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

【目的】利用慢病毒介导过表达技术,明确小鼠K26和KAP26.1基因过表达后对角蛋白关联蛋白基因KAP6.2、KAP7.1、KAP8.2、KAP11.1和对骨形态发生蛋白基因BMP-4、BMPR-IB的影响,探究小鼠绒毛细度变化的影响机制,达到人为调控(如慢病毒载体技术)使相关基因过表达,改善动物绒毛品质的目的,为哺乳动物绒毛细度调控的研究奠定理论基础,【方法】试验在辽宁省生物技术与分子药物研发重点实验室完成,小白鼠采自大连医科大学实验动物中心,选取出生后5周龄昆明种雄鼠。在Genebank查找到小鼠基因K26(Gene ID:NM_001033397)及KAP26.1(Gene ID:NM_027105.2)序列,根据目的基因序列设计引物,将质粒转入生长状态良好的293T细胞,构建小鼠K26和KAP26.1基因慢病毒过表达载体,将含有目的基因K26及KAP26.1的慢病毒表达载体分别转染小鼠皮肤成纤维细胞,用荧光显微镜观察其转染情况,确定慢病毒表达载体转染成功后,从病毒感染后的细胞中抽提总RNA,将RNA反转录成c DNA后放入Eppendorf Realplex荧光定量PCR仪,检测K26和KAP26.1基因过表达对KAP6.2、KAP7.1、KAP8.2、KAP11.1、BMP-4和BMPR-IB基因表达的影响。【结果】经RT-PCR检测,证明小鼠慢病毒载体p Lenti6.3-K26-IRES-EGFP和p Lenti6.3-K26.1-IRES-EGFP构建成功。经荧光场对比,发现目的慢病毒载体转染293T细胞72h时转染率最高。经PCR检测,证实包装好的目的慢病毒K26及KAP26.1转染小鼠成纤维细胞72 h后转染成功。经RT-PCR检测与SPSS Statistics 19软件分析目的基因表达量与阳性对照组(空载体质粒组,BLACK)、阴性对照组(小鼠表皮成纤维细胞,NC)表达量之间的显著性差异,发现K26基因过表达会导致KAP26.1基因上调,反之亦然,说明K26和KAP26.1基因之间存在一定的协同作用;K26和KAP26.1基因过表达后,均能导致KAP6.2、KAP7.1、KAP8.2、KAP11.1基因下调;K26基因过表达能使BMP-4基因表现为上调,而BMPR-IB基因表现为下调;KAP26.1基因过表达时,BMP-4和BMPR-IB基因均表现为上调。【结论】K26与KAP26.1基因在毛囊的内根鞘中起到协同作用,K26和KAP26.1基因的高表达会抑制KAP6.2、KAP7.1、KAP8.2、KAP11.1基因的表达,影响m TOR下游蛋白合成信号,进而起到调节绒毛粗细的作用;K26和KAP26.1基因过表达均能使BMP-4基因表现为上调,BMP-4是BMP信号通路的激活剂,能够激活BMP信号通路向下游转导,进而影响到毛囊的发育周期;K26基因过表达下调BMPR-IB基因的表达,而KAP26.1基因过表达上调BMPR-IB基因的表达,BMPR-IB基因是BMP信号的Ⅰ型受体,当BMPR-IB受体减少时,会抑制BMP信号向下游转导,从而使绒毛生长周期重新开始;当BMPR-IB受体增加时,会促进下游信号分子转录,进而影响毛囊发育周期。说明K26和KAP26.1基因过表达均能激活BMP信号通路,并由BMP和m TOR信号调节KAP6.2、KAP7.1、KAP8.2、KAP11.1、BMP-4和BMPR-IB基因的表达,但K26与KAP26.1基因对BMPR-IB基因的相反调节作用有待深入研究。

[ 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 KAPll. i, 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, KAPll.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 of KAP26.1 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 KAPll.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-1B 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, KAPll.I,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.