Involvement of Orai1 in tunicamycin-induced endothelial dysfunction

Endoplasmic reticulum (ER) stress is mediated by disturbance of Ca²⁺ homeostasis. The store-operated calcium (SOC) channel is the primary Ca²⁺ channel in non-excitable cells, but its participation in agent-induced ER stress is not clear. In this study, the effects of tunicamycin on Ca²⁺ influx in human umbilical vein endothelial cells (HUVECs) were observed with the fluorescent probe Fluo-4 AM. The effect of tunicamycin on the expression of the unfolded protein response (UPR)-related proteins BiP and CHOP was assayed by western blotting with or without inhibition of Orai1. Tunicamycin induced endothelial dysfunction by activating ER stress. Orai1 expression and the influx of extracellular Ca²⁺ in HUVECs were both upregulated during ER stress. The SOC channel inhibitor SKF96365 reversed tunicamycin-induced endothelial cell dysfunction by inhibiting ER stress. Regulation of tunicamycin-induced ER stress by Orai1 indicates that modification of Orai1 activity may have therapeutic value for conditions with ER stress-induced endothelial dysfunction.

Model construction of rat coronary artery smooth muscle cell endoplas-mic reticulum stress induced by thapsigargin / 中国病理生理杂志

AIM: To investigate the primary culture method for coronary artery smooth muscle cells (CASMCs), and to establish the endoplasmic reticulum stress ( ERS) model in CASMCs of SD rats.METHODS:CASMCs were cultured by tissue explant method .The morphological characteristics were observed under optical micro-scope.The marker proteins of CASMCs , including α-SMA and SM-MHC, were identified by immunofluorescence tech-nique.The protein expression levels of BiP and CHOP , the marker molecules of ERS, were determined by Western blot . RESULTS:The spindle-shaped CASMCs climbed out from the edge of coronary artery tissues after 6 d, and formed the typical hill and valleygrowth pattern of CASMCs at 9~10 d.The result of immunofluorescence technique showed that α-SMA and SM-MHC were positively expressed .The results of Western blot showed that the protein expression of BiP and CHOP in TG ( 1 and 2 μmol/L ) treatment groups was increased compared with control group .Compared with control group, the protein expression of BiP and CHOP was significantly increased after 1 μmol/L TG treatment for 24 and 48 h. CONCLUSION:CASMCs can be successfully cultured by tissue explant method .ERS model of CASMCs was established by 1 μmol/L TG treatment for 24 h.

The bifunctional effect of propofol on thromboxane agonist (U46619)-induced vasoconstriction in isolated human pulmonary artery

Propofol is known to cause vasorelaxation of several systemic vascular beds. However, its effect on the pulmonary vasculature remains controversial. In the present study, we investigated the effects of propofol on human pulmonary arteries obtained from patients who had undergone surgery. Arterial rings were mounted in a Multi-Myograph system for measurement of isometric forces. U46619 was used to induce sustained contraction of the intrapulmonary arteries, and propofol was then applied (in increments from 10–300 µM). Arteries denuded of endothelium, preincubated or not with indomethacin, were used to investigate the effects of propofol on isolated arteries. Propofol exhibited a bifunctional effect on isolated human pulmonary arteries contracted by U46619, evoking constriction at low concentrations (10–100 µM) followed by secondary relaxation (at 100–300 µM). The extent of constriction induced by propofol was higher in an endothelium-denuded group than in an endothelium-intact group. Preincubation with indomethacin abolished constriction and potentiated relaxation. The maximal relaxation was greater in the endothelium-intact than the endothelium-denuded group. Propofol also suppressed CaCl₂-induced constriction in the 60 mM K⁺-containing Ca²⁺-free solution in a dose-dependent manner. Fluorescent imaging of Ca²⁺ using fluo-4 showed that a 10 min incubation with propofol (10–300 µM) inhibited the Ca²⁺ influx into human pulmonary arterial smooth muscle cells induced by a 60 mM K⁺-containing Ca²⁺-free solution. In conclusion, propofol-induced arterial constriction appears to involve prostaglandin production by cyclooxygenase in pulmonary artery smooth muscle cells and the relaxation depends in part on endothelial function, principally on the inhibition of calcium influx through L-type voltage-operated calcium channels.

MEF2C mediates inhibitory effect of microRNA-214 on cardiomyocyte hypertrophy / 中国病理生理杂志

AIM:To investigate the effect of microRNA-214 ( miR-214) on cardiomyocyte hypertrophy and the expression of the potential target genes .METHODS:A cell model of hypertrophy was established based on angiotensin-Ⅱ( Ang-Ⅱ)-induced neonatal mouse ventricular cardiomyocytes ( NMVCs) .Dual luciferase reporter assay was performed to verify the interaction between miR-214 and the 3’ UTR of MEF2C.The expression of MEF2C and hypertrophy-related genes at mRNA and protein levels was determined by RT-qPCR and Western blot , respectively .RESULTS:The expression of ANP, ACTA1,β-MHC and miR-214 was markedly increased in Ang-Ⅱ-induced hypertrophic cardiomyocytes .Dual lu-ciferase reporter assay revealed that miR-214 interacted with the 3’ UTR of MEF2C, and miR-214 was verified to inhibit MEF2C expression at the transcriptional level .The protein expression of MEF2C was markedly increased in the hypertro-phic cardiomyocytes .Moreover, miR-214 mimic, in parallel to MEF2C siRNA, inhibited the expression of hypertrophy-re-lated genes in Ang-Ⅱ-induced NMVCs.CONCLUSION:MEF2C is a target gene of miR-214, which mediates the effect of miR-214 on attenuating cardiomyocyte hypertrophy .

Effect of circRNA_000203 on fibrotic phenotypes in mouse cardiac fibro-blasts / 中国病理生理杂志

AIM:To determine circular RNA (circRNA) profiles in the diabetic mouse myocardium , and to investigate the effect of circRNA_000203 on fibrotic phenotypes in cardiac fibroblasts .METHODS:Masson trichrome stai-ning was performed on the myocardium of the diabetic db /db mice and the non diabetic db/m control mice .circRNA ex-pression profile in the diabetic myocardium was detected by circRNAs microarray .The expression of circRNA_000203 was determined by real time fluorescence quantitative PCR ( RT-qPCR ) .Recombinant circRNA_000203 adenovirus was pre-pared for enforced the expression of circRNA_000203 in mouse cardiac fibroblasts.The expression of Col1a2, Col3a1andα-SMA was determined in circRNA_000203-modified cardiac fibroblasts , respectively .RESULTS:Masson trichrome stai-ning showed that fibrosis was increased in the diabetic mouse myocardium .The results of circRNA array detection revealed that circRNAs were dysregulated in the diabetic myocardium .circRNA_000203 was up-regulated in the diabetic myocardi-um.Significant over-expression of circRNA_000203 was achieved in the cardiac fibroblasts after infection with the recombi-nant circRNA_000203 adenovirus.The mRNA and protein expression of Col1a2, Col3a1 and α-SMA was significantly in-creased in the cardiac fibroblasts with over-expression of circRNA_000203.CONCLUSION:circRNA_000203 is up-regu-lated in the diabetic mouse myocardium .It has pro-fibrotic effect on the cardiac fibroblasts .

Endoplasmic reticulum stress induced by Thapsigargin in vascular smooth muscle cells of rat coronary artery / 中国病理生理杂志

AIM:To establish the endoplasmic reticulum stress ( ERS) cell model in vascular smooth muscle cells ( VSMCs) of Sprague-Dawley (SD) rats.METHODS:Under sterile condition, the coronary arteries were isolated from SD rats .The primary VSMCs were cultured by tissue-sticking method , and observed the basic morphological characteristics under optical microscope .The marker proteins of VSMCs including α-smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain ( SM-MHC) were identified by immuno-fluorescence technique .VSMCs were treated with thapsigargin (0.5, 1 and 2 μmol/L) for 24 h, and the expression levels of binding immunoglobulin protein (BiP) and C/EBP homologus protein (CHOP), the marker molecules of ERS, were detected using Western blotting.RESULTS:VSMCs climbed out from coronary artery tissues after about six days , and the cells had a nice state and formed the VSMC-like typical peak valley.The results of immunofluorescence technique show that the marker proteins of VSMCs ,α-SMA and SM-MHC were expressed significantly .The results of Western blotting show that the protein expression levels of BiP and CHOP were increased by thapsigargin in a dose-dependent manner .CONCLUSION:VSMCs can be successfully cultured by tissue-sticking method and built the ERS model induced by thapsigargin .

Role of tumor necrosis factor-αin the regulation of T-type calcium channel current in HL-1 cells / 中国病理生理杂志

AIM:Increasing evidence indicates that inflammation contributes to the initiation and perpetuation of atrial fibrillation ( AF) .Al-though tumor necrosis factor ( TNF)-αlevels are increased in patients with AF , the role of TNF-αin the pathogenesis of AF remains unclear.Recent research has revealed that T-type Ca2+currents ( ICa,T ) play an important role in the pathogenesis of AF .METH-ODS:In this study , we used the whole-cell voltage-clamp technique and biochemical assays to explore the role of TNF-αin the regula-tion of ICa,T in atrial myocytes.RESULTS:We found that compared with sinus rhythm (SR) controls, T-type calcium channel (TCC) subunit mRNA levels were decreased , while TNF-αexpression levels were increased , in human atrial tissue from patients with AF .In murine atrial myocyte HL-1 cells, after cultured for 24 h, 12.5, 25 and 50 μg/L TNF-αsignificantly reduced the protein expression levels of the TCC α1G subunit in a concentration-dependent manner .The peak current was reduced by the application of 12.5 or 25μg/L TNF-αin a concentration-dependent manner [from ( -15.08 ±1.11) pA/pF in controls to ( -11.89 ±0.83) pA/pF and (-8.54 ±1.55) pA/pF in 12.5 and 25 μg/L TNF-αgroups, respectively].TNF-αapplication also inhibited voltage-dependent inactivation of ICa,T shifted the inactivation curve to the left .CONCLUSION:These results suggest that TNF-αis involved in the path-ogenesis of AF, probably via decreasing ICa,T function in atrium-derived myocytes through impaired channel function and down -regula-tion of channel protein expression .This pathway thus represents a potential pathogenic mechanism in AF .

Dexmedetomidine inhibits vasoconstriction via activation of endothelial nitric oxide synthase

Despite the complex vascular effects of dexmedetomidine (DEX), its actions on human pulmonary resistance arteries remain unknown. The present study tested the hypothesis that DEX inhibits vascular tension in human pulmonary arteries through the endothelial nitric oxide synthase (eNOS) mediated production of nitric oxide (NO). Pulmonary artery segments were obtained from 62 patients who underwent lung resection. The direct effects of DEX on human pulmonary artery tension and changes in vascular tension were determined by isometric force measurements recorded on a myograph. Arterial contractions caused by increasing concentrations of serotonin with DEX in the presence or absence of L-NAME (endothelial nitric oxide synthase inhibitor), yohimbine (α2-adrenoceptor antagonist) and indomethacin (cyclooxygenase inhibitor) as antagonists were also measured. DEX had no effect on endothelium-intact pulmonary arteries, whereas at concentrations of 10⁻⁸~10⁻⁶ mol/L, it elicited contractions in endothelium-denuded pulmonary arteries. DEX (0.3, 1, or 3×10⁻⁹ mmol/L) inhibited serotonin-induced contraction in arteries with intact endothelium in a dose-dependent manner. L-NAME and yohimbine abolished DEX-induced inhibition, whereas indomethacin had no effect. No inhibitory effect was observed in endothelium-denuded pulmonary arteries. DEX-induced inhibition of vasoconstriction in human pulmonary arteries is mediated by NO production induced by the activation of endothelial α₂-adrenoceptor and nitric oxide synthase.

Cx43 is involved in electrical remodeling of atrial myocytes through regu-lating L-type calcium current / 中国病理生理杂志

AIM:To investigate whether the association of connexin 43 ( Cx43 ) and L-type calcium channel involved in the pathogenesis of atrial fibrillation ( AF) .METHODS:The biochemical assays and whole-cell patch-clamp technique were used to study the expression of Cx43 in human atrial tissue.The co-localization of Cx43 and L-type calcium channel, and the regulation of L-type calcium current in atrial myocytes were investigated.RESULTS:The expression of Cx43 at mRNA and protein levels was decreased in human atrial tissues of AF patients.In cultured atrium-derived myocytes ( HL-1 cells) , knockdown of Cx43 significantly inhibited the mRNA expression of L-type calcium channelα1c subunit, as well as L-type calcium current.Co-localization of Cx43 with L-type calcium channel α1c subunit in mouse atrial myocytes was observed.CONCLUSION:The decrease in Cx43 is involved in the pathogenesis of AF, probably through reducing the L-type calcium current in atrial myoctyes by co-localization with L-type calcium channel, thus representing the potential pathogenesis in atrial fibrillation.

Dexmedetomidine inhibits 5-HT-induced intrapulmonary artery vasoconstriction / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of dexmedetomidine on 5-HT-induced constrictions of isolated human intrapulmonary arteries and explore the mechanisms.</p><p><b>METHODS</b>Lung tissue was obtained from patients undergoing surgery for lung carcinoma. Intrapulmonary arteries were dissected and cut into rings, which were mounted in a Multi Myograph system to determine the effect of dexmedetomidine (0.3-3 nmol/L) on 5-HT-induced vasoconstrictions. The influences of the endothelium removal and various drugs including L-NAME, yohimbine and indomethacin were tested on the effects of dexmedetomidine.</p><p><b>RESULTS</b>Dexmedetomidine (0.1-100 nmol/L) did not obviously affect the resting tension of endothelium-intact human intrapulmonary arteries. 5-HT induced concentration-dependent contraction in endothelium-intact intrapulmonary arteries [pD2: 6.11∓0.05, Emax: (102.10∓1.96)%]. In the rings with intact endothelium, dexmedetomidine (0.3-3 nmol/L) significantly attenuated the Emax and pD2 of 5-HT-induced vasoconstriction [pD2: 5.94∓0.03, Emax: (79.96∓1.31)%]. 5-HT also induced concentration-dependent contraction in endothelium-denuded intrapulmonary arteries [pD2: 6.10∓0.07, Emax: (107.40∓3.20)%]. Dexmedetomidine produced no significant effects on the rings with denuded endothelium. The effects of dexmedetomidine on 5-HT-induced vasoconstriction was suppressed by L-NAME and yohimbine, but not by indomethacin.</p><p><b>CONCLUSION</b>Dexmedetomidine can inhibit 5-HT-induced vasoconstriction of isolated human intrapulmonary arteries probably through α2-adrenergic acceptor and NO released from the endothelium.</p>

Effects of stretch on transient outward potassium and inward rectifier po-tassium current in cultured neonatal rat atrial myocytes / 中国病理生理杂志

[ABSTRACT]AIM:Toinvestigatetheeffectsofmechanicalstretchontransientoutwardpotassiumcurrent(Ito), inward rectifier potassium current ( IK1 ) and action potential duration ( APD) of cultured neonatal rat atrial myocytes . METHODS:Neonatal rat atrial myocytes were isolated and cultured on silicone sheeting with or without stretch for 24 h. The silicone membrane area was increased by 12%in stretched group.The cells without stretch served as control .Ito, IK1 and APD were recorded by the technique of whole-cell patch clamp.RESULTS:Compared with control group, Ito density in stretched myocytes was significantly reduced [(1.6 ±0.4) pA/pF vs (12.1 ±2.9) pA/pF, P<0.01], whereas IK1 density was increased [(-10.8 ±0.8) pA/pF vs (-8.8 ±0.9) pA/pF, P<0.01].The APDs at 50%and 90%levels of repolarization ( APD50 and APD90 ) in the stretched cells were obviously decreased than those in non-stretched cells [(10.5 ±1.4) ms vs (15.5 ±2.4) ms, (30.0 ±2.8) ms vs (56.3 ±3.6) ms, P<0.01].CONCLUSION: Stretch stimulation leads to the reduction of Ito density, the increase in IK1 density and the shortness of APD in cultured rat atrial neonatal myocytes , which may contribute to atrial electrical remodeling induced by pressure overload .

Caspase-8 small hairpin RNA attenuates apoptosis of human bone mar-row mesenchymal stem cells under conditions of serum deprivation and hypoxia / 中国病理生理杂志

AIM:To investigate the effect of caspase-8 small hairpin RNA ( shRNA) on attenuating apoptosis of human mesenchymal stem cells ( hMSCs ) .METHODS: Two recombinant plasmids for over-expression of caspase-8 shRNA, pAd-Cap8 shRNA1 and pAd-Cap8 shRNA2, were constructed.Caspase-8 mRNA was determined in pAd-Cap8 shRNA-transfected human HEK293 cells by Q-PCR.The screened pAd-Cap8 shRNA was used to construct the recombinant adenovirus plasmid , which was linearized and transfected into HEK 293 cells for packaging and amplification of the recombi-nant adenovirus rAd-Cap8 shRNA.The expression of caspase-8 at mRNA and protein levels was determined by Q-PCR and Western blotting .Annexin V/PI staining and determination of caspase-8 activity were performed to assess apoptosis of hM-SCs under the conditions of serum deprivation and hypoxia .The mRNA expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), Bcl-2 and Bcl-xL was analyzed by Q-PCR.RESULTS:The pAd-Cap8 shRNA, which efficiently inhibited caspase-8 expression, was screened by Q-PCR.The recombinant adenovirus plasmid for caspase-8 shRNA was constructed and used to package and amplify the recombinant ad-enovirus ( rAd)-Cap8 shRNA successfully .rAd-Cap8 shRNA-mediated caspase-8 shRNA markedly inhibited caspase-8 ex-pression in hMSCs .Over-expression of caspase-8 shRNA by infection of rAd-Cap8 shRNA also efficiently decreased the ap-optotic rate and caspase-8 activity in hMSCs under the conditions of serum deprivation and hypoxia , with up-regulation of the mRNA expression of HGF, IGF-1 and Bcl-2.CONCLUSION:Caspase-8 shRNA attenuates hMSC apoptosis under the conditions of serum deprivation and hypoxia .

Reactivity of intrapulmonary arterial rings to thromboxane A2 and endothelin-1 is reduced in patients with chronic obstructive pulmonary disease / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the reactivity of intrapulmonary arterial rings to vasoactive substances as thromboxane A2 and endothelin-1 in patients with chronic obstructive pulmonary disease (COPD).</p><p><b>METHODS</b>Intrapulmonary arterial rings isolated from patients with normal lung function and COPD were mounted in a Multi Myograph system to determine the reactivity of the intrapulmonary arterial rings to 60 mmol/L KCl, thromboxane A2 analogue U46619 and endothelin-1 before and after preconditioning with the COX synthase inhibitor indomethacin.</p><p><b>RESULTS</b>The reactivity of intrapulmonary arterial rings to U46619 and endothelin-1 was significantly decreased in patients with COPD. The reactivity to U46619 was dramatically decreased in patients with normal lung function after application of indomethacin.</p><p><b>CONCLUSION</b>The reactivity of intrapulmonary arterial rings is significantly decreased in patients with COPD.</p>

Desmoplakin expression silencing affects cardiac voltage-gated sodium channel Nav1.5 in HL-1 cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the association of desmoplakin with the distribution and function of Nav1.5 by RNA silencing technology in HL-1 cells.</p><p><b>METHODS</b>HL-1 cells with desmoplakin expression suppression by RNA silencing were examined for desmoplakin and Nav1.5 protein expressions by Western blotting, and the distribution and co-location of desmoplakin and Nav1.5 protein were detected by immunofluorescence staining. Patch-clamp recording was applied to analyze the changes in whole-cell sodium current after desmoplakin silencing.</p><p><b>RESULTS</b>Compared with the untreated group and negative control group, the cells with desmoplakin silencing showed obviously reduced expressions of desmoplakin and Nav1.5 proteins. Co-localization of desmoplakin and Nav1.5 was detected at cell-cell contact in untreated and control conditions, and desmoplakin expression silencing induced a drastic redistribution of Nav1.5 with decreased peak current density (156.3∓6.2 vs 41.8∓3.1, n=6, P<0.05), a shift in voltage dependence of steady-state inactivation (-42 mV vs -61 mV, n=5, P<0.05), and prolonged time of recovery from inactivation.</p><p><b>CONCLUSION</b>Desmoplakin silencing caused redistribution of Nav1.5 protein and also changes in its electrophysiological properties in HL-1 cells.</p>

The level and source of inflammatory factors in patients with paroxysmal atrial fibrillation / 中国医师进修杂志

Objective To investigate the level and the source of inflammatory factors in patients with paroxysmal atrial fibrillation.Methods Thirty patients with paroxysmal atrial fibrillation were selected as observation group,and 20 cases of patients with paroxysmal supraventricular tachycardia were selected as control group.The blood samples of coronary sinus,right atria,left atria and femoral vein were consecutively collected during the procedure of radiofrequency ablation.The level of tumor necrosis factor (TNF)-α,soluble tumor necrosis factor receptor-1 (sTNFR1),and interleukin(IL)-6 was detected by ELISA separately and compared between two groups.Results The level of TNF-α and IL-6 of coronary sinus,right atria,left atria and femoral vein in observation group was significantly higher than that in control group [TNF-α:(4.45 ± 1.76) ng/L vs.(0.59 ± 0.36) ng/L,(6.67 ± 1.43) ng/L vs.(0.51 ± 0.30) ng/L,(8.35 ± 2.03) ng/L vs.(0.85 ± 0.50) ng/L,(9.97 ± 2.70) ng/L vs.(0.28 ± 0.29) ng/L,P=0.000;IL-6:(2.02 ± 0.87) ng/L vs.(1.04 ± 0.63) ng/L,(1.51 ± 0.68) ng/L vs.(0.74 ± 0.26) ng/L,(2.00 ± 0.51) ng/L vs.(0.88 ± 0.35) ng/L,(1.32 ±0.47) ng/L vs.(0.48 ±0.28) ng/L,P =0.000].The level of high sensitivity C reactive protein (hs-CRP) in observation group was significantly higher than that in control group [(2.41 ± 1.35) mg/L vs.(1.10 ±0.53) mg/L,P =0.002].The level of TNF-αof left atrium in observation group was significantly higher than that of other three sites (P=0.000).The level of IL-6 in the coronary sinus and femoral vein was significantly increased,compared with that in the right atria and left atria (P ＜ 0.05).The level of sTNFR 1 in the femoral vein,right atria and coronary sinus difference was not statistically significant (P ＞ 0.05),but was significantly higher than that in the left atria(P ＜ 0.05).The level of TNF-α,IL-6 and hs-CRP was correlated with the diameter of left atrium (LAD) (P ＜ 0.01 or ＜ 0.05).The level of sTNFR1 in left atria was positively correlated with LAD,and the level of sTNFR1 in right atria was negatively correlated with LAD (P ＜ 0.01).Conclusions The level of TNF-α,IL-6 and hs-CRP is increased in patients with paroxysmal atrial fibrillation.TNF-α and IL-6 may come from the heart and is related with the enlargement of left atrium.

Effect of ketamine on transient outward potassium current of isolated human atrial myocytes / 药学学报

The effects of ketamine on transient outward potassium current (I(to)) of isolated human atrial myocytes were investigated to understand the mechanism of part of its effects by whole-cell patch-clamp. Atrial myocytes were enzymatically isolated from specimens of human atrial appendage obtained from patients under going cardiac valve displacing. Ito is recorded in voltage-clamp modes using the patch-clamp technique at room temperature. Currents signals were recorded by an Axopatch 200B amplifier with the Digidata 1322A-pClamp 9.0 data acquisition system. Ketamine decreased I(to) of human atrial myocytes in a dose-dependent manner. The current-voltage curve was significantly lowered, 30, 100, 300, and 1000 micromol x L(-1) ketamine decreased respectively I(to) current density about (13.62 +/- 0.04)%, (38.92 +/- 0.05)%, (72.24 +/- 0.10)% and (83.84 +/- 0.05)% at the potential of 50 mV, with an IC50 of 121 micromol x L(-1). The I(to) activation curve, inactivation curve and the recovery curve were not altered by ketamine. So, ketamine concentration-dependently decreased I(to) of human atrial myocytes.

Change of TNF? level after the transfection of ?CREB eukaryotic expressive vector in neonatal rat cardiomyocytes / 中国病理生理杂志

AIM: To construct an eukaryotic expressive vector of cAMP response element binding protein (?CREB ) and observe the regulation effects of ?CREB transfection on TNF? transcription in cardiomyocytes. METHODS: ?CREB gene was obtained after PCR amplification from human heart cDNA library with CREB specific primers. After digestion and ligation, the complete cDNA was inserted into pAdTrack, a shuttle plasmid. Neonatal rat ventricular myocytes were dissociated and cultured. The myocytes were transfected with the vector by liposome-lipofectamine. The expression of transfected ?CREB was confirmed and evaluated with green fluorescent protein (GFP), competitive RT-PCR, and immunocytochemistry. Concentrations of TNF? in cultured supernatants of cardiomyocytes were measured with radioimmunoassay from control, forskolin (10 ?mol/L), transfected, and transfected with adding forskolin (10 ?mol/L) groups. RESULTS: The expression levels of ?CREB in transfected group (mRNA ratio to ?-actin, 1.00?0.05; positive protein 28.88%?9.05%) was significantly higher than that in control group (mRNA 0.76?0.04, P

Construction of recombinant adenovirus vector carrying human miR-133a and its expression in vitro / 中国病理生理杂志

AIM:To construct recombinant adenovirus vector carrying human miR-133a and study its expression in human mesenchymal stem cells(hMSCs).METHODS:The PCR product containing miR-133a was amplified from human genomic DNA and inserted into the adenoviral shuttle vector pAdTrack-CMV.Then the recombinant shuttle plasmid linearized by pmeⅠwas cotransformed into competent E.coli.BJ5183 with the adenoviral backbone plasmid pAdEasy-1 to generate the recombinant adenovirus vector rAd-mir-133a.rAd-mir-133a was then packaged and amplified in human embryonic kidney 293(HEK293) cells.The purified rAd-miR-133a was used to infect the hMSCs and the expression of miR-133a was detected by non-quantitative RT-PCR and real-time PCR.RESULTS:The recombinant adenovirus shuttle vector pAdTrack-CMV-miR-133a was constructed and verified by restriction endonuclease analysis and DNA sequence analysis.rAd-miR-133a was successfully packaged and amplified in HEK293 cells.The transcriptions of primary miR-133a and mature miR-133a were over-expressed in the hMSCs infected with rAd-miR-133a.CONCLUSION:The recombinant adenovirus vector carrying human miR-133a is successfully constructed,which lay a foundation for miR-133a function study.

siRNA suppresses the green fluorescent protein (GFP) gene expression in human umbilical vein endothelial cells / 中国病理生理杂志

AIM: To establish human umbilical vein endothelial cells (HUVECs) to express green fluorescent protein (GFP), and to study the suppression of GFP by siRNA in HUVECs. METHODS: Using lipofectamine 2000 to transform plasmid pN_3-EGFP encoding GFP into HUVECs. The HUVEC containing pN_3-EGFP, named HUVEC-GFP, was screened and selected by antibiotic G418. Using in vitro transcription T7 kit, GFPsiRNA targeting GFP mRNA and control-siRNA used as control were synthesized. The siRNAs were transfected into HUVEC-GFP with oligofectamine. 48 h later, the expression levels of GFP protein and mRNA in HUVEC-GFP were determined. RESULTS: The HUVEC-GFP was screened to express GFP in the presence of G418. The agarose gel electrophoresis analysis showed that the siRNAs prepared were integrated. 48 h after transfection with siRNAs, compared to control group, the level of GFP fluorescence was obviously decreased in the HUVEC-GFP transfected with GFPsiRNA. The results of RT-PCR detection showed that GFP mRNA expression was obviously suppressed by GFPsiRNA at the rate of 40%, and no obvious suppression of GFP mRNA expression was found in the HUVEC-GFP transfected with control siRNA. CONCLUSION: The siRNA targeting GFP mRNA, synthesized in vitro, efficiently suppresses the GFP expression in HUVECs.

Inward rectifier potassium current and mRNA expression of gene Kir2.1 in human atrial fibrillation / 中国病理生理杂志

AIM: To investigate alteration of inward re ctifier potassium current (I K1) in atrial myocytes and mRNA expression of gene Kir2.1 encoding I K1 in atrial myocardial tissue in patients with chronic atrial fibrillation (AF) compared to that with sinus rhythm (SR).METHODS: Single myocytes were isolated by enzymatic dissociation with the chunk method an d the ionic current was recorded using whole cell patch clamp technique. The sem i-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to measure the mRNA expression of Kir2.1 in atrial myocardial tissue, and the g ene GAPDH was used as an internal control.RESULTS: (1) The I K1 density was increased in AF group at hyperpolarizing pot entials, at -120 mV the current densities was (-5.71?0.65) pA/pF in AF group (n=28 cells from 7 patients) and (-4.26?1.22) pA/pF in SR group (n=35 cells from 9 patients) (P0.05).CONCLUSIONS: The increase in I K1 at hyperpolarizing potentials may be related to th e atrial electrophysiological remodeling in chronic human AF. The increased I K1 density in atrial myocytes in AF group without alteration of Kir2.1 mRNA expression in atrial tissue suggests that I K1 may be mediated at post-transcriptional levels.