反义端粒酶反转录酶对5-羟色胺诱导的肺动脉平滑肌细胞增殖抑制的机制研究

Studies on the mechanism of antisense telomerase reverse transcriptase in inhibiting the proliferation of pulmonary artery smooth muscle cells induced by 5-hydroxytryptamine

目的探讨端粒酶反转录酶(TERT)的反义寡核苷酸(ASODN)在抑制5-羟色胺(5-HT)诱导的肺动脉平滑肌细胞(PASMC)增殖过程中对细胞凋亡和细胞周期的影响。方法组织块法培养大鼠PASMC,并将细胞分为对照组(应用RPMI-1640培养液培养)、5-HT组(应用含10-6mol/L5-HT的培养液培养)、反义核酸组(应用含5-HT、反义TERT寡核苷酸的培养液培养)和正义核酸组(应用含5-HT、正义TERT寡核苷酸的培养液培养)。采用Hoechst染色、免疫荧光显微镜检测各组细胞凋亡形态,并采用流式细胞仪检测细胞凋亡和细胞周期,免疫组化法检测细胞核增殖抗原(PCNA)在细胞中的表达。结果Hoechst染色实验显示5-HT组凋亡细胞数(161±33)明显高于对照组(63±16,P<0.05),而与正义核酸组(177±48)比较差异无统计学意义(P>0.05);反义核酸组凋亡细胞数(289±58)高于5-HT组和正义核酸组(P<0.05)。流式细胞仪检测提示5-HT可促进正常细胞的早期和晚期凋亡,并促使细胞由G1期向S期转化。反义TERT对5-HT引起的细胞凋亡有协同作用,并引起G1期阻滞。免疫组化实验显示,5-HT组PASMC的PCNA表达较对照组增加(P<0.05);正义核酸组PCNA表达高于对照组(P<0.05),并与5-HT组表达相近;而反义TERT组PCNA的表达显著低于5-HT组和正义核酸组(P<0.05)。结论反义TERT可有效抑制5-HT诱导的PASMC增殖,为肺动脉高压的基因治疗提供了理论基础。

Objective To investigate the effect of antisense telomerase reverse transcriptase （TERT） on 5-hydroxytryptamine （5- HT） induced proliferation and cell cycle of pulmonary artery smooth muscle cells （PASMCs）. Methods PASMCs were cultured and divided into four groups： control group （cultured in RPMI 1640 culture medium）, 5-HT group （cultured in culture medium containing 5-HT）, antisense oligonucleotide （ASODN） group （cultured in culture medium containing 5-HT and ASODN TERT）, and sense oligonucleotide （SODN） group （cultured in culture medium containing 5-HT and SODN TERT）. The apoptosis of PASMC was observed by fluorescence microscopy with Hoechst staining. Apoptosis and cell cycle was analyzed with flow cytometry. Expression of proliferated cell nuclear antigen （PCNA） was determined by immunohistochemistry staining. Results Hoechst staining showed that apoptosis in 5-HT group （161± 33） was significantly higher than that in control group （63±16, P〈0. 05）, while no difference existed between 5-HT group and SODN group （177±48） ; apoptosis in ASODN group （289±58） was significantly higher than that in 5-HT group and SODN group. Flow cytometry analysis showed that 5-HT could promote early and late apoptosis d PASMC in control group, and it promoted cells in the G1 phases into S phases of the cell cycle. ASODN TERT can promote apoptosis induced by 5- HT and block cell proliferation in G1 phase. The immunohistochemistry staining showed that the expressions of PCNA protein in PASMC in 5-HT group and SODN TERT group increased significantly compared with that in control group, while no difference existed between 5-HT group and SODN group. The expression of PCNA protein in ASODN TERT group was significantly lower than that in 5-HT group and SODN group. Conclusion ASODN TERT can effectively inhibit the PASMC proliferation induced by 5- HT, and it may provide a rationale for gene therapy in patients with pulmonary hypertension.