• Journal of Periodontal and Implant Science
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• 제36권4호
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• pp.839-847
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• 2006

The aim of this study is to monitor reporter gene expression under osteocalcin gene promoter, using a real-time molecular imaging system, as tool to investigate osteoblast differentiation. The promoter region of mouse osteocalcin gene 2 (mOG2), the best-characterized osteoblast-specific gene, was inserted in promoterless luciferase reporter vector. Expression of reporter gene was confirmed and relationship between the reporter gene expression and osteoblastic differentiation was evaluated. Gene expression according to osteoblstic differentiation on biomaterials, utilizing a real-time molecular imaging system, was monitored. Luciferase was expressed at the only cells transduced with pGL4/mOGP and the level of expression was statistically higher at cells cultured in mineralization medium than cells in growth medium. CCCD camera detected the luciferase expression and was visible differentiation-dependent intensity of luminescence. The cells produced osteocalcin with time-dependent increment in BMP-2 treated cells and there was difference between BMP-2 treated cells and untreated cells at 14days. There was difference at the level of luciferase expression under pGL4/mOGP between BMP-2 treated cells and untreated cells at 3days. CCCD camera detected the luciferase expression at cells transduced with pGL4/mOGP on Ti disc and was visible differentiation-dependent intensity of luminescence This study shows that 1) expression of luciferase is regulated by the mouse OC promoter, 2) the CCCD detection system is a reliable quantitative gene detection tool for the osteoblast differentiation, 3) the dynamics of mouse OC promoter regulation during osteoblast differentiation is achieved in real time and quantitatively on biomaterial. The present system is a very reliable system for monitoring of osteoblast differentiation in real time and may be used for monitoring the effects of growth factors, drug, cytokines and biomaterials on osteoblast differentiation in animal.

• Journal of Chest Surgery
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• 제37권12호
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• pp.967-977
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• 2004

배경: DNA 메칠화란 유전자의 Promoter에 있는 CpG dinucleotide의 cytosine기에 메칠기가 붙는 현상을 말한다. CpG dinucleotide에 과메틸화가 일어나면 일부 유전자의 발현이 감소되며, 그 반대로 CpG dinucleotide의 메칠화가 억제되면 유전자 발현이 증가된다. DNA 메칠화 억제제인 5-aza-2'- deoxycytidine (ADC)을 폐암세포에 처치했을 때 암항원 유전자의 발현 유무와 이를 위한 최적 조건을 조사하고, 아울러 MHC와 B7의 발현과 세포 성장에 미치는 영향을 조사하여 암치료 백신에 ADC를 임상적으로 이용할 수 있는 지를 연구하였다. 대상 및 방법: 4개의 사람 폐암세포주 (NCIH1703, NCIH522, MRC-5 및 A549)에 ADC를 1 uM 농도로 처치한 후 48시간 뒤에 MAGE family, GAGE, NY-ESO-1, PSMA, CEA 및 SCC항원 유전자에 대한 RT-PCR을 실시하였고, 폐암세포에서 암항원의 발현을 증가시키는 최적의 ADC처치 조건을 규명하기 위하여 ADC농도와 처치 시간을 다양하게 하여 암세포를 자극한 후 암항원 유전자 발현성을 분석하였다. 또한 ADC 처리가 폐암 세포주의 MHC와 B7 발현을 증가시키는 가를 알아보기 위해 1 uM 농도의 ADC를 72시간 처치한 후 FACS 분석을 실시하였고, ADC가 세포성장에 미치는 영향을 알아보기 위하여, ADC를 0.2, 1 및 5 uM 농도로 96시간 처치 후 세포수를 측정하여 상대성장지수를 조사하였다. 결과: 세포주에 따라 차이는 있으나 MAGE, GAGE, NY-ESO-1 및 PSMA의 발현이 유도되었으며, MAGE아형 중에는 MAGE-1, -2, -3, -4, -6으로 나타났다. 그러나 비암항원인 CEA발현은 변화가 없었으며 SCC항원 유전자의 발현은 오히려 ADC처치에 의해 감소되었다. ADC 처치 후 24∼48 시간이 지난 뒤부터 암항원 유전자의 발현이 증가하였으며 ADC처리에 의해 유도된 유전자의 발현성은 ABC처치 후 최소 14일까지 유지되었다. 또 ADC를 0.2, 1, 5 uN 농도로 첨가하여 48시간 배양한 후 암항원 유전자 발현성을 측정한 결과 세포주에 따라 다소 차이는 있으나 대개 0.2 uM농도에서도 유전자 발현이 유도되었으며 1, 5 uM농도에서 매우 강하게 유도되었다. ADC 처리가 페암세포주의 MHC와 B7 발현을 증가시키는가를 알아보기 위해 1 uM 농도의 ADC를 72시간 처치한 후 FACS 분석을 실시한 결과 4개의 페암세포주에서 MHC 및 B7분자의 발현은 유도되지 않았다. 또 ADC농도가 세포성장에 미치는 영향을 알아보기 위하여 ADC를 0.2, 1, 5 uM농도로 96시간 처치 후 세포수를 측정하여 상대성장지수를 알아본 결과 ADC 처치 농도가 증가함에 따라 세포의 성장은 매우 감소하였다. 결론: 폐암세포주에서 ADC처치는 MAGE, GAGE 및 NY-ESO-1과 같은 세포독성 T 림프구 반응을 유도할 수 있는 암항원의 발현을 증가시킬 수 있으며, ADC의 세포독성과 항원 발현 유발시간을 분석할 때 1 uM 농도에서 48시간 처치한 후 ADC가 없는 배지에서 수일간 배양하는 것이 가장 효과적이라고 생각된다. 그러나, ADC를 처치하여도 MHC 및 B7의 발현의 변화는 없었으므로 ADC를 처치한 폐암세포를 암백신으로 사용하기 위해서는 MHC나 B7 및 cytokine의 발현을 증가시키는 추가적인 처치가 필요하다고 생각된다.

• Archives of Pharmacal Research
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• 제27권2호
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• pp.225-231
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• 2004

This study was designed to investigate the effect of Trolox, a hydrophilic analogue of vitamin E, on the alteration of vasoregulatory gene expression during hepatic ischemia and reperfusion (I/R). Rats were subjected to 60 min of hepatic ischemia in vivo. The rats were treated intravenously with Trolox (2.5 mg/kg) or the vehicle as a control 5 min before reperfusion. Liver samples were obtained 5 h after reperfusion for a RT-PCR analysis on the mRNA for the genes of interest. These mRNA peptides are endothelin-1 (ET -1), potent vasoconstrictor peptide, its receptor $ET_A$ and $ET_B$, vasodilator endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), heme oxygenase-1 (HO-1), tumor necrosis factor-$\alpha$ (TNF-$\alpha$) and cyclooxygenase-2 (COX-2). It was seen that serum alanine aminotransferase and lipid peroxi-dation levels were markedly increased after I/R and Trolox significantly suppressed this increase. In contrast, the glutathione concentration decreased in the I/R group, and this decrease was inhibited by Trolox. ET-1 mRNA expression was increased by I/R, an increase which was prevented by Trolox. The mRNA levels for $ET_A$ receptor was significantly decreased, whereas ET$_{B}$ receptor transcript increased in the I/R group. The increase in $ET_A$ was prevented by Trolox. The mRNA levels for iNOS and HO-1 significantly increased in the I/R group and Trolox attenuated this increase. There were no significant differences in eNOS mRNA expression among any of the experimental groups. The mRNA levels for COX-2 and TNF-$\alpha$ significantly increased in I/R group and Trolox also attenuated this increase. Our findings suggest that I/R induces an imbalanced hepatic vasoregulatory gene expression and Trolox ameliorates this change through its free radical scavenging activity.y.

• 미생물학회지
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• 제34권1_2호
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• pp.64-68
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• 1998

한국에서 분리된 전염성 조혈괴저바이러스(infectious hematopoietic necrosis virus, IHNV)인 IHNV-PRT의 특성을 규명하기 위하여 IHNV-PRT의 matrix 단백질인 M1 및 M2를 암호화하고 있는 cDNA를 클로닝하여 이들의 염기서열을 분석하였다. M1은 693 bp 크기의 open reading frame을 포함하였으며 이로부터 230개의 아미노산으로 구성된 25.9 kDa의 분자량을 가진 단백질이 합성될 수 있다. M2는 588 bp 크기의 open reading frame을 지니고 있으며 195개의 아미노산으로 구성된 21.9 kDa의 분자량을 지닌 단백질이 합성될 수 있다. IHNV-PRT의 M1과 M2의 아미노산 서열을 외국에서 분리된 IHNV들과 비교 분석한 결과 M1은 92-93%, M2는 97%의 상동성을 보였다. 이러한 사실은 IHNV의 M 단백질 유전자들은 IHNV의 strain에 관계없이 매우 보존되어 있음을 나타내준다.

• Journal of Ginseng Research
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• 제40권2호
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• pp.141-150
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• 2016

Background: Adipocyte-macrophage communication plays a critical role regulating white adipose tissue (WAT) inflammatory gene expression. Because WAT inflammation contributes to the development of metabolic diseases, there is significant interest in understanding how exogenous compounds regulate the adipocyte-macrophage crosstalk. An aqueous (AQ) extract of North American (NA) ginseng (Panax quinquefolius) was previously shown to have strong inflammo-regulatory properties in adipocytes. This study examined whether different ginseng extracts influence adipocyte-macrophage crosstalk, as well as WAT inflammatory gene expression. Methods: The effects of AQ and ethanol (EtOH) ginseng extracts ($5{\mu}g/mL$) on adipocyte and macrophage inflammatory gene expression were studied in 3T3-L1 and RAW264.7 cells, respectively, using real-time reverse transcription polymerase chain reaction. Adipose tissue organ culture was also used to examine the effects of ginseng extracts on epididymal WAT (EWAT) and inguinal subcutaneous WAT (SWAT) inflammatory gene expression. Results: The AQ extract caused significant increases in the expression of common inflammatory genes (e.g., Mcp1, Ccl5, Tnf-${\alpha}$, Nos2) in both cell types. Culturing adipocytes in media from macrophages treated with the AQ extract, and vice versa, also induced inflammatory gene expression. Adipocyte Ppar-${\gamma}$ expression was reduced with the AQ extract. The AQ extract strongly induced inflammatory gene expression in EWAT, but not in SWAT. The EtOH extract had no effect on inflammatory gene expression in either both cell types or WAT. Conclusion: These findings provide important new insights into the inflammo-regulatory role of NA ginseng in WAT.

• 대한한방내과학회지
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• 제25권4호
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• pp.324-336
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• 2004

Objectives : The purpose of this investigation is to evaluate the effects of Scutellaria baicalensis GEORGI on alteration in gene expression in a hypoxia model using cultured rat cortical cells. Methods : E18 rat cortical cells were grown in a Neurobasal medium containing B27 supplement. On 12 DIV, Scutellaria baicalensis GEORGI(20 ug/ml) was added to the culture media and left for 24 hrs. On 11 DIV, cells were given a hypoxic insult $(2%\;O_2/5%\;CO_2,\;37^{\circ}C,\;3\;hrs)$, returned to normoxia and cultured for another 24 hrs. Total RNA was prepared from Scutellaria baicalensis GEORGI-untreated (control) and -treated cultures and alteration in gene expression was analysed by microarray using rat 5K-TwinChips. Results : For most of the genes altered in expression, the Global M values were between -0.5 to +0.5. Among these, 1143 genes increased in their expression by more than Global M +0.1, while 1161 genes decreased by more than Global M -0.1. Effects on some of the genes whose functions are implicated in neural viability are as follows: 1) The expression of apoptosis-related genes such as Bad (Global M = 0.39), programmed cell death-2(Pdcd2) (Global M = 0.20) increased, while Purinergic receptor P2X(P2rxl) Global M = -0.22), Bc12-like1(Bc1211)(Global M = -0.19) decreased. 2) The expression of 'response to stress-related genes such as antioxidation-related AMP-activated protein kinase subunit gamma 1 gene (Prkag1) (Global M = 0.14), catalase gene (Global M = 0.14) and Heme Oxygenase(Hmoxl) increased. 3) The expression of Fos like antigen 2 (Fos12) expressed in neurons that survive ischemic insult increased (Global M = 0.97). Conclusions : these data suggest that Scutellaria baicalensis GEORGI increases the expression of antiapoptosis- and antioxidation- related genes in a way that can not yet be explained.

• Asian Pacific Journal of Cancer Prevention
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• 제16권6호
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• pp.2341-2345
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• 2015

This study aimed to investigate the relationship between prognosis and protein and mRNA expression of an apoptotic inhibitor gene, survivin, in patients with nasopharyngeal carcinoma. Furthermore, functions of the survivin gene in the CNE2 nasopharyngeal carcinoma cell line were assessed. Immunohistochemistry and in situ hybridization were used in detecting the survivin protein and mRNA in 44 nasopharyngeal carcinoma specimens, and 30 chronic nasopharyngitis samples as controls. Survivin gene expression in CNE2 cell line was suppressed with an shRNA (short hairpin RNA). The positive ratios of expression for survivin protein and mRNA in nasopharyngeal carcinoma were 79.5% and 75.0% respectively, obviously higher than in the control group (p<0.01), and there is very good consistency between the two methods. The mean survival time of patients with higher survivin protein or mRNA expression was shorter than in patients with lower levelsv(p<0.01). Proliferation of the CNE2 cell line was distinctly inhibited by the shRNA. The results indicate that overexpression of the survivin gene plays an important role in onset and development of nasopharyngeal carcinoma, and it may be helpful for prognostic appraisal.

• Environmental Analysis Health and Toxicology
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• 제27권
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• pp.11.1-11.8
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• 2012

Objectives: Hepatotoxicity of acetaminophen has been widely studied. However, the adverse effects on the heart have not been sufficiently evaluated. This study was performed to investigate cytotoxicity and alterations of gene expression in cultured cardiomyocytes ($H_9C_2$ cells) after exposure to acetaminophen. Methods: $H_9C_2$ cells were incubated in a 10 mM concentration of acetaminophen for the designated times (6, 12, and 24 hours), and cytotoxicity was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Alteration of gene expression was observed by microarray analysis, and RT-PCR was performed for the three representative oxidative stress-related genes at 24 hours after treatment. Results: It revealed that acetaminophen was toxic to cardiomyocytes, and numerous critical genes were affected. Induced genes included those associated with oxidative stress, DNA damage, and apoptosis. Repressed genes included those associated with cell proliferation, myocardial contraction, and cell shape control. Conclusions: These findings provide the evidences of acetaminophen-induced cytotoxicity and changes in gene expression in cultured cardiomyocytes of $H_9C_2$ cells.

• Asian Pacific Journal of Cancer Prevention
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• 제16권18호
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• pp.8197-8201
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• 2016

Background: To investigate the expression of the fragile histidine triad (FHIT) gene in acute lymphoblastic leukemia and its clinical significance. Materials and Methods: The level of expressed FHIT mRNA in peripheral blood from 50 patients with acute lymphoblastic leukemia (ALL) and in 50 peripheral blood samples from healthy volunteers was measured via RT-PCR. Correlation analyses between FHIT gene expression and clinical characteristics (gender, age, white blood count, immunophenotype of acute lymphoblastic leukemia and percentage of blast cells) of the patients were performed. Results: The FHIT gene was expressed at $2.49{\pm}7.37$ of ALL patients against $14.4{\pm}17.9$ in the healthy volunteers. The difference in the expression levels between ALL patients and healthy volunteers was statistically significant. The rate of gene expression did not significantly vary with immunophenotype subtypes. Gene expression was also found to be correlated with increase of total leukocyte and decrease in platelets, but not with age, gender, immunophenotyping or percentage of blast cells. Conclusions: FHIT gene expression is low in acute lymphoblastic leukemia and could be a useful marker to monitor minimal residual disease. This gene is also a candidate target for the immunotherapy of acute lymphoblastic leukemia.

• 한국발생생물학회지:발생과생식
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• 제16권1호
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• pp.31-38
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• 2012

Kisspeptin has been implicated in the process of puberty onset in various animal groups. This peptide is encoded by a gene, Kiss1 in avian and mammalian species. Contrary to these higher vertebrates, however, fish appeared to have another gene, Kiss2 that also codes for the precursor peptide of kisspeptin. To figure out biological significance of this gene during the puberty onset in fish, the expression profile of Kiss2 gene was investigated in the brain of Nile tilapia together with genes of GPR54, GnRH receptorI (rGnRHI) and GTH subunits ($LH{\beta}$ and $FSH{\beta}$). Expression of Kiss2 mRNA significantly increased at 2 weeks post hatch (wph) and 13 wph ($P$<0.05). This increase coincided with the increases of GPR54 and rGnRH I gene expression. Detection of $LH{\beta}$ and $FSH{\beta}$ subunit gene expression was possible later than 13 wph, indicating the activation of gonadotrophs in the pituitary. Data obtained from this study strongly suggest that, in addition to Kiss1 gene, Kiss2 gene is deeply associated with the onset of puberty by the activation of hypothalamus pituitary gonadal axis in Nile tilapia.