Influence of ovariectomy and estrogen replacement on gene expression profile of myocardium in rats

AIM:To investigate the influence of ovariectomy and estrogen replacement treatment on profile of gene expression in myocardium by cDNA microarray,and to characterize the targeting genes of estrogen.METHODS:cDNA microarray containing 1 400 rat cDNAs was used to study the genes differentially expressed in myocardium between sham (Ⅰ),ovariectomy (Ⅱ,OVX) and estrogen replacement treatment (Ⅲ,OVX+E2) group.Then down-regulated genes in myocardium of OVX rats were further confirmed by RT-PCR.RESULTS:177 genes were differentially expressed in myocardium between sham and OVX rats,with 91 genes up-regulated and 86 genes down-regulated in OVX rats.164 genes were differentially expressed in myocardium between OVX and OVX+E2 rats,with 113 genes up-regulated and 54 genes down-regulated in OVX rats.There were 54 genes differentially expressed in OVX compared to sham and OVX+E2.They are involved in membrane channels and transporters (18),cell receptors (9),intracellular transducers/effectors/modulator (7) and metabolism (6).Most of the genes (45) were down-regulated in OVX rats and up-regulated in OVX+E2 rats.RT-PCR test confirmed the results of cDNA microarray.CONCLUSIONS:Long-term estrogen replacement may influence the expression of genes involved in membrane channels and transporters,cell receptors,intracellular transducers/effectors/ modulator and metabolism.Long-term estrogen replacement has some beneficial effects on ionic concentration and cardiac function which partially comes from the results of influence of expression on Na+,K+-ATPase and Na+/H+ exchanger.Estrogen has an inhibitory effect on the expression of dopamine receptor,which partially clarify the myocardial protection of estrogen.     

Analysis of gene expression profile of multidrug resistant MCF/DOX cell line after benflumetol derivative LY980503 treatment

AIM:To investigate the effect of LY980503(a benflumetol derivative)on multidrug resistance of tumor cell line using DNA microarray.METHODS:Total RNA was extracted from multidrug resistant MCF/DOX cell line. cDNA microarray containing 320 cDNAs was used to detect the gene expression profile.RESULTS:9 down-regulated genes and 1 up-regulated gene were identified after multidrug resistant MCF/DOX cells were treated with LY980503.CONCLUSION:LY980503 can effectively reverse the resistance of MCF/DOX to DOX in vitro by adjusting the expression of multi-genes.     

Effect of nicotine in vitro on expressions of apoptosis-related genes in human lung adenocarcinoma cells by cDNA microarray

AIM: To investigate effect of nicotine on growth of human lung adenocarnoma cells and expressions of apoptosis-related gene. METHODS: Lung adenocarcinoma cell line, SPC-A-1, was cultured in the presence of various concentrations (1-1 000 μg/L) of nicotine for 48 hours. MTT was applied to evaluate effect of nicotine in vitro on growth of SPC-A-1 cell line. After SPC-A-1 cells were treated with 100 μg/L for 48 hours, cDNA expression profile microarray was used to detect the expressions of 451 apoptosis-related genes in SPC-A-1 cell line. RESULTS: Significant proliferation in SPC-A-1 cells treated with nicotine (1-10 μg/L) was observed, but this effect decreased with increase in concentration of nicotine in culture. Growth inhibition rate of 1, 10, 100, 1 000 μg/L of nicotine was 27%, -40%, -40% and -93%. Microarray detection showed that significantly different expressions appeared in 80 of 451 apoptosis-related genes. 29 apoptosis-promoted genes and 26 apoptosis-inhibited genes were up-regulated significantly (CY3/CY5>2.0), and 25 genes were significantly down-regulated (CY3/CY5<0.5). CONCLUSION: Nicotine may promote growth of human lung adenocarcinoma cell through regulating many apoptosis-related gene expressions.     

Gene and protein alterations in the hippocampus of rats with temporal lobe epilepsy

AIM:To examine the expression profiles of both genes and proteins in hippocampus of rats with temporal lobe epilepsy (TLE) for revealing the molecular mechanisms of TLE and looking for the candidate targets and new therapeutic approaches in clinical practice.METHODS:Rat temporal lobe epilepsy was induced by administration of lithium chloride and pilocarpine (LiCl-PILO).The expression spectra of genes and proteins were constructed through the techniques of cDNA microarray,two-dimensional (2D) electrophoresis and Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).Subsequently,the differentially expressed genes and proteins were identified and analyzed.RESULTS:There were 192 genes of differential expression observed in hippocampal tissues of LiCl-PILO-induced temporal lobe epilepsy,and 159 genes have been registered in Genbank database,in which 84 genes were up-regulated while 75 genes were down-regulated.78 protein spots of differential display were screened out,in which 31 proteins were detected to be down-regulated and 47 were up-regulated.Finally,5 proteins were identified.CONCLUSION:These genes and proteins found in our study may play pivotal roles in the pathogenic mechanisms of epilepsy and may promise new therapeutic targets for refractory epilepsy in the future.     

Cortical gene expression pattern in rat focal cerebral ischemia

AIM: To investigate the genes differential expression in cortex during rat focal cerebral ischemia.METHODS: cDNA microarray chips containing numerous cDNAs were used to investigate the gene expression pattern between samples of focal cerebral ischemia and sham-control operation rats. RESULTS: Two hundred and eleven genes differentially expressed were screened out, among these genes, up-and down-regulated genes were 199 and 12, respectively. CONCLUSIONS: The analysis of gene expression pattern of focal cerebral ischemia based on cDNA microarray can realize high-throughput screening of the genes associated with the focal cerebral ischemia. The differential expression of genes may be related to the pathogenesis of focal cerebral ischemic diseases.     

Effects of lovastatin on differentially expressed genes in HepG2 cells

AIM: To analyze the lovastatin-induced differential gene expression in HepG2 cells using a cDNA microarray assay. METHODS: Total RNA was extracted from the lovastatin-treated HepG2 cells and control group. cDNA was synthesized from RNA with Cy3/Cy5-labelled dCTP. Then the hybridization was conducted. The result was analyzed using Imagene and Genespring software. RT-PCR was carried to confirm the hybridization results. RESULTS: 30 genes were up-regulated while 11 genes were down-regulated in lovastatin-treated HepG2 cells, involved in some major functional areas including signal transduction, cell cycle regulation, tumor immunity, and so on. CONCLUSION: The analysis of differentially expressed genes in lovastatin-treated HepG2 cells is helpful to explore the mechanism of the anti-tumor activity of statins.     

Inhibitory effect of all-trans retinoic acid on proliferation of microvascular endothelial cells

AIM: To evaluate the effects of all-trans retinoic acid (atRA) on the proliferation in cultured mouse cerebral microvascular endothelial cells (bEnd.3). METHODS: Cultured cells were divided into five groups randomly, one as control group, the other four groups were 10-9, 10-8, 10-7 and 10-6 mol/L group. Effects of atRA on proliferation in bEnd.3 cells were detected by flow cytometry and immunocytochemitry of PCNA and MTT at 24 h, 48 h and 72 h. The effects of atRA (10-6 mol/L group) on the expressions of angiogenic genes in bEnd.3 cells were studied using microarray. RESULTS: The results of MTT and flow cytometry showed that all-trans retinoic acid at concentration of 10-6 mol/L significantly inhibited the proliferation of bEnd.3 cells. Immunocytochemical staining showed the expression of PCNA was markedly decreased in bEnd.3 cells at 24 h after treatment with atRA. Microarray results demonstrated that there were 11 down-regulated angiogenic genes and 2 up-regulated angiogenic genes in 10-6mol/L atRA group. CONCLUSION: All-trans retinoic acid at concentration of 10-6mol/L may significantly inhibit the proliferation of bEnd.3 cells treated for 24 h in vitro via down-regulation of angiogenic genes and PCNA expression.     

DNA microarray profiling to identify norepinephrine-response genes in A7r5 aortic smooth muscle cells

AIM: To define the gene expression changes of vascular smooth muscle cells (VSMCs) in response to norepinephrine (NE). METHODS: The expression adrenergic receptors (AR) were determined by radioligand binding assay in A7r5 cells. Gene expression profiles were identified by cDNA microarray after A7r5 cells were treated with NE for 24 h, and mRNA expressions of α_1A-AR and α_1B-AR were confirmed by real-time PCR. RESULTS: α₁-AR and β-AR existed in A7r5 cells. Seventy-five genes with changed expression in response to NE were screened out. These genes are involved in cell structure, cell/organism defense, metabolism, signal transduction and so on. α_1A-, α_1B-AR mRNA expression identified by microarray and realtime quantitive PCR displayed similar patterns. CONCLUSIONS: Gene expression profile in response to NE was analyzed comprehensively with the microarray technique. NE induces many kinds of different function genes in A7r5 cells, which may provide a novel insight into the particular role of NE that modulates multiple aspects of biological function in VSMCs.     

石榴离体培养再生体系的研究

对石榴休眠枝段、成熟叶片及当年生新梢离体培养 ,建立其再生体系。结果表明 ,以休眠枝段、成熟叶片为外植体均能形成愈伤组织并分化出不定芽 ;当年生新梢茎段培养 ,以MS +BA 2 0mg·L-1+NAA0 3mg·L-1对茎段腋芽的增殖最适宜。诱导生根 ,用 1/2MS为基本培养基附加NAA 0 5mg·L-1+活性炭0 1mg·L-1+蔗糖 2 0g·L-1,生根率达 95 8%。培养基中附加 0 1%活性炭 ,对促进生根均有显著效果。     

Preliminary comparison of inflammatory differential gene expression during atherosclerosis in ApoE-/- mice

AIM: To screen the expression of inflammatory genes associated with atherosclerosis (AS) in different weeks of ApoE^-/- mice using Agilent gene expression profile chip (AGEPC). METHODS: Male ApoE^-/- mice (n=60) were randomly divided into 3 groups:initial phase of AS (10 weeks old), early phase of AS (15 weeks old), and late phase of AS (25 weeks old). Homologous wild-type C57BL/6J mice were used for the control. The RNA samples of the arcus aortae from these mice were isolated. Total RNA from each sample was labeled with Cy3 and hybridized with AGEPC, and microarray detection was conducted. After washing, scaning, acquiring data, and standardized analysis, the expressed genes with default threshold of statistical significance of P≤0.05 and fold change ≥ 2.0 were selected. The expression of these genes were further verified by RT-qPCR. RESULTS: Compared with the control group, there were 895 differential genes in 10 weeks of ApoE^-/- mice, while 540 genes in 15 weeks, and 591 genes in 25 weeks, respectively. KEGG pathway and gene ontology (GO) analyses revealed that those diversely expressed genes related to inflammation were particularly arresting. Several selected genes including interleukin-12a (IL-12a), matrix metallopeptidase-12 (MMP-12), IL-1β, growth differentiation factor-15 (GDF-15) and interferon-γ (IFN-γ) were validated by RT-qPCR. Compared with the control group, the expression levels of IL-12a and MMP-12 were up-regulated while IL-1β was down-regulated in 10 weeks, the expression level of GDF-15 was up-regulated while the IL-12a and IL-1β levels were down-regulated in 15 weeks, and the levels of IL-12a, MMP-12 and GDF-15 were up-regulated in 25 weeks (P<0.05). Moreover, the increased level of IL-12a in 10 weeks, decreased level of IL-1β in 15 weeks, and increased levels of MMP-12 and GDF-15 in 25 weeks were even more statistically significant (P<0.01). CONCLUSION: The changes of inflammatory gene expression in different phases of AS suggest an important direction for medical intervention of AS.     

Differential expressions of ameloblastoma related genes determined by microarray

AIM: This study aims to screen the differentially expressed genes related to the pathogenesis of ameloblastoma by cDNA microarray. METHODS: The total RNAs were isolated from ameloblastoma and tooth germ, respectively. The RNAs were purified by oligotex. Both the mRNAs from two kinds of tissues were reversely transcribed to cDNA with the incorporation of fluorescent-labelled dUTP to prepare the hybridization probes. The mixed probes were hybridized to cDNA microarray. Tumor- related genes were screened through the analysis of fluroescent intensity. RESULTS: 722 genes exhibited significant changes in expression levels in the ameloblastoma in comparison with tooth germs tissues. 240 genes were overexpressed more than doubled (92 genes were more than 3-fold), and 482 genes were underexpressed to below 0.5 of the control level. CONCLUSION: Microarray technique facilitates large scale and rapid identification of potential target genes of ameloblastoma.     

Effect of rosiglitazone on expressions of insulin receptor substrate-1 and glucose transporter 4 in skeletal muscle of type 2 diabetic rats with hyperlipemia

AIM: To investigate the effect of rosiglitazone on the expressions of insulin receptor substrate-1 (IRS-1) and glucose transporter 4 (GLUT4) in skeletal muscles of type 2 diabetic rats with hyperlipemia, and to explore the different pharmacological mechanism. METHODS: The model of type 2 diabetic rats with hyperlipemia was established by injecting low dosage of streptozotocin (STZ) and feeding with high fat diet. Then the diabetic rats were divided into two groups: untreated diabetic group and rosiglitazone-intervened diabetic group. The course of treatment lasted for 4 weeks. The expressions of IRS-1 and the GLUT4 proteins in the cell membrane of isolated rats skeletal muscles were detected by Western blotting. RESULTS: The fasting blood glucose, insulin and triglyceride contents in rosiglitazone-intervened diabetic group were lower than those in untreated diabetic group, but they were still higher than those in control group. The result of Western blotting showed that the expression of GLUT4 protein in rosiglitazone-intervened diabetic group was increased compared with untreated diabetic group, but its level was still lower than that in control group. The protein expression and tyrosine phosphorylation of IRS-1 in rosiglitazone-intervened diabetic group were significantly higher than those in untreated diabetic group and their levels were lower than those in control group. CONCLUSION: The effect of rosiglitazone on GLUT4 protein may link to its ability to induce the protein expression and tyrosine phosphorylation of IRS-1 in skeletal muscles in type 2 diabetic rats.     

Overexpression of Jagged1 promotes aged rat-derived endothelial proge-nitor cells differentiating into mature endothelial cells

AIM: To investigate the effect of Jagged1 overexpression on endothelial cell-directional differentiation of aged rat-derived endothelial progenitor cells (EPC).METHODS: Mononuclear cells were obtained from bone marrow of young (1 to 2 months old) or aged (19 to 26 months old) Sprague-Dawley rats and cultured in DMEM/F12 medium supplemented with 10% FBS. EPC were characterized as double positive for DiI-ac-LDL uptake and lectin binding. The experiments were divided into control group, PIRES2-EGFP transfection group, PIRES2-EGFP-Jagged1 transfection group and young rat-derived EPC group in which transfection was not performed. The GFP expression positive cell number was acquired by fluorescence microscopy and the transfection efficiency was calculated. Immunofluorescence, RT-PCR and Western blotting were used to detect the mRNA and protein expression. In vitro vasculogenesis kit was used to test the tube formation ability of EPC.RESULTS: EGFP-Jagged1 transfection induced a significant increase in the expression of Jagged1 in aged rat-derived EPC (P<0.01). Compared with the control, Jagged1 overexpression markedly enhanced the mRNA expression of von Willebrand factor (vWF) and kinase insert domain receptor (KDR) of vascular endothelial grouth factor vWF in aged rat-derived EPC (P<0.01) and improved the EPC-related tube formation (P<0.01). No significant difference between Jagged1 transfection and young rat-derived EPC groups in vWF and KDR mRNA expression and the ability of tube formation was found. CONCLUSION: In endothelial cell-conditioning medium, Jagged1 overexpression significantly promotes aged rat-derived EPC differentiation into mature endothelial cells.     

Exercise training improves heart function through inducing autophagy and inhibitingapoptosis in aged rats

AIM：To study the mechanism of exercise training in improving old rat cardiac functions, and the effect of gradient exercise training on autophagy and apoptosis in aged rats. METHODS：The rats were randomly divided into 3 groups: young, old and old+exercise (old+Ex). Ultrasonic cardiogram was employed to determine the cardiac functions in the rats. Transmission electron microscope was applied to observe the changes of cardiomyocyte ultrastructure, autophagosome formation and mitochondrial morphology. Western blotting was used to observe the protein expression of Atg5, Beclin 1, microtubule-associated protein 1 light chain 3 (LC3) in cardiac tissues and cytochrome C (Cyt C) in the myocardial mitochondria. TUNEL was adopted to test the apoptosis and spectrophotometry was used to detect the opening of calcium-induced mitochondrial permeability transition pore (mPTP). RESULTS：(1) Compared with young group, the observation in old hearts under transmission electronic microscope found irregular arrangement in myofibrils, loose mitochondria matrix, rupture in mitochondrial membrane and mass deposition of lipofuscin granular in myofilament. In old group, the protein expression of Atg5 and Beclin 1 in the cardiac tissues decreased, the ratio of LC3Ⅱ to LC3Ⅰdropped, mitochondrial Cyt C expression declined, apoptotic index rose, and mitochondrial mPTP opening increased. Noticeable increases were found in left ventricular end-systolic diameter and left ventricular end-diastolic diameter, but left ventricular ejection fraction and left ventricular fractional shortening were decreased. (2) The ultra-structure of the hearts in old +Ex group showed clear sacromere structure, dense matrix and increased number of mitochondria, more autophagosomes and distinct decrease in lipofuscin granular deposition. In addition, the protein expression of Beclin 1 and Atg5 rose, conversion from LC3 I to LC3 II increased, apoptotic index decreased, mPTP opened less, the expression of mitochondrial Cyt C up-regulated, and a significant improvement was observed in left ventricle functions in old+Ex group as compared with old group. CONCLUSION：Exercise training may improve the heart functions in aged rat by upgrading cardiomyocyte autophagy and inhibiting cell apoptosis.