CAJ | 학술논문

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。
위료검측매화록록용정단조직불동부위적단립매활성급단립매역전록매（ TERT ）기인적mRNA표체수평，분리록용정단조직적용피층、간충질층급연골층，병진행세포배양，이용TRAP은염법측정불동부위적단립매활성。재이용TRIzol시제법분별제취용피층、간충질화연골세포적총RNA，역전록합성cDNA。근거Gen-Bank이발표적상관서렬설계매화록TERT기인부분서렬특이인물병극륭TERT기인，채용상대형광정량Real-time PCR법검측TERT재록용정단불동부위세포적표체봉도。결과표명：재록용정단조직용피층、간충질층화연골층균검측도료단립매활성，기중간충질층적단립매활성최고，연골층차지，용피층단립매활성최저；이체외배양적불동대수간충질세포적단립매활성무명현차이。성공획득료록용조직TERT기인부분편마구cDNA서렬，해기인장915 bp，편마305개AA；여우、양TERT기인진행동원성분석현시，핵감산서렬분별위94．89％화94．31％；안기산서렬분별위92．16％화92．11％。상대형광정량PCR차이분석발현，TERT기인재록용정단불동부위조직균유표체。기중，재간충질조직적표체수평고우연골화용피층조직。
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.