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目的研究肾综合征出血热(HFRS)患者外周血T淋巴细胞(PBL-T)活化诱导细胞死亡(activation-inducedcelldeath,AICD)的发生情况,探讨AICD在HFRS发病机制中的作用。方法以免疫磁珠负性分离PBL-T;应用植物血凝素(PHA)和离子霉素(ionomycin)体外培养诱导细胞活化;采用HE染色和脱氧核苷酸原位末端标记法(TUNEL)染色观察凋亡情况并用流式细胞仪检测PBL-T的凋亡率。应用Westernblot检测HFRS患者外周血T淋巴细胞caspase-3的表达情况,用RT-PCR对汉坦病毒的特异性基因片段进行检测。结果HFRS患者诱导组PBL-T凋亡率为(27.79±0.99)%,明显高于未诱导组的(17.16±1.14)%(P<0.01);无论是否诱导,患者PBL-T的凋亡率均高于健康人。HFRS患者诱导组PBL-Tcaspase-3的17000活性成分表达显著增加。扩增产物测序结果,有19例患者血清出现阳性条带,其中汉滩病毒7例,汉城病毒12例。结论以PHA和离子霉素可有效地诱导体外培养的PBL-T发生AICD。HFRS患者PBL-T经激活诱导后凋亡率明显提高。有79.2%的患者可以扩增到汉坦病毒特异性的基因片段。HFRS患者PBL-T凋亡相关蛋白caspase-3的表达增加,提示AICD可能与HFRS的发病机制有关。
Objective To investigate the activation-induced cell death (AICD) induced by peripheral blood T lymphocytes (PBL-T) in patients with hemorrhagic fever with renal syndrome (HFRS) and to explore the role of AICD in the pathogenesis of HFRS. Methods PBL-T was negatively isolated by magnetic beads. Cell activation was induced by PHA and ionomycin in vitro. HE staining and TUNEL staining The apoptosis was observed and the apoptosis rate of PBL-T was detected by flow cytometry. Western blot was used to detect the expression of caspase-3 in peripheral blood T lymphocytes of HFRS patients, and the specific gene fragments of Hantavirus were detected by RT-PCR. Results The apoptosis rate of PBL-T in induced group was (27.79 ± 0.99)%, which was significantly higher than that in non-induced group (17.16 ± 1.14)% (P <0.01). The apoptosis rate of PBL-T in HFRS patients Higher than healthy people. The expression of 17000 active components in PBL-Tcaspase-3 induced by HFRS patients was significantly increased. Sequencing results of amplification products showed positive bands in 19 patients, including Hantaan virus in 7 cases and Seoul virus in 12 cases. Conclusion PHA and ionomycin can effectively induce AICD in PBL-T cultured in vitro. The apoptosis rate of PBL-T induced by activation in HFRS patients was significantly increased. In 79.2% of patients, Hantavirus-specific gene fragments can be amplified. HFRS patients PBL-T apoptosis associated protein caspase-3 expression increased, suggesting that AICD may be related to the pathogenesis of HFRS.