体外转染NHE-1核酶对大鼠肺动脉平滑肌细胞增殖的影响

EFFECTS OF SODIUM HYDROGEN EXCHANGER-1 HAMMERHEAD RIBOZYME ON PROLIFERATION OF PULMONARY ARTERY SMOOTH MUSCLE CELLS IN RATS IN VITRO

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 (NHE 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 ,G41 8筛选后获取含有重组载体的细胞克隆 ,检测细胞NHE 1mRNA表达、2 2 Na摄取量、pHi和3H TdR掺入量。结果 :与转染 pLXSN空载体的细胞和正常对照组细胞比较 ,转染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降低和3H TdR掺入量减少 ,正常对照组和空载体组间无显著差异。结论 :NHE 1特异性锤头状核酶可对NHE 1mRNA进行特异性切割 ,减少其表达 ,从而诱导细胞酸化 。

Aim: To explore the effects of sodium hydrogen exchanger(NHE 1) specific hammerhead ribozyme on the expression and activity of NHE 1 and pHi in pulmonary artery smooth muscle cells (PASMCs) in rats, and its role in PASMCs proliferation. 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 NHE 1 hammerhead ribozyme, PRZ, was transfected into the cultured PASMCs. G418 resistant cell clones were screened with 60 μg/ml G418. Then,the expression of NHE 1 mRNA was detected by RT PCR, intracellular pH was measured with fluorescent probe BCECF, 22 Na and 3 H TdR incorporation were determined respectively. Results: Compared with the cells transfected with pLXSN and non transfected cells, NHE 1 mRNA, pHi value, 3 H TdR and 22 Na incorporation decreased significantly in cells transfected with recombinant vector PRZ. No significance was found between the pLXSN transfected group and non transfected group. Conclusion: NHE 1 hammerhead ribozyme can cleave the target RNA specifically, reduce the expression of NHE 1 mRNA, induce intracellular acidosis and consequently prohibit the proliferation of PASMCs.