The creation of the reconstruction of right ventricular outflow tract in rats / 中国胸心血管外科临床杂志

@#Objective To investigate the feasibility of animal model of the reconstruction of right ventricular outflow tract in rats. Methods A total of 15 female Sprague-Dawley (SD) rats underwent right ventricular outflow tract reconstruction surgery. Before the operation, the collagen scaffolds were treated with g 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride chemistry (EDC), and seeded with human bone marrow stem cells (h-MSCs). Three days after the surgery, 3 rats were randomly sacrificed to evaluate the transmural resection of right ventricular outflow tract. One or 3 months later, other 3 rats at each timepoint were sacrificed, stained with Masson’s Trichrome to observe the degradation of scaffold. Furthermore, 4 weeks after the surgery, 4 rats were sacrificed and the hearts were sliced. Anti-human mitochondria staining was used to identify the survival of seeding cells. Results The transmural resection of right ventricular outflow tract was feasible in rats at an acceptable mortality (13.3%). After EDC treatment, the degradation rate of collagen scaffold was extended greatly. The seeding cells were detected by anti-mitochandria immunofluorescent staining in all patches 4 weeks after the operation. Conclusion Rat model of right ventricular outflow tract reconstruction could be a stable, reliable and economical screening model for engineered heart tissue research.

The status and prospect of ultrasound and microbubble-mediated gene delivery in cardiovascular disease gene therapy / 中国胸心血管外科临床杂志

@#With the development of molecular and cellar cardiology, gene therapy to cardiovascular disease has become the hot spot and the direction of study. Now, preclinical studies on ultrasound-mediated gene delivery (UMGD) in cardiovascular disease have achieved some success, but it is still hindered by a series of practical challenges for clinical translation. Even so, UMGD still holds the promise to cardiovascular disease in gene therapy for its non-invasiveness, accuracy, safety and ability to deliver multiple genes with repeated deliveries. In this review, we will focus on the basic principle, the current development, the future prospect and drawbacks of UMGD in the therapeutic applications of cardiovascular disease.

From biological induction to engineering assembly—— the current status and perspective on vascularization of engineered heart tissue / 中国胸心血管外科临床杂志

@#The engineered heart tissues (EHTs) is regarded as a hope for myocardial repair and regeneration. But a series of “bottleneck” problems, such as vascularization, hinder their clinical translation. This review focuses on the strategies to vascularization of EHTs and encourages the emergence of novel EHTs that can meet clinic requirement properly.

Co-expression of the C-terminal domain of Yersinia enterocolitica invasin enhances the efficacy of classical swine-fever-vectored vaccine based on human adenovirus.

The use of adenovirus vector-based vaccines is a promising approach for generating antigen-specific immune responses. Improving vaccine potency is necessary in other approaches to address their inadequate protection for the majority of infectious diseases. This study is the first to reconstruct a recombinant replication-defective human adenovirus co-expressing E2 and invasin C-terminal (InvC) glycoproteins (rAd-E2-InvC). rAd-E2-InvC with 2×106 TCID50 was intramuscularly administered two times to CSFV-free pigs at 14 day intervals. No adverse clinical reactions were observed in any of the pigs after the vaccination. The CSFV E2-specific antibody titer was significantly higher in the rAd-E2-InvC group than that in the rAdV-E2 group as measured by NPLA and blocking ELISA. Pigs immunized with rAd-E2-InvC were completely protected against lethal challenge. Neither CSFV RNA nor pathological changes were detected in the tissues after CSFV challenge. These results demonstrate that rAd-E2-InvC could be an alternative to the existing CSF vaccine. Moreover, InvC that acts as an adjuvant could enhance the immunogenicity of rAdV-E2 and induce high CSFV E2-specific antibody titer and protection level.

Screening of cellular proteins that interact with the classical swine fever virus non-structural protein 5A by yeast two-hybrid analysis.

Classical swine fever virus (CSFV), the pathogen of classical swine fever (CSF), causes severe hemorrhagic fever and vascular necrosis in domestic pigs and wild boar. A large number of evidence has proven that non-structural 5A (NS5A) is not only a very important part of viral replication complex, but also can regulate host cell’s function; however, the underlying mechanisms remain poorly understood. In the current study, aiming to find more clues in understanding the molecular mechanisms of CSFV NS5A’s function, the yeast two-hybrid (Y2H) system was adopted to screen for CSFV NS5A interactive proteins in the cDNA library of the swine umbilical vein endothelial cell (SUVEC). Alignment with the NCBI database revealed 16 interactive proteins: DDX5, PSMC3, NAV1, PHF5A, GNB2L1, CSDE1, HSPA8, BRMS1, PPP2R3C, AIP, TMED10, POLR1C, TMEM70, METAP2, CHORDC1 and COPS6. These proteins are mostly related to gene transcription, protein folding, protein degradation and metabolism. The interactions detected by the Y2H system should be considered as preliminary results. Since identifying novel pathways and host targets, which play essential roles during infection, may provide potential targets for therapeutic development. The finding of proteins obtained from the SUVEC cDNA library that interact with the CSFV NS5A protein provide valuable information for better understanding the interactions between this viral protein and the host target proteins.

Ischemic postconditioning protects cardiomyocytes against ischemia/reperfusion injury by inducing MIP2

Cardiomyocytes can resist ischemia/reperfusion (I/R) injury through ischemic postconditioning (IPoC) which is repetitive ischemia induced during the onset of reperfusion. Myocardial ischemic preconditioning up-regulated protein 2 (MIP2) is a member of the WD-40 family proteins, we previously showed that MIP2 was up-regulated during ischemic preconditioning (IPC). As IPC and IPoC engaged similar molecular mechanisms in cardioprotection, this study aimed to elucidate whether MIP2 was up-regulated during IPoC and contributed to IPoC-mediated protection against I/R injury. The experiment was conducted on two models, an in vivo open chest rat coronary artery occlusion model and an in vitro model with H9c2 myogenic cells. In both models, 3 groups were constituted and randomly designated as the sham, I/R and IPoC/hypoxia postconditioning (HPoC) groups. In the IPoC group, after 45 min of ischemia, hearts were allowed three cycles of reperfusion/ischemia phases (each of 30 s duration) followed by reperfusion. In the HPoC group, after 6 h of hypoxia, H9c2 cells were subjected to three cycles of 10 minute reoxygenation and 10 minute hypoxia followed by reoxygenation. IPoC significantly reduced the infarct size, plasma level of Lactate dehydrogenase and creatine kinase MB in rats. 12 h after the reperfusion, MIP2 mRNA levels in the IPoC group were 10 folds that of the sham group and 1.4 folds that of the I/R group. Increased expression of MIP2 mRNA and attenuation of apoptosis were similarly observed in the HPoC group in the in vitro model. These effects were blunted by transfection with MIP2 siRNA in the H9c2 cells. This study demonstrated that IPoC induced protection was associated with increased expression of MIP2. Both MIP2 overexpression and MIP2 suppression can influence the IPoC induced protection.

Role of cell-surface nucleolin in lipopolysaccharide-stimulated expression and secretion of TNF-alpha and IL-1beta / 中南大学学报(医学版)

OBJECTIVE@#To explore the role of cell-surface nucleolin in lipopolysaccharide (LPS)-stimulated expression and secretion of TNF-alpha and IL-1beta in human THP-1 monocytes.@*METHODS@#Immuno-fluorescence assay and Western blot were used to identify the expression of nucleolin on the surface of THP-1 monocytes. Inactivation of nucleolin was induced by anti-nucleolin monoclonal antibody blockage, and the expression and secretion of TNF-alpha and IL-1beta were observed by using reverse transcription polymerase chain reaction (RT-PCR) and enzyme linked immuno-sorbent assay (ELISA)respectively in LPS-mediated human THP-1 monocyte inflammatory model.@*RESULTS@#Immuno-fluorescence showed that nucleolin was localized on the cell surface of THP-1 monocytes as indicated by dotted red fluorescence. Western blot assay indicated that nucleolin existed in the cell membrane fractions. RT-PCR assay showed that the expressions of TNF-alpha and IL-1beta mRNA significantly increased at 2 h and 3 h after the treatment with 1000 microg/L LPS. After 1 h pretreatment with anti-nucleolin antibody, the levels of TNF-alpha and IL-1beta mRNA decreased compared with an anti-nucleolin antibody untreated group and an irrelevant IgG+LPS group (P<0.05). ELISA assay showed that the pretreatment with anti-nucleolin antibody inhibited significantly the secretion of LPS-induced levels of TNF-alpha and IL-1beta after 4, 12 and 24 h treatment with 1000 microg/L LPS.@*CONCLUSION@#Nucleolin expresses on the cell surface of THP-1 monocytes and involves in the LPS-mediated expression and secretion of TNF-alpha and IL-1beta.

Endogenous myocardial protection: present questions and prospects / 生理学报

Since the findings of Murry and Currie et al. that ischemic preconditioning (IPC) and heat shock response (HSR) could protect evidently myocardium against ischemia-reperfusion injury in the middle of 1980s, endogenous myocardial protection has drawn widespread attentions. A great quantity of studies completed during the past 25 years made much progress in endogenous myocardial protection. Abundant research experiences have been accumulated and a basic theoretical framework has been established in this field. However, there are still many questions need to be solved. In this review, we focused on clarifying some hot questions and important future directions in IPC, heat shock proteins (HSPs), research models and strategies in endogenous myocardial protection.

Effect of heat shock protein 47 on the expression of collagen I induced by TGF-beta(1) in hepatic stellate cell-T6 cells / 中南大学学报(医学版)

OBJECTIVE@#To determine the effect of heat shock protein 47 (HSP47) on the expression of collagen I induced by transforming growth factor beta(1) (TGF-beta(1)) in hepatic stellate cell-T6 (HSC-T6) cells.@*METHODS@#We used 1 ng/mL and 10 ng/mL recombinant human TGF-beta(1) to stimulate the cultured HSC-T6 cells. Heat shock response (HSR) and antisense oligonucleotides of HSP47 were used to induce and block the expression of HSP47, respectively. The expressions of HSP47 and collagen I were detected by Western blot and the cell viability was observed by MTT assay.@*RESULTS@#Both HSP47 and collagen I were expressed in normal HSC-T6 cells. Collagen I and HSP47 expression could be induced by both 1 ng/mL and 10 ng/mL TGF-beta(1) and collagen I was expressed the most after the treatment with 10 ng/mL TGF-beta(1). Although HSR could not affect the synthesis of collagen I as it induced the HSP47 expression, HSR could promote the expression of collagen I induced by TGF-beta(1). With no effect on the cell viability, antisense oligonucleotides could significantly inhibit HSR-mediated HSP47 expression and TGF-beta(1)-induced collagen I synthesis.@*CONCLUSION@#Over-expression of HSP47 enhances TGF-beta(1)-induced expression of collagen I in HSC-T6 cells, and HSP47 may play important roles in the process of hepatic fibrosis.

HSF1 inhibits heat stress-induced apoptosis in Raw264.7 macrophages / 中南大学学报(医学版)

OBJECTIVE@#To observe the effect of heat shock factor 1 (HSF1) on heat stress-induced apoptosis in Raw264.7 macrophages.@*METHODS@#Raw264.7 cells transfected with pcDNA3.1 and pcDNA3.1-HSF1 were exposed to heat stress (42.5 degrees C +/- 0.5 degrees C) for 1 h and recovered at 37 degrees C for 6, 9, 12, and 24 h respectively. Flow cytometry (FCM), Hoechst 33258 staining and DNA ladder assays were performed to assess the apoptosis.@*RESULTS@#After heat stress, FCM showed that apoptotic cells were increased significantly and reached the peak at 9 h in Raw 264.7 cells transfected with pcDNA3.1, and were characterized with classical morphologic changes including apoptotic body and nuclear condensation. Agarose gel electrophoresis showed that "DNA ladder" could be observed clearly at 6, 9, and 12 h after the heat stress. But the overexpression of HSF1 could reduce the number of apoptotic cells and inhibit DNA fragmentation.@*CONCLUSION@#HSF1 can inhibit heat stress-induced apoptosis in Raw264.7 macrophages.

Immortalization of embryonic fibroblasts in heat shock transcription factor 1 knockout mouse / 中南大学学报(医学版)

OBJECTIVE@#To establish immortalized embryonic fibroblast lines in heat shock transcription factor 1 (HSF1) HSF1-/- and HSF1+/+ mice and to provide experimental models to study the function of HSF1.@*METHODS@#A mammalian expression vector (pSV3neo) containing the SV40 large T antigen was used to transfect the HSF1-/- and HSF1+/+ mouse embryonic fibroblast using Lipofectamine 2000. Colonies were screened by G418 and expanded to immortalized cell lines. PCR was used to detect the integration of the large T antigen with genome in the mouse embryonic fibroblast. Expression of SV40 large T antigen gene in expanded cells was identified by RT-PCR. HSP70 expression was examined by Western blot in the embryonic fibroblast lines.@*RESULTS@#The stable growth and serial propagation were observed in the HSF1-/- and HSF1+/+ cell lines for six months. The mRNA of SV40 T antigen gene expressed in the two cell lines. HSP70 expression could not be induced in the heat-treated HSF1-/- mouse embryo fibroblasts.@*CONCLUSION@#The immortalized cells of HSF1+/+ and HSF1-/- mouse embryo fibroblasts are successfully established.

Oxidative stress-induced accumulation of heat shock protein 70 within nucleolus / 中南大学学报(医学版)

OBJECTIVE@#To investigate the effect of oxidative stress on the accumulation of heat shock protein 70 (HSP70) within C2C12 myogenic cells.@*METHODS@#Heat shock response (42 degrees C for 1 h and recovery for 12 h at 37 degrees C) was used to induce the expression of heat shock protein 70. We constructed a recombinant plasmid of HSP70 with enhanced green fluorescent protein (EGFP). After being transfected transiently into C2C12 cells, immunoblotting was used to detect the expression of HSP70 induced by heat shock response and transfection. Immunocytochemistry, fluorescent microscopy and immunoblotting were used to detect the translocation of HSP70.@*RESULTS@#Immunoblotting showed that the overexpression of HSP70 was induced by heat shock response and transient transfenction. HSP70 localized within the cytoplasm of the normal cells, but HSP70 translocated from the cytoplasm to the nucleus and the nucleolus at 1 h after the treatment of oxidative stress (0.5 mmol/L H2O2) by using immunocytochemistry, fluorescent microscopy and immunoblotting for cellular partial proteins.@*CONCLUSION@#Oxidative stress may induce the accumulation of heat shock protein 70 within the nucleolus.

Characteristics and expression of Mip5, a novel gene associated with myocardial ischemia/reperfusion in rats / 中南大学学报(医学版)

OBJECTIVE@#To determine the characteristics of a novel gene Mip5 (GenBank accession number AY553870) and its expression under physiological and pathological conditions.@*METHODS@#The characteristics of Mip5 were analyzed by bioinformatic programs including BLAST, spidey, psort, ClustalW and so on. RT-PCR was performed to detect Mip5 expression.@*RESULTS@#Bioinformatic analysis showed that Mip5 gene lied in the 13th chromosome and contained 8 exons and 7 introns, its open reading frame contained 909 bp and its protein production was 302 amino acid residues including 6 kelth domains. Under normal conditions, MIP5 expressed abundantly in the heart, brain and kidney, but its expression could not be detected in the liver and muscle. Expression of Mip5 gene was increased significantly after ischemia-reperfusion compared with the sham groups, and reached its peak at 3 h and recovered at 12 h after the reperfusion. Conclusion Mip5 gene is a novel gene containing a putative open reading frame of 302 amino acids residues and may play an important role in rat cardiomyocytes suffering ischemia processing.

Effect of nucleolin down-regulation on the proliferation and apoptosis in C2C12 cells / 中南大学学报(医学版)

OBJECTIVE@#To clarify the effect of nucleolin on the proliferation and apoptosis in C2C12 cells.@*METHODS@#After inhibiting the expression of nucleolin using antisense oligonucleotides, the cellular proliferation was determined by MTT, and the apoptosis was detected by flow cytometry (FCM) assays and DNA ladder assays.@*RESULTS@#After being transfected with antisense oligonucleotides for 24 hours, Western blotting showed that the expression of nucleolin was repressed significantly. In cells treated with antisense oligonucleotides, the cellular proliferation was obviously inhibited; the apoptotic cell increased significantly; and the "DNA ladder" was clearly observed. But the sense and random oligonucleotides had no effect on the cellular proliferation and apoptosis.@*CONCLUSION@#The down-regulation of nucleolin can inhibit the cellular proliferation and initiate the apoptosis in C2C12cells.

Detection of severe acute respiratory syndrome probable patients' virus RNA in Hangzhou by using a two loci and a modified nested real-time reverse transcription polymerase chain reaction / 中华预防医学杂志

<p><b>OBJECTIVE</b>To detect the RNA of severe acute respiratory syndrome virus (SARS-CoV) by using reverse transcription polymerase chain reaction (RT-PCR) targeted for a two loci and a modified nested real-time RT-PCR as to improving the reliability and sensitivity of tests.</p><p><b>METHODS</b>A nested RT-PCR was used for detecting one fragment of SARS-CoV RNA in oropharyngeal swabs from 3 SARS probable patients, 4 SARS suspect patients and other 27 patients with fever in Hangzhou, and the nested RT-PCR product from one SARS probable patient was sequenced. Meanwhile in these 3 SARS probable patients, other three RT-PCR methods, including a hemi-nested RT-PCR targeted for another fragment of SARS-CoV RNA, a real-time RT-PCR and a modified nested real-time RT-PCR, were employed to detect SARS-CoV RNA.</p><p><b>RESULTS</b>Two positives were found in the 3 SARS probable patients, and none positive in 4 SARS suspect patients and other 27 patients with fever, using the nested RT-PCR. The sequence of the nested RT-PCR product from one SARS probable patient was identified with the counterpart of SARS-CoV genomes published in public database. The results of the hemi-nested RT-PCR, the real-time RT-PCR and the modified nested real-time RT-PCR in the 3 SARS patients were consistent with the one of the nested RT-PCR. During detecting specimen with low copies of RNA, a weak positive signal was produced after about 35 cycles in the real-time RT-PCR, but a strong positive signal was found only after 10 cycles in the modified nested real-time RT-PCR.</p><p><b>CONCLUSION</b>It might improve the reliability of test by employing RT-PCR targeted for two or more fragments in SARS-CoV genome. The modified nested real-time RT-PCR might have higher sensitivity than the routine real-time RT-PCR.</p>