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Objective To explore the effects of Na+ H-exchanger 1(NHE1) knockdown on ATP binding cassette transporter A1 (ABCA1) protein expression levels and cholesterol efflux in the hypoxic RAW264.7 cells.Methods The RAW264.7 cells were infected with lentiviral vectors expressing shRNA specific for NHE1(siNHE1) or scramble RNA (siNC).The expression of NHE1 at mRNA or protein level was detected by qRT-PCR and Western blotting respectively in the infected cells after 24 h in a hypoxia condition.In the meantime,the methods of SNARF-1,Fluo-4 NW andSuc-LLVY-aminoluciferin were employed to determine NHE1 activity,intracellular Ca2+ ([Ca2+]i) and calpain activity,respectively.Furthermore,ABCA1 protein levels were detected by Western blotting in the 24 h hypoxic cells.In parallel,the intracellular cholesterol content and cholesterol efflux were analyzed by the methods of combined enzymatic HLPC and 3 H-cholesterol.Results The hypoxia condition versus the normoxia condition up-regulated NHE1 mRNA and protein expression level and activity by 2.48 folds,1.28 folds and 61.96％ (all P＜0.05),and increased[Ca2+]i and calpain activity by 4.51 folds and 2.41 folds(all P＜0.05).Whereas the NHE1 mRNA and protein expression and activity at the presence of hypoxia were inhibited by siNHE1 with the inhibition ratio of 84.95％,60.75％ and 66.44％,respectively (all P＜0.05)and[Ca2+]i and calpain activity were reduced by 59.23％ and 54.66％ (P＜0.05).Furthermore,the ABCA1 protein level was 61.67％ lower in the hypoxic cells than in the normoxic cells (P＜0.05),and siNHE1 was increased by 56.52％ after treatment of Hypoxia.Hypoxia elevated intracellular total cholesterol and cholesterol ester by 74.57 ％ and 101.81％ (all P＜0.05).Treatment with siNHE1 in the hypoxia condition can reduce total cholesterol and cholesterol ester by 34.24 ％ 及 49.66 ％ (all P＜0.05).Hypoxia reduced the cholesterol efflux by 34.79％(P＜0.05),which were partially reversed by siNHE1.Conclusions NHE1 might play an important role in hypoxia-induced ABCA1 protein attenuation and reverse cholesterol transport dysfunction through[Ca2+]i/calpain pathway.
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Objective To establish a stable and fast method for primary culture of mouse cardiomyocytes. Methods Dishes were coated with polylysine firstly.A two-step approach was used to isolate and digest mouse cardiomyocytes cells (0.25%trypsin in 4°C overnight and 0.5 mg/mL to 1.0 mg/mL collagenase +5 mg/mL albumin collagen digestion liquid in 37°C for short-time digestion), then the cardiomyocytes were purified through differential adhesion for 70 min and 5-bromodeoxyuridine ( BrdU) .The cell morphology was observed under an inverted microscope. The survival rate of cardiacmyocytes was detected by trypan-blue staining and their purity was identified by α-actinin immunofluorescence staining.Results The cardiomyocytes were in good shape and pulsed spontaneously.The survival rate of the cardiomyocytes reached 98%and the purity was 95%.Conclusions This method described in this study is an ideal method for primary culture of mouse cardiomyocytes with a high survival rate and high purity.
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AIM:To examine the effects of hypoxia on sodium-hydrogen exchange 1 (NHE1) expression, in-tracellular Ca2+concentration ( [ Ca2+] i ) and calpain activity, and to explore the effect of amiloride on adenosine triphos-phate-binding cassette transporter A1 (ABCA1) degradation and its calpain-related mechanism.METHODS:RAW264.7 cells were exposed to hypoxia for 0 h, 12 h, 24 h and 48 h.The cell viability was measured by MTT assay and the expres-sion of NHE1 at mRNA and protein levels was detected by real-time PCR and Western blot.[ Ca2+] i was analyzed by flow cytometry.Calpain activity was assessed by the method of Suc-LLVY-aminoluciferin.Furthermore, the protein levels of ABCA1 in the RAW264.7 cells exposed to hypoxia for 24 h were determined after 6 h or 12 h treatment with NHE1 inhibi-tor amiloride in the presence of cycloheximide.ABCA1 protein levels and calpain activity were detected after 12 h incuba-tion with calpain inhibitor ALLN or intracellular calcium-chelating agent BAPTA.RESULTS: Hypoxia inhibited the cell viability in a time-dependent manner.Hypoxia up-regulated the mRNA and protein expression of NHE1, and increased [ Ca2+] i and calpain activity.Hypoxia increased the degradation of ABCA1 and amiloride slowed down the ABCA1 degra-dation.ALLN or BAPTA increased ABCA1 protein level and decreased calpain activity.CONCLUSION:NHE1 inhibitor amiloride attenuates the calpain-mediated degradation of ABCA1, indicating that hypoxia-induced NHE1 might, at least in part, participate in the ABCA1 degradation.
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Objective To explore the effects of transplantated bone marrow mesenchymal stem cells (MSCs) on myocardial fibrosis with the aid of ultrasound-mediated microbubbles (MB) and bispecific antibody(BiAb) combination.Methods With the aid of MB,isolated MSCs from male mice and the BiAb were transfused into female mice with isoproterenol-induced myocardial fibrosis via tail vein (MSCs + BiAb + MB group).BiAb was producted with anti-CD29 which can recognize MSCs and anti-myosin light chain antibody (AMLCA) which can specifically bind to injured myocardium.There were six groups investigated:MSC + BiAb + MB,MSC,BiAb,MB,MSC + BiAb,untreated,and control groups.Five weeks after treatment administration,the expressions of sex-determining region of Y-chromosome (SRY) in myocardium were detected by fluorescent quantitative PCR.The distribution of collagen was observed by sirius red staining.Heat shock protein-70 (HSP-70) in myocardium was detected by immunohistochemistry.Results The highest homing number of MSCs was in the MSCs + BiAb + MB group,MSCs + BiAb group ranked the second,and lowest in MSCs group.Compared to the untreated group,the MSCs + BiAb + MB,MSCs + BiAb and MSCs groups had less collagen deposition (P ＜0.05),and decreased level of HSP-70.Compared to those of the MSCs group,the collagen deposition were decreased in MSCs + BiAb + MB group (P ＜0.05).Conclusions MB and BiAb can promote the homing number of MSCs in mice.MB can further the homing rate and the repairing efficacy of MSCs.The homing MSCs can prevent the process of myocardial fibrosis.And HSP-70 was involved in the internal mechanism.
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Atherosclerosis is a kind of complex progressive inflammation.Exposure to atherogenic risk factors,particularly OxLDL,induces the activity of BKCa in endothelial cell,monocyte/macrophage(M?),vascular smooth muscle cell,platelet and other cells to activate,which precipitates dysfunction of the cells and therefore contributes to the development of atherosclerosis.This article briefly reviews the reseach progress in BKCa participating in the development of atherosclerosis.
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A summary will be made in this article concerning foreign seniors’ probation of Cardiology. These points as follows are vital to the successful teaching: making sufficient preparations before practice; analyzing the emphasis and difficulties, encouraging students to become the subject of class by making inquiry, doing physical examination by themselves and discussing the cases, paying great attention to the education of medical morality nurturance and correcting the students’ case records seriously.
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The current development of medical sciences focuses simply on the progress of natural sciences,while neglects the combination with humanities and social sciences,which leads to the absence or lack of medical humanistic spirit.This paper analyzes the absence of medical humanistic spirit contrasted by the rapid development of medical high-tech,the manifestations and reasons for the absence of medical humanistic spirit,and proposes to strengthen the humanistic quality of medical science education in order to solve problems brought by absence of medical humanistic spirit.