KR20120139311A - Composition producing lymph blood vessel - Google Patents

Composition producing lymph blood vessel Download PDF

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KR20120139311A
KR20120139311A KR1020110059046A KR20110059046A KR20120139311A KR 20120139311 A KR20120139311 A KR 20120139311A KR 1020110059046 A KR1020110059046 A KR 1020110059046A KR 20110059046 A KR20110059046 A KR 20110059046A KR 20120139311 A KR20120139311 A KR 20120139311A
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colony stimulating
stimulating factor
granulocyte
csf
granulocyte colony
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김원
박성광
강경표
이애신
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전북대학교산학협력단
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    • A—HUMAN NECESSITIES
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    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
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    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
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Abstract

PURPOSE: A lymphatic vascularization composition containing granulocyte-colony stimulating-factors is provided to induce capillarization, cell proliferation, and lymphatic sprout of human lymphatic vascular endothelial cells. CONSTITUTION: A lymphatic vascularization composition contains granulocyte-colony stimulating-factors as an active ingredient. The granulocyte-colony stimulating-factors include granulocyte macrophage colony stimulating factors(GM-CSF) and macrophage colony stimulating factors(MCSF). A composition for preventing or treating lymphedema contains the granulocyte-colony stimulating-factors as an active ingredient. A composition for preventing or treating lymphatic vessel loss or lymphopenia contains the granulocyte-colony stimulating-factors. An agent for enhancing ERK1/2 phosphorylation and Akt phosphorylation contains the granulocyte-colony stimulating-factors as an active ingredient.

Description

림프 혈관 생성 조성물{Composition producing lymph blood vessel}Composition producing lymph blood vessel

본 발명은 림프 혈관을 생성하는 조성물에 관한 발명이다.
The present invention relates to a composition for producing lymph vessels.

림프관은 조직의 간질로부터 조직액을 배액시키는 역할을 하며, 면역학적 방어기전에 중요한 역할을 한다. 특히, 림프관은 악성종양, 림프부종 및 염증성 질환의 병태생리학적 기전에 있어 중요한 역할을 한다. Lymphatic vessels serve to drain tissue fluid from tissue epilepsy and play an important role in immunological defense mechanisms. In particular, lymphatic vessels play an important role in the pathophysiological mechanisms of malignant tumors, lymphedema and inflammatory diseases.

림프관의 기능 장애가 생기면 세포외액과 거대분자물질의 이동에 문제가 생기며, 결국 조직의 부종, 면역기능장애 및 간질조직의 섬유화가 발생하게 된다. Lymphatic dysfunction causes problems in the transport of extracellular fluid and macromolecules, resulting in tissue edema, immune dysfunction and fibrosis of interstitial tissues.

이들 중 림프부종은 림프관이 손상되거나 막혀서 단백질이 비정상적으로 축적되어 부종과 염증을 유발시키는 것으로서, 유전성 또는 이차성 원인으로 발생한다.Among them, lymphedema is an abnormal accumulation of proteins due to damage or blockage of the lymphatic vessels, leading to edema and inflammation, which occurs as a hereditary or secondary cause.

이차성 원인으로 발생하는 림프부종은 수술, 방사선치료 등을 동반하는 악성종양의 치료 후에 많이 발생한다. 이러한 림프부종의 치료는 대부분 압박붕대를 이용하거나, 물리치료와 마사지 같은 보존적인 치료에 의존하고 있다. 이러한 림프부종의 치료를 위한 약물의 개발은 현재 미비한 수준이다. 다만 실험실적으로는 림프혈관생성을 유도하는 VEGF(vascular endothelial growth factor)-C/D, 앤지오포이에틴(Angiopoietin)과 같은 혈관생성인자가 림프혈관생성을 유도하는 강력한 물질로 알려져 있으며, 이러한 림프혈관생성 유도 효과로 인해 림프부종에 치료적 효과가 있음이 동물실험을 통해 알려지고 있는 수준이다. Lymphedema that occurs as a secondary cause often occurs after the treatment of malignant tumors, including surgery and radiation therapy. The treatment of these lymph edema mostly depends on the use of compression bandage or conservative treatment such as physiotherapy and massage. The development of drugs for the treatment of such lymphedema is currently incomplete. However, in the laboratory, angiogenic factors such as vascular endothelial growth factor (VEGF) -C / D and angiopoietin, which induce lymphangiogenesis, are known as potent substances that induce lymphangiogenesis. It is known from animal experiments that there is a therapeutic effect on lymphedema due to angiogenesis inducing effect.

그런데, 이러한 림프혈관생성은 통상적으로 악성세포가 전이되는 모세관 경로를 제공한다는 문제점을 가지고 있다. 그리하여 이러한 림프혈관을 증식하는 방식으로 림프부종을 치료하는 방식은 모세관 세포를 증식시켜 악성종양을 발생시킬 수 있다. However, such lymphangiogenesis typically has a problem in that it provides a capillary pathway through which malignant cells metastasize. Thus, the treatment of lymphedema by the way of proliferating such lymph vessels can proliferate capillary cells and cause malignant tumors.

한편, 과립구는 백혈구 중에서 원형질 내에 과립을 갖고 있는 백혈구의 대부분을 차지하는 세포인데, 과립구집락자극인자(granulocyte colony-stimulating factor: 이하, ‘G-CSF’라고 함)는 이러한 과립구의 집락을 자극하는 인자이다. On the other hand, granulocytes are the cells that occupy most of the leukocytes having granules in the plasma of the leukocytes, granulocyte colony-stimulating factor (hereinafter referred to as 'G-CSF') factors that stimulate the colonization of these granulocytes to be.

구체적으로 과립구집락자극인자(G-CSF, granulocyte-colony stimulating factor) 는 호중구 선조세포(neutrophil progenitor cell)에 특이적으로 작용하여 호중구의 증식과 분화를 촉진하고 호중구의 항체 의존성 세포 살생 능력을 증가시키는 한편, IgA-매개성 대식작용(phagocytosis)을 촉진하고 수퍼옥사이드(superoxide) 생성 능력을 증가시키는 작용이 있다. 따라서, 과립구집락자극인자(G-CSF)는 화학주성 펩티드(chemotactic peptide)에 대한 반응능력을 향상시켜 감염증 발생을 억제하고 발열의 빈도를 낮추는 역할을 하는 것으로 알려져 있다. 또한, 과립구집락자극인자(G-CSF)는 GM-CSF(granulocyte-macrophage CSF) 등의 다른 CSF에 비해 보다 분화된 골수세포에 작용하기 때문에 생체내에서 백혈병 모세포에 대한 영향이 적을 것으로 기대되고 있다. 이에 따라, G-CSF는 항암 화학요법, 항암제의 대량요법, 방사선요법과의 병합요법, 골수 이식 후 호중구의 회복을 촉진시키는 약물로 널리 사용되고 있다(Julie M. Vores et al. , Clinical Applications of Hemat opoietic Growth Factors. Journal of Clinincal Oncology, 13: 1023-1035, 1995).Specifically, granulocyte-colony stimulating factor (G-CSF) acts specifically on neutrophil progenitor cells to promote neutrophil proliferation and differentiation and increase neutrophil antibody-dependent cell killing ability. On the other hand, there is an action to promote IgA-mediated phagocytosis and increase the ability to produce superoxide. Therefore, granulocyte colony stimulating factor (G-CSF) is known to play a role in suppressing the occurrence of infection and reducing the frequency of fever by improving the ability to respond to chemotactic peptide (chemotactic peptide). In addition, granulocyte colony stimulating factor (G-CSF) is expected to have less effect on leukemia blast cells in vivo because it acts on more differentiated bone marrow cells than other CSF such as GM-CSF (granulocyte-macrophage CSF). . Accordingly, G-CSF has been widely used as a chemotherapy, chemotherapy with chemotherapy, radiation therapy, and drugs that promote neutrophil recovery after bone marrow transplantation (Julie M. Vores et al., Clinical Applications of Hemat opoietic Growth Factors.Journal of Clinincal Oncology, 13: 1023-1035, 1995).

그런데, 이와 같은 과립구집락자극인자를 이용하여, 림프 혈관 생성을 유도하여 림프 부종을 치료하는 방식은 현재 아직 시도되고 있지 못하고 있다.
However, a method of treating lymphedema by inducing lymphatic vessel formation using such granulocyte colony stimulating factors has not been attempted yet.

상기와 같은 종래 기술의 문제점을 해결하기 위해, 본 발명은 과립구집락자극인자를 이용하여 림프부종을 치료하기 위한 림프혈관생성을 유도하는 인자를 제공하고자 한다.
In order to solve the problems of the prior art as described above, the present invention is to provide a factor for inducing lymphangiogenesis for treating lymphedema using granulocyte colony stimulating factor.

위와 같은 과제를 해결하기 위한 본 발명의 한 특징에 따른 림프 혈관 생성 조성물은 과립구집락자극인자를 포함한다.Lymph angiogenesis composition according to a feature of the present invention for solving the above problems includes a granulocyte colony stimulating factor.

본 발명의 또 다른 특징에 따른 림프관 생성 유도제는 과립구집락자극인자를 포함한다.Lymphatic production inducing agent according to another feature of the present invention comprises a granulocyte colony stimulating factor.

본 발명의 또 다른 특징에 따른 림프부종 예방 또는 치료용 조성물은 과립구집락자극인자를 유효성분으로 포함한다.The composition for preventing or treating lymphedema according to another aspect of the present invention includes a granulocyte colony stimulating factor as an active ingredient.

본 발명의 또 다른 특징에 따른 림프관 손실 예방 또는 치료용 조성물은 과립구 집락자극인자를 유효성분으로 포함한다.Lymphatic tube loss prevention or treatment composition according to another feature of the present invention comprises a granulocyte colony stimulating factor as an active ingredient.

본 발명의 또 다른 특징에 따른 림프구 감소증 예방 또는 치료용 조성물은 과립구집락자극인자를 유효성분으로 포함한다.Lymphocytosis prevention or treatment composition according to another feature of the present invention comprises a granulocyte colony stimulating factor as an active ingredient.

본 발명의 또 다른 특징에 따른 림프관 형성 증진제는 과립구집락자극인자를 유효성분으로 포함한다.Lymphatic vessel formation enhancer according to another feature of the present invention comprises a granulocyte colony stimulating factor as an active ingredient.

본 발명의 또 다른 특징에 따른 ERK1/2 인산화 증진제는 과립구집락자극인자를 유효성분으로 포함한다.ERK1 / 2 phosphorylation enhancer according to another feature of the present invention comprises a granulocyte colony stimulating factor as an active ingredient.

본 발명의 또 다른 특징에 따른 Akt 인산화 증진제는 과립구 집락자극인자를 유효성분으로 포함한다.Akt phosphorylation enhancer according to another feature of the present invention comprises a granulocyte colony stimulating factor as an active ingredient.

본 발명에 따른 림프 혈관 생성 조성물은 과립구집락자극인자를 유효성분으로 포함하는 것으로서, 사람 림프혈관내피세포의 세포 유주, 모세관형성, 세포 증식 및 림프관 발아를 유도한다. The lymphatic vessel generating composition according to the present invention comprises granulocyte colony stimulating factor as an active ingredient, and induces cell migration, capillary formation, cell proliferation and lymphatic germination of human lymphatic vascular endothelial cells.

이러한 본 발명의 림프 혈관 생성 조성물에 포함되는 과립구집락자극인자는 사람 림프혈관내피세포에서 림프관 생성과 관련된 성장인자를 직접적으로 유도하는 것이 아니라, 과립구집락자극인자를 통하여 맵 키나아제(MAP kinase)와 Akt신호전달계를 통하여 사람 림프혈관내피세포의 세포 유주, 모세관형성, 세포 증식 및 림프관 발아를 유도한다. The granulocyte colony stimulating factor included in the lymphocyte-producing composition of the present invention does not directly induce growth factors related to lymphatic vessel formation in human lymphovascular endothelial cells, but is a map kinase (MAP kinase) and Akt through granulocyte colony stimulating factors. The signaling system induces cell migration, capillary formation, cell proliferation and lymphatic germination of human lymphovascular endothelial cells.

따라서 본 발명의 과립구집락자극인자를 포함하는 림프 혈관 생성 조성물은 림프 혈관을 생성하는 효과가 있으며, 더불어 림프관 생성 유도, 림프 부종의 예방 또는 치료, 림프관 손실 예방 또는 치료, 림프구 감소증 예방 또는 치료, 림프관 형성 증진의 효과가 있을 것으로 기대된다.
Therefore, the lymphocyte producing composition comprising granulocyte colony stimulating factor of the present invention has an effect of generating lymph vessels, and in addition, induction of lymphatic vessel production, prevention or treatment of lymphedema, prevention or treatment of lymphatic vessel loss, prevention or treatment of lymphocytosis, lymphatic vessels It is expected to have the effect of promoting formation.

도 1은 과립구집락자극인자에 의한 사람 림프혈관내피 세포에서의 세포 유주 효과를 보여주는 그래프이다.
도 2는 과립구집락자극인자가 사람 림프혈관내피 세포에서 모세관을 형성시키는 효과가 있음을 보여주는 사진과 그래프이다.
도 3은 과립구집락자극인자에 의한 사람 림프혈관내피 세포에서의 세포증식 효과를 XTT 분석(XTT assay)을 통해 보여주는 그래프이다.
도 4는 과립구집락자극인자에 의한 사람 림프혈관내피 세포에서 맵 키나아제(MAP kinase)의 인산화 효과를 보여주는 그림과 그래프이다.
도 5는 과립구집락자극인자에 의한 사람 림프혈관내피 세포에서 Akt 인산화 효과를 보여주는 그림과 그래프이다.
도 6은 과립구집락자극인자에 의한 사람 림프혈관내피세포에서 세포증식과 관련된 신호전달계는 맵 키나아제(MAP kinase) 신호전달계임을 보여주는 그래프이다.
도 7은 3차원 림프관 링 검사에서 과립구집락자극인자가 림프 혈관 발아를 유도하는 것을 보여주는 사진이다. 1 is a graph showing the effect of cell migration in human lymphatic vascular endothelial cells by granulocyte colony stimulating factor.
Figure 2 is a photograph and graph showing that the granulocyte colony stimulating factor has the effect of forming capillaries in human lymphatic vascular endothelial cells.
Figure 3 is a graph showing the effect of cell proliferation in human lymphatic vascular endothelial cells by granulocyte colony stimulating factor through XTT assay (XTT assay).
Figure 4 is a figure and graph showing the phosphorylation effect of MAP kinase (MAP kinase) in human lymphatic vascular endothelial cells by granulocyte colony stimulating factor.
Figure 5 is a figure and graph showing the effect of Akt phosphorylation in human lymphatic vascular endothelial cells by granulocyte colony stimulating factor.
Figure 6 is a graph showing that the signaling system associated with cell proliferation in human lymphatic vascular endothelial cells by granulocyte colony stimulating factor is a map kinase (MAP kinase) signaling system.
Figure 7 is a photograph showing that the granulocyte colony stimulating factor induces lymph vessel germination in the three-dimensional lymph vessel ring test.

이에 본 발명자들은 상기 종래기술들의 문제점을 극복하기 위해 예의 연구 노력한 결과, 림프 혈관의 생성에 과립구집락자극인자(granulocyte colony-stimulating factor: G-CSF)가 관여함을 확인하여 본 발명을 완성하였다.Accordingly, the present inventors have completed the present invention by confirming that granulocyte colony-stimulating factor (G-CSF) is involved in the production of lymph vessels as a result of earnest research efforts to overcome the problems of the prior art.

구체적으로는 본 발명은 사람 림프혈관내피세포에 과립구집락자극인자를 투여한 결과, 림프혈관내피세포의 세포유주, 세포의 증식, 모세관형성, 및 림프관 발아가 유도됨을 발견하였다. 그리고, 모세관 형성은 포스파티딜 이노시톨 3’-키나아제(phosphatidylinositol 3’-kinase), 맵 키나아제(MAP kinase) 및 Akt신호전달계에 의해 선택적으로 조절됨을 확인 하였다.Specifically, it was found that the granulocyte colony stimulating factor was administered to human lymphovascular endothelial cells to induce cell flow, lymphocyte proliferation, capillary formation, and lymphatic germination of lymphovascular endothelial cells. In addition, capillary formation was selectively regulated by phosphatidylinositol 3'-kinase, MAP kinase, and Akt signaling system.

이와 같이, 인간에게 과립구집락자극인자 투여 시 나타나는 기작은, 과립구집락자극인자가 림프혈관내피세포에서 새로운 림프관을 생성하는 림프혈관생성 물질임을 확인하여 준다. As such, the mechanism that appears when the granulocyte colony stimulating factor is administered to humans confirms that the granulocyte colony stimulating factor is a lymphangiogenic substance that generates new lymphatic vessels from lymphovascular endothelial cells.

본 발명에서 과립구집락자극인자로는 과립구대식세포 잡락자극인자(GM-CSF), 대식세포집락자극인자(M-CSF)등의 유사물질을 포함한다.Granulocyte colony stimulating factors in the present invention include granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF) and the like.

이하 본 발명을 바람직한 실시예를 참고로 하여 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있도록 상세히 설명한다. 그러나 본 발명은 여러 가지 상이한 형태로 구현될 수 있으며 여기에서 설명하는 실시예에 한정되는 것은 아니다.
Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

실험예Experimental Example 1: 세포배양 및 실험재료의 수득과정 1: Cell Culture and Obtaining Process Materials

사람 림프혈관내피세포를 론자(Basel, Switerland)에서 구입하고, 가습된 5% CO2 및 95% 공기의 대기 조건, 그리고 37℃의 온도 조건에서 5%(vol/vol) 소태아혈청(fetal bovine serum)이 첨가된 EBM-2배지에서 배양하여 준비하였다. 이때, 사람 림프혈관내피세포로서, 패시지(passage) 2~4 사이의 세포를 이용하였다. Human lymphovascular endothelial cells were purchased from Basel, Switerland, and 5% (vol / vol) fetal bovine serum at atmospheric conditions of humidified 5% CO2 and 95% air, and at 37 ° C. ) Was prepared by culturing in EBM-2 medium. At this time, cells between passages 2 to 4 were used as human lymphatic endothelial cells.

과립구 집락자극인자(granulocyte colony-stimulating factor: G-CSF)는 시그마알드리치(St.Louis, MO, USA)에서 구입하여 사용하였다. Granulocyte colony-stimulating factor (G-CSF) was purchased from Sigma Aldrich (St. Louis, MO, USA) and used.

또한 사람 VEGF-A 165는 R&D ststem(Minniapolis, MN, USA)에서 구입하였으며, MAPK/ERK억제제(PD98059), 포스파티딜 이노시톨 3’-키나아제(phosphatidylinositol 3’-kinase) 억제제(LY294002), 보르트만닌(wortmannin: WT), 젤라틴(gelatin) 및 항생제는 시그마 알드리치(St. Louis, MO, USA)에서 구입하였다.
Human VEGF-A 165 was also purchased from R & D ststem (Minniapolis, MN, USA), MAPK / ERK inhibitor (PD98059), phosphatidylinositol 3'-kinase inhibitor (LY294002), buttmannin Wortmannin (WT), gelatin and antibiotics were purchased from Sigma Aldrich (St. Louis, MO, USA).

실험예Experimental Example 2: 과립구  2: granulocytes 집락자극인자의Colony stimulating factor 사람  Person 림프혈관내피세포의Lymphatic endothelial cells 세포  cell 유주Yuju , 모세관형성 및 세포증식 유도, Capillary formation and cell proliferation

림프 혈관이 생성되기 위해서는 림프 혈관 내피 세포의 유주 및 증식이 일어나야 하므로, 과립구집락자극인자(granulocyte colony-stimulating factor: G-CSF)가 나타내는 사람 림프혈관내피세포의 림프관 생성 효과를 생체 밖에서 확인하기 위하여 세포 유주 검사, 모세관형성 및 세포 증식 검사를 시행하였다.
Since lymphocyte endothelial cells are required to grow and proliferate in order to produce lymphatic vessels, in order to confirm in vitro the lymphatic formation effect of human lymphovascular endothelial cells represented by granulocyte colony-stimulating factor (G-CSF). Cell shedding, capillary formation and cell proliferation were performed.

2-1: 세포 2-1: cells 유주Yuju 검사( inspection( MigrationMigration assayassay ))

사람 림프혈관내피세포의 세포 유주 검사는 변화된 보이덴(Boyden) 챔버(Neuro Probe, Cabin John, MD)를 이용하여 시행하였다. Cytotoxicity of human lymphovascular endothelial cells was performed using a modified Boyden chamber (Neuro Probe, Cabin John, MD).

사람 림프혈관내피 세포를 트립신 처리 후 EBM-2 배양액으로 두 차례 씻고 1% BSA가 있는 EBM-2 배양액으로 사람 림프혈관내피 세포를 재현탁한 후 보이덴(Boyden) 챔버의 위쪽에 5×103 개의 사람 림프혈관내피세포를 넣었다. After trypsin treatment, human lymphovascular endothelial cells were washed twice with EBM-2 medium and resuspended human lymphovascular endothelial cells with EBM-2 medium containing 1% BSA, followed by 5 × 103 humans on top of the Boyden chamber. Lymphatic endothelial cells were added.

그리고, 음성대조군(control buffer, CB), 시그마알드리치에서 구매한 50, 100ng/mL 농도의 과립구집락자극인자(granulocyte colony-stimulating factor: G-CSF) 및 양성대조군으로서 VEGF-A를 1% 소혈청알부민(bovine serum albumin; BSA)과 함께 각각 챔버의 아래쪽에 넣었다. 그리고, 8㎛의 구멍이 있는 폴리카르보네이트 필터(polycarboante filter)를 0.2% 젤라틴(gelatin, 시그마-알드리치사(St.Louis, MO, USA))으로 코팅한 후 상기 시료와 챔버 사이에 넣었다.In addition, 1% bovine serum of VEGF-A as a negative control group (CB), granulocyte colony-stimulating factor (G-CSF) and positive control group purchased from Sigma Aldrich at 50 and 100 ng / mL concentrations. Each with albumin (bovine serum albumin (BSA)) was placed at the bottom of the chamber. Then, a polycarbonate filter (polycarboante filter) having a hole of 8 μm was coated with 0.2% gelatin (gelatin, Sigma-Aldrich, St. Louis, MO, USA) and placed between the sample and the chamber.

VEGF-A는 일반적으로 림프혈관생성에 관여하는 인자로 알려져 있으며, 본 발명에서는 과립구집락자극인자의 림프혈관 생성효과를 확인하기 위해서 VEGF-A(30 ng/mL)을 처리한 세포를 양성대조군으로 사용하였다. 그후 필터는 메탄올로 고정하고 디프-퀵(Diff-Quik)액으로 염색한 후, 가습된 5% CO2, 95% air, 37 ℃ 상태에서 4시간 동안 배양하여 사람 림프혈관내피세포가 챔버의 아래쪽으로의 유주 정도를 관찰하였다.VEGF-A is generally known as a factor involved in lymphangiogenesis, and in the present invention, the cells treated with VEGF-A (30 ng / mL) as a positive control group in order to confirm the lymphangiogenic effect of granulocyte colony stimulating factor. Used. The filter was then fixed with methanol, stained with Diff-Quik solution, incubated for 4 hours in humidified 5% CO2, 95% air, and 37 ° C to allow human lymphatic endothelial cells to enter the bottom of the chamber. The degree of fermentation was observed.

배양 후, 필터의 위쪽에 남아있는 유주되지 않은 사람 림프혈관내피세포는 면봉으로 조심스럽게 제거하였으며, 유주된 사람 림프혈관내피세포는 200배 현미경하에서 관찰하고 유주된 사람 림프혈관내피세포의 개수를 측정하였다. After incubation, the non-leaved human lymphovascular endothelial cells remaining at the top of the filter were carefully removed with a cotton swab, and the cultured human lymphovascular endothelial cells were observed under a 200-fold microscope and the number of cultured human lymphovascular endothelial cells was measured. It was.

도 1로부터 과립구집락자극인자의 농도 의존적으로 사람 림프혈관내피세포의 세포 유주가 됨을 확인 할 수 있으며, 과립구집락자극인자의 농도가 50, 100ng/㎖ 인 경우, 통계적으로 유의하게 사람 림프혈관내피세포의 유주가 증가하였다. 구체적으로는 음성대조군(CB)과 비교할 때 과립구집락자극인자 100ng/㎖ 농도에서 약 5.9배 가량 세포유주를 증가시켰다. 양성대조군인 VEGF-A는 음성대조군에 비교하여 약 7.5배 가량 세포유주를 증가시켰다.
It can be seen from FIG. 1 that the concentration of granulocyte colony stimulating factor is a cell lineage of human lymphatic vascular endothelial cells, and when the concentration of granulocyte colony stimulating factor is 50, 100 ng / ml, it is statistically significant. Increasing amount of has increased. Specifically, the cell line was increased by about 5.9-fold at the concentration of 100 ng / ml granulocyte colony stimulating factor compared with the negative control group (CB). The positive control group VEGF-A increased cell lines by about 7.5-fold compared to the negative control group.

2-2: 모세관 형성 검사2-2: Capillary Formation Test

모세관 형성 검사 (capillary-like tube formation assay)는 ECM 겔(gel)에서 3차원 배양검사를 통하여 실시하였다. Capillary-like tube formation assay was performed through a three-dimensional culture test on the ECM gel (gel).

사람 림프혈관내피세포 2×104 세포/mL을 ECM 겔에 넣고, 0.2 mL씩을 24 웰 디쉬(well dish)에 넣은 후 가습된 5% CO2, 95% air, 37 ℃상태에서 2시간 배양, 젤이 굳도록 하였고, 이후, ECM 겔에 음성대조군 (control buffer, CB), 양성 대조군인 VEGF-A (30 ng/mL), 및 과립구집락자극인자(granulocyte colony-stimulating factor: G-CSF) 50, 100 ng/mL를 MARK/ERK억제제(PD98059, PD), 포스파티딜이노시톨 3’-키나아제(phosphatidylinositol 3’-kinase) 억제제인 LY294002(LY)와 보르트만닌(wortmannin: WT)와 함께 투여하여 관 형성을 유도하였고 16시간 후의 위상차를 현미경으로 관찰하였다.2 × 104 cells / mL of human lymphatic endothelial cells were placed in an ECM gel, 0.2 mL of each was placed in a 24-well dish, and cultured for 2 hours under humidified 5% CO 2, 95% air, and 37 ° C. The ECM gel was then treated with a negative control buffer (CB), a positive control VEGF-A (30 ng / mL), and a granulocyte colony-stimulating factor (G-CSF) 50, 100. ng / mL was administered in combination with MARK / ERK inhibitors (PD98059, PD), phosphatidylinositol 3'-kinase inhibitors LY294002 (LY) and wortmannin (WT) It was induced and the phase difference after 16 hours was observed under a microscope.

도 2으로부터, 과립구집락자극인자가 사람 림프혈관내피세포의 모세관을 형성하며, 농도를 50, 100ng/㎖로 달리하였을 때 모세관 형성이 농도 의존적으로 증가하는 것을 확인하였다. 구체적으로는 과립구집락자극인자를 100ng/㎖로 처리하였을 때 음성대조군(CB)와 비교하여 약 10배 가량 모세관형성을 증가시키는 것으로 확인되었다. 특히 사람 림프혈관내피세포의 모세관 형성에 관여하는 신호전달계를 확인하기 위해서 포스파티딜이노시톨 3’- 키나아제(phosphatidylinositol 3’-kinase) 억제제인 LY294002(LY, 50 umol/L)와 보르트만닌(wartmannin, WT, 30nmol/L)를 투여하였을 때 모세관 형성이 감소하였으며, 특히 ERK1/2 억제제인 PD98059 (PD, 50umol/L)를 투여하였을 때에는 모세관 형성이 거의 이루어지지 않음을 확인 할 수 있었다. 즉, 이러한 결과는 과립구집락자극인자를 이용한 사람 림프혈관내피 세포의 모세관 형성은 LY, WT, PD에 의해서 모세관 형성이 억제됨을 보여주는 것이다. 그리하여 과립구집락자극인자를 이용한 모세관 형성은 포스파티딜이노시톨 3’-키나아제(phosphatidylinositol 3’-kinase)와 ERK1/2경로를 통해 조절이 됨을 알 수 있었다.
From FIG. 2, it was confirmed that the granulocyte colony stimulating factor forms capillaries of human lymphatic endothelial cells, and capillary formation increases in concentration-dependently when the concentration is changed to 50 and 100 ng / ml. Specifically, it was confirmed that when the granulocyte colony stimulating factor was treated with 100ng / ml, capillary formation was increased by about 10 times compared to the negative control group (CB). In particular, the phosphatidylinositol 3'-kinase inhibitors LY294002 (LY, 50 umol / L) and wortmannin (wartmannin) were used to identify signaling pathways involved in capillary formation of human lymphatic endothelial cells. Capillary tube formation was reduced when WT, 30nmol / L) was administered, especially when PD98059 (PD, 50umol / L), an ERK1 / 2 inhibitor, was administered. That is, these results show that capillary formation of human lymphatic endothelial cells using granulocyte colony stimulating factor is inhibited by LY, WT, and PD. Thus, capillary formation using granulocyte colony stimulating factor was found to be regulated through phosphatidylinositol 3'-kinase and ERK1 / 2 pathway.

2-3: 세포 증식 검사(2-3: Cell proliferation test CellCell proliferationproliferation assayassay ))

세포 증식 검사는 사람 림프혈관내피세포를 트립신 처리 후, 96 웰 젤라틴-코팅 플레이트(well gelatin-coating plates)에 각각 5×103 개의 세포를 넣고, 24시간 동안 37 ℃에서 배양 후에 배양액을 제거하고, 0.5% FBS가 있는 배양액과 함께 음성대조군 (control buffer, CB), 과립구집락자극인자 (10, 50, 100 ng/mL), 양성대조군인 VEGF-A (30 ng/mL)를 각각 투여하였다. In the cell proliferation test, human lymphatic endothelial cells were trypsinized, 5 × 10 3 cells were put into 96 well gelatin-coating plates, and culture was removed at 37 ° C. for 24 hours, and then culture was removed. A negative control group (control buffer, CB), granulocyte colony stimulating factor (10, 50, 100 ng / mL), and a positive control group VEGF-A (30 ng / mL) were respectively administered with the culture medium containing 0.5% FBS.

투여 24시간 동안 37 ℃에서 배양 후 세포의 증식정도를 세포증식키트 II(Cell proliferation Kit II, XTT, Roche, Mannheim, Germany)을 이용하여 측정하였다. 상기 과립구집락자극인자(granulocyte colony-stimulating factor: G-CSF)는 시그마알드리치에서 구입하여 사용하였다. After culturing at 37 ° C. for 24 hours, the proliferation of the cells was measured using Cell Proliferation Kit II (Cell proliferation Kit II, XTT, Roche, Mannheim, Germany). The granulocyte colony-stimulating factor (G-CSF) was purchased from Sigma-Aldrich.

세포가 증식될수록 세포 내 미토콘드리아의 발색물질의 증가로 흡광도가 증가하므로, 사람 림프혈관내피세포의 증식은 흡광도를 통해 측정하였으며, 도 3는 이러한 과립구집락자극인자가 사람 림프혈관내피세포의 세포 증식 효과를 확인하기 위해 실시한 XTT검사의 결과를 보여주는 그래프이다. 구체적으로는 음성대조군(CB)에 비교하여 과립구집락자극인자는 50ng/㎖의 농도에서 약 1.2배의 세포증식 효과가 있음을 확인할 수 있으며, 양성대조군 VEGF-A는 음성대조군(CB)에 비교하여 약 1.4배의 세포 증식 효과가 있었다.
As cells proliferate, the absorbance increases due to the increase in the color of mitochondria in the cells. Thus, the proliferation of human lymphovascular endothelial cells was measured by absorbance. This is a graph showing the results of the XTT test conducted to confirm. Specifically, the granulocyte colony stimulating factor was found to have about 1.2-fold cell proliferation effect at a concentration of 50 ng / ml compared to the negative control group (CB), and the positive control group VEGF-A compared to the negative control group (CB). There was about 1.4-fold cell proliferation effect.

실험예Experimental Example 3:  3: 과립구집락자극인자의Of granulocyte colony stimulating factor 사람  Person 림프혈관내피세포에서In lymphatic endothelial cells MAPMAP 키나아제Kinase 및  And AktAkt 의 인산화 증가Phosphorylation of

일반적으로 혈관내피세포에서 혈관생성 (angiogenesis)에 관여하는 신호 전달계로 맵 키나아제(MAP kinase)와 Akt 신호전달계가 있다. 이러한 신호전달계가 과립구집락자극인자(granulocyte colony-stimulating factor: G-CSF)에 의한 림프혈관생성에도 활성화 되는지 확인하기 위해서 맵 키나아제이며 신호 전달물질인 ERK1(p44MAPK)와 ERK2 (p42MAPK) 및 Akt의 인산화 정도를 웨스턴 블럿을 통하여 확인하였다. MAPK/ERK 억제제 (PD98059)는 시그마-알드리치 사(Sigma-Aldrich) (St.Louis, MO, USA)에서 구입하였다.In general, MAP kinase and Akt signaling systems are involved in angiogenesis in angiogenesis. Phosphorylation of ERK1 (p44MAPK) and ERK2 (p42MAPK) and Akt, which are map kinases and signaling agents, to determine whether these signaling systems are also activated in lymphangiogenesis by granulocyte colony-stimulating factor (G-CSF) The extent was confirmed by Western blot. MAPK / ERK inhibitors (PD98059) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

사람 림프혈관내피세포를 1% 혈청이 포함된 EBM-2 배지에서 16 시간 동안 배양 후, 과립구집락자극인자를 100ng/mL투여하고, 각각 5, 10, 15, 30, 60분 후에 사람 림프혈관내피세포의 용해물을 얻고, 단백질을 추출하여 웨스턴 블럿을 시행하여 ERK1/2의 인산화를 확인하였다. Human lymphovascular endothelial cells were incubated for 16 hours in EBM-2 medium containing 1% serum, and then 100 ng / mL of granulocyte colony stimulating factor and human lymphovascular endothelial after 5, 10, 15, 30 and 60 minutes, respectively. Lysates of cells were obtained, proteins were extracted, and Western blot was performed to confirm phosphorylation of ERK1 / 2.

도 4는 상기 ERK1/2의 인산화 정도를 확인한 그래프이다. 즉 사람 림프혈관내피세포에 과립구 집락자극인자를 100ng/㎖투여하였을 때 ERK1/2의 인산화는 투여 10분에서 최대로 이루어졌으며, 이후 인산화 정도는 15분에서 감소하여 대조군과 비슷한 수준으로 감소하였다. 또한 ERK1/2 억제제인 PD98059 (PD, 50umol/L)를 투여하였을 때 ERK1/2의 인산화를 감소시킴을 확인하였다. 따라서 과립구 집락자극인자는 사람 림프혈관내피세포에서 ERK1/2의 인산화를 증가시킴을 확인할 수 있었다.4 is a graph confirming the degree of phosphorylation of the ERK1 / 2. In other words, when 100ng / ml granulocyte colony stimulating factor was administered to human lymphovascular endothelial cells, phosphorylation of ERK1 / 2 was maximal at 10 minutes after administration, and then the degree of phosphorylation was decreased at 15 minutes and was similar to that of the control group. In addition, it was confirmed that ERK1 / 2 inhibitor PD98059 (PD, 50umol / L) reduced the phosphorylation of ERK1 / 2. Therefore, it was confirmed that granulocyte colony stimulating factor increased the phosphorylation of ERK1 / 2 in human lymphovascular endothelial cells.

또한 도 5은 상기 Akt의 인산화 정도를 확인한 그래프이다. 즉 사람 림프혈관내피세포에 과립구집락자극인자를 100ng/㎖투여하였을 때 20분과 30분에 Akt의 인산화를 확인할 수 있으며, 구체적으로는 20분에 1.3배 증가하는 것을 확인 할 수 있다. 반면에 포스파티딜이노시톨 3’-키나아제(phosphatidylinositol 3’-kinase) 억제제인 LY와 WT를 투여하였을 때 Akt의 인산화를 억제하였으며, 이는 결과적으로 모세관형성이 감소되는 결과로 나타났다. 또한 과립구집락자극인자의 농도에 따라 Akt의 인산화 효과를 확인하기 위해서 각각 10, 50, 100ng/㎖ 로 투여하였을 때 농도 의존적으로 Akt의 인산화가 증가함을 확인하였고, 구체적으로는 50ng/㎖에서 Akt의 인산화가 최대로 증가함을 확인 할 수 있다. 반면에 포스파티딜이노시톨 3’-키나아제(phosphatidylinositol 3’-kinase)억제제인 LY294002(LY, 50 umol/L)와 보르트만닌(wortmannin, WT, 30nmol/L)를 투여하였을 때 Akt의 인산화를 감소시킴을 확인할 수 있다. 따라서 과립구집락자극인자는 사람 림프혈관내피세포에서 Akt의 인산화를 증가시킴을 확인할 수 있다. 5 is a graph confirming the degree of phosphorylation of the Akt. In other words, when 100ng / ml granulocyte colony stimulating factor was administered to human lymphatic endothelial cells, phosphorylation of Akt could be confirmed at 20 and 30 minutes, specifically, 1.3-fold increase at 20 minutes. On the other hand, the administration of phosphatidylinositol 3'-kinase inhibitors LY and WT inhibited phosphorylation of Akt, resulting in decreased capillary formation. In addition, in order to confirm the phosphorylation effect of Akt according to the concentration of granulocyte colony stimulating factor, it was confirmed that the phosphorylation of Akt increased in a dose-dependent manner when administered at 10, 50, and 100ng / ml, respectively. It can be seen that phosphorylation of maximally increases. In contrast, LY294002 (LY, 50 umol / L) and wortmannin (wortmannin, WT, 30 nmol / L), which are phosphatidylinositol 3'-kinase inhibitors, reduced Akt phosphorylation. can confirm. Therefore, it can be seen that the granulocyte colony stimulating factor increases the phosphorylation of Akt in human lymphatic endothelial cells.

또한 도 6은 과립구집락자극인자에 의한 사람 림프혈관내피세포에서 세포증식과 관련된 신호전달계는 맵 키나아제(MAP kinase) 신호전달계임을 보여주는 그래프이다. 즉, 포스파티딜이노시톨 3’-키나아제(phophatidylinositol 3’-kinase)억제제인 LY294002(LY, 50umol/L)와 보르트만닌(wortmannin, WT, 30nmol)를 동시에 투여하였을 때 LY294002는 과립구 집락자극인자에 의한 세포증식을 억제하는 효과를 보였다. 또한 ERK1/2 억제제인 ERK1/2 억제제인 PD98059 50umol/L를 투여하였을 때 과립구집락자극인자에 의한 세포 증식을 억제함을 확인하였다. 이는 과립구 집락자극인자에 의한 사람 림프혈관내피세포의 증식은 맵 키나아제(MAP kinase)신호전달계가 관여함을 보여주는 것이다.
6 is a graph showing that the signaling system associated with cell proliferation in human lymphatic vascular endothelial cells by granulocyte colony stimulating factor is a map kinase (MAP kinase) signaling system. In other words, when LY294002 (LY, 50umol / L) and wortmannin (wortmannin, WT, 30nmol), phosphatidylinositol 3'-kinase inhibitors, were administered at the same time, LY294002 was caused by granulocyte colony stimulating factor. Inhibitory effect on cell proliferation. In addition, when ERK1 / 2 inhibitor PD98059 50umol / L, which is an ERK1 inhibitor was administered, it was confirmed that the cell proliferation caused by granulocyte colony stimulating factor was inhibited. This suggests that MAP kinase signaling system is involved in the proliferation of human lymphatic endothelial cells by granulocyte colony stimulating factor.

실험예Experimental Example 4: 3차원 림프관 링 검사를 통한  4: 3D lymphatic tube ring test 과립구집락자극인자의Of granulocyte colony stimulating factor 림프관 발아 유도 확인 Confirmation of Lymphatic Germination Induction

림프관 링 검사는 림프관으로부터 림프혈관내피세포의 발아, 세포 증식, 세포 유주 및 림프혈관으로 분화 여부를 확인하는 검사로, 마우스의 흉관에서 과립구집락자극인자(granulocyte colony-stimulating factor: G-CSF)에 의해 나타나는 림프관의 발아를 관찰하였다. Lymphatic tube ring test is used to check the germination, cell proliferation, cell migration, and differentiation of lymphoid endothelial cells from lymphatic vessels to the granulocyte colony-stimulating factor (G-CSF). Germination of lymphatic vessels was observed.

구체적으로는 마우스의 흉관을 이용한 3차원 림프관 링 검사 (Thoracic duct collection and three-dimensional lymphatic ring assay)시, C57BL/6J 마우스의 흉관 조직을 미세수술기법을 통하여 얻은 후 얼음 위에서 혈청이 없는 EBM-2 배지상에 1 mm 길이로 잘라서 검사에 이용하였다. ECM 젤(gel)에 1 mm 길이의 마우스 흉관 조직을 넣고, 음성대조군(CB), 100 ng/mL의 과립구집락자극인자(G-CSF), 양성대조군인 VEGF-A 30 ng/mL을 넣고, 가습된 5% CO2, 95% air, 37 ℃ 상태에서 2시간 배양하였다. 그 후, 젤이 굳은 후 흉관에서 림프관이 자라나오는 정도를 현미경하에서 관찰하였다. Specifically, during thoracic duct collection and three-dimensional lymphatic ring assay, the thoracic duct tissue of C57BL / 6J mice was obtained by microsurgical technique, and then serum-free EBM-2 on ice. It was cut to 1 mm long on the medium and used for the inspection. Into the ECM gel (gel) 1 mm long mouse chest tube tissue, the negative control group (CB), 100 ng / mL granulocyte colony stimulating factor (G-CSF), positive control VEGF-A 30 ng / mL, Incubated for 2 hours at humidified 5% CO2, 95% air, 37 ℃. Thereafter, the degree of lymphatic growth in the chest tube after the gel solidified was observed under a microscope.

도 7는 3차원 림프관 링 검사에서 과립구집락자극인자가 림프혈관발아를 유도하는 것을 보여주는 사진이다. 음성대조군인 CB와 비교할 때 과립구집락자극인자는 림프관에서 림프혈관발아를 유도하였으며, 양성대조군인 VEGF-A와 비교할 때 림프혈관발아는 거의 동등한 효과를 보였다.
Figure 7 is a photograph showing that granulocyte colony stimulating factor induces lymph vessel germination in the three-dimensional lymphatic ring test. Granulocyte colony stimulating factor induced lymphocyte germination in lymphatic vessels as compared with negative control group CB. Lymphovascular germination showed almost the same effect as compared with positive control group VEGF-A.

통계학적 분석Statistical analysis

데이터를 평균±표준편차로 나타내었다. 아노바(ANOVA)를 이용한 유의차로 다중 비교하고 터키 사후검정을 이용한 개별 비교를 하였으며, 통계적 유의성은 p<0.05로 수행하였다.
Data are expressed as mean ± standard deviation. Multiple comparisons were made using ANOVA, and individual comparisons using the Turkish post hoc test. Statistical significance was performed at p <0.05.

상기에서는 본 발명의 바람직한 실시예에 대하여 설명하였지만, 본 발명은 이에 한정되는 것이 아니고 본 발명의 기술 사상 범위 내에서 여러 가지로 변형하여 실시하는 것이 가능하고 이 또한 첨부된 특허 청구 범위에 속하는 것은 당연하다.
While the present invention has been described in connection with what is presently considered to be practical exemplary embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, Do.

Claims (16)

과립구집락자극인자를 유효성분으로 포함하는 림프 혈관 생성 조성물.
Lymphatic vessel producing composition comprising granulocyte colony stimulating factor as an active ingredient.
제 1항에 있어서,
상기 과립구집락자극인자에는 유사물질인 과립구대식세포 잡락자극인자(GM-CSF)와 대식세포집락자극인자(M-CSF)를 포함하는 것을 특징으로 하는 림프 혈관 생성 조성물.
The method of claim 1,
The granulocyte colony stimulating factor is a lymphocyte-producing composition comprising granulocyte macrophage colony stimulating factor (GM-CSF) and macrophage colony stimulating factor (M-CSF) which are similar substances.
과립구집락자극인자를 유효성분으로 포함하는 림프관 생성 유도제.
Lymphatic vessel inducing agent comprising granulocyte colony stimulating factor as an active ingredient.
제 3항에 있어서,
상기 과립구집락자극인자에는 유사물질인 과립구대식세포 잡락자극인자(GM-CSF)와 대식세포집락자극인자(M-CSF)를 포함하는 것을 특징으로 하는 림프관 생성 유도제.
The method of claim 3,
The granulocyte colony stimulating factor is a lymphocyte generating inducer, characterized in that it comprises a granulocyte macrophage colony stimulating factor (GM-CSF) and macrophage colony stimulating factor (M-CSF) similar substances.
과립구집락자극인자를 유효성분으로 포함하는 림프부종 예방 또는 치료용 조성물.
Lymphedema prevention or treatment composition comprising a granulocyte colony stimulating factor as an active ingredient.
제 5항에 있어서,
상기 과립구집락자극인자에는 유사물질인 과립구대식세포 잡락자극인자(GM-CSF)와 대식세포집락자극인자(M-CSF)를 포함하는 것을 특징으로 하는 림프부종 예방 또는 치료용 조성물.
6. The method of claim 5,
The granulocyte colony stimulating factor is a composition for preventing or treating lymphedema, characterized in that it comprises a granulocyte macrophage colony stimulating factor (GM-CSF) and macrophage colony stimulating factor (M-CSF) similar substances.
과립구집락자극인자를 유효성분으로 포함하는 림프관 손실 예방 또는 치료용 조성물.
Lymphatic tube loss prevention or treatment composition comprising a granulocyte colony stimulating factor as an active ingredient.
제 7항에 있어서,
상기 과립구집락자극인자에는 유사물질인 과립구대식세포 잡락자극인자(GM-CSF)와 대식세포집락자극인자(M-CSF)를 포함하는 것을 특징으로 하는 림프관 손실 예방 또는 치료용 조성물.
8. The method of claim 7,
The granulocyte colony stimulating factor is a composition for preventing or treating lymphatic vessel loss, characterized in that it comprises a granulocyte macrophage colony stimulating factor (GM-CSF) and macrophage colony stimulating factor (M-CSF) that are similar substances.
과립구집락자극인자를 유효성분으로 포함하는 림프구 감소증 예방 또는 치료용 조성물.
Lymphocyte reduction prevention or treatment composition comprising granulocyte colony stimulating factor as an active ingredient.
제 9항에 있어서,
상기 과립구집락자극인자에는 유사물질인 과립구대식세포 잡락자극인자(GM-CSF)와 대식세포집락자극인자(M-CSF)를 포함하는 것을 특징으로 하는 림프구 감소증 예방 또는 치료용 조성물.
The method of claim 9,
The granulocyte colony stimulating factor is a composition for the prevention or treatment of lymphocyte reduction, characterized in that it comprises a granulocyte macrophage colony stimulating factor (GM-CSF) and macrophage colony stimulating factor (M-CSF) similar substances.
과립구집락자극인자를 유효성분으로 포함하는 림프관 형성 증진제.
Lymphatic vessel formation enhancer comprising granulocyte colony stimulating factor as an active ingredient.
제 11항에 있어서,
상기 과립구집락자극인자에는 유사물질인 과립구대식세포 잡락자극인자(GM-CSF)와 대식세포집락자극인자(M-CSF)를 포함하는 것을 특징으로 하는 림프관 형성 증진제.
12. The method of claim 11,
The granulocyte colony stimulating factor is a lymphocyte formation enhancer, characterized in that it comprises a granulocyte macrophage colony stimulating factor (GM-CSF) and macrophage colony stimulating factor (M-CSF) similar substances.
과립구집락자극인자를 유효성분으로 포함하는 ERK1/2 인산화 증진제.
ERK1 / 2 phosphorylation enhancer comprising a granulocyte colony stimulating factor as an active ingredient.
제 13항에 있어서,
상기 과립구집락자극인자에는 유사물질인 과립구대식세포 잡락자극인자(GM-CSF)와 대식세포집락자극인자(M-CSF)를 포함하는 것을 특징으로 하는 ERK1/2 인산화 증진제.
The method of claim 13,
The granulocyte colony stimulating factor ERK1 / 2 phosphorylation enhancer, characterized in that it comprises a granulocyte macrophage colony stimulating factor (GM-CSF) and macrophage colony stimulating factor (M-CSF) as a similar substance.
과립구집락자극인자를 유효성분으로 포함하는 Akt 인산화 증진제.
Akt phosphorylation enhancer comprising a granulocyte colony stimulating factor as an active ingredient.
제 15항에 있어서,
상기 과립구집락자극인자에는 유사물질인 과립구대식세포 잡락자극인자(GM-CSF)와 대식세포집락자극인자(M-CSF)를 포함하는 것을 특징으로 하는 Akt 인산화 증진제.
16. The method of claim 15,
The granulocyte colony stimulating factor Akt phosphorylation enhancer, characterized in that it comprises a granulocyte macrophage colony stimulating factor (GM-CSF) and macrophage colony stimulating factor (M-CSF) that is a similar substance.
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AU766238B2 (en) * 1998-03-09 2003-10-09 Caritas St. Elizabeth's Medical Center Of Boston, Inc. Compositions and methods for modulating vascularization
AU2001296521A1 (en) * 2000-10-02 2002-04-15 St. Elizabeth's Medical Center Of Boston, Inc. Use of lymphangiogenic agents to treat lymphatic disorders
KR100812884B1 (en) * 2000-11-27 2008-03-11 가부시키가이샤 디나벡크 겐큐쇼 Paramyxovirus vector encoding angiogenesis gene and use thereof
KR101226812B1 (en) * 2010-10-18 2013-01-25 전북대학교산학협력단 lymph duct formation revellent and lymph duct formation defect prevention and treatment composition using the same

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024023034A1 (en) * 2022-07-25 2024-02-01 Institut National de la Santé et de la Recherche Médicale Use of apelin for the treatment of lymphedema

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