摘要
目的:Visfatin是由内脏脂肪细胞分泌的一种脂肪细胞因子,它能够发挥拟胰岛素作用,并能促进脂肪细胞的分化、合成及积聚,与糖脂代谢有着密切的联系,但其确切生理功能及作用机制仍有待进一步研究。本实验成功构建了visfatin ShRNA腺病毒载体,并验证其对Hepal-6细胞visfatin mRNA表达的影响。旨在为进一步研究visfatin在影响糖脂代谢方面的作用机制提供一个简便可行的分子生物学研究平台。方法:将前期构建的pGenesil1.2-visfatin质粒双酶切,得到目的片段mU6-visfatin ShRNA,克隆入穿梭质粒pShuttle-Basic-EGFP,I-CeuI+I-SceI双酶切后转入pAdxsi载体,经筛选、鉴定后,通过脂质体转染HEK293细胞,包装后得到腺病毒Adxsi-GFP-mU6-visfatin,病毒颗粒法(viral particle,VP)和半数组织感染量法(50%tissue culture infective dose,TCID50)测定其滴度。感染hepal-6细胞后,实时定量PCR检测visfatin的mRNA表达。结果:经酶切及测序鉴定,结果与预期一致。VP法检测病毒滴度为3.20×1012VP/mL,TCID50法测得滴度为2×1011PFU/mL。重组病毒感染后,对小鼠Hepal-6细胞visfatin基因mRNA表达抑制率>70%。结论:成功构建了visfatin ShRNA重组腺病毒载体,并能有效抑制小鼠Hepal-6细胞visfatin基因表达,为后期进一步研究visfatin在糖脂代谢方面的作用机制提供了一个基因表达下调的生物学模型。
Objective： Visfatin is a kind of adipocytokin which is highly enriched in the visceral fat. Visfatin can imitates insulin actions and facilitates the differentiation, synthesis, accumulation of the adipose tissue. Visfatin has close relation with glucolipid metabolism but the exact physiological function and mechanism of action still needs further research. The experiment construct the RNA interfered adenovirus expression vector specific for visfatin gene and to observe the expression of visfatin in the Hepal-6 cells mediated by it. Aims to provide a simple and feasible molecular biology research platform for the further study of the mechanism which visfatin influenced glucolipid metabolism. Methods： Fragments mU6-visfatin ShRNA and pShuttle-Basic-EGFP were obtained by using EcoRI / HindlII to digest the plasmids pGenesil-l.2 and pShuttle-Basic-EGFP, then linked them together. The recombinants were cloned into adenovirus shuttle vector pAdxsi by standard procedure, then the newly constructed plasmids were transfected into 293 packaging cells to produce adenovirus, which were further multiplied and purified. Getting the titer of adenovius by measurting VP and TC1D50. The mRNA expression level of visfatin was evaluated by PCR after the recombinant adenoviruses infected Hepal-6 cells. Results： There were two inserted gene fragments which were 0.5 kb and 0.8 kb in recombinant shuttle plasmid and recombinant adenovirus vector respectively. The titer of adenovius were 3.20 x 10~2 VP/ml and 2 x 1011 PFU/ml. And the inhibition of visfatin mRNA expression in Hepal-6 cells is 71.90%. These results were all consistent with the expected. Conclusion： The recombinant adenovirus vector （Adxsi-GFP -mU6-visfatin） has been successfully constructed, and it could remarkably down regulated the expression ofvisfatin mRNA levels in tran- sfected Hepal-6 cells, which provides a visfatin mRNA down-regulation pattern for further research of the mechanism of visfatin and glucolipid metabolism.
出处
《现代生物医学进展》
CAS
2013年第12期2244-2248,共5页
Progress in Modern Biomedicine
基金
国家自然科学基金项目(30871199
81070640
81270913)
教育部博士点基金(20105503110002)
重庆市自然科学基金重点项目(cstc2012 jjB10022)
