KR101845161B1 - Methods, compositions and kits for regulationg differentiation of skin adult stem cell - Google Patents
Methods, compositions and kits for regulationg differentiation of skin adult stem cell Download PDFInfo
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Abstract
본 발명은 피부성체 줄기세포의 분화를 조절하는 방법에 관한 것으로서, SAHA를 첨가하여 인테그린과 제4형 콜라겐의 발현 증가를 통해 피부성체 줄기 세포의 분화를 조절하는 방법, 및 상기 방법을 적용한 조성물과 키트에 관한 기술이다.The present invention relates to a method for controlling the differentiation of adult stem cells, and more particularly, to a method for controlling the differentiation of adult stem cells by increasing the expression of integrin and type 4 collagen by adding SAHA, This is a description of the kit.
Description
본 발명은 피부성체 줄기세포의 분화를 조절하는 방법에 관한 것으로서, 더욱 상세하게는 suberoylanilohydroxamic acid(SAHA)를 처리하여 인테그린과 제4형 콜라겐의 발현 증가를 통해 피부성체 줄기 세포의 분화를 조절하는 방법에 관한 것이다. The present invention relates to a method for regulating differentiation of skin adult stem cells, and more particularly, to a method for regulating the differentiation of skin adult stem cells by increasing the expression of integrin and type 4 collagen by treating suberoylanilohydroxamic acid (SAHA) .
성체 줄기세포에 관한 연구는 배아 줄기세포가 가지고 있는 세포치료제로서의 윤리적인 문제를 해결하고 환자 본인의 세포를 이용하고자 하는 데서 출발하였다. 그러나 성체 줄기세포의 분리과정, 체외배양과정, 세포 주입 과정 중에 사용되는 여러 물질들에 대한 안전성의 문제들이 여전히 존재한다. Studies on adult stem cells have begun with the aim of solving ethical problems as a cell therapy agent of embryonic stem cells and using the cells of the patients themselves. However, there are still problems with the safety of various materials used during the process of adult stem cell isolation, in vitro culture, and cell infusion.
상기 문제점들로 인하여 연구자들은 줄기세포의 분화를 조절할 수 있는 방법과 기전에 관심을 갖게 되었다. 줄기세포의 분화를 조절할 수 있다면 해당 줄기세포나 그 줄기세포를 이용한 장기를 인공적으로 배양하여 신규 물질이나 제품의 안전성과 효능 실험 등을 효과적으로 수행할 수 있다. 이는 동물들을 대상으로 실험하거나 일반인이나 환자들을 대상으로 임상실험 하는 데 들어가는 시간과 비용을 줄일 수 있음을 의미한다.Due to these problems, researchers have been interested in methods and mechanisms to control stem cell differentiation. If the differentiation of the stem cells can be controlled, the stem cells or the organs using the stem cells can be artificially cultured to effectively perform the safety and efficacy tests of new substances or products. This means that it is possible to reduce the time and expense of experimenting with animals or conducting clinical trials in the general population or patients.
피부는 표피와 진피로 이루어져 있으며, 표피는 각질형성세포가 대부분을 차지하는 중층 편평 상피로서 진피로부터 기저층, 유극층, 과립층, 각질층 순의 4개 층으로 이루어져 있다. 기저층에서 각질형성세포의 분열이 일어난 후 각질형성 세포는 피부의 표면으로 이동하면서 여러 단계의 분화과정을 거치게 되는데 피부의 성체 줄기세포는 이러한 과정을 통하여 피부재생에 기여한다. 따라서, 피부성체 줄기세포의 줄기세포능을 오래 유지할 수 있다면 피부의 재생능력을 오래 유지할 수 있고, 피부를 보다 건강하게 만들 수 있을 것이다.The skin consists of the epidermis and dermis, and the epidermis is the middle layer squamous epithelium with the majority of the keratinocytes. The epidermis consists of four layers: the dermis, the basal layer, the superficial layer, the granular layer, and the stratum corneum. After cleavage of keratinocytes in the basal layer, keratinocytes migrate to the surface of the skin and undergo various stages of differentiation. Skin adult cells contribute to skin regeneration through this process. Thus, if stem cell ability of skin adult stem cells can be maintained for a long time, the skin regenerating ability can be maintained for a long time and the skin can be made healthier.
피부성체 줄기세포를 포함하는 성체 줄기세포의 분화 조절은 세포와 세포미세환경의 상호작용에 의해 조절되게 되므로 연구자들은 세포미세환경을 조절할 수 있는 방법을 연구해 왔다. Since adult stem cell differentiation control, including skin adult stem cells, is regulated by the interaction between the cell and the cell microenvironment, researchers have studied ways to control the cellular microenvironment.
또한 본 발명자들은 제4형 콜라겐에 대한 부착능에 따라 피부의 성체 줄기세포와 분화세포를 분리할 수 있는 방법을 개발한 바 있으며(Kim DS, Cho HJ, Choi HR, Kwon SB, Park KC. Isolation of human epidermal stem cells by adherence and the reconstruction of skin equivalents. Cellular and molecular life sciences. 2004 Nov;61(21):2774-81), 상기 방법에 따라 분리한 피부성체 줄기세포와 분화세포를 이용하여 성체 줄기세포의 분화를 조절하는 방법을 개발하였으며 이 모델을 이용한 결과 SAHA가 인테그린과 제4형 콜라겐의 발현을 증가시키는 효과를 통해 성체 줄기세포의 분화를 조절하는 효과를 확인하고 본 발명을 완성하였다.In addition, the present inventors have developed a method for separating adult stem cells and differentiated cells from skin according to the adherence to type 4 collagen (Kim DS, Cho HJ, Choi HR, Kwon SB, Park KC. 2004 Nov; 61 (21): 2774-81). Using the adult adult stem cells and differentiated cells isolated according to the above method, adult stem cells and adult stem cells We have developed a method to regulate the differentiation of cells. As a result of using this model, we confirmed the effect of SAHA in controlling the differentiation of adult stem cells through the effect of increasing the expression of integrin and type 4 collagen, and completed the present invention.
본 발명의 목적은 상기와 같은 과제를 해결하기 위해 suberoylanilohydroxamic acid(SAHA)를 이용하여 피부성체 줄기세포의 분화를 조절할 수 있는 방법을 제공하는 데 있다.It is an object of the present invention to provide a method for controlling the differentiation of skin adult stem cells using suberoylanilohydroxamic acid (SAHA) to solve the above problems.
또한, 본 발명은 목적은 피부성체 줄기세포의 분화를 조절할 수 있는 SAHA를 유효성분으로 포함하는 피부성체 줄기세포의 분화 조절용 조성물을 제공하는 데 있다.Another object of the present invention is to provide a composition for controlling the differentiation of skin adult stem cells, which comprises SAHA as an active ingredient capable of regulating differentiation of adult stem cells.
본 발명은 상기와 같은 과제를 해결하기 위하여 피부 모델의 인테그린과 제4형 콜라겐의 발현을 증가시키는 것을 특징으로 하는 피부성체 줄기세포의 분화 조절 방법을 제공한다. 본 발명의 피부성체 줄기세포의 분화 조절 방법에 있어서, 피부모델을 배양하고 SAHA를 처리한 결과 피부모델의 인테그린과 제4형 콜라겐의 발현이 크게 증가하는 것을 발견하였다. 또한, 피부성체 줄기세포의 표식자로 알려진 p63의 발현이 증가하는 것으로 볼 때 본 발명의 피부성체 줄기세포의 분화 조절 방법에 의하여 피부성체 줄기세포의 줄기세포능이 잘 유지되고 있음을 의미한다.The present invention provides a method for controlling the differentiation of skin adult stem cells, which comprises increasing the expression of integrin and type-4 collagen in a skin model to solve the above problems. In the method for regulating differentiation of skin adult stem cells according to the present invention, the skin model was cultured and treated with SAHA, and the expression of integrin and type-4 collagen in the skin model was significantly increased. In addition, the expression of p63, which is known as a marker of skin adult stem cells, is increased. This means that stem cell function of skin adult stem cells is well maintained by the method of controlling the differentiation of skin adult stem cells of the present invention.
본 발명의 피부성체 줄기세포의 분화 조절 방법에 있어서, SAHA를 이용하여 β1 인테그린 및 α6 인테그린과 제4형 콜라겐의 발현이 증가되는 것을 특징으로 한다.In the method for regulating differentiation of skin adult stem cells according to the present invention, the expression of? 1 integrin and? 6 integrin and type 4 collagen is increased using SAHA.
줄기세포능을 잘 유지하려면 줄기세포가 위치하는 공간, 즉 니치(stem cell niche)가 적절하게 유지되어야 한다. 피부성체 줄기세포는 기저막 바로 위에 존재하기 때문에 피부성체 줄기세포와 관련하여 중요한 기저막 구성 단백질 중의 하나인 제 4 형 콜라겐이 잘 발현되어야 한다. 또한, 피부성체 줄기세포가 니치 구조에 잘 붙어 있으려면 기저막 단백질과 결합할 수 있게 해주는 β1 인테그린과 α6 인테그린을 필요로 한다.To maintain good stem cell function, the space in which the stem cells are located, ie, the stem cell niche, must be maintained appropriately. Skin Adult Stem cells are located just above the basement membrane, so that type IV collagen, one of the important basement membrane constituent proteins in relation to skin adult stem cells, should be well expressed. In addition, skin adult stem cells require the
본 발명의 피부성체 줄기세포의 분화 조절 방법은 β1 인테그린 및 α6 인테그린과 제4형 콜라겐의 발현이 증가되어 기저막의 상태가 개선되고, 피부성체 줄기세포로서의 줄기세포능을 잘 유지하고 있음을 증명하는 것이다. The method of the present invention for controlling the differentiation of skin adult stem cells demonstrates that the expression of? 1 integrin and? 6 integrin and type 4 collagen is increased to improve the basement membrane state and maintain the stem cell function as a skin adult stem cell will be.
본 발명의 피부성체 줄기세포의 분화조절 방법에 있어서, 상기 SAHA를 이용하여 β1 인테그린, α6 인테그린, 제4형 콜라겐의 발현이 증가하고 이를 통해 줄기세포능이 잘 유지될 수 있음을 확인할 수 있었다. In the method of controlling the differentiation of skin adult stem cells according to the present invention, it was confirmed that the expression of [beta] 1 integrin, [alpha] 6 integrin and type 4 collagen is increased and the stem cell ability can be maintained by using SAHA.
본 발명은 또한, p63의 발현이 증가하는 피부성체 줄기세포의 분화 조절용 조성물을 제공한다.The present invention also provides a composition for regulating differentiation of skin adult stem cells in which the expression of p63 is increased.
본 발명은 또한, 본 발명의 피부성체 줄기세포의 분화 조절 조성물을 포함하는 피부성체 줄기세포 분화 조절용 키트를 제공한다.The present invention also provides a kit for regulating differentiation of skin adult stem cells comprising a composition for regulating differentiation of skin adult stem cells according to the present invention.
본 발명의 피부성체 줄기세포의 분화 조절 방법은 SAHA를 첨가함으로써 피부세포의 분화 속도를 조절하고 줄기세포능을 유지시켜서 피부세포의 증식능을 오래 지속시킬 수 있으며, 본 발명의 피부성체 줄기세포의 분화 조절 방법은 피부성체 줄기세포의 증식 분화를 조절하는 다양한 연구에서 활용할 수 있다. The method for controlling the differentiation of skin adult stem cells according to the present invention can control the differentiation rate of skin cells and maintain the stem cell function by adding SAHA to maintain the proliferative activity of skin cells for a long time. The control method can be used in various studies to control the proliferation differentiation of adult stem cells.
도 1은 SAHA를 다양한 농도로 첨가하여 배양한 실험군과 SAHA를 첨가하지 않고 배양한 대조군의 세포로 제조된 삼차원 인공피부모델에서 표피의 두께 변화를 보여주는 사진(A) 및 각질층(keratinocyte)의 수의 평균값을 보여주는 그래프(B)이다.
도 2는 SAHA를 다양한 농도로 첨가하여 배양한 실험군과 SAHA를 첨가하지 않고 배양한 대조군의 세포로 제조된 삼차원 인공피부모델에서 α6 인테그린, β1 인테그린, 및 제4형 콜라겐에 대한 발현 결과를 면역조직화학염색을 통해 보여주는 사진이다.
도 3은 SAHA를 다양한 농도로 첨가하여 배양한 실험군과 SAHA를 첨가하지 않고 배양한 대조군의 세포로 제조된 삼차원 인공피부모델에서 p63의 발현 결과를 면역조직화학염색을 통해 보여주는 사진(A) 및 총 표피세포 중 p63 양성 세포의 비율을 보여주는 그래프(B)이다.FIG. 1 is a photograph (A) showing the change in the skin thickness in the three-dimensional artificial skin model prepared from cells cultured with various concentrations of SAHA and cells cultured without SAHA, and the number of keratinocytes (B) showing the average value.
FIG. 2 shows the results of expression of the? 6 integrin,? 1 integrin, and type 4 collagen in the three-dimensional artificial skin model prepared from cells cultured with various concentrations of SAHA and the cells cultured without SAHA, It is a photograph showing through chemical dyeing.
FIG. 3 shows photographs (A) and (B) showing the expression of p63 in immunohistochemical staining in a three-dimensional artificial skin model prepared from cells cultured with various concentrations of SAHA and control cells cultured without SAHA (B) showing the ratio of p63 positive cells in epidermal cells.
이하에서는 본 발명을 실시예에 의하여 더욱 상세히 설명한다. 그러나, 이는 본 발명을 예시하는 것일 뿐, 본 발명이 하기 실시예에 의하여 한정되는 것은 아니다. Hereinafter, the present invention will be described in more detail by way of examples. However, it should be understood that the present invention is not limited by the following examples.
<< 실험예Experimental Example 1> 섬유아세포와 각질형성세포의 배양 1> Culture of fibroblasts and keratinocytes
이하의 실험예에서 다음의 실험 재료 및 방법을 사용하였다. 각질형성세포와 섬유아세포는 어린아이의 포경수술을 통하여 얻어진 포피에서 분리하고, 피부조직에서 잔여 피하 지방을 제거 후 잘게 절개하여 서몰리신(thermolysin) 용액에서 37℃에서 1시간 반응시켰다. 반응시킨 조직을 핀셋을 이용하여 표피와 진피로 분리하여 각각을 트립신(trypsin) 용액에 담아 37℃에서 15분간 반응 후 단일 세포로 분리되도록 진탕한 후, '레인왈드 및 그린(Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell. 1975 Nov;6(3):331-43)'의 방법을 따라 배양하였다.In the following experimental examples, the following experimental materials and methods were used. The keratinocytes and fibroblasts were separated from the foreskin obtained by circumcision of the young child, and the remaining subcutaneous fat was removed from the skin tissue, and then chopped in a thermolysin solution for 1 hour at 37 ° C. The reactioned tissues were separated into epidermis and dermis using tweezers, and each was put in a trypsin solution. After reacting at 37 ° C for 15 minutes, the cells were shaken to separate into single cells. Then, the cells were treated with Rheinwald JG, Green H 1975 Nov; 6 (3): 331-43). ≪ Desc / Clms Page number 13 >
분리한 각질형성세포는 각질형성세포 성장 배지(Clonetics KGM BulletKit, Lonza, Walkersville, MD, USA)에서 배양하였고, 섬유모세포는 10% FBS(Fetal Bovine Serum)가 포함된 DMEM(Dulbecco's modified Eagle's medium, Welgene, Daegu, Republic of Korea)사용하여 37℃ 배양기에서 7일 내지 10일간 배양하였다. The cells were cultured in keratinocyte growth medium (Clonetics KGM BulletKit, Lonza, Walkersville, MD, USA). Fibroblasts were cultured in DMEM (Dulbecco's modified Eagle's medium, Welgene , Daegu, Republic of Korea) at 37 ° C for 7 days to 10 days.
<실험예 2> 삼차원 인공피부모델 제조≪ Experimental Example 2 > Production of three-dimensional artificial skin model
<< 실험예Experimental Example 2-1> 진피 대용물의 제조 2-1> Preparation of dermal substitute
먼저 진피 대용물을 제조하기 위해 쥐꼬리로부터 분리 채취한 힘줄(tendon)을 4 ℃의 1:1000 빙초산(glacial acetic acid) 희석액 1 리터에 48시간 동안 교반하여 녹인 제1형 콜라겐 용액과 10 x DMEM과 완충액(0.05 N NaOH, 0.26 mM NaHCO3, 및 200 mM HEPES)을 8:1:1의 부피비로 혼합하여, 콜라겐 기질을 제조하였다. 이와 같이 얻어진 콜라겐 기질 3.5 mL 당 섬유아세포 오십만 개를 첨가하여 혼합하였다. 이후 상기 혼합액을 30 mm의 밀리셀(millicell, Millipore, Billerica, MA, USA)에 3.5 mL씩 넣어 37℃, 5% CO2 배양기에서 굳혀서 진피 대용물을 제조하였다.First, to prepare a dermis substitute, a tendon, which was collected from rat tail, was added to 1 liter of a 1: 1000 glacial acetic acid dilution solution at 4 ° C for 48 hours, a buffer solution (0.05 N NaOH, 0.26 mM NaHCO 3, and 200 mM HEPES) 8: 1: a mixture in a volume ratio of 1, to thereby prepare a collagen matrix. Five hundred thousand fibroblasts were added per 3.5 mL of the thus obtained collagen matrix and mixed. Then, 3.5 mL of the mixed solution was added to a 30 mm millicell (Millipore, Billerica, MA, USA), and the mixture was solidified at 37 ° C in a 5% CO 2 incubator to prepare a dermal substitute.
<< 실험예Experimental Example 2-2> 3차원 인공피부모델의 제조 2-2> Manufacture of 3D artificial skin model
이와 같이 제조된 진피 대용물 위에 배양한 각질형성세포를 뿌려 1일간 배지 침지시켜 배양한 다음 다시 12일간 공기에 노출시켜 배양함으로써 3차원 인공피부모델을 제조하였다. SAHA는 공기 노출을 시작하면서 첨가하기 시작하고 배지를 교환할 때마다 첨가해 주었다.The three-dimensional artificial skin model was prepared by cultivating the keratinocyte cultured on the thus prepared dermal substitute, culturing it for 1 day by immersing it in the medium, and then culturing it by exposure to the air for 12 days. SAHA was added at the start of air exposure and added every time the medium was exchanged.
이때, 배양 온도는 37℃로 하였고, 상기 배지는 5% FBS, 0.4 mg/mL 하이드로코르티손, 1 mM 이소프로테레놀, 25 mg/mL 아스코르빈산 및 5 mg/mL 인슐린이 포함된 DMEM 배지와, 함스 영양 혼합물 F12(Ham's nutrient mixture F12)을 3:1의 비율로 혼합한 혼합 용액을 사용하였다. 상기 침지 배양을 위한 배지에는 저농도 EGF (Epidermal Growth Factor, Invitrogen, 1 ng/mL)를 첨가하였고, 12일간의 공기 노출 배양 시에는 배지에 고농도 EGF(10 ng/mL)를 첨가하였다. 배지는 일주일에 3회 교환하였고, 상기 실험은 동일조건으로 최소 3번 반복 실시하였다. At this time, the incubation temperature was set to 37 캜, and the medium was DMEM medium containing 5% FBS, 0.4 mg / mL hydrocortisone, 1 mM isoproterenol, 25 mg / ml ascorbic acid and 5 mg / And Ham's nutrient mixture F12 at a ratio of 3: 1. EGF (10 ng / mL) was added to the culture medium for 12 days in air. The culture medium for the immersion culture was added with low EGF (Epidermal Growth Factor, Invitrogen, 1 ng / mL) The medium was changed three times a week and the experiment was repeated at least three times under the same conditions.
<< 실험예Experimental Example 2-3> 헤마톡실린 및 에오신(H&E) 염색 2-3> hematoxylin and eosin (H & E) staining
상기 실험예 2-2에서 얻어진 3차원 인공피부 모델 조직에 대해서 SAHA의 효과를 확인하기 위해 핵과 세포질을 쉽게 구별할 수 있게 해주는 헤마톡실린 및 에오신(H&E)으로 염색하고 그 결과를 도 1에 나타내었다. 도 1로부터 피부 모델 조직에 SAHA를 처리하여 표피가 두꺼워지는 것을 확인할 수 있다.The three-dimensional artificial skin model obtained in Experimental Example 2-2 was stained with hematoxylin and eosin (H & E) to allow easy discrimination between nuclei and cytoplasm in order to confirm the effect of SAHA. . From Fig. 1, it can be confirmed that the skin surface is thickened by treating SAHA in the skin model tissue.
<실험예 2-4> 면역조직화학 염색<Experimental Example 2-4> Immunohistochemical staining
상기 실험예 2-2에서 얻어진 3차원 인공피부 모델 조직에 대해서 면역조직화학염색을 수행하여 그 결과를 도 2 및 도 3에 나타내었다. 면역조직화학염색의 일차항체로서 p63 (#sc-8431 from Santa Cruz Biotechnology, Santa Cruz, CA, USA), α6 인테그린(#sc-6597 from Santa Cruz Biotechnology), integrin ㅯ1 (#sc-9970, from Santa Cruz Biotechnology), type IV collagen (239M-16, Cell Marque, Rocklin, CA)에 대한 항체를 이용하였다. 상기 p63는 피부에서 줄기세포 표식자로 알려졌고, α6 인테그린은 피부성체 줄기세포가 기저막 구조 단백질들과 결합할 수 있게 해주는 세포외 부착 수용체이다. 3-dimensional artificial skin model tissues obtained in Experimental Example 2-2 were subjected to immunohistochemical staining, and the results are shown in FIG. 2 and FIG. (# Sc-8431 from Santa Cruz Biotechnology, Santa Cruz, CA, USA), α6 integrin (# sc-6597 from Santa Cruz Biotechnology), integrin ㅯ 1 Santa Cruz Biotechnology), type IV collagen (239M-16, Cell Marque, Rocklin, CA). The p63 is known as a stem cell marker in the skin, and [alpha] 6 integrin is an extracellular adhesion receptor that allows skin adult stem cells to bind to basement membrane structural proteins.
도 2에서 SAHA를 첨가하면 피부성체 줄기세포 고유의 미세 환경인 니치(stem cell niche)의 주요 구조 단백질과 피부성체 줄기세포가 니치에 결합할 수 있게 해주는 α6 인테그린, β1 인테그린, 및 제4형 콜라겐의 발현이 함께 증가함으로써 줄기세포능이 유지되고 있음을 확인할 수 있다. In Fig. 2, the addition of SAHA results in a major structural protein of the stem cell niche, which is a microenvironment unique to skin adult stem cells, a6 integrin, beta1 integrin, and type 4 collagen The expression of the stem cells is increased and the stem cell ability is maintained.
도 3에서 SAHA를 첨가하면 p63의 발현이 증가함으로써 피부성체 줄기세포의 줄기세포능이 잘 유지되고 있음을 확인할 수 있다.In Fig. 3, the expression of p63 is increased by the addition of SAHA, thereby confirming that the stem cell ability of the skin adult stem cells is maintained well.
Claims (8)
A method of controlling the differentiation of adult stem cells by adding suberoylanilohydroxamic acid (SAHA) to cultured stem cells.
The method according to claim 1, wherein SAHA (suberoylanilohydroxamic acid) is added to increase the expression of? 6 integrin,? 1 integrin, and type 4 collagen.
The method according to claim 1, wherein SAHA (suberoylanilohydroxamic acid) is added to increase the expression of p63.
A composition for controlling differentiation of skin adult stem cells comprising SAHA (suberoylanilohydroxamic acid) as an active ingredient.
A kit for regulating the differentiation of skin adult stem cells comprising the composition according to claim 7.
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