本文關(guān)鍵詞： 賴氨酸 骨骼肌蛋白 重水 示蹤劑 分?jǐn)?shù)合成率　出處：《揚(yáng)州大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:給予大鼠一定濃度的~2H_2O作為示蹤劑標(biāo)記動(dòng)物體內(nèi)骨骼肌蛋白,以期建立氘標(biāo)記丙氨酸的檢測(cè)方法,為研究骨骼肌蛋白分?jǐn)?shù)代謝率提供方法保證;在日常飲食狀態(tài)下,分別觀察大鼠在較長時(shí)間內(nèi)攝入含有低(0.2%)、正常(0.9%)、高(1.6%)劑量的賴氨酸飼料,骨骼肌蛋白分?jǐn)?shù)合成率是否相同,分析骨骼肌蛋白代謝動(dòng)力學(xué)指標(biāo)是否對(duì)賴氨酸攝入量敏感,骨骼肌蛋白代謝動(dòng)力學(xué)是否一致。分析~2H_2O標(biāo)記法是否能有效測(cè)定骨骼肌蛋白的合成動(dòng)力學(xué)參數(shù),是否適用于長期標(biāo)記實(shí)驗(yàn),從而為進(jìn)一步進(jìn)行人體賴氨酸動(dòng)力學(xué)實(shí)驗(yàn)研究奠定基礎(chǔ)。方法:第一部分:(1)~2H_2O標(biāo)記大鼠身體水90min,收集血液;~2H_2O標(biāo)記大鼠骨骼肌一周,收集四肢肌肉,提取骨骼肌蛋白和血漿游離氨基酸。將身體水中的2H與丙酮發(fā)生交換,萃取丙酮。(2)酸水解法水解骨骼肌蛋白,收集水解后丙氨酸,同時(shí)收集血液游離丙氨酸。(3)對(duì)骨骼肌水解后丙氨酸經(jīng)0.22μm微孔膜對(duì)水解液進(jìn)行過濾,對(duì)血漿游離丙氨酸進(jìn)行分離和純化。(4)對(duì)丙氨酸進(jìn)行衍生化處理,身體水的萃取物經(jīng)過微孔膜過濾,分別經(jīng)GC/MS檢測(cè),確定了氘標(biāo)記丙氨酸檢測(cè)方法的可行性。第二部分:(1)參考AIN-93G各氨基酸成分比例設(shè)計(jì)三種飼料賴氨酸含量分別為低(0.2%)、正常(0.9%)、高(1.6%)。(2)健康成年雄性Sprague-Dawley(SD)大鼠72只,12周齡,體重320g左右,隨機(jī)分為3組,每組24只。分別給予低、正常和高劑量賴氨酸飼料,喂養(yǎng)8周,自由進(jìn)食,正常飲水。(3)自第9周開始,給予大鼠~2H_2O以標(biāo)記血液游離丙氨酸、骨骼肌蛋白以及身體水,標(biāo)記至第18周結(jié)束。18周內(nèi)每周稱量大鼠體重并記錄每組大鼠的進(jìn)食量。(4)在8,9,11,13,14,18周處死大鼠,收集血液和四肢肌肉,作為樣本,用凱氏定氮法測(cè)骨骼肌蛋白的含量,GC/MS檢測(cè)骨骼肌蛋白中的2H-丙氨酸的豐度、血漿游離丙氨酸中2H的豐度及身體水的2H豐度,觀察各豐度的變化情況。(5)分別繪制各組大鼠骨骼肌蛋白中2H示蹤劑的變化趨勢(shì)圖,計(jì)算2H標(biāo)記骨骼肌蛋白的代謝動(dòng)力學(xué)方程和蛋白質(zhì)的合成率,比較不同日糧賴氨酸組所得結(jié)果的異同,觀察身體水的2H豐度的變化情況,分析不同賴氨酸攝入水平對(duì)機(jī)體骨骼肌蛋白代謝動(dòng)力學(xué)具有怎樣的影響。結(jié)果:第一部分:(1)腹腔注射~2H_2O90min后,身體水中丙酮的分子量為58/59,血漿游離丙氨酸衍生物的分子量為99/100、158/159基團(tuán)的豐度均可測(cè)出,確定了身體水中2H豐度出峰時(shí)間為2.3min左右。(2)大鼠骨骼肌蛋白中丙氨酸衍生物的分子量為99/100、158/159基團(tuán)的豐度均可測(cè)出,通過與標(biāo)準(zhǔn)庫對(duì)比,確定骨骼肌丙氨酸衍生物的出峰時(shí)間為8.45min左右,且結(jié)果具有一致性。第二部分:(1)大鼠飼料的檢測(cè)結(jié)果,低、正常和高劑量飼料中賴氨酸含量分別為0.23g/100g、0.89g/100g和1.61g/100g,均符合實(shí)驗(yàn)要求。(2)三組大鼠的日均進(jìn)食量分別為21.64g、23.07g和23.78g,低劑量賴氨酸組大鼠的進(jìn)食量明顯減少,而正常劑量組與高劑量賴氨酸組大鼠的進(jìn)食量并未受到顯著影響。(3)高劑量賴氨酸組與正常劑量組大鼠體重均值分別是低劑量組大鼠體重均值的1.20倍和1.17倍,三組大鼠體重均值具有顯著性差異,隨賴氨酸攝入量增高,體重呈增加趨勢(shì)。(4)低、正常和高劑量組大鼠身體水中2H豐度均值分別為:0.1116、0.1190和0.1180,大鼠身體水被氘標(biāo)記,攝入~2H_2O后2H能夠在很短時(shí)間內(nèi)與全身各組織的水達(dá)到平衡并且穩(wěn)定。(5)三組大鼠的骨骼肌蛋白含量分別為18.28%、18.87%和18.77%,無顯著性差別。(6)低、正常和高劑量賴氨酸組的骨骼肌蛋白分?jǐn)?shù)合成率分別為11.6%,12.8%和18.8%。(7)骨骼肌蛋白合成動(dòng)力學(xué)方程為:低劑量賴氨酸組ft=0.1604×(1-e-0.116t);正常劑量賴氨酸組ft=0.158×(1-e-0.128t);高劑量賴氨酸組ft= 0.1464×(1—e-0.1878t)。結(jié)論:腹腔注射~2H_2O 90min后,即能檢測(cè)出血漿游丙氨酸和身體水被氘標(biāo)記,說明攝入~2H_2O后能夠成功的標(biāo)記血漿游丙氨酸并且2H能在很短時(shí)間內(nèi)與全身各組織的水達(dá)到平衡;每日給予4%~2H_2O作為日常飲水,持續(xù)1周后,即能檢測(cè)出骨骼肌蛋白中的丙氨酸被氘標(biāo)記,說明該方法能夠成功標(biāo)記骨骼肌蛋白。與正常劑量賴氨酸(0.9%)攝入量組大鼠骨骼肌蛋白分?jǐn)?shù)合成率相比,低劑量賴氨酸(0.2%)攝入量組大鼠骨骼肌蛋白分?jǐn)?shù)合成率略有降低,高劑量賴氨酸(1.6%)攝入量組提高了大鼠骨骼肌蛋白的分?jǐn)?shù)合成率。從骨骼肌蛋白分?jǐn)?shù)合成率、合成動(dòng)力學(xué)方程及曲線圖可知,骨骼肌蛋白的分?jǐn)?shù)合成率和代謝動(dòng)力學(xué)方程能敏感反映機(jī)體的高賴氨酸攝入狀態(tài),但對(duì)低賴氨酸攝入狀態(tài)不敏感。
[Abstract]:Objective: rats were given a certain concentration of ~2H_2O as tracer marker in animal skeletal muscle protein, in order to establish the detection method of deuterium labeled alanine, provide guarantee for the research of skeletal muscle protein metabolism scores; in the diet condition, rats were observed for a long time with low intake (0.2%), normal (0.9%) (1.6%), high lysine feed dosage, skeletal muscle protein fractional synthesis rate is the same, the analysis of skeletal muscle protein metabolism kinetics index of lysine intake sensitive skeletal muscle protein metabolism kinetics are consistent. Analysis of synthetic kinetic parameters of ~2H_2O labeling method can effectively determine whether skeletal muscle protein, is suitable for long term labeling experiments, which provides a basis for further study of human kinetics experiment of ammonia acid lysine. Methods: the first part: (1) ~2H_2O labeling of rat body water 90min, blood collection; ~2H_2O Rat skeletal muscle markers for a week, collecting the muscles of the limbs, extraction of skeletal muscle protein and plasma free amino acid. The exchange, 2H and acetone water extraction of acetone body. (2) acid hydrolysis of skeletal muscle protein, collected after hydrolysis of alanine, and collected blood free alanine. (3) of skeletal muscle after hydrolysis of alanine 0.22 mu of M microporous membrane of hydrolysate was filtered on plasma free alanine was isolated and purified. (4) derivatization of alanine, the body of water extract by microporous membrane filtration, were detected by GC/MS, to determine the feasibility of deuterium labeled alanine detection method. The second part: (1 the amino acid composition of the proportion of AIN-93G) reference design of three kinds of dietary lysine levels were low (0.2%), normal (0.9%), high (1.6%). (2) adult male Sprague-Dawley (SD) 72 rats, aged 12 weeks, weighing about 320G, were randomly divided into 3 groups, each group of 2 4. Were given low, normal and high dose of lysine amino acid feed, feeding for 8 weeks, free eating, normal drinking water. (3) since the beginning of the ninth week, the rats were given ~2H_2O with marker free blood alanine, skeletal muscle protein and water body, to mark the end of the eighteenth week.18 weeks weighing the weight of rats and the food intake of rats in each group were recorded. (4) at 8,9,11,13,14,18 weeks the rats were sacrificed, blood was collected and the muscles of the limbs, as samples, the content measurement of skeletal muscle protein nitrogen by the Kjeldahl method, the abundance of skeletal muscle protein was detected in GC/MS 2H- alanine, 2H abundance and abundance of plasma free amino acid C water body in 2H, to observe the changes in the abundance. (5) were drawing trend of each rat skeletal muscle protein 2H tracer kinetics equation, and the protein synthesis rate 2H markers of skeletal muscle protein, comparison of different dietary lysine group results The similarities and differences, changes in 2H abundance observed in body water, analysis of different lysine intake level of what is the effect of skeletal muscle protein metabolism kinetics. Results: the first part: (1) after intraperitoneal injection of ~2H_2O90min, the molecular weight of the body in water acetone was 58/59, the molecular weight of plasma free phenylalanine derivatives as abundance can be 99/100158/159 the group measured, determine the body water abundance of 2H peak time is about 2.3min. (2) the molecular weight of alanine derivatives in rat skeletal muscle protein in the 99/100158/159 group can be abundance determined by comparing with the standard library, determine the skeletal muscle alanine derivatives peak time is about 8.45min, and the results are consistent. The second part: (1) rat feed test results, low, normal and high dose dietary lysine content were 0.23g/100g, 0.89g/100g and 1.61g/100g, are in line with the experimental requirements. (2) of the three groups of rats daily food intake were 21.64g, 23.07g and 23.78g, lysine intake of low dose group rats significantly reduced food intake and normal dose group and high dose group of lysine in rats was not affected. (3) the average weight of high dose lysine amino acid the group with normal dose group rats were 1.20 times the average weight of low dose group rats and 1.17 times, the average weight of rats in the three groups have significant difference, with the increase of amino acid intake, body weight increased. (4) low, normal and high dose of body water rats 2H abundance respectively: 0.1116,0.1190 and 0.1180 rats, body water is deuterium labeled, after intake of ~2H_2O 2H can in a very short period of time and body tissues of water balance and stability. (5) protein in skeletal muscles of rats in the three groups were 18.28%, 18.87% and 18.77%, no significant difference between the low (6). Normal, and high dose of Lai Skeletal muscle protein fractional synthesis rate of ammonia acid group were 11.6%, 12.8% and 18.8%. (7) protein synthesis in skeletal muscle dynamics equation: low dose of lysine was ft=0.1604 * (1-e-0.116t); the normal dose of lysine was ft=0.158 * (1-e-0.128t); high dose lysine group (1 - 0.1464 x ft= e-0.1878t). Conclusion: intraperitoneal injection of ~2H_2O 90min, which can detect the plasma alanine and swim body of water by deuterium labeling, that after intake of ~2H_2O successfully mark plasma alanine and 2H can swim and body tissues of water balance in a very short period of time; as a daily dose of 4%~2H_2O daily drinking water, after 1 weeks that is, can detect protein in skeletal muscle by deuterium labeled alanine, indicating successful markers of skeletal muscle protein. The method with the normal dose of lysine (0.9%) intake group rats skeletal muscle protein fractional synthesis rate compared to the low dose of lysine intake (0.2%) The amount of skeletal muscle of rats protein fractional synthesis rate decreased slightly, the high dose of lysine intake group (1.6%) increased the fractional synthesis rate of skeletal muscle protein in rats. The skeletal muscle protein fractional synthesis rate, synthesis kinetics equation and the curve graph shows that the fractional synthesis rate and Metabolic Kinetic Equation of skeletal muscle protein can be sensitive reflect the high lysine intake of the body, but for the low lysine intake is not sensitive.

【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R151

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4 張琳靜;有氧運(yùn)動(dòng)訓(xùn)練和/或高脂飲食干預(yù)對(duì)大鼠骨骼肌FNDC5蛋白表達(dá)的影響[D];河北師范大學(xué);2016年

5 馮曉琪;有氧運(yùn)動(dòng)對(duì)自發(fā)性高血壓大鼠骨骼肌基礎(chǔ)自噬水平的影響及作用機(jī)制[D];陜西師范大學(xué);2016年

6 明鑫;有氧運(yùn)動(dòng)對(duì)Ⅱ型糖尿病大鼠骨骼肌TNF-α、IL-6及p38MAPK信號(hào)通路的影響[D];南京體育學(xué)院;2016年

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9 劉平平;TNF-α、脂聯(lián)素與實(shí)驗(yàn)性糖尿病大鼠骨骼肌病變的關(guān)系[D];山西醫(yī)科大學(xué);2007年

10 陳剛;耐力訓(xùn)練對(duì)高脂膳食大鼠以及GK大鼠骨骼肌PGC-1α、ERRα和NRF-2基因表達(dá)的影響[D];華東師范大學(xué);2010年

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