생은 혈관내피세포의 성장, 이동 , 분화, 모세혈관 형성 등의 복잡한 일련의 과정에 관여하는 과정을 의미한다.In the present invention, angiogenesis is a phenomenon in which endothelial cells of existing blood vessels break down extracellular matrix, migrate, divide, and differentiate to form new capillaries, and occur in physiological processes such as growth, reproduction, and wound healing. . Additionally, angiogenesis is also associated with pathological conditions such as growth of vascular endothelial cells, arthritis, and diabetes. Angiogenesis refers to a process involved in a complex series of processes such as growth, migration, differentiation, and capillary formation of vascular endothelial cells.
본 발명의 다른 일 양태는 지방 조직 유래 중간엽 줄기세포 (Adipose tissue-derived mesenchymal Stem Sells; ASC) 및 인간 기질혈관분획 (Human stromal Hascular Fraction; SVF)을 포함하는 중증하지 허혈 예방 또는 치료용 의약 조성물에 관한 것이다.Another aspect of the present invention is a pharmaceutical composition for preventing or treating severe lower extremity ischemia comprising adipose tissue-derived mesenchymal Stem Cells (ASC) and human stromal vascular fraction (SVF). It's about.
본 명세서 상 용어 "조성물 (agent)"는 인간으로부터 분리, 배양 및 특수한 조작을 통해 제조된 세포 및 조직으로 치료, 진단 및 예방의 목적으로 사용되는 의약품 (미국 FDA 규정)으로서, 세포 혹은 조직의 기능을 복원시키기 위하여 살아있는 자가, 동종, 또는 이종 세포를 체외에서 증식 선별하거나 다른 방법으로 세포의 생물학적 특성을 변화시키는 등의 일련의 행위를 통하여 이러한 세포가 질병의 치료, 진단 및 예방의 목적으로 사용되는 의약품을 의미한다.In this specification, the term "composition (agent)" refers to cells and tissues manufactured through isolation, culture, and special manipulation from humans, and is a medicine (US FDA regulations) used for treatment, diagnosis, and prevention purposes, and refers to the function of cells or tissues. These cells can be used for the purpose of treatment, diagnosis, and prevention of disease through a series of actions such as proliferating and selecting living autologous, allogeneic, or xenogeneic cells in vitro or changing the biological characteristics of cells by other methods in order to restore health. It means medicine.
본 발명의 조성물의 바람직한 투여량은 개체의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다. 투여는 하루에 한번 투여할 수도 있고, 수회에 나누어 투여할 수도 있으며, 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The preferred dosage of the composition of the present invention varies depending on the individual's condition and weight, degree of disease, drug form, administration route and period, but can be appropriately selected by a person skilled in the art. Administration may be administered once a day, or may be administered in several divided doses, and the above dosage does not limit the scope of the present invention in any way.
본 발명에 있어서 치료용 의약 조성물은 지방 조직 유래 중간엽 줄기세포 및 인간 기질혈관분획을 수용하기 위한 지지체, 바람직하게는 생분해성 지지체를 더 포함할 수 있다. 이때, 상기 생분해성 지지체는 피브린 글루, 히알루론산, 젤라틴, 콜라겐, 알긴산, 셀룰로오스, 펙틴, 키틴, 폴리글라이콜산, 또는 폴리유산과 같은 하이드로겔일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the pharmaceutical composition for treatment may further include a support, preferably a biodegradable support, for accommodating adipose tissue-derived mesenchymal stem cells and human stromal vascular fraction. At this time, the biodegradable support may be a hydrogel such as fibrin glue, hyaluronic acid, gelatin, collagen, alginic acid, cellulose, pectin, chitin, polyglycolic acid, or polylactic acid, but is not limited thereto.
본 발명에 있어서 치료용 의약 조성물은 약학적으로 허용가능한 담체를 더 포함할 수 있다. 상기 약학적으로 허용가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1종 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액 및 정균제 등 다른 통상의 첨가제를 첨가할 수 있다.In the present invention, the therapeutic pharmaceutical composition may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these ingredients, and if necessary, antioxidant, Other common additives such as buffers and bacteriostatic agents can be added.
본 발명에 있어서 치료용 의약 조성물은 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형으로 제제화 할 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the therapeutic pharmaceutical composition can be formulated into an injectable formulation such as an aqueous solution, suspension, emulsion, etc. by additionally adding a diluent, dispersant, surfactant, binder, and lubricant, but is not limited thereto.
본 발명에 있어서 지방 조직 유래 중간엽 줄기세포 및 인간 기질혈관분획은 1:9 내지 9:1 비율, 2:8 내지 8:2 비율, 3:7 내지 7:3의 비율, 예를 들어, 2:8의 비율로 혼합된 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, adipose tissue-derived mesenchymal stem cells and human stromal vascular fraction are used in a ratio of 1:9 to 9:1, 2:8 to 8:2, and 3:7 to 7:3, for example, 2 It may be mixed at a ratio of :8, but is not limited to this.
본 발명에 있어서 인간 기질혈관분획은 지방 전구 세포 (Preadipocytes), 내피세포 (EC), 혈관내피전구 세포 (EPC), 혈관 평활근 세포 (SMC), 혈관 주위 세포 (Pericytes), 벽세포, 대식세포 (Macrophages) 및 섬유 모세포로 이루어진 군에서 선택된 1종 이상의 세포로 혼합된 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the human stromal vascular fraction includes preadipocytes (Preadipocytes), endothelial cells (EC), vascular endothelial progenitor cells (EPC), vascular smooth muscle cells (SMC), pericytes, mural cells, and macrophages ( It may be a mixture of one or more types of cells selected from the group consisting of macrophages and fibroblasts, but is not limited thereto.
본 발명에 있어서 중증하지 허혈은 동맥경화성 말초동맥질환인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, critical limb ischemia may be arteriosclerotic peripheral artery disease, but is not limited thereto.
본 발명에 있어서 "예방"은 본 발명의 조성물의 투여로 중증하지 허혈을 억제하거나 진행을 지연시키는 모든 행위를 의미한다.In the present invention, “prevention” refers to any action that suppresses or delays the progression of critical limb ischemia by administering the composition of the present invention.
본 발명에 있어서 "치료" 및 "개선"은 본 발명의 조성물의 투여로 중증하지 허혈의 증상이 호전 또는 이롭게 변경되는 모든 행위를 의미한다.In the present invention, “treatment” and “improvement” refer to any action in which the symptoms of critical limb ischemia are improved or beneficially changed by administration of the composition of the present invention.
본 발명에 있어서 의약 조성물의 치료학적인 유효량 (therapeutically effective amount)은 연구자, 수의사, 의사 또는 기타 임상에 의해 생각되는 조직계, 동물 또는 인간에서 생물학적 또는 의학적 반응을 유도하는 유효 성분 또는 약학적 조성물의 양을 의미하는 것으로, 이는 치료되는 질환 또는 장애의 증상의 완화를 유도하는 양을 포함한다. 본 발명의 세포치료제 조성물에 포함되는 세포의 함량(수)은 원하는 효과에 따라 변화될 것임은 당업자에게 자명하다. 그러므로 최적의 세포 치료제 조성물 함량은 당업자에 의해 쉽게 결정될 수 있으며, 질환의 종류, 질환의 중증도, 조성물에 함유된 다른 성분의 함량, 제형의 종류, 및 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료 기간, 동시 사용되는 약물을 비롯한 다양한 인자에 따라 조절될 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the therapeutically effective amount of a pharmaceutical composition refers to the amount of the active ingredient or pharmaceutical composition that induces a biological or medical response in the tissue system, animal, or human as considered by researchers, veterinarians, doctors, or other clinicians. By this means, it includes amounts that lead to alleviation of the symptoms of the disease or disorder being treated. It is obvious to those skilled in the art that the content (number) of cells included in the cell therapy composition of the present invention will vary depending on the desired effect. Therefore, the optimal cell therapy composition content can be easily determined by a person skilled in the art, and can be determined based on the type of disease, the severity of the disease, the content of other ingredients contained in the composition, the type of formulation, and the patient's age, weight, general health, gender, and It may be adjusted according to various factors including diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs, but is not limited thereto.
본 발명에 있어서 조성물은 지방 조직 유래 중간엽 줄기세포 및 인간 기질 혈관 분획을 투여개체의 체중 kg 당 1 X 106 내지 1 X 107 세포수로 투여할 수 있다.In the present invention, the composition may be administered with adipose tissue - derived mesenchymal stem cells and human stromal vascular fraction at a cell number of 1
본 발명의 또 다른 일 양태 예에 따르면, 본 발명에 따른 세포 치료제 조성물을 포함하는 중증하지 허혈 예방 또는 치료용 개인 맞춤형 키트를 제공할 수 있으며, 상기 키트는 당업계에서 사용되고 있는 키트 제조방법에 따라 제조 할 수 있고, 다만 치료 효과를 유도할 수 있는 유효성분으로서 본 발명의 지방 조직 유래 중간엽 줄기세포 및 인간 기질 혈관 분획을 유효성분을 포함할 수 있다.According to another embodiment of the present invention, a personalized kit for preventing or treating severe lower extremity ischemia containing the cell therapy composition according to the present invention can be provided, and the kit is manufactured according to a kit manufacturing method used in the art. It can be manufactured and may contain the adipose tissue-derived mesenchymal stem cells and human stromal vascular fraction of the present invention as active ingredients that can induce a therapeutic effect.
본 발명은 지방 조직 유래 중간엽 줄기세포 및 인간 기질 혈관 분획을 포함하는 혈관신생 촉진용 의약 조성물에 관한 것으로, 본 발명의 혈관신생 촉진용 의약 조성물은 혼합하여 하지에 이식할 때 상승적인 혈관 신생에 우수한 효과를 나타내므로 혈관신생 촉진 용도로 유용하게 사용될 수 있다.The present invention relates to a pharmaceutical composition for promoting angiogenesis comprising adipose tissue-derived mesenchymal stem cells and human stromal vascular fraction. The pharmaceutical composition for promoting angiogenesis of the present invention provides synergistic angiogenesis when mixed and transplanted to the lower extremity. Because it has excellent effects, it can be usefully used to promote angiogenesis.
도 1은 본 발명의 일 실시예에 따른 혈관신생 유전자의 발현 수준을 비교한 결과를 보여주는 그래프이다.
도 2는 본 발명의 일 실시예에 따른 SVF 세포, ASC 세포 및 ASC+SVF 세포 를 주입한 후 마트리겔 (Matrigel) 플러그 (Plug)의 외형을 보여주는 도이다.
도 3은 본 발명의 일 실시예에 따른 SVF 세포 및 ASC+SVF 세포 내의 헤모글로빈 ?량을 정량화한 결과를 보여주는 그래프이다.
도 4는 본 발명의 일 실시예에 따른 각각의 비율에 따른 ASC 및 SVF 세포의 비율에 따른 헤모글로빈 함량을 정량화한 결과를 보여주는 그래프이다.
도 5는 본 발명의 일 실시예에 따른 SVF 세포 및 ASC+SVF 세포를 CM과 배양한 후 튜브형성을 관찰한 결과를 보여주는 도이다.
도 6은 본 발명의 일 실시예에 따른 SVF 세포 및 ASC+SVF 세포의 튜브 길이 및 분기점 수를 측정한 결과를 보여주는 그래프이다.
도 7은 본 발명의 일 실시예에 따른 SVF 세포 및 ASC+SVF 세포의 단층을 긁고 CM에 배양한 후 스크래피 상처를 관찰한 결과를 보여주는 도이다.
도 8은 본 발명의 일 실시예에 따른 SVF 세포 및 ASC+SVF 세포의 봉합율 (spave closure)(%)을 측정한 결과를 보여주는 그래프이다.
도 9은 본 발명의 일 실시예에 따른 SVF 세포 및 ASC+SVF 세포의 세포사멸도를 측정한 유세포 분석 결과를 보여주는 도이다.
도 10은 본 발명의 일 실시예에 따른 SVF 세포 및 ASC+SVF 세포의 세포사멸도를 측정한 결과를 보여주는 그래프이다.
도 11은 본 발명의 일 실시예에 따른 SVF 세포 및 ASC+SVF 세포의 LDH 수준을 측정한 결과를 보여주는 그래프이다.
도 12는 본 발명의 일 실시예에 따른 ASC+SVF 세포를 주사한 사지에서 혈액관류를 측정하여 나타낸 도이다.
도 13은 본 발명의 일 실시예에 따른 SVF 세포 및 ASC+SVF 세포를 주입한 팔다리를 비교한 결과를 보여주는 도이다.
도 14는 본 발명의 일 실시예에 따른 SVF 세포 및 ASC+SVF 세포의 사지 회수 비율(%)을 측정한 결과를 보여주는 그래프이다.
도 15는 본 발명의 일 실시예에 따른 SVF 세포 및 ASC+SVF 세포 주입 후 중증 하지허혈 회수를 보여주는 도이다.
도 16는 본 발명의 일 실시예에 따른 SVF 세포 및 ASC+SVF 세포 주입 후 사지 회수의 정략적인 분석 결과(%)를 보여주는 그래프이다.
도 17은 본 발명의 일 실시예에 따른 SVF 세포 및 ASC+SVF 세포 주입 후 근육의 퇴화 정도를 보여주는 도이다.
도 18은 본 발명의 일 실시예에 따른 SVF 세포 및 ASC+SVF 세포 주입 후 모세관 밀도를 보여주는 도이다.
도 19는 본 발명의 일 실시예에 따른 SVF 세포 및 ASC+SVF 세포 주입 후 모세관 밀도를 측정 결과를 보여주는 그래프이다.
도 20은 본 발명의 일 실시예에 따른 SVF 세포 및 ASC+SVF 세포 주입 후 하지 조직에서 혈관 신생 인자 발현 분포를 측정한 결과를 보여주는 그래프이다.Figure 1 is a graph showing the results of comparing the expression levels of angiogenic genes according to an embodiment of the present invention.
Figure 2 is a diagram showing the appearance of a Matrigel plug after injection of SVF cells, ASC cells, and ASC+SVF cells according to an embodiment of the present invention.
Figure 3 is a graph showing the results of quantifying the amount of hemoglobin in SVF cells and ASC+SVF cells according to an embodiment of the present invention.
Figure 4 is a graph showing the results of quantifying the hemoglobin content according to the ratio of ASC and SVF cells according to each ratio according to an embodiment of the present invention.
Figure 5 is a diagram showing the results of observing tube formation after culturing SVF cells and ASC+SVF cells with CM according to an embodiment of the present invention.
Figure 6 is a graph showing the results of measuring the tube length and number of branch points of SVF cells and ASC+SVF cells according to an embodiment of the present invention.
Figure 7 is a diagram showing the results of observing scrapie wounds after scraping a monolayer of SVF cells and ASC+SVF cells and culturing them in CM according to an embodiment of the present invention.
Figure 8 is a graph showing the results of measuring the spave closure (%) of SVF cells and ASC+SVF cells according to an embodiment of the present invention.
Figure 9 is a diagram showing the results of flow cytometry measuring the degree of apoptosis of SVF cells and ASC+SVF cells according to an embodiment of the present invention.
Figure 10 is a graph showing the results of measuring the degree of apoptosis of SVF cells and ASC+SVF cells according to an embodiment of the present invention.
Figure 11 is a graph showing the results of measuring the LDH level of SVF cells and ASC+SVF cells according to an embodiment of the present invention.
Figure 12 is a diagram showing blood perfusion measured in a limb injected with ASC+SVF cells according to an embodiment of the present invention.
Figure 13 is a diagram showing the results of comparing limbs injected with SVF cells and ASC+SVF cells according to an embodiment of the present invention.
Figure 14 is a graph showing the results of measuring the limb recovery ratio (%) of SVF cells and ASC+SVF cells according to an embodiment of the present invention.
Figure 15 is a diagram showing the recovery of severe limb ischemia after injection of SVF cells and ASC+SVF cells according to an embodiment of the present invention.
Figure 16 is a graph showing the quantitative analysis results (%) of limb recovery after injection of SVF cells and ASC+SVF cells according to an embodiment of the present invention.
Figure 17 is a diagram showing the degree of muscle degeneration after injection of SVF cells and ASC+SVF cells according to an embodiment of the present invention.
Figure 18 is a diagram showing capillary density after injection of SVF cells and ASC+SVF cells according to an embodiment of the present invention.
Figure 19 is a graph showing the results of measuring capillary density after injection of SVF cells and ASC+SVF cells according to an embodiment of the present invention.
Figure 20 is a graph showing the results of measuring the distribution of angiogenic factor expression in lower extremity tissues after injection of SVF cells and ASC+SVF cells according to an embodiment of the present invention.
이하 하나 이상의 구체예를 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 하나 이상의 구체예를 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes of one or more specific examples and the scope of the present invention is not limited to these examples.
제조예 1. 인간 기질혈관분획 분리Preparation Example 1. Isolation of human stromal vascular fraction
인간 기질혈관분획 (Stromal Vascular Fraction; 이하, SVF)은 가톨릭관동대학교 병원 기관심사위원회에서 승인한 가이드 라인을 사용하여 건강한 기증자로부터 사전 동의를 받은 후 피하 지방 조직 (subcutaneous fat tissue)에서 분리하였다. 각각의 3명의 기증자로부터 분리한 지방조직을 0.075% 콜라게나제 용액 (collagenase solution)으로 37 ℃에서 1시간 동안 처리한 다음 원심분리로 분리하였다. 세포 펠렛은 적혈구 용해 완충액에 재현탁한 후, 현탁된 세포를 100 μm 메쉬 필터를 사용하여 여과하여 인간 SVF 세포를 수득하였다.Human Stromal Vascular Fraction (SVF) was isolated from subcutaneous fat tissue after obtaining informed consent from healthy donors using guidelines approved by the institutional review board of Catholic Kwandong University Hospital. Adipose tissue isolated from each of the three donors was treated with 0.075% collagenase solution at 37°C for 1 hour and then separated by centrifugation. The cell pellet was resuspended in red blood cell lysis buffer, and the suspended cells were filtered using a 100 μm mesh filter to obtain human SVF cells.
제조예 2. 세포 배양Preparation Example 2. Cell Culture
인간 제대 정맥 내피 세포 (HUVEC)는 ATCC사 (Manassas, VA, USA)에서 구매하였고, 내피 성장 완전 배지 (endothelial growth complete media; EGM-2; Lonza Walkersville, MD, USA)에서 배양하였다.Human umbilical vein endothelial cells (HUVEC) were purchased from ATCC (Manassas, VA, USA) and cultured in endothelial growth complete media (EGM-2; Lonza Walkersville, MD, USA).
앞서 제조예 1과 동일한 3명의 공여자로부터 수득한 SVF 세포를 배양 플레이트에 도말하였고, 배양배지 (α-MEM, 10% 소 태아 혈청 (FBS), 100 U/mL의 페니실린 및 100 mg/mL의 스트렙토마이신 포함)에서 5% CO2존재하에, 37 ℃에서 유지하였다. 1주일 후, 부착성 지방줄기세포 (adherent Adipose-derived stem cell; 이하, ASC)가 배양 플레이트에서 관찰 되었으며, 비부착 세포 (non-adherent cell)를 제거하고 ASC를 배양 배지로 확장시켰다.SVF cells obtained from the same three donors as in Preparation Example 1 were plated on a culture plate and cultured in culture medium (α-MEM, 10% fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 mg/mL of streptocysteine. (including mycin) was maintained at 37°C in the presence of 5% CO 2 . One week later, adherent adipose-derived stem cells (ASCs) were observed in the culture plate, non-adherent cells were removed, and ASCs were expanded in culture medium.
실시예 1. 실시간 중합효소 연쇄반응 분석Example 1. Real-time polymerase chain reaction analysis
정량적 실시간 (qRT)-PCR 분석은 RNA-stat (Iso-Tex Diag-nostics, Friendswood, TX, USA)에서 사용된 세포로부터 총 RNA (total RNA)를 분리하는 방법 및 앞서 밝혀진 방법에 의해 수행되었다. 추출된 RNA는 이후 Taqman Reverse Transcription Reagents (Applied Biosystems, Foster City, Ca, USA에 의해 역전사 되었으며, 제조업체의 지침에 따라 수행되었다.Quantitative real-time (qRT)-PCR analysis was performed by isolating total RNA from used cells in RNA-stat (Iso-Tex Diag-nostics, Friendswood, TX, USA) and previously described methods. The extracted RNA was then reverse transcribed by Taqman Reverse Transcription Reagents (Applied Biosystems, Foster City, Ca, USA) and was performed according to the manufacturer's instructions.
합성된 cDNA는 인간/마우스 특이적 프라이머 및 프로브를 사용하여 qRT-PCR에 적용되었다. RNA 레벨 (level)은 ABI PRISM 7000 Sequence Detection System (applied Biosystems, Fostercity, CA, USA)을 사용하여 정량적으로 평가되었다. GAPDH 발현으로 정규화된 상대적인 mRNA 발현이 측정되었다. qRT-PCR을 위해 사용된 프라이머 및 프로브 세트는 하기 표 1과 같으며, Applied Biosystems사에서 구매하였다. The synthesized cDNA was subjected to qRT-PCR using human/mouse specific primers and probes. RNA levels were quantitatively assessed using the ABI PRISM 7000 Sequence Detection System (applied Biosystems, Fostercity, CA, USA). Relative mRNA expression normalized to GAPDH expression was measured. Primer and probe sets used for qRT-PCR are shown in Table 1 below and were purchased from Applied Biosystems.
qRT-PCR 결과는 도 1에 나타내었다.The qRT-PCR results are shown in Figure 1.
상기 도 1에서 확인할 수 있듯이, ASC 세포는 SVF 세포에 비해 더 높은 수준의 FGF-2를 발현하였다. 그리고 SVF 세포는 ASC 세포에 비해 더 높은 수준의 PDGF-b 및 VEGF-A를 발현하였다.As can be seen in Figure 1, ASC cells expressed a higher level of FGF-2 than SVF cells. And SVF cells expressed higher levels of PDGF-b and VEGF-A than ASC cells.
실시예 2. 인 비보 신생혈관생성 효능 평가Example 2. In vivo angiogenesis efficacy evaluation
7-10주 된 체중 18 내지 22g의 수컷 누드 마우스 (Joongang Laboratory Animal Inc. Seoul, South Korea)를 이소플루란 (인덕션 (induction): 450 mL 공기, 4.5 % 이소플루란, 유지관리: 200mL 공기, 2.0% 이소플루란, Baxter International, Inc., Deerfield, IL, USA)으로 마취시키고, 500 μL의 마트리겔 (Matrigel)을 2 x 105 세포와 함께 마우스에 피하로 주사하였다. 2주 후에, 마우스를 희생 시킨 후, 마트리겔 플러그 (plug)를 수확하였다. Drabkin의 시약 키트 (Sigma Aldrich, St. Louis, MO, USA)로 헤모글로빈 함량 수준을 조사하여 그 결과를 도 2, 3에 나타내었다.Male nude mice (Joongang Laboratory Animal Inc. Seoul, South Korea), 7–10 weeks old and weighing 18 to 22 g, were administered isoflurane (induction: 450 mL air, 4.5% isoflurane, maintenance: 200 mL air, The mouse was anesthetized with 2.0% isoflurane, Baxter International, Inc., Deerfield, IL, USA), and 500 μL of Matrigel was subcutaneously injected into the mouse along with 2 x 10 5 cells. After 2 weeks, the mice were sacrificed and Matrigel plugs were harvested. The hemoglobin content level was examined using Drabkin's reagent kit (Sigma Aldrich, St. Louis, MO, USA), and the results are shown in Figures 2 and 3.
도 2 및 3에서 확인할 수 있듯이, SVF 및 ASC 세포가 포함된 마트리겔 플러그는 SVF 세포만 포함하는 마트리겔 플러그와 비교하였을 때 더 높은 함량의 적혈구가 채워져 있는 것을 확인할 수 있었다. 반면, PBS 대조군에서는 적혈구가 관찰되지 않았다.As can be seen in Figures 2 and 3, the Matrigel plug containing SVF and ASC cells was confirmed to be filled with a higher content of red blood cells compared to the Matrigel plug containing only SVF cells. On the other hand, no red blood cells were observed in the PBS control group.
그 다음으로 SVF 및 ASC 세포의 비율에 따른 헤모글로빈의 함량을 비교하였다. 각각 ASC:SVF 세포의 비율을 5:5, 7:3, 3:7, 2:8 및 8:2의 비율로 조절하여 상기 방법과 동일하게 인 비보 신생혈관생성 효능 평가하여 그 결과를 도 4에 나타내었다.Next, the hemoglobin content according to the ratio of SVF and ASC cells was compared. The ratio of ASC:SVF cells was adjusted to 5:5, 7:3, 3:7, 2:8, and 8:2, respectively, and the in vivo angiogenesis efficacy was evaluated in the same manner as the above method, and the results are shown in Figure 4. shown in
도 4에서 확인할 수 있듯이, ASC 및 SVF 세포를 3:7 및 2:8의 비율로 혼합하였을 때 가장 높은 헤모글로빈의 함량을 나타내었다. 헤모글로빈의 함량이 늘어난 이유는 마트리겔내에 혈관 세포 형성에 가장 좋은 최적의 환경을 제공하였기 때문이며, 2:8의 비율이 가장 우수한 효과를 나타내는 이유는 SVF의 혈관 신생 효과가 크고 ASC의 FGF-2 영향이 시너지적인 효과를 보였기 때문이다.As can be seen in Figure 4, the highest hemoglobin content was observed when ASC and SVF cells were mixed at a ratio of 3:7 and 2:8. The reason why the hemoglobin content increased is because it provided the most optimal environment for the formation of blood vessel cells within Matrigel, and the reason why the ratio of 2:8 shows the best effect is because SVF has a large angiogenic effect and ASC has a strong effect on FGF-2. This is because it showed a synergistic effect.
실시예 3. 튜브 형성 및 스크래치 상처 봉합 평가Example 3. Evaluation of tube formation and scratch wound closure
시험관 내 혈관 형성 및 세포 이동 가능성을 평가하기 위해 마트리겔 튜브 형성 평가 및 스크래치 상처 봉합을 분석하였다.Matrigel tube formation was assessed and scratch wound closure was analyzed to evaluate in vitro angiogenesis and cell migration potential.
3-1. 튜브 형성 평가3-1. Tube formation assessment
SVF 및 ASC 세포 (1 x 106)를 T-75 세포 배양 플라스크에 접종하고, 저-포도당에서 자란 10% FBS, 100 U/ml 페니실린 및 100 mg/ml 스트렙토마이신 (깁코)를 포함하는 둘베코의 변형 독수리 배지 (LG-DMEM; Gibco, Grand Island, NT, USA)에서 세포의 대략적인 컨플루언스 (confluence)가 90%에 도달할 때까지 성장시켰다. 그 다음, 각 샘플을 1000 x g에서 5분간 원심분리하였고, 상층액을 수확하였다.SVF and ASC cells ( 1 Cells were grown in modified Eagle's medium (LG-DMEM; Gibco, Grand Island, NT, USA) until the approximate confluence of cells reached 90%. Then, each sample was centrifuged at 1000 x g for 5 minutes, and the supernatant was harvested.
튜브 형성 가능성을 평가하기 위해, 1x104 세포/웰 농도의 Human Umbilical Vein Endothelial Cells (HUVEC)을 1% FBS (대조군) 및 기저막 매트릭스 겔 (Matrigel, BD) 코팅 유리 슬라이드 (NUNC)의 SVF 및 ASC에서 유래된 각 CM을 함유하는 LG-DMEM에 포매하였다. 5시간 동안 배양한 후, 도립 현미경 (inverted microscopy)을 사용하여 대표 필드를 무작위로 촬영하고 각 샘플의 튜브 길이와 분기점을 이전에 설명한 방법으로 측정하여 그 결과를 도 5, 6에 나타내었다.To assess tube formation potential, Human Umbilical Vein Endothelial Cells (HUVEC) at a concentration of 1x104 cells/well were seeded in SVF and ASC on 1% FBS (control) and basement membrane matrix gel (Matrigel, BD) coated glass slides (NUNC). Each derived CM was embedded in LG-DMEM containing it. After culturing for 5 hours, representative fields were randomly photographed using an inverted microscope, and the tube length and branching point of each sample were measured by the previously described method, and the results are shown in Figures 5 and 6.
도 5, 6에서 확인할 수 있듯이, 튜브 길이 (tube length)와 분기점 수 (branch point numbers)는 SVF 세포의 CM이 있는 배양과 비교하여 A+S 세포의 CM이 있는 배양에서 유의하게 증가하였다.As can be seen in Figures 5 and 6, tube length and branch point numbers were significantly increased in cultures with CM of A+S cells compared to cultures with CM of SVF cells.
3-2. 스크래치 상처 분석3-2. Scratch wound analysis
Human Umbilical Vein Endothelial Cells (HUVEC)을 24 웰 배양 플레이트에서 1 x 105 세포/웰의 최종 밀도로 시딩하고 합류성 단층 (Confluent monolayers)을 형성하기 위해 24시간 동안 5% CO2에서 37 ℃에서 배양하였다. 멸균 피펫 팁을 사용하여 단층을 긁고 CM과 함께 인큐베이션을 하였다. 세포 이동성을 측정하기 위해 스크래치 및 CM 배양 후 6개의 임의 필드에서 이미지를 얻었다. 상처 부위는 NIH 이미지 프로그램 (http://rsb.info.nih.gov/nih-image/ (2021년 11월 10일 어세스함))로 측정하였고 그 결과를 도 7, 8에 나타내었다.Human Umbilical Vein Endothelial Cells (HUVEC) were seeded at a final density of 1 did. The monolayer was scraped using a sterile pipette tip and incubated with CM. To measure cell mobility, images were obtained from six random fields after scratch and CM incubation. The wound area was measured with the NIH image program ( http://rsb.info.nih.gov/nih-image/ (accessed on November 10, 2021)) and the results are shown in Figures 7 and 8.
도 7, 8에서 확인할 수 있듯이, ASC 및 SVF 세포 CM이 SVF 세포 CM에 비해 HUVEC 상처 봉합 속도를 유의하게 증가시켰음을 확인할 수 있었다.As can be seen in Figures 7 and 8, it was confirmed that ASC and SVF cell CM significantly increased the HUVEC wound closure rate compared to SVF cell CM.
실시예 4. 세포 보호 효과 평가Example 4. Evaluation of cell protection effect
세포 보호 효과를 평가하기 위해 세포사멸 세포의 비율 및 젖산 탈수소효소 (LDH)의 레벨을 측정하였다. To evaluate the cytoprotective effect, the proportion of apoptotic cells and the level of lactate dehydrogenase (LDH) were measured.
4-1. 세포사멸 세포의 비율 평가4-1. Assessment of the proportion of apoptotic cells
SVF (1 x 106) 세포 및 ASC (0.5 x 106) 세포와 SVF (0.5 x 106) 세포를 도말하고, 캄토테신 (Camptothecin; 6 μ, 시그마-알드리치, St-louis, MO, USA)를 4시간 동안 처리하였다. 세포사멸 (Apoptotic) 세포는 제조 업체의 프로토콜에 따라 propidium iodide (PI) 및 Annexin V-FITC 결합 분석 키트 II (BD PharMingen, San Diego, CA, USA)를 사용하여 측정하였고, 그 결과를 도 9, 10에 나타내었다.SVF (1 x 10 6 ) cells, ASC (0.5 x 10 6 ) cells and SVF (0.5 x 10 6 ) cells were plated and treated with camptothecin (6 μ, Sigma-Aldrich, St-louis, MO, USA). was treated for 4 hours. Apoptotic cells were measured using propidium iodide (PI) and Annexin V-FITC binding assay kit II (BD PharMingen, San Diego, CA, USA) according to the manufacturer's protocol, and the results are shown in Figure 9, It is shown in 10.
도 9, 10에서 확인할 수 있듯이, ASC +SVF 세포에서의 세포 사멸 세포의 비율은 SVF 세포에서보다 현저하게 낮았다 (도 9). 또한, 세포 사멸정도도 ASC + SVF 세포는 SVF 세포보다 낮은 사멸도를 나타내었다 (도 10).As can be seen in Figures 9 and 10, the percentage of apoptotic cells in ASC +SVF cells was significantly lower than that in SVF cells (Figure 9). In addition, ASC + SVF cells showed a lower degree of cell death than SVF cells (Figure 10).
4-2. 젖산탈수소효소 레벨 측정4-2. Measurement of lactate dehydrogenase level
세포의 변화를 측정하기 위해 세포를 무혈청 배지 (Serum-free medium)에 넣고 60분 동안 H2O2 (100 μ)로 처리하였다. 허혈로 인한 것과 유사한 세포 반응을 유도하기 위해 H2O2를 산화제로 사용하였다. 상층액의 LDH 수준은 LDH assay kit(Sigma Aldrich)로 측정하여 그 결과를 도 11에 나타내었다.To measure changes in cells, cells were placed in serum-free medium and treated with H 2 O 2 (100 μ) for 60 minutes. H 2 O 2 was used as an oxidizing agent to induce a cellular response similar to that caused by ischemia. The LDH level in the supernatant was measured using an LDH assay kit (Sigma Aldrich), and the results are shown in Figure 11.
도 10, 11에서 확인할 수 있듯이, ASC+SVF 세포는 H2O2에 대해 높은 수준으로 보호되는 것을 확인할 수 있었다.As can be seen in Figures 10 and 11, ASC+SVF cells were confirmed to be highly protected against H 2 O 2 .
실시예 5. 중증하지 허혈 모델 및 세포 이식의 유도Example 5. Induction of critical limb ischemia model and cell transplantation
ASC+SVF 세포의 치료 가능성을 조사하기 위해, 누드 마우스에서 하지 허혈을 유도하였다. 실험 프로토콜은 카톨릭관동대학교 동물 병원 동물관리위원회의 승인을 받았다. 또한 모든 절차는 미국 국립보건원 (NIH Publication No.85-23, 2011년 개정)에서 발간한 실험동물의 관리 및 사용에 관한 지침에 따라 수행되었다. 누드 마우스는 1 내지 10 주령의 수컷 생쥐를 사용하였다 (중앙 연구소, 서울, 한국). 마우스를 이소플루란 (Isoflurane)(유도: 450 mL 공기, 4.5 % 이소플루란, 유지: 200 mL 공기, 2.0 % 이소플루란, Baxter International, Inc., Deerfield, IL, USA)으로 마취하고 마취된 정도를 호흡수 및 발가락 꼬집기 반사 부족 (Lack of withdrawl reflex upon toe pinching)으로 확인하였다. 오른쪽 대퇴 동맥의 결찰에 의해 뒷다리 허혈이 유발되었다. PBS (대조군) 또는 1 x 106 ASC 또는 ASC+SVF (ASC; 0.5 x 106, SVF; 0.5 x 106) 세포 용액을 PBS에 준비한 후, 수술 후 중증하지 허혈 부위에 근육 주사를 하였다 (각 그룹에 대해 n=7). 부프레노르핀 (Buprenorphine) (0.1 mg/kg)은 수술이 끝날 때와 회복될 때까지 5시간마다 피하 주사되었다. 안락사는 티오펜탈 나트륨(40mg/kg)을 정맥 주사하여 수행되었다. 사지 회수는 절단이나 발과 발가락의 괴사 없이 사지를 유지하는 것으로 정의 되었다. 직렬 혈류를 측정하기 위해, 레이저 도플러 관류 이미지 (LDPI) 분석기 (Moor Instruments, Axminster, UK)를 사용하였으며, 그 결과를 도 12 내지 16에 나타내었다.To investigate the therapeutic potential of ASC+SVF cells, lower extremity ischemia was induced in nude mice. The experimental protocol was approved by the Animal Care Committee of Catholic Kwandong University Animal Hospital. Additionally, all procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, revised 2011). Nude mice were male mice aged 1 to 10 weeks (Central Research Institute, Seoul, Korea). Mice were anesthetized with isoflurane (induction: 450 mL air, 4.5% isoflurane; maintenance: 200 mL air, 2.0% isoflurane; Baxter International, Inc., Deerfield, IL, USA) and placed under anesthesia. The severity was confirmed by respiratory rate and lack of withdrawl reflex upon toe pinching. Hind limb ischemia was induced by ligation of the right femoral artery. Cell solutions were prepared in PBS ( control group) or 1 n=7 for group). Buprenorphine (0.1 mg/kg) was injected subcutaneously at the end of surgery and every 5 hours until recovery. Euthanasia was performed by intravenous injection of sodium thiopental (40 mg/kg). Limb salvage was defined as retention of the limb without amputation or necrosis of the foot or toes. To measure serial blood flow, a laser Doppler perfusion imaging (LDPI) analyzer (Moor Instruments, Axminster, UK) was used, and the results are shown in Figures 12 to 16.
도 12 내지 16에서 확인할 수 있듯이, 혈액관류는 ASC+SVF 세포를 주사한 사지에서 5일째에 유의하게 증가하였다 (도 12). SVF 세포 또는 PBS를 주입한 팔다리와 비교하여 ASC+SVF 세포가 주입된 사지에서 5일째에 유의하게 증가하였다 (도 13). 또한, ASC+SVF 세포가 처리된 사지는 SVF 세포 및 PBS를 주입한 것보다 더 높은 사지 회수 비율을 나타내었다 (도 14 내지 16).As can be seen in Figures 12 to 16, blood perfusion significantly increased on day 5 in the limbs injected with ASC+SVF cells (Figure 12). There was a significant increase on day 5 in the limbs injected with ASC+SVF cells compared to the limbs injected with SVF cells or PBS (FIG. 13). Additionally, limbs treated with ASC+SVF cells showed a higher limb recovery rate than those injected with SVF cells and PBS (FIGS. 14 to 16).
실시예6. 조직학적 분석Example 6. Histological analysis
6-1. 모세관 밀도 측정 및 현미경 관찰6-1. Capillary density measurements and microscopic observations
ASC+SVF 세포 치료의 치료효과의 기저 매커니즘을 파악하기 위해 H&E 염색을 수행하였다. 마우스 뒷다리의 내전근을 취하여 4 % 파라포름알데하이드 (paraformaldehyde)에 4시간 동안 고정하고, 15 % 자당 용액에서 밤새 배양하였다. 조직을 OCT 화합물 (Sakura Finetek USA, Torrance, CA, USA)에 포매하고, 액체 질소에서 급속 동결하고 10-20 μm의 두께 증분으로 절단하였다. 모세관 밀도 측정의 경우, 각 허혈성 조직 그룹에서 6개의 동결 절편을 비오틴화된 이소렉틴 B4 1차 항체 (anti-ILB4, 1:250; Vector Laboratory Inc., Burlingame, CA, USA)로 염색한 후, 스트렙타비딘 알렉사 플루오 488 (streptavidin Alexa Fluor 488) 2차 항체 (1:400; Invitrogen, Carlsbad, CA, USA)로 염색하였다. 5개의 조직 섹션에서 5개의 필드가 무작위로 선택되었으며, 각 필드에서 모세관의 수를 카운트 하였다. 그 다음, 형광 도립 현미경 및 공초점 현미경을 사용하여 촬영하고 그 결과를 도 17 내지 19에 나타내었다.H&E staining was performed to identify the underlying mechanism of the therapeutic effect of ASC+SVF cell therapy. The adductor muscle of the mouse hind limb was taken, fixed in 4% paraformaldehyde for 4 hours, and cultured in 15% sucrose solution overnight. Tissues were embedded in OCT compound (Sakura Finetek USA, Torrance, CA, USA), snap frozen in liquid nitrogen and sectioned in thickness increments of 10–20 μm. For capillary density measurements, six frozen sections from each ischemic tissue group were stained with biotinylated isolectin B4 primary antibody (anti-ILB4, 1:250; Vector Laboratory Inc., Burlingame, CA, USA). Staining was performed with streptavidin Alexa Fluor 488 secondary antibody (1:400; Invitrogen, Carlsbad, CA, USA). Five fields were randomly selected from five tissue sections, and the number of capillaries in each field was counted. Then, images were taken using a fluorescence inverted microscope and a confocal microscope, and the results are shown in Figures 17 to 19.
도 17 내지 19에서 확인할 수 있듯이, PBS 또는 SVF 세포-주입한 그룹에 비해 A+S 주입 그룹에서 감소된 근육 퇴화를 나타내었다 (도 17). 모세관 밀도는 PBS 또는 SVF 세포를 주입한 그룹보다 ASC+SVF 세포를 주입한 그룹에서 상당히 높게 나타났다 (도 18 및 19).As can be seen in FIGS. 17 to 19, the A+S injected group showed reduced muscle degeneration compared to the PBS or SVF cell-injected group (FIG. 17). Capillary density was significantly higher in the group injected with ASC+SVF cells than in the group injected with PBS or SVF cells (FIGS. 18 and 19).
6-2. qRT-PCR 및 웨스턴 블롯 분석6-2. qRT-PCR and Western blot analysis
마우스 하지 조직의 qRT-PCR 및 웨스턴 블롯 분석을 진행하여, 그 결과를 도 19에 나타내었다. qRT-PCR and Western blot analysis of mouse lower limb tissues were performed, and the results are shown in Figure 19.
ASC+SVF 세포 이식은 세포 이식 후 3일째에 SVF 세포가 이식된 하지 조직에 비해 FGF-2, IGF-1, HGF 및 VEGF-A를 유의하게 상향조절하였다 (도 19). 이러한 결과는 ASC+SVF 세포 주입은 혈관 재생을 위한 다중 혈관 신생 인자의 발현을 촉진하였음을 나타낸다.ASC+SVF cell transplantation significantly upregulated FGF-2, IGF-1, HGF, and VEGF-A compared to lower extremity tissue transplanted with SVF cells at 3 days after cell transplantation (FIG. 19). These results indicate that ASC+SVF cell injection promoted the expression of multiple angiogenic factors for vascular regeneration.
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