| Part1.Background and Objective:The apoptosis of cardiomyocytes plays an essential role in the development of congestive heart failure (CHF). Apoptosis of ventriculat myocytes is a causal mechanism of heart failure, and apoptosis of atrial myocytes related to CHF may create the substrate of atrial fibrillation (AF), CHF and AF form a vicious circle. However, the mechanisms of apoptosis regulation during CHF are elusive.Methods:We inserted a right ventricular lead via right internal jugular vein and implanted a pacemaker to the chest of dogs. Then the dogs’heart rhythm was240beats per minute for0,24hour,1week, and2weeks. At each time point, we ran some test and then harvested the hearts. The ECGs of dogs in each group were recorded. A pigtail catheter was inserted to the left ventricle for pressure. S1S2test was performed to get the AERP. The dogs were given1000Hz stimuli for1minute to induce atrial fibrillation. After the heart was harvested, real time RT-PCR and western blot (WB) were performed for PDCD4, p53and p47phox. TUNEL was performed to test the apoptosis. Masson was performed to test the fibrosis. NADPH activity, SOD, MDA and GSH were measured by biochemestry. BNP and caspase activity were also meatured by ELISA.Results:CHF was developed within the2-week pacing period, with elevated left ventricular end diastolic pressure (LVDP). AF was also developed in the VTP-induced heart failure model. However, there’s no significant reduction in AERP. miR-499,423,328and133a were significantly downregulated and miR-21was upregulated in the left atrial tissue. PDCD4, as the target gene of miR-499, was upregulated during the pacing period. There’s a increase in the number of atrial and ventricular myocyte nuclei undergoing apoptosis as measured by TUNEL. Fibrosis was seen in left atrial tissues, which is crutial in atrial remodeling. NADPH activity and MDA was increased after2weeks of VTP, showing the involvement of oxidative stress in VTP-induced CHF.Concludes:This project group had demonstrated that miR-499participated in inhibiting apoptosis by targeting PDCD4in VTP-induced heart failure. miR-499was downregualted in ventricular and atrial myocytes seperatively. At the same time, the modulating factors such as oxidative stress which in the upstream of miR-499regulation, and the potential target of miR-499such as PDCD4were elevated in the the VTP induced CHF model. The study will shed new light on the miR-499related signaling pathway for apoptosis resulting from CHF, and provide experiment evidence for the development of new drugs based on miR-499in the prevention and treatment of CHF and CHF-related AF. Part2.Background and Objective:MicroRNAs (miRNAs, miR) comprise a class of small, endogenous noncoding RNAs. Dysregulation of miRNA expression has been described in the physiopathologic mechanism for arrhythmia, especially atrial fibrillation resently. Atrial fibrillation (AF) is a highly prevalent arrhythmia. Several miRNAs have been reported to be involved in the structural and electrical remodeling in AF. As miRNAs are present in serum or plasma in a stable form, circulating miRNAs might be useful as promising clinical biomarkers. However, there’s little study on the circulating miRNA in AF patients by far. This study sought to determine expression profile of circulating miRNAs in persistent and paroxysmal atrial fibrillation patients using microRNA array and provide evidence for miRNAs’role in regulation of AF.Methods:To determine the expression of circulating miRNAs, we collected the blood from5patients with persistent atrial fibrillation,5patients with paroxysmal atrial fibrillation and5healthy volunteers. After centrifugation, total RNAs was isolated from the serum using TRIzol. RNA quality and quantity was measured, and RNA Integrity was determined. Then the RNA samples were labeled and hybridized on the miRCURYTM LNA Array. Following the washing steps the slides were scanned. Scanned images were then processed for data extraction, and expressed data were normalized. After normalization, differentially expressed miRNAs were identified. Finally, hierarchical clustering was performed to show distinguishable miRNA expression profiling among samples.Results:Thirteen miRNAs showed significantly differential expressions both between persistent and paroxysmal atrial fibrillation patients and healthy volunteers. Eight were up-regulated: hsa-miR-3169, hsa-miR-3612, hsa-miR-634, hsa-miR-376a, hsa-miR-517b, hsa-miR-377*, hsa-miR-590-3p, and hsa-miR-664; five were down-regulated:hsa-miR-1, kshv-miR-K12-5, hsa-miR-378c, hsa-miR-204, and hsa-miR-27a. In particular, the most significantly differentially expressed miRNAs--hsa-miR-3169and hsa-miR-3612, were elevated by>4-fold. However, there’s no report about the function of these two miRNAs by far. Besides, the up-regulation of miR-517b and miR-664and the down-regulation of miR-1was consistent with former studys, while several significantly differentially expressed miRNAs in atrial fibrillation reported by former studys, such as miR-328and miR-499, were not significantly differentially expressed in this study.Concludes:Several circulating miRNAs are significantly up-regulated or down-regulated in patients with persistent and paroxysmal atrial fibrillation. MiRNAs may shed new light on the molecular mechanism of AF. Besides, this study indicates circulating miRNAs as a potential preventative and therapeutic target for AF. Part3.Background and Objective:Adipose-derived mesenchymal stem cells (ADMSCs) are considered as a good cell source for regenerative medicine with their self-renew capacity, multilineage differentiation and immunomodulatory potency. Based on this background, the aim of this study was to evaluate the influence of Rehmannia glutinosa Oligosaccharide (RGO), a traditional Chinese medicine, on human ADMSCs’proliferation, H2U2-induced apoptosis, and secretion of Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in vitro.Methods:Human ADMSCs were isolated and cultured in vitro. Then flow cytometry was carried out to characterize the cells, and MTT assay was performed to detect the proliferation. H2O2-induced apoptosis was evaluated by flow cytometry. VEGF and HGF production were detected by ELISA kits.Key findings:Human ADMSCs were positive for CD90and CD29, but negative for CD31, CD34and CD45. The results also indicate that RGO can promote the proliferation and alleviate H2O2-induced apoptosis of human ADMSCs. The mechanism of RGO’s protective effect may involve the up-regulation of VEGF and HGF.Significance:The present study indicates that RGO may increase the viability and proliferative capacity and alleviate H2O2-induced apoptosis of human ADMSCs via the paracrine release of VEGF and HGF. These results indicate that RGO application will enhance stem cell viability and improve their effects in cell therapy. |
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