摘要
目的 探讨锤头状核酶对大鼠肺动脉平滑肌细胞Na+ /H+ 交换器 (NHE) 1表达和活性、pHi的影响。方法 构建含锤头状核酶序列的 pLXSN反转录病毒重组载体 ,将其转染体外培养的大鼠肺动脉平滑肌细胞 ,G4 18筛选后获取含有重组载体的细胞克隆。检测细胞NHE 1mRNA表达、pHi和Na+ /H+ 交换活性变化。结果 与转染 pLXSN空载体的细胞和正常对照组细胞比较 ,转染了重组载体的肺动脉平滑肌细胞中NHE 1mRNA表达减少 ,Na+ /H+ 交换活性降低 ,同时伴有pHi降低。结论 所设计的锤头状核酶可对NHE 1mRNA进行特异性切割 ,减少其表达 ,使其活性下降 ,从而诱导细胞酸化。
Objective To explore the effect of hammerhead ribozyme on the expression and activity of NHE 1 and pHi in pulmonary artery smooth muscle cells (PASMCs) of rats in vitro. Methods Based on the secondary structure of NHE 1 mRNA in rats,NHE 1 specific hammerhead ribozyme was designed with the assistance of computer. The recombinant vector of retroviral plasmid pLXSN and hammerhead ribozyme, PRZ, was transfected into the cultured PASMCs. G418 resistant cell clones were screened with 100ug/ml G418. Then, the expression of NHE 1 mRNA was detected by RT PCR, intracellular pH(pHi) value and Na+/H+ exchange activity after intracellular acid loading were measured with fluorescent probe BCECF. Results Compared with the cells transfected with pLXSN and non transfected cells, NHE 1 mRNA, pHi value and Na+/H+ exchange activity decreased significantly in cells transfected with recombinant vector PRZ. No significance was found between the pLXSN transfected group and non transfected group. Conclusion Hammerhead ribozyme can cleave the target NHE 1 mRNA specifically, reduce the expression of NHE 1 mRNA and induce intracellular acidosis.
出处
《重庆医学》
CAS
CSCD
2002年第11期1074-1076,共3页
Chongqing medicine
基金
国家自然科学基金资助项目 (39870 352 )
