促血小板生成素的体外心肌细胞保护作用研究

Protective effect of thrombopoietin on myocardial cells in vitro

目的研究促血小板生成素(thrombopoietin,TPO)对体外心肌细胞的保护作用。方法建立H9C2细胞株;利用差速贴壁分离法提取新生大鼠原代心肌细胞。用多柔比星诱导H9C2细胞株及原代心肌细胞凋亡,使用TPO干预该过程。采用下列方法研究TPO对体外心肌细胞的保护作用:(1)用甲基噻唑基四唑(methyl thiazolyl tetrazolium,MTT)法检测不同TPO浓度下的多柔比星诱导H9C2细胞株凋亡的存活率;(2)动态观察TPO干预前、后24 h及48 h的原代心肌细胞集落跳动心率的影响;(3)特异荧光探针JC-1染色H9C2细胞,予流式细胞仪检测其荧光强度,计算各组的凋亡率。结果 (1)MTT法示多柔比星使心肌细胞存活率降至73.8%±1.09%;TPO浓度50、100 ng/mL作用后的存活率明显提高,分别为84.6%±3.59%、85.6%±4.43%,差异有统计学意义(P<0.05、P<0.01)。(2)多柔比星使原代心肌细胞集落第48 h心率降低,明显低于多柔比星+TPO组(TPO浓度为100 ng/mL),差异有统计学意义(15.2%±8.45%vs.47.8%±11.45%,P<0.01)。(3)TPO使由多柔比星诱导的细胞凋亡率明显下降,由17.8%±8.8%降低至11.3%±6.36%,差异有统计学意义(P<0.05)。结论 TPO对体外心肌细胞有直接保护作用,其保护作用是通过对抗细胞凋亡或诱导血管形成实现的。

Objectives To investigate the protective effect of thrombopoietin （TPO） on myocardial cells in vitro. Methods H9C2 cell line was maintained in Iscove＇s Dulbecco＇s medium （IMDM） supplemented with 10% calf serum. Beating cells from heart ventricles of neonatal heart were cultured at an in-vitro system. Apoptosis of the cell line above was induced by treatment of doxorubicin （DOX） and was blocked by TPO. Cell survival rate of H9C2 was measured by the methyl thiazolyl tetrazolium （MTr） assay. Changes of beating rate of neonatal myocardial cells were captured by digital camera and was calculated. Flow eytometry was employed to study anti-apoptotic effect of TPO by staining JC-1 protein to H9C2 cell. Results MTr assay demonstrated that DOX reduced cell survival rate to 73.8%＋_1.09%. TPO concentration of 50 ng/mL and 100 ng/mL increased cell survival rate to 84.6%±3.59%（P〈0.05）, 85.6±4.43% （P 〈0.01 ） at a dose-dependent manner. Beating rate of primary neonatal myocardial cells also decreased to 15.2±8% at the 48th h, and 100 ng/mL TPO improved beating rate to 47.8%±11.45% （P〈0.01）. TPO decreased apoptosis rate from 17.8%±8.8% to 11.3±6.% （P 〈0.01）. Conclusions TPO has protective effect on myocardial cells in vitro. Anti-apoptosis is one of the mechanisms by which TPO protects injured heart.