中文摘要：

目的：构建含不同分子区域的caveolin-1真核表达质粒，转染caveolin-1基因敲除小鼠（Car-1^-/-）的肾小球系膜细胞，观察能否逆转系膜细胞周期阻滞。方法：首先从pLHCX-N—FLAG2／caveolin-1全长质粒中扩增cave—olin-1N末端（第1—82位氨基酸）、N末端＋脚手架区（第1—101位氨基酸）的cDNA片段，同时在引物两端加上限制性酶切位点HindBI及XhoI，再与复制缺陷型逆转录病毒表达载体pLHCX—N—FLAG3连接。完成酶切和测序鉴定后将pLHCX—N—FLAG3／cav-1—82和pLHCX—N—FLAG3／cav-1—101分别转染HEK293T细胞，包装产生有感染力的病毒颗粒，再分别感染Cav-1十系膜细胞，流式细胞分析观察能否逆转细胞周期阻滞。结果：成功构建含eaveolin-1N末端、N末端＋脚手架区片段的重组真核表达载体，流式细胞分析发现pLHCX-N—FLAG3／cav-1—82和pLH—CX-N—FLAG3／cav-1—101基因导入Cav-1^-/-系膜细胞，可逆转G0／G1细胞周期阻滞。Vcaveolin-1可能主要通过N末端第1～82位氨基酸区域参与细胞周期进程，具体机制还需进一步研究。

英文摘要：

Objective： To construct caveolin-1 eukaryotic expression plasmid with different regions and to investigate its effect on mesangial cells from caveolin-1 knockout （Cav-1^-/-） mice after transfection. Methods： First, caveolin-1 N-terminal （1-82 amino acids） and N-terminal plus scaffold area （1-101 amino acids） cDNA fragments were amplified from pLHCX-N-FLAG2/caveolin-1 fulllength plasmid, and the HindⅢ and Xho I restriction sites were added respectively at both ends of primers. The PCR product was ligated with replication-defective retroviral expression vector pLHCX-N-FLAG3 after digestion by HindⅢ and Xho I. pLHCX-N-FLAG3/cav-1-82 and pLH- CX-N-FLAG3/cav-1-101 were respectively transfected into HEK293T cells after sequencing and packaged as infectious virus particles, and then were infected into Cav-1^-/-mesangial cells. Flow cytometry was used to observe whether the infection can reverse cell cycle arrest or not. Results： Eukaryotic expression vector of caveolin-1 N-terminal and N-terminal plus scaffold area was successfully constructed. Flow cytometry results showed that pLHCX-N-FLAG3/cav-1-82 and pLH- CX-N-FLAG3/cav-1-101 transfection can reverse G0/G1 arrest in Cav-1^-/- mesangial cells. Conclusion： Caveolin-1 peptide fragments including 1-82 and 1-101 amino acids can reverse G0/G1 arrest in Cav-1^-/- mesangial cells, indicating that caveolin-1 may involve in cell cycle control mainly through N-terminal regions of 1 to 82 amino acid.