布比卡因神经毒性大鼠海马基因表达谱的研究

发布时间：2018-02-20 18:31

本文关键词： 布比卡因神经毒性海马基因芯片大鼠　出处：《青岛大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的观察布比卡因引起大鼠神经毒性海马基因表达谱的变化,在基因水平探究布比卡因中枢神经毒性的机制。方法健康雄性8周SD大鼠6只,随机分为布比卡因组(n=3)及对照组(n=3),各组组内大鼠随机编号。大鼠适应性喂养1周后,置于自制麻醉箱内七氟醚吸入麻醉,尾静脉穿刺置管,布比卡因组泵注0.75%布比卡因,直至大鼠出现烦躁、共济失调、惊厥发作等毒性反应,脑电图出现惊厥波形后立即取材,对照组泵注生理盐水。大鼠断颈处死,解剖显微镜下取海马组织加入RNase-Free的生理盐水,清洗,液氮冷冻保存。海马组织总RNA采用Trizol试剂一步法提取,RNA的纯度和含量用紫外分光光度计测量。根据Oligotex m RNA Midi Kit纯化m RNA。以四种脱氧核苷酸作原料,以m RNA为模板,在逆转录酶作用下合成单链c DNA;再用单链c DNA为模板,以四种脱氧核苷酸作原料,继续合成DNA另一条链,两条DNA链合成双链c DNA。然后以c DNA为模板,以Cy3-DTP、Cy5-DTP为原料分别标记布比卡因组和对照组转录的c RNA。用RNeasy试剂盒纯化,片段化后,将探针混合与测试芯片Test 3预杂交进行质控;符合要求后与Affymetrix大鼠全基因组芯片置于杂交舱内杂交。布比卡因组大鼠出现惊厥时,记录各大鼠的布比卡因用量。采用SPSS 17.0统计软件处理,计量资料以均数±标准差((?)±s)表示。Gene Chip Scanner 3000扫描芯片检测信号,Microarray Suite Version 5.0分析软件分析基因是否表达,以及在对照组和布比卡因组之间表达程度是否有差异。结果(1)布比卡因用量:布比卡因组大鼠出现惊厥表现时输注布比卡因用量达到(5.50±0.66)mg/kg。(2)基因表达差异结果:与对照组比较,布比卡因组海马组织差异表达基因有107条,其中80条基因表达上调,包括Arid5a、Atf3、Bax、Caspase-3、Egr1、Fcgr3、Fgl2、Fos、Hspb1、Jun、Klf10、Lp1、Litaf、Mt、Nans、Nr4a1、Pde4b、Plaa、Ptgs2、Timpl等基因,27条基因表达下调,其中包括Bcl-2、Eef2k、Fmo5、Grm3、Lrrfip2、Npy5r、Orc4、PI3K、Rrag B、Sdpr、Tpmt、Strbp、Wasl、Znf386等基因。(3)基因生物学信息分析:这些差异性表达基因按照其编码蛋白质的功能可分为凋亡调控相关基因、信号传导相关基因、转录相关基因、代谢相关基因、酶类相关基因等。结论(1)布比卡因神经毒性诱导大鼠海马基因表达发生明显变化,提示布比卡因神经毒性是一个多基因参与的结果。(2)通过分析差异表达基因编码蛋白质的功能,发现信号传导相关基因和细胞凋亡相关基因占了差异表达基因的大多数,可以认为其神经毒性是通过多条信号通路多靶位导致的神经细胞凋亡。我们目前的研究为以后对布比卡因神经毒性引起变化的基因更加深入的研究打下了基础。
[Abstract]:Objective to observe the changes of gene expression profile in neurotoxic hippocampus of rats induced by bupivacaine and to explore the mechanism of neurotoxicity of bupivacaine at the gene level. Rats in each group were randomly divided into bupivacaine group (n = 3) and control group (n = 30). Rats in each group were randomly numbered. After one week of adaptive feeding, rats were given sevoflurane inhalation anesthesia in a self-made anesthesia box, caudal vein puncture tube was inserted, and bupivacaine group was injected with 0.75% bupivacaine by pump. The rats were subjected to irritability, ataxia, convulsion and other toxic reactions, the electroencephalogram (EEG) was taken immediately after the convulsion wave appeared, and the control group was injected with saline by pump. The rats were killed by cervical amputation, and the hippocampal tissues were taken out under anatomic microscope to add normal saline to RNase-Free. The purity and content of total RNA extracted from hippocampus by Trizol reagent were measured by UV spectrophotometer. Four deoxynucleotides were used as raw materials and m RNA as template, according to Oligotex m RNA Midi Kit. Single strand c DNA was synthesized by reverse transcriptase, and then using single strand c DNA as template and four kinds of deoxynucleotides as raw materials, another strand of DNA was synthesized, and two chains of DNA were used to synthesize double stranded c DNA. Then, c DNA was used as template. The c RNAs transcribed from bupivacaine group and control group were labeled with Cy3-DTP- Cy5-DTP respectively. The probes were purified by RNeasy kit. The probes were mixed with the test chip Test 3 for quality control. When the rats in bupivacaine group had convulsion, the amount of bupivacaine was recorded. The data were processed by SPSS 17.0 software and measured with mean 卤standard deviation. Gene Chip Scanner 3000 scan chip detection signal microarray Suite Version 5.0 analysis software analysis of gene expression, Results: the dose of bupivacaine: the bupivacaine dosage reached 5.50 卤0.66 mg / kg 路L ~ (-2) when the rats in the bupivacaine group showed convulsion symptoms, the results showed that: compared with the control group, the expression of bupivacaine was higher than that of the control group (P < 0.01), and whether there was a difference in the expression of bupivacaine between the control group and the bupivacaine group. In bupivacaine group, there were 107 differentially expressed genes in hippocampal tissue, 80 of which were up-regulated, including the down-regulated expression of 27 genes in Arid5aAtf3Ptgs2Timpl and other genes, including Caspase-3, Egr1, Fcgr3, Fgl2, Fosmb1, Junchlf10, Lp1, LitafnansNr4a1, Pde4bPlaaGs2, Timpl, etc. This includes the analysis of gene biological information of Bcl-2Eef2khe Fmo5GMT, Lrfip2Npy5rC4, PI3KN, Rrag, etc.) gene information analysis: these differentially expressed genes can be classified into apoptosis-related genes, signal transduction related genes, transcriptional related genes, metabolism-related genes according to the functions of their encoded proteins, and so on, and these differentially expressed genes can be classified into apoptosis-related genes, signal transduction related genes, transcriptional related genes, metabolism-related genes, and so on, which can be classified as apoptosis regulation related genes, signal transduction related genes, transcriptional related genes, metabolism-related genes, etc. Conclusion bupivacaine neurotoxicity induces significant changes in gene expression in hippocampus of rats, suggesting that bupivacaine neurotoxicity is a result of multiple gene involvement. It was found that signal transduction related genes and apoptosis-related genes accounted for the majority of differentially expressed genes. It can be concluded that the neurotoxicity of bupivacaine is induced by multiple signal pathways and multiple targets. Our current studies have laid a foundation for further research on the genes involved in the neurotoxicity of bupivacaine.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R614

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