KR20230071602A - Colorectal cancer specific targeting exosome composition and use thereof - Google Patents
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Abstract
Description
본 발명은 재조합 발현벡터로부터 형질전환된 엑소좀을 포함하는 대장암 특이적 표적 엑소좀 조성물 및 이의 용도에 관한 것이다.The present invention relates to a colorectal cancer-specific targeting exosome composition comprising an exosome transformed from a recombinant expression vector and a use thereof.
대장은 크게 결장과 직장으로 구분되며 결장에 생기는 암은 결장암, 직장에 생기는 암은 직장암으로, 대장암 또는 결장직장암으로 통칭하기도 한다. 결장직장암은 대장 및 직장(결장직장)의 거의 모든 암은 대장(결장) 및 직장의 내벽에서 발생하는 샘암종으로, 장 또는 직장 내벽 표면에서 암이 성장에 의하여 발생하며 인접 림프절에도 침입할 수 있으며, 결장직장암 발생률은 40 ~ 50세경에 급격히 증가하기는 특성이 존재한다.The large intestine is largely divided into colon and rectum, and cancer that occurs in the colon is colon cancer, cancer that occurs in the rectum is called rectal cancer, and it is also commonly referred to as colon cancer or colorectal cancer. Colorectal cancer is an adenocarcinoma that occurs in the inner wall of the large intestine (colon) and rectum. Almost all cancers of the large intestine and rectum (colorectal) are caused by cancer growth on the surface of the inner wall of the intestine or rectum, and can invade adjacent lymph nodes. There is a characteristic that the incidence of colorectal cancer increases rapidly around the age of 40 to 50 years.
엑소좀은 그 크기가 30-100nm에 불과하지만, 원세포에 들어있는 단백질, 핵산, 지질 등 여러 물질을 포함하고 있는 물질로서, 면역세포나 줄기세포 등 우리 지방유래줄기세포가 분비하는 나노입자이다. 엑소좀의 세포간 정보 전달체 역할을 하는 기능이 알려지면서 차세대 항암물질로 주목받고 있다.Exosomes are only 30-100 nm in size, but contain various substances such as proteins, nucleic acids, and lipids contained in original cells. They are nanoparticles secreted by our adipose-derived stem cells, such as immune cells and stem cells. . As the function of exosomes as intercellular information carriers is known, they are attracting attention as next-generation anticancer substances.
이에, 본 발명자들은 약물전달물질로서의 대장암 특이적 표적 세포외소포체를 개발하기 위하여 예의 노력한 결과, 지방유래줄기세포로부터 추출된 엑소좀이 대장암 표적 항암제의 기능이 있음을 확인함으로써, 본 발명을 완성하였다.Accordingly, the present inventors made diligent efforts to develop a colorectal cancer-specific target extracellular vesicle as a drug delivery material, and as a result, it was confirmed that the exosomes extracted from adipose-derived stem cells have the function of a colorectal cancer-targeted anticancer drug, thereby concluding the present invention. completed.
본 발명의 목적은 줄기세포의 세포외소포체로 발현되는 유전자 내에 대장암 세포의 수용체와 결합하는 리간드를 발현시키는 유전자가 삽입된 재조합 발현벡터로 형질전환된 세포외소포체를 포함하는 대장암의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.An object of the present invention is to prevent or prevent colorectal cancer including extracellular vesicles transformed with a recombinant expression vector into which a gene expressing a ligand that binds to a receptor of a colorectal cancer cell is inserted into a gene expressed in the extracellular vesicle of stem cells; It is to provide a pharmaceutical composition for treatment.
본 발명의 또 다른 목적은 (a) 지방유래줄기세포의 세포외소포체로 발현되는 유전자 내에 대장암 세포의 수용체와 결합하는 리간드를 발현시키는 유전자를 도입하는 단계; (b) 상기 유전자가 도입된 세포를 배지에서 배양하여 세포외소포체를 수득하는 단계; 및 (c) 상기 수득된 세포외소포체에 대장암 유발 유전자의 발현을 억제시킬 수 있는 핵산을 형질주입시키는 단계;를 포함하는 대장암 예방 또는 치료용 약학적 조성물 제조방법을 제공하는 것이다.Another object of the present invention is (a) introducing a gene that expresses a ligand that binds to a receptor of colorectal cancer cells into a gene expressed in the extracellular endoplasmic reticulum of adipose-derived stem cells; (b) culturing the cells into which the gene has been introduced in a medium to obtain extracellular vesicles; And (c) transfecting the obtained extracellular vesicle with a nucleic acid capable of suppressing the expression of a colorectal cancer-inducing gene;
본 출원에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 출원에서 개시된 다양한 요소들의 모든 조합이 본 출원의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 출원의 범주가 제한된다고 볼 수 없다.Each description and embodiment disclosed in this application can also be applied to each other description and embodiment. That is, all combinations of various elements disclosed in this application fall within the scope of this application. In addition, the scope of the present application is not to be construed as being limited by the specific descriptions described below.
본 발명의 일 측면은 줄기세포의 세포외소포체로 발현되는 유전자 내에 대장암 세포의 수용체와 결합하는 리간드를 발현시키는 유전자가 삽입된 재조합 발현벡터로 형질전환된 세포외소포체를 포함하는 대장암의 예방 또는 치료용 약학적 조성물을 제공한다.One aspect of the present invention is the prevention of colorectal cancer including extracellular vesicles transformed with a recombinant expression vector into which a gene expressing a ligand that binds to a receptor of a colorectal cancer cell is inserted into a gene expressed in the extracellular vesicle of stem cells. Or it provides a pharmaceutical composition for treatment.
본 발명에서의 "발현벡터"는 선형 DNA 벡터, 플라스미드 DNA 벡터 및 재조합 바이러스 벡터로 이루어진 군에서 선택될 수 있으나, 이에 제한되는 것은 아니며 당업계에서 형질전환을 위해 사용되는 통상적인 벡터들은 모두 본 발명의 방법에 사용될 수 있다. 본 발명의 일 구체예에 있어서, 상기 발현벡터는 pDisplay 벡터일 수 있다.The "expression vector" in the present invention may be selected from the group consisting of a linear DNA vector, a plasmid DNA vector, and a recombinant viral vector, but is not limited thereto, and conventional vectors used for transformation in the art are all of the present invention. can be used in the method of In one embodiment of the present invention, the expression vector may be a pDisplay vector.
본 발명에서의 "세포외소포체"는 세포에서 유래되는 막으로 둘러싸인 작은 구체를 의미하는 것으로, 상기 세포외소포체는 자연계, 예컨대 식물, 동물, 미생물로부터 유래된 세포외 소포체 또는 인공적으로 제조된 세포외소포체일 수 있다. 또한, 상기 세포는 자연계 생물 개체로부터 분리된 세포인 것일 수 있다.In the present invention, "extracellular vesicle" refers to a small sphere surrounded by a membrane derived from a cell, and the extracellular vesicle is an extracellular vesicle derived from nature, such as plants, animals, and microorganisms, or artificially prepared extracellular It may be an endoplasmic reticulum. In addition, the cells may be cells isolated from natural organisms.
상기 "세포외소포체"는 엑소좀(exosome), 엑토솜(ectosome) 또는 미세소포(microvesicle)일 수 있으나, 본 발명의 일 구체예에 있어서, 상기 "세포외소포체"는 엑소좀일 수 있다.The "extracellular vesicles" may be exosomes, ectosomes or microvesicles, but in one embodiment of the present invention, the "extracellular vesicles" may be exosomes.
본 발명에서의 "엑소좀"은 다양한 세포들로부터 분비되는 막 구조의 작은 소낭으로서, 다낭체와 원형질막의 융합이 일어나 세포 밖 환경으로 방출되는 소낭을 의미한다. 본 발명의 일 구체예에 있어서, 상기 엑소좀은 지방유래줄기세포로부터 유래 또는 분리한 것일 수 있다.In the present invention, "exosome" is a small membrane-structured vesicle secreted from various cells, and refers to a vesicle released into the extracellular environment after fusion of the polycystic body and the plasma membrane. In one embodiment of the present invention, the exosomes may be derived from or isolated from adipose-derived stem cells.
본 발명의 일 구체예에 있어서, 상기 엑소좀은 50 내지 150 nm의 직경을 갖는 것일 수 있다. 또한, 상기 엑소좀은 1 내지 50 μL/ml의 농도로 포함될 수 있다.In one embodiment of the present invention, the exo-some may have a diameter of 50 to 150 nm. In addition, the exosomes may be included at a concentration of 1 to 50 μL/ml.
본 발명에서의 "줄기세포(stem cells)"는 미분화 상태에서 적절한 조건을 맞춰주면 다양한 조직 세포로 분화될 수 있는 만능세포로서 다양한 유래의 세포를 의미한다."Stem cells" in the present invention refers to cells of various origins as pluripotent cells capable of differentiating into various tissue cells when appropriate conditions are met in an undifferentiated state.
상기 줄기세포는 골수유래줄기세포, 제대혈유래줄기세포 또는 지방유래줄기세포일 수 있다. 본 발명의 일 구체예에 있어서, 상기 줄기세포는 지방유래줄기세포일 수 있다. 또한, 상기 골수유래줄기세포, 제대혈유래줄기세포 또는 지방유래줄기세포는 인체 또는 동물유래 줄기세포인 것일 수 있다.The stem cells may be bone marrow-derived stem cells, cord blood-derived stem cells or adipose-derived stem cells. In one embodiment of the present invention, the stem cells may be adipose-derived stem cells. In addition, the bone marrow-derived stem cells, cord blood-derived stem cells, or adipose-derived stem cells may be human or animal-derived stem cells.
본 발명에서의 "지방유래줄기세포"는 지방 조직으로부터 유래한 줄기세포로서, 다분화능 및 자기증식능을 가진 세포를 의미하며, 본 발명의 지방유래줄기세포는 지방흡입술 및 다양한 외과적 수술을 통해 수득할 수 있으나, 이에 한정되지 않는다."Adipose-derived stem cells" in the present invention are stem cells derived from adipose tissue, and refer to cells having pluripotency and self-renewal ability, and the adipose-derived stem cells of the present invention are obtained through liposuction and various surgical operations. It can, but is not limited to this.
본 발명의 일 구체예에 있어서, 상기 엑소좀은 줄기세포 등 호스트세포의 엑소좀으로 발현하는 유전자 내에 대장암 세포의 수용체와 결합하는 리간드를 발현시키도록 형질전환되어 대장암 세포에 표적으로 작용할 수 있는 것을 의미한다.In one embodiment of the present invention, the exosome is transformed to express a ligand that binds to a receptor of a colon cancer cell in a gene expressed as an exosome of a host cell such as a stem cell to act as a target for colon cancer cells. means there is
또한, 본 발명의 일 구체예에 있어서, 상기 엑소좀은 대장암 유발 유전자의 발현을 억제시킬 수 있는 핵산이 형질주입된 것일 수 있다.In addition, in one embodiment of the present invention, the exosome may be transfected with a nucleic acid capable of suppressing the expression of a colorectal cancer-inducing gene.
본 발명에서의 "형질주입"은 동물세포에 DNA 또는 RNA를 직접 도입하여 세포의 유전형질을 변이시키는 방법을 의미하며, 이는 당해 기술분야에 공지된 방법, 예를 들어, 칼슘 인산염 형질주입법(calcium phosphate transfection), 리포펙션법(lipofection), 전기천공법(electroporation), 미량주사법(microinjection), 마이크로프로젝틸법(microprojectile)을 이용할 수 있으나, 이에 제한되지 않는다. 진핵생물의 경우 DNA인산칼슘침전법 또는 리포펙션, PEI 등의 상품화된 시약과 DNA를 혼합하여 세포를 처리하는 방법을 이용할 수 있다."Transfection" in the present invention refers to a method of directly introducing DNA or RNA into animal cells to mutate the genetic traits of cells, which is a method known in the art, for example, calcium phosphate = transfection (calcium phosphate transfection) Phosphate transfection), lipofection, electroporation, microinjection, and microprojectile may be used, but are not limited thereto. In the case of eukaryotes, a method of treating cells by mixing DNA with commercially available reagents such as DNA calcium phosphate precipitation, lipofection, or PEI may be used.
본 발명에서의 "핵산"은 줄기세포의 세포외소포체로 발현되는 유전자 내에 삽입되어 세포외소포체로 발현 시 대장암 세포의 수용체와 결합하는 펩타이드를 코딩할 수 있는 유전자를 포함하는 것을 의미한다."Nucleic acid" in the present invention means a gene that can encode a peptide that is inserted into a gene expressed in the extracellular endoplasmic reticulum of stem cells and binds to a receptor of colon cancer cells when expressed in the extracellular endoplasmic reticulum.
본 발명에서의 "핵산"은 대장암 유발 유전자의 발현을 억제시킬 수 있는 핵산으로서, 발암 유전자를 발현하는 핵산과 결합 또는 발암 유전자와 직접 결합하여 발현을 억제시키는 방식으로 대장암 유발 유전자의 발현을 억제시킬 수 있다."Nucleic acid" in the present invention is a nucleic acid capable of suppressing the expression of a colorectal cancer-inducing gene, and inhibits the expression of a colorectal cancer-inducing gene by combining with a nucleic acid expressing an oncogene or directly binding to an oncogene to inhibit expression. can suppress.
본 발명에서의 "핵산"은 miRNA, siRNA, shRNA, 안티센스 RNA 및 리보자임(ribozyme)으로 이루어진 군으로부터 선택된 하나 이상의 핵산일 수 있으나, 본 발명의 일 구체예에 있어서, 상기 "핵산"은 miRNA 및 siRNA로 이루어진 군으로부터 선택된 하나 이상의 핵산을 포함한다."Nucleic acid" in the present invention may be one or more nucleic acids selected from the group consisting of miRNA, siRNA, shRNA, antisense RNA and ribozyme, but in one embodiment of the present invention, the "nucleic acid" is miRNA and It includes one or more nucleic acids selected from the group consisting of siRNAs.
또한, 본 발명의 또 다른 측면은 (a) 지방유래줄기세포의 세포외소포체로 발현되는 유전자 내에 대장암 세포의 수용체와 결합하는 리간드를 발현시키는 유전자를 도입하는 단계; (b) 상기 유전자가 도입된 세포를 배지에서 배양하여 세포외소포체를 수득하는 단계; 및 (c) 상기 수득된 세포외소포체에 대장암 유발 유전자의 발현을 억제시킬 수 있는 핵산을 형질주입시키는 단계;를 포함하는 대장암 예방 또는 치료용 발현벡터 제조방법을 제공한다.Another aspect of the present invention is (a) introducing a gene that expresses a ligand that binds to a receptor of colorectal cancer cells into a gene expressed in the extracellular vesicles of adipose-derived stem cells; (b) culturing the cells into which the gene has been introduced in a medium to obtain extracellular vesicles; and (c) transfecting the obtained extracellular vesicle with a nucleic acid capable of suppressing the expression of a colorectal cancer-inducing gene.
본 발명에서의 "세포외소포체"는 엑소좀(exosome), 엑토솜(ectosome) 또는 미세소포(microvesicle)일 수 있으나, 본 발명의 일 구체예에 있어서, 상기 "세포외소포체"는 엑소좀일 수 있다."Extracellular vesicles" in the present invention may be exosomes, ectosomes or microvesicles, but in one embodiment of the present invention, the "extracellular vesicles" may be exosomes there is.
본 발명에서의 "배지"는 지방유래줄기세포 배양배지인 것일 수 있다. 본 발명의 일 구체예에 있어서, 상기 지방유래줄기세포 배양배지는 지방유래줄기세포를 FBS(inactivated fetal bovine serum) 및 P/S(penicillin-streptomycin)을 첨가한 DMEM high low glucose 배지 또는 FBS-Free DMEM low glucose로 배지일 수 있다.The "medium" in the present invention may be an adipose-derived stem cell culture medium. In one embodiment of the present invention, the adipose-derived stem cell culture medium is DMEM high-low glucose medium or FBS-Free to which FBS (inactivated fetal bovine serum) and P/S (penicillin-streptomycin) are added. The medium can be DMEM low glucose.
본 발명에서의 “예방”은 본 발명에 따른 약학적 조성물의 투여에 의해 대장암을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다."Prevention" in the present invention refers to all activities that suppress or delay the onset of colon cancer by administration of the pharmaceutical composition according to the present invention.
본 발명에서의 “치료”는 본 발명에 따른 약학적 조성물의 투여에 의해 대장암에 대한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다."Treatment" in the present invention refers to all activities that improve or beneficially change the symptoms of colon cancer by administration of the pharmaceutical composition according to the present invention.
본 발명에서의 "대장암(colorectal cancer)"은 대장에 생긴 암세포로 이루어진 악성 종양을 의미한다. 대장암은 병리학적으로 대부분 융종(polyp)의 형태에서 발전된 선암(adenocarcinoma)이며 드물게 유암종(carcinoid), 림프종(lymphoma) 등에 의한 암이 발생하기도 한다. 부위별로는 크게 결장암과 직장암으로 나뉜다.In the present invention, "colorectal cancer" means a malignant tumor composed of cancer cells formed in the large intestine. Colon cancer is pathologically most adenocarcinoma developed in the form of a polyp, and cancer caused by carcinoid, lymphoma, etc. may occur in rare cases. By site, it is largely divided into colon cancer and rectal cancer.
본 발명의 줄기세포의 세포외소포체로 발현되는 유전자 내에 대장암 세포의 수용체와 결합하는 리간드를 발현시키는 유전자가 삽입된 재조합 발현벡터로 형질전환된 세포외소포체를 포함하는 약학적 조성물은 세포외소포체로서 엑소좀을 약물전달물질로서 활용하여 표적 세포인 대장암 세포에 선별적으로 결합하여 대장암에 특이적으로 작용할 수 있다. 따라서, 상기 표적 엑소좀은 대장암 치료 분야, 특히 임상적 적용 기술로 응용될 수 있다.A pharmaceutical composition comprising extracellular vesicles transformed with a recombinant expression vector into which a gene expressing a ligand that binds to a receptor of colon cancer cells is inserted into a gene expressed in the extracellular endoplasmic reticulum of stem cells of the present invention. As such, exosomes can be used as a drug delivery material to selectively bind to target cells, colorectal cancer cells, and act specifically on colorectal cancer. Therefore, the target exosome can be applied to the field of colorectal cancer treatment, particularly clinical application technology.
도 1은 발현벡터를 통하여 엑소좀에 타겟팅 리간드를 발현시키는 모습을 나타낸 모식도이다.
도 2는 엑소좀에 대한 구조 분석을 위하여 유세포분석기 (FACS)을 이용하여 분석한 결과이다.
도 3은 HCT116 및 HCT29에서의 실시간 PCR 분석 결과이다.
도 4는 본 발명에 따른 표적 엑소좀의 대장암세포주인 HCT116에서의 wound healing assay에 따른 cell migration을 나타낸 결과이다.
도 5는 BALB/C Nude mouse에 대장암세포주 HCT116 이식하여 구축된 대장암 동물 는모델에서의 외형 관찰 결과 중 종양의 크기를 나타낸 것이다.
도 6은 BALB/C Nude mouse에 대장암세포주 HCT116 이식하여 구축된 대장암 동물 모델에서의 외형 관찰 결과 중 종양의 크기를 나타낸 것이다.
도 7은 BALB/C Nude mouse에 대장암세포주 HCT116 이식하여 구축된 대장암 동물 모델에서의 외형 관찰 결과 중 체중의 변화를 나타낸 것이다.
도 8은 BALB/C Nude mouse에 대장암세포주 HCT116 이식하여 이식한 대장암 동물 모델에서의 각 인자 별 발현 분석을 나타낸 것이다.
도 9는 BALB/C Nude mouse에 대장암세포주 HCT116 이식하여 유도한 대장암 동물 모델에서의 Ecad 발현 분석을 IHC로 확인한 결과이다.
도 10은 BALB/C Nude mouse에 대장암세포주 HCT116 이식하여 유도한 대장암 동물 모델에서의 Snail발현 분석을 IHC로 확인한 결과이다.1 is a schematic diagram showing the expression of a targeting ligand in exosomes through an expression vector.
2 is a result of analysis using flow cytometry (FACS) for structural analysis of exosomes.
3 is a real-time PCR analysis result in HCT116 and HCT29.
4 shows the results of cell migration according to the wound healing assay in HCT116, a colon cancer cell line of the target exosome according to the present invention.
5 shows the size of a tumor among the external observation results in a colorectal cancer animal model constructed by transplanting the colorectal cancer cell line HCT116 into a BALB/C Nude mouse.
6 shows the size of a tumor among the external observation results in a colorectal cancer animal model constructed by transplanting the colorectal cancer cell line HCT116 into a BALB/C Nude mouse.
7 shows changes in body weight among external observation results in a colorectal cancer animal model constructed by transplanting the colorectal cancer cell line HCT116 into a BALB/C Nude mouse.
8 shows expression analysis of each factor in a colorectal cancer animal model transplanted by transplanting the colorectal cancer cell line HCT116 into a BALB/C Nude mouse.
9 shows the result of IHC analysis of Ecad expression in a colorectal cancer animal model induced by transplanting the colorectal cancer cell line HCT116 into a BALB/C Nude mouse.
10 shows the result of IHC analysis of Snail expression in a colorectal cancer animal model induced by transplanting the colorectal cancer cell line HCT116 into a BALB/C Nude mouse.
이하, 실시예를 통하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for exemplifying the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.
실시예 1. 지방유래줄기세포 유래의 표적 엑소좀 제작 및 확인Example 1. Adipose-derived stem cell-derived "target exosome" production and confirmation
본 발명의 대장암에 특이적으로 작용하는 표적 엑소좀을 제작하기 위하여 지방유래줄기세포를 사용하여 엑소좀을 추출하였다. 참고적으로, 본 발명에서는 표적 엑소좀을 생산하기 위한 donor cells로서 지방유래줄기세포를 사용하였지만, NK세포와 같은 면역세포도 가능하므로, 이에 한정하지 않는다.In order to prepare target exosomes that specifically act on colorectal cancer of the present invention, exosomes were extracted using adipose-derived stem cells. For reference, in the present invention, adipose-derived stem cells were used as donor cells for producing target exosomes, but immune cells such as NK cells are also possible, so it is not limited thereto.
지방유래줄기세포를 제조사의 지침에 따라 형질감염시약(lipofectamine, invitrogen)을 사용하여 pDisplay 벡터(invitrogen)에 APRIL를 표적하는 대장암세포 표적 리간드를 인코딩하는 아미노산 서열(AAAPLAQPHMWA)을 인코딩 하는 염기서열(GATCTGCAGCCGCTCCTCTTGCACAACCCCACATGTGGGCTG)로 형질도입(transduction)시켰다. 상기 벡터를 클로닝한 후, 지방유래줄기세포에 삽입하여 표적 엑소좀을 제작하였다. Adipose-derived stem cells were transfected using transfection reagents (lipofectamine, invitrogen) according to the manufacturer's instructions, and the nucleotide sequence (GATCTGCAGCCGCTCCTCTTGCACAACCCCACATGTGGGCTG ) was transduced. After cloning, the vector was inserted into adipose-derived stem cells to prepare target exosomes.
제작된 표적 엑소좀을 추출하기 위하여 지방유래줄기세포를 10% FBS(inactivated fetal bovine serum) 및 1% P/S(penicillin-streptomycin)을 첨가한 DMEM low glucose 배지에서 37℃, 5% CO2조건으로 배양하였다.To extract the prepared target exosome, adipose-derived stem cells were prepared in DMEM low glucose medium supplemented with 10% FBS (inactivated fetal bovine serum) and 1% P/S (penicillin-streptomycin) at 37°C and 5% CO 2 condition. cultured with.
24시간 경과 후 상기 배지를 FBS-Free DMEM low glucose로 배지를 교환하였으며, 배지 교환 후 24시간 경과 후 지방유래줄기세포의 배양 배지를 걷어 분별원심분리(differential centrifugation) 방법으로 1000rpm조건으로 원심분리하여 세포를 제거하였다.After 24 hours, the medium was exchanged with FBS-Free DMEM low glucose, and after 24 hours after the medium exchange, the culture medium of adipose-derived stem cells was removed and centrifuged at 1000 rpm by differential centrifugation. cells were removed.
상기 세포가 제거된 배양 배지를 thermo exosome 추출 kit(Thermo Fisher scientific)을 사용하여 제조사의 지침에 따라 대장암 표적 엑소좀을 확보하였다.Colon cancer target exosomes were obtained from the culture medium from which the cells were removed using a thermo exosome extraction kit (Thermo Fisher scientific) according to the manufacturer's instructions.
여기에, 상기 과정을 통하여 확보한 표적 엑소좀에 Exo-Fect kit(SBI System biosciences)을 사용하여 PIN-siRNA를 표적 엑소좀 내에 형질주입하였으며 이를 통해 대장암 수용체에 표적이 가능한 엑소좀을 추가적으로 제작하였다.Here, the PIN-siRNA was transfected into the target exosomes using the Exo-Fect kit (SBI System biosciences) in the target exosomes obtained through the above process, and through this, additional exosomes capable of targeting the colorectal cancer receptor were produced did
실시예 2. 지방유래줄기세포 유래의 표적 엑소좀의 특성 분석Example 2. Analysis of characteristics of target exosomes derived from adipose-derived stem cells
본 발명의 대장암에 특이적으로 작용하는 표적 엑소좀에 대한 구조 분석을 위하여 유세포분석기 (FACS)을 이용하여 분석하여, 엑소좀의 FACs 분석 결과를 도 2에 나타내었다.In order to analyze the structure of target exosomes that specifically act on colorectal cancer of the present invention, flow cytometry (FACS) was used to analyze them, and the results of FACs analysis of exosomes are shown in FIG. 2 .
유세포분석기 (FACS)를 이용하여 엑소좀 표면 마커인 CD81, CD63, 및 CD9의 발현이 확인됨에 따라 분리된 엑소좀은 전형적인 엑소좀의 특성을 갖는 것을 알 수 있다(도 2).As the expression of CD81, CD63, and CD9, which are exosome surface markers, was confirmed using flow cytometry (FACS), it can be seen that the isolated exosomes have typical characteristics of exosomes (FIG. 2).
실시예 3. 지방유래줄기세포 유래의 표적 엑소좀의 실시간 PCR 분석Example 3. Real-time PCR analysis of target exosomes derived from adipose-derived stem cells
대장암 세포주 별 발현에 차이를 확인하기 위하여, 대장암 세포주 및 대장암 표적 엑소좀에 장착할 유전자를 선별하였다. 대장암 세포주로서 HCT116 및 HCT29를 선별하고, 표적 엑소좀에 장착할 유전자는 대장암에서 EMT(Epithelial to Mesenchymal Transition)를 촉진한다고 알려져 있는 PIN1을 선정하였다.In order to confirm the difference in expression of each colorectal cancer cell line, a gene to be loaded into the colorectal cancer cell line and the colorectal cancer target exosome was selected. HCT116 and HCT29 were selected as colorectal cancer cell lines, and PIN1, which is known to promote EMT (Epithelial to Mesenchymal Transition) in colorectal cancer, was selected as a gene to be inserted into the target exosome.
실시간 PCR(RealtimePCR)분석을 위하여, cDNA kit(TOYOBO)를 사용하여 10ng의 RNA를 특이적 프라이머를 사용하여 첫번째 cDNA가닥으로 전환시킨 후 SYBR green(TOYOBO)를 사용하여 thermo PCR machine으로 실시간 PCR(RealtimePCR)을 95℃ 20sec, 95℃ 10min(1cycle), 95℃ 10sec, 60℃ 1min (40cycle), 95℃ 15sec, 60℃ 1min, 95℃ 15sec (1cycle)의 조건으로 수행한 후, 결과를 관찰하였다(도 3).For real-time PCR (RealtimePCR) analysis, 10 ng of RNA was converted into the first cDNA strand using specific primers using a cDNA kit (TOYOBO), and then real-time PCR (RealtimePCR ) was performed under conditions of 95 °
그 결과, 도 3에 나타낸 바와 같이, HCT116에서 PINsiRNA의 발현이 증가하는 경향을 확인할 수 있었다. 따라서, HCT116에서 PINsiRNA를 장착한 대장암 표적 엑소좀의 항종양 효과를 확인하는 세포주로 사용할 수 있음을 확인하였다.As a result, as shown in FIG. 3, it was confirmed that PINsiRNA expression increased in HCT116. Therefore, it was confirmed that HCT116 can be used as a cell line to confirm the antitumor effect of the colorectal cancer target exosome loaded with PINsiRNA.
실시예 4. 지방유래줄기세포 유래의 표적 엑소좀의 종양생성 억제능 평가Example 4. Evaluation of tumorigenesis inhibitory ability of target exosomes derived from adipose-derived stem cells
비표적 엑소좀과 표적엑소좀에 의한 세포의 전이능을 관찰하기 위하여 HCT116 대장암세포주에 처리하여 wound healing assay를 진행하였다. 먼저, 대조군(Control)으로서 무처리(Ct), 비표적엑소좀(엑소좀) 및 표적엑소좀(표적엑소좀)을 준비하고 여기에 Negative control(Mock) 군 및 PIN-siRNA를 장착(siPIN)한 군을 준비하였다.In order to observe the metastatic ability of cells by non-target exosomes and target exosomes, HCT116 colorectal cancer cell line was treated and wound healing assay was performed. First, as a control (Control), untreated (Ct), non-targeting exosomes (exosomes) and target exosomes (targeting exosomes) are prepared, and a negative control (Mock) group and PIN-siRNA are attached to them (siPIN) A group was prepared.
실험결과, wound healing assay(0hr 및 48hr)를 수행했을 때, PINsiRNA 장착한 대장암 표적엑소좀에 의해서 유의하게 cell migration이 억제되어 wound healing을 억제하는 효과가 두드러지게 나타남을 확인할 수 있었다(도 4).As a result of the experiment, when wound healing assay (0 hr and 48 hr) was performed, it was confirmed that cell migration was significantly inhibited by the PINsiRNA-equipped colon cancer target exosome, and the effect of inhibiting wound healing was remarkably displayed (FIG. 4). ).
실시예 5. 대장암 동물 모델을 이용한 지방유래줄기세포 유래 표적 엑소좀의 외형 관찰 평가Example 5. Observation and evaluation of adipose-derived stem cell-derived target exosomes using colorectal cancer animal models
BALB/C Nude mouse(in vivo)에 대장암세포주 HCT116을 5x106으로 이식하여 대장암을 유도한 뒤, 본 발명의 표적 엑소좀을 이용한 대장암의 치료 효능을 확인하고자 하였다.Colon cancer was induced by transplanting 5x10 6 colon cancer cell line HCT116 into a BALB/C Nude mouse (in vivo), and then the efficacy of the treatment of colon cancer using the target exosome of the present invention was confirmed.
대조군(Control)으로서 무처리(Ct), 비표적엑소좀(엑소좀) 및 표적엑소좀(표적엑소좀)을 준비하고 여기에 Negative control(Mock) 군 및 PIN-siRNA를 장착(siPIN)한 군을 준비하고, 각각을 상기 구축된 종양모델에 Tail vein injection 방식으로 2주간 주입한 후, 마우스를 희생하여 대장암의 항 종양효과에 엑소좀이 미치는 영향을 확인하였다.As a control (Control), non-treatment (Ct), non-targeting exosomes (exosomes) and target exosomes (targeting exosomes) were prepared, and a negative control (Mock) group and a PIN-siRNA attached (siPIN) group were prepared. were prepared, and each was injected into the constructed tumor model by tail vein injection method for 2 weeks, and then the mouse was sacrificed to confirm the effect of exosomes on the antitumor effect of colorectal cancer.
그 결과, 구축된 대장암 동물 모델에서의 외형 관찰 결과에 의하여 PIN1siRNA를 장착한 표적엑소좀을 처리한 군에서 종양의 크기를 확연하게 감소되어 종양 증식이 억제됨을 확인할 수 있었다(도 5 및 도 6). 또한, 엑소좀 처리에 의한 마우스 체중의 변화는 나타나지 않았음을 알 수 있었다(도 7).As a result, it was confirmed that the size of the tumor was significantly reduced in the group treated with the target exosome equipped with PIN1siRNA according to the external observation results in the constructed colorectal cancer animal model, and thus tumor growth was suppressed (FIGS. 5 and 6). ). In addition, it was found that there was no change in the body weight of the mouse by the exosome treatment (FIG. 7).
실시예 7. 대장암 동물 모델을 이용한 지방유래줄기세포 유래 표적 엑소좀의 효능 평가Example 7. Efficacy evaluation of adipose-derived stem cell-derived target exosomes using colorectal cancer animal models
BALB/C Nude mouse(in vivo)에 대장암세포주 HCT116을 5x106으로 이식하여 대장암을 유도한 뒤, 본 발명의 표적 엑소좀을 이용한 대장암의 치료 효능을 확인하고자 하였다.Colon cancer was induced by transplanting 5x10 6 colon cancer cell line HCT116 into a BALB/C Nude mouse (in vivo), and then the efficacy of the treatment of colon cancer using the target exosome of the present invention was confirmed.
대조군(Control)으로서 무처리(Ct), 비표적엑소좀(엑소좀) 및 표적엑소좀(표적엑소좀)을 준비하고 여기에 Negative control(Mock) 군 및 PIN-siRNA를 장착(siPIN)한 군을 준비하고, 각각을 상기 구축된 종양모델에 Tail vein injection 방식으로 2주간 주입한 후, 마우스를 희생하여 대장암의 항 종양효과에 엑소좀이 미치는 영향을 확인하였다.As a control (Control), non-treatment (Ct), non-targeting exosomes (exosomes) and target exosomes (targeting exosomes) were prepared, and a negative control (Mock) group and a PIN-siRNA attached (siPIN) group were prepared. were prepared, and each was injected into the constructed tumor model by tail vein injection method for 2 weeks, and then the mouse was sacrificed to confirm the effect of exosomes on the antitumor effect of colorectal cancer.
EMT(Epithelial to Mesenchymal Transition) 기전에 관여하는 인자인 E-cad, N-cad, vimentin, Snail 및 PIN1의 발현 수준을 비교함으로써, 변화를 관찰하였다(도 7). 그 결과, 구축된 종양 조직에서 관련인자의 발현을 확인한 결과, PIN1siRNA장착한 표적엑소좀 군에서 E-cad의 발현 수준이 증가하고 N-cad, vimentin, Snail의 발현 수준이 감소되어 EMT가 억제됨을 확인할 수 있었다(도 8).Changes were observed by comparing the expression levels of E-cad, N-cad, vimentin, Snail, and PIN1, which are factors involved in the EMT (Epithelial to Mesenchymal Transition) mechanism (FIG. 7). As a result, as a result of confirming the expression of related factors in the constructed tumor tissue, the expression level of E-cad increased and the expression level of N-cad, vimentin, and Snail decreased in the target exosome group loaded with PIN1siRNA, indicating that EMT was inhibited. It was confirmed (FIG. 8).
또한, 면역조직화학염색법(IHC: immunohistochemistry)을 통하여 PINsiRNA장착군(tExo+PINsiRNA)에서 Ecad의 발현은 증가하며, Snail의 발현은 감소하는 경향을 확인할 수 있었다(도 9 및 10).In addition, through immunohistochemistry (IHC), it was confirmed that the expression of Ecad increased and the expression of Snail decreased in the PINsiRNA-loaded group (tExo + PINsiRNA) (Figs. 9 and 10).
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.
<110> The Catholic University of Korea Industry-Academic Cooperation Foundation <120> Colorectal cancer specific targeting exosome composition and use thereof <130> DPC215627 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> incoding sequence <400> 1 gatctgcagc cgctcctctt gcacaacccc acatgtgggc tg 42 <110> The Catholic University of Korea Industry-Academic Cooperation Foundation <120> Colorectal cancer specific targeting exosome composition and use its <130> DPC215627 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 42 <212> DNA <213> artificial sequence <220> <223> encoding sequences <400> 1 gatctgcagc cgctcctctt gcacaacccc acatgtgggc tg 42
Claims (10)
(b) 상기 유전자가 도입된 세포를 배지에서 배양하여 세포외소포체를 수득하는 단계; 및
(c) 상기 수득된 세포외소포체에 대장암 유발 유전자의 발현을 억제시킬 수 있는 핵산을 형질주입시키는 단계;를 포함하는 대장암 예방 또는 치료용 발현벡터 제조방법.(a) introducing a gene that expresses a ligand that binds to a receptor of colorectal cancer cells into a gene expressed in the extracellular vesicles of adipose-derived stem cells;
(b) culturing the cells into which the gene has been introduced in a medium to obtain extracellular vesicles; and
(c) transfecting the obtained extracellular vesicle with a nucleic acid capable of suppressing the expression of a colorectal cancer-inducing gene; a method for preparing an expression vector for preventing or treating colorectal cancer.
The method of preparing an expression vector for preventing or treating colorectal cancer according to claim 9, wherein the extracellular vesicles are exosomes.
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