局麻药神经毒性比较：酰胺类—布比卡因与酯类—普鲁卡因

发布时间：2018-03-13 12:14

本文选题：局麻药　切入点：布比卡因　出处：《南方医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：局部麻醉药(简称局麻药)根据其化学结构,分为酰胺类和酯类,常用于临床麻醉。然而,大量研究表明,在多种类型细胞实验中,长时间或高浓度使用局麻药,会产生细胞毒性。在临床应用,也有关于局麻药毒性作用的报道。尽管局麻药临床浓度所致毒性发生率很低,但一旦发生,会产生严重并发症。尤其是脊髓麻醉后产生的短暂神经综合征、持续性腰骶神经病变,以及最严重并发症马尾综合征,多年来引起广泛关注。对这些并发症的发生,局麻药的神经毒性被认为是主要因素。然而,局麻药神经毒性的确切机制,目前仍未可知。既往已有关于多种局麻药神经毒性大小的研究,但是对两种类型局麻药(酰胺类-布比卡因,酯类-普鲁卡因)所致神经毒性机制是否具有差异的全面性探索,目前国内外仍未见报道。基于此,我们设计本课题。目的1.探索布比卡因和普鲁卡因对SH-SY5Y细胞活力的影响。2.比较布比卡因和普鲁卡因对SH-SY5Y细胞和DRG神经元细胞产生神经毒性机制的异同。方法1.分别用不同浓度布比卡因和普鲁卡因处理SH-SY5Y细胞,随后使用CCK-8实验和LDH实验分别检测细胞活力和细胞毒性,用曲线拟合计算两种药物LD50值,选取该值作为后续细胞模型的药物浓度。2.分别使用布比卡因和普鲁卡因LD50值浓度处理SH-SY5Y细胞。随后,使用Rhod-2-AM探针检测线粒体内钙离子含量,JC-1试剂盒检测线粒体膜电位变化,两者共同反映细胞内线粒体功能;使用DCFH-DA探针检测细胞内过氧化物含量,DHE探针检测细胞内超氧化物含量,两者共同反映细胞内氧化应激水平;使用彗星实验检测DNA损伤,Western Blot实验检测DNA损伤标志蛋白p-γ-H2AX含量,两者共同反映DNA损伤;使用TUNEL实验检测细胞凋亡,Western Blot 实验检测凋亡相关蛋白 cleaved caspase3、cleaved caspase9 含量,两者共同反映细胞凋亡情况。上述检测也在DRG神经元上进行验证。结果1.布比卡因和普鲁卡因均明显降低SH-SY5Y细胞活力,并呈剂量依赖性。2.布比卡因和普鲁卡因均显著诱导细胞线粒体内钙超载、线粒体膜电位下降、氧化应激爆发、DNA损伤以及凋亡产生(P0.05)。在细胞线粒体损伤和凋亡水平上,两药物组间无显著差异(P0.05)。但布比卡因比普鲁卡因诱导细胞产生更多超氧化物,早期DNA损伤程度更严重,而普鲁卡因比布比卡因诱导细胞产生更多过氧化物(P0.05)。结论布比卡因和普鲁卡因产生神经毒性的机制可能涉及细胞线粒体功能失调、氧化应激爆发、DNA损伤以及凋亡。两种药物诱导产生超氧化物和过氧化物含量不同,提示我们不同类型局麻药产生神经毒性机制可能通过不同途径。我们可基于各自不同的毒性机制,实现个体化和靶向性治疗。
[Abstract]:According to its chemical structure, local anesthetics are divided into amides and esters, which are often used in clinical anaesthesia. However, a large number of studies have shown that in many types of cell experiments, local anesthetics are used for a long time or at a high concentration. In clinical use, there are also reports of the toxic effects of local anesthetics. Although the incidence of toxicity caused by the clinical concentration of local anesthetics is very low, once that happens, Severe complications, especially transient nerve syndrome following spinal anesthesia, persistent lumbosacral neuropathy, and cauda equina syndrome, the most serious complications, have caused widespread concern over the years. The neurotoxicity of local anesthetic drugs is considered to be the main factor. However, the exact mechanism of neurotoxicity of local anesthetic drugs is still unknown. However, whether there are differences in neurotoxic mechanisms between two types of local anesthetic drugs (amide-bupivacaine, ester-procaine) has not been reported at home and abroad. Objective 1. To explore the effects of bupivacaine and procaine on the viability of SH-SY5Y cells. 2. To compare the neurotoxic mechanisms of bupivacaine and procaine on SH-SY5Y cells and DRG neurons. Do not treat SH-SY5Y cells with different concentrations of bupivacaine and procaine. CCK-8 test and LDH test were used to detect cell viability and cytotoxicity respectively. The LD50 values of the two drugs were calculated by curve fitting. The concentration of Bupivacaine and procaine LD50 were used to treat SH-SY5Y cells respectively. Then, the intracellular calcium content in mitochondria was detected by Rhod-2-AM probe and the change of mitochondrial membrane potential was detected by JC-1 kit. The DCFH-DA probe was used to detect the content of superoxide in the cells and the level of oxidative stress in the cells was reflected by the two probes. Comet assay was used to detect DNA damage and Western Blot assay was used to detect DNA damage marker protein p- 纬 -H2AX, both of which reflected DNA damage, TUNEL assay was used to detect apoptosis and cleaved caspase3cleaved caspase9 was detected by TUNEL assay. The results showed that: 1. Bupivacaine and procaine significantly decreased the viability of SH-SY5Y cells. In a dose-dependent manner, bupivacaine and procaine could significantly induce intracellular calcium overload, decrease of mitochondrial membrane potential, oxidative stress outbreak of DNA damage and apoptosis. There was no significant difference between the two groups (P 0.05), but bupivacaine induced more superoxide in cells than procaine, and the degree of DNA damage was more serious in the early stage of Bupivacaine than in procaine. Compared with bupivacaine, procaine can induce more peroxides (P0.05). Conclusion the mechanism of neurotoxicity of bupivacaine and procaine may be related to the dysfunction of cell mitochondria. Oxidative stress breaks out DNA damage and apoptosis. The two drugs induce the production of superoxide and peroxide. It is suggested that the neurotoxic mechanisms of different types of local anesthetic drugs may be produced by different ways, and we can realize individualized and targeted therapy based on different toxic mechanisms.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R614

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