Endocrinology and Metabolism

The Korean Endocrine Society (대한내분비학회)
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The Effect of Oxidative Stress on the Proliferation and Differentiation of Human Bone Marrow Stromal Cell-Derived Osteoblasts

산화스트레스가 사람 골수기질세포 유래 조골세포의 증식과 분화에 미치는 영향

Oh, Eun-Sook;Baek, Ki-Hyun;Lee, Won-Young;Oh, Ki-Won;Kim, Hye-Su;Han, Je-Ho;Lee, Kwang-Woo;Son, Ho-Young;Kang, Sung-Koo;Kang, Moo-Il
오은숙;백기현;이원영;오기원;김혜수;한제호;이광우;손호영;강성구;강무일

  • Published : 20060600
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Abstract

Background: The objectives of our study were to assess the effects of oxidative stress on the proliferation, differentiation and apoptosis of human bone marrow stromal cell (BMSC)-derived osteoblasts and to explore pathways by which osteoblast cell apoptosis was induced. Methods: Mononuclear cells including BMSCs were cultured to osteoblastic lineage. Different doses of hydrogen peroxide (H2O2) were added to the culture media. The colony forming units-fibroblastic (CFU-Fs) were stained with crystal violet and alkaline phosphatase (ALP). The MTT assay was done to see the effect of H2O2 on cell viability. The effect of H2O2 on osteocalcin gene expression was determined by RT-PCR. The matrix calcification measurement was performed. FACS analysis was performed to determine the osteoblasts apoptosis. Caspase-3, -8 and 9 activity assay and cytochrome c release were measured. Results: The size and number of ALP (+) CFU-Fs were also decreased by H2O2 treatment. When compared with the control group, H2O2 significantly decreased the total number of cells of each culture well during MTT assay. H2O2 significantly diminished expression of osteocalcin mRNA. N-acetylcystein (NAC) blocked the diminution of cell viability and the inhibition of osteocalcin mRNA expression by H2O2. H2O2 reduced matrix calcification. FACS analysis revealed H2O2 increased percentage of apoptotic cells. Addition of H2O2 resulted in the increase of caspase-9 and -3 activity but not caspase-8, and release of cytochrome c to the cytosol. Conclusion: These data suggest that, in primary human BMSCs, oxidative stress inhibits proliferation of stromal cells and inhibits the differentiation to osteoblastic lineage. In addition, oxidative stress induces apoptosis of human BMSC-derived osteoblasts and this may be mediated by mitochondrial pathway of apoptotic signal.

연구배경: 최근 세포주와 동물실험에서 산화스트레스가 조골세포의 분화를 억제함이 보고 되고 있고, 사람연구에서 도 증가된 산화스트레스가 골밀도의 감소와 연관됨이 보고 되고 있다. 이에 본 연구는 산화스트레스가 인체 골수에 존재하는 조골세포의 전구세포인 골수기질세포로부터 조골세포로 증식, 분화 및 세포자멸사에 미치는 영향을 살펴보고자 하였다.방법: 정상 골수공여자로부터 골수를 채취, 단핵세포를 분리한 후 조골세포로 분화를 유도하는 배양 조건에서 다양한 농도의 H2O2 및 항산화제인 N-acetylcystein (NAC)를 처리 하였다. 일차배양에서는 CFU-Fs를 계수하여 골수 내 기질세포의 수를 추산하였고 알카리성 포스파타제 염색을 통하여 조골세포로의 분화를 관찰하였다. 계대 배양 후 이차배양에서는 오스테오칼신 mRNA의 발현과 칼슘 침착을 관찰하여 H2O2 농도에 따른 분화능 차이를 비교하였으며, MTT 측정을 하여 세포의 활성도를 관찰하였다. 또한 H2O2가 인체골수기질세포의 세포자멸사에 관여하는지를 살펴보고자 FACS analysis를 시행하였고, 미토콘드리아 cytochrome c 의 세포질 내로의 유출과 caspase-3, caspase-8, caspase-9의활동도를 비교하였다.결과: 일차배양 15일에 평균 CFU-F의 수, 평균 면적 및 총 면적은 0.3 mM H2O2 처리군에서 대조군에 비하여 유의하게 감소되었다. 또한 알카리성 포스파타제 염색 양성인 CFU-F의 평균 개수와 평균 크기도 0.3 mM H2O2 처리군에서 의미 있게 감소되었다. 이차배양에서 시행한 MTT 검사에서 0.3 mM과 0.5 mM의 H2O2 처리군에서 의미 있게 세포활성도가 감소되었다. 항산화제인 NAC와 동시에 처리한경우 H2O2처리에 의해 감소된 세포활성도가 의미 있게 회복되었다. 이차배양 중에 계측된 오스테오칼신 mRNA의 발현은 0.5 mM의 H2O2 처리군에서 의미 있게 감소되었다.조골세포 분화의 후기 지표인 기질 무기화는 0.5 mM의 H2O2 처리군에서 의미 있게 감소되었다. H2O2 처리군에서 Annexin V-FITC와 PI 이중염색을 이용하여 FACS analysis로 관찰한 결과 세포자멸사된 세포의 비율이 증가되었다. Caspase-3과 caspase-9의 활성은 H2O2 처리군에서 의미 있게 증가되었으나, caspase-8의 활성도에는 변화가 없었다. H2O2 처리는 미토콘드리아 내 cytochrome c의 세포질 내로의 유출을 유도하였다. 결론: 산화스트레스는 인체 골수기질세포로부터 조골세포로의 증식과 분화를 저해하였고, 이와 같은 과정에는 미토콘드리아 경로를 통한 세포자멸사 기전이 관여하는 것으로 여겨진다.

Keywords

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