[논문]인체 섬유아세포 및 케라티노사이트에 대한 지방줄기세포 분비물의 세포생물학적 기능

이재설; 이종환

doi:10.4014/kjmb.1204.04001

인체 섬유아세포 및 케라티노사이트에 대한 지방줄기세포 분비물의 세포생물학적 기능
Cell Biological Function of Secretome of Adipose-Derived Stem Cells on Human Dermal Fibroblasts and Keratinocytes 원문보기

한국미생물·생명공학회지 = Korean journal of microbiology and biotechnology, v.40 no.2, 2012년, pp.117 - 127

이재설 (동의대학교 바이오물질제어학과) , 이종환 (동의대학교 바이오물질제어학과)

초록
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피부재생에 대한 지방줄기세포 배양상등액(ADSC-CM)의 효능에 대한 연구를 진행하였다. ADSC-CM이 피부재생에 기여하는 기작은 명확하지 못하지만, ADSC-CM은 다양한 분비물을 포함하고 있고 따라서 피부트러블 처리를 위한 훌륭한 재료이다. 저 산소 상태에서 생산된 ADSC-CM, 즉 advanced adipose-derived stem cell protein extract (AAPE)는 피부재생에 보다 좋은 재료이다. 본 연구는 피부 재생에 결정적 역할을 하는 인체 primary 세포인 섬유아세포(HDF)와 케라티노사이트(HK)를 이용하여 AAPE의 효능을 검증하였다. 0.32 ${\mu}g/ml$ AAPE에서 콜라겐 합성이 관찰 되었으며 AAPE는 stress fiber 형성을 강화하였다. DNA microarray 결과에서는 세포증식, 세포이동, 세포부착, 상처반응에 관여하는 133개의 유전자 발현이 조절되는 것을 알았다. Antibody array를 통해 CD54, FGF-2, GM-CSF, IL-4, IL-6, VEGF, TGF-${\beta}2$, TGF-${\beta}3$, MMP-1, MMP-10, 그리고 MMP-19와 같은 MMP, 성장인자, 사이토카인등 25개의 알려진 단백질이 포함되어 있다는 것을 알았다. 따라서, AAPE는 HK의 세포생물학적 기능을 활성화 할 수 있다고 사료되며 HDF에서는 콜라겐 합성을 유도하였다. 이러한 결과는 AAPE가 피부재생에 임상적 적용이 가능하리라는 것을 의미한다.

Abstract ▼ AI-Helper

The beneficial effects of adipose-derived stem cell conditioned media (ADSC-CM) for skin regeneration have previously been reported, despite the precise mechanism of how ADSC-CM promotes skin regeneration remaining unclear. ADSC-CM contains various secretomes and this may be a factor in it being a good resource for the treatment of skin conditions. It is also known that ADSC-CM produced in hypoxia conditions, in other words Advanced Adipose-Derived Stem cell Protein Extract (AAPE), has excellent skin regenerative properties. In this study, a human primary skin cell was devised to examine how AAPE affects human dermal fibroblast (HDF) and human keratinocyte (HK), which both play fundamental roles in skin regeneration. The promotion of collagen formation by HDFs was observed at 0.32 mg/ml of AAPE. AAPE treatment significantly stimulated stress fiber formation. DNA gene chips demonstrated that AAPE in HKs (p<0.05) affected the expression of 133 identifiable transcripts, which were associated with cell proliferation, migration, cell adhesion, and response to wounding. Twenty five identified proteins, including MMP, growth factor and cytokines such as CD54, FGF-2, GM-CSF, IL-4, IL-6, VEGF, TGF-${\beta}2$, TGF-${\beta}3$, MMP-1, MMP-10, and MMP-19, were contained in AAPE via antibody arrays. Thus, AAPE might activate the HK biological function and induce the collagen synthesis of HDF. These results demonstrate that AAPE has the potential to be used for clinic applications aimed at skin regeneration.

주제어

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문제 정의

They also produce extracellular matrix proteins, and adhesive molecules. This study investigated whether or not locally applied AAPE can accelerate skin regeneration process in vitro system. The collagen neo-synthesis of HDF, which is one major cell type present in skin, was increased by AAPE treatment.

제안 방법

The conditioned media were collected and micro-filtered, followed by quantitated total protein production. Finally, for fresh use, 4 ml vials containing equal protein concentration were freeze-dried as a single lot sample preparation of AAPE (Prostemics Research Institute, Sungnam, Korea) for this study.
For this analysis with GeneChip Human Gene 1.0 ST array (Affymetrix, Santa Clara, CA, US) containing 28,869 gene-level probe set, total cellular RNA was isolated from HK incubated with AAPE (1.25 µg/ml) for 24 h using Trizol reagent (Invitrogen, Foster, CA, US) according to manufacturer's directions.
In order to visualize protein expression for proteome analysis, an explorer antibody array chip coated with 656 antibodies was conducted to identify proteins in AAPE (Fig. 5). Protein level measurements were clustered by the ratio of hormone (Fig.
Thus, cell biological function of both cells is essential for performing regenerative processes on the skin surface. In this study, we examined the cell biological function of AAPE on HDF and HK in vitro, and the components of AAPE through antibody array analysis.
5). Protein level measurements were clustered by the ratio of hormone (Fig. 5A), cytokine (Fig. 5B), cell cycle (Fig. 5C), cancer marker (Fig. 5D), apoptosis (Fig. 5E), angiogenesis (Fig. 5F), stem cell (Fig. 5G), and signal transduction (Fig. 5H) level by comparing the signal intensity of soluble protein expression in AAPE to a negative control. Furthermore, a cytokine-focused antibody array with 77 antibodies was conducted to identify proteins that may be involved in mediating the effect of AAPE on proliferation, migration or regeneration (Fig.
After detecting, the slide was washed 2 times with 30 ml of washing solution. The slide scanning was performed using Revolution^TM 4200 microarray scanner (Vidar Systems, Herndon, VA, US) and ArraySifter Express 1.3 (Vidar Systems). The slide was scanned at 10 µm resolution, optimal laser power and PMT.

대상 데이터

The RNA samples were submitted to the Advanced Medical Technology Center for Diagnosis & Prediction (School of Medicine, Kyungpook National University, Taegu, Korea) for array preparation and analysis.
The freeze dried protein sample was submitted to the Ebiogen (Kyung Hee business center, Kyung Hee University, Seoul, Korea) for array preparation and analysis. The explorer antibody microarray slide (Fullmoon biosystems, Sunnyvale, CA, US) with 656 antibodies and the cytokine focused microarray slide with 77 antibodies were treated 30 ml of blocking solution and incubated on shaker for 45 minutes at room temperature.

데이터처리

Each value represents the mean ± S.D. of three experiments, and values containing asterisks are significantly from the nontreatment group (Cont.) at p<0.01(**) by Dunnett’s t-test.
The amount HDF proliferation was significantly less in AAPE (more than 0.625 µg/ml) treated group compared to the group treated with AAPE (from 0 to 0.313µg/ml) by Student’s t-test.
The list was filtered first for the absent genes, secondly for a fold change cutoff of 2, and thirdly for p value of ≤0.05 using Welch's t-test.

이론/모형

Portions of the array illustrating the differential expression of proteins between HGF and sample antibody are shown. Heat map were analyzed by Genowiz 4.0TM as described in Materials and Methods. The median streptavidin-Cy3 fluorescence from all microsphere subsets was exported.

성능/효과

Analysis of the array results revealed high protein expression level of VEGF, TGF-β2, CD54 and TGF-β3 compared to HGF, shows low protein expression level of FGF-1, FGF-2, IL-4, IL-6, G-CSF, GM-CSF compared to HGF (Fig. 6).
Our results demonstrate that AAPE in HKs (p < 0.05) affected expression of 133 identified transcripts regulated minimally by greater than or equal to a 2-fold change.

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저자의 다른 논문 :

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