| Backgrounds: Rennin angiotensin aldosterone system (RAS) plays a crucial role in the initiation and development of cardiac remodeling. Despite great success with traditional therapy, such as angiotensin converting enzyme inhibitors (ACEI) and angiotensin II receptor 1 (AT1) antagonists, there are still many patients with poorly controlled high blood pressure and target organs dysfunction due to many inherent disadvantages including compliance, side effects, and lack of complete reversal of pathophysiological aspects of this disease. With the development of molecular biological theory and technology, especially with the great progression in target gene identification and construction, gene therapy for hypertension becomes a new available selection. Antisense oligonucleotides (AS-ODNs) are one of the most commonly used methods, which have been explored during recent years. They can be produced relatively cheaply, rapidly, and in large quantities. They are safe, nonimmunogenic, and do not integrate into the host genome, and it is easy to control. Compared with traditional receptor antagonists, they do not cross the blood brain barrier and placenta, therefore it would not have central effects or do harm to fetus when given peripherally. Recently, several studies have shown that delivery of angiotensin II type receptor 1 antisense (AT1-AS) by nonviral or viral vector mediated-systems resulted in an impressive lowering of blood pressure accompanied by attenuation of some pathophysiological events observed in the target organs in hypertensive rats, but did not reduce blood pressure in normotensive individuals. It is likely that antisense is useful for reducing overactive hormonal systems, but does not interfere with the normal physiological functions of RAS. However, most of these studies laid particular emphasis on hemodynamics changes. Until now, there was little evidencing indicates that all of the effectiveness came from cardiomyocytes that transfected by AS-ODNs, but not from fibroblast, vascular myocytes or other cells.Objective: We tested the hypothesis that AT1 -AS-ODNs can transfect culturedneonatal cardiomyocytes and block AT1 gene expression. We try to study the AT1 associated signal transduction passway and cell growth such as DNA content, apoptosis, cellular viability when AT1 gene expression were blocked by AS-ODNs in the presence or absence of excessive AngII.Methods: Neonatal rat cardiomyocytes were digested and separated by collagenase II and trypsin, then purification with differential anchoring. Immunohistochemistry and phase-contrast microscope were used to comprehend the purity, vitality and morphology. Cardiomyocytes were transfected by AS-ODNs encapsulated with or without oligofectamine. Cellular distribution and transfection efficiency of transfection complex were monitored and quantified by using fluorescein-labeled oligonucleotides. MTT experiment, together with phase-contrast microscope was used to evaluate the toxicity of transfection complex on cardiomyocytes. Western blot was used for AT1 protein expression detection. c-Jun N-terminal protein kinase (JNK) activity was characterized by immune complex kinase assay. c-Jun protein expression was examined by immunocytochemistry. DNA content and cell cycle, as well as apoptosis index were detected by flow cytometric assay. Atrial natriuretic factor (ANP) expression was identified by radioimmunoassay.Results: Part I Immunohistochemistry results showed that more than 90% cells were a -sarcometin actin stained positive cells, indicating that the cultures were pure, vigorous and typical cardiomyocytes. Fluorescein-labeled oligonucleotides were incubated with myocytes for time ranging from 0 to 24h in the presence or absence of oligofectamine. In the absence of oligofectamine, partial cell surface was stained brightly at 30 min, and the transfection efficiency was about 23%. At the time of 60min, some fluorescent drops could be seen in the nucleus, and the efficiency reached the peak value (30%). 2h later...
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