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OBJECTIVE: To construct the SERCA gene interference lentivirus expression vector and establish stable transfected PC 12 cell line. METHODS: The interference sequence targeting at rat SERCA gene was designed and synthesized. pGag/Pol, pRev, and pVSV-G were co-transfected into 293T cells. The lentivirus particles were packaged and generated. The virus titer was detected. PC 12 cells were transfected for establishing the stable cell line; RT-PCR and Western blot were used to detect SERCA gene and protein expression in stable PC 12 cells,and the RESULTS were compared with those in the control group. RESULTS: The lentivirus expression vector targeted at SERCA was successfully constructed and the virus titer was 3×108 U•mL-1. Stable transfected PC 12 cells line was established. The effective interference verification revealed that shSERCA could significantly reduce the mRNA and protein levels of SERCA (P<0.01). CONCLUSION: The shRNA lentiviral expression vector of SERCA gene is successfully constructed and the PC 12 cell line stably interfering with SERCA expresion is established.
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Objective To investigate the mechanism of Thapsigargin in the proliferation of SW480 and provide experimental basis for development of anti-signaling therapy for patients with colon cancer by detection of intracellular calcium concentration in Thapsigargininduced human colon cell line SW480,.Methods Level of[Ca2+]was measured by fluorescence measurement with fura-2/AM,a fluorescent index of calcium.The suppression rate of SW480 cells was calculated by MTY assay.Results Thapsigargin promoted the levels of [Ca2+]from(133.26±5.17,197.46±4.62)to(436.63±5.21,766.41±3.12),and increased the suppression rate from(10.67±5.28)to(31.46±7.21).When W7 Was combined used with Thapsigargin,no significant changes were found in the levels of[Ca2+]and increment of the suppression rate.Condusion Thapsigargin might promote the proliferation of SW480 cells and lead to apoptosis.but the signaling pathway still remain to he studied.
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Objective To observe the effects of 1,3-dipropyl-8-cyclopentylxanthine(DPCPX),an adenosine A1 receptor antagonist,on brain neurons damage induced by hypoxia and reoxygenation(H/R),and to elucidate the relevant mechanisms.Methods An in vitro cultured rat cerebral cortical neuronal H/R damage model was established;the effects of DPCPX were detected at final concentrations of 0(control),25,50,100nmol/L on the lactate dehydrogenase(LDH) release from normoxic neurons and H/R neurons which were treated with hypoxia for 8,12,24 hours followed by reoxygenation for 24 hours;the changes of malondialdehyde(MDA) content,activities of xanthine oxidase(XO) and Ca2+-ATPase in H/R neurons which were treated with hypoxia for 12 hours and reoxygenation for 24 hours brought by administration of DPCPX at the concentration of 100nmol/L were also determined by use of specific reagents.Results With addition of 100nmol/L DPCPX,the LDH release from H/R neurons which were treated with hypoxia for 12 hours and reoxygenation for 24 hours was significantly increased compared with that in control group(P
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Objective To investigate the mechanism of intracellular calcium and other ions disturbance by measuring the activity of Ca 2+ adenosine triphosphatase (Ca 2+ ATPase) and Na + K + adenosine triphosphatase (Na + K + ATPase) Methods Model of fetal rats ischemia and reperfusion was established The duration of ischemia was 15,30,45 and 60mins respectively;after ischemia for 15 mins, reperfusion for 1,4,8,15 and 24 hours There were 7 11 fetal rats sacrificed at different time points respectively, 12 rats in sham for control The mitochondria and endoplasmic reticulium (microsomia) were estracted and the activity of the enzyme was measured Results In the ischemia group: with the development of ischemia, the activity of Ca 2+ ATPase in mitochondria decreased gradually ( P
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AIM: To explore the relationship between the alteration in gene expression of sarcoplasmic reticulum Ca~(2+)-ATPase (SERCA) and phospholamban (PLB) in spontaneously hypertensive rats (SHR). METHODS: 294 samples of total RNA were obtained from the tissue of ventriculum , aortic smooth muscle, liver and kidney in SHR and normotensive rats (WKY). RNA array was used to determine the mRNA levels of SERCA and PLB. RESULTS: Compared with age-matched WKY rats, the systolic blood pressure increased higher in 6-week-old SHR (P
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AIM: The changes of myocardial nuclear membrane Ca 2+ -ATPase function was investigated in ischemia/reperfusion injury. METHODS: The model of myocardial ischemia/reperfusion injury was established in rats. Myocardial nuclei were purified with sucrose density centrifugation,the activity of Ca 2+ -ATPase was measured and calcium uptake was assayed with [ 45 Ca 2+ ] . RESULTS: Plasma levels of malondialdehyde (MDA) and free fatty acid (FFA) in myocardial ischemia/reperfusion injury increased significantly( P
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Objective To study the effects of desflurane on Ca 2+-ATPase activity isolated rat cardiac myocyte plasma membrane ,for the mechanisms of its inhibitory myocardial function.Methods The effects of different concentration desflurane(0%-11%) on Ca 2+-ATPase activity were studied at different calcium concentration(04-20?mol/L) and at 25℃ or 37℃Results Ca 2+-ATPase activity wan depressed by desflurane,the greater inhibitory effect,the higher desflurane concentration,and more severely at low calcium concentration or at 37℃ than at high calcium concentration or at 25℃Conclusions At clinical concentration desflurane produces tha inhibitory effect on rat cardiac myocyte plasma membrane Ca 2+-ATPase activity dose-dependently,which may in part explain its depression on myocardial function.
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29.3% decrease in ejection fraction).4 of 11 were used as heart failure group(HF,n=4).9 HF beagles were randomized to receive either a recombinant adeno-associated viral carrying the SERCA2a gene(HF+SERC A2a,n=5) or the reporter gene enhanced green fluorescent protein(HF+EGFP,n=4) by thoracotomy.All HF beagles paced by 180 beats/min in order to maintain failing state.Thirty days after infection,parameters of systolic and diastolic function were measured by doppler echocardiography and hemodynamic monitor in all beagles.RESULTS:At 30 days after gene transfer,symptoms of HF+SERCA2a dogs improved.Echocardiogram parameters were superior to those in HF+EGFP group(P
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AIM: To observe the changes of sarcoplasmic reticulum Ca~(2+)-ATPase (SERCA), phospholamban (PLB) during heart failure after acute myocardial infarction (AMI) in rats and the effect of carvedilol. METHODS: Rats were randomly assigned to normal control group, sham-operation group, AMI group and carvedilol (CAR) group. 6 weeks later, in vivo hemodynamic, morphometry and SERCA, PLB mRNA and protein expression of myocytes were measured in all animals. RESULTS: In comparison with sham-operation group, LV end diastolic pressure (LVEDP) and weight of ventricles were increased, while maximal rate of rise and fall (?dp/dt) of LV pressure were decreased in AMI group. After treatment with carvedilol, these parameters were all improved. The mRNA and protein expression of SERCA were downregulated (P0.05). CONCLUSIONS: The changes of SERCA and PLB may be the important mechanism of contractile dysfunction in heart failure after AMI. Carvedilol is effective in preventing LV dysfunction after AMI. The molecular mechanism may be related with normalization of SERCA expression.
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AIM: To investigate the effect of phospholamban antisense RNA (asPLB) on the activity of sarco-endoplasmic reticulum (SR) Ca 2+-ATPase, and the change of intracellular free Ca 2+ concentration ([Ca 2+]i) in rat cardiomyocytes by adeno-associated virus(AAV) vector. METHODS: rAAV-asPLB and rAAV-LacZ were constructed by AAV Helper-Free System. RT-PCR and Western blotting were used to determine the mRNA and protein expression of PLB. The activity of SR Ca 2+-ATPase and the [Ca 2+]i were measured. RESULTS: Compared to controls, the PLB mRNA and protein expression reduced in rat cardiomyocytes transfected with rAAV-asPLB. The activity of Ca 2+-ATPase was increased. In rest state, the level of [Ca 2+]i in rAAV-asPLB transfected group was decreased. The level of [Ca 2+]i was increased when induced by isoproterenol. CONCLUSION: rAAV-asPLB vector disrupts the expression of PLB, enhances the activity of Ca 2+-ATPase, reduces the resting [Ca 2+]i and enhances the isoproterenol-induced [Ca 2+]i.
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100% elevation of PKC activity in the retina, and administration of D ? tocopherol prevented the elevation of PKC activity and diabetes induced decrease of both Na + K + ATPase and Ca 2+ ATPase activities. D ? tocopherol achieved a complete prevention of augmented pericyte and endothelial cell profile areas and basement membrane thickening in the superficial and deep capillaries bed of diabetic retina but had no effect on blood glucose and HbA 1c . Conclusion Diabetes induced histopathological abnormalities are mostly mediated by PKC. D ? tocopherol reduces the ultrastructural lesions in retinal capillary bed induced by hyperglycemia.