【作者】 梁海涛；

【导师】 娄玉杰；

【作者基本信息】 吉林农业大学 ， 动物营养与饲料科学， 2004， 硕士

【摘要】 本试验以初生1日龄健康军牧1号仔猪为试验动物，研究不同铜浓度对CuZn-SOD酶活性及mRNA表达的影响。实验一利用24孔细胞培养板培养细胞，细胞培养液中添加10％小牛血清，同时分别添加0、15.6、31.2、46.8、62.4、78、93.6μmol／LCu，观察细胞形态，测定细胞培养液中CuZn-SOD酶活性。结果显示：各个试验组的酶活性均显著增加，62.4μmol／Lcu时酶的活性最高(P＜0.05)。 实验二利用RT-PCR方法测定肝细胞CuZn-SODmRNA表达量。细胞培养液中添加10％小牛血清，同时分别添加0、31.2、62.4、93.6μmol／LCu，利用Trizol法从肝细胞中提取总RNA，采用RT-PCR方法扩增出SODcDNA，与pGEM-T载体相连接，转化大肠杆菌JM109，通过PCR鉴定及测序分析，证明所扩增DNA片段为目的片段。PCR产物经琼脂糖凝胶电泳，用β-actin作内参，用凝胶成像分析仪检测SODmRNA的相对表达量。结果显示，SODcDNA序列与基因库中的相一致，重合性达98.8％。细胞培养液中添加31.2、62.4、93.6μmol／LCu，肝细胞中SODmRNA的表达量均显著高于对照组(p＜0.05)，以62.4μmol／LCu组效果最为明显。 从两个实验的结果我们可以推测，铜作为CuZn-SOD的活性中心，不仅可以直接提高SOD的酶活性，而且可以对其基因表达进行调控，提高转录水平，使SODmRNA表达量增加，为后期的翻译提供了条件，这对机体的抗氧化功能有重要意义，为铜的抗氧化机制提供了分子依据。更多还原

【Abstract】 The experiment was conducted to evaluate the effects of different copper levels on CuZn-SOD activities and expression of CuZn-SOD gene mRNA in hepatic cells in newborn pigs. In the first experiment, hepatic cells were cultured with 10% blood serum media supplemented with 0, 15.6, 31.2, 46.8, 62.4, 78, 93.6umol/L Cu in 24 aperture culture plate . The results were as follows: CuZn-SOD activities in hepatic cells was significantly increased in culture media added to 0,15.6,31.2,46.8, 62.4, 78, 93.6nmol/L Cu and were highest at 62.4umol/Lcu(P<0.05).In the second experiment, to meaure the expression of CuZn-SOD gene mRNA in hepatic cells, the quantitative reverse transcription-polymerase chain reaction(RT-PCR) was constructed. Hepatic cells was cultured with 10% blood serum media supplemented with 0, 31.2, 62.4, 93.6umol/L Cu in 24 aperture culture plate. The total RNA in hepatic cells was extracted by the Trizol way, CuZn-SOD cDNA was amplified,being ligated with pGEM vector, and the expression of the CuZn-SOD gene mRNA was measured with the RT-PCR. The results were as follows: sequence of the cloned CuZn-SOD gene was identical to Genbank. The expression of CuZn-SOD gene mRNA was enhanced in culture media added to 31.2, 62.4, 93.6umol/L Cu. The optimal copper concentration was 62.4umol/L Cu.We can confer from the above results suggests that as the center of the CuZn-SOD activity Cu can not only increase the activities ,but also control the expression of the gene and enhance the transcription,As the results the increase of transcription offer condition for the translation, which is very important to resist oxidation and offer molecule thereunder for the mechanism of resisting-oxidation of Cu.更多还原