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Preliminary Function Analysis for Selenoprotein W in Chicken Skeletal Muscle and Its Response to Selenium

Author: RuanHongFeng
Tutor: XuShiWen
School: Northeast Agricultural University
Course: Clinical Veterinary Medicine
Keywords: Chicken Skeletal muscle Differentiation Selenium Selenoprotein W mRNA RNA interference
CLC: S831.5
Type: Master's thesis
Year: 2011
Downloads: 58
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Abstract

Selenium (Se), essential trace elements in human and animal plays an important biological function in poultry growth and development, reproduction, immune function and disease resistance. Selenium exists mainly in the form of selenoproteins and its biological role. Selenoprotein W (Selenoprotein W, SelW) is a skeletal muscle and myocardium abundant selenoproteins in normal animals, while not detected in the muscle of animals suffering from selenium deficiency-related diseases, and later found the body selenium status with the skeletal muscle SelW changes closely related, and SelW with muscle metabolism and diseases that may be relevant. At present research about SelW mainly focused on mammals (including primates, rodents, sheep, pigs, etc.), the study of the biological function of SelW family of proteins is in its infancy, but its role in the the poultry muscle system development its study of the regulation of gene expression has not been reported. In this study, a sterile cell culture techniques to establish a reliable chicken embryo primary cultured myoblasts and purity identification of cultured cells by immunofluorescence techniques; chicks and chick myoblasts as test objects, using quantitative PCR method, determination of different selenium levels on chicken skeletal muscle (, including winged muscle, chest and leg) and myoblasts SelW transcription level, at the same time through the use of RNAi technology interference to myoblasts SelW gene level, and to detect SelW interference into muscle-related adjustment factor (Myf-5, Myogenin, MRF4 and P21) gene level, the cell cycle and intracellular total GSH content, the results showed that: 1 type I collagenase digestion, Percoll separation was further purified chick embryo muscle cell proliferation culture medium proliferation; well differentiated in differentiation culture medium to promote cell fusion and formation of myotubes. Desmin and α-Sacromeric Actin Immunocytochemistry myoblasts positive reaction, the positive rate of 95%, high purity myoblasts obtained for the the subsequent myoblast plus Selenium reaction and knockout trial to lay the foundation. 2 feed selenium concentrations (0.033 mg · kg ^{-1 , 0.150 mg · kg ^{-1 , 1.500 mg · kg ^{-1 ) can the impact of different ages (d, 15 d, 25 d, 35 d) the selenium content and SelW the transcriptional level in chicken muscle tissue, sensitivity and fast response ability of different chicken muscle tissue selenium content, including breast and leg sensitive to changes in the content of selenium;, the selenium content of the culture medium (final concentration of 10-8M ,10-7M ,10-6M) also can regulate the level of transcription in vitro cultured chick embryo myoblasts SelW vivo test similar results. The prompt SelW certain role in chicken muscle tissue metabolism. 3 the success of this experiment will Stealth RNA (S-RNA) was transfected into primary cultured chick myoblasts, the optimum condition is added to each well of six-well plates 30 nM of S-RNA and 3.0 μl LipofectamineTM RNAiMAX higher transfection efficiency; successfully screened out the efficient chicken SelW, the interference sequence SelW _S tealth (268) the myoblasts SelW mRNA expression levels decreased to 20% of the control group; and found that by interfering with mRNA outside of the open reading frame (ORF) in the untranslated region selenocysteine ??insertion element (SECIS) area can also get the interference effect. 4 In SelW RNAi after 12 h, 24 h, 36 h and 48 h, myoblasts in myoblast differentiation factor expression levels were detected, found Myogenin and MRF4 mRNA at 12 h and 36 h, lower than that of the control group , P21 lower than the control group (P lt; 0.05) at 24 h and 48 h. To prompted SelW involved in the regulation of muscle cell differentiation processes;, Myf-5 transcription level was significantly reduced myoblast cell cycle detection found G ₀ / G ₁ of cells The proportion increased proportion of G ₂ / M phase and S phase cells decreased, speculated that the cell proliferation ability by SelW interference. In point of time to get efficiency of maximum SelW interference (interference after 24 h), the GSH content myoblasts also significantly reduced, suggesting that reduced antioxidant capacity of cells.}}}

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