OBJECTIVE: To construct the co-expressing lentivirus vector of human glucocorticoid receptor (GR) beta and green fluorescent protein (GFP). METHOD: cDNA encoding hGRbeta obtained from the expression library of fetal brain using gene recombinant technology was cloned into pGC-LV by double digests technology. The constructed lentivirus vector of pGC-FU-GRbeta that was confirmed the sequencing was correct, transfected 293T cells through lipofectamine 2000. The constructed lentivirus vector was identified by fluorescence detection and Western Blot method. The packed lentivirus vector was used to infect 293T cells. The titer of virus was tested by real-time quantitative PCR. RESULT: A recombinant lentivirus vector co-expressing hGRbeta and GFP gene was constructed successfully. After transfection, a large number of 293T cells with green fluorescence were observed under fluorescent microscope, and the expression of GRbeta and GFP fusion protein was detected by Western Blot. The virus titer was 2.00E+8 TU/ml tested by Real-time PCR. CONCLUSION Successful construction of hGRbeta and GFP co-expressing lentivirus vector provides a stable vector for investigating the relationship between GRbeta and hormonal sensitivity or resistance in the therapy of chronic rhinosinusitis whose choice drug are glucocorticoids.
Cell Line ; DNA, Complementary ; genetics ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Humans ; Lentivirus ; genetics ; Receptors, Glucocorticoid ; genetics ; Transfection

DNA Mutational Analysis ; Drug Resistance ; genetics ; Genetic Variation ; Glucocorticoids ; pharmacology ; Humans ; Mutation ; Receptors, Glucocorticoid ; classification ; genetics ; physiology

Human monocyte leukemia cell line THP-1 was stimulated with lipopolysaccharide (LPS) to simulate the sepsis model and the expression of human glucocorticoid receptor-alpha (GR-alpha) mRNA in montocytes with endotoxin tolerance was investigated. THP-1 cells were cultured in serum-free medium, randomly divided into groups A, B, C, D and E, and stimulated with 0, 10, 10, 100, 0 ng/mL LPS for 24 h followed with 100, 100, 10, 100, 0 ng/mL LPS for another 24 h respectively. The expression of GR-alpha mRNA was detected by semi-quantitative reverse transcriptional polymerase chain reaction. Tumor necrosis factor-alpha (TNF-alpha) was determined by enzyme linked immunosorbent assay (ELISA). The results showed that the A values of GR-alpha/beta-actin in groups A, B, C, D and E was 0.607 +/- 0.006, 0.368 +/- 0.005, 0.484 +/- 0.008, 0.509 +/- 0.004 and 0.564 +/- 0.014 respectively with the difference being significant among the groups (P < 0.05). The GR-alpha mRNA expression was negatively correlated with the TNF-alpha expression (P < 0.01). It was concluded that the down-regulation of the expression of GR-alpha mRNA in endotoxin tolerance THP-1 cells might play an important role in the development of endotoxin tolerance in THP-1 cells.
Drug Tolerance ; Endotoxins/*pharmacology ; Leukemia, Monocytic, Acute/*metabolism ; Leukemia, Monocytic, Acute/pathology ; Monocytes/*metabolism ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Receptors, Glucocorticoid/*biosynthesis ; Receptors, Glucocorticoid/genetics ; Tumor Cells, Cultured

OBJECTIVE: To construct mesangial cell lines over- or under- expressing glucocorticoid receptor beta (GRbeta), to investigate the effect of GRbeta on glucocorticoid biological function, and to determine whether the overexpression of GRbeta explains the glucocorticoid-resistant in glomerular mesangial cells (GMCs). METHODS: The recombinant human sense or anti-sense gene of GRbeta was transferred into the rat GMCs by retrovirus-mediated stable transfection technique. Expression of hGRbeta mRNA in GMCs was determined by reverse transcription of total RNA followed by quantitative reverse transcription-polymerase chain reaction (RT-PCR), and the product of RT-PCR was then analyzed by gene sequencing. The expression of hGRbeta protein in GMCs was tested by Western blot. The inhibitory rate of dexamethasone-mediated cells on lipopolysaccharide (LPS)-stimulated GMC proliferation was detected to assess the effect of GRbeta at different expression levels on the glucocorticoid action. The cell proliferative activity in different cells with different levels of GRbeta was tested by MTT chromatometry. The change of cell cycle was analyzed by flow cytometry. RESULTS: RT-PCR and gene sequencing showed that the recombinant sense and anti-sense genes were correctly integrated into genomic DNA of mesangial cells. The protein expression tested by Western blot showed that GRbeta in cells inserted with the sense hGRbeta gene was higher than those cells inserted with the anti-sense hGRbeta gene (109.74+/-10.63 vs. 19.08+/-1.01, P<0.05). The inhibitory rate of cell proliferation induced by dexamethasone was lower in GMCs transfected with sense hGRbeta gene than those transfected with anti-sense hGRbeta gene (18.47%+/-2.12% vs. 60.33%+/- 5.29%,P<0.05). Under the inhibition of dexamethasone, the decreased cell number of S-stage cells was significantly lower, and the increased cell number of G1- stage cells was significantly higher in GMCs transfected with sense hGRbeta gene than those of non-transfected cells. CONCLUSION The overexpression of GRbeta may inhibit the glucocorticoid action in GMCs. The GRbeta level in mesangial cells may be an important factor in determining whether they are sensitive or resistant to glucocorticoid.
Animals ; Cell Line ; Dexamethasone ; pharmacology ; Glucocorticoids ; pharmacology ; Male ; Mesangial Cells ; metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, Glucocorticoid ; genetics ; metabolism ; Transfection

OBJECTIVEThe expression of glucocorticoid receptor (GR) expression in obstructive sleep apnea hypopnea syndrome (OSAHS) in children is currently unknown. The aim of this study was to determine the GR-alpha and GR-beta status in the adenoidal tissues in children with OSAHS.

METHODSThirty-four pediatric patients (aged 3-14 years, median 7.8 years) had sleep study with polysomnography before adenoidectomy. According to the criteria of apnea hypopnea index (AHI) > or = 5 /h or/and apnea index (AI) > or = 1/h, they were divided into OSAHS and non-OSAHS sub groups. The study was based on fluorescent quantitative PCR (FQ-RT-PCR) for the mRNA expression of GR-alpha and GR-beta in the adenoidal tissues in children.

RESULTSGR isoforms mRNA encoding for expression of both GR-alpha and GR-beta were detected in the adenoids of all children. GR-alpha mRNA level [(9.40 +/- 3.06) x 10(5) cDNA copies/microg total RNA] in the adenoidal tissues in OSAHS was lower than those in the non-OSAHS [(1.60 +/- 0.26) x 10(6) cDNA copies/microg total RNA] (F = 40.285, P < 0.001), whereas no differences found for GR-beta [(1.57 +/- 0.35) x 10(4) cDNA copies/microg total RNA, (1.52 +/- 0.18) x 10(4) cDNA copies/microg total RNA]. GR-alpha/GR-beta ratio was 62.3 +/- 20. 3 in OSAHS and 107.4 +/- 24.4 in non-OSAHS. AHI or AI was not related to the mRNA levels of GR-alpha and GR-beta in OSAHS or non-OSAHS.

CONCLUSIONSGR-alpha and GR-beta were detectable in the adenoidal tissues in children. These data indicated that the relationship between the expressions of GR and the clinical significance in OSAHS need further and profound investigation.

Adenoids ; metabolism ; Adolescent ; Child ; Child, Preschool ; Female ; Humans ; Male ; RNA, Messenger ; genetics ; Receptors, Glucocorticoid ; metabolism ; Sleep Apnea, Obstructive ; metabolism

Animals ; Brain ; growth & development ; metabolism ; Emotions ; Glucocorticoids ; therapeutic use ; Humans ; Memory ; RNA, Messenger ; analysis ; Receptors, Glucocorticoid ; analysis ; genetics ; physiology

OBJECTIVETo explore the association between plasma fat and glucose, cortisol and adrenocorticotropic hormone (ACTH) levels and genotypes of GR and ACTHR genes in healthy Chinese Han subjects.

METHODSTwo hundred healthy subjects were analyzed for GR and ACTHR gene polymorphisms using PCR-restriction fragment length polymorphism method. Plasma lipid, glucose, cortisol, ACTH levels were determined and correlated with the genotypes.

RESULTSNo significant difference was found between plasma lipid and glucose levels and various GR and ACTHR genotypes. Subjects with AG genotype of GR 5556A/G polymorphism had lower plasma cortisol levels than AA genotype. Compared with subjects with GG genotype of GR 4534-4536GAG/AAA [GAGAGG (GluArg)>GAAAAG(GluLys)] polymorphism, those with AG genotype had significantly lower plasma cortisol levels. Subjects with CC and CG genotypes of GR 6294C/G polymorphism also had significantly lower plasma cortisol levels compared with those with GG genotype. With regard to plasma ACTH levels, those with TT genotype of ACTHR 2T/C polymorphism were significantly lower than CC and CT genotypes, and those with AG genotype for GR 5556 A/G polymorphism were also significantly lower than AA genotype.

CONCLUSIONThere was no difference in plasma cortisol and glucose levels between subjects with GR and ACTHR gene variants. GR gene variants (5556A/G, 4534-4536GAG/AAA and 6294C/G polymorphisms) may influence plasma cortisol level, and ACTHR 2T/C, GR 5556A/G polymorphisms may decrease plasma ACTH level.

Adrenocorticotropic Hormone ; blood ; genetics ; Adult ; Blood Glucose ; genetics ; metabolism ; Female ; Genotype ; Humans ; Hydrocortisone ; blood ; genetics ; Lipids ; blood ; genetics ; Male ; Middle Aged ; Polymorphism, Genetic ; Receptors, Glucocorticoid ; genetics

BACKGROUNDGeneralized glucocorticoid resistance syndrome is a rare familial or sporadic condition characterized by generalized, partial, target-tissue insensitivity to glucocorticoids. This syndrome is partially caused by mutations in the human glucocorticoid receptor (hGR) gene. The clinical spectrum of generalized glucocorticoid resistance is broad, ranging from fatigue or no symptoms to severe hypertension with hypokalemic alkalosis. The purpose of this study was to explore the genetic disorder of glucocorticoid resistance syndrome.

METHODSWe identified a 56-year-old male patient diagnosed with generalized glucocorticoid resistance syndrome accompanied with an adrenocortical adenoma. This asymptomatic patient referred to Peking Union Medical College Hospital for treatment of his adrenal incidentaloma. Endocrinological evaluation consistently revealed his elevated serum cortisol level. Total RNA was extracted from the patient's peripheral blood mononuclear leukocytes (PBMLs) and entire coding region of hGR alpha was amplified by reverse transcription (RT)-PCR. To confirm the possible mutation identified by sequencing RT-PCR products, genomic DNA sequence of hGR gene from the patient and 50 healthy controls was analyzed by PCR and directly sequencing.

RESULTSA heterozygotic (C→T) substitution at nucleotide position of 1667 (exon 5) in GR alpha gene was found in this patient by sequencing of RT-PCR products of hGR gene. This substitution was also identified at genomic DNA level and it was absent in 100 chromosomes from 50 unrelated health controls. This substitution resulted in a threonine to isoleucine substitution (ACT→ATT) at amino acid 556 in the ligand-binding domain of GR alpha.

CONCLUSIONGeneralized glucocorticoid resistance in this patient might be caused by a novel heterozygotic mutation in the ligand-binding domain of the GR alpha.

Adrenocortical Adenoma ; genetics ; Drug Resistance ; genetics ; Endocrine System Diseases ; genetics ; Glucocorticoids ; pharmacology ; Humans ; Male ; Middle Aged ; Point Mutation ; Receptors, Glucocorticoid ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVEThe nephrotic syndrome is defined by heavy proteinuria, edema, hypoalbuminemia, and hyperlipidemia. Idiopathic nephrotic syndrome (INS) mainly occurs in children, which is generally treated with glucocorticoids. The majority of patients are steroid-sensitive (SSINS) while steroid-resistance occurs in a subset of NS children (SRINS). Although intensive efforts have been undertaken to study the associations between SRINS and renal pathological changes, pharmacokinetics, and the GR density and binding affinity, the mechanisms underlying steroid-resistance are still not elucidated entirely. The authors hypothesized that it might be associated with polymorphisms in the glucocorticoid receptor gene (NR3C1). The study aimed to screen the NR3C1 gene for polymorphisms in genomic DNA samples from SRINS, SSINS children and control group, and to analyze the association of the polymorphisms in the NR3C1 gene and SRINS of children.

METHODSTotally 39 SRINS and 67 SSINS children (81 males and 25 females with the mean age of 7 years) were involved in the study. Umbilical cord blood of 62 normal neonates and peripheral blood of 2 healthy volunteers were selected as controls. Genomic DNA was isolated from peripheral blood lymphocytes of all subjects. All the NR3C1-coding exons and intron-flanking portions were amplified by polymerase chain reaction (PCR). For polymorphism screen, PCR products were analyzed by denaturing high performance liquid chromatography (DHPLC). DNA fragments with aberrant elution profiles were re-amplified and sequenced directly.

RESULTSTwelve aberrant elution profiles were identified with DHPLC in SRINS, SSINS and controls. Among them, 6 previously reported polymorphisms and 6 novel polymorphisms were confirmed by sequencing (198G > A, 200G > A, IVSD-16G > T, 1896C > T, 2166C > T, 2430T > C; novel, 1206C > T, 1374A > G, IVSG-68_IVSG-63delAAAAAA, 2193T > G, IVSH-9C > G, 2382C > T), and 3 groups of SNPs were in complete linkage disequilibrium, which resulted in 3 different haplotypes ([198G > A + 200G > A], [1374A > G + IVSG-68_IVSG-63delAAAAAA + IVSH-9C > G + 2382C > T], [1896C > T + 2166C > T + 2430T > C]). The last two genotypes were first reported. The genotype frequencies of the 2 novel haplotypes were 10.3% vs 1.5% in SRINS and SSINS, and 15.4% vs 7.5% in SRINS and SSINS, respectively. Other polymorphisms were relatively rare detectable both in patients and controls.

CONCLUSIONTwelve polymorphisms in the NR3C1 gene were detected with the technique of DHPLC, of which six polymorphisms were identified at the first time. Two types of newly found haplotypes were associated with steroid-resistant idiopathic nephrotic syndrome of children, which might be responsible for steroid-resistance in partial idiopathic nephrotic syndrome of children.

Adolescent ; Child ; Child, Preschool ; Chromatography, High Pressure Liquid ; methods ; Drug Resistance ; genetics ; Female ; Humans ; Male ; Nephrotic Syndrome ; genetics ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Receptors, Glucocorticoid ; genetics

BACKGROUNDGlucocorticoid receptor (GR) is believed to be a major factor in brain maturation and in modulation of a series of brain activity. Hippocampal neurons are abundant in glucocorticoid receptor, and there is significant change in GR expression under certain pathological state. Epilepsy is a special pathological state of the central nervous system. This study aimed to explore the role of GR in epilepsy by observing the change and functions of GR in hippocampus with a basolateral amygdale-electrical kindled rat epilepsy model.

METHODSFirstly, we established the basolateral amygdale-electrical kindled rat epilepsy model. Then GR mRNA expression in the hippocampus was assayed by semi-quantitative reverse transcription-PCR in this experiment. In addition, the processes of epileptic seizures were observed and electroencephalograms were recorded. One-way analysis of variance (ANOVA) was employed for comparing means of multiple groups, followed Fisher's least significant difference (LSD) for paired comparison.

RESULTSThe rats were successfully kindled after an average of (13.50 ± 3.99) times electrical stimulation, in which it was showed that GR mRNA expression reduced obviously as compared with the control group and the sham groups (P < 0.001). The down-regulation of GR mRNA expression was abated or reversed by some anti-epilepsy drugs (P < 0.001 compared with the epilepsy group), accompanied by attenuation of seizures and improvement of electroencephalograms.

CONCLUSIONSDown-regulation of hippocampal GR mRNA expression may be related to the kindling. Anti-epilepsy drugs exposure can retard this change.

Amygdala ; metabolism ; Animals ; Epilepsy ; genetics ; Kindling, Neurologic ; genetics ; Male ; Rats ; Rats, Sprague-Dawley ; Receptors, Glucocorticoid ; genetics ; Reverse Transcriptase Polymerase Chain Reaction