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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(Cav-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-1 N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HindⅢ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-/-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caveolin-1 N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。
OBJECTIVE: To construct a caveolin-1 eukaryotic expression plasmid containing different molecular regions and transfected into mesangial cells of caveolin-1 knockout mice (Cav-1 - / -) to observe whether it can reverse the cell cycle of mesangial cells Blocking. Methods: The cDNAs of caveolin-1 N-terminal (amino acids 1-82) and N-terminal + scaffold (amino acids 1-101) were amplified from pLHCX-N-FLAG2 / caveolin- At the same time, restriction endonuclease HindⅢ and XhoⅠ were added to both ends of the primer, and then ligated with replication-defective retrovirus expression vector pLHCX-N-FLAG3. After digestion and sequencing, pLHCX-N-FLAG3 / cav-1-82 and pLHCX-N-FLAG3 / cav-1-101 were transfected into HEK293T cells respectively and packaged to produce infectious virus particles. -1 - / - mesangial cells, flow cytometry analysis can reverse the cell cycle arrest. Results: The recombinant eukaryotic expression vector containing caveolin-1 N-terminus and N-terminal + scaffolding region was successfully constructed. The expression of pLHCX-N-FLAG3 / cav-1-82 and pLH-CX-N-FLAG3 / cav -1-101 gene into Cav-1 - / - mesangial cells can reverse G0 / G1 cell cycle arrest. Vcaveolin-1 may participate in cell cycle progression mainly through amino acid residues 1-82 of N terminus. The mechanism needs further study.