铁对Caco-2细胞及肉仔鸡锰吸收的影响及其机制
发布时间：2018-08-12 20:05
【摘要】：本论文通过两个试验分别研究了铁对Caco-2细胞及肉仔鸡锰吸收的影响及其机制?试验一研究了铁处理对Caco-2细胞锰吸收的影响及其机制。本试验包括2个小试验。试验1研究了不同接种密度对Caco-2细胞吸收模型分化及去极化评价指标的影响,确定了细胞模型适用于吸收和转运试验的时间?将Caco-2细胞按高密度(1×105 cells/cm2)?中密度(5×104 cells/cm2)和低密度(3×104 cells/cm2)三个接种密度,分别接种于带聚碳酸酯微孔滤膜插槽的Transwell 6孔板上,培养29天,通过测定跨膜电阻值(TEER)?碱性磷酸酶活性(ALP)和酚红透过率,评价细胞单层的完整性?极化和透过性?结果表明:高?中?低密度接种分别于第12?15和18天TEER值超过600Ω/cm2,第27?29和29天TEER值开始下降;高?中?低密度接种分别于第15?18和21天上、下侧碱性磷酸酶活性的比值达到10倍以上,第29天开始降低;高?中?低密度接种分别于第9?12和15天酚红透过率小于0.5%?综合上述三项指标,高密度接种细胞在第15天?中密度接种细胞在第18天?低密度接种细胞在第21天开始,可以用于吸收和转运试验,27天以后细胞活力就开始下降,不适合做吸收和转运细胞模型?试验2研究了铁处理对Caco-2细胞锰吸收的影响及其机制?采用单因子完全随机试验设计,共5个处理组,分别为正常含血清培养基、正常含血清培养基加铁100μmol/L、正常含血清加铁200μmol/L、无血清培养基添加铁100μmol/L和无血清培养基加铁200μmol/L,每个处理6个重复?分别在细胞培养的第18~23天在培养基里以次氮基三乙酸络合铁的形式添加铁,铁处理120h结束后,在所有细胞培养液中以无机硫酸锰的形式添加800μmol/L锰,37℃?50r/min水浴振荡器上振荡孵育120 min,收集细胞及上、下层培养液,测定其中铁和锰含量;分别在铁处理72和120h后收集细胞,测定二价金属转运蛋白1(DMT1)、膜转铁蛋白1(FPN1)和十二指肠细胞色素还原酶b(Dcytb)mRNA及蛋白表达量。结果表明:与不加铁对照组相比,无血清培养基中添加铁处理组细胞中铁含量显著高于对照组(P0.09),有血清培养基中添加铁处理组细胞中铁含量无显著提高(P0.31);无血清培养基中添加铁处理组和有血清培养基中添加铁200μmol/l处理组细胞对锰的摄取量(p0.03)和吸收量(p0.02)均显著降低;铁处理72h有血清培养基中添加铁对dmt1mrna(p0.38)和fpn1(p0.43)mrna无显著影响,铁处理120h有血清培养基中添加铁200μmol/l和无血清培养基中添加铁组dmt1(p0.01)和dcytbmrna(p0.05)水平均显著降低,无血清培养基中添加铁处理72和120h均显著降低dmt1(p0.01)和fpn1(p0.05)mrna水平,对dcytbmrna水平无显著影响(p0.14);铁处理72和120h对fpn1蛋白表达量均无显著影响(p0.89),无血清培养基中添加铁组dmt1蛋白表达量显著降低(p0.08),有血清培养基中添加铁组dmt1蛋白表达量无显著变化(p0.44)。本研究结果提示,血清影响铁处理细胞对锰的摄取和吸收以及细胞中dmt1、fpn1和dcytb的表达,铁处理细胞中dmt1和fpn1基因表达水平降低伴随着摄取和吸收锰量的降低,表明铁通过dmt1和fpn1调控细胞对锰的摄取和跨膜吸收。试验二研究了铁处理对肉仔鸡锰吸收的影响及其机制。采用单因子完全随机设计,共4个处理组,分别为不加铁基础饲粮组(0mg/kg)、以硫酸亚铁(feso4·7h2o)形式添加100、250和500mg/kg组?将336只1日龄商品代罗斯308肉公雏,按体重随机分为4个处理组,每组6个重复,每个重复14只鸡,饲养至28日龄。分别在8、15、22和28日龄早晨空腹称重、采血和屠宰取组织器官。28日龄对添加铁水平为100和500mg/kg组鸡进行原位结扎十二指肠灌注8.74mmol/l锰?结果表明,除添加铁500mg/kg组1~14日龄日增重(p0.10)及1~7日龄采食量显著(p0.03)显著低于对照组外,饲粮铁水平对其他各周生长性能指标无显著影响(p0.16);饲粮铁水平对各周龄血浆总铁结合力?血红蛋白浓度及红细胞压积均无显著影响(p0.16),但显著影响血浆铁和血浆转铁蛋白饱和度(p0.09),且各周龄血浆铁和血浆转铁蛋白饱和度均随饲粮铁水平增加而提高(p0.01);饲粮铁水平除对第21和28日龄心脏铁含量无显著影响(p0.23)外,显著影响第7和14日龄心脏及各日龄肝脏、十二指肠、胰腺和骨灰铁含量(p0.02),且这些组织铁含量均随饲粮铁水平增加而提高(p0.01);除各日龄肝脏和心脏锰含量无显著变化外(p0.13),各日龄十二指肠、胰腺和骨灰锰含量均随饲粮铁水平增加而降低(p0.01);饲粮添加铁除降低第7日龄胰腺锌含量外(p0.01),对其他日龄胰腺和各日龄其他所测组织锌含量、各日龄所测组织铜含量均无显著影响(p0.13);饲粮铁水平显著影响各日龄十二指肠粘膜dmt1和fpn1mrna水平(p0.10),各日龄dmt1和fpn1mrna水平均随饲粮铁添加水平升高而降低(P0.10);饲喂添加铁500 mg/kg饲粮组肉仔鸡原位结扎十二指肠中锰的吸收率明显低于饲喂添加铁100 mg/kg饲粮组(P0.04)?本试验结果提示,饲粮铁通过调控十二指肠粘膜DMT1和FPN1的表达影响肉仔鸡对锰的吸收。综上所述,体外细胞培养Caco-2细胞和肉仔鸡饲养及灌注均表明,铁处理通过调控细胞或肠道DMT1和PFPN1基因表达降低锰的摄取和吸收,从分子水平揭示了铁对锰吸收的影响及其机制,为进一步研究锰吸收的分子机制奠定了基础。
[Abstract]:Two experiments were conducted to study the effects of iron on the absorption of manganese by Caco-2 cells and broiler chickens. Experiment 1 was conducted to study the effect of iron treatment on the absorption of manganese by Caco-2 cells and its mechanism. Caco-2 cells were incubated on a Transwell 6-well plate with a polycarbonate microporous membrane slot for 29 days at three inoculation densities of high density (1 X105 cells / cm2)? Medium density (5 x104 cells / cm2) and low density (3 x104 cells / cm2). The results showed that the TEER value exceeded 600_/cm2 on the 12th? 15th and 18th day, and began to decrease on the 27th? 29th and 29th day, respectively; the high? Medium? Low density inoculated cells on the 15th? 18th and 21st days, and the lower side of the cell was alkaline phosphatase. The activity ratio was more than 10 times and began to decrease on the 29th day, and the phenol red transmissivity was less than 0.5% on the 9th, 12th and 15th days respectively. Considering the above three indexes, the high density inoculated cells on the 15th day? The medium density inoculated cells on the 18th day? The low density inoculated cells on the 21st day could be used for absorption and transport tests, and the high density inoculated cells on the 27th day. In experiment 2, the effect of iron treatment on manganese uptake by Caco-2 cells and its mechanism were studied. A single factor complete randomized trial design was used. Five treatment groups were used: normal serum medium, normal serum medium with iron 100 micromol/L, normal serum with iron 200. Iron was added in serum-free medium and serum-free medium with 100 micromol/L iron and 200 micromol/L iron. Six treatments were repeated each time. Iron was added in the form of iron complexes with nitrite-triacetic acid on the 18th to 23rd days of cell culture. After 120 hours of iron treatment, 800 micromol/L iron was added in the form of inorganic manganese sulfate in all cell cultures. Manganese, 37? 50R / min water bath oscillator for 120 minutes, collected cells and upper and lower culture media, determined the content of iron and manganese; after 72 and 120 hours of iron treatment, collected cells, measured the expression of divalent metal transporter 1 (DMT1), membrane transferrin 1 (FPN1) and duodenal cytochrome reductase B (Dcytb) mRNA and protein. Ming: Compared with the control group, the iron content in the serum-free medium was significantly higher than that in the control group (P Manganese uptake (p0.03) and uptake (p0.02) were significantly decreased; iron supplementation in serum medium had no significant effect on DMT1 mRNA (p0.38) and fpn1 mRNA (p0.43) after 72 h of iron treatment, but the levels of DMT1 (p0.01) and Dcytb mRNA (p0.05) in serum medium and serum-free medium were significantly decreased after 120 h of iron treatment. The levels of DMT1 (p0.01) and fpn1 (p0.05) mRNA were significantly decreased after 72 and 120 hours of iron treatment in serum-free medium, but the levels of Dcytb mRNA were not significantly affected (p0.14); the expression of fpn1 protein was not significantly affected after 72 and 120 hours of iron treatment in serum-free medium (p0.89), and the expression of DMT1 protein in serum-free medium was significantly decreased (p0.08). There was no significant change in the expression of DMT1 protein (p0.44). The results suggested that serum affected the uptake and absorption of manganese by iron-treated cells and the expression of dmt1, fpn1 and Dcytb in iron-treated cells. The effects of iron treatment on manganese uptake and its mechanism in broilers were studied in experiment 2. A single-factor complete randomized design was used to study the effects of iron treatment on manganese uptake in broilers. The chickens were randomly divided into 4 groups, each with 6 replicates, and each replicate consisted of 14 chickens, fed to 28 days of age. The chickens were weighed on an empty stomach in the morning of 8, 15, 22 and 28 days, and their blood and organs were collected and slaughtered. The daily gain (p0.10) and intake (p0.03) of 1-14 days old and 1-7 days old in group A were significantly lower than those in control group, but the iron level in diet had no significant effect on the growth performance of other weeks (p0.16); the iron level in diet had no significant effect on plasma total iron binding capacity, hemoglobin concentration and hematocrit (p0.16), but had significant effect on plasma iron and hematocrit (p0.16). Plasma transferrin saturation (p0.09), and plasma iron and plasma transferrin saturation increased with the increase of dietary iron level (p0.01); Dietary iron level had no significant effect on heart iron content at 21 and 28 days of age (p0.23), but had significant effect on liver, duodenum, pancreas and bone ash iron content at 7 and 14 days of age (p The content of iron in the duodenum, pancreas and ashes decreased with the increase of dietary iron level (p0.01), except that the content of manganese in liver and heart had no significant change (p0.13), and the content of manganese in the duodenum, pancreas and ashes decreased with the increase of dietary iron level (p0.01). Zinc content in pancreas and other tissues of all age groups had no significant effect on copper content (p0.13), dietary iron level significantly affected the levels of DMT1 and fpn1 mRNA in duodenal mucosa of all age groups (p0.10), and the levels of DMT1 and fpn1 mRNA in all age groups decreased with the increase of dietary iron level (p0.10); dietary iron supplementation 500 mg / kg diet significantly affected the levels of DMT1 and fpn1 mRNA in duodenal mucosa of all age groups (p0.10). The absorption rate of manganese in duodenum ligated in situ of broilers in group A was significantly lower than that in group B fed 100 mg/kg diet supplemented with iron (P 0.04)? The results suggested that dietary iron affected the absorption of manganese by regulating the expression of DMT1 and FPN1 in duodenal mucosa. Iron treatment reduces the uptake and absorption of manganese by regulating the expression of DMT1 and PFN1 genes in cells or intestines, revealing the effect of iron on manganese uptake and its mechanism at the molecular level, laying a foundation for further study of the molecular mechanism of manganese uptake.
【学位授予单位】：河北科技师范学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S831.5
本文编号：2180214
[Abstract]:Two experiments were conducted to study the effects of iron on the absorption of manganese by Caco-2 cells and broiler chickens. Experiment 1 was conducted to study the effect of iron treatment on the absorption of manganese by Caco-2 cells and its mechanism. Caco-2 cells were incubated on a Transwell 6-well plate with a polycarbonate microporous membrane slot for 29 days at three inoculation densities of high density (1 X105 cells / cm2)? Medium density (5 x104 cells / cm2) and low density (3 x104 cells / cm2). The results showed that the TEER value exceeded 600_/cm2 on the 12th? 15th and 18th day, and began to decrease on the 27th? 29th and 29th day, respectively; the high? Medium? Low density inoculated cells on the 15th? 18th and 21st days, and the lower side of the cell was alkaline phosphatase. The activity ratio was more than 10 times and began to decrease on the 29th day, and the phenol red transmissivity was less than 0.5% on the 9th, 12th and 15th days respectively. Considering the above three indexes, the high density inoculated cells on the 15th day? The medium density inoculated cells on the 18th day? The low density inoculated cells on the 21st day could be used for absorption and transport tests, and the high density inoculated cells on the 27th day. In experiment 2, the effect of iron treatment on manganese uptake by Caco-2 cells and its mechanism were studied. A single factor complete randomized trial design was used. Five treatment groups were used: normal serum medium, normal serum medium with iron 100 micromol/L, normal serum with iron 200. Iron was added in serum-free medium and serum-free medium with 100 micromol/L iron and 200 micromol/L iron. Six treatments were repeated each time. Iron was added in the form of iron complexes with nitrite-triacetic acid on the 18th to 23rd days of cell culture. After 120 hours of iron treatment, 800 micromol/L iron was added in the form of inorganic manganese sulfate in all cell cultures. Manganese, 37? 50R / min water bath oscillator for 120 minutes, collected cells and upper and lower culture media, determined the content of iron and manganese; after 72 and 120 hours of iron treatment, collected cells, measured the expression of divalent metal transporter 1 (DMT1), membrane transferrin 1 (FPN1) and duodenal cytochrome reductase B (Dcytb) mRNA and protein. Ming: Compared with the control group, the iron content in the serum-free medium was significantly higher than that in the control group (P Manganese uptake (p0.03) and uptake (p0.02) were significantly decreased; iron supplementation in serum medium had no significant effect on DMT1 mRNA (p0.38) and fpn1 mRNA (p0.43) after 72 h of iron treatment, but the levels of DMT1 (p0.01) and Dcytb mRNA (p0.05) in serum medium and serum-free medium were significantly decreased after 120 h of iron treatment. The levels of DMT1 (p0.01) and fpn1 (p0.05) mRNA were significantly decreased after 72 and 120 hours of iron treatment in serum-free medium, but the levels of Dcytb mRNA were not significantly affected (p0.14); the expression of fpn1 protein was not significantly affected after 72 and 120 hours of iron treatment in serum-free medium (p0.89), and the expression of DMT1 protein in serum-free medium was significantly decreased (p0.08). There was no significant change in the expression of DMT1 protein (p0.44). The results suggested that serum affected the uptake and absorption of manganese by iron-treated cells and the expression of dmt1, fpn1 and Dcytb in iron-treated cells. The effects of iron treatment on manganese uptake and its mechanism in broilers were studied in experiment 2. A single-factor complete randomized design was used to study the effects of iron treatment on manganese uptake in broilers. The chickens were randomly divided into 4 groups, each with 6 replicates, and each replicate consisted of 14 chickens, fed to 28 days of age. The chickens were weighed on an empty stomach in the morning of 8, 15, 22 and 28 days, and their blood and organs were collected and slaughtered. The daily gain (p0.10) and intake (p0.03) of 1-14 days old and 1-7 days old in group A were significantly lower than those in control group, but the iron level in diet had no significant effect on the growth performance of other weeks (p0.16); the iron level in diet had no significant effect on plasma total iron binding capacity, hemoglobin concentration and hematocrit (p0.16), but had significant effect on plasma iron and hematocrit (p0.16). Plasma transferrin saturation (p0.09), and plasma iron and plasma transferrin saturation increased with the increase of dietary iron level (p0.01); Dietary iron level had no significant effect on heart iron content at 21 and 28 days of age (p0.23), but had significant effect on liver, duodenum, pancreas and bone ash iron content at 7 and 14 days of age (p The content of iron in the duodenum, pancreas and ashes decreased with the increase of dietary iron level (p0.01), except that the content of manganese in liver and heart had no significant change (p0.13), and the content of manganese in the duodenum, pancreas and ashes decreased with the increase of dietary iron level (p0.01). Zinc content in pancreas and other tissues of all age groups had no significant effect on copper content (p0.13), dietary iron level significantly affected the levels of DMT1 and fpn1 mRNA in duodenal mucosa of all age groups (p0.10), and the levels of DMT1 and fpn1 mRNA in all age groups decreased with the increase of dietary iron level (p0.10); dietary iron supplementation 500 mg / kg diet significantly affected the levels of DMT1 and fpn1 mRNA in duodenal mucosa of all age groups (p0.10). The absorption rate of manganese in duodenum ligated in situ of broilers in group A was significantly lower than that in group B fed 100 mg/kg diet supplemented with iron (P 0.04)? The results suggested that dietary iron affected the absorption of manganese by regulating the expression of DMT1 and FPN1 in duodenal mucosa. Iron treatment reduces the uptake and absorption of manganese by regulating the expression of DMT1 and PFN1 genes in cells or intestines, revealing the effect of iron on manganese uptake and its mechanism at the molecular level, laying a foundation for further study of the molecular mechanism of manganese uptake.
【学位授予单位】：河北科技师范学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S831.5
【参考文献】
相关硕士学位论文 前1条
1 马春艳;22-42日龄肉鸡玉米—豆粕型饲粮铁适宜水平的研究[D];中国农业科学院;2014年
，本文编号：2180214
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