大蒜新素对人巨细胞病毒感染的人胚肺成纤维细胞凋亡的影响

Effect of allitridin on apoptosis of human embryonic lung fibroblast infected by human cytomegalovirus

目的研究大蒜新素对人巨细胞病毒(HCMV)诱导感染的人胚肺成纤维细胞(HELF)凋亡的动态变化及凋亡调控基因bcl-2和fasmRNA表达的影响。方法用流式细胞术测定HELF在高、低感染复数(MOI分别为2.5、0.25)HCMV感染后1、12、24、36、48、72、96h凋亡细胞比率;大、中、小剂量大蒜新素(9、6和3μg/mL)处理正常的和感染的HELF细胞(MOI为2.5)后上述各时间点凋亡细胞比率。用原位杂交法检测大剂量大蒜新素处理感染细胞(MIO为0.25)72h后bcl-2和fasmRNA表达强度变化,并与正常细胞和感染细胞对比分析。结果正常细胞凋亡比率保持恒定低水平(凋亡率平均2.68%);低、高感染复数感染细胞随时间延长凋亡细胞逐渐增多,凋亡率峰值分别达8.85%(96h)和25.63%(72h);各剂量大蒜新素处理的正常细胞凋亡率增加,呈剂量依赖性,峰值分别为4.88%、6.47%和8.35%;但各剂量药物处理感染细胞后却使细胞凋亡比率下降,大剂量药物处理72h时效应最为明显,凋亡细胞比率由25.63%降至16.24%。正常细胞高表达bcl-2,低表达fas基因;感染HCMV后,bcl-2表达显著下调,fas表达明显增强;大蒜新素处理感染细胞后使bcl-2表达强度明显高于感染细胞,fas表达水平明显低于感染细胞,但仍未恢复到正常细胞水平。结论HCMV是HELF凋亡的强诱导剂;

Object To study the effect of allitridin on the dynamic changes of apoptosis and the expression of apoptosis modulating genes, bcl 2 and fas mRNA in human embryonic lung fibroblast (HELF) infected by human cytomegalovirus (HCMV). Methods By using flow cytometry, the apoptostic cell rates in high and low MOI (2.5 and 0.25) infected cells at 1, 12, 24, 36, 48, 72, and 96 h after HCMV infection and in infected cells (MOI=2.5) treated with different doses of allitridin (9, 6, and 3 μg/mL) at above time course after treatment of drug were measured. The levels of bcl 2 and fas mRNA expression in infected cells (MOI=0.25) treated with high dose of allitridin for 72 h were determined by insitu hybridization method, compared with infected and normal cells. Results The apoptostic cell rates were persistently low in normal HELF cells (mean: 2.68%) and increased as time in low and high MOI infected cells, peak reached 8.85% (96 h) and 25.63% (72 h), respectively. Allitridin also induced apoptosis of normal cells in a dose dependent manner, the peak was 4.88%, 6.47%, and 8.35%, respectively. Significantly, different doses of allitridin could inhibit apoptosis of high MOI infected cells. High dose of drug had the highest efficacy at 72 h, apoptostic cell rate decreased from 25.63% to 16.24%. High bcl 2 mRNA expression and low fas mRNA expression in normal HELF cells were observed, and in HCMV infected cells, the expression of bcl 2 mRNA were down regulated and that of fas mRNA were up regulated. Allitridin was significantly able to up regulate the expression of bcl 2 gene and down regulate fas gene in HCMV infected cells, although they still couldn't return to the levels of normal cells. Conclusion HCMV is a strong inducer of apoptosis for HELF cells, which is associated with its role of altering the expression of apoptosis regulating genes, bcl 2 and fas. Allitridin, although it induces apoptosis of normal cells, can markedly inhibit apoptosis induced by HCMV, and this inhibition may be related to interfering with the action of HCMV on the expression of bcl 2 and fas mRNA.