心肌细胞条件性 Ppara 敲除小鼠模型中他莫昔芬安全有效剂量的筛选

摘要:目的 探 究 不 同 的 他 莫 昔 芬 ( tamoxifen) 给 药 剂 量 对 小 鼠 心 肌 细 胞 过 氧 化 物 酶 体 增 殖 物 激 活 受 体 α( peroxisome proliferator-activated receptor α, PPARα)基因 Ppara 敲除效率及心脏功能的影响,建立稳定有效的可诱导型心肌 细 胞 特 异 性 Ppara 敲 除 小 鼠。 方 法 Pparafl / fl 小 鼠 与 ^Myh6-ERT2Cre 小 鼠 进 行 交 配 及 回 交 得 到Ppara ^{fl / fl}, ^myh6-ERT2Cre 小鼠及同窝对照Ppara^{fl / fl}小鼠。 将 Ppara^{fl / fl},^myh6-ERT2Cre 小鼠随机分为对照组( 腹腔注射含 20% 乙醇的他莫昔芬玉米油溶液) 、A 组(单次腹腔注射他莫昔芬 2 mg / 只) 、B 组( 20 mg / kg, 连续注射他莫昔芬 5 d) 和 C组(40 mg / kg, 连续注射他莫昔芬 5 d) ,n = 8。 两周后,采用小动物超声监测小鼠的心功能, 病理 Masson 三色染色观察心肌纤维化的情况,Real-time qPCR 和 Western blot 检测心肌组织中 PPARα 的表达改变。 结果 与对照组相比,A 组小鼠的心功能与对照组没有显著差异,但 Real-time qPCR 显示心肌中 Ppara 的敲除效率不佳;B 组,心脏超声监测和 Masson 染色结果显示此给药剂量对小鼠心脏功能没有影响,但 Real-time qPCR 和 Western blot 均显示心肌组织中 Ppara 敲除充分;而 C 组,虽然 Real-time qPCR 结果显示心肌组织中 Ppara 的敲除充分,但心脏超声和Masson 染色结果显示此给药剂量对小鼠的心脏功能有一定程度的损伤。 结论 连续 5 d 给予Ppara^{fl / fl}, ^myh6-ERT2Cre 小鼠腹腔注射 20 mg / kg 他莫昔芬可以充分诱导心肌细胞中 Ppara 的敲除,成功建立可诱导型心肌细胞特异性 Ppara敲除( Ppara△ CM )小鼠。

Establishment of an Inducible Cardiomyocyte-specific Ppara-deficient Mice#br#

Abstract: Objective To establish a stable and effective inducible cardiomyocyte-specific Ppara ( gene ofperoxisome proliferator - activated receptor α, PPARα ) -deficient mice model, the effects of different tamoxifen administration on PPARα knockout efficiency and cardiac function in these inducible cardiomyocyte-specific Pparadeficient mice. Method Ppara^{fl / fl}, ^myh6-ERT2Cre mice and the littermate control ( Ppara^{fl / fl} ) mice were obtained by mating and backcross between Pparafl / fl mice and ^Myh6-ERT2Cre mice. Group A ( mice were intraperitoneally injected with a single dose of 2 mg tamoxifen) , group B ( mice were intraperitoneally injected with 20 mg / kg· d tamoxifen for 5 days) and group C ( mice were intraperitoneally injected with 40 mg / kg· d tamoxifen for 5 days) . Two weeks after administration of tamoxifen, the cardiac function was monitored by echocardiography, myocardial fibrosis was observed with Masson staining and the PPARα expression was detected by Real-time qPCR andWestern blot. Result Compared with the vehicle group, the cardiac function of mice in group A was not affected, as Real-time qPCR showed that the knockdown efficiency of Ppara in the myocardium was insufficient. While, in group B and group C, results of Real-time qPCR and Western blot confirmed the loss of PPARα in cardiomyocytes, and the echocardiography and histology indicated that the cardiac function of mice was normal in group B, but injured in group C. Conclusion These data suggested that intraperitoneal injection of 20 mg / kg tamoxifen for 5 consecutive days on Ppara^{fl / fl}, ^myh6-ERT2Cre mice can induce Ppara knockdown in cardiomyocytes sufficiently, leading to a successful establishment of inducible cardiac-specific Ppara deficient mice model.