• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제36권4호
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• pp.243-249
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• 2010

Tissue engineered bone (TEB) can replace an autogenous bone graft requiring an secondary operation site as well as avoid complications like inflammation or infection from xenogenic or synthetic bone graft. Adult mesenchymal stem cells (MSC) for TEB are considered to have various ranges of differentiation capacity or multipotency by the donor site and age. This study examined the effect of age on proliferation capacity, differentiation capacity and bone morphogenetic protein-2 (BMP-2) responsiveness of human bone marrow stromal cells (hBMSC) according to the age. In addition, to evaluate the effect on enhancement for osteoblast differentiation, the hBMSC were treated with Trichostatin A (TSA) and 5-Azacitidine (5-AZC) which was HDAC inhibitors and methyltransferase inhibitors respectively affecting chromatin remodeling temporarily and reversibly. The young and old group of hBMSC obtained from the iliac crest from total 9 healthy patients, showed similar proliferation capacity. Cell surface markers such as CD34, CD45, CD90 and CD105 showed uniform expression regardless of age. However, the young group showed more prominent transdifferentiation capacity with adipogenic differentiation. The osteoblast differentiation capacity or BMP responsiveness was low and similar between young and old group. TSA and 5-AZC showed potential for enhancing the BMP effect on osteoblast differentiation by increasing the expression level of osteogenic master gene, such as DLX5, ALP. More study will be needed to determine the positive effect of the reversible function of HDAC inhibitors or methyltransferase inhibitors on enhancing the low osteoblast differentiation capacity of hBMSC.

• Clinical and Experimental Reproductive Medicine
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• 제49권3호
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• pp.175-184
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• 2022

Objective: The aim of this study was to evaluate the impacts of platelet-rich plasma (PRP) and conditioned medium (CM) derived from endometrial stromal cells on mouse preantral follicle culture in a two-dimensional system to produce competent mature oocytes for fertilization. Methods: In total, 240 preantral follicles were isolated from female mouse ovarian tissue and divided into four groups. The preantral follicles were isolated three times for each group and then cultured, respectively, in the presence of alpha minimum essential medium (control), PRP, CM, and PRP+CM. The in vitro growth, in vitro maturation, and cleavage percentage of the preantral follicles were investigated. Immunocytochemistry (IHC) was also conducted to monitor the meiotic progression of the oocytes. Additionally, the mRNA expression levels of the two folliculogenesis-related genes (Gdf9 and Bmp15) and two apoptosis-related genes (Bcl2 and Bax) were investigated using real-time polymerase chain reaction. Results: In the PRP, CM, and PRP+CM groups, the preantral follicle maturation (evaluated by identifying polar bodies) were greater than the control group. The cleavage rate in the CM, and PRP+CM groups were also greater than the control group. IHC analysis demonstrated that in each treatment group, meiotic spindle was normal. In the PRP+CM group, the gene expression levels of Bmp15, Gdf9, and Bcl2 were greater than in the other groups. The Bax gene was more strongly expressed in the PRP and control groups than in the other groups. Conclusion: Overall, the present study suggests that the combination of CM and PRP can effectively increase the growth and cleavage rate of mouse preantral follicles in vitro.

• Reproductive and Developmental Biology
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• 제30권2호
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• pp.143-156
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• 2006

Major characteristics of embryonic stem cells (ESCs) are sustaining of sternness and pluripotency by self-renewal. In this report, transcriptional profiles of the molecules in the developmentally important signaling pathways including Wnt, BMP4, $TGF-\beta$, RTK, Hh, Notch, and JAK/STAT signaling pathways were investigated to understand the self-renewal of mouse ESCs (mESCs), J1 line, and compared with the NIH3T3 cell line and mouse embryonic fibroblast (MEF) cells as controls. In the Wnt signaling pathway, the expression of Wnt3 was seen widely in mESCs, suggesting that the ligand may be an important regulator for self-renewal in mESCs. In the Hh signaling pathway, the expression of Gli and N-myc were observed extensively in mESCs, whereas the expression levels of in a Shh was low, suggesting that intracellular molecules may be essential for the self-renewal of mESCs. IGF-I, IGF-II, IGF-IR and IGF-IIR of RTK signaling showed a lower expression in mESCs, these molecules related to embryo development may be restrained in mESCs. The expression levels of the Delta and HESS in Notch signaling were enriched in mESCs. The expression of the molecules related to BMP and JAK-STAT signaling pathways were similar or at a slightly lower level in mESCs compared to those in MEF and NIH3T3 cells. It is suggested that the observed differences in gene expression profiles among the signaling pathways may contribute to the self-renewal and differentiation of mESCs in a signaling-specific manner.

• Asian-Australasian Journal of Animal Sciences
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• 제27권12호
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• pp.1695-1704
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• 2014

Maternal malnutrition during pregnancy may give rise to female offspring with disrupted ovary functions in adult age. Neonatal ovary development predisposes adult ovary function, yet the effect of maternal nutrition on the neonatal ovary has not been described. Therefore, here we show the impact of maternal protein restriction on the expression of folliculogenic and steroidogenic genes, their regulatory microRNAs and promoter DNA methylation in the ovary of neonatal piglets. Sows were fed either standard-protein (SP, 15% crude protein) or low-protein (LP, 7.5% crude protein) diets throughout gestation. Female piglets born to LP sows showed significantly decreased ovary weight relative to body weight (p<0.05) at birth, which was accompanied with an increased serum estradiol level (p<0.05). The LP piglets demonstrated higher ratio of bcl-2 associated X protein/B cell lymphoma/leukemia-2 mRNA (p<0.01), which was associated with up-regulated mRNA expression of bone morphogenic protein 4 (BMP4) (p<0.05) and proliferating cell nuclear antigen (PCNA) (p<0.05). The steroidogenic gene, cytochrome P450 aromatase (CYP19A1) was significantly down-regulated (p<0.05) in LP piglets. The alterations in ovarian gene expression were associated with a significant down-regulation of follicle-stimulating hormone receptor mRNA expression (p<0.05) in LP piglets. Moreover, three microRNAs, including miR-423-5p targeting both CYP19A1 and PCNA, miR-378 targeting CYP19A1 and miR-210 targeting BMP4, were significantly down-regulated (p<0.05) in the ovary of LP piglets. These results suggest that microRNAs are involved in mediating the effect of maternal protein restriction on ovarian function through regulating the expression of folliculogenic and steroidogenic genes in newborn piglets.

• 한국발생생물학회지:발생과생식
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• 제11권2호
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• pp.85-96
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• 2007

중간엽 줄기세포(MSC)를 체외배양할 때 사용하는 소태아혈청 (FBS)의 생물학적 불안전성은 이를 임상적으로 사용하는데 있어 제한점으로 작용한다. 본 연구에서는 소태아혈청 및 인간의 제대혈청을 사용하여 인간의 양막유래 중간엽 줄기세포 (HAM)를 각각 배양한 후, 세포의 성장속도와 유전자 및 단백질의 발현 양상을 비교하였다. 제왕절개 후 얻은 양막으로부터 HAM을 분리하여 10% FBS, 5% HCS 혹은 10% HCS가 각각 첨가된 DMEM 배양액에서 배양하였으며, 초기와 후기 계대의 세포를 얻어 이들의 생물학적 특성을 비교 분석하였다. 역전자 중합효소반응 결과, 혈청의 종류에 상관없이 배양된 세포들은 모두 OCT-4, Rex-1, SCF, FGF-5, BMP-4, nestin, NCAM, GATA-4, HLA-ABC 유전자를 발현하였으며, 이러한 발현은 초기 및 후기 계대의 세포에서도 마찬가지로 나타났다. 세포면역화학 반응 결과, FBS 혹은 HCS를 첨가한 배양액에서 배양된 HAM은 4번째 계대에서 collagen I, II, III, XII, fibronectin, $\alpha$-smooth muscle actin, vimentin, CK18, CD54, FSP, TRA-1-60, SSEA-3, -4, HLA ABC 단백질을 뚜렷하게 발현하였다. 그러나 desmin 단백질은 FBS가 첨가된 배양액에서 배양된 HAM에서만 발현되었고 vWF 단백질은 HCS가 첨가된 배양액에서 배양된 HAM에서만 발현되었다. 결론적으로 유전자와 단백질의 발현양상을 살펴본 결과, HCS가 첨가된 배양액에서 배양된 HAM은 전형적인 인간성체줄기세포의 특징을 나타내고 있으며, FBS가 첨가된 배양액과 비교하여 동등한 성장 촉진 효과를 가지는 것으로 보인다.

• 생명과학회지
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• 제29권3호
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• pp.303-310
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• 2019

비만은 체내 과도한 지방 축적으로 인하여 지방세포 자체에서 염증성 사이토카인이 증가로 인하여 세포의 기능을 약화시킨다. 규칙적인 운동은 지방분해와 갈색지방 증가로 인해 비만의 치료방법 중 핵심 전략으로 적용되고 있다. 고강도 운동은 염증성 사이토카인과 근형질세망 스트레스 발생을 초래하여 지방대사에 부정적인 결과를 초래하는 것으로 알려져 있다. 그러나 비만모델에서 중강도 운동과 고강도 운동에 의한 인플라마좀, 대식세포 침윤, 갈색지방 관련 바이오 마커의 비교연구는 이루어진 바 없다. 따라서 이 연구의 목적은 중강도 유산소 운동과 고강도 운동을 비교하여 인플라마좀(NLRP3, ASC), 대식세포 침윤인자 M1 (CD11c, CD86), M2 (CD206), 갈색지방($PGC1{\alpha}$, BMP7, PRDM, UCP1) 관련 변인에 우선적인 효과가 있는지 비교분석하고자 하였다. 이 연구의 목적을 위해 1) 정상식이 그룹(normal diet control, NC; n=10), 2) 60% 고지방식이 그룹(high-fat diet control, HC; n=10), 3) 중강도 운동 그룹(high fat diet with moderate intensity exercise, HME; n=10), 4) 고강도 운동그룹(high fat diet with high intensity exercise, HIE; n=10)으로 나누어 실시하였다. 중강도 운동 그룹은 고지방식이 그룹과 비교하여 NLRP3, F480, CD11c, CD8의 발현이 유의하게 낮아졌다. 중강도 운동은 CD206, $PGC1{\alpha}$, BMP7, PRDM이 유의하게 증가하였다. 고강도 운동은 NLRP3, CD11c and CD86은 유의하게 감소한것으로 확인되었다. 그러나 고강도 운동은 $PGC1{\alpha}$, BMP7는 증가한다. 이러한 결과는 중강도 운동은 인플라마좀, 대식세포 M1, M2 침윤과 갈색지방 세포 관련 요인의 개선이 효과적인 것으로 나타났다.

• Molecules and Cells
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• 제37권5호
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• pp.406-411
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• 2014

The initial step of atrioventricular (AV) valve development involves the deposition of extracellular matrix (ECM) components of the endocardial cushion and the endocardialmesenchymal transition. While the appropriately regulated expression of the major ECM components, Versican and Hyaluronan, that form the endocardial cushion is important for heart valve development, the underlying mechanism that regulates ECM gene expression remains unclear. We found that zebrafish crip2 expression is restricted to a subset of cells in the AV canal (AVC) endocardium at 55 hours post-fertilization (hpf). Knockdown of crip2 induced a heart-looping defect in zebrafish embryos, although the development of cardiac chambers appeared to be normal. In the AVC of Crip2-deficient embryos, the expression of both versican a and hyaluronan synthase 2 (has2) was highly upregulated, but the expression of bone morphogenetic protein 4 (bmp4) and T-box 2b (tbx2b) in the myocardium and of notch1b in the endocardium in the AVC did not change. Taken together, these results indicate that crip2 plays an important role in AV valve development by downregulating the expression of ECM components in the endocardial cushion.

• 대한치과교정학회지
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• 제34권2호
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• pp.143-152
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• 2004

하악과두 연골이 발생하고 분화하는 과정에서 나타내는 특성을 규명하기 위하여, 발생 16, 18일과 출생 1일, 5일, 10일, 20일 및 30일 후의 ICR생쥐의 하악과두를 형태학적으로 분석하고, 생쥐 I형, II형, X형 교원질, Indian hedgehog (IHH) 및 BMP-4 등의 mRNA 발현을 in-situ hybridization 방법으로 연구하였다. 1. 생쥐 I형 및 II형 교원질 mRNA는 하악과두의 발생 및 성장과정에서 모두 발현되었다. I형 교원질 mRNA는 휴지층과 증식층의 상부에서 관찰된 반면 II형 교원질은 휴지층과 증식층 그리고 비대연골층의 상부에서 관찰되었다. 2. 하악과두 연골은 성장에 따라 비대연골층이 계속 증가하는 소견을 보였으며, 비대 연골층의 세포들은 특징적으로 X형 교원질 mRNA의 발현을 보였다. 3. BMP-4 mRNA는 하악과두 연골 원기와 골화중인 하악골체에서 모두 발현되었다. 4. IHH mRNA는 하악과두의 발생과정에서 증식 연골층의 하부와 비대연골층의 상부에서 선택적으로 관찰되었다.

• Nutrition Research and Practice
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• 제11권3호
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• pp.190-197
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• 2017

BACKGROUND/OBJECTIVES: Gallus gallus domesticus (GD) is a natural mutant breed of chicken in Korea with an atypical characterization of melanin in its tissue. This study investigated the effects of melanin extracts of GD on osteoblast differentiation and inhibition of osteoclast formation. MATERIALS/METHODS: The effects of the melanin extract of GD on human osteoblast MG-63 cell differentiation were examined by evaluating cell viability, osteoblast differentiation, and expression of osteoblast-specific transcription factors such as bone morphogenetic protein 2 (BMP-2), small mothers against decapentaplegic homologs 5 (SMAD5), runt-related transcription factor 2 (RUNX2), osteocalcin and type 1 collagen (COL-1) by reverse transcription-polymerase chain reaction and western blotting analysis. We investigated the inhibitory effect of melanin on the osteoclasts formation through tartrate-resistant acid phosphatase (TRAP) activity and TRAP stains in Raw 264.7 cell. RESULTS: The melanin extract of GD was not cytotoxic to MG-63 cells at concentrations of $50-250{\mu}g/mL$. Alkaline phosphatase (ALP) activity and bone mineralization of melanin extract-treated cells increased in a dose-dependent manner from 50 to $250{\mu}g/mL$ and were 149% and 129% at $250{\mu}g/mL$ concentration, respectively (P < 0.05). The levels of BMP-2, osteocalcin, and COL-1 gene expression were significantly upregulated by 1.72-, 4.44-, and 2.12-fold in melanin-treated cells than in the control cells (P < 0.05). The levels of RUNX2 and SMAD5 proteins were higher in melanin-treated cells than in control vehicle-treated cells. The melanin extract attenuated the formation of receptor activator of nuclear factor kappa-B ligand-induced TRAP-positive multinucleated RAW 264.7 cells by 22%, and was 77% cytotoxic to RAW 264.7 macrophages at a concentration of $500{\mu}g/mL$. CONCLUSIONS: This study provides evidence that the melanin extract promoted osteoblast differentiation by activating BMP/SMADs/RUNX2 signaling and regulating transcription of osteogenic genes such as ALP, type I collagen, and osteocalcin. These results suggest that the effective osteoblastic differentiation induced by melanin extract from GD makes it potentially useful in maintaining bone health.

• Journal of Animal Science and Technology
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• 제63권2호
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• pp.426-439
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• 2021

It is already well known that castration improves marbling quality but exact timing of castration is still highly debated in beef cattle production industry. After castration, blood hormonal changes occur in steer and objective of this study was to investigate the effects of growth hormone (GH) levels on adipocyte differentiation in stromal vascular cells (SVCs) and transdifferentiation into adipocytes in C2C12 myoblasts. Total GH concentrations were measured via enzyme-linked immunosorbent assay (ELISA) in 24 male calves and 4 female calves. Cell proliferation, cellular triglyceride (TG) accumulation, and the cell's lipolytic capability were measured in C2C12 myoblasts and SVCs. Myogenic, adipogenic, and brown adipocyte-specific gene expression was measured via real-time polymerase chain reaction (PCR) using SYBR green. Serum GH levels were the highest in late-castrated calves. Treatment with 5 ng/mL GH resulted in greater TG accumulation as well as increased CCAAT-enhancer-binding protein (C/EBP)α and peroxisome proliferator-activated receptor (PPAR)γ expression compared to that after treatment with 15 ng/mL GH. Treatment with 5 ng/mL GH also resulted in lower myogenin (myo)G and myoD expression compared to that after treatment with 15 ng/mL GH. The expression of bone morphogenetic protein (BMP) 7 after treatment with 5 ng/mL GH was higher than that after treatment with 15 ng/mL GH. But carcass characteristics data showed no significant difference between early and late castrated steers. Therefore, our results indicate that castration timing does not seem to be inevitable determinate of carcass qualities, particularly carcass weight and marbling score in Hanwoo beef cattle.