次乌头碱对乳大鼠原代培养心肌细胞的毒性作用

目的探讨次乌头碱(HA)对心肌细胞的毒性作用。方法制备乳大鼠原代心肌细胞,用锥虫蓝染色法检测心肌细胞存活率,用小鼠抗大鼠α横纹肌肌动蛋白和兔抗大鼠纤连蛋白多克隆抗体免疫化学染色法鉴定心肌细胞。制备的原代细胞培养3～4d,分别加入HA0,3,6,30,60和120μmol·L-1培养5,15,30和60min,记录每组细胞搏动频率;以HA体外引起心肌细胞搏动消失的浓度及作用时间为依据,将心肌细胞分为培养基对照,二甲亚砜(DMSO)对照,HA30,60和120μmol·L-1组,分别连续培养5,15,30和60min,比色法检测乳酸脱氢酶(LDH)漏出率,倒置显微镜下观察细胞形态的变化。结果与HA0μmol·L-1组比较,HA30μmol·L-1与心肌细胞作用5,15,30和60min时细胞搏动频率增加,由40±26,42±27,38±19和(20±10)min-1分别增加到114±27,98±32,100±32和(49±13)min-1(n=12,P<0.01);HA120μmol·L-1立即导致心肌细胞停搏。不同浓度的HA随着作用时间的延长,导致LDH漏出率不同程度的增加,其中HA30,60和120μmol·L-1作用60min时细胞LDH漏出率分别为(29±8)%,(42±10)%和(57±9)%与同一时间点DMSO组比较有明显差异(P<0.01)。倒置显微镜观察发现,随着HA浓度的增加和作用时间的延长,心肌细胞有死亡现象。结论 HA对心肌细胞有毒性作用,主要表现为细胞搏动频率加快,细胞膜稳定性破坏;HA120μmol·L-1与心肌细胞作用超过120min会引起少量心肌细胞死亡。

Toxicity of hypaconitine on primary cultured myocardial cells of neonatal rats

OBJECTIVE To explore the toxicity of hypaconitine (HA) to myocardial cells in vitro. METHODS The myocardial cells of primary neonatal rats were generated and cultured. The cell survival rate was detected using the trypan blue staining method, and myocardial cells were identified by anti-α-sarcomeric actin and anti-fibronectin antibodies with immunochemical staining assay. When cells were cultured for 3-4 d，HA 3, 6, 30, 60 and 120 μmol·L^-1 was given to myocardial cells respectively, and the three fields under the inverted microscope were randomly selected and observed to record myocardial cells beating rate after being cultured with HA for another 5, 15, 30 and 60 min, respectively. According to the above results, the myocardial cells were divided into 5 groups: medium control, DMSO control, HA 30, 60 and 120 μmol·L^-1 groups. When the cells were cultured with HA for 15, 30 and 60 min, the release rate of lactate dehydrogenase (LDH) was assayed with biochemical method and the morphological changes of cells were studied under the inverted microscope. RESULTS Compared with HA 0 μmol·L^-1 group, the cell beating rate increased from 40±26，42±27，38±19 and (20±10)min^-1 to 114±27，98±32, 100±32 and （49±13）min^-1 respectively when the myocardial cells were cultured with HA 30 μmol·L^-1 for 5, 15，30 and 60 min. The cell stopped beating immediately under HA 120 μmol·L^-1. LDH release was increased with HA concentration and duration of incubation. LDH was (29±8)％,（42±10）％ and (57±9)％ after cells were cultured with HA 30, 60 and 120 μmol·L^-1 for 60 min. There was statistical sigificant difference compared with DMSO control group(P<0.01). Under the inverted microscope, it was found that the myocardial cell death occurred with the increase of HA concentration and length of culture. CONCLUSION HA has toxicity on myocardial cells, including faster beating rate of cells and impaired stability of cell membrane. Some myocardial cells may be induced to death when cultured with HA 120 μmol·L^-1 for 120 min.