鐵對Caco-2細胞及肉仔雞錳吸收的影響及其機制
發(fā)布時間：2018-08-12 20:05
【摘要】：本論文通過兩個試驗分別研究了鐵對Caco-2細胞及肉仔雞錳吸收的影響及其機制?試驗一研究了鐵處理對Caco-2細胞錳吸收的影響及其機制。本試驗包括2個小試驗。試驗1研究了不同接種密度對Caco-2細胞吸收模型分化及去極化評價指標的影響,確定了細胞模型適用于吸收和轉(zhuǎn)運試驗的時間?將Caco-2細胞按高密度(1×105 cells/cm2)?中密度(5×104 cells/cm2)和低密度(3×104 cells/cm2)三個接種密度,分別接種于帶聚碳酸酯微孔濾膜插槽的Transwell 6孔板上,培養(yǎng)29天,通過測定跨膜電阻值(TEER)?堿性磷酸酶活性(ALP)和酚紅透過率,評價細胞單層的完整性?極化和透過性?結(jié)果表明:高?中?低密度接種分別于第12?15和18天TEER值超過600Ω/cm2,第27?29和29天TEER值開始下降;高?中?低密度接種分別于第15?18和21天上、下側(cè)堿性磷酸酶活性的比值達到10倍以上,第29天開始降低;高?中?低密度接種分別于第9?12和15天酚紅透過率小于0.5%?綜合上述三項指標,高密度接種細胞在第15天?中密度接種細胞在第18天?低密度接種細胞在第21天開始,可以用于吸收和轉(zhuǎn)運試驗,27天以后細胞活力就開始下降,不適合做吸收和轉(zhuǎn)運細胞模型?試驗2研究了鐵處理對Caco-2細胞錳吸收的影響及其機制?采用單因子完全隨機試驗設(shè)計,共5個處理組,分別為正常含血清培養(yǎng)基、正常含血清培養(yǎng)基加鐵100μmol/L、正常含血清加鐵200μmol/L、無血清培養(yǎng)基添加鐵100μmol/L和無血清培養(yǎng)基加鐵200μmol/L,每個處理6個重復?分別在細胞培養(yǎng)的第18~23天在培養(yǎng)基里以次氮基三乙酸絡(luò)合鐵的形式添加鐵,鐵處理120h結(jié)束后,在所有細胞培養(yǎng)液中以無機硫酸錳的形式添加800μmol/L錳,37℃?50r/min水浴振蕩器上振蕩孵育120 min,收集細胞及上、下層培養(yǎng)液,測定其中鐵和錳含量;分別在鐵處理72和120h后收集細胞,測定二價金屬轉(zhuǎn)運蛋白1(DMT1)、膜轉(zhuǎn)鐵蛋白1(FPN1)和十二指腸細胞色素還原酶b(Dcytb)mRNA及蛋白表達量。結(jié)果表明:與不加鐵對照組相比,無血清培養(yǎng)基中添加鐵處理組細胞中鐵含量顯著高于對照組(P0.09),有血清培養(yǎng)基中添加鐵處理組細胞中鐵含量無顯著提高(P0.31);無血清培養(yǎng)基中添加鐵處理組和有血清培養(yǎng)基中添加鐵200μmol/l處理組細胞對錳的攝取量(p0.03)和吸收量(p0.02)均顯著降低;鐵處理72h有血清培養(yǎng)基中添加鐵對dmt1mrna(p0.38)和fpn1(p0.43)mrna無顯著影響,鐵處理120h有血清培養(yǎng)基中添加鐵200μmol/l和無血清培養(yǎng)基中添加鐵組dmt1(p0.01)和dcytbmrna(p0.05)水平均顯著降低,無血清培養(yǎng)基中添加鐵處理72和120h均顯著降低dmt1(p0.01)和fpn1(p0.05)mrna水平,對dcytbmrna水平無顯著影響(p0.14);鐵處理72和120h對fpn1蛋白表達量均無顯著影響(p0.89),無血清培養(yǎng)基中添加鐵組dmt1蛋白表達量顯著降低(p0.08),有血清培養(yǎng)基中添加鐵組dmt1蛋白表達量無顯著變化(p0.44)。本研究結(jié)果提示,血清影響鐵處理細胞對錳的攝取和吸收以及細胞中dmt1、fpn1和dcytb的表達,鐵處理細胞中dmt1和fpn1基因表達水平降低伴隨著攝取和吸收錳量的降低,表明鐵通過dmt1和fpn1調(diào)控細胞對錳的攝取和跨膜吸收。試驗二研究了鐵處理對肉仔雞錳吸收的影響及其機制。采用單因子完全隨機設(shè)計,共4個處理組,分別為不加鐵基礎(chǔ)飼糧組(0mg/kg)、以硫酸亞鐵(feso4·7h2o)形式添加100、250和500mg/kg組?將336只1日齡商品代羅斯308肉公雛,按體重隨機分為4個處理組,每組6個重復,每個重復14只雞,飼養(yǎng)至28日齡。分別在8、15、22和28日齡早晨空腹稱重、采血和屠宰取組織器官。28日齡對添加鐵水平為100和500mg/kg組雞進行原位結(jié)扎十二指腸灌注8.74mmol/l錳?結(jié)果表明,除添加鐵500mg/kg組1~14日齡日增重(p0.10)及1~7日齡采食量顯著(p0.03)顯著低于對照組外,飼糧鐵水平對其他各周生長性能指標無顯著影響(p0.16);飼糧鐵水平對各周齡血漿總鐵結(jié)合力?血紅蛋白濃度及紅細胞壓積均無顯著影響(p0.16),但顯著影響血漿鐵和血漿轉(zhuǎn)鐵蛋白飽和度(p0.09),且各周齡血漿鐵和血漿轉(zhuǎn)鐵蛋白飽和度均隨飼糧鐵水平增加而提高(p0.01);飼糧鐵水平除對第21和28日齡心臟鐵含量無顯著影響(p0.23)外,顯著影響第7和14日齡心臟及各日齡肝臟、十二指腸、胰腺和骨灰鐵含量(p0.02),且這些組織鐵含量均隨飼糧鐵水平增加而提高(p0.01);除各日齡肝臟和心臟錳含量無顯著變化外(p0.13),各日齡十二指腸、胰腺和骨灰錳含量均隨飼糧鐵水平增加而降低(p0.01);飼糧添加鐵除降低第7日齡胰腺鋅含量外(p0.01),對其他日齡胰腺和各日齡其他所測組織鋅含量、各日齡所測組織銅含量均無顯著影響(p0.13);飼糧鐵水平顯著影響各日齡十二指腸粘膜dmt1和fpn1mrna水平(p0.10),各日齡dmt1和fpn1mrna水平均隨飼糧鐵添加水平升高而降低(P0.10);飼喂添加鐵500 mg/kg飼糧組肉仔雞原位結(jié)扎十二指腸中錳的吸收率明顯低于飼喂添加鐵100 mg/kg飼糧組(P0.04)?本試驗結(jié)果提示,飼糧鐵通過調(diào)控十二指腸粘膜DMT1和FPN1的表達影響肉仔雞對錳的吸收。綜上所述,體外細胞培養(yǎng)Caco-2細胞和肉仔雞飼養(yǎng)及灌注均表明,鐵處理通過調(diào)控細胞或腸道DMT1和PFPN1基因表達降低錳的攝取和吸收,從分子水平揭示了鐵對錳吸收的影響及其機制,為進一步研究錳吸收的分子機制奠定了基礎(chǔ)。
[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.
【學位授予單位】：河北科技師范學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S831.5
本文編號：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.
【學位授予單位】：河北科技師范學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S831.5
【參考文獻】
相關(guān)碩士學位論文 前1條
1 馬春艷;22-42日齡肉雞玉米—豆粕型飼糧鐵適宜水平的研究[D];中國農(nóng)業(yè)科學院;2014年
，本文編號：2180214
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