中国农业科学2017，Vol.50Issue(10)：1893-1903,11.DOI:10.3864/j.issn.0578-1752.2017.10.014
慢病毒介导小鼠K26/KAP26.1基因过表达对相关基因的调控作用
Regulation of Related Genes by Lentivirus-Mediated K26/KAP26.1 Gene Overexpression in Mice
摘要
Abstract
[Objective] Using the lentivirus-mediated overexpression technique,the aims of this study were at determining the effects of K26 and KAP26.1 gene overexpression on keratin-associated protein genes KAP6.2,KAP7.1,KAP8.2,and KAP11.1,and bone morphogenetic protein genes BMP4 and BMPR1B,and exploring the mechanism by which these genes influence hair fineness in mice,to achieve the goal of improving the quality of animal hair,to realize artificial regulation (such as lentivirus-mediated technology) of overexpression of some genes,and to lay a theoretical foundation for investigating the artificial regulation of mammalian hair fineness.[Method] In October,the experiment was conducted in Liaoning Provincial Key Laboratory of Biotechnology and Drug Discovery.The Kunming species mice aged five weeks were obtained from Experimental Animal Center of Dalian Medical University.The mice gene sequences of K26 (Gene ID:NM_001033397) and KAP26.1 (Gene ID:NM_027105.2) were retrieved from Genbank,and the primers were designed according to the sequences of target genes.The healthy 293T cells were transfected with the plasmid to establish the vectors of mice K26 and KAP26.1 gene lentivirus overexpression,respectively,which were transfected into mice skin fibroblasts.Transfection efficiency was observed by quantitative fluorescence microscopy.After determining the success of lentivirus overexpression,the total RNA was extracted from the transfected cells,and after reverse transcription,the obtained cDNA was measured with the Eppendorf Realplex florescent quantitative PCR to detect the influence of K26 and KAP26.1 genes overexpression on KAP6.2,KAP7.1,KAP8.2,KAP11.1,BMP-4 and BMPR-IB gene expression [Result] Proved by RT-PCR detection,mice lentivirus vectors pLenti6.3-K26-IRES-EGFP and pLenti6.3-K26.1-IRES-EGFP were established successfully.Compared by fluorescent field,the highest transfection rate of 293T cell transfected lentivirus vectors was reached after 72hr.Confirmed by PCR detection,packaged K26 and KAP26.1 lentivirus vectors transfected into mice fibroblast successfully after 72hr.Through RT-PCR detection and analyzed by SPSS 19 software,expression levels showed significant difference among the target genes,positive control group (empty plasmid,BLACK) and negative control group (mice skin fibroblasts,NC),indicating that after K26 overexpression,the expression level ofKAP26.1 was up-regulated,and vice versa.This finding suggested synergy between K26 and KAP26.1.After K26 and KAP26.1 overexpression,the expression levels of KAP6.2,KAP7.1,KAP8.2,and KAP11.1 were down-regulated.After K26 overexpression,BMP4 gene expression increased,while BMPR1B gene expression decreased.After KAP26.1 overexpression,expression levels of BMP4 and BMPR1B both were up-regulated.[Conclusion] K26 and KAP26.1 genes had a synergistic effect on the inner root sheath of the hair follicle by influencing the downstream protein synthesis signal of mTOR pathway.The high expression of K26 and KAP26.1 genes could inhibit the expression of KAP6.2,KAP7.1,KAP8.2,and KAP11.1 genes and thereby regulated hair fineness.Both K26 and KAP26.1 overexpression could up-regulate the expression level of BMP-4 gene which is the activator of BMP signaling pathway and could activate the BMP signaling pathway and then affected the growth of hair follicle cycle.K26 gene overexpression could down-regulate BMPR-IB gene expression,while KAP26.1 gene overexpression up-regulate BMPR-IB gene expression.BMPR-IB gene is the receptor I of the BMP signal.When BMPR-IB receptors decreased,the BMP downstream signal transduction will be inhibited,and then restarted hair growth cycle.When BMPR-IB receptors increased,the downstream signaling molecules transcription will be promoted,and then affected hair follicle growth cycle.Both K26 and KAP26.1 overexpression could activate BMP signaling pathway,and the expressions of KAP6.2,KAP7.1,KAP8.2,KAP11.1,BMP-4 and BMPR-IB genes were in turn regulated by the mTOR and BMP signaling pathways.But the opposite regulation effects of K26 and KAP26.1 genes on BMPR-IB gene still need to be further explored.关键词
慢病毒介导/K26/KAP26.1基因/角蛋白关联蛋白基因/BMP信号通路Key words
lentivirus-mediated/K26/KAP26.1 gene/keratin-associated protein gene/BMP signaling pathway引用本文复制引用
金梅,康琳,孙东禹,朴君,朴敬爱,赵凤琴..慢病毒介导小鼠K26/KAP26.1基因过表达对相关基因的调控作用[J].中国农业科学,2017,50(10):1893-1903,11.基金项目
国家自然科学基金(31172188)、大连市科技计划(2013B12NC090)、辽宁省教育厅科研项目(L201683652) （31172188）
