【作者】 李春艳；

【导师】 马美湖；

【作者基本信息】 华中农业大学 ， 农产品加工及贮藏工程， 2014， 硕士

【摘要】 鸡蛋中的卵黄高磷蛋白(phosvitin)是已知所有蛋白质中磷酸化程度最高的蛋白质,含磷量占蛋黄总磷量的80%,是鸡胚骨骼发育中矿物质磷的主要来源。本文以phosvitin作为研究对象,围绕着phosvitin对鸡胚胎骨骼发育的影响开展了系列工作。主要研究内容和研究成果摘要如下：研究了鸡胚胎发育期间与骨骼发育相关指标的变化规律。发现鸡胚胎发育9-10d是一个转折点：蛋清pH在9d胚龄以后,逐渐趋于稳定,保持在pH7.5左右；蛋黄pH在10d胚龄时突然上升,达到最大值8.19,之后呈小幅度下降,在pH7.5附近相对稳定；蛋黄总磷含量在10d胚龄时出现急剧下降,这与胚胎在9d胚龄开始骨化有很大相关性。比较分析蛋黄磷含量的降低速率、碱性磷酸酶活力变化以及鸡胚体长的增长发现三者的变化趋势基本一致,先上升后下降。在14-16d总磷下降速率最快,平均达到0.027mg/g.d, ALP活力也达到最大值1.12U/gprot,体长增长率的变化有一定滞后,在17d胚龄增长率达到最大,1.17cm/d,这与蛋黄中的磷被转移到胚胎然后被胚胎骨骼发育利用需要一定时间有关。研究了胚胎发育的9-20d phosvitin的变化。变性凝胶电泳和非变性凝胶电泳的实验结果分析发现了胚胎发育期间phosvitin分子量逐渐下降,磷氮比的测定结果和对红外原始图谱的分析都证明了孵化期间phosvitin磷酸根含量下降,通过红外光谱和圆二色谱数据分析发现phosvitin二级结构在孵化期间γ-无规则卷曲和β-转角结构向α-螺旋结构发生了转化,phosvitin向有序性变化。综合分析P/N比、二级结构、与上一章中的ALP、体长增长率,这些指标的变化最快最高的阶段在时间和区间上都表现出一致性,在13-16d胚龄phosvitin的磷酸根含量降低最明显,蛋白质结构有序性增强,γ-无规则卷曲和p-转角结构向α-螺旋结构发生了转化,碱性磷酸酶、鸡胚体长大幅度的增加。证明了在phosvitin与鸡胚骨骼发育密切相关,并且是通过脱磷酸化提供鸡胚骨骼形成需要的磷。研究了0、50、100、500、1000μg/mL phosvitin处理MC3T3-E1细胞24h、48h后对细胞生长影响。CCK-8法检测phosvitin对MC3T3-E1细胞的增殖活力的影响,流式细胞仪法分析了phosvitin对细胞细胞周期和细胞凋亡的影响,结果表明：低浓度蛋白可以促进增殖,抑制细胞凋亡；高浓度蛋白会抑制细胞增殖,诱导细胞凋亡。phosvitin浓度100μg/mL时效果最好。100μ/mL phosvitin处理MC3T3-E1细胞48h后：细胞增殖活力达到108.61%,细胞增殖率达到23.49%,显著高于对照组及高浓度组；细胞正在凋亡率为6.42%,已经凋亡率为2.47%,显著低于对照组和高浓度组。研究了空白、Pv. Pv+Vc、Pv+β-GP、Vc+β-GP5种矿化诱导剂对MC3T3-E1细胞矿化的影响。实验首先确立诱导矿化实验中最佳接种密度为1×104/cm2。对诱导矿化期间细胞内ALP活力、矿化结节含量以及相关基因表达量结果分析发现phosvitin单独诱导不能引起细胞矿化但是可以促进细胞成骨活动,21d时ALP活力达到1.0U/gprot,显著高于阴性对照组,并且显著上调Col-I mRNA、OCN mRNA的表达,对Runx-2mRNA. BMP-2mRNA表达量的影响较弱,茜素红染色结果呈阴性。Pv+P-GP组可以诱导矿化,21d时ALP活力达到2.1U/gprot,稍低于阳性对照组,对Runx-2mRNA、BMP-2mRNA、Col-Ⅰ、mRNA、OCN mRNA都有显著上调作用,茜素红染色结果呈阳性,与阳性照组相比均无显著差异。我们推测诱导矿化的过程中phosvitin替代了Vc的作用,作为强抗氧化剂／还原剂参与合成胶原,进而促进成骨活动。Phosvitin可能是通过上调Col-Ⅰ mRNA和OCNmRNA的表达,促使其下游产物ALP表达水平增加,进而诱导矿化活动增强。但是phosvitin对成骨细胞影响详细的细胞学和分子学机制仍需进一步研究确定。更多还原

【Abstract】 Phosvitins possess the highest level of phosphorylation of all known proteins in eggs, and it contains approximately80%of an egg yolk’s phosphorous. Phosvitin serves as the main source of phosphorus for chicken embryo bone formation. The research object of this diploma paper was phosvitin. We focused on the effects of phosvitin on chicken embryonic skeletal development and carried out a series of work. The main research content and research results are summarized as follows:We studied the variation of skeletal-related indicators during incubation. Experimental results show that9-10d embryo age was a turning point:Albumen pH gradually stabilized after9d, keep at pH7.5; yolk pH had a sudden rise in10d, reached a maximum8.19, and after a slight decline it was relatively stable in7.5; Yolk phosphorus content appeared sharp decline in10d,these were associated with that embryos begined to form bone at9d. Comparative analysis of Phosphorus loss、ALP activity and body length’s growth rate Found that the three are basically the same trends, First increased and then decreased. The fastest Phosphorus loss occurred at days13-16, reaching a maximum of0.027mg/g.d, ALP activity initially increased, reaching a maximum of1.12U/g prot, the growth rate increased significantly at days17-18, at1.1cm/d The growth rate growth rate lagged behind phosphorus loss and ALP activity, these were because yolk phosphorus transferred to embryos and then use by embryonic bone development needes time.We studied the changes of phosvitin during9-20d embryo age. The results of Gel electrophoresis showed that phosvitin’s molecular weight decreased, during embryonic development; Analysis of P/N ratio and infrared spectra proved that phosvitin’s phosphate levels decreased during incubation; through analysis infrared spectroscopy date and circular dichroism date, we found thatγ-random coil and β-turn structure transformed to the a-helix structure during incubation, phosvitin tends ordering. In summary, there were correlated changes in the P/N ratio and secondary structure of phosvitin, ALP activity, and body length growth. ALP activity and body length growth slightly lagged behind changes in the P/N ratio and the secondary structure of phosvitin. Phosvitin’s phosphate content decreased, the γ-random coil and β-turn gradually transformed into a-helixes, and the protein’s secondary structure tended to become more orderly; these changes mainly occurred on days13-16. Bone formation of the chicken embryos occurred primarily on days14-18, whereas ALP activity and body length growth increased substantially. All of these findings demonstrate that the phosphorus in phosvitin is involved in chicken embryo bone formation through dephosphorylation.Cultured MC3T3-E1cell were treated with phosvitin in different concentrations(0、50、100、500、1000ug/mL) for24h、48h. Cell proliferation was assessed by CCK-8colorimetric assay. Cell cycle and apoptosis were assessed by flow cytometry analysis. The results showed that:low concentrations of protein can be found to promote proliferation and inhibit apoptosis, high concentrations of proteins inhibit cell proliferation and induce apoptosis. After the cells were treated with100μg/mL phosvitin for48h, cell proliferation activity reached108.61%, the cell proliferation rate reached23.49%, which is significantly higher then control and high concentrations. At the same time, cells undergoing apoptosis was6.42%, the apoptosis rate was2.47%, which is significantly lower than the control group and the high concentration group.Cultured MC3T3-E1cell were treated with different Mineralization inducers (control、 Pv、Pv+Vc、Pv+β-GP、Vc+β-GP), ALP activity in cells, mineralized nodules content and related gene expression levels was measured during induction of mineralization. The experiments first established optimal seeding density is1×104/cm2. The results show that phosvitin alone can not induce cell mineralization but can promote osteoblast cell activity: ALP activity reached1.0U/gprot at21d, significantly higher than the negative control group, and the expression level of Col-I mRNA, OCN mRNA increased significantly, while the expression level of Runx-2mRNA, BMP-2mRNA almost no change, and alizarin red staining was negative. Pv+β-GP groups can induce mineralization, ALP activity reached2.1U/gprot at21d, slightly lower than the positive control groups, and the expression level of Runx-2mRNA, BMP-2mRNA, Col-I mRNA, OCN mRNA all increased significantly, and alizarin red staining results were positive. Compared with the positive control group, the measurement results of Pv+β-GP groups were not significantly different. We speculate that phosvitin replaced Vc as a powerful antioxidant/reducing agent involved in the synthesis of collagen, thereby promoting osteoblast activity during the induction of mineralization. Phosvitin probably through upregulating the expression of Col-I mRNA and OCN mRNA, promote the expression levels of ALP increased, thus enhancing the mineralization activities. But the detailed cytological and molecular mechanisms of phosvitin’s impact on osteoblasts need further study.更多还原