哺乳小白鼠在不同泌乳阶段乳铁蛋白（Lactoferrin）基因表达的差异及铁对其表达影响的研究
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摘要
本课题研究了ICR哺乳小白鼠在不同泌乳阶段乳铁蛋白(LF)mRNA表达的差异，采用体内和体外试验相结合的方式研究了铁对小白鼠乳铁蛋白基因表达的影响。
1．首先以哺乳小白鼠乳腺组织为材料，提取乳腺组织总RNA，根据已报道的小白鼠LF基因序列设计引物，通过反转录和PCR技术克隆出小白鼠乳铁蛋白(murine Lactoferrin)基因部分序列，其编码299个氨基酸组成的多肽，是成熟肽的主体部分。
2．以泌乳后第1、9、17、和25天的ICR哺乳小白鼠为试验对象，以半定量RT-PCR法为检测手段，研究小白鼠在不同泌乳阶段乳铁蛋白基因表达的变化规律。根据已报道的小白鼠LF和看家基因β-肌动蛋白(β-actin)基因序列，分别设计LF和β-actin的引物，以本实验室构建的优化的半定量RT-PCR法，来检测小白鼠乳铁蛋白基因mRNA表达量。
试验结果发现，在小白鼠的整个泌乳阶段，LF基因的表达量存在差异，即在泌乳后第1天到17天呈下降的趋势，而在泌乳后第17天到25天呈升高的趋势。
3．体外原代培养哺乳期ICR小白鼠的乳腺细胞，配制不同的FeSO_4浓度梯度，用MTT法检测不同浓度的Fe~(2+)对乳腺细胞增殖的影响，结果发现：2.5μg／ml Fe~(2+)对乳腺细胞的促增殖作用最为显著；选择2.5μg／ml Fe~(2+)为最佳的铁离子浓度，作用于原代培养的ICR小白鼠的乳腺细胞，并用0.5μg／ml雌激素作为阳性对照，加诱导物刺激后分别培养12小时、24小时和48小时，之后提取细胞总RNA，反转录成cDNA保存。采用已构建的优化半定量RT-PCR法，适当调整PCR的参数，检测特定时间段Fe~(2+)对小白鼠乳铁蛋白(mLF)转录水平表达量影
2004年浙江大学硕士学位论文
响。
体外细胞培养试验结果表明:铁和雌激素都提高了原代培养的小白鼠乳腺细
胞LF mRNA的表达量。(l)加雌激素和铁两种诱导物分别作用于原代培养的乳腺
细胞12小时后，与对照组相比，LF mRNA相对于旦一actin的表达量分别提高
了54.1%(p<住05)，105.42%(p<0.01);(2)加雌激素和铁两种诱导物分别作
用24小时后，与对照组相比，LF mRNA相对于日一ac七如的表达量分别提高了
36·63%(p>0.05)，43.19%(p>0.05);(3)加雌激素和铁两种诱导物分别作用
48小时后，与对照组相比，LF mRNA相对于日一aCtin的表达量分别提高了
27.83%(P>0.05、、89.27%(P<0.05)。
飞.挑选60只ICR雌性小白鼠(交配后12天)进行饲养试验，试验分成处
理1(对照组)、处理2(加FeSO4组)两个组(每组设3个重复，每个重复10
只夕。两处理组同时饲喂小鼠纯合日粮，自由饮水和采食。处理1组的纯合日粮
中不含Fe，处理2组纯合日粮中含有120 Ing Fe/Kg，饲养试验持续35天，在
饲养试验过程中，在泌乳后第1、9、17、和25天每个处理分别挑选3只小白鼠
处死，取乳腺组织，提取总RNA，同样采用民FPCR法，检测FeSO4对哺乳小
白鼠在不同泌乳阶段乳铁蛋白mRNA表达量的影响。
体内饲养试验结果表明:在小白鼠日粮中添加铁可提高LF mRNA的表达量，
与对照组相比，泌乳后第l天提高了85.90%(p<0.01)，泌乳后第9天提高了
17.97(p>0.05)，泌乳后第17天提高了13.79(p>0.05)，泌乳后第25天提
高了55%(p<0.01)。
In this article we researched the developmental gene expression of lactoferrin and effect of iron on gene regulation of lactoferrin in mammary gland of mice in vivo and vitro.
1.Total RNA was extracted from mammary gland of lactating mice, primer was designed and synthesized according to the murine lactoferrin gene reported in Genbank. A portion of the sequence of lactoferrin gene was cloned through RT-PCR and coded 299 amino acid residues, which constituted the major part of mature lactoferrin.
2. The female ICR mice at day 1, 9, 17 and 25 of lactation were used to determine effect of different lactating stages on mRNA expression of lactoferrin. The primers for LF and β-actin were designed respectively according to the known mouse LF gene and housekeeping gene β-actin sequence, An optimized semi-quantitative RT-PCR was constructed to evaluate expression of LF-mRNA in different lactating stages in mice.
The result showed that the amount of lactoferrin gene expression changed with lactation stages in the whole lactating stage. Lactoferrin mRNA had strong expression at day 1 after parturition. Then, the amount decreased steadily at day 9 and at day 17 after parturition. Stronger lactoferrin mRNA expression was observed from the day 17 to 25.
64
2004
3. Mouse mammary epithlium cell was selected for primary culture. The effect of iron (FeSCU) of different concentrations on cell proliferation was determined. The data demonstrated that the proliferation of cell was most significant at 2 g/ml of Fe2+. 2.5 g/ml FeSO4 was selected to add to the mammary epithlium cell cultured in vitro. 0.5.g/ml estrogen was selected as control, the cell was cultured for twelve, twenty-four and forty-eight hours respectively after adding the inducements. Then the total RNA was extracted from the cell, and the RNA was converted in single cDNA. The optimized semi-quantitative RT-PCR constructed was used to evaluate the effect of Fe2+ on the expression of LF-mRNA at indicated times.
The experiment in vitro demonstrated that iron and estrogen improved the levels of lactoferrin mRNA in the mammary epithelium. (1) After mammary epithelium was incubated with estrogen and iron for 12 hours, the ratio of LF/ 3 -actin increased by 54.1% (p < 0.0-5), 105.42% (p < 0.01) compared with control respectively. (2) After 24 hours, the ratio of LF/ P -actin increased by 36.63% (p > 0.05 ), 43.19%(p > 0.05). (3) After 48 hours, the ratio of LF/ P -actin increased by 27.83% (p > 0.05), 89.27% Cp<0.05) .
4. Total 60 female-mice at day 12 after mating were devided into two treatments (control and adding FeSO4) at random and were kept in individual cages and begun provide purified diet with food and water ad libitum. The diet of control group contained Omg Fe/kg and the diet of treatment group contained 120 mg Fe/kg. The feeding trial period lasted 35 days. During feeding experiment, three mice each treatment were chosen at day 1, 9 17 and 25 of lactation respectively. Mice were killed by decapitation and mammary gland tissues were aseptically removed and total RNA was extracted. The effect of iron on lactoferrin mRNA expression of mice at different lactating stages was determined by semi-quantitative RT-PCR.
The experiment in vivo demonstrated that dietary iron improved the levels of lactoferrin mRNA. In contrast to control, the ratio of LF/ {3 -actin increased by 85.90%(p < 0.01) at day 1 after parturition, and increased by 17.97(p > 0.05) 13.79
(p 0.05 X 55% (p 0.01) at day 9 17, 25 after parturition respectively.
1．首先以哺乳小白鼠乳腺组织为材料，提取乳腺组织总RNA，根据已报道的小白鼠LF基因序列设计引物，通过反转录和PCR技术克隆出小白鼠乳铁蛋白(murine Lactoferrin)基因部分序列，其编码299个氨基酸组成的多肽，是成熟肽的主体部分。
2．以泌乳后第1、9、17、和25天的ICR哺乳小白鼠为试验对象，以半定量RT-PCR法为检测手段，研究小白鼠在不同泌乳阶段乳铁蛋白基因表达的变化规律。根据已报道的小白鼠LF和看家基因β-肌动蛋白(β-actin)基因序列，分别设计LF和β-actin的引物，以本实验室构建的优化的半定量RT-PCR法，来检测小白鼠乳铁蛋白基因mRNA表达量。
试验结果发现，在小白鼠的整个泌乳阶段，LF基因的表达量存在差异，即在泌乳后第1天到17天呈下降的趋势，而在泌乳后第17天到25天呈升高的趋势。
3．体外原代培养哺乳期ICR小白鼠的乳腺细胞，配制不同的FeSO_4浓度梯度，用MTT法检测不同浓度的Fe~(2+)对乳腺细胞增殖的影响，结果发现：2.5μg／ml Fe~(2+)对乳腺细胞的促增殖作用最为显著；选择2.5μg／ml Fe~(2+)为最佳的铁离子浓度，作用于原代培养的ICR小白鼠的乳腺细胞，并用0.5μg／ml雌激素作为阳性对照，加诱导物刺激后分别培养12小时、24小时和48小时，之后提取细胞总RNA，反转录成cDNA保存。采用已构建的优化半定量RT-PCR法，适当调整PCR的参数，检测特定时间段Fe~(2+)对小白鼠乳铁蛋白(mLF)转录水平表达量影
2004年浙江大学硕士学位论文
响。
体外细胞培养试验结果表明:铁和雌激素都提高了原代培养的小白鼠乳腺细
胞LF mRNA的表达量。(l)加雌激素和铁两种诱导物分别作用于原代培养的乳腺
细胞12小时后，与对照组相比，LF mRNA相对于旦一actin的表达量分别提高
了54.1%(p<住05)，105.42%(p<0.01);(2)加雌激素和铁两种诱导物分别作
用24小时后，与对照组相比，LF mRNA相对于日一ac七如的表达量分别提高了
36·63%(p>0.05)，43.19%(p>0.05);(3)加雌激素和铁两种诱导物分别作用
48小时后，与对照组相比，LF mRNA相对于日一aCtin的表达量分别提高了
27.83%(P>0.05、、89.27%(P<0.05)。
飞.挑选60只ICR雌性小白鼠(交配后12天)进行饲养试验，试验分成处
理1(对照组)、处理2(加FeSO4组)两个组(每组设3个重复，每个重复10
只夕。两处理组同时饲喂小鼠纯合日粮，自由饮水和采食。处理1组的纯合日粮
中不含Fe，处理2组纯合日粮中含有120 Ing Fe/Kg，饲养试验持续35天，在
饲养试验过程中，在泌乳后第1、9、17、和25天每个处理分别挑选3只小白鼠
处死，取乳腺组织，提取总RNA，同样采用民FPCR法，检测FeSO4对哺乳小
白鼠在不同泌乳阶段乳铁蛋白mRNA表达量的影响。
体内饲养试验结果表明:在小白鼠日粮中添加铁可提高LF mRNA的表达量，
与对照组相比，泌乳后第l天提高了85.90%(p<0.01)，泌乳后第9天提高了
17.97(p>0.05)，泌乳后第17天提高了13.79(p>0.05)，泌乳后第25天提
高了55%(p<0.01)。
In this article we researched the developmental gene expression of lactoferrin and effect of iron on gene regulation of lactoferrin in mammary gland of mice in vivo and vitro.
1.Total RNA was extracted from mammary gland of lactating mice, primer was designed and synthesized according to the murine lactoferrin gene reported in Genbank. A portion of the sequence of lactoferrin gene was cloned through RT-PCR and coded 299 amino acid residues, which constituted the major part of mature lactoferrin.
2. The female ICR mice at day 1, 9, 17 and 25 of lactation were used to determine effect of different lactating stages on mRNA expression of lactoferrin. The primers for LF and β-actin were designed respectively according to the known mouse LF gene and housekeeping gene β-actin sequence, An optimized semi-quantitative RT-PCR was constructed to evaluate expression of LF-mRNA in different lactating stages in mice.
The result showed that the amount of lactoferrin gene expression changed with lactation stages in the whole lactating stage. Lactoferrin mRNA had strong expression at day 1 after parturition. Then, the amount decreased steadily at day 9 and at day 17 after parturition. Stronger lactoferrin mRNA expression was observed from the day 17 to 25.
64
2004
3. Mouse mammary epithlium cell was selected for primary culture. The effect of iron (FeSCU) of different concentrations on cell proliferation was determined. The data demonstrated that the proliferation of cell was most significant at 2 g/ml of Fe2+. 2.5 g/ml FeSO4 was selected to add to the mammary epithlium cell cultured in vitro. 0.5.g/ml estrogen was selected as control, the cell was cultured for twelve, twenty-four and forty-eight hours respectively after adding the inducements. Then the total RNA was extracted from the cell, and the RNA was converted in single cDNA. The optimized semi-quantitative RT-PCR constructed was used to evaluate the effect of Fe2+ on the expression of LF-mRNA at indicated times.
The experiment in vitro demonstrated that iron and estrogen improved the levels of lactoferrin mRNA in the mammary epithelium. (1) After mammary epithelium was incubated with estrogen and iron for 12 hours, the ratio of LF/ 3 -actin increased by 54.1% (p < 0.0-5), 105.42% (p < 0.01) compared with control respectively. (2) After 24 hours, the ratio of LF/ P -actin increased by 36.63% (p > 0.05 ), 43.19%(p > 0.05). (3) After 48 hours, the ratio of LF/ P -actin increased by 27.83% (p > 0.05), 89.27% Cp<0.05) .
4. Total 60 female-mice at day 12 after mating were devided into two treatments (control and adding FeSO4) at random and were kept in individual cages and begun provide purified diet with food and water ad libitum. The diet of control group contained Omg Fe/kg and the diet of treatment group contained 120 mg Fe/kg. The feeding trial period lasted 35 days. During feeding experiment, three mice each treatment were chosen at day 1, 9 17 and 25 of lactation respectively. Mice were killed by decapitation and mammary gland tissues were aseptically removed and total RNA was extracted. The effect of iron on lactoferrin mRNA expression of mice at different lactating stages was determined by semi-quantitative RT-PCR.
The experiment in vivo demonstrated that dietary iron improved the levels of lactoferrin mRNA. In contrast to control, the ratio of LF/ {3 -actin increased by 85.90%(p < 0.01) at day 1 after parturition, and increased by 17.97(p > 0.05) 13.79
(p 0.05 X 55% (p 0.01) at day 9 17, 25 after parturition respectively.
引文
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Sheng-Hsiang Li, Yee-Hsiung Chen. Various forms of mouse lactoferrins: purification and characterization. Journal of chromatography B: Biomedical Sciences and Applications, 1999, 726(1-2): 45-52.
Shimizu K., Matsuzawa H., Okada K., et al. Lactoferrin-mediated protection of the host from murine cytomegalovirus infection by a T-cell-dependent augmentation of natural killer cell activity. Arch. Firol., 1996, 141: 1875-1889.
Sigman M., Lnnerdal B. Response of rat mammary gland transferring receptors to maternal dietary iron during pregnancy and lactation. Am. J. Clin. Nutr., 1990, 52: 446-450.
Tsui S., Hirata Y., Mukai F., et al. Comparison of lactoferrin content
in colostrums between different cattle breed. J. Dairy Sci., 1990, 125-128.
Ushida Yoshihiko, Sekine Kazunori, Kuhara Tetsuya, et al. Inhibitory effects of bovine lactoferrin on intestinal polyposis in the ApcMin mouse. Cancer Letters, 1998,134(2): 141-145.
Ward Pauline P., Hanpo Chu, Xiaodong Zhou, et al. Expression and characterization of recombinant murine lactoferrin. Gene, 1997, 204(1-2): 171-176.
Ying Yang, Jingfeng Li, Anna Szeles, et al. Consistent downregulation of human lactoferrin gene, in the common eliminated region 1 on 3p21.3, following tumor growth in severe combined immunodeficient (SCID) mice. Cancer Letters., 2003, 191(2): 155-164.
Zucali J R, Broxmeyer H E, Levy D, et al. hactoferrin decreases monocvte-induced fibroblast production of myeloid colony-stimulation activity by suppressing monocyte release of interleukin-1. Blood, 1989,74:1531-1536.
白洁，邵宗鸿．铁代谢异常和缺铁性贫血．中华血液学杂志，2002，23(11)：614-615．
曹阳，包永明，安利佳．乳铁蛋白研究现状．食品科学，2002，23(12)：132-138．
费学刚，安云庆．乳铁蛋白及其生物学功能．国外医学免疫学分册，1996，1：36-38．
龚广予，巫庆华，吴正钧．乳铁蛋白的生理功能．中国乳品工业，2001，29(1)：20-23．
刘旭新．微量元素铁代谢的研究进展．广东微量元素科学，2001，8(1)：11-15．
闰有旺，蔡连捷．铁的生物学功能及代谢．化学教学，2002，12：39-40．
桑亚新，王向红，孙记录．乳铁蛋白及其生理功能研究进展．河北农业大学学报，2003，26：154-156．
司徒镇强，吴军正．细胞培养．西安：世界图书出版社，1996．93-95．
唐传核，曹劲松，彭支英．乳铁蛋白最新研究进展-活性多肽以及生理功能(Ⅱ)．
中国乳品工业，2000，28(5)：44-47．
吴兴发，欧阳五庆，张申卫．乳源生物活性物质．中国兽医科技，2001，31(4)：42-43．
伍喜林，杨凤，汪明锋等．乳铁蛋白的营养生理作用及其作为饲料添加剂的应用．中国畜牧兽医，2002，29(4)：20-23．
薛庆善．体外培养的原理与技术．北京：科学出版社，2001．401-403．
杨帆，陈燕忠，张纪兴．利用乳腺生物反应器生产人乳铁蛋白．广东药学院学报，2001，17(2)：123-124．
姚笠．乳铁蛋白-一种多功能免疫调节．国外医学免疫学分册，1995，19(6)：317-318．
张丹风，陆东林．乳铁蛋白及其生理功能．草食家畜，2002，2：3-6．
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S.H. Wang, T.S. Yang. Expression, characterization, and purification of recombinant porcine lactoferrin in pichia pastoris. Protein Expression and Purification, 2002, 25:41-49.
Sanchez L., Calvo M. and Brock J.H. Biological role of lactoferrin. Arch. Dis. Child., 1992, 67: 657-661.
Segars Frances M., Kinkade Jr, Joseph M. Radioimmunoassay for murine lactoferrin, a protein marker of myeloid and mammary epithelial secretory cell differentiation. Journal of immunological methods, 1997,14(1): 1-14.
Sheng-Hsiang Li, Yee-Hsiung Chen. Various forms of mouse lactoferrins: purification and characterization. Journal of chromatography B: Biomedical Sciences and Applications, 1999, 726(1-2): 45-52.
Shimizu K., Matsuzawa H., Okada K., et al. Lactoferrin-mediated protection of the host from murine cytomegalovirus infection by a T-cell-dependent augmentation of natural killer cell activity. Arch. Firol., 1996, 141: 1875-1889.
Sigman M., Lnnerdal B. Response of rat mammary gland transferring receptors to maternal dietary iron during pregnancy and lactation. Am. J. Clin. Nutr., 1990, 52: 446-450.
Tsui S., Hirata Y., Mukai F., et al. Comparison of lactoferrin content
in colostrums between different cattle breed. J. Dairy Sci., 1990, 125-128.
Ushida Yoshihiko, Sekine Kazunori, Kuhara Tetsuya, et al. Inhibitory effects of bovine lactoferrin on intestinal polyposis in the ApcMin mouse. Cancer Letters, 1998,134(2): 141-145.
Ward Pauline P., Hanpo Chu, Xiaodong Zhou, et al. Expression and characterization of recombinant murine lactoferrin. Gene, 1997, 204(1-2): 171-176.
Ying Yang, Jingfeng Li, Anna Szeles, et al. Consistent downregulation of human lactoferrin gene, in the common eliminated region 1 on 3p21.3, following tumor growth in severe combined immunodeficient (SCID) mice. Cancer Letters., 2003, 191(2): 155-164.
Zucali J R, Broxmeyer H E, Levy D, et al. hactoferrin decreases monocvte-induced fibroblast production of myeloid colony-stimulation activity by suppressing monocyte release of interleukin-1. Blood, 1989,74:1531-1536.
白洁，邵宗鸿．铁代谢异常和缺铁性贫血．中华血液学杂志，2002，23(11)：614-615．
曹阳，包永明，安利佳．乳铁蛋白研究现状．食品科学，2002，23(12)：132-138．
费学刚，安云庆．乳铁蛋白及其生物学功能．国外医学免疫学分册，1996，1：36-38．
龚广予，巫庆华，吴正钧．乳铁蛋白的生理功能．中国乳品工业，2001，29(1)：20-23．
刘旭新．微量元素铁代谢的研究进展．广东微量元素科学，2001，8(1)：11-15．
闰有旺，蔡连捷．铁的生物学功能及代谢．化学教学，2002，12：39-40．
桑亚新，王向红，孙记录．乳铁蛋白及其生理功能研究进展．河北农业大学学报，2003，26：154-156．
司徒镇强，吴军正．细胞培养．西安：世界图书出版社，1996．93-95．
唐传核，曹劲松，彭支英．乳铁蛋白最新研究进展-活性多肽以及生理功能(Ⅱ)．
中国乳品工业，2000，28(5)：44-47．
吴兴发，欧阳五庆，张申卫．乳源生物活性物质．中国兽医科技，2001，31(4)：42-43．
伍喜林，杨凤，汪明锋等．乳铁蛋白的营养生理作用及其作为饲料添加剂的应用．中国畜牧兽医，2002，29(4)：20-23．
薛庆善．体外培养的原理与技术．北京：科学出版社，2001．401-403．
杨帆，陈燕忠，张纪兴．利用乳腺生物反应器生产人乳铁蛋白．广东药学院学报，2001，17(2)：123-124．
姚笠．乳铁蛋白-一种多功能免疫调节．国外医学免疫学分册，1995，19(6)：317-318．
张丹风，陆东林．乳铁蛋白及其生理功能．草食家畜，2002，2：3-6．