Sensitivity to tumor development by TALEN-mediated Trp53 mutant genes in the susceptible FVB/N mice and the resistance C57BL/6 mice

윤우빈,김지은,이미림,최준영,박진주,송보람,강병철,남기택,이한웅,황대연 한국실험동물학회 2021 Laboratory Animal Research Vol.37 No.4

Background This study was undertaken to compare the sensitivities of mice strains during tumor induction by transcription activator-like effector nucleases (TALEN)-mediated Trp53 mutant gene. Alterations of their tumorigenic phenotypes including survival rate, tumor formation and tumor spectrum, were assessed in FVB/N-Trp53em2Hwl/Korl and C57BL/6-Trp53em1Hwl/Korl knockout (KO) mice over 16 weeks. Results Most of the physiological phenotypes factors were observed to be higher in FVB/N-Trp53em2Hwl/Korl KO mice than C57BL/6-Trp53em1Hwl/Korl KO mice, although there were significant differences in the body weight, immune organ weight, number of red blood cells, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), total bilirubin (Bil-T) and glucose (Glu) levels in the KO mice relative to the wild type (WT) mice. Furthermore, numerous solid tumors were also observed in various regions of the surface skin of FVB/N-Trp53em2Hwl/Korl KO mice, but were not detected in C57BL/6-Trp53em1Hwl/Korl KO mice. The most frequently observed tumor in both the Trp53 KO mice was malignant lymphoma, while soft tissue teratomas and hemangiosarcomas were only detected in the FVB/N-Trp53em2Hwl/Korl KO mice. Conclusions Our results indicate that the spectrum and incidence of tumors induced by the TALEN-mediated Trp53 mutant gene is greater in FVB/N-Trp53em2Hwl/Korl KO mice than C57BL/6-Trp53em1Hwl/Korl KO mice over 16 weeks.

Comparison of scopolamine-induced cognitive impairment responses in three different ICR stocks

윤우빈,최현준,김지은,박지원,강미주,배수지,이영주,최유상,김길수,정영석,조준용,황대연,송현근 한국실험동물학회 2018 Laboratory Animal Research Vol.34 No.4

Cognitive impairment responses are important research topics in the study of degenerative brain diseases as well as in understanding of human mental activities. To compare response to scopolamine (SPL)-induced cognitive impairment, we measured altered parameters for learning and memory ability, inflammatory response, oxidative stress, cholinergic dysfunction and neuronal cell damages, in Korl:ICR stock and two commercial breeder stocks (A:ICR and B:ICR) after relevant SPL exposure. In the water maze test, Korl:ICR showed no significant difference in SPL-induced learning and memory impairment compared to the two different ICRs, although escape latency was increased after SPL exposure. Although behavioral assessment using the manual avoidance test revealed reduced latency in all ICR mice after SPL treatment as compared to Vehicle, no differences were observed between the three ICR stocks. To determine cholinergic dysfunction induction by SPL exposure, activity of acetylcholinesterase (AChE) assessed in the three ICR stocks revealed no difference of acetylcholinesterase activity. Furthermore, low levels of superoxide dismutase (SOD) activity and high levels of inflammatory cytokines in SPL-treated group were maintained in all three ICR stocks, although some variations were observed between the SPLtreated groups. Neuronal cell damages induced by SPL showed similar response in all three ICR stocks, as assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, Nissl staining analysis and expression analyses of apoptosis-related proteins. Thus, the results of this study provide strong evidence that Korl:ICR is similar to the other two ICR. Stocks in response to learning and memory capacity.

Overexpression of N141I PS2 increases γ-secretase activity through up-regulation of Presenilin and Pen-2 in brain mitochondria of NSE/hPS2m transgenic mice

윤우빈,박진주,김지은,성지은,이현아,이재호,배창준,황대연 한국실험동물학회 2016 Laboratory Animal Research Vol.32 No.4

Alzheimer’s disease (AD) is known to induce alterations of mitochondrial function such as elevation of oxidative stress and activation of apopotosis. The aim of this study was to investigate the effects of human Presenilin 2 mutant (hPS2m) overexpression on the γ-secretase complex in the mitochondrial fraction. To achieve this, alterations of γ-secretase complex expression and activity were detected in the mitochondrial fraction derived from brains of NSE/hPS2m Tg mice and Non-Tg mice. Herein, the following were observed: i) overexpression of the hPS2m gene significantly up-regulated the deposition of Aβ-42 peptides in the hippocampus and cortex of brain, ii) overexpression of hPS2m protein induced alterations of γ-secretase components such as main component protein and activator protein but not stabilization-related proteins, iii) changes in γ-secretase components induced by overexpression of hPS2m protein up-regulated γ-secretase activity in the mitochondrial fraction, and iv) elevation of γ-secretase activity induced production of Aβ-42 peptides in the mitochondrial fraction. Based on these observations, these results indicate that alteration of γ-secretase activity in cells upon overexpression of hPS2m is tightly linked to mitochondrial dysfunction under the specific physiological and pathological conditions of AD.

The effects of biomaterials in growing medium on the response of Zelkova serrata in a containerized production system

윤우빈,한시호,서정민,AUNGAUNG,Huong Thi Thuy Dao,안지영,박병배,조민석 충남대학교 농업과학연구소 2019 Korean Journal of Agricultural Science Vol.46 No.4

Changes in the physical and chemical properties of soil materials during the nursing process have a great influence on the quality of containerized seedlings and on growth and survival after planting. In this study, the effect of biomaterials and their mixed ratios on the growth of Zelkova serrata seedlings in a containerized seedling production system was investigated. Mushroom sawdust, pine bark, and carbonized rice husk were used as biomaterials. The mixed ratios were 10% and 20% volume ratio of the growing medium volume, including the untreated controls. There was no significant difference in the height growth of the Zelkova serrata seedlings according to the biomaterials. The root collar diameter was the highest with the 20% carbonized rice husk and the lowest with the mushroom sawdust. The difference between the highest quality index and the lowest quality index was 30% in the order of the carbonized rice husk, pine bark, control, and mushroom sawdust, but there was no statistical significance. In this study, if the growing medium mixed with biomaterials does not reduce the seedling growth compared with the control, it is considered that the biomaterial can replace a part of the growing media. Therefore, the results show that some of the growing media can be replaced with carbonized rice husk or pine bark when producing Zelkova serrata seedlings.

Chemosensitivity to doxorubicin in primary cells derived from tumor of FVB/N-Trp53tm1Hw1 with TALEN-mediated Trp53 mutant gene

윤우빈,김지은,진유정,노유정,송희진,설아윤,김태렬,민경선,박은서,박기호,강현구,최연식,황대연 한국실험동물학회 2023 Laboratory Animal Research Vol.39 No.4

Background: To evaluate the chemosensitivity to doxorubicin (DOX) in two primary cells derived from a tumor of FVB/N-Trp53tm1Hw1 knockout (KO) mice with TALEN-mediated Trp53 mutant gene, we evaluated the cell survivability, cell cycle distribution, apoptotic cell numbers and apoptotic protein expression in solid tumor cells and ascetic tumor cells treated with DOX. Results: The primary tumor cells showed a significant (P < 0.05) defect for UV-induced upregulation of the Trp53 protein, and consisted of different ratios of leukocytes, fibroblasts, epithelial cells and mesenchymal cells. The IC50 level to DOX was lower in both primary cells (IC50 = 0.12 μM and 0.20 μM) as compared to the CT26 cells (IC50 = 0.32 μM), although the solid tumor was more sensitive. Also, the number of cells arrested at the G0/G1 stage was significantly decreased (24.7–23.1% in primary tumor cells treated with DOX, P < 0.05) while arrest at the G2 stage was enhanced to 296.8–254.3% in DOX-treated primary tumor cells compared with DOX-treated CT26 cells. Furthermore, apoptotic cells of early and late stage were greatly increased in the two primary cell-lines treated with DOX when compared to same conditions for CT26 cells. However, the Bax/Bcl-2 expression level was maintained constant in the primary tumor and CT26 cells. Conclusions: To the best of our knowledge, these results are the first to successfully detect an alteration in chemosensitivity to DOX in solid tumor cells and ascetic tumor cells derived from tumor of FVB/N-Trp53tm1Hw1 mice TALEN-mediated Trp53 mutant gene.

The Effects of Biochar Made by Oaks on the Growth and Seedling Quality Index of Prunus sargentii in a Containerized Production System

윤우빈,Loth Meng,한시호,Aung Aung,조민석,박병배 한국토양비료학회 2019 한국토양비료학회지 Vol.52 No.1

The purpose of this study is to investigate the effects of biochar mixed ratios made by oaks on the growth andquality index of Prunus sargentii seedlings in a containerized seedling production system. We treated biocharmade by oak charcoal with 10% and 20% volume ratio of the artificial soil including the untreated control andapplied two levels of 0.5 and 1.0 g/L fertilization treatments. Generally, growth parameters including height,root collar diameter, and dry weight were decreased by biochar treatments and there were no fertilizationeffects. However, 10% biochar treatment significantly increased quality index by 26% compared to that ofcontrol. Our results suggested the oak charcoal, a kind of biochar, can be substituted up to 10% of the artificialsoil in the containerized P. sargentii seedling production system and expect to increase the survival rate in thebeginning of the plantation.

미더덕껍질과 천연고분자 혼합물을 이용한 마스크팩시트의 제조방법

윤우빈(Woobin Yun),이예찬(Yechan Lee),김다솜(Dasom Kim),김지은(Jieun Kim),성지은(Jieun Sung),이현아(Hyunah Lee),손홍주(Hongju Son),황대연(Daeyoun Hwang),정영진(Youngjin Jung) 한국염색가공학회 2017 韓國染色加工學會誌 Vol.29 No.1

Characterization the response of Korl:ICR mice to loperamide induced constipation

김지은,윤우빈,성지은,이현아,최준영,최연식,정영석,김길수,황대연 한국실험동물학회 2016 Laboratory Animal Research Vol.32 No.4

Animal models of constipation induced with drugs and diet have been widely employed to investigate therapeutic effects and the action mechanism of drugs against this disease. ICR mice were selected to produce this disease model through oral administration of loperamide (Lop), even though SD rats are commonly utilized in studies of constipation. To compare the responses of ICR mice obtained from three different sources to constipation inducers, alterations in stool number, histopathological structure, mucin secretion and opioid-receptor downstream signaling pathway were measured in Korl:ICR (Korea FDA source), A:ICR (USA source) and B:ICR (Japan source) injected with low and high concentrations of Lop (LoLop and HiLop). The number, weight and moisture content of stools decreased significantly in the Lop treated group of all ICR relative to the Vehicle treated group. Additionally, decreased mucosa layer thickness, muscle thickness, and mucin secretion were observed in the transverse colon of Lop treated ICR mice, while a similar number of goblet cells and crypt of lieberkuhn were detected in the same group. Furthermore, a similar change in the level of Gα expression and PKC phosphorylation was detected in the Lop treated group relative to the vehicle treated group, while some differences in the change pattern were observed in the B:ICR group. Therefore, these results of the present study provide strong additional evidence that Korl:ICR, A:ICR and B:ICR derived from different sources have a similar overall response to constipation induced by Lop injection, although there were a few differences in the magnitude of their responses.

Successful development of squamous cell carcinoma and hyperplasia in RGEN-mediated p27 KO mice after the treatment of DMBA and TPA

최준영,윤우빈,김지은,이미림,박진주,송보람,김혜령,박지원,강미주,강병철,이한웅,황대연 한국실험동물학회 2018 Laboratory Animal Research Vol.34 No.3

To evaluate the carcinogenicity of p27 knockout (KO) mice with RNA-guided endonuclease (RGENs)- mediated p27 mutant exon I gene (IΔ), alterations in the carcinogenic phenotypes including tumor spectrum, tumor suppressor proteins, apoptotic proteins and cell cycle regulators were observed in p27 (IΔ) KO mice after treatment with 7,12-Dimethylbenz[a]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13- acetate (TPA)(DT) for 5 months. The target region (544~571 nt) in exon I of the p27 gene was successfully disrupted in p27 (IΔ) KO mice using the RGEN-induced non-homologous end joining (NHEJ) technique. After DT exposure for 5 months, a few solid tumors (identified as squamous cell carcinoma) developed on the surface of back skin of DT-treated p27 (IΔ) KO mice. Also, squamous cell hyperplasia with chronic inflammation was detected in the skin dermis of DT-treated p27 (IΔ) KO mice, while the Vehicle+p27 (IΔ) KO mice and WT mice maintained their normal histological skin structure. A significant increase was observed in the expression levels of tumor suppressor protein (p53), apoptotic proteins (Bax, Bcl-2 and Caspase-3) and cell-cycle regulator proteins (Cyclin D1, CDK2 and CDK4) in the skin of DT-treated p27 (IΔ) KO mice, although their enhancement ratio was varied. Taken together, the results of the present study suggest that squamous cell carcinoma and hyperplasia of skin tissue can be successfully developed in new p27 (IΔ) KO mice produced by RGEN-induced NHEJ technique following DT exposure for 5 months.

Synergic Laxative Effects of an Herbal Mixture of Liriope platyphylla, Glycyrrhiza uralensis, and Cinnamomum cassia in Loperamide-Induced Constipation of Sprague Dawley Rats

김지은,윤우빈,이미림,최준영,박진주,김혜령,송보람,홍진태,송현근,황대연 한국식품영양과학회 2019 Journal of medicinal food Vol.22 No.3

Constipation is an acute or chronic illness attributed to various causes, ranging from lifestyle habits to side effects of a disease. To improve the laxative effects of some traditional medicines, herbal mixtures of Liriope platyphylla, Glycyrrhiza uralensis, and Cinnamomum cassia (LGC) were evaluated for their mechanism of action and therapeutic effects in loperamide (Lop)-induced constipated Sprague Dawley rats by examining alterations in excretion parameters, histological structure, mucin secretion, and related protein levels. Food intake and water consumption were constant for all animals. We observed that the Lop+LGC-treated group had significantly greater excretion of stool and urine than was observed in the Lop+Vehicle-treated group. Administration of LGC in the constipation model restored the intestinal transit ratio to normal levels, and increased the number of goblet cells, mucosal layer, and muscle thickness. Mucin secretion was greater in the Lop+LGC-treated group than in the Lop+Vehicle-treated group, and the expression of MUC2 and AQP8 genes were also increased. In addition, reverse transcription polymerase chain reaction and Western blot revealed an increase in the muscarinic acetylcholine receptors (mAChRs) in the Lop+LGC-treated group compared to the Lop+Vehicle-treated group. Furthermore, compared with the Lop+Vehicle-treated group, treatment with LGC reduced the phosphorylation of PKC and PI3K, and expression of Gα protein, but increased levels of IP3. Our results suggest that the traditional herbal mixture of LGC induces a potent laxative effect in Lop-induced constipation through mucosal tissue changes and mucin production. We also demonstrated that the laxative effect of LGC is closely related to the expression of mAChR and its downstream signals, suggesting the possibility of developing a constipation-laxative agent using LGC.