三氧化二砷对人冠状动脉平滑肌细胞作用的体外观察

Effects of arsenic trioxide on human coronary smooth muscle cells： experiment in vitro

目的探讨三氧化二砷对人冠状动脉平滑肌细胞（HCSMC）的促凋亡作用及其对损伤动脉再狭窄防治的意义。方法将人冠状动脉平滑肌细胞传代培养，将细胞分为正常对照组、三氧化二砷（浓度分别为1．0、2．0、3．0、4．0及5．0μmol／L）组，通过透射电镜、DNA电泳、原位细胞凋亡检测试剂盒（TUNEL）、四甲基偶氮唑盐（MTT）检测细胞活性观察三氧化二砷促细胞凋亡作用，Western印迹检测凋亡相关基因Bcl-2和Bax的表达变化。结果随三氧化二砷浓度的增加能够抑制HCSMC的增殖并且该作用与时间及三氧化二砷浓度呈正相关，观察第7天、浓度为5．0μmol／L时细胞生存数为[（4.41±0．10）×10^5／ml，正常对照组（30．11±0．93）×10^5／ml差异有统计学意义，P〈0．05]，透射电镜下可见凋亡小体、DNA电泳可见典型凋亡后梯形条带、TUNEL可见凋亡细胞由（16．0±3．1）％增至（38．7±2．7）％（P〈0．05），三氧化二砷上调凋亡促进基因Bax的表达而下调凋亡抑制基因Bcl—2表达。正常对照组均不见典型上述变化。结论三氧化二砷可促进HCSMC的凋亡，抑制其过度增殖。

Objective To study the apoptotic effects of arsenic trioxide on human coronary smooth muscle cells （HCSMCs）. Methods HCSMCs were cultured and randomly divided into 5 groups to be treated by arsenic trioxide of the concentrations 1.0, 2.0, 3.0, 4.0, and 5.0 μmol/L for 48 h. Cell growth curve was drawn by MTF method. DNA electrophoresis was used to observe the apoptosis. Western blotting was conducted to examine the protein expression of Bax, an apoptosis-promoting gene, and Bcl-2, an apeptosis-inhibiting gene. Other HCSMCs were cultured with 4.0 μmo]/L arsenic trioxide for 48 h, then transmission electron microscopy was used to observe the ultra-structure and TUNEL was used to detect the percentage of apoptotic cells. HCSMCs not treated with arsenic trioxide were used as control group. Results Arsenic trioxide of different concentrations inhibited the proliferation of HCSMCs dose and time-dependently. When the concentration of arsenic trioxide was 5.0 μmol/L the number of living cells was （4.41 ±0.10） × 10^5/ml, significantly lower than that of the control group [（30. 11 ± 0. 93） × 10^5/ml, P 〈0.05]. Apoptosis bodies were observed under the transmission electron microscope. DNA electrophoresis showed hazy ladders, especially when the concentrations were 3.0 - 4.0 μmol/L. When the concentration was 5.0 μmol/L the number of necrotic cells increased remarkably and apoptosis became less significant. TUNEL showed that the apoptotic cells increased from 16.0% ± 3.1% to 38.7% ± 2.7% （ P 〈 0.05 ） . MTT test showed that arsenic trioxide decreased the absorbence of HCSMCs dose and time-dependently. Western blotting showed that the Bcl-2 expression was decreased and the expression of Bax increased after treatment of arsenic trioxide. Conclusion Arsenic trioxide exerts an apoptotic effect on HCSMCs.