鹿茸顶端组织端粒酶活性检测及TERT基因的差异表达

摘要

为了检测梅花鹿鹿茸顶端组织不同部位的端粒酶活性及端粒酶逆转录酶（ TERT ）基因的mRNA表达水平，分离鹿茸顶端组织的茸皮层、间充质层及软骨层，并进行细胞培养，利用TRAP银染法测定不同部位的端粒酶活性。再利用TRIzol试剂法分别提取茸皮层、间充质和软骨细胞的总RNA，逆转录合成cDNA。根据Gen-Bank已发表的相关序列设计梅花鹿TERT基因部分序列特异引物并克隆TERT基因，采用相对荧光定量Real-time PCR法检测TERT在鹿茸顶端不同部位细胞的表达丰度。结果表明：在鹿茸顶端组织茸皮层、间充质层和软骨层均检测到了端粒酶活性，其中间充质层的端粒酶活性最高，软骨层次之，茸皮层端粒酶活性最低；而体外培养的不同代数间充质细胞的端粒酶活性无明显差异。成功获得了鹿茸组织TERT基因部分编码区cDNA序列，该基因长915 bp，编码305个AA；与牛、羊TERT基因进行同源性分析显示，核苷酸序列分别为94．89％和94．31％；氨基酸序列分别为92．16％和92．11％。相对荧光定量PCR差异分析发现，TERT基因在鹿茸顶端不同部位组织均有表达。其中，在间充质组织的表达水平高于软骨和茸皮层组织。%In order to detect the telomerase activity of different parts of top tissue of sika deer antler and mRNA expression of TERT gene, the skin of antler, mesenchymal layer and layer of cartilage were separated from the antler tip tissue and cul-tured.TRAP silver staining was improved to detect the telomerase activity of different parts.And total RNA of cartilage layer and the skin layer of antler and mesenchymal layer were isolated by TRIzol reagent to use reverse transcription for synthesizing the cDNA.According to the correlation sequences that GenBank published, sika TERT gene partial sequence special primers were designed and the gene was cloned.The relative fluorescence quantitative real-time PCR was used to detect the expression abundance of TERT in the cell, which is in the different parts of the antler top.The telomerase activi-ty was detected on skin of antler, mesenchymal layer and layer of cartilage from the antler tip tissue respectively.Com-pared with their telomerase activity, the telomerase activity of mesenchymal is the highest, the telomerase activity of the layer of cartilage is the higher and the telomerase activity of the antler cortex is the lowest.Telomerase activity of different generation mesenchyma cells cultured in exterior has no significant difference.We obtained the partial coding cDNA se-quence of TERT gene.The gene length is 915 bp, encoded with 305-amino-acids-long peptide.Compared with other two species, the homology of TERT to cattle and sheep are 948.9 %and 94.31%, respectively, but the homology of protein are 92.16%and 92.11%, respectively.By fluorescence quantification, TE RT cDNA is distinctly up-regulated in skinlay-er, mesenchyme layer and cartilage layer from growing antler tip.In the mesenchymal tissue, the expression level of TERT gene is higher than in the cartilage tissue and the antler skin tissue.