论文总字数：17508字

摘 要

蟾蜍的干燥皮作为传统中药一直在临床中有所使用，蟾皮有着镇痛、消炎、抑制肿瘤的功效，随着人们对药物安全的重视程度的提高，以及蟾皮药物的抗肿瘤活性受到了人们的关注，蟾皮在肿瘤的治疗中配合化疗、放疗出现的越来越多，其毒性反应的研究也随之而来，主要探讨蟾皮提取物对细胞的抑制作用和抑制机制。本篇论文的实验目的是研究蟾皮80%乙醇提取物对大鼠心肌细胞H9c2中Na -K -ATP酶的毒性，以此来衡量蟾皮80%乙醇提取物对大鼠心肌细胞的心脏毒性。实验方法是使用蟾皮乙醇提取物去干预大鼠心肌细胞，使用Na -K -ATP酶活性检测试剂盒去检测心肌细胞中Na -K -ATP酶活性，Na -K -ATP酶活性降低，发现蟾皮乙醇提取物会抑制Na -K -ATP酶活性。Na -K -ATP酶的功能在于能够调节钙离子浓度，并且调节能量，蟾皮中的活性成分通过与酶中的α亚基结合，造成心肌细胞的能量紊乱，钙超载，从而产生心脏毒性。实验建立了空白对照组，6个浓度梯度的蟾皮提取物干预组以及两个浓度的地高辛阳性对照组，计算出蟾皮提取物对Na -K -ATP酶活性的抑制率，对数据进行拟合，得出蟾皮乙醇提取物的半数抑制浓度。现不仅对蟾皮提取物的心脏毒性进行了研究，也对蟾皮类药物毒性处理方法以及此类药物的未来发展做出思考与讨论。

关键词：蟾皮 Na -K -ATP酶 蟾酥 心脏毒性 抑制

Study on the toxicity of ethanol extract from toad skin to Sodium-Potassium Pump

Abstract

The dry skin of toad has been used in clinic as a traditional Chinese medicine. Toad skin has analgesic, anti-inflammatory and tumor inhibiting effects. As more and more attention has been paid to drug safety and anti-tumor activity of toad-skin drugs, more and more toad-skin is used in combination with radiotherapy and chemotherapy in the treatment of tumors, and studies on its toxicity have been carried out. These studies mainly explore the inhibitory effect of toad-skin extract on cells and its inhibitory mechanism. In this experiment, ethanol extract from toad skin was used to intervene rat cardiomyocytes, and the activity of sodium-potassium pump in cardiomyocytes was detected by sodium potassium pump activity detection kit. The results showed that the activity of sodium-potassium pump was decreased, and it was found that ethanol extract from toad skin could inhibit the activity of sodium-potassium pump. The sodium-potassium pump can regulate the concentration of calcium ions as well as the intracellular energy. The active components in toad skin bind to the α subunit in the sodium-potassium pump causing energy disorder and calcium overload in cardiomyocytes resulting in cardiac toxicity. The blank control group, the intervention group with six concentration gradients and the digoxin positive control group with two concentration levels were established in the experiment and the inhibition rate of toad skin extract on the activity of sodium-potassium pump was calculated. After data processing, half of the inhibitory concentration of toad skin ethanol extract was obtained. This paper not only studies the cardiotoxicity of toad-skin extract, but also discusses the toxicity treatment of toad-skin drugs and the future development of these drugs.

Key words：toad skin; Na -K -ATP ase; Venenum Bufonis; cardiotoxity; inhibition

目 录

摘 要 I

Abstract II

1、绪论 1

1.1蟾皮提取物的研究背景 1

1.2蟾皮药物心脏毒性 2

1.3心脏毒性的研究方法 3

1.4蟾皮提取工艺及药物剂型 4

1.5 Na -K -ATP酶的介绍 4

1.6 Na -K -ATP酶活力检测方法 5

1.7本论文的意义 6

2、实验材料与方法 7

2.1实验材料： 7

2.2实验仪器： 7

2.3实验步骤： 8

2.3.1细胞培养 8

2.3.2药品配制： 8

2.3.3加药干预： 9

2.3.4细胞收集： 9

2.3.5细胞计数： 9

2.3.6 Na -K -ATP酶活力检测： 9

2.3.7数据处理: 12

3、结果与讨论 13

3.1细胞形态观察 13

3.2吸光度值 13

3.3酶活力值 14

3.4半数抑制浓度 15

3.5讨论 15

4、展望 16

参考文献 17

致谢 20

1、绪论

1.1蟾皮提取物的研究背景

蟾皮是蟾蜍科（Bufonidae）动物的干燥皮^[1]。蟾皮在我国一直被作为传统中药材之一。20世纪80年代就有科学家对蟾皮的药理作用进行了研究^[2]，蟾皮有着强心苷、镇痛消肿、抗肿瘤多种药理作用，所以在临床治疗中，蟾皮也经常被用于各种疾病，例如癌症胃癌、肝癌、乳腺癌的治疗中，蟾皮通过抑制癌细胞的DNA对癌细胞造成抑制作用，并且蟾皮中的蟾毒灵还可以阻断细胞的信号传导通路，起到诱导细胞凋亡的作用^[3]。

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