Yak-kong and Soybean Induced Expression of Osteoprotegerin in MG-63 Human Osteoblastic Cells Requires Estrogen Receptor-$\beta$

Kim, Jin-Young,Cho, Yun-Hi The Korean Nutrition Society 2005 Nutritional Sciences Vol.8 No.3

Phytoestrogens, especially Yak-kong or soybean-derived isoflavones have been traditionally used as a supplement of estrogen for preventing postmemopausal osteoporosis in oriental folk medicine. In our previous study, the treatment of Yak-kong and soybean increased estrogen receptor-a (ERa) expression and proliferation of MG-63 osteoblastic cells. In contrast, the increase of estrogen receptor-$\beta$ (ER$\beta$) expression in proliferating MG-63 cells with Yak-kong and soybean treatment was less pronounced, which suggested that ER$\beta$ may play a role rather in the regulation of bone cell differentiation To determine the role of ER$\beta$ in Yak-kong or soybean mediated regulation of bone cell differentiation, we established MG-63 cell lines stably expressing either ER$\beta$ or antisense ER$\beta$ RNAs. Increased expression of ER$\beta$ did not affect ERa expression and proliferation of MG-63 cells. However, increased expression of ER$\beta$ in MG-63 cells (ER$\beta$-MG63 cells) selectively enhanced Yak-kong or soybean induced expression of osteoprotegerin (OPG), a novel soluble glycoprotein which is secreted from osteoblasts and mediates the signal for osteoclast differentiation. Inhibition of ER$\beta$ expression by antisense ER$\beta$ RNAs (As-ER$\beta$-MG63) caused these cells to insensitize Yak-kong or soybean induced expression of OPG but increased MG-63 cell proliferation. Furthermore, the comparable effects between Yak-kong and the combined treatment of genistein and daidzein at $0.5{\times}l0^{-8}$ M, which is a concentration of these two isoflavones similar to Yak-kong at 0.001 mg/mL, on OPG expression in ER$\beta$-MG63 cell demonstrate that the enhanced expression of OPG with Yak-kong treatment is mediated by the synergistic effect of low leveled isoflavones in the extracts. Together, coupled with low level of ER expression in osteoclasts, our data demonstrate that ER$\beta$ in osteoblasts plays an important role in Yak-kong and soybean mediated inhibition of osteoclast differentiation indirectly by enhancing the expression of OPG.

Transglutaminase-2 Is Involved in Expression of Osteoprotegerin in MG-63 Osteosarcoma Cells

( Hye Ja Lee ),( Chang Hoon Lee ) 한국응용약물학회 2013 Biomolecules & Therapeutics(구 응용약물학회지) Vol.21 No.3

Osteoprotegerin (OPG) is a secreted glycoprotein and a member of the tumor necrosis factor receptor superfamily. It usually functions in bone remodeling, by inhibiting osteoclastogenesis through interaction with a receptor activator of the nuclear factor κB (RANKL). Transglutaminases-2 (Tgase-2) is a group of multifunctional enzymes that plays a role in cancer cell metastasis and bone formation. However, relationship between OPG and Tgase-2 is not studied. Therefore, we investigated the involvement of 12-O-Tetradecanoylphorbol 13-acetate in the expression of OPG in MG-63 osteosarcoma cells. Interleukin-1β time-dependently induced OPG and Tgase-2 expression in cell lysates and media of the MG-63 cells by a Western blot. Additional 110 kda band was found in the media of MG-63 cells. 12-O-Tetradecanoylphorbol 13-acetate also induced OPG and Tgase-2 expression. However, an 110 kda band was not found in TPA-treated media of MG-63 cells. Cystamine, a Tgase-2 inhibitor, dose-dependently suppressed the expression of OPG in MG-63 cells. Gene silencing of Tgase-2 also signifi cantly suppressed the expression of OPG in MG-63 cells. Next, we examined whether a band of 110 kda of OPG contains an isopeptide bond, an indication of Tgase-2 action, by monoclonal antibody specifi c for the isopeptide bond. However, we could not fi nd the isopeptide bond at 110 kda but 77 kda, which is believed to be the band position of Tgase-2. This suggested that 110 kda is not the direct product of Tgase-2`s action. All together, OPG and Tgase-2 is induced by IL-1β or TPA in MG-63 cells and Tgase-2 is involved in OPG expression in MG-63 cells.

Effect of Adrenomedullin on Proliferation and Mitogen-Activated Protein Kinase Activity in Osteosarcoma Cell Lines (MG63, U2OS)

Jung Ryul Kim(김정렬),Byung Wan Choi(최병완),Jong Hyuk Park(박종혁),Kyu Hyung Kim(김규형) 대한정형외과학회 2008 대한정형외과학회지 Vol.43 No.6

목적: 아드레노메듀린이 인간 골육종 세포주에 작용하여 세포 증식에 영향을 주는지 알아보고, 증식을 일으키는 기전에 유사분열 물질 활성화 단백 활성효소, 특히 세포외 신호 조절 활성 효소 1/2가 작용하는지 알아보고자 하였다. 대상 및 방법: MG63과 U2OS 골육종 세포주에 대한 세포 증식 검사를 실시하였고, 골육종세포에 대한 아드레노메듈린의 효과를 알아보기 위해 세포외 신호 조절 활성 효소 1/2에 대한 Western blot 분석을 실시하였다. 아드레노메듈린이 골육종 세포주에서 유사분열 물질 활성화 단백 활성효소 작용기전을 통해 세포 증식을 일으키는지 알아보기 위해 세포외 신호 조절 활성 효소의 억제제인, PD98059을 사용하였다. 결과: 아드레노메듈린을 MG63과 U2OS 골육종 세포주에 투여했을 때 투여양에 비례하여 세포 증식이 일어났다. Western blot 분석 소견에서 세포의 신호 조절 활성 효소 1/2가 강하게 활성화되어 세포 증식은 유사분열물질 활성화 단백 활성효소 작용기전을 통해 일어남을 알 수 있었고, 세포외 신호 조절 활성 효소의 억제제인, PD98059에 의해서 세포 증식은 억제되었다. 결론: 아드레노메듈린은 MG63과 U2OS 골육종 세포주의 세포 증식에 중요한 역할을 하며, 그 작용 기전에 있어서 세포외 신호 조절 활성 효소에 의한 신호 전달이 중요한 역할을 한다고 생각된다. Purpose: The aims investigated how how human osteosarcoma cell proliferation and the MAP kinases cascade are regulated, in the MG63 and U2OS human osteosarcoma cell lines after stimulating them with adrenomedullin (AM) with particular focus on extracellular signal-regulated kinase 1/2 (ERK 1 and 2) activation. Materials and Methods: A cell proliferation assay was used to examined the effects of AM on the osteosarcoma cell lines (MG63 and U2OS). The effects of AM on ERK1/2 were examed by Western blot analysis. The roles of ERK 1/2 in the AM-induced proliferative signaling pathways in the two cell types were examed using PD98059, a selective inhibitor of the mitogen activated protein-ERK kinase (MEK) pathway. Results: The addition of AM to the medium containing the osteosarcoma MG63 and U2OS cells induced proliferation in a dose-dependent manner. AM strongly activated ERK 1/2 mediated cell proliferation signaling, which was prevented using PD98059. Conclusion: These results suggest that AM plays an important role in the proliferation of human osteosarcoma MG63 and U2OS cells, and ERK kinase pathway plays a signal transduction role in AM treated human osteosarcoma MG63 and U2OS cell lines.

mTOR Signal Transduction Pathways Contribute to TN-C FNIII A1 Overexpression by Mechanical Stress in Osteosarcoma Cells

Zheng, Lianhe,Zhang, Dianzhong,Zhang, Yunfei,Wen, Yanhua,Wang, Yucai Korean Society for Molecular and Cellular Biology 2014 Molecules and cells Vol.37 No.2

Osteosarcoma is the most common primary malignant bone tumor with a very poor prognosis. Treating osteosarcoma remains a challenge due to its high transitivity. Tenascin-C, with large molecular weight variants including different combinations of its alternative spliced FNIII repeats, is specifically over expressed in tumor tissues. This study examined the expression of Tenascin-C FNIIIA1 in osteosarcoma tissues, and estimated the effect of mechanical stimulation on A1 expression in MG-63 cells. Through immunohistochemical analysis, we found that the A1 protein was expressed at a higher level in osteosarcoma tissues than in adjacent normal tissues. By cell migration assay, we observed that there was a significant correlation between A1 expression and MG-63 cell migration. The relation is that Tenascin-C FNIIIA1 can promote MG-63 cell migration. According to our further study into the effect of mechanical stimulation on A1 expression in MG-63 cells, the mRNA and protein levels of A1 were significantly up-regulated under mechanical stress with the mTOR molecule proving indispensable. Meanwhile, 4E-BP1 and S6K1 (downstream molecule of mTOR) are necessary for A1 normal expression in MG-63 cells whether or not mechanical stress has been encountered. We found that Tenascin-C FNIIIA1 is over-expressed in osteosar-coma tissues and can promote MG-63 cell migration. Furthermore, mechanical stress can facilitate MG-63 cell migration though facilitating A1 overexpression with the necessary molecules (mTOR, 4E-BP1 and S6K1). In con-clusion, high expression of A1 may promote the meta-stasis of osteosarcoma by facilitating MG-63 cell migration. Tenascin-C FNIIIA1 could be used as an indicator in metastatic osteosarcoma patients.

mTOR Signal Transduction Pathways Contribute to TN-C FNIII A1 Overexpression by Mechanical Stress in Osteosarcoma Cells

Lianhe Zheng,Dianzhong Zhang,Yunfei Zhang,Yanhua Wen,Yucai Wang 한국분자세포생물학회 2014 Molecules and cells Vol.37 No.2

Osteosarcoma is the most common primary malignant bone tumor with a very poor prognosis. Treating osteosarcoma remains a challenge due to its high transitivity. Tenascin-C, with large molecular weight variants including different combinations of its alternative spliced FNIII repeats, is specifically over expressed in tumor tissues. This study examined the expression of Tenascin-C FNIIIA1 in osteosarcoma tissues, and estimated the effect of mechanical stimulation on A1 expression in MG-63 cells. Through immunohistochemical analysis, we found that the A1 protein was expressed at a higher level in osteosar-coma tissues than in adjacent normal tissues. By cell migration assay, we observed that there was a significant correlation between A1 expression and MG-63 cell migra-tion. The relation is that Tenascin-C FNIIIA1 can promote MG-63 cell migration. According to our further study into the effect of mechanical stimulation on A1 expression in MG-63 cells, the mRNA and protein levels of A1 were significantly up-regulated under mechanical stress with the mTOR molecule proving indispensable. Meanwhile, 4E-BP1 and S6K1 (downstream molecule of mTOR) are necessary for A1 normal expression in MG-63 cells whether or not mechanical stress has been encountered. We found that Tenascin-C FNIIIA1 is over-expressed in osteosar-coma tissues and can promote MG-63 cell migration. Furthermore, mechanical stress can facilitate MG-63 cell migration though facilitating A1 overexpression with the necessary molecules (mTOR, 4E-BP1 and S6K1). In con-clusion, high expression of A1 may promote the meta-stasis of osteosarcoma by facilitating MG-63 cell migration. Tenascin-C FNIIIA1 could be used as an indicator in metastatic osteosarcoma patients.

초피 추출물의 항돌연변이 및 MG-63 암세포 증식억제 효과

김소희,박건영 한국산업미생물학회 1993 한국미생물·생명공학회지 Vol.21 No.6

초피나무의 과피 부분의 메탄올 추출물과 이를 더욱 분리한 핵산, 클로르포름, 에틸 아세테이트, 부탄올, 수층분획물들의 항돌연변이 효과를 Ames mutagenicity test와 SOS chromotest를 이용하여 검토하고 사람의 골육암 세포인 MG-63 cell의 증식에 대한 억제 효과를 실험하였다. 초피의 메탄올 추출물은 Ames mutagenicity test 에서 aflatoxin B_1(AFB_1)과 N-methyl-N’-nitro-N-nitrosoguanidine(MNNG)의 돌연변이유발성을 크게 저해하였는데(p<0.01), AFB_1에 대하여는 시료에 대한 20% 농도 추출물의 첨가로 거의 90% 이상까지도 억제시킬 수 있었다. 한편 초피의 메탄올 추출물은 SOS chromotest에서도 MNNG의 SOS response를 억제시키는 강한 항돌연변이 효과를 나타내었다. 아울러 그의 활성 분획물을 찾기 위하여 메탄올 추출물을 극성이 다른 용매들로 분획한 분획물들중에는 특히 헥산분획물이 가장 큰 항돌연변이 효과를 나타내어 AFB_1에 대하여는 98% 이상을, MNNG에 대하여는 64%까지 돌연변이성을 억제시켰으며, 부탄올추출물도 비교적 큰 항돌연변이 효과가 있었다. 그리고 초피의 메탄올 추출물은 100 ㎍의 첨가로 사람의 골육암 세포인 MG-63의 증식을 98%까지 억제시키는 항암 효과를 보였다. 메탄올 추출물에서 분리한 헥산분획물과 클로르포름분획물(10% 농도)도 암세포에 대하여 현저한 효과를 나타내어 MG-63 cell의 증식을 각각 98%, 96%까지 억제시켰다. The inhibitory effects of various extracts from Chinese pepper on the mutagenicity and the growth of MG-63 human osteosarcoma cells were studied. Chinese pepper was extracted with methanol and then the methanol extract was further fractionated by using hexane, chloroform, ethyl acetate and butanol. The methanol extract of Chinese pepper revealed the strong antimutagenic activity on the aflatoxin B_1(AFB_1) and N_methyl-N’-nitrosoguanidine(MNNG) in Ames mutagenicity test and SOS chromotest. Among the solvent extracted fractions from the methanol extract, the hexane fraction exhibited the greatest antimutagenic effect suppressing the mutagenicity of AFB_1 and MNNG with ihibition rate of 98 and 64 percent, respectively. The methanol extract of Chinese pepper also showed the inhibitory effect on the growth of MG-63 human osteosarcoma cells. The hexane fraction and the chloroform fraction from the methanol extract of Chinese pepper were most effective and inhibited the growth of MG-63 cells by 98 and 96 percent after 6 days of incubation at 37℃, respectively.

MMP-1 and TIMP-1 production in MG-63 cells stimulated with Prevotella nigrescens Lipopolysaccharide

Yang, Won-Kyung,Kim, Mi-Ri,Shon, Won-Jun,Lee, In-Bog,Cho, Byeong-Hoon,Um, Chung-Moon,Son, Ho-Hyun 大韓齒科保存學會 2004 Restorative Dentistry & Endodontics Vol.29 No.5

The purpose of this study is to monitor the secretion of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) produced by human osteosarcoma cell line (MG63) stimulated with Prevotella nigrescens lipopolysaccharides (LPS). and to compare the level of secretion before and after the treatment of calcium hydroxide on P. nigrescens LPS. LPS was extracted and purified from anaerobically cultured P. nigrescens. MG63 cells were stimulated by the LPS (0, 1, 10㎍/ml) or LPS(10㎍/ml) pretreated with 12.5 mg/ml of Ca(OH)₂ for 3 days. Total RNA was isolated from the cell. and real-time quantitative polymerase chain reaction (PCR) was performed for quantification of MMP-1 and TIMP-1. The results were as follows. 1. MMP-1 mRNA expression at 48 hr was highly increased by stimulation with P. nigrescens LPS. The increase was dose-dependent. 2. When stimulated with 1㎍/ml of LPS. TIMP-1 mRNA expression was highly increased at 24 hr and 48 hr. However. TIMP-1 expression was suppressed at higher concentration (10 ㎍/ml). 3. When P. nigrescens LPS was pretreated with Ca(OH)₂, MMP-1 and TIMP-1 gene expression was downregulated. The results of this study suggest that transcriptional regulation of MMP-1 and TIMP-1 by P. nigrescens LPS could be one of the important mechanisms in bone resorption of periapical inflammation. The result of calcium hydroxide on MMP-1 and TIMP-1 gene expression suppression shows that calcium hydroxide detoxified bacterial LPS and thus should be used the medication of choice for intracanal dressings in root canal infected with black-pigmented bacteria. 본 연구의 목적은 Prevotella nigrescens lipopolysaccharides (LPS)로 자극된 MG63 osteosarcoma 세포에서 생성, 분비되는 기질금속단백효소인 MMP-1과 그 억제제인 TIMP-1을 측정할 뿐아니라 수산화칼슘으로 처리한 P. nigrescens LPS에 의한 기질금속단백효소와 그 억제제의 분비수준의 변화를 알아보는데 있다. 혐기성 조건에서 배양한 P. nigrescens로부터 LPS를 추출하여 순수정제한 다음 0, 1 그리고 10㎍/ml의 LPS 농도로 MG63 세포를 자극하거나 또는 수산화칼슘으로 처리한 10㎍/ml의 LPS로 세포를 다양한 자극하여 다양한 시간이 경과한 다음 세포로부터 분비되는 MMP-1과 TIMP-1의 RNA 수준을 real time-PCR 방법으로 측정하였다. 실험결과 MMP-1의 mRNA수준은 48시간에서 최고에 달하였고 그 분비정도는 LPS의 농도에 비례하였다. TIMP-1 mRNA는 1㎍/ml의 세균성 LPS 자극시 24시간 및 48시간에서 높은 증가를 보였으나 고농도인 10㎍/ml의 LPS로 자극한 경우 오히려 그 발현이 억제되었다. 또한 수산화칼슘으로 전처리한 P. nigrescens LPS로 자극한 MG 63 세포에서는 MMP-1과 TIMP-1의 분비가 억제되었다. 이러한 결과를 통해 볼 때 P. nigrescens LPS에 의한 MMP-1과 TIMP-1의 발현조절이 치근단 질환에서 발생하는 치조골 흡수 기전중 하나로 사료된다. 뿐만 아니라 P. nigrescens에 의해 분비되는 기질금속단백효소를 매개로 하는 염증반응 감소에 수산화칼슘이 효과적으로 작용하는것으로 확인되어 치근단 질환에 관여하는 세균성 LPS를 제거하기 위해 임상적으로 사용되는 근거가 될 수 있다.

흑염소와 약용식물 복합 증탕추출액 및 증류액이 조골세포 증식과 파골세포 형성에 미치는 영향

송효남,임강현,권인숙 한국영양학회 2015 Journal of Nutrition and Health Vol.48 No.2

Purpose: The effects of water extract and distillate from the mixture of black goat meat and medicinal herb on MG-63 osteoblast proliferation and mouse bone marrow derived osteoclast formation were investigated. Methods: Proximate composition, volatile basic nitrogen (VBN), mineral content, free amino acid composition and free fatty acid composition in black goat meat were determined. Water extract and distillate were prepared with three groups; goat meat only (BG-E, BGD), six herbs added group (BG-E6, BG-D6), and eight herbs added group (BG-E8, BG-D8). Osteoblast proliferation, mineralization and calcium uptake activity of MG-63 cells were measured and tartrate resistant acid phosphatase activity of osteoclasts was analyzed. Results: Black goat meat had remarkably low fat and high level of calcium. Glutamic acid was the most abundant amino acid. Herbs added extract groups (BG-E6 and BG-E8) showed increased MG-63 cell proliferation in a concentration dependent manner, while all the distillates did not show the effect. All extracts and distillates showed significantly increased osteoblast mineralization depending on the concentration. In particular, herb added extract, BG-E6, increased 170.3% of control and the distillate of BG-D and BG-D6 increased up to 168.5% and 159.8%, respectively. Calcium uptake activities of all water extracts showed remarkable increase of BG-E6 and BG-E8 up to 615.5% and 628.1% of control, respectively. Ditillates had no effect except BG-D6. All water extracts significantly reduced the activity of tartrate-resistant acid phosphatase (TRAP) in osteoclasts derived from mouse bone marrow. Conclusion: Combination of black goat meat and medicinal herb increased the MG-63 cell proliferation and effectively inhibited osteoclast differentiation in both water extracts and distillate of them, which implies that they could be used as potent functional food materials for bone health. 골기능 개선용 흑염소 액상제품을 개발하기 기초연구로서 흑염소 및 약용식물 복합추출물이 MG-63 조골세포 및마우스 골수세포 유래 파골세포의 분화에 미치는 영향을분석하였다. 흑염소 원료육의 일반성분, 휘발성 염기질소, 무기질함량, 유리아미노산 조성 및 지방산 조성 등의 영양성분을 분석하여 기초자료를 마련하였다. 흑염소에 첨가할 약용식물의 종류와 배합을 달리한 두 그룹의 한약재 첨가군에 대해 증탕추출액과 증류액을 제조하여 총 6개 시료군 (흑염소육 (BG-E, BG-D), 6종 한약재 첨가군 (BG-E6, BG-D6) 및 8종 한약재 첨가군 (BG-E8, BG-D8)을 대상으로 골강화 활성을 분석하였다. 식품공전상 식품의 원료로사용이 가능한 원료 중 황기, 홍화씨, 당귀, 황정, 속단, 우슬각각 2/2/2/2/1/1의 배합비를 지닌 한약재 6종 첨가군 및 동일배합에 녹용과 녹각을 각각 0.3/1.2 로 추가배합한 한약재 8종 첨가군을 사용하였다. 조골세포 MG-63의 증식 촉진 활성에 대해 시료별 및 농도별로 유의적인 차이가 나타났으며 한약재 무첨가 흑염소군보다 6종 및 8종 등 한약재첨가량이 많을수록 활성이 증가하였다. 증탕추출액과 달리 증류액은 모두 유의한 효과가 없었다. 조골세포의 골석회화 촉진활성시험 결과 대조군에 비해 모든 시료 처리군에서 칼슘함량이 유의적으로 증가하여 MG-63 세포의 석회화 결절 형성을 농도 의존적으로 유의하게 증가시켰다. BG-E6는 대조군에 비해 석회화 형성을 170.3% 증가시켰고, 증류액인 BG-D와 BG-D6는 각각 168.5% 및 159.8%의증가를 나타내었다. 시료별 차이에 있어 한약재 첨가군이무첨가군보다 높았고, 증탕추출액이 증류액보다 높은 활성을 보였다. 세포의 칼슘 흡수량을 측정한 결과 모든 증탕추출액에서 활성이 증가하였고 특히 BG-E6와 BG-E8은각각 615.5%와 628.1%로 유의적으로 가장 높은 증가율을보였다. 증류액은 BG-D6의 1/10 농도군외에 효과가 없었다. 마우스 골수세포 유래 파골세포의 증식억제 실험결과TNF-α 만을 처리한 대조군에 비해 모든 시료군이 TRAP 활성을 억제하는 경향을 나타내었다. 특히 BG-D 및 BGE6, BG-E8은 유의하게 파골세포로의 분화를 억제하였다. 종합적으로 흑염소육을 비롯하여 황기, 홍화씨, 당귀, 황정, 속단, 우슬, 녹용 및 녹각 등 한약재의 복합추출물은 골 기능 강화에 매우 효과적인 기능성 원료가 될 수 있을 것으로사료된다.

THE EFFECT OF SEVERAL ROOT-END FILLING MATERIALS ON MG63 OSTEOBLAST-LIKE CELLS

Lee, Jeong-Ho,Shon, Won-Jun,Lee, WooCheol,Baek, Seung-Ho 大韓齒科保存學會 2010 Restorative Dentistry & Endodontics Vol.35 No.3

The purpose of this study was to compare mineral trioxide aggregate (MTA; Dentsply, Tulsa Dental, Tulsa, OK, USA), which is widely used as root-end filling material, with DiaRoot BioAggregate (DB; Innovative BioCaramix Inc, Vancouver, BC, Canada), newly developed product, by using MG63 osteoblastlike cells. MTA, DB, and Intermediate Restorative Material (IRM; Dentsply Caulk, Milford, DE, USA) were used for root-end filling material while tissue culture plastic was used for control group. Each material was mixed and, the mixtures were left to set for 24 hours. MG63 cells were seeded to each group and then they were cultured for attachment for 4 hours. Following the attachment of cells to the root-end filling material, early cellular response was observed. After another 12 hours’culture, the level of attachment between cells and material was observed and in order to identify the effect of each material to bone formation, transforming growth factor beta1 (TGFβ1) and osteocalin (OC) were estimated by using enzymelinked immunosorbent assay (ELISA), and the amount of alkaline phosphatase (ALP) was also measured. The data were analyzed using one-way ANOVA. As a result, only at OC and the number of cells which were attached to materials, there was no statistical difference between MTA and DB. At other items, there was statistically significant difference in all groups. Although DB has not shown exactly the same cellular response like that of MTA, the number of attached cells shows that biocompatibility of the material and OC indicates bone formation rate. Therefore, if DB is used for root end filling material, it is expected to lead to similar results to MTA. 본 연구의 목적은 현재 치근단 역충전재로 널리 사용되고 있는 MTA와 새롭게 개발된 제품인 DB를 MG63 세포를 사용하여 비교하는 것이다. 치근단 역충전재료로는 MTA, DB, IRM을 사용하였고 대조군으로는 tissue culture plastic을 사용하였다. 각 재료를 혼합하고 혼합물의 경화가 일어나도록 24시간 동안 놓아두었다. MG63 세포를 각 군에 뿌려준 후 세포가 재료에 부착될 수 있도록 4시간 배양하였다. 치근단 역충전재를 세포에 접촉시킨 후 세포수준의 초기 반응을 관찰하였다. 12시간 더 배양한 후 세포가 각 재료에 붙어 있는 정도를 관찰하고, 각 재료가 골형성에 미치는 영향을 알아보기 위해 ELISA를 이용하여 TGFβ1, OC를 측정하였고 ALP의 양도 측정하였다. 결과는 일원배치분산분석법으로 통계처리하였다. 그 결과, 재료에 부착이 일어난 세포의 수 항목과 OC 항목에서만 MTA와 DB간에 통계적으로 차이가 없었다. 다른 항목들에서는 모든 군 간에 통계적으로 유의한 차이가 있었다. DB가 MTA와 완전히 같은 세포반응을 보이지는 않았지만 부착이 일어난 세포의 수는 재료의 생체적합성을 나타내며 OC는 골형성 정도를 나타내므로 DB가 역충전 재료로 사용된다면 MTA와 유사한 결과를 보일 것으로 예측된다.

Serum Deprivation-Induced Human GM3 Synthase (hST3Gal V) Gene Expression Is Mediated by Runx2 in Human Osteoblastic MG-63 Cells

Yoon, Hyun-Kyoung,Lee, Ji-Won,Kim, Kyoung-Sook,Mun, Seo-Won,Kim, Dong-Hyun,Kim, Hyun-Jun,Kim, Cheorl-Ho,Lee, Young-Choon MDPI 2016 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.17 No.1

<P>Serum deprivation (SD) is well known to induce G0/G1 cell cycle arrest and apoptosis in various cells. In the present study, we firstly found that SD could induce G1 arrest and the differentiation of human osteoblastic MG-63 cells, as evidenced by the increase of osteoblastic differentiation markers, such as bone morphogenetic protein-2 (BMP-2), osteocalcin and runt-related transcription factor 2 (Runx2). In parallel, gene expression of human GM3 synthase (hST3Gal V) catalyzing ganglioside GM3 biosynthesis was upregulated by SD in MG-63 cells. The 5′-flanking region of the hST3Gal V gene was functionally characterized to elucidate transcriptional regulation of hST3Gal V in SD-induced MG-63 cells. Promoter analysis using 5′-deletion constructs of the hST3Gal V gene demonstrated that the −432 to −177 region functions as the SD-inducible promoter. Site-directed mutagenesis revealed that the Runx2 binding sites located side-by-side at positions −232 and −222 are essential for the SD-induced expression of hST3Gal V in MG-63 cells. In addition, the chromatin immunoprecipitation assay also showed that Runx2 specifically binds to the hST3Gal V promoter region containing Runx2 binding sites. These results suggest that SD triggers upregulation of hST3Gal V gene expression through Runx2 activation by BMP signaling in MG-63 cells.</P>