• 제목/요약/키워드: Real-Time Polymerase Chain Reaction

검색결과 809건 처리시간 0.032초

Zearalenone exposure affects the Wnt/β-catenin signaling pathway and related genes of porcine endometrial epithelial cells in vitro

  • Song, Tingting;Yang, Weiren;Huang, Libo;Yang, Zaibin;Jiang, Shuzhen
    • Animal Bioscience
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    • 제34권6호
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    • pp.993-1005
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    • 2021
  • Objective: Zearalenone (ZEA) has estrogen-like effects. Our previous study has shown that ZEA (0.5 to 1.5 mg/kg) could induce abnormal uterine proliferation through transforming growth factor signaling pathway. To further study the other regulatory networks of uterine hypertrophy caused by ZEA, the potential mechanism of ZEA on porcine endometrial epithelial cells (PECs) was explored by the Illumina Hiseq 2000 sequencing system. Methods: The PECs were treated with ZEA at 0 (ZEA0), 5 (ZEA5), 20 (ZEA20), and 80 (ZEA80) µmol/L for 24 h. The collected cells were subjected to cell cycle, RNA-seq, real-time quantitative polymerase chain reaction, immunofluorescence, and western blot analysis. Results: The proportion of cells in the S and G2 phases decreased (p<0.05), but the proportion of cells in the G1 phase increased (p<0.05) in the ZEA80 treatment. Data analysis revealed that the expression of Wnt pathway-related genes, estrogen-related genes, and mitogen-activated protein kinase pathway-related genes increased (p<0.05), but the expression of genetic stability genes decreased (p<0.05) with increasing ZEA concentrations. The relative mRNA and protein expression of WNT1, β-catenin, glycogen synthase kinase 3β (GSK-3β) were increased (p<0.05) with ZEA increasing, while the relative mRNA and protein expression of cyclin D1 (CCND1) was decreased (p<0.05). Moreover, our immunofluorescence results indicate that β-catenin accumulated around the nucleus from the cell membrane and cytoplasm with increasing ZEA concentrations. Conclusion: In summary, ZEA can activate the Wnt/β-catenin signaling pathway by up-regulating WNT1 and β-catenin expression, to promote the proliferation and development of PECs. At the same time, the up-regulation of GSK-3β and down-regulation of CCND1, as well as the mRNA expression of other pathway related genes indicated that other potential effects of ZEA on the uterine development need further study.

어린 돌기해삼 Apostichopus japonicus (Echinodermata; Holothuroidea) 방류 방법에 따른 열충격단백질90 유전자의 발현 분석 (Heat Shock Protein 90 Gene Expression in Juvenile Sea Cucumber Apostichopus japonicus (Echinodermata; Holothuroidea) according to Releasing Methods)

  • 이동한;이승헌;정동빈;손영창
    • 한국해양생명과학회지
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    • 제7권1호
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    • pp.29-36
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    • 2022
  • 돌기해삼 Apostichopus japonicus는 주요 양식 대상 무척추동물로서 우리나라 연안 해역에 서식하고 있다. 본 연구는 방류 방법에 따른 단기간의 생리학적 스트레스 정도를 평가하기 위하여 heat shock protein 90 (HSP90) 유전자의 발현 변화를 실시간 정량적 중합효소연쇄반응법으로 조사하였다. 어린 돌기해삼을 비닐봉지에 산소 포장하여 30분간 수송하거나 방류 해역의 간조기에 1시간 공기 중에 노출된 실험군의 HSP90 유전자 발현은 대조군의 HSP90 유전자 발현에 비하여 통계학적으로 유의미하게 증가하였다(수송 후 실험군 p=0.001; 간조기 실험군 p=0.032). 어린 돌기해삼을 방류 후 6시간까지 분석한 결과, 선상에서 씨뿌림 방식으로 방류된 6시간째의 개체 및 호스를 통과하여 수중으로 방류된 2~6시간째의 HSP90 유전자 발현율은 대조군에 비하여 약간 감소하는 경향을 보였다(씨뿌림 실험군 p=0.069; 호스 방류군 p=0.093). 한편, 잠수부에 의해 수중에서 방류된 어린 돌기해삼은 방류 후 시간이 경과할수록 HSP90 유전자 발현율은 증가하는 패턴이 관찰되었다(p=0.061). 이상의 결과는 방류된 어린 돌기해삼의 단기간 스트레스 반응 연구와 효과적인 방류 방법의 개발에 HSP90 유전자 발현이 유용하게 사용될 수 있음을 시사한다.

β-Asarone이 Lipopolysaccharide에 의한 생쥐 해마의 염증성 사이토카인 발현과 학습 및 기억 장애에 미치는 영향 (Effects of β-Asarone on Pro-Inflammatory Cytokines and Learning and Memory Impairment in Lipopolysaccharide-Treated Mice)

  • 최문숙;곽희준;권기중;황지모;신정원;손낙원
    • 대한본초학회지
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    • 제28권6호
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    • pp.119-127
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    • 2013
  • Objectives : ${\beta}$-Asarone (BAS) is an active ingredient in Acori Rhizoma. This study investigated anti-neuroinflammatory and memory ameliorating effects of BAS in systemic lipopolysaccharide (LPS)-treated C57BL/6 mice. Methods : BAS was administered orally at doses of 7.5, 15, and 30 mg/kg for 3 days prior to LPS (3 mg/kg, intraperitoneal) injection. Pro-inflammatory cytokine mRNA, including tumor necrosis factor-${\alpha}$ (TNF-ㅍ), interleukin (IL)-$1{\beta}$ and IL-6, was measured in hippocampus tissue using real-time polymerase chain reaction at 4 h after the LPS injection. An ameliorating effect of 30 mg/kg BAS on learning and memory impairment in the LPS-treated mice was verified using the Morris water maze test. Results : BAS significantly attenuated up-regulation of TNF-${\alpha}$, IL-$1{\beta}$, and IL-6 mRNA in hippocampus tissue of the LPS-treated mice. In acquisition training test, BAS improved learning performance of the LPS-treated mice with a significant decrease of escape latency to the platform. In memory retention test, BAS also ameliorated memory impairment of the LPS-treated mice with a significant increase of swimming time in zones neighboring to the platform, number of target heading, and memory score. Conclusion : The results suggest that inhibition of pro-inflammatory cytokines and neuroinflammation in the hippocampus by BAS could be one of the mechanisms for BAS-mediated ameliorating effect on learning and memory impairment in LPS-treated mice.

VITEK 2 시스템과 Multiplex Real-time PCR을 이용한 반코마이신 내성 장알균(VRE)과 내성관련 유전자 검출 (Detection of Vancomycin-Resistant Enterococci and Related Genes Using VITEK 2 System and Multiplex Real-time PCR Assay)

  • 정민경;유영빈;김상하;김성현;김영권
    • 대한임상검사과학회지
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    • 제49권4호
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    • pp.401-406
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    • 2017
  • 이 연구에서 VITEK 2 시스템을 사용하여 $6{\mu}g/mL$ vancomycin이 첨가된 Enterococcosel (EV6 agar에서 배양한 327개의 검체 중 74개의 분리균(22.6%)으로 확인하였다. E. faecium은 55주(74.3%), E. gallinarum 16주(21.6%), E. casseliflavus 2주(2.7%) 및 E. avium 1주(1.4%)로 확인되었다. E. faecium의 55가지 표현형 중 vanA가 42주(76.4%), vanB가 9주(16.4%), vanC 표현형이 4주(7.3%)로 나타났다. E. gallinarum 16주와 E. casseliflavus 2주 모두 vanC 표현형을 보였으며 E. avium 1주는 vanB 표현형을 나타내었다. EV4에서만 증식된 E. faecium 1주는 VITEK2 시스템을 이용한 항균제 감수성 검사 결과 vancomycin과 teicoplanin에 모두 감수성이었고 vancomycin 내성 표현형 유전자는 PCR에 의해 검출되지 않았다. 총 327 검체를 $6{\mu}g/mL$ vancomycin (EV6 broth)을 첨가 한 Enterococoscosel broth에서 배양하여 120 균주(36.7%)가 분리되었다. 120균주에서 다중 중합 효소 연쇄반응에 의한 반코마이신 내성 유전형 실험을 실시한 결과, vanA가 51주(42.5%), vanA와 vanC가 5주(4.2%), vanC가 18주(15%), 나머지 46주(38.3%)에서는 vancomycin 내성 유전형 유전자는 검출되지 않았다.

Claritromycin Resistance and Helicobacter pylori Genotypes in Italy

  • Francesco Vincenzo De;Margiotta Marcella;Zullo Angelo;Hassan Cesare;Valle Nicolar Della;Burattini Osvaldo;D'Angel Roberto;Stoppino Giuseppe;Cea Ugo;Giorgio Floriana;Monno Rosa;Morini Sergio;Panella Carmine
    • Journal of Microbiology
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    • 제44권6호
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    • pp.660-664
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    • 2006
  • The relationship between H. pylori clarithromycin resistance and genetic pattern distribution has been differently explained from different geographic areas. Therefore, we aimed to assess the clarithromycin resistance rate, to evaluate the bacterial genetic pattern, and to search for a possible association between clarithromycin resistance and cagA or vacA genes. This prospective study enrolled 62 consecutive H. pylori infected patients. The infection was established by histology and rapid urease test. Clarithromycin resistance, cagA and vacA status, including s/m subtypes, were assessed on paraffin-embedded antral biopsy specimens by TaqMan real time polymerase chain reaction (PCR). Primary clarithromycin resistance was detected in 24.1 % of cases. The prevalence of cagA was 69.3%, and a single vacA mosaicism was observed in 95.1 % cases. In detail, the s1m1 was observed in 23 (38.9%) patients, the s1m2 in 22 (37.2%), and the s2m2 in 14 (23.7%), whereas the s2m1 combination was never found. The prevalence of cagA and the vacA alleles distribution did not significantly differ between susceptible and resistant strains. Primary clarithromycin resistance is high in our area. The s1m1 and s1m2 are the most frequent vacA mosaicisms. There is no a relationship between clarithromycin resistance and bacterial genotypic pattern and/or cagA positivity.

2005년 봄부터 2006년 가을까지 국내 중부지역의 enterovirus 감염의 분자유전학 조사와 임상양상 (Molecular identification and clinical features of enteroviral infection in children of central Korea: An overview of enteroviral epidemiology between spring 2005 and autumn 2006)

  • 노의정;진용만;정은희;장영표;박우성;박귀성;지영미
    • Clinical and Experimental Pediatrics
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    • 제52권11호
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    • pp.1234-1240
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    • 2009
  • 목 적:Enteroviruse (EV)는 영아와 소아에서 가장 흔한 감염의 원인으로 경한 발열성 질환에서 뇌수막염이나 치명적인 심근염까지 다양한 질환을 일으키는 원인이다. 본 연구에서는 국내 중부지역의 EV 유병율에 대해 알아보고자 하였다. 방 법:2005년 4월부터 2006년 10월까지 국내 중부지역에서 발열성 질환이나 뇌수막염이 의심되어 입원한 소아에서 얻은 검체를 모아 RT-PCR을 시행하여 EV를 확인하였고, genotype과 phylogenetic 분석을 위해 direct sequencing을 시행하였다. 결 과:뇌척수액과 대변검체에서 PCR을 시행한 305명의 환아 중 51명(16.7%)에서 EV가 양성을 보였다. 그중 다음의 serotype이 44명에서 확인되었다: Echovirus (ECV) 18 (18 cases, 35.2%), Coxsackievirus (CVB) 5 (13 cases, 25.4%), ECV25 (5 cases, 9.8%), ECV9 (4 cases, 7.8%), ECV5 (3 cases, 5.8%), EV74 (1 case, 1.9%). 2005년 6월과 8월 사이에는 ECV18과 CVB5가 가장 많이 검출되었다. 2006년 7월과 8월 사이에는 ECV25가 가장 많이 검출되었다. 결 론:지속적인 enterovirus 감염의 감시가 필요하며 임상적인 의의가 밝혀져야 할 것이다. 특히 EV74가 처음으로 국내에서 발견되었기에 이를 보고하는 바이다.

Effects of Bacillus subtilis KN-42 on Growth Performance, Diarrhea and Faecal Bacterial Flora of Weaned Piglets

  • Hu, Yuanliang;Dun, Yaohao;Li, Shenao;Zhao, Shumiao;Peng, Nan;Liang, Yunxiang
    • Asian-Australasian Journal of Animal Sciences
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    • 제27권8호
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    • pp.1131-1140
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    • 2014
  • This research focused on the effects of different doses of Bacillus subtilis KN-42 on the growth performance, diarrhea incidence, faecal bacterial flora, and the relative number of Lactobacillus and Escherichia coli in faeces of weaned piglets to determine whether the strain can serve as a candidate antimicrobial growth promoter. A total of 360 piglets (initial body weight $7.14{\pm}0.63$ kg) weaned at $26{\pm}2$ days of age were randomly allotted to 5 treatment groups (4 pens per treatment with 18 pigs per pen) for a 28-day trial. Dietary treatments were basal diet without any antimicrobial (negative control; NC), basal diet supplemented with 120 mg/kg feed of neomycin sulfate (positive control; PC) and basal diet supplemented with $2{\times}10^9$ (L), $4{\times}10^9$ (M) and $20{\times}10^9$ (H) CFU/kg feed of B. subtilis KN-42. During the overall period, average daily gain and feed efficiency of piglets were higher in groups PC, M, and H than those in group NC (p<0.05), and all probiotics and antibiotics groups had a lower diarrhea index than group NC (p<0.05). The 16S rDNA gene-based methods were used to analyze faecal bacterial flora on day 28 of experiment. The result of denaturing gradient gel electrophoresis analysis showed that supplementation of B. subtilis KN-42 to the diet changed the bacterial communities, with a higher bacterial diversity and band number in group M than in the other four groups. Real-time polymerase chain reaction analysis showed that the relative number of Lactobacillus were higher in groups PC and H than in group NC (p<0.05), and the supplemented B. subtilis KN-42 to the diet also reduced the relative number of E. coli (p<0.05). These results suggest that dietary addition of B. subtilis KN-42 can improve the growth performance and gastrointestinal health of piglets.

Differential Expression of PPARγ, FASN, and ACADM Genes in Various Adipose Tissues and Longissimus dorsi Muscle from Yanbian Yellow Cattle and Yan Yellow Cattle

  • Ji, Shuang;Yang, Runjun;Lu, Chunyan;Qiu, Zhengyan;Yan, Changguo;Zhao, Zhihui
    • Asian-Australasian Journal of Animal Sciences
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    • 제27권1호
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    • pp.10-18
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    • 2014
  • The objective of this study was to investigate the correlation between cattle breeds and deposit of adipose tissues in different positions and the gene expressions of peroxisome proliferator-activated receptor gamma ($PPAR{\gamma}$), fatty acid synthase (FASN), and Acyl-CoA dehydrogenase (ACADM), which are associated with lipid metabolism and are valuable for understanding the physiology in fat depot and meat quality. Yanbian yellow cattle and Yan yellow cattle reared under the same conditions display different fat proportions in the carcass. To understand this difference, the expression of $PPAR{\gamma}$, FASN, and ACADM in different adipose tissues and longissimus dorsi muscle (LD) in these two breeds were analyzed using the Real-time quantitative polymerase chain reaction method (qRT-PCR). The result showed that $PPAR{\gamma}$ gene expression was significantly higher in adipose tissue than in LD in both breeds. $PPAR{\gamma}$ expression was also higher in abdominal fat, in perirenal fat than in the subcutaneous fat (p<0.05) in Yanbian yellow cattle, and was significantly higher in subcutaneous fat in Yan yellow cattle than that in Yanbian yellow cattle. On the other hand, FASN mRNA expression levels in subcutaneous fat and abdominal fat in Yan yellow cattle were significantly higher than that in Yanbian yellow cattle. Interestingly, ACADM gene shows greater fold changes in LD than in adipose tissues in Yan yellow cattle. Furthermore, the expressions of these three genes in lung, colon, kidney, liver and heart of Yanbian yellow cattle and Yan yellow cattle were also investigated. The results showed that the highest expression levels of $PPAR{\gamma}$ and FASN genes were detected in the lung in both breeds. The expression of ACADM gene in kidney and liver were higher than that in other organs in Yanbian yellow cattle, the comparison was not statistically significant in Yan yellow cattle.

Effects of dietary supplementation of lipid-coated zinc oxide on intestinal mucosal morphology and expression of the genes associated with growth and immune function in weanling pigs

  • Song, Young Min;Kim, Myeong Hyeon;Kim, Ha Na;Jang, Insurk;Han, Jeong Hee;Fontamillas, Giselle Ann;Lee, Chul Young;Park, Byung-Chul
    • Asian-Australasian Journal of Animal Sciences
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    • 제31권3호
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    • pp.403-409
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    • 2018
  • Objective: The present study was conducted to investigate the effects of a lipid-coated zinc oxide (ZnO) supplement Shield Zn (SZ) at the sub-pharmacological concentration on intestinal morphology and gene expression in weanling pigs, with an aim to gain insights into the mechanism of actions for SZ. Methods: Forty 22-day-old weanling pigs were fed a nursery diet supplemented with 100 or 2,500 mg Zn/kg with uncoated ZnO (negative control [NC] or positive control [PC], respectively), 100, 200, or 400 mg Zn/kg with SZ for 14 days and their intestinal tissues were taken for histological and molecular biological examinations. The villus height (VH) and crypt depth (CD) of the intestinal mucosa were measured microscopically following preparation of the tissue specimen; expression of the genes associated with growth and immune function was determined using the real-time quantitative polymerase chain reaction. Results: There was no difference in daily gain, gain:feed, and diarrhea score between the SZ group and either of NC and PC. The VH and VH:CD ratio were less for the SZ group vs NC in the jejunum and duodenum, respectively (p<0.05). The jejunal mucosal mRNA levels of insulin-like growth factor (IGF-I) and interleukin (IL)-10 regressed and tended to regress (p = 0.053) on the SZ concentration with a positive coefficient, respectively, whereas the IL-6 mRNA level regressed on the SZ concentration with a negative coefficient. The mRNA levels of IGF-I, zonula occludens protein-1, tumor necrosis $factor-{\alpha}$, IL-6, and IL-10 did not differ between the SZ group and either of NC and PC; the occludin and transforming growth $factor-{\beta}1$ mRNA levels were lower for the SZ group than for PC. Conclusion: The present results are interpreted to suggest that dietary ZnO provided by SZ may play a role in intestinal mucosal growth and immune function by modulating the expression of IGF-I, IL-6, and IL-10 genes.

LETM1 Promotes Gastric Cancer Cell Proliferation, Migration, and Invasion via the PI3K/Akt Signaling Pathway

  • Zhang, Yunfeng;Chen, Lele;Cao, Yifan;Chen, Si;Xu, Chao;Xing, Jun;Zhang, Kaiguang
    • Journal of Gastric Cancer
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    • 제20권2호
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    • pp.139-151
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    • 2020
  • Purpose: Globally, there is a high incidence of gastric cancer (GC). Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) is reported to play a vital role in several human malignancies. However, there is limited understanding of the role of LETM1 in GC. This study aims to investigate the effects of LETM1 on proliferation, migration, and invasion of GC cells. Materials and Methods: The expression levels of LETM1 in the normal gastric mucosal epithelial cells (GES-1) and GC cells were analyzed by quantitative real-time polymerase chain reaction and western blotting. CCK-8, wound healing, and Transwell invasion assays were performed to evaluate the effect of LETM1 knockdown or overexpression on the proliferation, migration, and invasion of the GC cells, respectively. Additionally, the effect of LETM1 knockdown or overexpression on GC cell apoptosis was determined by flow cytometry. Furthermore, the effect of LETM1 knockdown or overexpression on the expression levels of PI3K/Akt signaling pathway-related proteins was evaluated by western blotting. Results: The GC cells exhibited markedly higher mRNA and protein expression levels of LETM1 than the GES-1 cells. Additionally, the knockdown of LETM1 remarkably suppressed the GC cell proliferation, migration, and invasion, and promoted the apoptosis of GC cells, which were reversed upon LETM1 overexpression. Furthermore, the western blotting analysis indicated that LETM1 facilitates GC progression via the PI3K/Akt signaling pathway. Conclusions: LETM1 acts as an oncogenic gene to promote GC cell proliferation, migration, and invasion via the PI3K/Akt signaling pathway. Therefore, LETM1 may be a potential target for GC diagnosis and treatment.