嘌呤核苷酸代谢电化学检测系统的建立及应用研究

发布时间：2018-10-13 16:54

【摘要】：目的研究仓鼠肺细胞（V79细胞）的电化学行为，考察不同因素对V79细胞电化学信号的影响，建立细胞内嘌呤核苷酸代谢电化学检测系统。并将此系统用于药物对V79细胞次黄嘌呤鸟嘌呤磷酸核糖转移酶（HGPRT）基因突变的研究。为HGPRT基因突变电化学检测方法的建立提供理论依据。方法本文以V79细胞为实验细胞模型，采用循环伏安法研究了V79细胞的电化学行为，采用高效液相色谱法对V79细胞的电化学信号进行了归属；采用电化学方法和常规HGPRT基因突变检测方法对照研究了药物致V79细胞HGPRT基因位点的突变。结果V79细胞裂解液有两个电化学信号峰，分别归属于鸟嘌呤和黄嘌呤的氧化（0.7V），以及腺嘌呤和次黄嘌呤的氧化（1.03V）；检测最佳条件：pH值7.4，50℃恒温水浴裂解30min；对甲磺酸乙酯（EMS）致V79细胞HGPRT基因位点突变研究结果显示，在EMS浓度为1.6mg·mL-1时， V79细胞加药组信号I峰面积在给药后第4天开始，明显高于对照组，信号II峰面积在给药后第5天开始，明显高于对照组；不同EMS给药浓度研究发现，在药物浓度0.7~1.6mg·mL-1范围内，随着药物浓度的增加，突变率增加，加药组与对照组的两个电化学信号峰面积差值则随剂量增加而加大。结论本课题采用电化学法检测到V79细胞有两个明显信号峰，以此两个信号峰为指标，建立了细胞内嘌呤电化学检测系统，并将此系统用于V79细胞HGPRT基因突变研究中，，结果与常规HGPRT基因突变结果具有一致性。电化学法检测基因突变在给药第4天即可看到信号的明显变化，可缩短常规HGPRT基因突变长达15天的检测时间，并提高了检测结果的客观性。
[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.
【学位授予单位】：佳木斯大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

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