【作者】 周荣艳；

【导师】 彭健；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2004， 硕士

【摘要】 本研究建立了饲料添加剂中游离谷氨酰胺(Glutamine，Gln)和丙氨酰谷氨酰胺(Alanyl-glutamine，Ala-Gln)的高效液相色谱分析方法；以Gln和Ala-Gln为试验材料，用体外培养技术研究了Gln和Ala-Gln对早期断奶仔猪小肠粘膜上皮细胞和肠系膜淋巴结细胞增殖的影响：以Gln和百傲康为试验材料研究了其对28日龄早期断奶仔猪生产性能和血液生化指标的影响，为早期断奶仔猪日粮中利用Gln或Gln二肽作为免疫促进剂，改善仔猪的生产性能提供科学依据。具体研究结果如下： 1．本试验对测定饲料添加剂中游离Gln和Ala-Gln的高效液相色谱分析方法进行研究。采用Spherisorb，C18色谱柱为固定相，OPA为柱前衍生剂，乙腈：磷酸缓冲液(50mmol／L)=6／94为流动相，进行单泵洗脱，激发波长为230nm，发射波长为389nm，荧光检测器检测Gln的含量，结果线性范围为200 μmol／L-6400 μmol／L，线性回归方程为Y=1793.4X+274241，r=0.9999，批内变异系数为1.39％。采用Spherisorb C18色谱柱为固定相，邻苯二甲醛(OPA)为柱前衍生剂，乙腈：磷酸缓冲液(12.5mmol／L)=15／85(pH，4.35)为流动相，进行单泵洗脱。激发波长为338nm，发射波长为450nm。荧光检测器检测Ala-Gln的含量，线性范围为6.25-100mmol／L，线性回归方程为：Y=28186X-9905.1，r=0.9997，批内变异系数为5.33％，回收率为98％。利用此方法测定百傲康中游离Gln和Ala-Gln含量分别为21.1％和2.65％。结果证实本测定方法快速、简单、无需梯度洗脱，分离效果好，灵敏度和精密度较高，可用于饲料添加剂中游离Gln和Ala-Gln二肽分析。 2．研究Gln及Ala-Gln对原代培养仔猪肠上皮细胞增殖的影响。采用胶原酶Ⅺ型和中性蛋白酶Ⅰ型联合作用消化分离肠上皮细胞。将获得的细胞接种于含不同浓度Gln或Ala-Gln的培养基中进行培养，培养72h后，噻唑兰(MTT)显色法测定贴壁生长的肠上皮细胞活细胞数量。采用此方法消化可以快速获得大量有活性的细胞。培养液中Gln浓度大于0.5mmol／L时肠上皮细胞快速增殖。Ala-Gln浓度在0.25mmol／L时具有显著的促进作用，增加浓度至2mmol／L时，细胞增殖率达到最大。同浓度Gln和Ala-Gln组差异不显著(p＞0.05)。结果表明Gln和Ala-Gln均可以刺激体外原代培养的仔猪肠上皮细胞增殖，且作用相似。 3．采用体外淋巴细胞转化试验研究Gln及Ala-Gln对早期断奶仔猪肠系膜淋巴结细胞增殖的影响。摘取28日龄断奶仔猪肠系膜空、回肠淋巴结，分离淋巴细胞，接种于含不同浓度Gln或Ala-Gln的培养液中进行培养，37℃培养72h后，MTT法测定细胞增殖。Gln浓度为2mmol／L时，淋巴细胞刺激指数最大；Ala-Gln浓度为1mmol／L时，淋巴细胞刺激指数最大。增加Gln或Ala-Gln浓度并没有增加其效应。在一定的华中农业大学2004届硕士学位论文浓度范围内Ala-Gln与Gln对于细胞培养液中MLN细胞增殖的作用相似。本试验表明Gin和Ala一Gln均可以促进丝裂原PHA刺激的淋巴细胞增殖，在一定浓度范围内Ala一Gln可以发挥Gln的作用。 4.选择大长母猪所产仔猪42头，公母各半，仔猪28日龄一次性断奶后随机分为3组，每组2个重复，每个重复7头，仔猪进行单因子试验设计，试验组分别添加0.7%Gln和l%百傲康。研究日粮添加Gln和百傲康对早期断奶仔猪生产性能、腹泻率和血液生化指标的影响。添加Gln和百傲康对日增重、采食量、料肉比、腹泻率和血清总蛋白影响不显著沙0.05)，但可以降低断奶后第2天血清尿素氮水平印<。.1)，显著降低血清皮质醉浓度勿<0 .05)旧粮添加Gln可以维持血浆Gln水平印<0 .05)。结果表明日粮添加Gin和百傲康可以缓解早期断奶仔猪应激，。更多还原

【Abstract】 A high performance liquid chromatography method for determining the concentration of free Glutamine (Gin) and Alanyl-Glutamine (Ala-Gin) in feed additive was established in this study. We have investigated the effect of Gin or Ala-Gin on the intestinal epithelial cell and mesenteric lymph node (MLN) cell proliferation in vitro and performance and blood biochemincal index of the 28 d weaned piglets. These provide a scientific data that Gin or Ala-Gin was supplemented in the early-weaned piglets diet as immunity promoter to alleviate early-weaned stress. The main results are followed.1. we have investigated the high performance liquid chromatography method for determining the concentration of free Gin and Ala-Gin in feed additive. Gin was derived with o-phthaldialdehyde (OPA) and diluted with phosphate solution buffer (50mmol/L) containing 6% acetonitrile on reverse phase CIS column. The derivation was detected at excitaion wave 230nm,emission wave 389nm with fluorescence detector. The result was showed as follows: calibration on curves for Gin was linear from 200 moI/L to 6400 mol/L; linear formula is Y=1793.4X+274241,r=0.9999 and CV was 1.39%. Ala-Gin was derived with o-phthaldialdehyde (OPA) and diluted with phosphate solution buffer (12.5mmol/L, pH 4.35) containing 15% acetonitrile on reverse phase CIS column. The derivation was detected at excitaion wave 338nm,emission wave 450nm with fluorescence detector. The result was showed as follows: calibration on curves for Ala-Gin was linear from 6.25 to lOOmmol/L; linear formula is Y=28186X-9905.1,r=0.9997and CV was 5.33%; The recovery was 98%. The concent of free Gin and Ala-Gin in Biocom are 21.1% and 2.65% respectively. We draw a conclusion from the experiment that the method is simple, quick and efficient and is suit to determining the concentration of free Gin and Ala-Gin in feed additive.2. To investigate the effect of Gin and Ala-Gin on the proliferation of piglet intestinal epithelial cells, we use collagenase/dispase digestion. The cells were cultured in DMEM (non-Gin) containing different concentration of Gin or Ala-Gin at 37 for 72h.Cell proliferation was determined by MTT colormetric assay. A large number of viable and pure pig intestinal epithelial cells were separated rapidly by collagenase XI /dispase Idigestion.The rapid proliferation was presented as the concentration of Gin exceeding 0.5mmol/L. The significant promotion was presented at 0.25mmol/L Ala-Gln.The maximal proliferation rate wds appeared at 2mmol/L Ala-Gin. There is no significant difference between the same concentration of Gin and Ala-Gin (p>0.05). These results suggest that Gin and Ala-Gin improve the proliferation of piglet’s intestinal epithelial cells and have similar effects.3. To investigate the effect of Gin and Ala-Gin on MLN cell proliferation of early-weaned piglets we use lymphocyte proliferation response in vitro. The jejunal and ileal MLN were dissected from the piglet weaned at 28 day and lymphocytes were prepared in sterile, the cells were plated in the RPMI 1640 (non-Gin) containing different concentration Gin of Ala-Gin with or without PHA and cultured at 37 for 72h .Cell proliferation was determined by MTT colormetric assay. The maximal stimulation index (SI) was presented as the concentration of Gin is 2 mmol/L and that of Ala-Gin is 1 mmol/L. The SI did not increase with the increase of the concentration of Gin or Ala-Gin. The results suggest that Gin and Ala-Gin are both promote the proliferation of MLN with PHA-stimulated and have sililar effect in the appropriate concentration.4. Forty-two 28 day old weaned piglets (LandranceX Yorkshire, 8.37 ?.13kg BW) were selected and divided into 3 groups with 2 repetitions and included 28 day experiment period to study the effect of Gin and Biocom (a protein source containing Ala-Gin) on the growth performance and blood biochemical index of the weaned piglet. The result showed that daily weight gain, feed intake, feed conversion, diarrhea rate and serum total protein was no difference with Gin o更多还原