發(fā)布時(shí)間：2018-02-20 18:31

  本文關(guān)鍵詞： 布比卡因 神經(jīng)毒性 海馬 基因芯片 大鼠　出處：《青島大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的觀察布比卡因引起大鼠神經(jīng)毒性海馬基因表達(dá)譜的變化,在基因水平探究布比卡因中樞神經(jīng)毒性的機(jī)制。方法健康雄性8周SD大鼠6只,隨機(jī)分為布比卡因組(n=3)及對(duì)照組(n=3),各組組內(nèi)大鼠隨機(jī)編號(hào)。大鼠適應(yīng)性喂養(yǎng)1周后,置于自制麻醉箱內(nèi)七氟醚吸入麻醉,尾靜脈穿刺置管,布比卡因組泵注0.75%布比卡因,直至大鼠出現(xiàn)煩躁、共濟(jì)失調(diào)、驚厥發(fā)作等毒性反應(yīng),腦電圖出現(xiàn)驚厥波形后立即取材,對(duì)照組泵注生理鹽水。大鼠斷頸處死,解剖顯微鏡下取海馬組織加入RNase-Free的生理鹽水,清洗,液氮冷凍保存。海馬組織總RNA采用Trizol試劑一步法提取,RNA的純度和含量用紫外分光光度計(jì)測(cè)量。根據(jù)Oligotex m RNA Midi Kit純化m RNA。以四種脫氧核苷酸作原料,以m RNA為模板,在逆轉(zhuǎn)錄酶作用下合成單鏈c DNA;再用單鏈c DNA為模板,以四種脫氧核苷酸作原料,繼續(xù)合成DNA另一條鏈,兩條DNA鏈合成雙鏈c DNA。然后以c DNA為模板,以Cy3-DTP、Cy5-DTP為原料分別標(biāo)記布比卡因組和對(duì)照組轉(zhuǎn)錄的c RNA。用RNeasy試劑盒純化,片段化后,將探針混合與測(cè)試芯片Test 3預(yù)雜交進(jìn)行質(zhì)控;符合要求后與Affymetrix大鼠全基因組芯片置于雜交艙內(nèi)雜交。布比卡因組大鼠出現(xiàn)驚厥時(shí),記錄各大鼠的布比卡因用量。采用SPSS 17.0統(tǒng)計(jì)軟件處理,計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差((?)±s)表示。Gene Chip Scanner 3000掃描芯片檢測(cè)信號(hào),Microarray Suite Version 5.0分析軟件分析基因是否表達(dá),以及在對(duì)照組和布比卡因組之間表達(dá)程度是否有差異。結(jié)果(1)布比卡因用量:布比卡因組大鼠出現(xiàn)驚厥表現(xiàn)時(shí)輸注布比卡因用量達(dá)到(5.50±0.66)mg/kg。(2)基因表達(dá)差異結(jié)果:與對(duì)照組比較,布比卡因組海馬組織差異表達(dá)基因有107條,其中80條基因表達(dá)上調(diào),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條基因表達(dá)下調(diào),其中包括Bcl-2、Eef2k、Fmo5、Grm3、Lrrfip2、Npy5r、Orc4、PI3K、Rrag B、Sdpr、Tpmt、Strbp、Wasl、Znf386等基因。(3)基因生物學(xué)信息分析:這些差異性表達(dá)基因按照其編碼蛋白質(zhì)的功能可分為凋亡調(diào)控相關(guān)基因、信號(hào)傳導(dǎo)相關(guān)基因、轉(zhuǎn)錄相關(guān)基因、代謝相關(guān)基因、酶類相關(guān)基因等。結(jié)論(1)布比卡因神經(jīng)毒性誘導(dǎo)大鼠海馬基因表達(dá)發(fā)生明顯變化,提示布比卡因神經(jīng)毒性是一個(gè)多基因參與的結(jié)果。(2)通過(guò)分析差異表達(dá)基因編碼蛋白質(zhì)的功能,發(fā)現(xiàn)信號(hào)傳導(dǎo)相關(guān)基因和細(xì)胞凋亡相關(guān)基因占了差異表達(dá)基因的大多數(shù),可以認(rèn)為其神經(jīng)毒性是通過(guò)多條信號(hào)通路多靶位導(dǎo)致的神經(jīng)細(xì)胞凋亡。我們目前的研究為以后對(duì)布比卡因神經(jīng)毒性引起變化的基因更加深入的研究打下了基礎(chǔ)。
[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 鹵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 鹵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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R614

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