• Korean Journal of Animal Reproduction
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• v.24 no.4
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• pp.365-373
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• 2000

The purpose of this study was to investigate the effects of the fusion pulses and fusion media on fusion rate and the development of embryos produced by somatic cell nuclear transfer in Hanwoo (Korean cattle). Nuclear donor cumulus and fetal fibroblast cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum at 38.5$^{\circ}C$ in a humidified atmosphere of 5% $CO_2$in air. The in vitro matured oocytes were enucleated and then the isolated donor cells were introduced. The cumulus cell and cytoplast were fused using one pulse of 70 volts for 40$mutextrm{s}$, two pulses of 70 volts for 40$mutextrm{s}$ and one pulse of 180 volts for 15$mutextrm{s}$. The fetal fibroblast cell and cytoplast were fused using one pulse of 180 volts for 15$mutextrm{s}$ or 30$mutextrm{s}$. The cumulus cell and cytoplast were fused using mannitol and Zimmerman cell fusion medium (ZCFM) as a fusion medium. The fused embryos were activated after the fusion with 10 $\mu$M calcium ionophore for 5 min and 2 mM 6-dimethyl- aminopurine for 3 h. The nuclear transfer embryos were cultured in 500 ${mu}ell$ well of modified CR1aa supplemented with 3 mg/$m\ell$ BSA in th $\varepsilon$ four well dish cove red with mineral oil. After 3 days culture, culture medium was changed into modified CRlaa medium containing 1.5 mg/$m\ell$ BSA and 5% FBS for 4 days. The incubation environment was 5% $CO_2$, 5% $O_2$, 90% $N_2$ at 38.5$^{\circ}C$. When the cumulus cells were fused with enucleated oocytes by three different fusion pulses, one pulse of 180 volts for 15 $mutextrm{s}$ yielded the highest fusion rate and developmental rate to blastocyst among the pulses (P＜0.05). When the fetal fibroblast cells were fused with enucleated oocytes, one pulse of 180 volts for 30$mutextrm{s}$ yielded significantly higher fusion rate compared with that for 15 $mutextrm{s}$(P＜0.05). The present result indicates that the fusion rate between karyoplast and cytoplast was affected by the cell type and the optimal fusion condition was different according to cell type or size. When the fusion was conducted by the use of mannitol and ZCFM, the fusion rate was 71.2% and 65.8%, respectively. The developmental rates to blastocyst were 37.8% and 39.8%, respectively. There was no significant difference between two fusion media in the developmental rate of cumulus cell nuclear transfer embryos. These results indicate that optimal electric current should be selected according to cell type.

• Asian-Australasian Journal of Animal Sciences
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• v.27 no.12
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• pp.1671-1677
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• 2014

Ras homolog enriched in brain (Rheb) and FK506 binding protein 38 (FKBP38) are two important regulatory proteins in the mammalian target of rapamycin (mTOR) pathway. There are contradictory data on the interaction between Rheb and FKBP38 in human cells, but this association has not been examined in cashmere goat cells. To investigate the interaction between Rheb and FKBP38, we overexpressed goat Rheb and FKBP38 in goat fetal fibroblasts, extracted whole proteins, and performed coimmunoprecipitation to detect them by western blot. We found Rheb binds directly to FKBP38. Then, we constructed bait vectors (pGBKT7-Rheb/FKBP38) and prey vectors (pGADT7-Rheb/FKBP38), and examined their interaction by yeast two-hybrid assay. Their direct interaction was observed, regardless of which plasmid served as the prey or bait vector. These results indicate that the 2 proteins interact directly in vivo. Novel evidence is presented on the mTOR signal pathway in Cashmere goat cells.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.140-140
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• 2003

The study evaluated the effect of donor cell treatments for G0/Gl synchronization and the donor ceil type on development and incidence of apoptosis in cloned cattle embryos. Primary cultures were established from a female fetus on day 50 of gestation and adult ear skin biopsies. Cells were randomly allocated into 3 experimental treatment groups after 6~8 passages. Group 1 (Confluent), cells were cultured in DMEM supplemented with 10% FBS until 90% confluent. Group 2 (Serum-starvation), cells were cultured in DMEM Supplemented With 0.5% FBS for 5 days. Group 3 (Roscovitine), Cells were cultured in DMEM supplemented with 10% FBS and 30 $\mu$M Roscovitine for 12 h. Cell cycle and apoptosis were analyzed using flow cytometry after labelling with DAPI and YO-PRO-1. At 19 h post-maturation (hpm), enucleated oocytes were reconstructed with donor cells and fused by a single DC pulse (1.6 kV/cm, 60 $\mu$sec). (중략)

• The Journal of Korean Academy of Prosthodontics
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• v.44 no.1
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• pp.112-123
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• 2006

Purpose: The biocompatibility and bio-adhesive property of a dental implant abutment are important for proper soft tissue healing and maintenance of osseointegration of implant. However, studies of soft tissue healing and mucosal attachment of various materials of implant abutment other than titanium are still needed. In this study, cell attachment, proliferation, cytotoxicity of human gingival fibroblast for ceramic, gold alloy, Ni-Cr alloy and, commercially available pure titanium as a control were evaluated, using MTS and scanning electron microscopy. Materials and Methods: Specimen was designed to disc, 4mm diameter and 1mm thickness, made of ceramic, gold alloy, Ni-Cr alloy and commercially available pure titanium. Primary culture of human gingival fibroblasts were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 1% antibiotics. Cells were inoculated in the multiwell plates placed the specimen disc. Cell Titer 96 AQucous One Solution Cell Proliferation Assay were done after 1hour 3hours, 24hours, 3days, 5days of incubation. The discs were processed for scanning electron micrography to evaluate cell attachment and morphologic change. Results: The results were obtained as fellows. 1. The ceramic showed high cell attachment and proliferation and low cytotoxicity, which is as much bioadhesive and biocompatible as titanium. 2. The gold alloy represented limited proliferation of human gingival fibroblast and the highest cytotoxicity among tested materials (p<0.05). 3. The Ni-Cr alloy limited the proliferaion of the human gingival fibroblast compared to titanium(p<0.05) but cytotoxicity on the bottom of well was not so considerable, compared to titanium. 4. On the scanning electron micrographs , the ceramic showed good attachment and proliferation of human gingival fibroblast, which was similar to titanium. But gold alloy and Ni-Cr alloy showed the shrinkage of gingival fibroblast both after 24 hours and 3 days. On 5th day, small amount of the human gingival fibroblast proliferation was observed on the Ni-Cr alloy, while the shrinkage of gingival fibroblast was still observed on the gold alloy. Conclusions: These results suggest that the ceramic abutment is as biocompatible as titanium to make proper mucosal seal. The gold alloy has a high cytotoxicity to limit proliferation of gingival fibroblast, which suggest limited use on the anterior tooth where soft tissue healing is recommeded.

• Journal of Periodontal and Implant Science
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• v.33 no.2
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• pp.277-288
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• 2003

It has been suggested that increased number and activity of phagocytes in periodontitis lesion results in a high degree of reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, nitric oxide and peroxynitrite. There are few reports on the relationship between ROS and MMPs expressions in gingival fibroblast. We studied to elucidate whether and how ROS, especially nitric oxide affects the MMP expression. Human gingival fibroblasts and HTl080 cells (human fibrosarcoma sell line as reference) were grown in DMEM supplemented with 10 mM HEPES, 50 mg/L gentamicin, and 10% heat inactivated fetal bovine serum with addition of various reactive oxygen species (ROS). Culture media conditioned by cells were examined by gelatin zymography. HT1080 cells expressed proMMP-2 and proMMP-9, but human gingival fibroblasts (HGF) produced only proMMP-2. Hydrogen peroxide upregulated MMP-9 expression in HT1080 cells, whereas in human gingival fibroblast SNP treatment showed marked increase in MMP-2 level compared to other ROS. These results suggest that the effects of ROS on MMPs expressions are cell-type specific. RT-PCR for MMP-2 and TIMP-2 m-RNA were performed using total RNA from cultured cells under the influence various kinase inhibitors. In HT1080 cells, treatment with FPTI III (Ras processing inhibitor) and LY294002 (PI3-kinase inhibitor) resulted in inhibition of MMP-2 and MMP-9 expressions, suggesting that Ras/P13-kinase pathway is important for MMPs expression in HT1080 cells. In gingival fibroblasts, treatment with FPTI III and PDTC (NF-kB inhibitor) showed marked decrease in MMP-2 regardless of the of SNP , suggesting that Ras/NF-kB could be the key pathway for NO-induced MMP-2 expression in gingival fibroblasts. This study showed that ROS, especially nitric oxide, could be the critical mediator of periodontal disease progression through control of MMP-2 expression in gingival fibroblasts possibly via Ras/NF-kB pathway.

• The korean journal of orthodontics
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• v.24 no.4 s.47
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• pp.933-943
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• 1994

The present study was undertaken to determine the effect of tensile force on DNA and protein biosynthesis in bone cells, and to identify the cell type(s) which primarily respond to external physical force among the heterogenous bone cell populations. As a prerequisite for this study, two bone cell populations which retain fibroblastic and osteoblastic feature were isolated from fetal rat calvaria with sequential enzyme digestion scheme. Tensile force was delivered to each bone cell population by two acrylic resin plates connected with a orthodontic expansion screw during culture period. Rate of DNA and protein synthesis in each bone cell population were assessed by the incorporated radioactivity of $[^3H]-thymidine$ into DNA and $[^3H]-proline$ into fraction of collagenase-digestible protein and noncollagenous protein, respectively. DNA synthesis of osteoblast-like calvarial cell populations was increased significantly by the application of tensile force for 24 hours. In contrast, no alteration in DNA synthesis of fibroblast-like populations could be observed in response to applied force. Tensile force induced the change in protein synthesis of bone cell populations with the same pattern. Total protein and collagen synthesis were increased whithin 24 hours in osteoblast-like populations, but not in fibroblast-like populations by tensile force application. These findings indicate that physical force can affect cellullar activity of the particular cell population, not all cell Populations residing in bone and osteoblasts respond more sensitively than fibroblasts. So osteoblasts can modulate the behavior of other bone cells including osteoclasts by producing several local regulating factors of bone metabolism. In this context, preferential responsiveness of osteoblasts to applied tensile force observed in this study suggests that osteoblasts may play an important role in regulation of physical force-induced remodelling process.

• Korean Journal of Agricultural Science
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• v.33 no.1
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• pp.35-41
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• 2006

This study was conducted to investigate the developmental ability of interspecies cloned embryos after nuclear transfer of goat fetal fibroblast cells into porcien oocytes. Recipient porcine and goat oocytes were obtained from slaughterhouse and matured in vitro according to established protocols. Enucleation was accomplished by aspirating the first polar body and cytoplasm and a single donor cell was individually microinjected into vitelline space of the enucleated oocyte. The reconstructed oocytes were electrically fused with 0.3M mannitol fusion medium. After electro-fusion, interspecies reconstituted embryos were cultured in PZM-3 for 7 days. In porcine interspecies nuclear transfer with goat fetal fibroblast cells, the cleavage rate of reconstituted embryos were 58.9% which was no significant different from that in porcine nuclear transfer embryos (67.4%). However, the developmental rate into blastocyst stage was 5.4% in interspecies nuclear transfer which was significantly lower than that in porcine intraspecies nuclear transfer (13.6%). When the developmental ability of porcine interspecies nuclear transfer with goat cells was compared with goat intraspecies nuclear transfer, the cleavage rate of embryos were 59.2% and the developmental rate into morular and blastocyst stage was 13.6% in interspecies nuclear transfer which were significantly lower than those in intraspecies nuclear transfer embryos. This result indicated that porcine interspecies nuclear transfer with goat fetal fibroblast cells showed the developmental potential in vitro with lower cleavage and developmental rate compared with intraspecies nuclear transfer.

• The Korean Journal of Physiology and Pharmacology
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• v.19 no.1
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• pp.35-42
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• 2015

In cardiovascular disorders, understanding of endothelial cell (EC) function is essential to elucidate the disease mechanism. Although the mouse model has many advantages for in vivo and in vitro research, efficient procedures for the isolation and propagation of primary mouse EC have been problematic. We describe a high yield process for isolation and in vitro culture of primary EC from mouse arteries (aorta, braches of superior mesenteric artery, and cerebral arteries from the circle of Willis). Mouse arteries were carefully dissected without damage under a light microscope, and small pieces of the vessels were transferred on/in a Matrigel matrix enriched with endothelial growth supplement. Primary cells that proliferated in Matrigel were propagated in advanced DMEM with fetal calf serum or platelet-derived serum, EC growth supplement, and heparin. To improve the purity of the cell culture, we applied shearing stress and anti-fibroblast antibody. EC were characterized by a monolayer cobble stone appearance, positive staining with acetylated low density lipoprotein labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate, RT-PCR using primers for von-Willebrand factor, and determination of the protein level endothelial nitric oxide synthase. Our simple, efficient method would facilitate in vitro functional investigations of EC from mouse vessels.

• Journal of Embryo Transfer
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• v.21 no.1
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• pp.13-20
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• 2006

The purpose of this study was to develope the transgenic cattle expressing hFSH into the urine using the nuclear transfer. To produce the interest gene in urine, the specific vector was ligated with hFSH gene undo. maUII promoter. The fetal fibroblast cells (KbFF) were isolated from a 45-day male fetus. The hFSH gene was co-transfected with pcDNA3 (neo) vector to KbFF cells by electroporation. The gene-transfected cells were cultured with G-418 selection medium for 2 weeks. Selected colonies were confirmed by PCR. For nuclear transfer, enucleated bovine oocytes were transferred with hFSH transfected or nontransfected fetal fibroblasts. The cleavage and blastocyst formation rates were significantly lower (p<0.05) in cloned embryos transfected with hFSH gene (68.7% and 15.7%) than in those non-transfected (67.6% and 24.5 %), respectively. Apoptosis analysis showed no difference between hFSH transfected and non-transfected blastocysts (p>0.05). The blastocysts were transfected to 77 (control 24, hFSH 53) recipient cows. Two calves were born (1.9%) following transfer with NT embryos transfected with hFSH gene, but they were confirmed not to be transgenic calves. This result shows that the hFSH colonies were mixed with transfected and non transfected cells. Further research will be needed for selection and establishment of gene transfected cells.

• Anatomy and Cell Biology
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• v.56 no.4
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• pp.508-517
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• 2023

In cancer patients, chemo/radio therapy may cause infertility by damaging the spermatogenesis affecting the self-renewal and differentiation of spermatogonial stem cells (SSCs). In vitro differentiation of stem cells especially mesenchymal stem cells (MSCs) into germ cells has recently been proposed as a new strategy for infertility treatment. The aim of this study was to evaluate the proliferation and differentiation of SSCs using their co-culture with Sertoli cells and conditioned medium (CM) from adipose tissue-derived MSCs (AD-MSCs). Testicular tissues were separated from 2-7 days old neonate Wistar Rats and after mechanical and enzymatic digestion, the SSCs and Sertoli cells were isolated and cultured in Dulbecco's modified eagle medium with 10% fetal bovine serum, 1X antibiotic, basic fibroblast growth factor, and glial cell line-derived neurotrophic factor. The cells were treated with the CM from AD-MSCs for 12 days and then the expression level of differentiation-related genes were measured. Also, the expression level of two major spermatogenic markers of DAZL and DDX4 was calculated. Scp3, Dazl, and Prm1 were significantly increased after treatment compared to the control group, whereas no significant difference was observed in Stra8 expression. The immunocytochemistry images showed that DAZL and DDX4 were positive in experimental group comparing with control. Also, western blotting revealed that both DAZL and DDX4 had higher expression in the treated group than the control group, however, no significant difference was observed. In this study, we concluded that the CM obtained from AD-MSCs can be considered as a suitable biological material to induce the differentiation in SSCs.