蛋雞子宮內(nèi)膜細胞鈣轉(zhuǎn)運及蛋殼鈣化早期鈣沉積機制的研究
發(fā)布時間：2019-06-11 23:23
【摘要】：目的:研究孕酮對蛋殼鈣化初期與鈣沉積相關的基質(zhì)蛋白和基因表達的影響,初步探討鈣化起始時的鈣沉積機制。方法:本研究以產(chǎn)蛋高峰期蛋雞作為實驗動物,體外培養(yǎng)蛋雞子宮內(nèi)膜細胞,測定細胞生長曲線,檢測不同濃度的激素對細胞體外增殖和鈣離子濃度的影響,并利用實時熒光定量PCR技術(RTQ-PCR)檢測不同處理組細胞中Cabp-d28k、Ca2+-ATPase、碳酸酐酶(CA)、TRPV6、骨橋蛋白(OPN)、卵鐵傳遞蛋白(Ovotransferrin)、卵清蛋白(Ovalbumin)、Ovocleidin 17和凝集素(Clusterin)等基因的表達量變化。在此基礎上對蛋雞進行孕酮和孕酮受體拮抗劑處理,蛋殼鈣化初期(排卵后4.5-5.5h)采集子宮組織和蛋殼,掃描電子顯微鏡(SEM)觀察蛋殼超微結(jié)構(gòu)變化,測定子宮中鈣離子濃度及相關基因表達量的變化規(guī)律,對基因的表達量變化與超微結(jié)構(gòu)的相關性進行分析,并采用免疫組化和免疫印跡(Western-Blotting)分析Cabp-d28k和Ovalbumin蛋白在子宮中的變化。結(jié)果:本研究成功培養(yǎng)了蛋雞子宮內(nèi)膜上皮細胞和基質(zhì)細胞,并測得兩種細胞的生長曲線;添加100n M的孕酮能顯著(P0.05)促進上皮細胞和基質(zhì)細胞的體外增殖;激素對體外培養(yǎng)的細胞中鈣離子濃度的影響不顯著(P0.05),但雞體經(jīng)孕酮處理后子宮組織中的鈣離子含量顯著(P0.05)下降。不同處理對子宮內(nèi)膜細胞和子宮組織中基因的表達量影響為:孕酮處理后Cabp-d28k、CA和OPN m RNA在子宮中的表達量顯著(P0.05)或極顯著(P0.01)下降,OVA、Clu和OC17m RNA的表達量顯著(P0.05)或極顯著(P0.01)升高;孕酮受體拮抗劑處理后TRPV6 m RNA的表達量顯著(P0.05)下降,OVA、OVO、Clu和OC17 m RNA的表達量顯著(P0.05)或極顯著(P0.01)升高。與對照組和拮抗劑處理相比,孕酮處理后蛋殼內(nèi)膜層和乳突層變厚,纖維直徑和結(jié)節(jié)直徑變大,乳突間隙和纖維間隙變小,差異均達到顯著(P0.05)或極顯著(P0.01)水平,且孕酮處理后Cabp-d28k、OVA、OVO以及OPN等基因的表達量變化與蛋殼超微結(jié)構(gòu)有相關性;免疫組化和Western Blotting分析結(jié)果表明,Cabp-d28k和Ovalbumin蛋白在不同處理組的子宮中均有表達,但Cabp-d28k蛋白在孕酮處理組中的表達量低于對照組和孕酮受體拮抗劑處理組,Ovalbumin蛋白則相反。結(jié)論:蛋殼鈣化初期孕酮顯著(P0.05)抑制了Cabp-d28k和OPN在子宮中的表達,使蛋殼的乳突面積、乳突直徑和纖維間隙減小;孕酮顯著(P0.05)促進了OVA和OVO的表達,使蛋殼的纖維徑和結(jié)節(jié)直徑增加;且使蛋殼內(nèi)膜層厚度和乳突層厚度增加。研究認為蛋殼鈣化初期孕酮可通過調(diào)控蛋雞子宮中與鈣化相關的基因和蛋白的表達,使蛋殼纖維內(nèi)膜層和乳突層結(jié)構(gòu)更致密,排列更有序,從而使增加蛋殼強度。本研究為蛋殼鈣化時鈣沉積的機制的進一步研究提供了理論依據(jù)和實驗基礎。
[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.
【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S831
本文編號：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.
【學位授予單位】：石河子大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S831
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