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 .

Objective To analyze the features of onset, clinical pathological characteristics about the primary bladder mucinous adenocarcinoma.Methods From January 1990 to June 2015, we retrospectively reviewed the data from 15 patients diagnosed as primary bladder mucinous adenocarcinoma, including 10 male patients and 5 female patients.Their mean age was 58 years old, ranged from 41 to 78 years old.Among the fifteen patients, the initial symptoms included hematuria in 13 cases, lower abdominal pain in 1 case and urinary irritation symptom in 1 case.The ultrasound and CT scan revealed bladder tumors, which the size ranged from 2 to 6 cm.The location of bladder tumors included front wall in 12 cases, trigone zone in 2 cases and top wall in 1 case.Nine cases was suspected as tumor from urachal remnants.Eleven patients underwent partial cystectomy, three patients accepted the radical cystectomy and one case accepted transurethral resection of bladder tumor (TURBT).Result Pathological diagnosis was bladder mucinous adenocarcinoma in all patients, including nine from urachal remnant and the others from urothelimn.The tumor exhibited the mushroom liked prominence, which was associated with surface ulceration and infiltrated into the depth of bladder.Meanwhile, it was covered with thick mucinous substances.The histologic examination revealed the presence of andenoid structure, composed by various degree of diferenfiated mucinous cells.In cases with adenocarcinomas from urachus, the residue of urachal tissue could be noticed.The bladder mucous was intact or ulcerated.No sign of metaplasia was observed.In the pathological diagnosis, the classification included grade Ⅲ in 3 cases, grade Ⅱ in 7 cases and grade Ⅰ in 5 cases.Ten persons reported the information of the follow up.Eight of them, whose tumor originated from urachus, accepted bladder-sparing surgery.One died from acute myocardial infarction after 23 months postoperatively.And one died from cerebral hemorrhage 45 months postoperatively.The others have been followed up from 8 to 65 months with no sign of recurrence.In two cases with urothelial carcinoma, one was found the new urothelial carcinoma 50 months after TURBT and one died from cancer metastasis 29 months after partial cystectomy.Conclusions Primary mucinous adenocarcinoma of the bladder possess the relatively high malignant tendency.The hematuria is the main initial symptoms.The histologic examination revealed the presence of different differentiated mucinous cells and formed the andenoid structure.The case with urachal remnant adenocarcinoma has the better prognosis than other types.

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.

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.

Objective To study the effect of human G-coupled protein kinase 4(GRK4) A142V overexpression on angiotensin Ⅱ1 type(AT1 ) receptor and its-mediated proliferation of rat vascular smooth muscle cells .Methods We constructed a lentiviral vec-tor carrying human GRK4-EGFP gene and observed its expression in A10 cells .Expression of AT1 receptor were determined by im-munoblotting ,GRK4 activity were checked by spectrophotometry ;the linkage between GRK4 and AT1 receptor were determined by co-immunoprecipitation .[3 H] thymidine incorporation was used to detect changes of cell proliferation .Results As compared with the control cells ,A142V-transfected cells had higher GRK4 activity and higher AT1 receptor expression ;there was linkage between GRK4 and AT1 receptor ,the co-immunoprecipitation levels were lower in A142V cells .The basal levels of VSMC proliferation was higher in A142V cells ,Ang Ⅱ increased VSMC proliferation to a greater extent in A 142V cells .Conclusion GRK4 A142V ,via in-creasing GRK4 activity ,increases AT1 receptor expression and function in vascular smooth muscle cell proliferation .

[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 .

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.

Objective To evaluate the clinical presentations,pathologic characteristics,and the diagnosis and treatment of eosinophilic cystitis.Methods Two cases of eosinophilic cystitis were analyzed,and the relevant literature was reviewed.Case 1,a 63-year-old man,complained of voiding urgency and frequency,dysuria,intermittent hematuria,and lower abdominal pain.Cystoscopic examination showed that an about 5cm?3cm abnormal focus was 0.2-0.4 cm above bladder mucosa at the fundus of the bladder.Case 2,a 42-year-old woman,complained of voiding urgency and frequency,dysuria,and suprapubic pain with intermittent hematuria.Cystoscopic examination showed that about 4 cm?3 cm?2 cm abnormal foci of cauliflower-like were 0.3-0.4 cm above bladder mucosa at the fundus and neck of the bladder.Imaging examinations(Bultrasound,CT and IVU) showed diffuse thickening of bladder wall and tumor-like lesions.They both underwent transurethral resection of the lesions and were treated with oral steroids and antibiotics.Results Postoperative pathology confirmed eosinophilic cystitis in both cases.The follow-up was 6 months.The voiding symptoms disappeared,and imaging examinations showed no space-occupying lesions of the bladder in them.Up to now,there has no evidence of relapse.Conclusions Eosinophilic cystitis is a very rare tumor-like inflammatory disease.It is easily misdiagnosed as bladder neoplasm.Cystoscopy and biopsy are gold standard for the diagnosis.Current treatment modalities include transurethral resection of the bladder lesion along with nonspecific medical therapy.

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

AIM: To determine the effects of valsartan on calcium channel and sodium-calcium exchanger current in isolated ventricular myocytes of congestive heart failure (CHF) rats. METHODS: Eight weeks after coronary ligation, the rats with heart failure were confirmed by measuring the hemodynamic parameters and divided randomly into the group treating with valsartan (CHF-T, 20 mg/kg) and placebo (CHF-C). Sham-operated group rats served as negative controls (PS). Twelve weeks later, 6 rats were selected randomly for the study of ion channel. Single ventricular myocytes of rats were isolated by enzymatic dissociation. The whole-cell patch-clamp recording technique was used to record calcium channel current and sodium-calcium exchanger current. RESULTS: (1) In the hemodynamic variables, HR and blood pressure were not significantly different in three groups. Compared CHF-C with PS group, LVEDP and Cm increased, LVSP and ?d p /d t max decreased ( P 0 05). (4) Na +-Ca 2+ exchanger current in CHF-C group increased significantly. Na +-Ca 2+ exchanger current in CHF-T group was smaller significantly than that in CHF-C group. However, CHF-T group and PS group were not significantly different. CONCLUSION: Administration of valsartan is effective in preventing from cardiac function deterioration, increases calcium channel current and decreases Na +-Ca 2+ exchanger current in ventricular myocytes of heat failure rats.