蛋鸡子宫内膜细胞钙转运及蛋壳钙化早期钙沉积机制的研究
发布时间：2019-06-11 23:23
【摘要】：目的:研究孕酮对蛋壳钙化初期与钙沉积相关的基质蛋白和基因表达的影响,初步探讨钙化起始时的钙沉积机制。方法:本研究以产蛋高峰期蛋鸡作为实验动物,体外培养蛋鸡子宫内膜细胞,测定细胞生长曲线,检测不同浓度的激素对细胞体外增殖和钙离子浓度的影响,并利用实时荧光定量PCR技术(RTQ-PCR)检测不同处理组细胞中Cabp-d28k、Ca2+-ATPase、碳酸酐酶(CA)、TRPV6、骨桥蛋白(OPN)、卵铁传递蛋白(Ovotransferrin)、卵清蛋白(Ovalbumin)、Ovocleidin 17和凝集素(Clusterin)等基因的表达量变化。在此基础上对蛋鸡进行孕酮和孕酮受体拮抗剂处理,蛋壳钙化初期(排卵后4.5-5.5h)采集子宫组织和蛋壳,扫描电子显微镜(SEM)观察蛋壳超微结构变化,测定子宫中钙离子浓度及相关基因表达量的变化规律,对基因的表达量变化与超微结构的相关性进行分析,并采用免疫组化和免疫印迹(Western-Blotting)分析Cabp-d28k和Ovalbumin蛋白在子宫中的变化。结果:本研究成功培养了蛋鸡子宫内膜上皮细胞和基质细胞,并测得两种细胞的生长曲线;添加100n M的孕酮能显著(P0.05)促进上皮细胞和基质细胞的体外增殖;激素对体外培养的细胞中钙离子浓度的影响不显著(P0.05),但鸡体经孕酮处理后子宫组织中的钙离子含量显著(P0.05)下降。不同处理对子宫内膜细胞和子宫组织中基因的表达量影响为:孕酮处理后Cabp-d28k、CA和OPN m RNA在子宫中的表达量显著(P0.05)或极显著(P0.01)下降,OVA、Clu和OC17m RNA的表达量显著(P0.05)或极显著(P0.01)升高;孕酮受体拮抗剂处理后TRPV6 m RNA的表达量显著(P0.05)下降,OVA、OVO、Clu和OC17 m RNA的表达量显著(P0.05)或极显著(P0.01)升高。与对照组和拮抗剂处理相比,孕酮处理后蛋壳内膜层和乳突层变厚,纤维直径和结节直径变大,乳突间隙和纤维间隙变小,差异均达到显著(P0.05)或极显著(P0.01)水平,且孕酮处理后Cabp-d28k、OVA、OVO以及OPN等基因的表达量变化与蛋壳超微结构有相关性;免疫组化和Western Blotting分析结果表明,Cabp-d28k和Ovalbumin蛋白在不同处理组的子宫中均有表达,但Cabp-d28k蛋白在孕酮处理组中的表达量低于对照组和孕酮受体拮抗剂处理组,Ovalbumin蛋白则相反。结论:蛋壳钙化初期孕酮显著(P0.05)抑制了Cabp-d28k和OPN在子宫中的表达,使蛋壳的乳突面积、乳突直径和纤维间隙减小;孕酮显著(P0.05)促进了OVA和OVO的表达,使蛋壳的纤维径和结节直径增加;且使蛋壳内膜层厚度和乳突层厚度增加。研究认为蛋壳钙化初期孕酮可通过调控蛋鸡子宫中与钙化相关的基因和蛋白的表达,使蛋壳纤维内膜层和乳突层结构更致密,排列更有序,从而使增加蛋壳强度。本研究为蛋壳钙化时钙沉积的机制的进一步研究提供了理论依据和实验基础。
[Abstract]:Aim: to study the effect of progesterone on the expression of matrix proteins and genes related to calcium deposition in the early stage of eggshell calcification, and to explore the mechanism of calcium deposition at the beginning of calcification. Methods: the intimal cells of laying hens were cultured in vitro, the cell growth curves were measured, the effects of different concentrations of hormones on cell proliferation and calcium concentration in vitro were detected, and the Cabp-d28k,Ca2-ATPase, carbonic anhydrase (CA), TRPV6, osteopontin (OPN), egg ferritin (Ovotransferrin), in different treatment groups was detected by real-time fluorescence quantitative PCR (RTQ-PCR). The expression of ovalbumin (Ovalbumin), Ovocleidin 17 and lectin (Clusterin) was changed. On this basis, progesterone and progesterone receptor antagonists were used to treat laying hens. Uterine tissue and eggshell were collected at the initial stage of eggshell calcification (4.5 h after ovulatory). The ultrastructure of eggshell was observed by scanning electron microscope (SEM), the concentration of calcium ion in uterus and the expression of related genes were measured, and the correlation between gene expression and ultrastructure was analyzed. The changes of Cabp-d28k and Ovalbumin proteins in uterus were analyzed by immunohistochemistry and immunoblotting (Western-Blotting). Results: the intimal epithelial cells and stroma cells of laying hens were cultured successfully, and the growth curves of the two kinds of cells were measured. Progesterone supplemented with 100nM could significantly (P 0.05) promote the proliferation of epithelial cells and stroma cells in vitro; the effect of hormones on the concentration of calcium ion in cultured cells was not significant (P 0.05), but the content of Ca ~ (2 +) in uterine tissue of chickens treated with progesterone decreased significantly (P 0.05). The effects of different treatments on the expression of genes in endometrial cells and uterine tissues were as follows: after progesterone treatment, the expression of Cabp-d28k,CA and OPN m RNA in uterus decreased significantly (P05) or extremely significantly (P01), while the expression of OVA,Clu and OC17m RNA increased significantly (P05) or extremely significantly (P01). After treatment with progesterone receptor antagonist, the expression of TRPV6 m RNA decreased significantly (P 0.05), while the expression of OVA,OVO,Clu and OC17 m RNA increased significantly (P 0.05) or very significantly (P 0.01). Compared with the control group and antagonist treatment, the intima and mastoid layer became thicker, the fiber diameter and nodular diameter became larger, and the mastoid space and fiber space became smaller after progesterone treatment, the difference was significant (P 0.05) or extremely significant (P 0.01). The expression of Cabp-d28k,OVA,OVO and OPN genes after progesterone treatment was correlated with the ultrastructure of eggshell. The results of immunohistochemistry and Western Blotting analysis showed that Cabp-d28k and Ovalbumin proteins were expressed in the uterus of different treatment groups, but the expression of Cabp-d28k protein in progesterone treatment group was lower than that in control group and progesterone receptor antagonist treatment group, while Ovalbumin protein was opposite. Conclusion: progesterone significantly decreased the expression of Cabp-d28k and OPN in uterus and decreased the mastoid area, mastoid diameter and fiber space in the early stage of eggshell calcification, progesterone significantly promoted the expression of OVA and OVO, increased the fiber diameter and nodular diameter of eggshell, and increased the intimal layer thickness and mastoid layer thickness of eggshell. It is considered that progesterone in the early stage of eggshell calcification can increase the strength of eggshell by regulating the expression of genes and proteins related to calcification in the uterus of laying hens, so that the intima and mastoid layer of eggshell fiber can be more compact and arranged in a more orderly manner. This study provides a theoretical and experimental basis for the further study of the mechanism of calcium deposition in eggshell calcification.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S831
本文编号：2497543
[Abstract]:Aim: to study the effect of progesterone on the expression of matrix proteins and genes related to calcium deposition in the early stage of eggshell calcification, and to explore the mechanism of calcium deposition at the beginning of calcification. Methods: the intimal cells of laying hens were cultured in vitro, the cell growth curves were measured, the effects of different concentrations of hormones on cell proliferation and calcium concentration in vitro were detected, and the Cabp-d28k,Ca2-ATPase, carbonic anhydrase (CA), TRPV6, osteopontin (OPN), egg ferritin (Ovotransferrin), in different treatment groups was detected by real-time fluorescence quantitative PCR (RTQ-PCR). The expression of ovalbumin (Ovalbumin), Ovocleidin 17 and lectin (Clusterin) was changed. On this basis, progesterone and progesterone receptor antagonists were used to treat laying hens. Uterine tissue and eggshell were collected at the initial stage of eggshell calcification (4.5 h after ovulatory). The ultrastructure of eggshell was observed by scanning electron microscope (SEM), the concentration of calcium ion in uterus and the expression of related genes were measured, and the correlation between gene expression and ultrastructure was analyzed. The changes of Cabp-d28k and Ovalbumin proteins in uterus were analyzed by immunohistochemistry and immunoblotting (Western-Blotting). Results: the intimal epithelial cells and stroma cells of laying hens were cultured successfully, and the growth curves of the two kinds of cells were measured. Progesterone supplemented with 100nM could significantly (P 0.05) promote the proliferation of epithelial cells and stroma cells in vitro; the effect of hormones on the concentration of calcium ion in cultured cells was not significant (P 0.05), but the content of Ca ~ (2 +) in uterine tissue of chickens treated with progesterone decreased significantly (P 0.05). The effects of different treatments on the expression of genes in endometrial cells and uterine tissues were as follows: after progesterone treatment, the expression of Cabp-d28k,CA and OPN m RNA in uterus decreased significantly (P05) or extremely significantly (P01), while the expression of OVA,Clu and OC17m RNA increased significantly (P05) or extremely significantly (P01). After treatment with progesterone receptor antagonist, the expression of TRPV6 m RNA decreased significantly (P 0.05), while the expression of OVA,OVO,Clu and OC17 m RNA increased significantly (P 0.05) or very significantly (P 0.01). Compared with the control group and antagonist treatment, the intima and mastoid layer became thicker, the fiber diameter and nodular diameter became larger, and the mastoid space and fiber space became smaller after progesterone treatment, the difference was significant (P 0.05) or extremely significant (P 0.01). The expression of Cabp-d28k,OVA,OVO and OPN genes after progesterone treatment was correlated with the ultrastructure of eggshell. The results of immunohistochemistry and Western Blotting analysis showed that Cabp-d28k and Ovalbumin proteins were expressed in the uterus of different treatment groups, but the expression of Cabp-d28k protein in progesterone treatment group was lower than that in control group and progesterone receptor antagonist treatment group, while Ovalbumin protein was opposite. Conclusion: progesterone significantly decreased the expression of Cabp-d28k and OPN in uterus and decreased the mastoid area, mastoid diameter and fiber space in the early stage of eggshell calcification, progesterone significantly promoted the expression of OVA and OVO, increased the fiber diameter and nodular diameter of eggshell, and increased the intimal layer thickness and mastoid layer thickness of eggshell. It is considered that progesterone in the early stage of eggshell calcification can increase the strength of eggshell by regulating the expression of genes and proteins related to calcification in the uterus of laying hens, so that the intima and mastoid layer of eggshell fiber can be more compact and arranged in a more orderly manner. This study provides a theoretical and experimental basis for the further study of the mechanism of calcium deposition in eggshell calcification.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S831
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