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The Study of Prevention and Treatment of Myocardial Fibrosis by Pyk2-Related Non-Kinase

Author: YouKai
Tutor: HaoJia
School: Third Military Medical University
Course: Surgery
Keywords: Gene therapy Calcium-dependent tyrosine kinase Calcium-dependent kinase- associated tyrosine kinase Myocardial fibrosis Adenovirus vectors
CLC: R54
Type: Master's thesis
Year: 2011
Downloads: 17
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Abstract

1 Background and Objective: Myocardial fibrosis (myocardial fibrosis) is an important and common pathological process, which in cardiac decompensation from compensatory to transition from reversible myocardial remodeling irreversible change to play a vital effect. In prevention of myocardial fibrosis gene therapy means, target selection and gene transfer method is critical. Study found that cardiac fibroblasts (cardiac fibroblasts, CFs) myocardial interstitial fibrosis as the final effector cells, is likely to become a new cardiac remodeling and heart failure treatment target cells. In CFs find a suitable meeting point signaling network as a target, which may explore myocardial interstitial fibrosis prevention measures is of great significance and application value. Calcium-dependent tyrosine kinase (calcium-dependent proline-rich tyrosine kinase-2, Pyk2) in intracellular signal transduction plays an important role, and we have confirmed previous experiments Pyk2 in the process of myocardial fibrosis plays a prominent role in the regulation of . Calcium-dependent tyrosine kinase-related non-kinase (Pyk2-related non-kinase, PRNK) Pyk2 gene is generated as a different splicing Expression of c-terminal domain of individual homologous proteins, in particular cell may be selectively Pyk2 regulates function. Based on the above background information, we passed in the experiment carried PRNK adenovirus from the cell and animal models two levels of initial observation adenovirus vector technology can be effective and stable will PRNK transferred to the target cell; whether overexpression PRNK effectively inhibit Pyk2 phosphorylation, thereby slowing the development of myocardial fibrosis; preliminary study will be with PRNK adenovirus targeting prevention of myocardial fibrosis inhibiting Pyk2 as means of gene therapy possibilities for in-depth study and Pyk2 PRNK influence on myocardial fibrosis and molecular mechanisms provide a theoretical basis. (2) Gene synthesis method first PRNK cDNA coding region and inserted into an adenovirus vector, the gene construct with PRNK recombinant adenovirus genome; transfected 293T cells for packaging and amplification, purification; measuring virus droplets degrees, OK restriction enzyme digestion, the lesions observed 293T cells and fluorescence expression, Western blot were identified PRNK whether high expression. Then PRNK adenoviral vector transfection to angiotensin Ⅱ (angiotention Ⅱ, Ang Ⅱ)-induced rat cardiac fibroblasts. MTT (MTT method) and flow cytometry CFs proliferation and apoptosis changes, and by chemical assay and enzyme-linked immunosorbent assay (ELISA method) to detect cell culture supernatant hydroxyl prolyl acid and type Ⅰ, Ⅳ collagen secretion changes. CFs detected by Western blot in PRNK, phosphorylated Pyk2 protein expression. Finally, the preparation of abdominal aortic stenosis model in rats, divided into four groups: sham operation group 10; PRNK adenovirus intervention group 10, a week after modeling intravenously PRNK adenovirus 1ml; drug intervention group 10 only, a week after modeling give PPARγ agonist rosiglitazone 100 mg · kg ^{-1 · d ^{-1 , intraperitoneal injection; surgery group 10, the establishment of large rat model of abdominal aortic coarctation. After five weeks, measuring left ventricular hypertrophy index. Myocardial collagen Van Gieson staining measurements of myocardial collagen volume fraction (collagen volume fraction, CVF) and myocardial perivascular collagen area ratio (perivascular collagen area, PVCA); myocardial collagen MASSON France by special staining blue dye collagen content. TUNEL assay of apoptosis in myocardial tissue changes. Myocardial tissue detected by ELISA type Ⅰ, Ⅳ collagen secretion changes. Western blot analysis of rats after gene introduction myocardial tissue PRNK and phosphorylated Pyk2 protein expression. 3 Results (1) successfully synthesized PRNK functional fragment, and build a fragment containing the adenovirus vector. The recombinant adenovirus expressing PRNK can effectively target cell proteins. (2) PRNK adenovirus infection at a higher efficiency of infection in vitro cultured rat cardiac fibroblasts and stable expression. (3) the use of PRNK CFs adenovirus infection, the cell culture supernatant hydroxyproline, Ⅰ collagen, Ⅳ collagen detection values ??were significantly lower than the control group. A MTT colorimetric values ??and flow cytometry demonstrated PRNK significantly inhibited angiotensin Ⅱ induced proliferation of CFs and promote apoptosis. (4) the use of PRNK CFs adenovirus infection can be highly expressed PRNK, and significantly inhibited by the Ang Ⅱ induced Pyk2 phosphorylation. (5) The PRNK adenovirus vector via the tail vein into myocardial interstitial fibrosis, may cause PRNK rats left ventricular weight / weight loss. (6) The PRNK adenovirus vector via the tail vein into myocardial interstitial fibrosis after, CVF and PVCA measured values, blue gray collagen were significantly lower than the control group. Cardiac fibroblast apoptosis rate was significantly higher. (7) PRNK adenovirus vector via the tail vein into myocardial interstitial fibrosis after myocardial hydroxyproline, Ⅰ-type, Ⅳ collagen detection was significantly lower than the control group. (8) PRNK adenovirus vector via the tail vein into myocardial interstitial fibrosis after myocardial tissue protein highly expressed PRNK while Pyk2 phosphorylation levels down. (9) PRNK adenovirus intervention group and give PPARγ agonist rosiglitazone group, serum and myocardial tissue type Ⅰ, Ⅳ collagen secretion and other indicators were no significant differences, were significantly lower than the control group. 4 Conclusions (1) In this study, successfully constructed PRNK adenovirus vector and CFs and cardiac tissue in highly expressed gene, indicating that adenovirus technology can be used as the mechanism and prevention of myocardial fibrosis effective means of gene transfer. (2) PRNK adenoviral vectors can significantly inhibit the angiotensin Ⅱ-induced cardiac fibroblast proliferation, decreased cell synthesis and secretion Ⅰ, Ⅳ collagen and hydroxyproline content, promote apoptosis. Description to PRNK targeted inhibition of Pyk2 may be prevention of myocardial interstitial fibrosis of new ideas and effective way. (3) The PRNK adenoviral vectors were injected rat model of myocardial interstitial fibrosis in vivo, can be highly expressed in cardiac tissue, through competitive inhibition of Pyk2 phosphorylation, thereby inhibiting myocardial fibrosis and myocardial hypertrophy also have some inhibition. Preliminary results of this study will be verified with PRNK adenovirus targeted inhibition of Pyk2 prevention of myocardial fibrosis as a means of gene therapy is feasible.}}

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