Developmental Gene Expression Of Lactoferrin At Different Lactating Stages And Effect Of Iron On Gene Regulation Of Lactoferrin In Mammary Gland Of Mice

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 25.6420043. 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.