Experimental study of compound BAM15 alleviating cryopreservation-induced primary hepatocyte injury in rats
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摘要:
目的 探讨化合物BAM15对大鼠离体肝脏原代细胞冷保存损伤的影响。 方法 采用胶原酶灌注法提取大鼠肝脏原代细胞，根据细胞培养条件的不同，将细胞分为4组：A组（含250 nmol/L BAM15的Hibernate细胞培养液）；B组（含500 nmol/L BAM15的Hibernate细胞培养液）；C组(含1 000 nmol/L BAM15的Hibernate细胞培养液）；对照组（Hibernate细胞培养液）。各组细胞均冷保存12 h。荧光显微镜下观察肝脏原代细胞的纯度。同时测定各组细胞的增殖能力、凋亡情况以及线粒体活性氧（ROS）变化情况。 结果 B组和C组的细胞增殖能力均强于对照组（均为P < 0.05）。A、B、C组的细胞凋亡率分别为（33.7±2.2）%、（19.7±1.1）%、（28.7±1.2）%，均明显低于对照组[（82.7±4.2）%，均为P < 0.05]。A、B、C组的细胞内ROS阳性率分别为（11.8±4.0）%、（7.6±1.3）%、（8.9±1.6）%，均明显低于对照组[（27.4±4.5）%，均为P < 0.05]。 结论 化合物BAM15能有效减轻大鼠肝脏原代细胞的冷损伤，其保护机制可能与BAM15减少冷缺血期间ROS生成有关。 Abstract:Objective To investigate the effect of compound BAM15 on the primary hepatocyte injury induced by cold storage in rats. Methods The primary rat hepatocytes were extracted by collagenase perfusion method. According to different cell culture conditions, the cells were divided into 4 groups: group A (Hibernate cell culture solution containing 250 nmol/L BAM15), group B (Hibernate cell culture solution containing 500 nmol/L BAM15), group C (Hibernate cell culture solution containing 1 000 nmol/ L BAM15), control group (Hibernate cell culture solution). The cells of each group were cryopreserved for 12 h. The purity of primary hepatocytes was observed under fluorescence microscope. The changes in the cell proliferation ability, cell apoptosis rate and mitochondrial reactive oxygen species (ROS) were measured in each group. Results The cell proliferation ability in groups B and C was significantly higher than that in the control group (both P < 0.05). The apoptosis rates in groups A, B and C were (33.7±2.2)%, (19.7±1.1)% and (28.7±1.2)%, which were significantly lower than (82.7±4.2)% in the control group (all P < 0.05). The positive rates of intracellular ROS in groups A, B and C were (11.8±4.0)%, (7.6±1.3)% and (8.9±1.6)%, remarkably lower than (27.4±4.5)% in the control group (all P < 0.05). Conclusions Compound BAM15 can effectively mitigate the primary hepatocyte injury in rats induced by cryopreservation. The underlying mechanism is probably associated with the role of BAM15 in reducing ROS generation during cold ischemia. -
Key words:
- Compound BAM15 /
- Cold storage /
- Primary hepatocyte /
- Proliferation /
- Apoptosis /
- Oxidative stress /
- Liver transplantation
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图 1 大鼠肝脏原代细胞的提取步骤
A图为通过大鼠肝门静脉插管；B图为Hank' s平衡盐溶液灌注后肝脏开始变得苍白；C图为胶原酶灌注后肝脏变软
Figure 1. Extraction steps of primary liver cells of rats
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