铜对猪肝细胞CuZn-SOD酶活性及mRNA表达的影响
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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。
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.
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。
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.
引文
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[28] 韦安阳，周春兰．微量元素铜、铁、锰、锗与自由基[J]．广东微量元素科学，2001，8(6)：15-16
[29] 赵秀英，陈俊梅，阎惠平等．乙肝患者血清超氧化物歧化酶与丙二醛水平测定及干扰素治疗对其
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[30] 柳明洙，崔勋．大鼠血浆、肝脏和腓肠肌亚细胞组分中超氧化物歧化酶、谷光甘肽过氧化物酶活性和丙二醛含量分布[J]．延边大学医学学报，1997，20(1)：5-7
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[33] 金海玲，韩虹．原发性肝癌血清脂质过氧化物和超氧化物歧化酶的变化．中国综合临床，2000，16(4)：4
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[2] 武书庚，齐广海，郑君杰．微量元素铜的研究综述[J]．饲料工业，1999，20(12)：5-8
[3] 张录强，铜对人体及动物的生物学效应[J]．生物学通报，1999，34(8)：23
[4] 吴建设，呙于明，杨汉春等．微量元素铜的营养与免疫研究进展[J]．国外畜牧科技，1999，32(1)：33-34
[5] 吴建设，呙于明，杨汉春等．微量元素铜的营养与免疫研究进展[J]．国外畜牧科技，1999，26(1)：5-9
[6] 赵昕红，李德发，田福刚．高锌和高铜对仔猪生长性能、免役功能和抗氧化酶活性的影响[J]．中国农业大学学报，1999，4(1)：91-96
[7] Miller E R,D E Ullrey,D J Ellis,B L Schoepke,J A Hoefer.Comparison of copper sulfate and a selected antibiotic for growing-finishing swine[J].J Anim Sci, 1969, 29:140
[8] Lloyd L E.D E McDonald,E W Crampton.Fundamentals of nutrition(2nd Ed.).W H Freeman and Co., San Francisco, 1978, 253
[9] Cromwell G L,T S Stanly,W D Willians.Efficacyof copper as a growth promotant and ets interrelationship with sulfur and antibiotics for swine.Distillers Feed Conference, 1981, 36:15
[10] 褚勇，吴建设，孙淑珍．高铜改善猪生产性能及其机理研究进展[J]．饲料博览，1998，10(3)：3-14
[11] Miller E R, H D Stoew, P K Ku,G M Hill.Copper and zinc in swine nutrition. In:National Feed Ingredients Association Literature Review on Copper and Zinc in Animal Nutrion.National Feed Ingredients Association,West Des Moines, IA 1979
[12] Zhou W, E T Kornegay,H van Laar,J W GM Swinkles,E A Wong, M D Lindemann. The role of feed consumption and efficiency in copper-stimulated growth[J]. J Anim Sci. 1994, 72:2385-2394
[13] Dove C R, K D Haydon.The effect of copper and fat addition to the the diets of weanling swine on growth performance and serum fatty acids [J]. J Anim. Sci.
1992, 70:805
[14] Dove C R. The effect of copper addition and various fat source to the diets of weanling swine on growth performance and serum fatty acid profiles. 1993, 71:2187
[15] 田允波，曾书琴．高铜改善猪生产性能和促生长机理的研究进展．黑龙江畜牧兽医，2000，11：36-37
[16] Dove C R. The effect of copper level on nutrient utilization of weanling pigs.J Anim. Sci. 1995, 73:166
[17] 刘晓波，罗绪刚，张荣强．高剂量铜对猪促生长作用机理的研究进展动物营养学报，1997，9(3)：1-6
[18] LaBella F, T Dular, S Vivian,et al. Pituitary hormone releasing of inhibiting activity of metal ions present in hypothalamic extracts[J].Biochem. Biophys. Res. Commun.1973, 52:786
[19] Zhou W, Kornegay E T, Lindemann M D, et al.Stimulation of growth by intravenous injection of copper in weanling pigs[J]. J Anim Sci, 1994, 72:2395-2403
[20] 程忠刚，林映才，许梓荣．高铜促生长机理综述[J]．兽药与饲料添加剂，2001，3：33-35
[21] 刘国文，王哲．促生长激素轴与铜促生长的关系[J]．动物医学进展．2000，21(3)：22-24
[22] Maria C Linder, Lisa Wooten, Philip Cerveza etc.Copper transport[J], Am J Nutr 1998, 67(suppl):965s-971s
[23] 高亚利，刘浩，刘天云．发育过程中铜在肝脏内的储存及运输细胞毒性作用(一)[J]．国外医学医学地理分册，1997，18(2)：68-70
[24] 刘一凡，石学敏，韩景献等．针刺对快速老化模型小鼠肝抗氧化酶活性及基因表达水平的影响[J]．山东中医药大学学报，2001，25(5)：382-386
[25] 向华，卫文仲，谭华荣．人铜锌超氧化物歧化酶基因的克隆和在乳酸乳球菌中的表达[J]．生物工程学报，2001，16(1)：6-9
[26] 胡晓珂，于文功，路新枝．甘糖酯对大鼠肝脏CuZn—SODmRNA表达和酶活的诱导作用[J]．药学学报，2000，37(1)：23-26
[27] 王德海，程国富．铜抗氧自由基作用的研究进展[J]．微量元素与健康研究，2001，18(4)：70-73
[28] 韦安阳，周春兰．微量元素铜、铁、锰、锗与自由基[J]．广东微量元素科学，2001，8(6)：15-16
[29] 赵秀英，陈俊梅，阎惠平等．乙肝患者血清超氧化物歧化酶与丙二醛水平测定及干扰素治疗对其
影响[J]．首都医科大学学报，2000，22(3)：260-261
[30] 柳明洙，崔勋．大鼠血浆、肝脏和腓肠肌亚细胞组分中超氧化物歧化酶、谷光甘肽过氧化物酶活性和丙二醛含量分布[J]．延边大学医学学报，1997，20(1)：5-7
[31] 季红光，王恩美．缺铜对大鼠自由基防御系统酶组分和血清溶血素的影响[J]．营养学报，1990，12(2)：146-157
[32] 李光植，黄瑛，王琳．远志对D-半乳糖致小鼠红细胞中超氧化物歧化酶、肝组织谷胱甘肽过氧化物酶活性影响的实验研究[J]．黑龙江医药科学，2000，23(1)：4
[33] 金海玲，韩虹．原发性肝癌血清脂质过氧化物和超氧化物歧化酶的变化．中国综合临床，2000，16(4)：4
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