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Junbo Wu,Kang Sun Park,Lili Zhuang,Dong Eon Kim,Eun Do Lee,Chi Sun Yun,Eun Ji Lee,Beom Sik Kim,Min Kyu Kim 한국수정란이식학회 2017 한국수정란이식학회 학술대회 Vol.2017 No.05
Somatic cell nuclear transfer (SCNT) is the technique which generates embryos by transferring diploid nucleus into an enucleated oocyte, it has produced specific animals successfully in a variety of species. However, the developmental capacity of SCNT embryos is still relatively lower than that of embryos produced in vivo. Oocyte is a kind of lipid rich cells, its quality limits the efficiency of embryo production. L-carnitine is a co-enzyme facilitating the transportation of long chain fatty acids across the inner mitochondria membrane where fatty acids are used for generating adenosine triphosphate (ATP) via beta-oxidation. It also has antioxidant actions which may protect mitochondrial membranes and DNA against damage induced by reactive oxygen species (ROS). Whether L-carnitine is functional in bovine SCNT embryos are unknown. Therefore, the objective of this study was to examine the effects of L-carnitine on oocyte maturation and developmental competence of subsequent SCNT embryos. L-carnitine was supplemented during IVM, then intracellular ROS and GSH levels, mitochondrial activity, gene expression of COCs were analyzed at the end of IVM. SCNT embryos were produced subsequently, apoptosis detection and gene expression evaluation were performed in blastocysts. In the results, treatments with 1.5 mM and 3 mM L-carnitine significantly improved maturation rates (P<0.05). Treatments with 3 mM L-carnitine effectively induced improvement in nuclear maturation, intracellular GSH levels and mitochondrial activity, as well as a reduction in intracellular ROS levels (P<0.05). mRNA levels of CPT1A, ACAA1, ACAA2, AREG, EREG, SOD1, GPX4, GLUT1 and CDC2 transcripts were effectively up-regulated by 3 mM L-carnitine treatments (P < 0.05). Similarly, 3mM L-carnitine induced an increase in blastocyst developmental rates and an improvement in blastocyst quality (P<0.05). Our study indicates that L-carnitine treatment during IVM improves oocyte nuclear maturation and subsequent SCNT embryo development.
Lili Zhuang,Bo Myeong Lee,Junbo Wu,Eun Do Lee,Chi Sun Yun,Kuk Bin Ji,Ji Hye Lee,Ju Lan Chun,Min Kyu Kim 한국수정란이식학회 2016 한국수정란이식학회 학술대회 Vol.2016 No.10
Prolonged communication between oocytes and the surrounding somatic cells is one of the unique reproductive physiology in canine. Paracrine Kit ligand (KITL) signaling is a well-known communication between granulosa cells and the oocyte. KITL is a cytokine growth factor secreted by granulosa cells that signals via the c-kit receptor expressed by oocytes. Paracrine factors, including growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), exert their effects by binding with the kinase receptors expressed on the granulosa cells. However, the regulations of GDF9 and BMP15 in the canine KITL expression are currently poorly understood. Therefore, we investigated the effects of GDF9 and BMP15 on the expression of KITL in canine ovarian granulosa cells in vitro. In Annexin V assay recombinant GDF9 and BMP15 did not induce apoptosis in the cultured ovarian granulosa cells. When treated, FSH significantly increased KITL expression, and hCG suppressed its expression. When both FSH and hCG were treated, the expression of KITL was affected by GDF9 and BMP15 in dose and time dependent manner in the luteal granulosa cells. GDF9 (10 ng/mL) significantly decreased KITL expression after12 h. BMP15 (10 ng/mL) significantly also decreased KITL expression after 24 h. Western blot and immunochemistry results indicate that GDF9 activated Smad2/3. After blocking ALK 4/5/7 receptors by SB, GDF9 failed to activate Smad2/3, also BMP15 did not activate Smad1/5/8 after blocking ALK 2/3/6 receptors by DM. So GDF9 exerts its effects via using ALK 4/5/7 receptors to activate SMAD2/3 signaling, and BMP15 binds ALK 2/3/6 receptors to activate SMAD1/5/8 signaling. The expression of KITL was not changed by SB or DM treatment. However, the effect of GDF9 and BMP15, which decreased the expression of KITL, was suppressed by SB or DM treatment. In conclusion, this study provides the first evidence that recombinant GDF9 and BMP15 decrease KITL expression in canine ovarian granulosa cells.
Establishment of Efficient System in the Microinjection of TALEN mRNA into Porcine Embryos
Don Buddika Oshadi Malaweera,Sisitha Ramachandra,Junbo Wu,Seungkyu Oh,Seunghwan Kim,Seokjoong Kim,Goo Jang,Jongki Cho 한국동물생명공학회(구 한국동물번식학회) 2013 발생공학 국제심포지엄 및 학술대회 Vol.2013 No.1
To investigate the efficient laboratory techniques for the injection of TALENs mRNA into porcine parthenogenesis and IVF embryos we had explores the injection time, volume and concentration, for the efficient blastocyst production has been identified within the experimental period. In experiment 1, to investigate injection time, compared 4 different time durations (1 hr, 2 hrs, 4 hrs & 5 hrs) after post activation of parthenogenetic embryos. There is no significant difference between four groups regarding the percentage of cleavages (55%). But comparatively higher percentage (20%) of blastocyst can be observed in the groups of 1hr and 2hrs injection after post activation. Even though observed higher blastocysts, only EGFP expressed blastocysts (2.2%) taken from the treatment of injection after 4 hrs of post activation. In experiment 2, compared two different concentration of EGFP injection in to embryos. 20 ng/μL and 50 ng/μL two concentrations were injected to the parthenogenetic embryos after 4 hrs of post activation and observed blastocysts after 7 days of incubation. There were significant difference between two treatments of concentrations considering percentages of cleavages and blastocysts. 50 ng/μL concentration injected embryos capable enough to cleaved up to 39% and produces blastocysts 10%, meanwhile 20 ng/ μL concentration illustrates 62% cleavage and 16% blastocysts percentages. Only 20 ng/μL injected embryos capable enough to express EGFP (3.1%) after 7 days of incubation. These results implies that 20 ng/μL concentration and injection 4hrs after post activation more effective injection conditions for microinjection.
Ubiquitin D Promotes Progression of Oral Squamous Cell Carcinoma via NF-Kappa B Signaling
Song, An,Wang, Yi,Jiang, Feng,Yan, Enshi,Zhou, Junbo,Ye, Jinhai,Zhang, Hongchuang,Ding, Xu,Li, Gang,Wu, Yunong,Zheng, Yang,Song, Xiaomeng Korean Society for Molecular and Cellular Biology 2021 Molecules and cells Vol.44 No.7
Ubiquitin D (UBD) is highly upregulated in many cancers, and plays a pivotal role in the pathophysiological processes of cancers. However, its roles and underlying mechanisms in oral squamous cell carcinoma (OSCC) are still unclear. In the present study, we investigated the role of UBD in patients with OSCC. Quantitative real-time polymerase chain reaction and Western blot were used to measure the expression of UBD in OSCC tissues. Immunohistochemistry assay was used to detect the differential expressions of UBD in 244 OSCC patients and 32 cases of normal oral mucosae. In addition, CCK-8, colony formation, wound healing and Transwell assays were performed to evaluate the effect of UBD on the cell proliferation, migration, and invasion in OSCC. Furthermore, a xenograft tumor model was established to verify the role of UBD on tumor formation in vivo. We found that UBD was upregulated in human OSCC tissues and cell lines and was associated with clinical and pathological features of patients. Moreover, the overexpression of UBD promoted the proliferation, migration and invasion of OSCC cells; however, the knockdown of UBD exerted the opposite effects. In this study, our results also suggested that UBD promoted OSCC progression through NF-κB signaling. Our findings indicated that UBD played a critical role in OSCC and may serve as a prognostic biomarker and potential therapeutic target for OSCC treatment.
Fracture analysis of a welded front axle tube structure from a mini-truck
Weimin Guo,Ning Ding,Na Xu,Long Liu,Nan Li,Junbo Shi,Chi-Man Lawrence Wu 대한기계학회 2020 JOURNAL OF MECHANICAL SCIENCE AND TECHNOLOGY Vol.34 No.1
The failure (fracture) mechanism of a welded front axle tube structure made of C45E4 steel from a mini truck was analyzed. The fracture occurred on the right side of the right support frame with the fracture surface perpendicular to the tube axis. SEM examination showed that the fracture surface could be divided into three areas: intergranular area, cleavage area and dimple area. Crack initiation site of the failed front axle tube was at the front weld joint fixing the right support frame on the axle tube. The crack propagated in two opposite directions along the circumference of the tube and converged at the dimple area. Intergranular fracture was found to be in heat affected zone (HAZ). With higher magnification, fine dimples, intergranular and trans-granular fracture characteristics were observed in the crack initiation site. By metallurgical examination, Widmanstätten ferrites, which could decrease the toughness and strength of the weld joint, were observed in the columnar grains. The hardness of HAZ coarse grain area (623 VHN) was far higher than HAZ fine grain area (310 VHN) and base metal (225 VHN). As the weld process indicates, neither pre-weld nor post-weld treatment was carried out. A non-uniform temperature distribution around the weld joint could generate large thermal residual tensile stress in HAZ; thus, the material was very unstable. It could fracture for very small or even no external stress. Hydrogen atoms would be released during welding and microstructures with the highest hardness are the most susceptible of hydrogen assisted cracking. It is concluded that the fracture was caused by hydrogen assisted brittleness under the induction of weld residual stress. Post-weld aging treatment (PWAT) is recommended to release the residual stress generated during welding process. In this case, PWAT was carried out on the failed weld joint and Vickers hardness of HAZ coarse grain area, HAZ fine grain area and base metal decreased to 232 VHN, 205 VHN and 125 VHN, respectively. That indicates that the PWAT procedure could effectively soften the material and relieve residual stress.
Improvement of Porcine SCNT Embryo Development using Histone Deacetylase Inhibitors
Jin Hee Lee,Keun Jung Kim,Ji Hye Lee,Eun Young Kim,Kil Woo Han,Junbo Wu,Lili Zhuang,Bo Myung Lee,Dong Hee Kim,Ju Lan Chun,Jun Jong Baek,Jae Sam Lim,Jung Youn Jo,Min Kyu Kim 한국동물생명공학회(구 한국동물번식학회) 2015 발생공학 국제심포지엄 및 학술대회 Vol.2015 No.10