摘要
目的 :本研究运用差异显示技术研究动脉血管平滑肌细胞在钙化过程中基因表达的改变 ,探讨与动脉钙化相关的基因。方法 :体外培养牛主动脉平滑肌细胞 ,在培养环境中加入 10mmol/L的 β 磷酸甘油酯 ,诱导细胞钙化 ,作为动脉钙化模型 ,分别提取对照细胞和钙化细胞的总RNA ,用荧光标记的引物进行DD PCR扩增 ,电泳显示差异表达的cDNA ,再用反向Northernblot对这些差异cDNA进行鉴定确认 ,并对确认的差异cDNA片段进行克隆测序。结果 :DD PCR显示 6 5个表达差异的片段 ,经过回收、扩增和反向Northernblot有 7个片断确定有持续的差异表达。经过测序和同源性比较 ,发现有 3个片段为新的基因片段。结论 :初步确定 7个与血管钙化相关的cDNA片段 ,其中
Aim: The process of vascular calcification involves various genetic alterations which may play a very important role in the vascular calcification. Vascular smooth muscle cells undoubtedly composed the main part of vascular cells, and are involved in vascular calcification. So bovine artery smooth muscle cell(BASMC) was used to investigate the gene changes during BASMC's calcification. Me-(thods:) Bovine artery smooth muscle cells cultured in vitro was induced calcified by β-Glycerophosphate(β-GP). Using DD-PCR technique to screening differentially expressed genes and those differentially expressed bands were re-examed by reverse Northern blot. All the ESTs were sequenced and BLAST with GenBank. Results: Total 65 cDNAs were isolated as differentially expressed genes and 40 of them were successfully reamplified. Using reverse-Northern blot, seven of these 40 cDNAs were reproducibly expressed differentially between the two cells. Three of them are new bands and have not been reported before. Conclusion: This is the first time using DD-PCR to screen differentially expressed genes of BASMC calcification. Seven related ESTs were identified relating to BASMC calcification.
出处
《中国应用生理学杂志》
CAS
CSCD
北大核心
2004年第3期272-275,F006,共5页
Chinese Journal of Applied Physiology
基金
国家自然科学基金资助项目 ( 98G0 3 3 9770 869)
关键词
动脉钙化
血管平滑肌细胞
差异显示
表达序列标签
artery calcification
vascular smooth muscle cell
differential display
expressed sequence tags
