KR101820264B1 - Composition for preventing or treating cancer comprising exosome derived from stem cells - Google Patents

Composition for preventing or treating cancer comprising exosome derived from stem cells Download PDF

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KR101820264B1
KR101820264B1 KR1020170008390A KR20170008390A KR101820264B1 KR 101820264 B1 KR101820264 B1 KR 101820264B1 KR 1020170008390 A KR1020170008390 A KR 1020170008390A KR 20170008390 A KR20170008390 A KR 20170008390A KR 101820264 B1 KR101820264 B1 KR 101820264B1
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cells
exosome
stem cells
ovarian cancer
composition
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김진회
최윤정
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건국대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising exosome derived from stem cells. The exosome is extracted from a conditioned medium (CM) cultured from stem cells, inhibits proliferation of cancer cells by being introduced into cancer cells and activates death of cancer cells, thereby being usefully used for preventing or treating ovarian cancer.

Description

줄기세포 유래 엑소좀을 포함하는 암의 예방 또는 치료용 조성물{Composition for preventing or treating cancer comprising exosome derived from stem cells}TECHNICAL FIELD [0001] The present invention relates to a composition for preventing or treating cancer including stem cell-derived exosomes,

본 발명은 줄기세포 유래 엑소좀을 포함하는 암의 예방 또는 치료용 조성물에 관한 것이다. The present invention relates to a composition for preventing or treating cancer comprising stem cell-derived exosomes.

줄기세포(stem cell)란 미분화된 세포로서 자기 복제 능력을 가지면서 두 개 이상의 서로 다른 종류의 세포로 분화하는 능력을 갖는 세포를 말한다. 줄기세포는 분화능에 따라 만능 줄기세포(totipotent stem cell), 전분화능 줄기세포(pluripotent stem cells), 다분화능(다능성) 줄기세포(multipotent stem cells)로 분류할 수 있으며, 세포학적 유래에 따라 배아 줄기세포와 성체 줄기세포로 분류할 수 있다. 배아 줄기세포는 착상 전 수정란이나 발생중인 태아 생식기 조직 등에서 유래하는 반면, 성체 줄기세포는 성체 내에서 존재하는 각 기관, 예를 들면 골수, 뇌, 간, 췌장 등에서 유래한다.Stem cells are undifferentiated cells that have the ability to self-replicate and have the ability to differentiate into two or more different types of cells. Stem cells can be divided into totipotent stem cells, pluripotent stem cells, and multipotent stem cells depending on their differentiation ability. According to their cytological origins, embryonic stem cells can be classified into embryonic stem cells, Stem cells and adult stem cells. Embryonic stem cells originate from pre-implantation embryos or from the developing fetal genital tissue, whereas adult stem cells originate from each organ present in the adult, such as bone marrow, brain, liver, pancreas, and the like.

엑소좀은 지름이 30 내지 200 nm의 막-결합 마이크로 소포체로서, RNA, 단백질, DNA 및 지질을 포함하는 중용한 구성요소를 포함하고 있는 것으로 알려져있다. 엑소좀은 이웃 세포로 포함될 수 있는 능력에도 불구하고 모든 세포에서 분비되는 것으로 보고되고 있으며, 특히, 세포-분비의 microRNA(miRNA: 18~22 뉴클레오타이드)는 엑소좀에 의해 주로 옮겨지고, mRNA 사이런싱을 통해 유전자 발현의 전사 후 조절에 중요한 역할을 하는 것으로 연구되었다.Exosomes are membrane-bound microfibrils of 30-200 nm in diameter and are known to contain moderately soluble components including RNA, proteins, DNA and lipids. Although exosomes are reported to be secreted by all cells despite their ability to be included in neighboring cells, cell-secreted microRNA (miRNA: 18-22 nucleotides) is mainly exported by exosomes, Transcriptional regulation of gene expression through gene expression.

1. 한국등록특허 제10-1662405호1. Korean Patent No. 10-1662405

본 발명의 목적은 줄기세포에서 추출된 엑소좀을 유효성분으로 포함하는 암의 예방 또는 치료용 약학 조성물을 제공하는 것이다.It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of cancer comprising an exosome extracted from stem cells as an active ingredient.

본 발명의 또 다른 목적은 줄기세포에서 추출된 엑소좀을 유효성분으로 포함하는 암 전이 억제용 약학 조성물을 제공하는 것이다.It is still another object of the present invention to provide a pharmaceutical composition for inhibiting cancer metastasis, which comprises exosome extracted from stem cells as an active ingredient.

상기 목적을 달성하기 위하여, 본 발명은 줄기세포에서 추출된 엑소좀을 유효성분으로 포함하는 암의 예방 또는 치료용 약학 조성물을 제공한다. In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising exosome extracted from stem cells as an active ingredient.

본 발명의 일실시예에 있어서, 상기 엑소좀은 줄기세포를 배양한 배양액(conditioned medium, CM)에서 추출된 것일 수 있다. In one embodiment of the present invention, the exosome may be extracted from a conditioned medium (CM) in which stem cells have been cultured.

본 발명의 일실시예에 있어서, 상기 엑소좀은 50 내지 150 nm의 지름을 가진 것일 수 있고, 바람직하게는 70 내지 100 nm의 지름을 가진 것일 수 있으나 이에 제한되는 것은 아니다. In one embodiment of the present invention, the exosome may have a diameter of 50 to 150 nm, preferably 70 to 100 nm, but is not limited thereto.

본 발명의 일실시예에 있어서, 상기 줄기세포는 제대, 제대혈, 골수, 지방, 근육, 신경, 피부, 양막 및 태반으로 이루어진 그룹에서 선택되는 어느 하나의 조직에서 유래된 줄기세포인 것일 수 있다. In one embodiment of the present invention, the stem cell may be a stem cell derived from any one tissue selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane and placenta.

본 발명의 일실시예에 있어서, 상기 암은 난소암인 것일 수 있다. In one embodiment of the present invention, the cancer may be an ovarian cancer.

본 발명의 일실시예에 있어서, 상기 엑소좀은 암세포 내로 유입(내재화)되는 것일 수 있다. In one embodiment of the present invention, the exosome may be introduced (internalized) into cancer cells.

본 발명의 일실시예에 있어서, 상기 엑소좀은 암세포의 증식을 억제하고 암세포의 사멸을 활성화하는 것일 수 있다. In one embodiment of the present invention, the exosome may inhibit the proliferation of cancer cells and activate the death of cancer cells.

또한, 본 발명은 줄기세포에서 추출된 엑소좀을 유효성분으로 포함하는 암 전이 억제용 약학 조성물을 제공한다. The present invention also provides a pharmaceutical composition for inhibiting cancer metastasis comprising exosome extracted from stem cells as an active ingredient.

본 발명의 일실시예에 있어서, 상기 엑소좀은 줄기세포를 배양한 배양액(conditioned medium, CM)에서 추출된 것일 수 있다. In one embodiment of the present invention, the exosome may be extracted from a conditioned medium (CM) in which stem cells have been cultured.

본 발명의 일실시예에 있어서, 상기 엑소좀은 50 내지 150 nm의 지름을 가진 것일 수 있고, 바람직하게는 70 내지 100 nm의 지름을 가진 것일 수 있으나 이에 제한되는 것은 아니다. In one embodiment of the present invention, the exosome may have a diameter of 50 to 150 nm, preferably 70 to 100 nm, but is not limited thereto.

본 발명의 일실시예에 있어서, 상기 줄기세포는 제대, 제대혈, 골수, 지방, 근육, 신경, 피부, 양막 및 태반으로 이루어진 그룹에서 선택되는 어느 하나의 조직에서 유래된 줄기세포인 것일 수 있다. In one embodiment of the present invention, the stem cell may be a stem cell derived from any one tissue selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane and placenta.

본 발명의 일실시예에 있어서, 상기 암은 난소암인 것일 수 있다. In one embodiment of the present invention, the cancer may be an ovarian cancer.

본 발명에 따른 줄기세포에서 추출된 엑소좀은 줄기세포를 배양한 배양액(conditioned medium, CM)에서 추출되었고, 암세포 내로 유입되어 암세포의 증식을 억제하고 암세포의 사멸을 활성화시킴으로서, 특히 난소암의 예방 또는 치료에 유용하게 사용될 수 있다.The exosome extracted from stem cells according to the present invention was extracted from a conditioned medium (CM) in which stem cells were cultured, and was introduced into cancer cells to inhibit the proliferation of cancer cells and activate the death of cancer cells, Or can be used therapeutically.

도 1은 hAMSC-CM가 in vitro에서 A2780 난소암 세포의 증식을 억제하는 효과에 대한 결과를 나타낸 것이다. (a)는 hAMSC-CM으로 처리한 후 A2780 세포의 생존율의 퍼센트를 보여주는 결과이고(*p < 0.05 및 **p < 0.01), (b)는 hAMSC-CM로 처리한 후 72시간에 JC-1 (monomer/aggregate) 비율을 MMP 어세이로 측정한 결과를 나타낸 것이며(**p < 0.01), (c)는 hAMSC-CM로 처리한 후 72시간에 ROS 형성을 측정한 결과를 나타낸 것이고(*p < 0.05), (d)는 25% CM을 처리한 후 세포주ㄱ 분석에 대한 결과를 나타낸 것이며, (e)는 hAMSC-CM으로 처리한 후 72시간에 A2790에서 세포사멸 비율을 TUNEL(terminal deoxynucleotidyl transferase dUTP nick-end-labelling) 어세이로 측정한 결과를 나타낸 것이고, (f)는 내재적 세포사멸 관련 신호 분자인 BAX, cytosolic cytochrome-c, CAS9, CAS3, 및 BCL2에 대한 면역블럿에 대한 결과를 나타낸 것이다(*p < 0.05, **p < 0.01 및 ***p < 0.001).
도 2는 hAMSC-CM 유래의 엑소좀에 의한 A2780 암세포의 억제에 대한 결과를 나타낸 것이다. (a)는 hAMSC-CM에서 분리된 구형 엑소좀을 TEM으로 이미지화한 것이고, (b)는 엑소좀의 지름 크기의 분포도를 나타내는 결과이며, (c)는 엑소좀 및 세포분해물에서 단백질 크기에 따른 분포를 나타내는 SDS-PAGE의 Coommassie Blue 염색에 대한 결과를 나타낸 것이고, (d)는 엑소좀 유래 단백질에서 엑소좀-특이적 마커(α-CD63)의 발현 여부를 웨스턴 블럿팅으로 측정한 결과를 나타낸 것이며, (e)는 A2780 세포로의 엑소좀의 내재화를 나타내는 결과이고, (f)는 엑소좀 처리 후 상처-치료 어세이 결과를 나타내는 것이며, (g) 엑소좀 처리 후 콜로니 형성능에 대한 결과를 나타낸 것이고, (h)는 엑소좀 처리 후 플레이팅 효율에 대한 퍼센트를 나타낸 결과이며, (i)는 엑소좀 처리 후 생존율(survival fraction)의 퍼센트를 나타낸 결과이고, (j)는 엑소좀 처리 후 세포생존율(cell viability)을 나타낸 결과이며, (k)는 프로테아제-분해의 엑소좀 분해물로 처리한 후 세포생존율(cell viability)를 나타낸 결과이고, (l)은 RNase-분해의 엑소좀 분해물로 처리한 후 세포생존율(cell viability)를 나타낸 결과이다(*p < 0.05 및 **p < 0.01).
도 3은 엑소좀을 처리한 A2780 암세포에서 세포사멸 활성에 대한 메커니즘을 나타낸 것이다. (a)는 전-세포사멸인자의 상향조절 및 항-세포사멸인자 BCL2의 하향조절에 대한 웨스턴블럿팅 결과를 나타낸 것이고, (b)는 hAMSC-CM 유래의 엑소좀에 의한 A2780 세포의 증식 억제를 나타내며, 상기 엑소좀은 MSC-분비의 miRNA의 전달체로서 기능하는 것이며, 엑소좀에서 방출되는 miRNA에 의하여 암세포의 증식이 억제되고 암전이를 억제할 수 있다는 개요도를 나타낸 것이다. Figure 1 shows the results of the effect of hAMSC-CM inhibiting the proliferation of A2780 ovarian cancer cells in vitro . (a) shows the percentage of A2780 cell survival after treatment with hAMSC-CM (* p <0.05 and ** p <0.01), (b) 1 (monomer / aggregate) ratio was measured by MMP assay (** p <0.01), and (c) shows the result of measurement of ROS formation at 72 hours after treatment with hAMSC-CM ( p <0.05), (d) shows the result of cell line analysis after treatment with 25% CM, (e) shows the cell death rate in A2790 at 72 hours after treatment with hAMSC- deoxynucleotidyl transferase dUTP nick-end-labeling assays, and (f) the results for immunoblots for intrinsic apoptosis-related signaling molecules BAX, cytosolic cytochrome-c, CAS9, CAS3, and BCL2 (* P <0.05, ** p <0.01 and *** p <0.001).
Figure 2 shows the results of inhibition of A2780 cancer cells by exosomes from hAMSC-CM. (a) shows TEM image of spherical exosome isolated from hAMSC-CM, (b) shows the distribution of diameter of exosome, and (c) (D) shows the result of Western blotting of the exosome-specific marker (? -CD63) expression in the exosome-derived protein. (E) shows the results of internalization of exosome into A2780 cells, (f) shows wound-healing assay results after exosomal treatment, and (g) (H) represents the percentage of plating efficiency after exosome treatment, (i) represents the percentage of survival fraction after exosome treatment, (j) represents the percentage of survival fraction after exosome treatment, Cell viability (K) is the result of cell viability after treatment with protease-degraded exosomal degradation product. (1) shows the cell viability after treatment with exogenous degradation product of RNase- viability) (* p <0.05 and ** p <0.01).
Figure 3 shows the mechanism of apoptotic activity in A2780 cancer cells treated with exosomes. (a) shows Western blotting results for upregulation of pro-apoptotic factors and down-regulation of anti-apoptotic factor BCL2, (b) inhibition of A2780 cell proliferation by hAMSC- , And the exosome functions as a carrier of MSC-secreting miRNA, and shows the outline of inhibition of cancer cell proliferation and inhibition of cancer metastasis by miRNA released from exosome.

본 발명의 용어, "줄기세포"는 미분화된 세포로서 자기 복제 능력을 가지면서 두 개 이상의 서로 다른 종류의 세포로 분화하는 능력을 갖는 세포를 말한다. 본 발명의 줄기세포는 자가 또는 동종 유래 줄기세포일 수 있으며, 인간 및 비인간 포유류를 포함한 임의 유형의 동물 유래일 수 있고, 상기 줄기세포가 성체로부터 유래된 것이든 배아로부터 유래된 것이든 이에 한정되지 않는다. 본 발명의 줄기세포는 배아 줄기세포 또는 성체 줄기세포를 포함하며, 바람직하게는 성체 줄기세포이다. 상기 성체 줄기세포는 중간엽 줄기세포, 인간 조직 유래 중간엽 기질세포(mesenchymal stromal cell), 인간 조직 유래 중간엽 줄기세포, 다분화능 줄기세포 또는 양막상피세포일 수 있으며, 바람직하게는 중간엽 줄기세포이나, 이에 한정되지 않는다. 상기 중간엽 줄기세포는 제대, 제대혈, 골수, 지방, 근육, 신경, 피부, 양막 및 태반 등으로부터 유래된 중간엽 줄기세포일 수 있으나, 이에 한정되지 않는다.The term "stem cell" of the present invention refers to a cell having the ability to self-replicate as an undifferentiated cell and capable of differentiating into two or more different kinds of cells. The stem cells of the present invention may be autologous or allogeneic stem cells, and may be derived from any type of animal, including human and non-human mammals, whether stem cells derived from an adult or stem cells derived from an embryo Do not. The stem cells of the present invention include embryonic stem cells or adult stem cells, preferably adult stem cells. The adult stem cells may be mesenchymal stem cells, mesenchymal stromal cells derived from human tissues, mesenchymal stem cells derived from human tissues, multipotential stem cells or amniotic epithelial cells, preferably mesenchymal stem cells But is not limited thereto. The mesenchymal stem cells may be mesenchymal stem cells derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane and placenta, but are not limited thereto.

본 발명의 용어, "엑소좀(exosome)"은 다양한 세포들로부터 분비되는 막 구조의 작은 소낭을 의미하며, 다낭체와 원형질막의 융합이 일어나 세포 밖 환경으로 방출되는 소낭을 의미한다. 엑소좀의 지름은 50 내지 150 nm의 지름을 가진 것일 수 있고, 바람직하게는 70 내지 100 nm의 지름을 가진 것일 수 있으나 이에 제한되는 것은 아니다. The term "exosome" of the present invention means a small follicle having a membrane structure that is secreted from various cells, and refers to a follicle which is fused with a polycapellum and a plasma membrane and released into the extracellular environment. The diameter of the exosome may be 50-150 nm in diameter, preferably 70-100 nm in diameter, but is not limited thereto.

본 발명의 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체를 더 포함할 수 있다. 예를 들어, 주사제의 경우에는 보존제, 무통화제, 가용화제 또는 안정화제 등을, 국소 투여용 제제의 경우에는 기제(base), 부형제, 윤활제 또는 보존제 등을 추가로 포함할 수 있다.The compositions of the present invention may further comprise suitable carriers routinely used in the manufacture of pharmaceutical compositions. For example, a preservative, an anhydrous agent, a solubilizing agent or a stabilizer may be added in the case of an injection, and a base, an excipient, a lubricant or a preservative in the case of a preparation for topical administration.

본 발명의 조성물은 개체에게 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁 내 경막 또는 뇌혈관 내 주사에 의해 투여될 수 있으며, 바람직하게는 치료가 필요한 개체의 직접 생착 또는 이식하는 것이 가능하나 이에 한정되지는 않는다.The compositions of the present invention may be administered to a subject in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-dermal or intracerebral injection, preferably by direct engraftment of the subject in need of treatment or Although transplantation is possible, it is not limited to this.

본 발명의 조성물은 암 치료를 위하여 단독으로, 또는 수술, 방사선 치료, 호르몬 치료, 화학 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for cancer treatment.

이하, 본 발명을 실시예를 통하여 더욱 상세히 설명하기로 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

실시예Example 1. 재료 및 방법 1. Materials and Methods

1.1. 시약1.1. reagent

ExoQuick-TC exosome-isolation reagent (EXOTC50A-1), SeraMir exosome RNA-isolation kit (RA808A-1), exosome-specific marker anti-α CD63 antibody (EXOABCD63A-1), 및 florescence exosome-preparation reagent (EXDC300A-1)은 System Biosciences (SBI, Palo Alto, CA, USA)으로부터 구입하였다. Penicillin-streptomycin (PS) solution, trypsin-EDTA solution, RPMI medium, 및 α -MEM media는 Life Technologies GIBCO (Grand Island, NY, USA)에서 구입하였다. FBS(Fetal bovine serum)는 Sigma-Aldrich (St. Louis, MO, USA)에서 구입하였다. 면역블럿에 사용된 항체의 경우 phosphorylated p53 (Cell Signaling Technology, Beverly, MA, USA), BAX, BCL2, CASP9, CASP3, β -actin (Abcam, Cambridge, MA), 및 cytochrome c (ENZO Diagnostics Inc., Farmingdale, NY, USA)가 사용되었다. Specific marker anti-α CD63 antibody (EXOABCD63A-1), and a fluorescence exosome-preparation reagent (EXDC300A-1), exo-specific exonuclease ) Were purchased from System Biosciences (SBI, Palo Alto, Calif., USA). Penicillin-streptomycin (PS) solution, trypsin-EDTA solution, RPMI medium, and α-MEM media were purchased from Life Technologies GIBCO (Grand Island, NY, USA). FBS (Fetal bovine serum) was purchased from Sigma-Aldrich (St. Louis, MO, USA). BAX, BCL2, CASP9, CASP3, β-actin (Abcam, Cambridge, MA), and cytochrome c (ENZO Diagnostics Inc., Cambridge, Mass.) Were used for the immunoblot antibody. Farmingdale, NY, USA) was used.

1.2. CM(Conditioned medium) 준비1.2. Conditioned medium preparation

hAMSCs(human adipose-derived mesenchymal stem cells)은 주식회사 마리아바이오텍(서울, 한국)에서 기부된 것을 사용하였고, 10% FBS와 100 U/ml PS가 포함된 α-MEM 배지를 사용하고 37℃, 5% CO2 인큐베이터에서 배양되었다. hAMSC-CM은 6번째 및 7번째 사이의 계대배양의 세포에서 획득되었다. 간단하게, 2x106 세포가 10-cm 배양 디쉬에 접종되고, 적당한 부착을 위하여 37℃, 5% CO2 인큐베이터에서 오버나잇되었다. 배지는 새롭게 갈아주었고, 세포는 추가로 48시간 동안 배양되었을 때, 이에 따른 배지를 hAMSC-CM으로 간주하였다. pH 안정성은 전기적 pH 미터 및 pH 페이퍼를 사용하여 확인하였다. 전체 1L의 CM을 모으고, 필터하여, 혼합시킨 후, 15ml 및 50 ml의 튜브에 나누어 20℃에 보관하였다. Human adipose-derived mesenchymal stem cells (hAMSCs) were purchased from MARIA BIOTECH Co., Ltd. (Seoul, Korea) and cultured in α-MEM medium containing 10% FBS and 100 U / CO 2 incubator. hAMSC-CM was obtained from cells in the sixth and seventh subculture. Briefly, 2x10 &lt; 6 &gt; cells were inoculated into 10-cm culture dishes and over-nested at 37 [deg.] C in a 5% CO 2 incubator for proper attachment. The medium was freshly changed, and when the cells were cultured for an additional 48 hours, the resulting medium was regarded as hAMSC-CM. pH stability was confirmed using an electrical pH meter and pH paper. A total of 1 L of CM was collected, filtered and mixed, and then divided into 15 ml and 50 ml tubes and stored at 20 캜.

1.3. CM 정량 결정 및 세포-생존율 1.3. CM quantification and cell-survival rate 어세이Assay

A2780 세포 증식에서 hAMSC-CM 의 효과를 확인하기 위하여, 새로운 배지에 1 내지 30%의 CM 추가제를 사용하여 세포생존율 어세이가 수행되었다. A2780 세포는 96-well flat-bottomed culture plates에 접종되었고, 24시간 동안 37℃, 5% CO2 인큐베이터에서 배양되었다. 배지는 대조군(fresh) 또는 실험군(0 내지 30% CM) 배지로 교환되었고, 세포 생존율은 cell counting kit-8 (CCK-8; Dojindo Laboratories, Tokyo, Japan)을 사용하여 확인되었다. 흡광도는 microtiter plate reader (Multiskan FC; Thermo Fisher Scientific, Waltham, MA, USA)를 사용하여 450 nm에서 측정되었다. 상기로부터 얻어진 결과를 근거로, 새로운 배지에 25% CM의 추가하는 것으로 모든 실험에 적용되었다. To confirm the effect of hAMSC-CM on A2780 cell proliferation, cell viability assays were performed using 1 to 30% CM additive in fresh medium. A2780 cells were seeded in 96-well flat-bottomed culture plates and cultured in a 5% CO 2 incubator at 37 ° C for 24 hours. The medium was replaced with fresh or experimental medium (0-30% CM) medium and cell viability was confirmed using cell counting kit-8 (CCK-8; Dojindo Laboratories, Tokyo, Japan). Absorbance was measured at 450 nm using a microtiter plate reader (Multiskan FC; Thermo Fisher Scientific, Waltham, Mass., USA). Based on the results obtained above, the addition of 25% CM to the fresh medium was applied to all experiments.

1.4. 1.4. ROSROS (reactive oxygen reactive oxygen speicesspeices ) 확인) Confirm

ROS 형성은 H2-DCFDA(2′,7′-dichlorodihydrofluorescein diacetate; Sigma-Aldrich)을 사용하여 정량하였다. 세포는 10 μM H2-DCFDA 를 처리하고 30분 동안 37℃, 5% CO2 인큐베이터에서 배양되었다. 이후, 세포는 PBS(phosphate-buffered saline)으로 씻어지고, 재부양되었고, Gemini EM (SpectraMAX; Molecular Devices, Sunnyvale, CA, USA)를 사용하여 각각 515 nm 및 488 nm에서 fluorescence emission 및 excitation이 측정되었다. ROS formation was quantified using H2-DCFDA (2 ', 7'-dichlorodihydrofluorescein diacetate; Sigma-Aldrich). Cells were treated with 10 μM H2-DCFDA and incubated for 30 min at 37 ° C in a 5% CO 2 incubator. Cells were then washed and resuspended in PBS (phosphate-buffered saline) and fluorescence emission and excitation were measured at 515 nm and 488 nm using Gemini EM (SpectraMAX; Molecular Devices, Sunnyvale, CA, USA) .

1.5. 1.5. MMPMMP (( mitochondrialmitochondrial membrane potential) 확인  membrane potential

MMP는 cationic fluorescent indicator JC-1 (Molecular Probes, Eugene, OR, USA)으로 확인되었다. 세포는 10 μM JC-1가 처리된 후 15분 동안 37℃에서 배양되었고, PBS로 씻어지고 재부양되었으며, 형광강도(fluorescence intensity)가 측정되었다. MMP는 JC-1 모노머와 JC-1 집합체에 대한 형광강도의 비율로서 표현되었다. JC1 집합체는 583 nm emssion에서 온전한 미토콘드리아에 대하여 red fluorescence으로 나타나고, JC1 모노머는 525 nm emission과 488 nm excitation에서 세포질에 대하여 green fluorescence로 나타났다. MMP was identified as a cationic fluorescent indicator JC-1 (Molecular Probes, Eugene, OR, USA). Cells were treated with 10 μM JC-1, incubated at 37 ° C for 15 min, washed and resuspended in PBS, and fluorescence intensity was measured. MMP was expressed as a ratio of fluorescence intensity to JC-1 monomer and JC-1 aggregate. JC1 aggregates appeared as red fluorescence against intact mitochondria at 583 nm emssion, and JC1 monomers showed green fluorescence for cytoplasm at 525 nm emission and 488 nm excitation.

1.6. 세포주기1.6. Cell cycle

A2780 세포는 6-well plates (30,000 cells/cm2)에 접종되고, 오버나잇시킨 후, 배지는 대조군(fresh) 또는 실험군(25% CM) 배지로 24시간 마다 교환하였다. 세포는 세포주기 분석을 위하여 48시간 및 72시간에 모아지고, PBS로 씻었고, 70% 에탄올로 고정하였으며, 20℃에 저장하였다. 고정된 세포는 PBS로 씻어지고, RNaseA 용액으로 처리된 후, PI(propidium iodide)로 염색되었다. 세포주기 분석은 BD FACSCalibur (BD Biosciences, San Jose, CA, USA)을 사용하여 수행되었다. 기초 데이터는 ModFit software trial version (http://www.vsh.com/products/mflt/index.asp)을 사용하여 분석되었다. A2780 cells were inoculated into 6-well plates (30,000 cells / cm2), and after over-nipping, the medium was changed every 24 hours in fresh or experimental (25% CM) medium. Cells were collected at 48 and 72 hours for cell cycle analysis, washed with PBS, fixed with 70% ethanol and stored at 20 ° C. Immobilized cells were washed with PBS, treated with RNase A solution and stained with PI (propidium iodide). Cell cycle analysis was performed using BD FACSCalibur (BD Biosciences, San Jose, Calif., USA). The baseline data was analyzed using the ModFit software trial version (http://www.vsh.com/products/mflt/index.asp).

1.7. 1.7. TMRTMR (( tetramethylrhodamine테트라 메틸 히드 아민 )-) - 레드Red 어세이Assay . .

A2780 세포는 6-well plates (30,000 cells/cm2)에 접종되고, 오버나잇시킨 후, 배지는 대조군(fresh) 또는 실험군(25% CM) 배지로 24시간 마다 교환하였다. 72시간에, 세포는 모아지고, 세포자멸 비율은 제조사의 지시에 따라 in situ cell-death detection kit (TMR red; Roche Diagnostics, Indianapolis, IN, USA)을 사용하여 확인하였다. A2780 cells were inoculated into 6-well plates (30,000 cells / cm2), and after over-nipping, the medium was changed every 24 hours in fresh or experimental (25% CM) medium. At 72 h, cells were harvested and apoptotic rate was determined using in situ cell-death detection kit (TMR red; Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's instructions.

1.8. 1.8. 면역블롯팅Immunoblotting

세포를 분해하기 위해 방사능 면역침전(radio-immunoprecipitation) 버퍼가 사용되었고, 단백질 양은 BCA protein assay kit (Thermo Scientific)로 측정되었다. 단백질은 12% SDS-PAGE를 사용하여 전기영동을 진행하였고, PVDF(polyvinylidene fluoride) 멤브레인으로 블럿팅하였다. 멤브레인은 5% 스킴 밀크에서 RT(room temperature)에서 2시간 동안 처리하고, 1차 항체를 넣은 후 4℃에서 오버나잇시킨 후, horseradish peroxidase-conjugated 1차 항체를 넣은 후 RT에서 1시간 동안 처리하였다. 면역-반응은 enhanced chemiluminescence reagents로 확인되었다. Radioactive immunoprecipitation buffer was used to break down the cells, and the amount of protein was measured with the BCA protein assay kit (Thermo Scientific). Proteins were electrophoresed using 12% SDS-PAGE and blotted with PVDF (polyvinylidene fluoride) membrane. Membranes were treated with 5% skim milk at room temperature for 2 hours, then incubated at 4 ° C with primary antibody, horseradish peroxidase-conjugated primary antibody, and incubated for 1 hour at RT . Immune responses were identified as enhanced chemiluminescence reagents.

1.9. 1.9. 엑소좀Exosome 분리 및 특성 Separation and characterization

엑소좀은 제조사(ExoQuick-TC kit)의 지시에 따라 분리되었다. 간단하게, ExoQuick-TC reagent가 hAMSC-CM에 1:5 비율로 더해지고, 튜브를 거꾸로 하여 여러 번 혼합시켰다. 혼합물은 4℃에서 오버나잇시키고, RT에서 30분 동안 1500 g 에서 원심분리하였다. TEM(transmission electron microscope)을 위해, 엑소좀-TEM grid가 제조사(Exosome-TEM-easy kit; 101Bio, Palo Alto, CA, USA)의 지시대로 준비되었고, 엑소좀 입자는 Hitachi H7600 electron microscope (Hitachi, Tokyo, Japan)를 사용하여 100 kV 가속 전압에서 확인하였다. The exosomes were separated according to the manufacturer's instructions (ExoQuick-TC kit). Briefly, ExoQuick-TC reagent was added to the hAMSC-CM in a 1: 5 ratio, and the tubes were mixed upside down several times. The mixture was over-knit at 4 &lt; 0 &gt; C and centrifuged at 1500 g for 30 min at RT. (TEM) easy kit (101Bio, Palo Alto, Calif., USA), and exosome particles were prepared on a Hitachi H7600 electron microscope (Hitachi, Tokyo, Japan) at an accelerating voltage of 100 kV.

1.10. 세포생존율 1.10. Cell survival rate 어세이Assay

CM의 1 ml에서 유래한 엑소좀은 1 U 엑소좀으로 정의하였다. 생존율 어세이를 위하여 세포는 1 U/ml의 엑소좀으로 처리되었다. 프로테아제 또는 RNase-분해의 엑소좀을 사용할 때, 대조군 배지는 실험적 에러를 피하기 위하여 동일양의 프로테아제 또는 RNase 용액을 추가하였다. 프로테아제-분해의 엑소좀은 37℃에서 30분 동안 100 μl의 트립신(0.25%)으로 5 U 엑소좀을 분해함으로써 준비되었다. RNase-분해의 엑소좀은 37℃에서 30분 동안 100 μl의 RNaseA solution (100 μg/ml)으로 5 U 엑소좀을 분해함으로써 준비되었다. Exosomes derived from 1 ml of CM were defined as 1 U exosomes. Cells were treated with 1 U / ml exosomes for survival assay. When using exosomes of protease or RNase-degradation, the control medium was supplemented with the same amount of protease or RNase solution to avoid experimental errors. The proteasome-degraded exosomes were prepared by digesting 5 U exosomes with 100 μl trypsin (0.25%) for 30 min at 37 ° C. RNase-degraded exosomes were prepared by dissolving 5 U exosomes with 100 μl of RNaseA solution (100 μg / ml) for 30 min at 37 ° C.

1.11. 상처 치료 1.11. Wound treatment 어세이Assay

상처 치료 능력을 확인하기 위하여, 세포(30,000 cells/cm2)는 12-well plate에서 접종되었고, 37℃, 5% CO2 인큐베이터에서 오버나잇으로 배양되었다. 상처는 10-μl 파이펫 팁(pipette tips)으로 만들었고, 대조군(fresh) 또는 다른 실험군(25% CM, 1 U/ml 엑소좀, 또는 1 U/ml 엑소좀+25% CM)으로 실험이 진행되었다. Cells (30,000 cells / cm 2 ) were inoculated on a 12-well plate and incubated overnight at 37 ° C in a 5% CO 2 incubator. The wound was made with 10-μl pipette tips and the experiment was run with either fresh or other experimental groups (25% CM, 1 U / ml exosomes, or 1 U / ml exosomes + 25% CM) .

1.12. 1.12. 콜로니Colony 형성  formation 어세이Assay

콜로니 형성 어세이를 위하여, 1000 개의 세포가 60-cm cell-culture dishes에 접종되었고, 37℃, 5% CO2 인큐베이터에서 오버나잇으로 배양되었다. 배지는 대조군(fresh) 또는 다른 실험군(25% CM, 1 U/ml 엑소좀, 또는 1 U/ml 엑소좀+25% CM)으로 교환되었고, 세포는 2주 동안 배양되었다. 콜로니를 포함하는 디쉬는 1% 메탄올 및 1% 포르말린이 포함된 0.05% (w/v) crystal-violet solution으로 RT에서 20분 동안 염색되었다. 콜로니 사진은 ultraviolet table을 사용하여 찍었고, 콜리니는 OpenCFU software (http://opencfu.sourceforge.net/)를 사용하여 카운팅하였다. PE(cell-plating efficiency) 및 SF(survival fraction)은 하기의 공식으로 계산되었다. For colony formation assays, 1000 cells were inoculated into 60-cm cell-culture dishes and incubated overnight at 37 ° C in a 5% CO 2 incubator. The medium was replaced with fresh or another experimental group (25% CM, 1 U / ml exosomes, or 1 U / ml exosomes + 25% CM) and cells were incubated for 2 weeks. Colony-containing dishes were stained with 0.05% (w / v) crystal-violet solution containing 1% methanol and 1% formalin for 20 minutes at RT. Colony pictures were taken using an ultraviolet table, and Colinie was counted using OpenCFU software (http://opencfu.sourceforge.net/). The cell-plating efficiency (PE) and survival fraction (SF) were calculated by the following formula.

Plating efficiency (PE) = (Number of colonies/Number of cells seeded) x 100Plating efficiency (PE) = (Number of colonies / Number of cells seeded) x 100

Surviving fraction(SF) = (PE of treated cells/PE of control cells) x 100Surviving fraction (SF) = (PE of treated cells / PE of control cells) x 100

1.13. 형광-1.13. Neon- 엑소좀Exosome 준비 및 암세포로의 내재화(internalization) Preparation and Internalization into Cancer Cells

제조사(EXDC300A-1, SBI, Palo Alto, CA, USA)의 프로토콜에 따라 형광 엑소좀이 준비되고, 암세포에 의한 hAMSC-CM 유래의 엑소좀의 내재화가 결정되었다. 간단하게, 50 μl의 10× Exo-Red가 500 μl의 엑소좀 서스펜션(in PBS)에 더해지고, 혼합되었다. 혼합물은 10분 동안 37℃에서 놓이고 반응을 멈추게 하기 위하여 100 μl의 ExoQuick-TC reagent가 더해졌으며, 30분 동안 얼음에 두었다. 혼합물은 14,000 rpm에서 3분 동안 원심분리되었고, 레이블된 엑소좀 펠릿은 재부양되고, 암세포로 전달되었다. 내재화는 형광 현미경으로 확인되었다. Fluorescent exosomes were prepared according to the protocol of the manufacturer (EXDC300A-1, SBI, Palo Alto, CA, USA) and the internalization of exosome from hAMSC-CM by cancer cells was determined. Briefly, 50 μl of 10 × Exo-Red was added to 500 μl of exosomal suspension (in PBS) and mixed. The mixture was placed at 37 ° C for 10 minutes and 100 μl of ExoQuick-TC reagent was added to stop the reaction and placed on ice for 30 minutes. The mixture was centrifuged at 14,000 rpm for 3 minutes and the labeled exosomal pellet was resuspended and transferred to cancer cells. Internalization was confirmed by fluorescence microscopy.

1.14. 통계적 분석1.14. Statistical analysis

실험은 세 번 수행되었고, 통계적 분석은 one-way analysis of variance를 사용하여 수행되었다. 대조군과 처리된 세포 사이의 유의성의 차이는 Student t test에 의하여 결정되었다. 유의성은 p < 0.05, p < 0.01 및 p < 0.001로서 결정되었다. Experiments were performed three times and statistical analysis was performed using one-way analysis of variance. The difference in significance between the control and treated cells was determined by Student t test. Significance was determined as p <0.05, p <0.01 and p <0.001.

실시예Example 2.  2. hAMSChAMSC -CM 처리에 의한 난소암 세포의 증식 억제 효과Effect of inhibition of proliferation of ovarian cancer cells by -CM treatment

hAMSC 유래의 CM은 향상된 산화적 스트레스를 통해 세포 증식을 변경하였고, MMP(mitochondrial membrane potential)을 감소시켰다. CM은 배양액의 pH를 변경시키지 않고, 25% CM 에서 가장 효과적으로 세포증식을 억제하였음을 확인하였는데, 특히, 25% CM을 처리한 후 48시간에서는 세포 생존율을 대조군에 비해 20% 감소시키고, 72시간에서는 40%로 감소시켰음을 확인하였다(도 1a). 또한, CM 처리에 따른 세포 사멸의 효과를 평가하기 위하여, 본 발명자들은 CM-처리된 A2780 세포에서 ROS 와 MMP를 측정하였고, 그 결과, 25% CM 처리에서 JC-1 모노머/집합체 비율이 대조군에 비해 65% 향상되었다(도 1b). 즉, 미토콘드리아 막 전위가 불안정하였음을 확인하였다. ROS의 생성에서도 대조군에 비해 30% 증가됨을 확인하였다(도 1c). 세포주기 분석에서도 CM 처리 후 48시간 후에는 S-phase 세포의 수가 증가하였고, G0/G1 phase 및 G2/M phase 세포는 감소됨을 확인하였다(도 1d). 그러나, CM 처리 후 72시간에서는 S phase 및 G2/M phase 세포가 증가하였고, GO/G1 phase 세포가 유의적으로 감소되었다. CM from hAMSCs altered cell proliferation through improved oxidative stress and reduced mitochondrial membrane potential (MMP). CM was found to inhibit cell proliferation most effectively at 25% CM without changing the pH of the culture medium. Specifically, cell viability was reduced by 20% compared to the control at 48 hours after treatment with 25% CM, (Fig. 1 (a)). In order to evaluate the effect of CM treatment on cell death, we measured ROS and MMP in CM-treated A2780 cells. As a result, in the 25% CM treatment, the ratio of JC-1 monomer / (Fig. 1B). In other words, it was confirmed that the mitochondrial membrane potential was unstable. ROS production was also increased by 30% compared to the control (Fig. 1C). In the cell cycle analysis, it was confirmed that the number of S-phase cells was increased and the number of G0 / G1 phase and G2 / M phase cells were decreased 48 hours after CM treatment (FIG. However, at 72 hours after CM treatment, S phase and G2 / M phase cells were increased and GO / G1 phase cells were significantly decreased.

따라서, hAMSC-CM은 암세포의 증식을 억제하고, 세포주기를 억제하는 것을 확인하였다. Thus, it was confirmed that hAMSC-CM inhibits the proliferation of cancer cells and inhibits the cell cycle.

실시예Example 3.  3. hAMSChAMSC -CM 처리에 의한 암세포에서 내재적 세포사멸 경로의 활성 효과Activation of intracellular apoptosis pathway in cancer cells by -MM treatment

hAMSC- 유래의 CM 처리 후 세포사멸 유도를 결정하기 위하여, 본 발명자들은 TMR(tetramethylrhodamine)-red 어세이를 수행하여, 사멸된 세포의 퍼센트를 측정하였다. FACS(Fluorescence-activated cell sorting) 분석 결과, 대조군에 비하여 CM 처리 후 사멸된 세포의 퍼센트에서 유의하게 증가됨을 확인하였다(도 1e).To determine the induction of apoptosis after hAMSC-derived CM treatment, we performed a TMR (tetramethylrhodamine) -red assay to determine the percentage of apoptotic cells. Fluorescence-activated cell sorting (FACS) analysis showed that the percentage of cells killed after CM treatment was significantly increased compared with the control group (Fig. 1e).

또한, 본 발명자들은 CM-처리된 A2780 세포에서 내재적 세포사멸 경로-관련 항-세포사멸 분자 및 전-세포사멸 분자의 발현을 확인하는 실험을 수행하였다. CM-처리된 A2780 세포에서, 항-세포사멸 단백질인 BCL2의 발현은 대조군 세포에 비해 하향조절된 반면, 인산화된-p53, BAX, CASP9, CASP3, 및 세포질의 Cytochrome-c를 포함하는 몇 개의 전-세포사멸 분자의 발현은 상당히 상향조절되거나 활성화되었다. 추가적으로, BAX/BCL2 비율 및 활성-/전-CASP3 비율은 대조군에 배해 CM-처리된 세포에서 몇 배로 증가되었음을 확인하였다(도 1f).In addition, the present inventors performed an experiment to confirm the expression of endogenous apoptotic pathway-related anti-apoptotic molecules and pro-apoptotic molecules in CM-treated A2780 cells. In CM-treated A2780 cells, the expression of BCL2, an anti-apoptotic protein, was down-regulated compared to control cells, while several expressions including phosphorylated-p53, BAX, CASP9, CASP3 and cytochrome c- The expression of apoptotic molecules was significantly up-regulated or activated. In addition, BAX / BCL2 ratio and active - / pre-CASP3 ratios were increased several fold in CM-treated cells in the control group (Fig. 1F).

따라서, CM의 처리는 암세포에서 세포사멸을 유도함을 확인하였다.Therefore, it was confirmed that the treatment of CM induces apoptosis in cancer cells.

실시예Example 3.  3. hAMSChAMSC -CM 유래의 -CM-derived 엑소좀의Exosomatic 동정, 특성 및 A2780 세포 내로의  Identification, characterization and expression of A2780 엑소Exo 좀의 내재화Internalization of Joss

본 발명자들은 hAMSC-CM 유래의 엑소좀이 CM-처리된 A2780 세포에서 관찰된 세포사멸의 유도에 관련이 있는지를 확인하고자 하였다. 도 2a는 TEM(transmission electron microscopy)에서 확인한 hAMSC-CM 유래의 엑소좀을 보여주고 있다. 엑소좀은 50 내지 150 nm의 크기를 가진 구(sphere)로 나타났고, 다수는 70 내지 100 nm인 것으로 확인되었다(도 2b). 다음으로, 본 발명자들은 엑소좀으로부터 전체 단백질을 추출하였고, Coommassie Blue 염색으로 결과를 확인하였다(도 2c). 도 2d에서와 같이, 엑소좀 단백질 마커인 CD63이 엑소좀-유래의 분해물에서 확인되었다. 끝으로, 본 발명자들은 엑소좀이 A2780 암세포로 내재화될 수 있는지 조사하였다. 엑소좀은 엑소좀 RNA 에 결합을 확인할 수 있는 Exo-red 염색 키트를 사용하여 레이블되었다. 그 결과, CM-유래의 엑소좀은 A2780 암세포로 내재화되었음이 확인되었다(도 2e).We sought to determine whether exosome from hAMSC-CM is involved in the induction of apoptosis observed in CM-treated A2780 cells. 2A shows exosomes derived from hAMSC-CM as confirmed by transmission electron microscopy (TEM). The exosomes appeared as spheres with a size of 50-150 nm and the majority were found to be 70-100 nm (Fig. 2b). Next, the present inventors extracted whole proteins from exosomes and confirmed the results by Coommassie Blue staining (Fig. 2C). As in Figure 2D, the exosomal protein marker CD63 was identified in the exosome-derived degradation product. Finally, we investigated whether exosomes could be internalized into A2780 cancer cells. Exosomes were labeled using an Exo-red staining kit to confirm binding to exosomal RNA. As a result, CM-derived exosomes were confirmed to be internalized with A2780 cancer cells (Fig. 2E).

실시예Example 4.  4. hAMSChAMSC -CM 유래의 -CM-derived 엑소좀에To exosome 의한  by 세포생존율Cell survival rate , 상처-회복 능력 및 콜로니 형성 능력의 감소, Wound-healing ability and reduction of colony forming ability

난소암 세포에서 hAMSC-CM-유래의 엑소좀 내재화의 영향을 결정하기 위하여, 본 발명자들은 A2780, SKOV-3 및 CAOV-3 난소암 세포에 hAMSC-CM 유래의 엑소좀을 처리하였다. 그 결과, A2780, SKOV-3 세포에서는 엑소좀 처리에 의한 억제 효과가 나타났으나, CAOV-3 세포에서는 특별한 변화가 없었다. 본 발명자들은 A2780 세포에서 상처-회복 능력(도 2f) 및 콜로니 형성능력(도 2g)을 실험하였다. 그 결과, 도 2h, 2i 및 2j에서와 같이, 대조군에 비해 암세포로 엑소좀의 내재화 후에 세포-플레이팅 효율, 세포-생존 부분 및 세포 생존율에서의 상당한 감소가 나타났다. In order to determine the effect of hAMSC-CM-derived exosome internalization in ovarian cancer cells, we treated exosomes from hAMSC-CM on A2780, SKOV-3 and CAOV-3 ovarian cancer cells. As a result, A2780 and SKOV-3 cells showed an inhibitory effect by exosome treatment, but there was no specific change in CAOV-3 cells. We tested wound-healing ability (Figure 2f) and colony forming ability (Figure 2g) in A2780 cells. As a result, there was a significant reduction in cell-plating efficiency, cell-survival fraction and cell survival rate after internalization of exosome with cancer cells as compared to the control group, as in Figs. 2h, 2i and 2j.

실시예Example 5.  5. hAMSChAMSC -CM 유래의 -CM-derived 엑소좀의Exosomatic 내재화에 의한 A2780 세포에서의 전-세포사멸 분자의 상향조절 효과 Up-regulation of pro-apoptotic molecules in A2780 cells by internalization

본 발명자들은 CM-유래의 엑소좀이 내재화된 A2780 세포에서 대조군에 비해 인산화된-p53, BAX, CASP9, CASP3, 및 세포질의 Cytochrome-c를 포함하는 몇 개의 전-세포사멸 신호전달 분자가 상향조절됨을 웨스턴 블럿팅 실험을 통해 확인하였다. 또한, 항-세포사멸 BCL2는 대조군에 비해 하향조절되었다(도 3). We have shown that several pro-apoptotic signaling molecules including up-regulated phosphorylated-p53, BAX, CASP9, CASP3, and cytochrome-c are upregulated in A2780 cells with CM-derived exosomes internalized Were confirmed by Western blotting experiments. In addition, anti-apoptotic BCL2 was downregulated compared to the control (Figure 3).

따라서, CM-유래 엑소좀의 내재화는 A2780 세포에서 내재적 세포사멸 경로의 활성을 이끌 수 있음을 확인하였다. Thus, it was confirmed that the internalization of CM-derived exosomes can lead to the intrinsic apoptosis pathway in A2780 cells.

Claims (12)

인간 양막 중간엽 줄기세포(Human Amniotic Membrane Mesenchymal Stem Cell, hAMSC)의 배양액(conditioned medium, CM)에서 추출한 엑소좀을 유효성분으로 포함하는 난소암의 예방 또는 치료용 약학 조성물로서,
상기 배양액은 총 조성물 부피 대비 1-50 부피%으로 포함하는 것을 특징으로 하는, 난소암의 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating ovarian cancer comprising, as an active ingredient, exosome extracted from a conditioned medium (CM) of Human Amniotic Membrane Mesenchymal Stem Cell (hAMSC)
The pharmaceutical composition for preventing or treating ovarian cancer, wherein the culture liquid is contained in an amount of 1-50% by volume based on the total volume of the composition. 삭제delete 제 1 항에 있어서,
상기 엑소좀은 50 내지 150 nm의 지름을 가진 것을 특징으로 하는 조성물. The method according to claim 1,
Wherein the exosome has a diameter of 50 to 150 nm. 삭제delete 삭제delete 제 1 항에 있어서,
상기 엑소좀은 난소암 세포 내로 유입(내재화)되는 것을 특징으로 하는 조성물. The method according to claim 1,
Wherein the exosomes are introduced (internalized) into ovarian cancer cells. 제 1 항에 있어서,
상기 엑소좀은 난소암 세포의 증식을 억제하고 난소암 세포의 사멸을 활성화하는 것을 특징으로 하는 조성물.The method according to claim 1,
Wherein said exosome inhibits the proliferation of ovarian cancer cells and activates the death of ovarian cancer cells. 인간 양막 중간엽 줄기세포(Human Amniotic Membrane Mesenchymal Stem Cell, hAMSC)의 배양액(conditioned medium, CM)에서 추출한 엑소좀을 유효성분으로 포함하는 난소암 전이 억제용 조성물로서,
상기 배양액은 총 조성물 부피 대비 1-50 부피%으로 포함하는 것을 특징으로 하는, 난소암 전이 억제용 조성물.A composition for inhibiting ovarian cancer metastasis comprising, as an active ingredient, exosome extracted from a conditioned medium (CM) of human amniotic membrane mesenchymal stem cell (hAMSC)
The composition for inhibiting ovarian cancer metastasis according to claim 1, wherein the culture medium contains 1-50% by volume of the total composition. 삭제delete 제 8 항에 있어서,
상기 엑소좀은 50 내지 150 nm의 지름을 가진 것을 특징으로 하는 조성물. 9. The method of claim 8,
Wherein the exosome has a diameter of 50 to 150 nm. 삭제delete 삭제delete
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WO2020230954A1 (en) * 2019-05-14 2020-11-19 (유)스템메디케어 Method for preparing immune-tolerant extracellular vesicle containing lactate dehydrogenase b and peroxisome proliferator-activated receptor gamma coactivator 1-alpha, and anti-cancer composition using extracellular vesicle
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KR20200012587A (en) 2018-07-27 2020-02-05 강원대학교산학협력단 Composition for prevention or treatment of chronic obstructive pulmonary disease comprising PTD-bFGF or incresed exosome by PTD-bFGF
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WO2022045813A1 (en) * 2020-08-26 2022-03-03 한양대학교 에리카산학협력단 Pharmaceutical composition for treating, preventing, or inhibiting metastasis of lung cancer, comprising exosome as active ingredient
WO2022050720A1 (en) 2020-09-04 2022-03-10 재단법인대구경북과학기술원 Extracellular vesicle expressing cytokine and antibody, method for producing same, and use thereof
KR20220031505A (en) 2020-09-04 2022-03-11 재단법인대구경북과학기술원 Extracellular vesicle expressing cytokines and antibodies, Method of preparing the same, and Use of the same
KR20220040092A (en) * 2020-09-23 2022-03-30 대한민국(농림축산식품부 농림축산검역본부장) Exosomes Derived From Canine For Inhibiting Proliferation of Canine Solid Cancer
KR102601831B1 (en) 2020-09-23 2023-11-14 대한민국 Exosomes Derived From Canine For Inhibiting Proliferation of Canine Solid Cancer
KR20230022800A (en) 2021-08-05 2023-02-16 경북대학교 산학협력단 Composition comprising miRNA derived from extracellular vesicles of activated T cells as an active ingredient and use thereof
KR20230022808A (en) 2021-08-05 2023-02-16 경북대학교 산학협력단 Composition comprising miRNA derived from extracellular vesicles of T cells as an active ingredient and use thereof
KR20230021248A (en) 2021-08-05 2023-02-14 경북대학교 산학협력단 Pharmaceutical composition for combined administration comprising the extracellular vesicles of T cells and an anticancer agent as active ingredients
KR20240043664A (en) 2022-09-26 2024-04-03 재단법인대구경북과학기술원 Composition for treating cancer diseases comprising extracellular vesicles derived from natural killer cells with surface expression of cytokines and antibodie

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