• 생체재료학회지
• /
• 제22권4호
• /
• pp.271-278
• /
• 2018

Background: The chondrogenic differentiation of mesenchymal stem cells (MSCs) is regulated by many factors, including oxygen tensions, growth factors, and cytokines. Evidences have suggested that low oxygen tension seems to be an important regulatory factor in the proliferation and chondrogenic differentiation in various MSCs. Recent studies report that synovium-derived mesenchymal stem cells (SDSCs) are a potential source of stem cells for the repair of articular cartilage defects. But, the effect of low oxygen tension on the proliferation and chondrogenic differentiation in SDSCs has not characterized. In this study, we investigated the effects of hypoxia on proliferation and chondrogenesis in SDSCs. Method: SDSCs were isolated from patients with osteoarthritis at total knee replacement. To determine the effect of oxygen tension on proliferation and colony-forming characteristics of SDSCs, A colony-forming unit (CFU) assay and cell counting-based proliferation assay were performed under normoxic (21% oxygen) or hypoxic (5% oxygen). For in vitro chondrogenic differentiation, SDSCs were concentrated to form pellets and subjected to conditions appropriate for chondrogenic differentiation under normoxia and hypoxia, followed by the analysis for the expression of genes and proteins of chondrogenesis. qRT-PCR, histological assay, and glycosoaminoglycan assays were determined to assess chondrogenesis. Results: Low oxygen condition significantly increased proliferation and colony-forming characteristics of SDSCs compared to that of SDSCs under normoxic culture. Similar pellet size and weight were found for chondrogensis period under hypoxia and normoxia condition. The mRNA expression of types II collagen, aggrecan, and the transcription factor SOX9 was increased under hypoxia condition. Histological sections stained with Safranin-O demonstrated that hypoxic conditions had increased proteoglycan synthesis. Immunohistochemistry for types II collagen demonstrated that hypoxic culture of SDSCs increased type II collagen expression. In addition, GAG deposition was significantly higher in hypoxia compared with normoxia at 21 days of differentiation. Conclusion: These findings show that hypoxia condition has an important role in regulating the synthesis ECM matrix by SDSCs as they undergo chondrogenesis. This has important implications for cartilage tissue engineering applications of SDSCs.

• 한국동물생명공학회지
• /
• 제36권4호
• /
• pp.299-306
• /
• 2021

Vitrification methods are commonly used for mammalian reproduction through the long-term storage of blastocyst produced in vitro. However, the survival and quality of embryos following vitrification are significantly low compared with blastocyst from in vitro production (IVP). This study evaluates that the survival of frozen-thawed bovine embryos was relevant to mitochondrial superoxide derived mitochondrial activity. Here we present supplementation of the cryopreservation medium with Mito-TEMPO (0.1 µM) induced a significant (p < 0.001; non-treated group: 56.8 ± 8.7%, reexpanded at 24 h vs Mito-TEMPO treated group: 77.5 ± 8.9%, re-expanded at 24 h) improvement in survival rate of cryopreserved-thawed bovine blastocyst. To confirm the quality of vitrified blastocyst after thawing, DNA fragmentation of survived embryos was examined by TUNEL assay. As a result, TUNEL positive cells rates of frozen-thawed embryos were lower in the Mito-TEMPO treated group (4.2 ± 1.4%) than the non-treated group (7.1 ± 3.5%). In addition, we investigated the intracellular ROS and mitochondrial specific superoxide production using DCF-DA and Mito-SOX staining in survived bovine embryos following vitrification depending on Mito-TEMPO treatment. As expected, intracellular ROS levels and superoxide production of vitrified blastocysts after cryopreservation were significantly reduced (p < 0.05) according to Mito-TEMPO supplement in freezing medium. Also, mitochondrial activity measured by MitoTracker Orange staining increased in the frozen-thawed embryos with Mito-TEMPO compared with non-treated group. These results indicate that the treatment of Mito-TEMPO during cryopreservation might induce reduction in DNA fragmentation and apoptosis-related ROS production, consequently increasing mitochondrial activation for developmental capacity of frozen-thawed embryos.

• Journal of Veterinary Science
• /
• 제25권2호
• /
• pp.31.1-31.8
• /
• 2024

Background: Recently, there has been a growing interest in stem cells for human medicine. Limited feline endometrial mesenchymal stem cell (fEM-MSC) research in veterinary medicine necessitates reporting for future feline disease research and therapy. Objectives: This study aimed to isolate fEM-MSCs from feline endometrial tissues and evaluate their morphology, proliferative ability, differentiation ability, and immunophenotype. Methods: Feline endometrial tissues were obtained from the ovariohysterectomies of healthy cats and isolated using an enzymatic method. The morphology and proliferative ability of the isolated cells were assessed using a doubling time (DT) assay from passages 3 to 6 (P3 - P6). We measured pluripotency gene expressions of cells in P2 using quantitative real-time polymerase chain reaction (qRT-PCR). To investigate MSC characteristics, a trilineage differentiation assay was conducted in P4, and cells in P4 were immunophenotyped using flow cytometry. Results: fEM-MSCs showed a typical spindle-shaped morphology under a microscope, and the DT was maintained from P3 to P6. fEM-MSCs could differentiate into adipocytes, osteoblasts, and chondrocytes, and expressed three pluripotency markers (OCT4, SOX2, and NANOG) by qRT-PCR. Immunophenotypic analysis showed that the fEM-MSCs were CD14 ^-, CD34 ^-, CD45 ^-, CD9⁺, and CD44⁺. Conclusions: In this study, the feline endometrium was a novel source of MSCs, and to the best of our knowledge, this is the first report on the isolation method and characteristics of fEM-MSCs.

• Korean Chemical Engineering Research
• /
• 제40권6호
• /
• pp.746-751
• /
• 2002

석탄 연소기술에서 타 연소로에 비해서 유동층 연소는 황산화물과 질소산화물 배출을 줄이는 기술이다. 석회석의 소성으로 생성되는 CaO에 의한 황산화물의 제거와 저온 연소와 공기 다단계 주입에 의한 NOx를 줄일 수 있다는 것이 유동층 연소로의 큰 장점이지만, 상대적으로 $N_2O$의 배출은 매우 높다. $N_2O$는 지구온난화 가스일 뿐만 아니라 성층권내의 오존층을 파괴하는 물질이기도 하다. CaO는 $N_2O$ 분해를 위한 촉매 물질로 알려져 있다. 본 연구는 CaO를 충진시킨 고정층 반응기에서 CaO에 의한 $N_2O$의 분해특성에 관하여 수행하였으며, 유동층 연소온도와 가스조성에서 온도변화에 대한 $N_2O$의 분해특성, CaO 충진량의 변화와 $CO_2$, NO, $O_2$ 농도변화에 따른 $N_2O$ 분해특성에 관하여 수행하였다. 또한 실험 결과로부터 CaO표면에서 $N_2O$분해반응에 대한 반응속도식을 나타낼 수 있었다. 결과로서 온도가 증가함에 따라 $N_2O$ 분해반응이 증가하였으며, $CO_2$의 농도를 변화시킬 경우 $CO_2$ 농도가 증가할수록 $N_2O$ 분해반응이 감소하였다. NO 존재시와 비교하였을 때 $N_2O$의 분해반응이 감소함을 알 수 있었다. 반응속도론적으로 해석한 결과 $CO_2$ 농도에 대한 $N_2O$ 분해반응의 반응속도식을 다음과 같이 나타내었다. 본 연구 결과 CaO는 $N_2O$분해 반응에서 좋은 촉매 기능을 지니고 있음을 알 수 있었다. $\frac{d[N_2O]}{dt}=\frac{3.86{\times}10^9{\exp}(-15841/R)K_{N_2O}[N_2O]}{(1+K_{N_2O}[N_2O]+K_{CO_2}[CO_2])}$

• 동의생리병리학회지
• /
• 제33권2호
• /
• pp.131-140
• /
• 2019

The objective of present study is to evaluate concentration-dependent in vitro anti-osteoarthritic (OA) effects of synergic mixed formula consisted of dried pomegranate juice concentrate powder, Eucommiae Cortex aqueous extract and Achyranthis Radix aqueous extract 5:4:1 (g/g) mixture on the primary cultured rat articular chondrocytes. First, any cytotoxic effect of mixture was observed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium Bromide) assay. Next, cyto-protective effect of test substances was evaluated by using the recombinant human interleukin $(rhIL)-1{\alpha}$ induced chondrocytes. In addition, anti-inflammatory effects were also observed on the lipopolysaccaride (LPS) treated chondrocytes through prostaglandin $E_2(PGE_2)$ productions and 5-lipoxygenase (LPO) activities, and inhibitory effects on matrix metalloproteinase (MMP)-2 and MMP-9 activities were observed on $rhIL-1{\alpha}$ treated chondrocytes with their extracellular matrix (ECM) related mRNA expressions. No obvious cytotoxic effects of mixture were demonstrated. Inflammatory damages of chondrocytes and related ECM degradations induced by treatment of LPS or $rhIL-1{\alpha}$ were significantly and concentration-dependently inhibited by pretreatment of mixture from a concentration level of 0.001 mg/ml to 1 mg/ml. In addition, mixture showed $IC_{50}$ for $rhIL-1{\alpha}-induced$ MMP-2 and MMP-9 activities as 44.01 and $162.47{\mu}g/ml$, and also showed $EC_{50}$ for $rhIL-1{\alpha}-induced$ inhibition of collagen type II, SOX9 and aggrecan mRNA expression as 8.61, 10.79 and $4.47{\mu}g/ml$, respectively. It is observed that mixture showed concentration-dependent anti-inflammatory and cytoprotective ECM preserved effects on the primary cultured rat articular chondrocytes without cytotoxicity.

• Asian-Australasian Journal of Animal Sciences
• /
• 제27권2호
• /
• pp.266-277
• /
• 2014

Somatic cell nuclear transfer (SCNT) has generally demonstrated that a differentiated cell can convert into a undifferentiated or pluripotent state. In the SCNT experiment, nuclear reprogramming is induced by exposure of introduced donor nuclei to the recipient cytoplasm of matured oocytes. However, because the efficiency of SCNT still remains low, a combination of SCNT technique with the ex-ovo method may improve the normal development of SCNT embryos. Here we hypothesized that treatment of somatic cells with extracts prepared from the germinal vesicle (GV) stage Siberian sturgeon oocytes prior to their use as nuclear donor for SCNT would improve in vitro development. A reversible permeability protocol with $4{\mu}g/mL$ of digitonin for 2 min at $4^{\circ}C$ in order to deliver Siberian sturgeon oocyte extract (SOE) to porcine fetal fibroblasts (PFFs) was carried out. As results, the intensity of H3K9ac staining in PFFs following treatment of SOE for 7 h at $18^{\circ}C$ was significantly increased but the intensity of H3K9me3 staining in PFFs was significantly decreased as compared with the control (p<0.05). Additionally, the level of histone acetylation in SCNT embryos at the zygote stage was significantly increased when reconstructed using SOE-treated cells (p<0.05), similar to that of IVF embryos at the zygote stage. The number of apoptotic cells was significantly decreased and pluripotency markers (Nanog, Oct4 and Sox2) were highly expressed in the blastocyst stage of SCNT embryos reconstructed using SOE-treated cells as nuclear donor (p<0.05). And there was observed a better development to the blastocyst stage in the SOE-treated group (p<0.05). Our results suggested that pre-treatment of cells with SOE could improve epigenetic reprogramming and the quality of porcine SCNT embryos.

• 생명과학회지
• /
• 제14권6호
• /
• pp.967-974
• /
• 2004

골조직은 골아세포, 파골세포, 골세포 등으로 구성되며, 골개조시 여러 인자가 세포증식, 분화, 활성화 및 골대사 조절에 관여한다. 이때 조골세포의 활성은 골형성에 중요하므로, 본 연구에서는 MC3T3-E1 조골세포주를 이용하여 식용자원인 복분자의 조골세포의 증식과 분화활성에 미치는 영향을 조사하였다. 복분자 추출물이 조골세포의 성장에 미치는 영향을 MTT 검색법으로 조사한 결과, 복분자 추출물 $10\;{\mu}g/mL$ 처리시 대조군과 비교하여 $142\%$ 증가하여 조골세포에 대해 높은 성장률을 보였다. 복분자 추출물이 ALP 효소 활성에 미치는 영향을 3일 간격으로 배지교환 및 시료처리를 하면서 27일 동안 배양시간에 따른 변화를 측정하여 조사하였다. 그 결과, 농도 1, 10, $100\;{\mu}g/mL$ 처리시 $100\;{\mu}g/mL$을 제외한 나머지 분획물들이 시간이 지남에 따라 ALP 활성을 증가시켰고, $10\;{\mu}g/mL$ 농도의 추출물은 27일째에 대조군에 비해 약 2.6배 이상, 양성대조군에 비해 약 1.5배 이상 ALP 활성을 증가시켰다. 복분자 추출물은 다시 ALP 효소 염색법과 Alizarin Red 염색으로 조골세포의 ALP활성유도, 분화와 석회화 형성능을 재확인하였으며 골기질 유전자의 발현의 변화도 확인하였다. 따라서 종래의 골질환에 좋다고 알려진 식품인 복분자 추출물이 양성대조군에 비해 빠르게 세포 증식과 분화를 유도하고 있어 앞으로 복분자에 대한 좀 더 깊은 분자생물학 수준 등의 구체적인 연구들과 기작연구가 지속적으로 이루어져야 할 것이라 추측된다.

• 한국신재생에너지학회:학술대회논문집
• /
• 한국신재생에너지학회 2009년도 추계학술대회 논문집
• /
• pp.513-513
• /
• 2009

최근 바이오디젤의 원료로 미세조류가 많은 관심을 끌고 있다. 미세조류는 물, 이산화탄소와 태양광을 이용해 광합성 성장이 가능하며, 지질(오일) 성분이 풍부하여 바이오디젤의 원료로 이용할 수 있다. 미세조류는 단위 면적당 오일 생산량이 곡물류의 50-100배 이상이며, 이산화탄소를 기질로 이용하므로 온실가스 직접 저감이 가능하다. 또한 배양시 비경작지나 황무지를 사용할 수 있으므로 기존 식용작물과 경쟁하지 않으며, 하수, 해수, 폐수 등 다양한 물자원을 이용할 수 있다. 본 연구에서는 고농도 $CO_2$에 내성을 지닌 Chlorella sp. KR-1을 대상으로 1 L 규모 기포탑 광생물반응기에서 균체 성장 및 지질(바이오오일) 합성에 대한 $CO_2$ 농도, 가스 공급속도, 질산염 농도 등 환경 및 영향 조건의 영향을 조사하였다. 가스 공급속도 0.4 L/min에서 $CO_2$ 농도를 0.03-20% 범위에서 조사하였을 때 최대 균체성장은 $CO_2$ 10%에서 관찰되었다. 균체내 지방산 함량은 $CO_2$ 농도 0.03%에서 가장 낮았고, 5-20% 범위에서는 $CO_2$ 농도 증가에 따라 감소하는 경향이 관찰되었다. 가스 공급속도를 0.2 L/min에서 0.8 L/min으로 증가시켰을 때 최대 균체농도는 0.6-0.8 L/min의 범위에서 관찰되었고, 생체내 최대 지방산 함량은 0.4 L/min에서 관찰되었다. Nitrate 농도 1-20 mM 범위에서 최종 균체농도는 nitrate 농도 증가에 따라 10mM까지 증가하였으나 그 이상에서는 증가하지 않았다. 반면 지방산 함량은 nitrate 농도 증가에 따라 감소하는 경향이 관찰되었다. 본 실험에서 얻은 최대 지방산 생산량은 1,100 mg/L이었으며, 주요 지방산은 C10:0(1.7%), C16:0(28.5%), C18:0(11%), C18:1n9c(25.9%), C18:2n6c(26.3%), C18:3n3(6.6%)이었다. 현재 100 L 규모 광생물반응기에서 석탄발전소 배가스($CO_2$ 12-15%, SOx 50ppm, NOx 100ppm)를 이용한 바이오오일 생산 실험이 진행 중이다.

• Journal of Veterinary Science
• /
• 제23권2호
• /
• pp.40.1-40.13
• /
• 2022

Background: Somatic cell nuclear transfer (SCNT) is used widely in cloning, stem cell research, and regenerative medicine. The type of donor cells is a key factor affecting the SCNT efficiency. Objectives: This study examined whether urine-derived somatic cells could be used as donors for SCNT in pigs. Methods: The viability of cells isolated from urine was assessed using trypan blue and propidium iodide staining. The H3K9me3/H3K27me3 level of the cells was analyzed by immunofluorescence. The in vitro developmental ability of SCNT embryos was evaluated by the blastocyst rate and the expression levels of the core pluripotency factor. Blastocyst cell apoptosis was examined using a terminal deoxynucleotidyl transferase dUTP nick end-labeling assay. The in vivo developmental ability of SCNT embryos was evaluated after embryo transfer. Results: Most sow urine-derived cells were viable and could be cultured and propagated easily. On the other hand, most of the somatic cells isolated from the boar urine exhibited poor cellular activity. The in vitro development efficiency between the embryos produced by SCNT using porcine embryonic fibroblasts (PEFs) and urine-derived cells were similar. Moreover, The H3K9me3 in SCNT embryos produced from sow urine-derived cells and PEFs at the four-cell stage showed similar intensity. The levels of Oct4, Nanog, and Sox2 expression in blastocysts were similar in the two groups. Furthermore, there is a similar apoptotic level of cloned embryos produced by the two types of cells. Finally, the full-term development ability of the cloned embryos was evaluated, and the cloned fetuses from the urine-derived cells showed absorption. Conclusions: Sow urine-derived cells could be used to produce SCNT embryos.

• 해양환경안전학회지
• /
• 제29권6호
• /
• pp.697-703
• /
• 2023

최근 대기오염으로 인한 환경오염을 줄이고자 국제 산업계의 노력의 일환으로 국제해사기구(IMO)의 규제 발효등으로 이어지고 있다. IMO는 EEXI,EEDI,CII 등 선박에서 나오는 대기오염을 줄이기 위해 각종 규제를 발효시키고 선박에서 소모되는 전력을 줄여 에너지를 절약하는 방안을 추진하고 있다. 선박에서 사용되는 전력의 대부분은 전동기가 차지한다. 선박에 설치된 전동기 중 큰 부하를 차지하는 기관실 송풍기는 수요와 관계없이 정속운전으로 운전하기 때문에 주파수제어를 통한 에너지절감을 기대할 수 있다. 본 연구에서는 발전기의 과급기에 연소공기를 공급하는 발전기 송풍기의 전동기 주파수를 제어하여 에너지 절감에 대한 실효성을 입증하였다. 송풍기 주파수 입력에 따른 과급기출구 온도의 출력 데이터를 기반으로 시스템을 모델링하고, 과급기 출구온도를 목표값으로 하여 주파수를 제어하는 PI 제어계를 형성하여 과급기 설계기준 출구온도를 유지하면서 송풍기의 주파수 제어를 통해 연간 15,552kW 전력소모량을 절감하였다. 송풍기 팬 주파수 제어를 통한 에너지절감액의 유효성은 하계(4월~9월) 및 동계(3월~10월) 기간동안 검증하였으며 이를 토대로 실습선의 연간 6,091천원의 유류비 절약과 이산화탄소 8.5Ton, SOx 2.4kg, NOx 7.8kg의 대기오염물질 저감을 달성하였다.