Alterations of cardiac sympathetic norepinephrine transporter expression in rats with volume overload

AIM: To determine the alterations of myocardial 1-adrenergic receptor (1-AR) and cardiac sympathetic norepinephrine transporter (NET) mRNA expression, which is upstream modulator of 1-AR, in rats with longterm volume overload (VOL). METHODS: Left ventricular systolic (LVSP) and end diastolic pressure (LVEDP) of rats with VOL induced by aortacaval fistula operation and control group were measured at 314,30and 60d after the operation, the mRNA at the time points was measured by RT-PCR and Northern blot analysis and quantified by densitometry. RESULTS: The cardiac sympathetic NET specific expression is in the cardiac sympathetic ganglia. Be compared with the control group, LVSP of VOL rats decreased most dramatically by 24% (P<0.05) at 3 d, after that LSVP increased gradually and were higher than control group at 60d. LVEDP increased initially compared with the control (P<0.05), the latter recovered to the control levels. RT-PCR and Northern blot showed that in VOL rats the NET mRNA did not decreased significantly from 3to 30d, and decreased remarkably at 60d after the operation (P<0.05). The pattern of 1-AR mRNA expression were similar to the NET. CONCLUSION: The results suggest decreased levels of NET mRNA may contribute to cardiac sympathetic dysfunction in heart failure due to longterm VOL, which may lead to decreased myocardial 1-AR mRNA. We conclude that the normal NET mRNA expression may play a critical role to maintain sensitivity of 1-AR to adrenergic stimuli and cardiac contractility.     

Effects of Gax gene transfection on proto-oncogene expression and proliferation of PASMCs in rats under hypoxia conditions

AIM: To explore the effects of Gax gene transfection on expressions of c-fos and c-jun mRNA and proliferation of pulmonary arterial smooth muscle cells (PASMCs) in rat under hypoxia.METHODS: PASMCs were transfected with Gax gene by Ad-Gax.Under normal oxygentention (21% O₂) or hypoxia (2.5% O₂) for 12 h condition,expressions of Gax mRNA and protein in PASMCs were detected by RT-PCR and immunocytochemistry.The expressions of c-fos and c-jun mRNA were evaluated by RT-PCR.［³H］-TdR incorporation was used to measure the PASMCs proliferation.RESULTS: The Gax overexpression in transfection group was confirmed by RT-PCR and immunocytochemistry.Under normal oxygentention or hypoxia,the c-fos and c-jun mRNA levels in transfection group were lower than those in the non-transfection group,respectively (P<0.05).［³H］-TdR incorporation in the transfection group was lower than that in non-transfection group (P<0.05,P<0.01).CONCLUSION: Gax overexpression might inhibit the PASMCs proliferation induced by hypoxia through downregulating the expressions of c-fos and c-jun.     

Effect on the expression of proto-onogene c-mpl in megakaryocytes line-HEL by interleukin-13

AIM: To investigate the relevance of the proliferation of megakaryocytic cell line-HEL stimulated by the recombinant human interleukin-13 (IL-13) to the expression of pro-oncogene c-mpl in HEL cells. METHODS: MTT colorimetric assay and reverse transcrition polymerase chain reaction (RT-PCR) are separately used in this study to observe the effect on the proliferation of HEL cells and the expression of c-mpl mRNA in HEL cells by rhIL-13. RESULTS: RhIL-13 stimulated the proliferation of HEL cells and upregulated the expression of c-mpl mRNA in HEL cells. CONCLUSION: Our results suggest that rhIL-13 stimulated the proliferation of HEL cells and provide the evidence that its mechanism is partly because of increasing the pro-oncogene c-mpl expression in HEL cells.     

Effects and mechanism of fenofibrate and pioglitazone on ventricular remodelingin in pressure overload rats

AIM: To study the effects and mechanism of peroxisome proliferator-activated receptors (PPARs) ligands,fenofibrate and pioglitazone,on ventricular remodeling in pressure overload rats.METHODS: A pressure overload model was established by the constriction of abdominal aorta in Wistar rats.The hemodynamics and ventricular remodeling parameters,plasma and myocardial renin activity,angiotensin Ⅱ and aldosteron,the mRNA expression of angiotensin Ⅱ type 1 receptor (AT1) were investigated in the constriction of abdominal aorta group (CAA group,n=7) at 12-week after operation and treated experimental groups in which rats were treated with fenofibrate (F group,n=8),pioglitazone (P group,n=7),concomitant fenofibrate and pioglitazone (F+P group,n=6) for 12 weeks since 2 days after operation.The sham-operated rats served as controls (n=8).RESULTS: The ratio of left ventricular weight to body weight,mean arterial pressure,left ventricular systolic pressure,left ventricular end diastolic pressure,left ventricular systolic pressure and heart rate were significantly lower,the maximum left ventricular pressure rising and declining rates(±dp/dtmax) were significantly higher in all treated experimental groups than those in CAA group.Fenofibrate or pioglitazone had no effect on plasma and myocardial levels of renin,angiotensin Ⅱand aldosteron.The mRNA expression of AT1 was downregulated in treated groups except F group.CONCLUSION: PPAR ligands have no effect on plasma and myocardial levels of renin,angiotensin Ⅱand aldosteron,but fenofibrate and pioglitazone inhibit ventricular remodeling,decrease preload and afterload,increase ±dp/dtmax in pressure overload rats.The expression of mRNA of AT1 is downregulated in myocardium of pressure overload rats by the PPARγ signaling pathway.     

Correlation between myocardial interstitial collagen remodeling and changes in hemodynamics in rats with myocardial infarction

AIM: To study the relationship between cardiac extracellular matrix remodeling and cardiac function after myocardial infarction. METHODS: We observed sequential changes in collagen contents and collagen Ⅰ/Ⅲ ratios in infarct zone (IZ) and non-infarct zone (NIZ) and their relationship to the parameters of left ventricular systolic and diastolic function in the rat model of myocardial infarction induced by ligation of left main coronary artery. RESULTS: Collagen conteants in IZ and NIZ after 3d of myocardial infarction were significantly higher than those in sham group at corresponding time (P<0.05, P<0.01). Collagen Ⅰ/Ⅲ ratio in IZ decreased on day 3, significantly increased after 7 d (P<0.01). Collagen Ⅰ/Ⅲ ratio in NIZ increased significantly afte14 d. Correlated analysis between collagen contents in IZ or NIZ and collagen type Ⅰ/Ⅲ ratio and maximal ascending velocity (+p'_max) or maximal descending velocity of the left ventricular pressure (-p'_max) was performed and the negative correlation between collagen contents in NIZ and +P'_max (r=-0.589, P>0.05) and -P'_max (r=-0.788, P<0.01) was found. Collagen content in IZ positively correlated to the +P'_max (r=0.70, P<0.50), but not to -P'_max (r=-0.29, P>0.05). Collagen type Ⅰ/Ⅲ ratios in NIZ correlated negatively to the +P'_max (r=-0.504, P>0.05) and -P'_max (r=-0.545, P>0.05), but there were no relationship between collagen type Ⅰ/Ⅲ ratios in IZ and +P'_max or -P'_max in IZ. CONCLUSION: Collagen deposition in IZ after myocardial infarction was of benefit to improvement of systolic function. Collagen deposition in NIZ was harmful to systolic and diastolic function.     

Relationship between blood pressure and fibrinogenic expression of fibrinogen Bβ-chain gene polymorphisms

AIM: To study the correlation between blood pressure and 6 common fibrinogen (Fg) Bβ-chain gene polymorphisms (Bβ-854G/A,-455G/A,-249C/T,-148C/T, 448G/A, Bcl-1G/A), and to investigate the effect of blood pressure on plasma fibrinogen concentration and the functional expression of fibrinogen. METHODS: 1 391 subjects from Kailuan Group Corporation were enrolled for medical examination and questionnaire survey. Venous blood were collected in the early morning to measure Fg concentration, fibrin monomer polymerized velocity (FMPV), absorbance maximum (A_max) and FMPV/A_max. The blood pressure was also measured and the subjects were accordingly divided into hypertension group (HG) and the normal blood pressure group (NG). The 6 gene polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: No differences of the genotype frequencies of the 6 sites between hypertension group (HG) and normal blood pressure group (NG) were found (P>0.05). No differences of Fg concentration and FMPV/A_max between HG group and NG group were observed (P>0.05). However, FMPV and A_max in HG group were lower than those in NG group (P<0.05). In HG and NG group, the Fg concentration, FMPV, A_max, FMPV/A_max in the subjects with Bβ-854 mutated genotype were all higher than those in the subjects with wild genotype (P<0.05). In NG group, the Fg concentration and FMPV in the subjects with Bβ Bcl-1 mutated genotype were higher than those in the subjects with wild genotype (P<0.05). Fg, FMPV and A_max in the subjects with Bβ-854 and Bcl-1 mutated genotype in HG group were lower than those in NG group (P<0.05). CONCLUSION: Fg Bβ-854 is one of the main gene situs of regulating the functional expression of fibrinogen. Bcl-1 mutated genotype increases plasma Fg concentration and FMPV. Hypertension decreases the effect of this regulation, and affects the process of fibrin monomer aggregation into dimer, resulting in a special type of coagulation dysfunction.     

Gene transfer and expression of recombined human proinsulin in hepatoma cells of rats

AIM:To explore the recombined human proinsulin gene containing glucose reaction element (GLRE) expression in transfected CBRH7919 cells. METHODS:The packaged retrovirus encoding genetically modified human proinsulin PLXSN-(GLRE)3-BP-1MpINS3 and PLXSN-(GLRE)3-BP-1MpINS2 were transfected into CBRH7919 cells. Insulin values in cells after transient and steady expression screened by G418 at different glucose levels were detected. Chromosome DNA was isolated from transfected and untransfected cells and polymerase chain reaction (PCR) was performed. PCR products were analyzed by electrophoresis. RESULTS:38 h after transfection, at the glucose levels of 0-25 mmol/L, the levels of insulin produced by cells including PLXSN-(GLRE)3-BP-1MpINS3 were (3.57±0.21)U/L, (5.30±0.20)U/L, (16.27±0.87)U/L, (23.23±1.12)U/L, respectively (P<0.05). One month after transfection, under above glucose levels, insulin values were (3.57±0.21)U/L, (5.30±0.20)U/L, (16.27±0.87)U/L, (23.23±1.12)U/L (P<0.05). CBRH7919 cells including PLXSN-(GLRE)3-BP-1MpINS2 secreted detectable insulin value at the level of 25 mmol/L, they were (2.10±0.23)U/L and (2.05±0.17)U/L, respectively. PCR products of transfected cells showed target band, but control cells did not. CONCLUSIONS:Recombined proinsulin gene was transfected successfully in CBRH7919 cells. The cells combined human proinsulin gene has the ability of producing insulin with increase in glucose concentration in vitro.     

Effect of erythropoietin on expression of myocardial NADPH oxidase in pressure overload rats

AIM:To explore the effect of erythropoietin （EPO） on the expression of myocardial NADPH oxidase （Nox） in the pressure overload rats. METHODS:Male SD rats (n=36) were used to establish a pressure overload myocardial hypertrophy model by abdominal aorta ligation. The animals were divided into model group, control group (sham, without narrowing abdominal aorta, the rest of the operation was the same as the model) and recombinant human erythropoietin (rhEPO) treatment group (intraperitoneal injection of rhEPO postoperatively, 4 000 U/kg, twice a week). After 8 weeks, the cardiac ultrasound imaging and hemodynamic evaluation were conducted to determine the cardiac functions. Masson staining was used to observe the degree of myocardial fibrosis. The expression of Nox2 and Nox4 at mRNA and protein levels was detected by real-time quantitative PCR and Western blotting. The protein levels of myocardial inflammatory factors CD45, F4/80 and TGF-β were determined by Western blotting. RESULTS:Compared with model group, the left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular end-systolic pressure (LVESP) and left ventricular pressure maximum rising and falling rates (±dp/dtmax) increased significantly in EPO treatment group (P<0.01). At the same time, left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD) and left ventricular end-diastolic pressure (LVEDP) were decreased in EPO treatment group (P<0.01). EPO reduced the degree of myocardial fibrosis caused by pressure overload (P<0.01) and decreased the expression of Nox2 and Nox4 at mRNA and protein levels in the myocardium (P<0.05 or P<0.01), and reduced the protein expression of myocardial inflammatory factors CD45, F4/80 and TGF-β. CONCLUSION: EPO inhibits rat myocar-dial fibrosis induced by pressure overload, improves heart functions by decreasing NADPH oxidase activity and inhibiting myocardial oxidative stress levels and myocardial inflammatory reaction.     

Hypothalamic expression of orexin A and lipid metabolism disorder in rats with alimentary obesity

AIM: To evaluate the effect of orexin A in rat hypothalamus on lipid metabolism disorder in rats with alimentary obesity induced by high-fat diet.METHODS: The rat model of alimentary obesity was induced by high-fat diet. The levels of insulin, triglyceride (TG) and total cholesterol (TC) in the serum were detected by luminescent immunoassay and enzymic method. The mRNA expression of orexin A in rat hypothalamus was determined by real-time PCR.RESULTS: There were statistically significant differences of weight, body fat content, and Lee's index between high-fat diet group and control group after 8-week feeding of high-fat diet. Compared to control animals, the levels of insulin, TG and TC in the rats with alimentary obesity significantly increased by 50％, 94％ and 43％, respectively (P<0.05). The expression of orexin A in rat hypothalamus significantly decreased by 57％, and had significant negative correlation with Lee's index, insulin, TG and TC. Their correlation coefficients were r=-0.798 (P<0.05), r=-0.868 (P<0.05), r=-0.981(P<0.05) and r=-0.815 (P<0.05), respectively. CONCLUSION: Alimentary obesity and lipid metabolism disorder induced by high-fat diet are correlated with down-regulation of orexin A expression in rat hypothalamus.     

Relationship between zinc and zinc transporter expression in breast cancer MCF-7 cells

AIM: To study the changes of zinc transporter gene expression in MCF-7 cell line exposed to ZnCl₂ and TPEN. METHODS: Human breast cancer cell line MCF-7 was exposed to different concentrations of ZnCl₂ (0, 50, 100, 150, 200 μmol/L) and TPEN (0, 5, 10, 15 μmol/L), respectively. Twelve hours later, the cell viability was measured by MTT and levels of zinc transporter mRNA by RT-PCR. Zinquin was used to estimate the intracellular zinc concentrations. RESULTS: MCF-7 cells viability rate was significantly decreased when exposed to ZnCl₂ (with 150 μmol/L and 200 μmol/L) and TPEN. The intracellular zinc concentration was significantly increased when exposed to ZnCl₂ and decreased when exposed to TPEN. ZnT-1 mRNA level was increased along with the increasing concentration of ZnCl₂ but decreased when exposed to TPEN. The expressions of ZIP2 and ZIP10 were increased along with the increasing concentration of TPEN. CONCLUSION: ZnT-1 gene expression is induced by zinc supplement and repressed by zinc deficiency. ZIP2 and ZIP10 gene expressions are induced by zinc deficiency.     

Relationship between apoptosis,proliferation and expression,mutation of related genes in breast cancer

AIM:To study the relationship between apoptosis, proliferation and expression,mutation of related genes in breast cancer.METHODS:Methods of TUNEL, immunohistochemical S-P and PCR-SSCP were used respectively to study apoptotic index (AI), mitotic index(MI), expression of Bcl-2,p53,c-erbB-2,PCNA,Ki67,TopoⅡ and mutation of p53 in 54 cases of breast cancer.RESULTS:AI and MI were 9.40±3.78 and 5.96±2.36, respectively. There was a significant direct correlation between them(r=0.46.P＜0.01).High expression of Bcl-2,PCNA,Ki67,TopoⅡ coincided with high AI,MI(P＜0.01). High expression of p53,c-erbB-2 and mutation of p53 coincided with high MI(P＜0.01). Type of p53 mutation coincided with AI(P＜0.05).CONCLUSION:Disturbance of gene control between apoptosis and proliferation is related with expression,mutation of related genes in breast cancer.     

Variation of brain derived neurotrophic factor and its mRNA expression in infancy rats after experimental bacterial meningitis

AIM: To investigate the expression and effect of brain derived neurotrophic factor (BDNF) mRNA and its protein in infancy rats after exposure to bacterial meningitis. METHODS: Three week old rats were used to construct the models of bacterial meningitis (n=30) and mormal(n=18). At 24 h, 48 h, 5 d after inoculating, the expression of BDNF mRNA and its protein were detected by in situ hybridization and immunohistochemical staining methods, respectively. RESULTS: The increase in BDNF mRNA expression was detected by in situ hybridization at 24 h in experiment(0.13320±0.02750) compared to control(0.06269±0.01147)(P＜0.01). Expression of BDNF mRNA was declined at 48 h, but its expression was still stronger than that in controls at 5 d(P＜0.05). The expression of BDNF protein was enhanced and reached to its zenith at 24 h in this experiment (0.16896±0.02717) (P＜0.01), compared to controls(0.08700±0.03413), and it declined after 48 h, then restored to the control levels at 5 d(P＞0.05). Meanwhile, in the brain from the experiment rats, strong positive hybridization and immunoreactivity were observed in the infiltrated inflammatory cells in leptomeninges, subarachnoid cavity, ventricles and brain parenchyma. CONCLUSIONS: These results support the hypothesis that BDNF might play a neuroprotective role in brain damage process in bacterial meningitis attacked rats. BDNF might take part in imune response. These results also support the hypothesis that BDNF has some relationships with other inflammatory mediators during acute inflammatory response of bacterial meningitis.     

Alterations in gene expression of series key enzymes in mevalonic acid pathway detected by RNA array in spontaneously hypertensive rats

AIM: To explore the alteration in gene expressions of several enzymes in mevalonic-acid (MVA) pathway in SHR.METHODS: 294 samples of total RNA were extrated from the tissues of ventriculum, aortic smooth muscle, liver and kidney in SHR and WKY. RNA array was used to determine the mRNA level of several enzymes in mevalonic acid pathway.RESULTS: (1) Systolic blood pressure of SHR surpassed that in WKY since the 6th week (P<0.01). (2) The level of cholesterol in serum was significantly lower in SHR than that in WKY (P<0.01), while that in tissue between two bands had no difference. (3) The gene overexpressions of four enzymes in the four tissues of SHR (P<0.01), including farnesyl diphosphate synthase (FDS), isopentenyl diphosphate isomerase (IDI), farnesyltransferase alpha subunit (FT1), farnesyltransferase beta subunit (FT2), which catalyze the vital mid-productions in MVA pathway, were observed. (4) Gene overexpressions of some enzymes assayed in MVA pathway in SHR renal tissue were observed since 4th week (P<0.01), including 3-hydroxy-3-methylglutaryl -coenzyme A reductase (HMGR), mevalonate kinase (MVK), mevalonate decarboxylase (MVD), squalene synthetase (SQS) and squalene epoxidase (SQ). (5) The gene expressions of these enzymes in ventriculum, aortic smooth muscle and liver showed no difference between two band rats, eg HMGR, MVK, MVD, SQS, SQ.CONCLUSION: The gene expressions of several enzymes in mevalonic acid non-cholesterol pathway in SHR ascend and the mechanism will be further explored.     

Zacopride attenuates pressure overload-induced ventricular remodeling in rats

AIM:To investigate the protective effect of zacopride (ZAC) on the pressure-overload left ventricular remodeling in the rats induced by coarctation of abdominal aorta. METHODS:Male Sprague-Dawley (SD) rats with pressure overload were induced by the coarctation of abdominal aorta. The model rats were intraperitoneally administered with ZAC, chloroquine (Chlor), and zacopride+chlorquine (ZAC+Chlor). The study duration was 8 weeks. The cardiac structure and function were assessed by echocardiography. The heart weight/body weight (HW/BW) ratio and the left ventricular weight/body weight (LVW/BW) ratio were calculated. The changes of structure and shape in myocardial tissue were observed with HE staining. The ultrastructure of the myocytes was observed under transmission electron microscope. The inward rectifier potassium channel (I_K1) protein expression was determined by Western blot. The mRNA expression of Kir2.1 was detected by RT-PCR. RESULTS:Compared with vehicle group, ZAC improved cardiac function, as indicated by the decreased left ventricular end-diastolic dimension (LVEDD) and left ventricular end systolic dimension (LVESD) (P<0.05), and the increased left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) (P<0.01). The HW/BW and LVW/BW ratios were significantly decreased, and the cross-sectional area of the cardiomyocytes was significantly less in ZAC group than that in vehicle group (P<0.01). The ultrastructure of the myocytes was significantly improved. Chlor blocked the protective effect of zacopride on the pressure-overload left ventricular remodeling. The protein level of I_K1 and mRNA expression of Kir2.1 in the cardiac tissues in ZAC group were significantly increased compared with vehicle group (P<0.01). CONCLUSION:I_K1 agonist ZAC significantly attenuates pressure overload-induced ventricular remodeling in rats.     

香蕉WAT1基因的鉴定及表达分析

【目的】基于香蕉基因组数据筛选Walls are thin 1(WAT1)基因,分析它们的序列及表达特性。【方法】以拟南芥WAT1为参考序列,通过本地Blast筛选获得香蕉WAT1基因,分析其核苷酸、启动子及编码蛋白特性,并利用实时定量PCR技术研究其在不同组织部位、不同激素和逆境胁迫处理下的表达情况。【结果】筛选获得5个香蕉WAT1基因(命名为MaWAT1-1~5)。蛋白亚细胞定位预测结果显示,MaWAT1-1、MaWAT1-2、MaWAT1-4主要定位在液泡和细胞膜上,MaWAT1-3主要定位在细胞质和细胞膜,MaWAT1-5定位在细胞膜和叶绿体。基因结构分析和系统进化树分析均将MaWAT1s分为两组,MaWAT1-1、MaWAT1-2、MaWAT1-4聚为一组(含6个外显子和5个内含子),MaWAT1-3和MaWAT1-5归为一组(含7个外显子和6个内含子)。启动子顺式作用元件分析结果显示:MaWAT1s启动子含有大量激素和胁迫响应相关元件。实时定量PCR结果显示,MaWAT1-4在叶片中表达量最高,MaWAT1-1在根和假茎中表达量最高,其余均在根中表达量最高;大多数MaWAT1s的表达受GA3、SA、盐胁迫和干旱等显著诱导,受高温显著抑制,同时部分成员的表达受IAA、ABA、JA、低温、机械损伤和枯萎病影响显著。【结论】MaWAT1s的表达受多种激素和逆境影响显著,可能在香蕉生长发育和抗逆防御反应中发挥着重要作用。     

全基因组基因表达分析技术及其在果树上的应用

基因表达分析对于揭示基因功能、了解基因间相互关系、阐明植物生长发育过程中的生理生化调控机制起着至关重要的作用。综述了mRNA差异显示、cDNA-AFLP、基因芯片、EST数据分析、基因表达系列分析(SAGE)等全基因组基因表达分析技术的原理特点及在果树上的应用情况,并介绍了利用全基因组基因表达分析技术开展果树研究的思路。