ABSTRACT
Coronary artery disease (CAD) is a multifactorial disease in which inflammation plays a central role.This study aimed to investigate the association of inflammatory markers such as the neutrophil to lymphocyte ratio (NLR),the Global Registry of Acute Coronary Events (GRACE) score with in-hospital mortality of elderly patients with acute myocardial infarction (AMI) in an attempt to explore the prognostic value of these indices for elderly AMI patients.One thousand consecutive CAD patients were divided into two groups based on age 60.The laboratory and clinical characteristics were assessed retrospectively by reviewing the medical records.The NLR and GRACE score were calculated.In the elderly (≥60 years),patients with non-ST-elevation myocardial infarction (NSTEMI) and ST-elevation myocardial infarction (STEMI) had significantly higher NLR than did those with unstable angina (UA) and stable angina pectoris (SAP) (P＜0.01).The NLR was considerably elevated in older AMI patients compared with their younger counterparts (＜60 years) (P＜0.05).In elderly AMI patients,the NLR was considerably higher in the high-risk group than in both the low-risk and medium-risk groups based on the GRACE score (P＜0.05 and P＜0.01,respectively),and the NLR was positively correlated with the GRACE score (r=0.322,P＜0.001).Either the NLR level or the GRACE score was significantly higher in the death group than in the surviving group (P＜0.05).By curve receiver operator characteristic curve (ROC) analysis,the optimal cut-off levels of 9.41 for NLR and 174 for GRACE score predicted in-hospital death [ROC area under the curve (AUC) 0.771 and 0.787,respectively,P＜0.001].It was concluded that an elevated NLR is a potential predictor of in-hospital mortality in elderly patients with AMI.
ABSTRACT
Coronary artery disease (CAD) is a multifactorial disease in which inflammation plays a central role.This study aimed to investigate the association of inflammatory markers such as the neutrophil to lymphocyte ratio (NLR),the Global Registry of Acute Coronary Events (GRACE) score with in-hospital mortality of elderly patients with acute myocardial infarction (AMI) in an attempt to explore the prognostic value of these indices for elderly AMI patients.One thousand consecutive CAD patients were divided into two groups based on age 60.The laboratory and clinical characteristics were assessed retrospectively by reviewing the medical records.The NLR and GRACE score were calculated.In the elderly (≥60 years),patients with non-ST-elevation myocardial infarction (NSTEMI) and ST-elevation myocardial infarction (STEMI) had significantly higher NLR than did those with unstable angina (UA) and stable angina pectoris (SAP) (P＜0.01).The NLR was considerably elevated in older AMI patients compared with their younger counterparts (＜60 years) (P＜0.05).In elderly AMI patients,the NLR was considerably higher in the high-risk group than in both the low-risk and medium-risk groups based on the GRACE score (P＜0.05 and P＜0.01,respectively),and the NLR was positively correlated with the GRACE score (r=0.322,P＜0.001).Either the NLR level or the GRACE score was significantly higher in the death group than in the surviving group (P＜0.05).By curve receiver operator characteristic curve (ROC) analysis,the optimal cut-off levels of 9.41 for NLR and 174 for GRACE score predicted in-hospital death [ROC area under the curve (AUC) 0.771 and 0.787,respectively,P＜0.001].It was concluded that an elevated NLR is a potential predictor of in-hospital mortality in elderly patients with AMI.
ABSTRACT
Objective To explore the prognostic value of biomarkers in type 2 diabetic patients with acute myocardial infarction (AMI), this study was to investigate the associations between the neutrophil to lymphocyte ratio (NLR), the Global Registry of Acute Coronary Events (GRACE) score and in-hospital mortality. MethodsSeven hundred and seven consecutive AMI patients were divided into diabetic group (DM-AMI group), impaired glucose tolerance group (IGT-AMI group), and normal glycemic group (NGT-AMI group). The laboratory and clinical characteristics were assessed retrospectively from the medical records. The NLR and GRACE score were calculated. Results In AMI patients, the DM-AMI group had significantly higher NLR and GRACE scores compared with those from the IGT-AMI group and NGT-AMI group (P>=Neutrophil to lymphocyte ratio; In-hospital mortality; Acute myocardial infarction; Diabetes mellitus, type 2
ABSTRACT
Posterior ischemic optic neuropathy is a kind of ischemic optic neuropathy, the incidence rate of which is lower with less obviously clinical features, less positive signs and more difficultly diagnosis when compared with anterior ischemic optic neuropathy.Meanwhile, therapeutic method of posterior ischemic optic neuropathy has remained controversial.This article will summarize the research development of the auxiliary examination, diagnosis, differential diagnosis and therapeutic method of posterior ischemic optic neuropathy.
ABSTRACT
To compare the clinical effect of Qianliekang and finasteride in the treatment of benign prostatic hyperplasia to explore the effectiveness of traditional Chinese medicine for the therapy of benign prostatic hyperplasia. Methods:Totally 36 Wistar rats were selected, and then divided into 3 groups randomly with 12 ones in each, namely Qianliekang group, finasteride group and the control group. After 14 days of castration, the three groups were all treated with subcutaneous injection of 5 mg kg-1 testosterone propi-onate, and Qianliekang group was additionally treated with intragastric administration at 10-fold adult dose, finasteride group was trea-ted with intragastric administration at the dose of 0. 1 mg·kg-1 , and the control group was treated with the same amount of distilled water. The rats were sacrificed after the 21-day treatment, and the wet weight of prostate was determined, the prostate volume was measured and the pathological changes in prostate tissue were observed under a light microscope. Results:The wet weight of prostate in Qianliekang group and finasteride group was (0. 467 ± 0. 061) g and(0. 408 ± 0. 058) g, respectively, the prostate volume was (0. 371 ± 0. 059)ml and(0. 365 ± 0. 054)ml, respectively, and the above indicators were significantly lower than those in the control group(P<0. 05). Conclusion:Qianliekang can effectively inhibit benign prostatic hyperplasia in the model rats, and the mechanism may be related to the proliferation inhibition of prostate cells.
ABSTRACT
Asiaticoside is a compound extracted from traditional Chinese medicine Centella asiatica, and mainly used in wound healing and scar repair in clinical, with notable efficacy. However, its poor transdermal absorption and short action time restrict its wide application. In this experiment, the reserve-phase-extrusion-lyophilization method was conducted to prepare the lyophilized asiaticoside-loaded flexible nanoliposomes (LAFL). Its characteristics including electron microscope structure, particle size, Zeta potential, entrapment rate, drug-loading rate, stability and drug release were determined with the intelligent transdermal absorption instrument. LAFL were white spheroids, with pH, particle size and zeta potential of 7. 03, 70. 14 nm and - 36. 5 mV, respectively. The average entrapment rate of the 3 batch samples were 31. 43% , and the average asiaticoside content in 1 mg lyophilized simple was 0. 134 mg. The results indicated that LAFL have good physicochemical properties and pharmaceutical characteristics, with an improved transdermal performance.
Subject(s)
Animals , Liposomes , Chemistry , Nanoparticles , Chemistry , Triterpenes , ChemistryABSTRACT
Objective To investigate the relationship between the mutation of the exon 4 of leptin receptor gene(4Lys 109 Arg)and left ventricular function and renal hemodynamics in patients with hypertension.Methods From December 2005 to December 2006,digoxin marked primer enzyme coloration technique was used to detect the 4Lys 109 Arg polymorphism.The color Doppler echocardiography was used to detect the left ventricular function and the renal hemodynamics.The study covered 90 patients with hypertension (hypertension group)and 52 controls (control group).Results The frequencies of 4Lys109Arg in hypertension group and control group had no significant difference.The hypertension with GG genotype was significant difference in left ventrieular ejection fraction(LVEF),E/A,left ventrieular fractional shortening(LVFS),resistance index(BI),pulsatility index(PI)(P<0.05).The hypertension with GA genotype had significant difference in LVEF,E/A,LVFS(P<0.05).In hypertension group,LVEF,E/A,LVFS.RI and PI had significant difference between GG and AA genotype(P<0.05).In control goup,RI had significant difference between GG and AA genotype(0.65 ±0.02 vs 0.63±0.02,P<0.05).Conclusion The renal hemodynamics and the left ventricular function are associated with 4Lys109Arg polymorphism.
ABSTRACT
The aim of the present study was to investigate the role of peroxisome proliferator-activated receptor γ (PPARγ) signal transduction pathway in the expression of ATP binding cassette transporter A1 (ABCA1) and acyl-CoA:cholesterol acyltransferase 1 (ACAT1) induced by visfatin and to discuss the mechanism of foam cell formation induced by visfatin. THP-1 monocytes were induced into macrophages by 160 nmol/L phorbol myristate acetate (PMA) for 48 h, and then the macrophages were exposed to visfatin and PPARγ activator rosiglitazone, respectively. The expressions of PPARγ, ABCA1 and ACAT1 mRNA and protein were determined by RT-PCR and Western blot respectively. The contents of total cholesterol (TC) and free cholesterol (FC) were detected by enzyme fluorescence analysis. The content of cholesterol ester (CE) was calculated by the difference between TC and FC. The results showed that visfatin decreased the mRNA and protein expressions of PPARγ and ABCA1, increased the mRNA and protein expressions of ACAT1, and increased the contents of FC and CE in a concentration-dependent manner. These above effects of visfatin were inhibited by rosiglitazone in a concentration-dependent manner. These results suggest that visfatin may down-regulate the ABCA1 expression and up-regulate the ACAT1 expression via PPARγ signal transduction pathway, which decreases the outflow of FC, increases the content of CE, and then induces foam cell formation.
Subject(s)
Humans , ATP Binding Cassette Transporter 1 , ATP-Binding Cassette Transporters , Genetics , Metabolism , Acetyl-CoA C-Acetyltransferase , Genetics , Metabolism , Cell Differentiation , Cell Line , Cholesterol Esters , Metabolism , Foam Cells , Cell Biology , Macrophages , Cell Biology , Monocytes , Cell Biology , Nicotinamide Phosphoribosyltransferase , Pharmacology , PPAR gamma , Physiology , RNA, Messenger , Genetics , Metabolism , Signal Transduction , Thiazolidinediones , PharmacologyABSTRACT
AIM: To investigate the signal transduction mechanism of Chlamydia pneumoniae (Cpn) in down-regulating the expression of ATP binding cassette A1 (ABCA1) and ATP binding cassette G1 (ABCG1),the key molecules in cholesterol efflux and atherogenesis,from THP-1-derived macrophages. METHODS: Cpn was propagated in Hep-2 cells. THP-1 monocytes were induced into macrophages by 160 nmol/L phorbol myristate acetate (PMA) for 48 h,and were randomly allocated into 4 groups to incubate continually: control group,50 mg/L low density lipoprotein (LDL); Cpn infection group,Cpn (1×10~6 IFU) and 50 mg/L LDL; Cpn and SP600125 (a special JNK inhibiter) group,THP-1 macrophages were previously treated with different concentrations (1-20 μmol/L) of SP600125 for 1 h,and then infected with Cpn (1×10~6 IFU) and 50 mg/L LDL; SP600125 group,SP600125(20 μmol/L)and 50 mg/L LDL. The expressions of ABCA1/ABCG1 and peroxisome proliferator-activated receptor γ (PPARγ) from each group were detected then. The cholesterol efflux was detected by enzyme-fluorescence. The expressions of ABCA1/ABCG1 and PPARγ mRNA and protein were determined by RT-PCR and Western blotting,respectively. RESULTS: Cpn not only down-regulated the ABCA1/ABCG1 expression,but also down-regulated the expression of PPARγ,which regulated the ABCA1/ABCG1 genes transcriptions. However,the mentioned effects of Cpn infection were restrained by the special JNK inhibitor SP600125 in a dose-dependent manner. CONCLUSION: Chlamydia pneumoniae may down-regulate ABCA1/ABCG1 expression from THP-1-derived macrophages via JNK-PPARγ signal transduction pathway.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of Ghrelin on the expression of acyl coenzyme A:cholesterol acyltransferases-1 (ACAT-1) in THP-1 derived foam cells.</p><p><b>METHODS</b>The human monocytic leukemia cell line (THP-1) was chosen in our study. The differentiation of THP-1 cells into macrophages was induced by phorbol 12-myristate 13-acetate. Macrophages were then incubated with oxidized LDL (ox-LDL) to generate foam cells. Ghrelin and [D-Lys3]-GHRP-6, the special antagonist of growth hormone secretagogue receptor (GHS-R), were treated during foam cells formation. The ACAT-1 protein and mRNA levels were detected by Western blot and RT-PCR. The effect of variance of cholesterol content was measured by zymochemistry via-fluorospectrophotometer.</p><p><b>RESULTS</b>Ghrelin reduced the content of cholesterol ester in foam cells obviously. ACAT-1 protein and mRNA levels were also decreased. The antagonist of GHS-R inhibited the effects of Ghrelin on ACAT-1 expression in dose-dependent manner. The ACAT-1 mRNA levels of the GHS-R specific antagonist groups (10(-5), 5 x 10(-5), 10(-4) mol/L) were 1.14 +/- 0.04, 1.58 +/- 0.03, 2.40 +/- 0.16, significantly higher than that of the Ghrelin group (0.89 +/- 0.05). And the protein expressions were 1.25 +/- 0.09, 1.77 +/- 0.11, 2.30 +/- 0.09, also higher than that of the Ghrelin group (0.86 +/- 0.08).</p><p><b>CONCLUSIONS</b>Ghrelin might interfere atherosclerosis by down-regulating the expression of ACAT-1 via GHS-R pathway.</p>
Subject(s)
Humans , Acetyl-CoA C-Acetyltransferase , Metabolism , Acyl Coenzyme A , Metabolism , Blotting, Western , Cell Line, Tumor , Cholesterol , Metabolism , Down-Regulation , Foam Cells , Metabolism , Ghrelin , Physiology , RNA, Messenger , Metabolism , Receptors, Ghrelin , Physiology , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , SpectrophotometryABSTRACT
Objective To investigate the role of c-Jun N-terminal kinase (JNK) signal transduction pathway on the up-regulation of the expression of acyl-coenzyme A: cholesterol acyltransferasel (ACAT1) induced by Chlamydia pneumoniae (C. pn), and to discuss the mechanism of macrophages-derived foam cell formation induced by C. pn. Methods C. pn was propagated in Hep-2 cells. THP-1 monocytes were induced into macrophages by 160 nmol/L phorbol myristate acetate(PMA) for 48 h, and were randomly allocated into four groups to be incubated continually: control group, C. pn infection group, C. pn and SP600125 (a special JNK inhibitor)group and SP600125 group. Lipid droplets in cytoplasm were observed by oil red O staining. The contents of intracellular cholesterol ester were detected by enzyme fluorescence analysis. The expressions of ACAT1 mRNA and protein were determined by reverse transcriptase polymerase chain reaction(RT-PCR) and Western blot, respectively. Results Compared with the control group, the expressions of ACAT1 mRNA and protein were up-regulated in C. pn infection group [(4.16±0.26) vs. (2.17±0.18), (1.20±0.10)vs. (0.61±0.03), both P<0.05], and C. pn-induced foam cell formation was observed. The expressions of ACAT1 mRNA and protein and the foam cell formation were inhibited by SP600125 in a concentration-dependent manner (r = - 0.92, P<0.05; r= - 0. 96, P<0.05, respectively). Conclusions The up-regulation of ACAT1 expression is induced by C. pn via JNK signal transduction pathway, which is involved in the mechanism of C. pn-induced macrophage-derived foam cell formation.
ABSTRACT
<p><b>OBJECTIVE</b>To probe into indication of living-related liver transplantation (LRLT) for Wilson's Disease.</p><p><b>METHODS</b>From January 2001 to February 2007, thirty-seven living-related liver transplants were performed. A retrospective analysis was carried on outcome of those patients. The indications for LRLT were acute hepatic failure in 3 patients and chronic advanced liver disease in 32 patients including 13 patients with Wilsonian neurological manifestations. Two patients presented with severe Wilsonian neurological manifestations even though their liver functions were stable. According to the scoring system for evaluation of the neurological impairment in Wilson disease based on neurological signs and functions (total score was 30), the pre-transplantation score of those patients with neurological manifestations was 15.9 +/- 4.3 (n = 15).</p><p><b>RESULTS</b>Thirty-seven patients were followed up for 20 - 93 months. The survival rates of post-transplant patients and grafts at 1, 3, and 5 year were 91.9%, 83.8%, 75.7%, and 86.5%, 78.4%, 75.7%, respectively. Postoperative surgical complications occurred in 2 donors with bile leakage required drainage, in 2 recipients with hepatic thrombosis underwent retransplantation of cadaveric liver and in 1 recipient with hepatic stenosis required balloon dilatation. Neurological function was improved in all recipients and the score of posttransplantation at 6, 12, 18, 24, and 30 month was 17.5 +/- 3.7 (n = 13); 21.0 +/- 4.3 (n = 12); 23.9 +/- 3.9 (n = 10); 26.6 +/- 2.2 (n = 10) and 28.1 +/- 1.9 (n = 7) respectively.</p><p><b>CONCLUSIONS</b>Patients with acute hepatic failure or patients with severe liver disease unresponsive to chelation treatment should be treated with LRLT. Early transplantation in patients with an unsatisfactory response medical treatment may prevent irreversible neurological deterioration even though their liver function is stable.</p>
Subject(s)
Adolescent , Adult , Child , Female , Humans , Male , Young Adult , Follow-Up Studies , Hepatolenticular Degeneration , General Surgery , Liver Failure , General Surgery , Liver Transplantation , Living Donors , Nervous System Diseases , Retrospective Studies , Transplantation, Homologous , Treatment OutcomeABSTRACT
<p><b>OBJECTIVE</b>To study the efficacy of anti-telomerase siRNA in hepatocellular carcinoma both in vitro and in vivo.</p><p><b>METHODS</b>Lentvirus vectors contained anti-telomerase siRNA were conducted with a high performance homologous recombination system, and then were transduced into human hepatocellular carcinoma HepG2 cells. The telomerase activity was detected by RT-PCR, HepG2 cell proliferation was determined by MTT assay, and apoptosis was detected by TUNEL assay. The in vivo experiment was carried out by inoculation of HepG2 cells into nude mice and the tumor growth was measured and analyzed.</p><p><b>RESULTS</b>The growth of transfected HepG2 cells was significantly inhibited and the inhibition rate was 57.5% at the 8th day after transfection. The telomerase activity was significantly suppressed in vitro. The growth of transfected human hepatocellular HepG2 tumor in the nude mice was also significantly inhibited.</p><p><b>CONCLUSION</b>The results of this study demonstrate that the growth of hepatocellular carcinoma cells is effectively inhibited by transfection of anti-telomerase siRNA both in vitro and in vivo.</p>
Subject(s)
Animals , Female , Humans , Mice , Apoptosis , Carcinoma, Hepatocellular , Therapeutics , Cell Proliferation , Genetic Therapy , Methods , Genetic Vectors , Hep G2 Cells , Lentivirus , Genetics , Liver Neoplasms , Therapeutics , Mice, Inbred BALB C , Mice, Nude , Neoplasm Transplantation , RNA, Messenger , Metabolism , RNA, Small Interfering , Recombinant Proteins , Genetics , Metabolism , Telomerase , Genetics , Metabolism , Transfection , Tumor BurdenABSTRACT
<p><b>OBJECTIVE</b>To investigate the expression changes of acyl-coenzyme A: cholesterol acyltransferase 1 (ACAT1) on Chlamydia pneumoniae (C.pn) induced foam cell formation.</p><p><b>METHODS</b>Human monocytic cell line (THP-1) was induced into macrophages by 160 nmol/L phorbol myristate acetate (PMA) for 48 h, and were randomly allocated into four groups: negative control group (50 microg/ml LDL for 48 h); positive control group (50 microg/ml ox-LDL for 48 h); C.pn infection group (50 microg/ml LDL plus 1 x 10(5), 4 x 10(5), 5 x 10(5) and 1 x 10(6) IFU C.pn for 48 h or 1 x 10(6) IFU C.pn for 0, 24, 48 and 72 h); ACAT inhibitor 58-035 plus C.pn infection group (1, 5, 10 microg/ml ACAT inhibitor 58-035 pretreatment for 1 h, 50 microg/ml LDL and 1 x 10(6) IFU C.pn for 48 h). The mRNA and protein expressions of ACAT1 were determined by RT-PCR and Western blot, respectively. Lipid droplets in cytoplasm were observed by oil red O staining. The contents of intracellular cholesteryl esters were detected by enzyme-fluorescence.</p><p><b>RESULTS</b>The mRNA and protein expressions of ACAT1 were significantly up-regulated in positive control cells compared those in negative control cells and further upregulated by C.pn infection in a time-dependent and concentration-dependent manner (all P < 0.05). There were significantly increases in the accumulation of lipid droplets and the ratio of cholesteryl ester to total cholesterol in positive control cells as compared with negative control cells and these were further aggravated by C.pn (at the concentrations of 5 x 10(5) and 1 x 10(6) IFU for 48 h) and C.pn infection induced increases in the accumulation of lipid droplets and the ratio of cholesteryl ester to total cholesterol could be significantly attenuated by ACAT inhibitor 58-035 (all P < 0.05).</p><p><b>CONCLUSION</b>Chlamydia pneumoniae induces THP-1-derived foam cell formation by up-regulating the expression of ACAT1.</p>
Subject(s)
Humans , Cell Line, Tumor , Chlamydophila pneumoniae , Foam Cells , Cell Biology , Metabolism , Monocytes , Cell Biology , Sterol O-Acyltransferase , Metabolism , Up-RegulationABSTRACT
Objective To investigate the mechanisms of Chlamydia pneumoniae (C. pn)-induced foam cell formation, the expression of ATP binding cassette transporter AI ( ABCA1 ) and perexisome prolif-erator-activated receptor γ (PPARγ) were examined. Methods THP-1 monneytes were induced into mac-rophages after the addition of 160 nmol/L phorbol myristate acetate (PMA) for 72 h. THP-1-dorived macro-phages when co-cuhured 50 mg/L low density lipoprotein (LDL) were designated randomly in four groups: control (uninfected) group, C. pn infection group, rosiglitazone + C. pn infection group and rosiglitazone group. Lipid droplets in cytoplasm were observed by oil red O staining. The contents of intracellnlur choles-terol ester were detected by enzyme-flnoreseence. The expression of ABCA1, PPARγ, mRNA and protein were determined by RT-PCR and Western blot, respectively. Results C. pn down-regulated the expression of ABCA1, PPARγ at mRNA and protein levels in a concentration-dependent manner in THP-1-derived mac-rophages when co-incubated with LDL. Resiglitazone not only concentration-dependently alleviated the down-regulation of ABCA1 expression by C. pn infection (P<0.05), but also markedly suppressed the accumula- tion of lipid droplets and cholesteryl ester by C. pn at higher concentrations ( 10 and 20 μaol/L). Condu-sion C. pn induces foam cell formation by down-regulating the expression of ABCA1 via PPART pathway, which may provide a new evidence for the development and progression of atherosclerosis initiated by C. pn infection.
ABSTRACT
Objective:To investigate the effects of Chlamydia pneumoniae(Cpn) on SR-A1 and CD36 expression in THP-1-derived macrophages and role of c-Jun NH_2-terminal signal transduction pathway in the process.Methods:Cpn was propagated in Hep-2 cells.THP-1 monocytes were induced into macrophages by 160 nmol/L phorbol myristate acetate(PMA)for 48h,and were randomly allocated into four groups to be incubated continually: control group;Cpn infection group;Cpn and SP600125(a JNK inhibiter)group and SP600125 group.Lipid droplets in cytoplasm were observed by oil red O staining.The contents of intracellular cholesterol ester were detected by enzyme-fluorescence.The expression of SR-A1 and CD36mRNA and protein were determined by RT-PCR and Western blot, respectively. Results:THP-1-derived macrophages infected with Cpn resulted in large accumulation of lipid droplets and foam cell formation when co-cultured with LDL.Meanwhile,the expression of SR-A1 mRNA and protein were up-regulated by Cpn infection (P<0.05).However,the expressions of CD36 mRNA and protein in THP-1-derived macrophages infected with Cpn were unchanged.Moreover,the up-regulation of SR-A1 and foam cell formation induced by Cpn could be restrained by the JNK inhibiter SP600125 in a dose-dependent manner,and SP600125 had little impact on the expression of CD36 in THP-1-derived macrophages infected with Cpn.Conclusion:The up-regulation of SR-A1 but not CD36 expression is involved in mechanisms of Cpn inducing foam cell formation.And Chlamydia pneumoniae up-regulates the expression of SR-A1 via the JNK signal transduction pathway.This may be a novel mechanism for the foam cell formation induced by Cpn.
ABSTRACT
The DNA segment of the human acyl coenzyme A: cholesterol acyltransferasel (ACAT1) gene P7 promoter was amplified by PCR from human monocytic leukemia cell line (THP-1) and cloned to TA vector, then the positive clone was confirmed by restriction enzymes and sequencing. The targeted segment was subcloned to Firefly luciferase report vector pGL3-Enhancer. The recombinant plasmid pGL3E-P7 was transfected transiently into THP-1, then the expression of luciferase could be detected in THP-1 by pGL3E-P7 transfection. We successfully constructed luciferase reporter vector containing P7 promoter of the human ACAT1 gene, and established a new means to study the transcriptional regulation mechanisms of ACAT1 during atherosclerosis.
Subject(s)
Humans , Cell Line, Tumor , Chromosomes, Human, Pair 7 , Genetics , Gene Expression Regulation , Genes, Reporter , Genetics , Genetic Vectors , Genetics , Leukemia, Monocytic, Acute , Pathology , Luciferases, Firefly , Genetics , Metabolism , RNA, Messenger , Genetics , Metabolism , Recombinant Proteins , Genetics , Metabolism , Sterol O-Acyltransferase , Genetics , Metabolism , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To study the mechanism of enhancement of the CTL activity in mice co-expressing of CD80, CD86 and CD137L genes.</p><p><b>METHODS</b>The mice were randomly divided into five groups, named A, B, C, D and E. The group A and B were control groups (CG). H22-BAL B/c HCC mouse model was established by subcutaneous injection with hepatocellular carcinoma cells of cell line H22-Wt (group A), H22-neo (group B), H22-CD80/CD86(+) (group C), H22-CD137L(+) (group D) and H22-CD80/CD86/CD137L(+) (group E), respectively. On the 14th, 35th, 56th and 84th day after the first inoculation of tumor cells, TUNEL staining and DNA ladder examination were used to detect apoptosis of splenic T lymphocytes in all groups at each post-inoculation time point. Electrophoretic mobility-shift assay (EMSA) method was used to detect the activity of nuclear factor kappaB (NF-kappaB) in splenic T lymphocytes in each group at each time point post-inoculation.</p><p><b>RESULTS</b>Apoptosis was found in a great number of T lymphocytes in CG on the 14th day, much more than that in group C and E. The number of apoptotic T cells in group C had a significant difference compared with that in the group E from 14th to 84th day (P = 0.003). DNA ladder analysis showed typical positive results in group C and E. The significant apoptosis fragments were found in group C on 21st, 35th and 84th days. NF-kappaB activity of T cells in groups C and E was remarkably higher than that of groups CG and D, with higher in group D than that of CG (P = 0.002), and with no significant difference between group C and E on 14th day. The activity in group E was stable and remarkably higher than that of group C on 56th and 84th days after the first inoculation.</p><p><b>CONCLUSION</b>H22-CD80/CD86/CD137L(+) induces higher NF-kappaB activity of the host T cells by synergistic action of CD28 and CD137, which may be one of the mechanisms of enhancement of the host CTL activity induced by co-expression of CD80, CD86 and CD137L genes.</p>
Subject(s)
Animals , Female , Mice , 4-1BB Ligand , Metabolism , Apoptosis , B7-1 Antigen , Metabolism , B7-2 Antigen , Metabolism , CD28 Antigens , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Liver Neoplasms, Experimental , Allergy and Immunology , Metabolism , Pathology , Lymphocyte Activation , Mice, Inbred BALB C , NF-kappa B , Metabolism , Random Allocation , Spleen , Pathology , T-Lymphocytes , Metabolism , Pathology , T-Lymphocytes, Cytotoxic , Allergy and ImmunologyABSTRACT
In order to explore the effect and mechanisms of tumor necrosis factor-α (TNF-α) on the activity of the acyl coenzyme A: cholesteryl acyltransferase (ACAT), THP-1 monocytes were cultured and induced to differentiate into macrophages with phorbol ester. TNF-α (60 ng/mL) was added at different time points into the macrophage-containing medium and the ACAT enzyme activity was measured by quantifying the incorporation of [1-14C] oleoyl CoA into cholesteryl esters. The expression of ACAT-1 protein and mRNA was respectively detected by Western blotting and RT-PCR in THP-1 macrophages 24 h after treatment with TNF-α (60 ng/mL). The results indicated that ACAT activity in THP-1 macrophages treated with TNF-α was increased in a time-dependent manner. The expression levels of ACAT-1 protein and mRNA were significantly increased in THP-1 macrophages after treatment with TNF-α (P＜0.05). It was suggested that TNF-α could increase the activity of ACAT in THP-1 macrophages by up-regulating the expression of ACAT-1 gene.