【摘要】：當(dāng)前,卵巢靜止已成為集約化牛場(chǎng)高產(chǎn)奶牛產(chǎn)后常見(jiàn)的卵泡生長(zhǎng)異常的疾病。其特征為卵巢或相關(guān)的卵巢結(jié)構(gòu)出現(xiàn)無(wú)周期性特征的靜止?fàn)顟B(tài)。奶牛產(chǎn)后卵巢上卵泡生長(zhǎng)受到諸多因素的影響。盡管能量負(fù)平衡被公認(rèn)為奶牛產(chǎn)后卵巢靜止的主要病因,但是奶牛產(chǎn)后發(fā)生卵巢靜止時(shí),機(jī)體整體蛋白表達(dá)的情況,以及與該病的關(guān)系還未見(jiàn)報(bào)道。為此,本試驗(yàn)應(yīng)用蛋白組學(xué)中熒光差異雙向凝膠電泳(Fluorescence Two-dimensional Differential Gel Electrophoresis,2D-DIGE)與基質(zhì)輔助激光解析離子化飛行時(shí)間質(zhì)譜(MatrixAssisted Laser Desorption/Lonization Time of Flight Mass Spectrometry,MALDI-TOF-MS)聯(lián)用技術(shù),結(jié)合生物信息學(xué)分析,獲得了能量負(fù)平衡所致的卵巢靜止奶牛血清差異表達(dá)蛋白,探究了這些差異表達(dá)蛋白與卵巢靜止之間的關(guān)系,為今后深入探索奶牛產(chǎn)后卵巢靜止發(fā)生機(jī)制提供科學(xué)依據(jù)。本試驗(yàn)在黑龍江某集約化奶牛場(chǎng)選取了年齡、胎次相近的經(jīng)產(chǎn)奶牛,在產(chǎn)后14~21d根據(jù)血清中β-羥丁酸(β-hydroxybutyrate,BHBA)、游離脂肪酸(Non esterified fatty acid,NEFA)和葡萄糖(Glucose,Glc)含量將其分為能量正平衡和能量負(fù)平衡組。兩組奶牛跟蹤至產(chǎn)后60~90d,根據(jù)有無(wú)發(fā)情表現(xiàn),結(jié)合B超與直腸檢查卵巢卵泡狀態(tài),最終確定了30頭能量負(fù)平衡的卵巢靜止奶牛為實(shí)驗(yàn)組(T)和30頭能量正平衡的發(fā)情奶牛為健康對(duì)照組(C)。隨機(jī)選取兩組(T或C)奶牛各15頭,應(yīng)用2D-DIGE/MS技術(shù)檢測(cè)兩組奶牛血清樣品獲得兩組奶牛的差異表達(dá)蛋白。通過(guò)生物信息學(xué)分析,確定差異蛋白與卵巢靜止的關(guān)系。應(yīng)用Western-blot技術(shù)對(duì)血清中AMBP蛋白復(fù)合體(alpha-1-microglobulin/bikunin precursor,α1微球蛋白/bikunin蛋白前體)進(jìn)行驗(yàn)證。兩組(T或C)中剩余15頭奶牛,檢測(cè)血清中生殖激素和AMBP蛋白復(fù)合體含量,并進(jìn)行彼此的相關(guān)性分析,結(jié)合回歸分析和受試者工作特征曲線(xiàn)(receiver operating characteristic curve,ROC),確定AMBP蛋白復(fù)合體對(duì)卵巢靜止的預(yù)警作用。結(jié)果顯示:(1)通過(guò)2D-DIGE/MALDI-TOF-MS檢測(cè)和差異分析,發(fā)現(xiàn)卵巢靜止組和發(fā)情組奶牛之間有12種差異表達(dá)蛋白質(zhì),其中AMBP蛋白復(fù)合體(Protein AMBP)、α-S1-酪蛋白(Alpha-S1-casein)、血紅蛋白(Hemoglobin subunit beta)和結(jié)合珠蛋白(Haptoglobin)等4種蛋白表達(dá)上調(diào),另外載脂蛋白A1(Apolipoprotein A-I)、載脂蛋白A4(Apolipoprotein A-IV)、α-1-酸性糖蛋白(Alpha-1-acid glycoprotein)、α-2-HS-糖蛋白(Alpha-2-HS-glycoprotein)、亮氨酸富集α-2-糖蛋白1(Leucine-rich alpha-2-glycoprotein 1)、補(bǔ)體3d(Complement component 3d(Fragment))、補(bǔ)體C3(Complement C3)和絲氨酸蛋白酶抑制劑E2C(Endopin 2C)等8種蛋白表達(dá)下調(diào)。通過(guò)生物信息學(xué)分析發(fā)現(xiàn)這些差異表達(dá)蛋白的基因功能主要與類(lèi)固醇和脂質(zhì)代謝,酶活性和抗氧化作用有關(guān);參與的主要通路是脂肪和維生素消化與吸收,PPAR信號(hào)通路和補(bǔ)體與凝血級(jí)聯(lián)。AMBP蛋白的Western-blotting結(jié)果與2D-DIGE的一致,都上調(diào)。(2)能量負(fù)平衡的卵巢靜止組奶牛產(chǎn)后60~90d血清NEFA含量均顯著高于健康發(fā)情組(p0.05),血清雌激素(Estrogen,E2)含量顯著低于發(fā)情奶牛(p0.05),血清AMBP蛋白復(fù)合體濃度極顯著高于健康發(fā)情組奶牛(p0.01);血清中E2濃度與試驗(yàn)動(dòng)物分組情況呈極顯著正相關(guān)(p0.01),AMBP蛋白復(fù)合體與試驗(yàn)動(dòng)物分組呈極顯著負(fù)相關(guān)(p0.01);血清NEFA與E2含量呈顯著負(fù)相關(guān)(p0.01);通過(guò)回歸分析和ROC分析確定了當(dāng)奶牛血清中AMBP4268.59 ng/L時(shí),奶�；悸殉察o止的風(fēng)險(xiǎn)增大。結(jié)論:本研究應(yīng)用蛋白質(zhì)組學(xué)技術(shù)獲得了能量負(fù)平衡所致的卵巢靜止奶牛血清差異表達(dá)蛋白,探討了差異表達(dá)蛋白質(zhì)與奶牛產(chǎn)后卵巢靜止之間的關(guān)系,確定了AMBP蛋白復(fù)合體在奶牛產(chǎn)后卵巢靜止發(fā)生的風(fēng)險(xiǎn)預(yù)警作用,為今后深入揭示能量負(fù)平衡所致的卵巢靜止發(fā)生機(jī)理提供了新的方向。
[Abstract]:At present, ovarian motility has become a common postpartum follicular growth disorder in high-yielding dairy cows in intensive farms. It is characterized by a stationary state of ovaries or related ovarian structures with no periodic characteristics. However, the expression of whole body protein and its relationship with the disease have not been reported in postpartum dairy cows with ovarian motility. In this study, fluorescence Two-dimensional Differential Gel Electrophoresis (2D-DIGE) and matrix-assisted laser resolution were applied to isolate the disease. Matrix Assisted Laser Desorption/Lonization Time of Flight Mass Spectrometry (MALDI-TOF-MS) combined with bioinformatics analysis was used to obtain differentially expressed proteins in ovarian resting cow serum induced by negative energy balance. In this study, we selected the age and parity of the same type of dairy cows from an intensive dairy farm in Heilongjiang Province. According to the serum levels of beta-hydroxybutyrate (BHBA), non-esterified fatty acid (NEFA) and glucose (Glucose, Glc) from 14 to 21 days postpartum. Two groups of dairy cows were followed up to 60-90 days postpartum. According to oestrus and ovarian follicular status, 30 calorie-balanced ovarian stationary cows were selected as experimental group (T) and 30 calorie-balanced calorie cows as healthy control group (C). Fifteen dairy cows in each group (T or C) were used to detect the differentially expressed proteins in the serum of the two groups. The relationship between the differentially expressed proteins and ovarian motility was determined by bioinformatics analysis. The serum AMBP protein complex (alpha-1-microglobulin/bikunin precursor, alpha-1 microspheres) was detected by Western blot. Serum levels of reproductive hormones and AMP protein complexes were measured in 15 dairy cows in two groups (T or C), and their correlation was analyzed. Combined with regression analysis and receiver operating characteristic curve (ROC), the resting ovarian function of AMBP protein complexes was determined. The results showed that: (1) Through two-dimensional DIGE/MALDI-TOF-MS detection and differential analysis, 12 differentially expressed proteins were found between ovarian resting group and estrus group, including AMBP protein complex (Protein AMBP), alpha-S1-casein (Alpha-S1-casein), hemoglobin subunit beta (Hemoglobin subunit beta) and Haptoglobin (Haptoglobin). Apolipoprotein A-I, Apolipoprotein A-IV, Alpha-1-acid glycoprotein, Alpha-2-HS-glycoprotein, Leucine-rich alpha-2-glycoprotein 1, Complement 3D Bioinformatics analysis showed that the gene functions of these differentially expressed proteins were mainly related to steroid and lipid metabolism, enzyme activities and antioxidant activities, and the main pathways involved were fat and vitamin digestion and absorption, and PPA. Western-blotting results of AMBP protein were consistent with those of 2D-DIGE. (2) Negative energy balance ovarian motility group had significantly higher serum NEFA content than healthy estrus group (p0.05), lower serum estrogen (Estrogen, E2) content than estrus cows (p0.05), and serum AMBP protein complex. Body concentration was significantly higher than that of healthy estrous cows (p0.01); E2 concentration in serum was significantly positively correlated with the grouping of experimental animals (p0.01); AMBP protein complex was significantly negatively correlated with the grouping of experimental animals (p0.01); serum NEFA and E2 content was significantly negatively correlated (p0.01); regression analysis and ROC analysis showed that the serum of dairy cows was positively correlated with the grouping of experimental animals (p0.01). CONCLUSION: In this study, the serum differentially expressed proteins in ovarian quiescent cows induced by negative energy balance were obtained by proteomics, and the relationship between differentially expressed proteins and postpartum ovarian quiescent in dairy cows was discussed. The risk warning function of resting ovarian motility provides a new direction for further revealing the mechanism of resting ovary caused by negative energy balance.
【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S858.23

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