【作者】 袁中彪；

【导师】 陈代文；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2004， 博士

【摘要】 CCK是由小肠近端Ⅰ细胞和中枢神经细胞分泌的一种脑肠肽，具有刺激胰酶分泌和胆囊收缩、降低胃排空、调节小肠运动、促进胰岛中胰岛素和生长抑素释放的作用，同时CCK也是动物体内的一种饱感因子，参与调节动物的采食。动物体内CCK的分子形式较多，其中CCK₈具有CCK的全部生物学功能。本研究通过考察CCK₈主动免疫对猪生产性能、营养物质消化率、内分泌激素、胰腺的发育、胰酶活性及基因表达的影响来认识CCK主动免疫的营养生理效应及其可能机制。研究共包括二个试验。 试验一 CCK₈主动免疫对猪的生产性能及CCK基因表达的影响 试验选用体重为27.55±3.15kg的杜洛克×长白猪×约克夏（DLY）三元杂交阉猪10头，随机分为2组，每组5个重复，每个重复1头猪。试验组用370μgCCK₈与人血清白蛋白（hSA）的交联物（CCK₈-hSA）分别在试验第1、29、43和57天进行免疫，对照组用等量人血清白蛋白免疫。试验期为77天。整个试验期间，试猪自由采食和饮水。在试验的第71天，从前腔静脉采血，测定血清CCK₈的含量和CCK抗体滴度。试验结束时屠宰所有试验猪，迅速取出垂体和空肠前段，经液氮速冻后，-70℃下保存，测定CCKmRNA的含量。 结果显示： ①370μgCCK₈主动免疫后，与对照组相比，试验猪全期日增重和采食量分别提高了14.29％（P=0.23）和9.34％（P=0.34）； ②370μgCCK₈主动免疫后，试验猪血清中CCK₈含量与对照组相比降低了19.77％（P＜0.15），抗体滴度则升高了67.63％（P＜0.01）； ③试验猪的空肠和垂体中CCKmRNA的含量显著低于对照组（P＜0.05）。 结果表明，370μgCCK₈主动免疫能提高猪血清中CCK抗体滴度、抑制猪空肠和垂体中CCK基因的表达，从而降低血清中CCK₈的浓度，趋于提高猪的采食量和生长速度。 试验二 不同水平CCK₈主动免疫对猪生产性能、胰腺功能、CCK及受体基因表达的影响试验选用体重为27.66±1.71kg的DLY三元杂交阉猪15头，随机分为3个处理，每个处理5个重复，每个重复1头猪。处理1、2和3的猪分别用由0μg、250μg和500μgCCK₈与人血清白蛋白交联后形成的抗原进行免疫，免疫的时间分别为试验第1、29、43和59天。试验期为74天。整个试验期间，试验猪自由采食和饮水。在试验的第1、15、29、胆囊收缩素主动免疫对猪的营养生理效应及其机制的研究书、59和73天，从前腔静脉采血，测定血清中CCK抗体滴度，或测定血清中CCKS、胰岛素和瘦素的含量。试验结束时屠宰所有试验猪，迅速取出胰腺，纵向分为两部分，一部分用4%的甲醛溶液固定，用于胰腺的组织学检查，另一部分胰腺与取出垂体和空肠样品经液氮速冻后，于一70℃保存，侧定胰酶的活性及CCKmRNA和CCK一A受体爪RNA的含量。 结果显示: ①ccK主动免疫对猪生产性能的影响存在着剂量效应关系，250雌CcKS主动免疫能显著提高猪的全期采食量和日增重，与对照组相比分别提高了H.76%（P<0.05）和14.62%（P<0 .05）:而500林gCCK主动免疫有降低猪采食量和日增重的趋势; ②ccK主动免疫显著降低血清中胰岛素、瘦素和CcK。的浓度。250陀CC城主动免疫后，猪第73天的血清中胰岛素、瘦素和cCK:的含量与o林gCC丸组相比分别降低了21.39%（p<0·05）·30.58%（p<0.05）和29.00%（p<0.05）:500协gCCK:主动免疫使其分别降低了23.57%（P<0.05）、29.48%（P<0.05）和37.72%（P<0.05）。 ③CcK主动免疫能显著提高胰淀粉酶和胰脂肪酶的比活力‘250协g和500陀CCKs主动免疫与对照相比，胰淀粉酶和胰脂肪酶的比活力分别提高了7.15%（P<0 .05）、1 15.15%（P<0.05）和12.90%（P<0.05）、94.70%（p<0.05）;500卜gCCKs主动免疫显著提高胰蛋白酶的比活力，与对照组和250林gCCKs组相比分别提高了85.97%（p<0.05）和35.97%（P<0.05）。 ④250腿CCKS主动免疫对胰腺组织学结构无显著影响。500件gCCK:主动免疫引起胰腺组织病变，主要表现为胰腺组织空泡变性、细胞增生坏死、胰腺充血、出血以及酶原颗粒减少。 ⑤CCK主动免疫后显著降低猪空肠、垂体和胰腺中CCK基因和CCK一A受体基因的表达。与对照相比，250林gCCK:主动免疫使猪空肠、垂体和胰腺中CCK基因和CCK受体基因的mRNA含量分别降低了37.02%（P<0.05）、30.02%（p<0.05）、22.400，0（P<0.05）和25.300，0（P<0.05）、26.06%（P<0.05）、20.43%（P<0.05）;500协gCeK:主动免疫则使它们分别降低了44 .97%（p<0 .05）、一8.67%（p<0.05）、24.09%（p<0.05）和22.72%（P<0.05）、10.48%（P<0.05）、16.49%（P<0.05）O 结果表明，250协gCCK:主动免疫显著提高猪生产性能，其机制是CCK主动免疫产生的特异性抗体中和了部分内源性CCK，同时通过抑制CCK基因和CCK受体墓因的表达降低了内源CCK的分泌量，减弱了CCK信号传导作用，从而部分解除了CCK对采食的抑制作用。250陀CCKs主动免疫对胰腺发育和养分消化率无显著影响。500阵CCK四川农业大学博士学位论文主动免疫可进一步提高抗体滴度和抑制基因表达，但有降低猪生产性能的趋势。这表明不同程度的CCK:主动免疫对猪生产性能的影响存在差异，免疫过度可能会损伤猪胰腺的健康。 上述两个试验结果表明，CCK主动免疫可?更多还原

【Abstract】 The brain-gut peptides cholecystokinin (CCK) is synthesized by enteroendocrine I cell of the proximal small intestine and by central neurons. The release of CCK from the former is associated with the stimulation of pancreatic enzyme secretion and gallbladder contraction, the inhibition of gastric emptying, the regulation of intestine motion, the acceleration of insulin and somatostation releases from pancreatic islets. Meanwhile, the release of CCK from the latter is a satiety factor, which regulates the feed intake of animal. CCK occurs in various molecular forms in animal, but the cholecystokinin octapeptide (CCK8) has the whole biological function of CCK. To know the nutritional and physiological functions and the underlying mechanisms of active immunization against cholecystokinin in pigs, two experiments were conducted to study the effects of active immunization against cholecystokinin octapeptide on performance, nutrient digestibility, endocrine hormones, pancreatic development, pancreatin activity, pancreatic tissue lesion and the gene expressions of CCK and CCK receptor in pigs.Exp. 1 Effects of Active Immunization against Cholecystokinin octapeptide on Growth Performance and CCK Gene Expression in Pigs.10 DLY pigs with body weight of 27.55 1.71 kg were distributed randomly into 2 treatments with 5 replicates of 1 pig. The treated group was immunized with 370 g CCK8 conjugated to human serum albumin (hSA). Primary inoculation was on day I and booster inoculations were on day 29, 43 and 57. The control group was immunized against hSA by the same protocol. In the whole test, pigs were fed ad libitum. Blood samples were collected on day 71 and assayed for the content of CCKg and antibody titre of CCK. All pigs were slaughtered on day 77. Proximal jejunum and pituitary gland were rapidly removed, freezed in liquid nitrogen and stored under -70 C for the assay of CCKmRNA. The results showed: (1). The average day weight (ADG) was 14.29%(P=0.23) greater and feed intake (FI) was 9.34%(P=0.34) greater for CCK immunization group than the control group. (2). The content of CCK8 in treated group decreasedby 19.77% (P<0.15) and the liter of CCK8 antibody increased by 67.63% (P<0.01) compared with the control group. (3) The levels of CCK gene expressions in jejunum and pituitary of the treated group decreased significantly (P<0.05) compared with the control group. The date indicated that active immunity at the dose of 370 g CCKg had a tendency to increase feed intake and growth rate of pigs, due to the decrease of CCK8 content in serum resulted from the increase of CCK antibody litre in serum and the inhibition of CCK gene expression in proximal jejunum and pituitary gland.Exp.2. Effects of Active Immunization against CCK8 at two Doses on Performance, Pancreatic Functions and Gene Expressions in Pigs.A single factor design was conducted to explore the effects of CCKg active immunization at 2 doses on performance, pancreatic functions and gene expressions in pigs. 15 DLY pigs of 27.66 1.71 kg of body weight were distributed randomly inlo 3 treatments with 5 replicates of 1 pig. The CCK8 level of group 1, 2 and 3 was 0 g, 250 g and 500 g conjugated to hSA respectively. Primary inoculation was done on day 1. Booster inoculations were on day 29, 43 and 59. In the whole test, pigs intook food and drank water at liberty. Blood samples were collected on day 1, 15, 29, 43, 59 and 71 and assayed for antibody litre of CCK in surem, or for the contenl of CCKg, insulin and leptin in serum. All pigs were slaughtered on day 74 and took out the proximal jejunum, pancreas and pituitary gland rapidly. A part of pancreas was fixed with 4% formaldehyde solution for examining pancreatic histomorphology, and the other part of pancreas, jejunum and pituitary gland were frozen in liquid nitrogen, and stored under-70’C for assaying the content of CCK mRNA and CCK-A receptor mRNA. The results showed: (1). It existed the dose response of CCK active immunization on growth performance in pigs. FI was 11.76%(P<0.05) greater and ADG was 14.更多还原