【摘要】：目的研究倉(cāng)鼠肺細(xì)胞（V79細(xì)胞）的電化學(xué)行為，考察不同因素對(duì)V79細(xì)胞電化學(xué)信號(hào)的影響，建立細(xì)胞內(nèi)嘌呤核苷酸代謝電化學(xué)檢測(cè)系統(tǒng)。并將此系統(tǒng)用于藥物對(duì)V79細(xì)胞次黃嘌呤鳥(niǎo)嘌呤磷酸核糖轉(zhuǎn)移酶（HGPRT）基因突變的研究。為HGPRT基因突變電化學(xué)檢測(cè)方法的建立提供理論依據(jù)。 方法本文以V79細(xì)胞為實(shí)驗(yàn)細(xì)胞模型，采用循環(huán)伏安法研究了V79細(xì)胞的電化學(xué)行為，采用高效液相色譜法對(duì)V79細(xì)胞的電化學(xué)信號(hào)進(jìn)行了歸屬；采用電化學(xué)方法和常規(guī)HGPRT基因突變檢測(cè)方法對(duì)照研究了藥物致V79細(xì)胞HGPRT基因位點(diǎn)的突變。 結(jié)果V79細(xì)胞裂解液有兩個(gè)電化學(xué)信號(hào)峰，分別歸屬于鳥(niǎo)嘌呤和黃嘌呤的氧化（0.7V），以及腺嘌呤和次黃嘌呤的氧化（1.03V）；檢測(cè)最佳條件：pH值7.4，50℃恒溫水浴裂解30min；對(duì)甲磺酸乙酯（EMS）致V79細(xì)胞HGPRT基因位點(diǎn)突變研究結(jié)果顯示，在EMS濃度為1.6mg·mL-1時(shí)， V79細(xì)胞加藥組信號(hào)I峰面積在給藥后第4天開(kāi)始，明顯高于對(duì)照組，信號(hào)II峰面積在給藥后第5天開(kāi)始，明顯高于對(duì)照組；不同EMS給藥濃度研究發(fā)現(xiàn)，在藥物濃度0.7~1.6mg·mL-1范圍內(nèi)，隨著藥物濃度的增加，突變率增加，加藥組與對(duì)照組的兩個(gè)電化學(xué)信號(hào)峰面積差值則隨劑量增加而加大。 結(jié)論本課題采用電化學(xué)法檢測(cè)到V79細(xì)胞有兩個(gè)明顯信號(hào)峰，以此兩個(gè)信號(hào)峰為指標(biāo)，建立了細(xì)胞內(nèi)嘌呤電化學(xué)檢測(cè)系統(tǒng)，并將此系統(tǒng)用于V79細(xì)胞HGPRT基因突變研究中，，結(jié)果與常規(guī)HGPRT基因突變結(jié)果具有一致性。電化學(xué)法檢測(cè)基因突變?cè)诮o藥第4天即可看到信號(hào)的明顯變化，可縮短常規(guī)HGPRT基因突變長(zhǎng)達(dá)15天的檢測(cè)時(shí)間，并提高了檢測(cè)結(jié)果的客觀性。
[Abstract]:Objective to study the electrochemical behavior of hamster lung cells (V79 cells) and to investigate the effects of different factors on the electrochemical signals of V79 cells and to establish an electrochemical detection system for the metabolism of purine nucleotides in V79 cells. The system was used to study the mutation of (HGPRT) gene of Hypoxanthine guanine phosphotransferase in V79 cells. It provides a theoretical basis for the establishment of electrochemical detection method for mutation of HGPRT gene. Methods the electrochemical behavior of V79 cells was studied by cyclic voltammetry and the electrochemical signals of V79 cells were assigned by high performance liquid chromatography (HPLC). The mutation of HGPRT gene in V79 cells was studied by electrochemical method and conventional HGPRT gene mutation detection method. Results there were two electrochemical signal peaks in V79 cell lysate, belonging to oxidation of guanine and xanthine (0.7V) and oxidation of adenine and Hypoxanthine (1.03V). The results of HGPRT locus mutation in V79 cells induced by ethyl mesylate (EMS) showed that when EMS concentration was 1.6mg mL-1, the signal I peak area of V79 cells was significantly higher than that of the control group at the 4th day after administration. The peak area of signal II was significantly higher than that of control group at the 5th day after administration, and the mutation rate increased with the increase of drug concentration in the range of 0.7~1.6mg mL-1 concentration. The difference of peak area between the two groups was increased with the increase of dose. Conclusion there are two obvious signal peaks in V79 cells detected by electrochemical method. Using these two peaks as indicators, an electrochemical detection system for purine in V79 cells was established, and the system was applied to the study of HGPRT gene mutation in V79 cells. The results were consistent with the results of conventional HGPRT gene mutation. The detection of gene mutation by electrochemical method can see the obvious change of signal on the 4th day of administration, which can shorten the detection time of conventional HGPRT gene mutation for 15 days, and improve the objectivity of the detection result.
【學(xué)位授予單位】：佳木斯大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R96

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