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Molecular Cloning, Sequence Analysis and Prokaryotic Expression of Ovine Activin Receptor Type IIB(ActRIIB) Gene 被引量:1
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作者 张雪梅 安静 +1 位作者 张宁 刘明军 《Agricultural Science & Technology》 CAS 2014年第10期1644-1648,共5页
Objective] This study aimed to clone ovine activin receptor type llB (Ac-tRIIB) gene, construct the prokaryotic expression vector and express the target gene in vitro, thus providing basis for further function verif... Objective] This study aimed to clone ovine activin receptor type llB (Ac-tRIIB) gene, construct the prokaryotic expression vector and express the target gene in vitro, thus providing basis for further function verification. [Method] The template cDNA which was reversely transcribed from total RNA of sheep liver tissue, was subjected to polymerase chain reaction (PCR) using specific primers of ActRIIB. The ful-length cDNA of ovine ActRIIB was obtained by pMD18-T cloning and sequencing for bioinformatics analysis. Ovine ActRIIB encoding sequence was subcloned into prokaryotic expression vector pET41a with restriction sites BamHl/Notl, and then transformed into BL21 (DE3). The induced products by lPTG were analyzed with SDS-PAGE and Western Blot. [Result] The amplified ful-length cDNA of ovine Ac-tRllB gene was 1 564 bp in length (Genbank accession number: JX422071.1) with an open reading frame of 1 539 bp, encoding 512 amino acides. Ovine ActRllB shared the highest homology (99.6%) with bovine ActRllB. ActRllB had highly ho-mologous C-terminal domains and belonged to the TGFβ family. After prokaryotic expression, an approximately 92 kD His-tagged ActRllB recombinant protein was obtained, which was consistent with the excepted result. [Conclusion] Cloning and successful expression of ovine ActRIIB laid solid foundation for further investigation of its biological function. 展开更多
关键词 SHEEP actriib gene Sequence analysis Prokaryotic expression
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四君子汤改善C57小鼠恶病质状态肌肉消耗的疗效及机制研究 被引量:4
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作者 朱健 吉兆奕 +4 位作者 焦拥政 朱晓云 袁国兴 吴洁 《环球中医药》 CAS 2022年第5期752-758,共7页
目的观察四君子汤缓解或改善C57小鼠恶病质状态肌肉消耗的疗效及机制。方法C57BL/6小鼠皮下注射lewis肺腺癌肿瘤细胞建立肿瘤恶病质模型,按照随机数字表法分为正常组、模型组、四君子组1 g/(mL·d)和地屈孕酮组予甲地孕酮溶液0.024 ... 目的观察四君子汤缓解或改善C57小鼠恶病质状态肌肉消耗的疗效及机制。方法C57BL/6小鼠皮下注射lewis肺腺癌肿瘤细胞建立肿瘤恶病质模型,按照随机数字表法分为正常组、模型组、四君子组1 g/(mL·d)和地屈孕酮组予甲地孕酮溶液0.024 g/(kg·d),每组6只,连续给药14天。检测各组小鼠的体质量、肿瘤大小、脾/肝脏质量、骨骼肌质量,电镜下观察骨骼肌纤维形态的变化,酶联免疫吸附分析(enzyme linked immunosorbent assay,ELISA)法检测血清炎症因子肿瘤坏死因子(tumor necrosis factor,TNF-α)、激活素-A(Activin-A)水平,蛋白免疫印迹(western blot,WB)法及实时荧光定量PCR(real-time quantitative PCR,qRT-PCR)检测肌萎缩基因Myostatin、Activin-A、MuRF-1表达水平。结果(1)与模型组相比,四君子组的瘤体质量降低,肝脏质量降低,骨骼肌绝对质量增加(P<0.05)。(2)与正常组相比,模型组小鼠腓肠肌肌细胞排列疏松,肌纤维细胞缩小,细胞间隙增大,肌纤维细胞大小差异较大。与模型组相比,四君子组小鼠腓肠肌肌细胞排列相对紧密,细胞间隙缩小。(3)与正常组相比,模型组和四君子组小鼠血清中TNF-α、Activin-A水平明显升高(P<0.05);与模型组相比,地屈孕酮组和四君子组Activin-A呈低表达(P<0.05)。(4)qRT-PCR检测显示,与模型组相比,四君子组Myostatin、MuRF1的mRNA表达明显降低(P<0.05);Western blot结果显示四君子汤能够降低肌萎缩相关蛋白Activin-A的表达,结果与mRNA表达结果一致。结论四君子汤对荷瘤小鼠恶病质状态下的肌肉消耗有明显的缓解或改善作用,可能通过降低血清中Activin-A表达水平,改善肝肿大以及骨骼肌萎缩症状,降低Myostatin、MuRF1的mRNA表达,调节Myostatin/Actvin-ActRIIB信号途径抑制肺癌恶病质小鼠骨骼肌萎缩。 展开更多
关键词 C57BL/6小鼠 lewis细胞 肿瘤恶病质 骨骼肌萎缩 Myostatin/Actvin-actriib信号通路 四君子汤
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