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 1表达和活性、pHi的影响 ,及其与细胞增殖的关系。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 ,G418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、pHi恢复速率、2 2 Na摄取量和3H TdR掺入量变化。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、2 2 Na摄取量减少 ,同时伴有pHi降低、pHi恢复速率下降和3H TdR掺入量减少。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 effects of hammerhead ribozyme on the expression and activity of NHE 1 and pHi in pulmonary artery smooth muscle cells (PASMCs) of rats and its role in PASMCs proliferation in vitro . Methods According to 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 100 μg/ml G418. Then, the expression of NHE 1 mRNA was detected by RT PCR, intracellular pH(pHi) value and recovery rate of pHi after intracellular acid loading were measured by fluorescent probe BCECF. 22 Na uptake and 3H TdR incorporation were determined, respectively. Results Compared with the cells transfected with pLXSN and non transfected cells, NHE 1 mRNA, pHi value, pHi recovery rate, 22 Na uptake and 3H TdR incorporation 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, induce intracellular acidosis and consequently suppress the proliferation of PASMCs.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2003年第5期385-387,共3页
Journal of Third Military Medical University
基金
国家自然科学基金资助项目 ( 39870 352 )
