摘要
本论文共分4个试验研究了壳聚糖对肉鸡免疫功能的影响及其机制。试验1-试验3利用体内法,采用单因子完全随机试验设计,将1日龄健康艾维茵肉仔鸡(公母各半)240只,随机分为6个日粮处理组,每个处理组5个重复,每个重复组8只鸡,试验期42天。以玉米和豆粕为主要原料配制试验基础日粮,对照组饲喂基础日粮,其他5种试验日粮分别是在基础日粮中添加50、200、500、1000和2000 mg/kg的壳聚糖配制而成。试验1研究了壳聚糖对肉鸡体液免疫功能和细胞免疫功能的影响,进一步验证了壳聚糖对肉鸡免疫功能的促进作用。试验2主要从内分泌角度探讨了壳聚糖对肉鸡免疫功能的影响机制。试验3主要通过研究壳聚糖对肉鸡小肠组织中iNOS mRNA和cPLA_2mRNA相对表达的影响,探讨了壳聚糖对肉鸡免疫功能影响的分子机理。试验4利用体外细胞培养法,研究了不同浓度壳聚糖(0,40,80,160和320μg/ml)对肉鸡腹腔巨噬细胞分泌功能及iNOS mRNA和cPLA2mRNA相对表达的影响,从细胞水平和分子水平进一步探讨了壳聚糖对肉鸡免疫功能的影响机制。
在本研究的试验条件下初步得出以下结果:
①日粮中添加壳聚糖可提高肉鸡的体液免疫功能与细胞免疫功能。壳聚糖添加量为500-1000 mg/kg时,新城疫抗体滴度、T淋巴细胞亚群含量、血清免疫球蛋白浓度均呈不同程度增加,肉鸡免疫功能较强;壳聚糖添加量为2000mg/kg时,肉鸡免疫功能呈降低趋势。
②日粮壳聚糖对肉鸡血清免疫细胞因子IL-1、IL-2和相关激素的影响呈显著二次曲线剂量依赖关系(P<0.05)。壳聚糖的添加水平为500-1000 mg/kg时,血清IL-1、IL-2及TNF-α浓度较高,ACTH及COR浓度较低。当日粮壳聚糖添加量提高到2000 mg/kg时,ACTH及COR浓度升高,IL-1、IL-2、TNF-α浓度均呈不同程度的降低趋势。提示壳聚糖对肉鸡免疫功能的影响与免疫细胞因子和相关激素的水平发生改变有关。
③利用体内法从分子水平研究了日粮壳聚糖对肉鸡免疫功能的影响机制。研究表明:日粮壳聚糖可提高血清NO含量及iNOS活性,增强小肠iNOS mRNA表达,血清NO含量、iNOS活性与小肠iNOS mRNA表达均随壳聚糖添加量的增加而增加,并呈显著的二次剂量依赖关系(P<0.05);其中,以添加500-1000 mg/kg时较高,添加2000 mg/kg时则有降低趋势。日粮中添加壳聚糖对肉鸡血清PLA2、AA和PGE_2浓度的增加及小肠cPLA_2 mRNA表达均有显著的促进效果。随壳聚糖添加量的增加,上述各项指标均呈显著的二次曲线增加(P<0.05);其中以添加500-1000 mg/kg时效果较好,但添加2000mg/kg时促进效果呈降低趋势。
④体外法研究结果表明:壳聚糖可显著增强肉鸡腹腔巨噬细胞iNOS mRNA表达(P<0.05),对NO及iNOS的分泌均有一定促进效果;壳聚糖可显著促进腹腔巨噬细胞cPLA_2 mRNA表达与cPLA_2活性(P<0.05),对PLA_2、AA、PGE_2及LTB_4的分泌均有一定促进效果。提示壳聚糖可促进肉鸡巨噬细胞的分泌功能。
⑤以上结果提示,壳聚糖影响肉鸡免疫功能的分子机制可能是:壳聚糖通过诱导iNOS基因的表达,增加iNOS的合成,调节iNOS的活性,促进NO分泌,进而调节肉鸡的免疫功能;壳聚糖通过调控肉鸡cPLA_2 mRNA表达与cPLA_2活性,促进cPLA_2分解膜磷脂产生AA,进而调节肉鸡的免疫功能。
⑥综合多项试验结果初步得出,壳聚糖刺激肉鸡免疫功能的日粮适宜添加量为500-1000 mg/kg。'
The present study was conducted to investigate the effects of chitosan on immune functions in broiler chickens and the underlying molecular mechanisms. The first three experiments were conducted by using the experimental technique in vivo. A total of 240 one-day-old Arbor Acre mixed-sex broiler chickens were randomly allotted to six dietary treatments with five replicates in each treatment, and with eight chickens in each replicate. The basal diet was mainly composed of corn and soybean. The broiler chickens in the six treatments were fed with the basal diets supplemented with 0 (control), 50, 200, 500, 1000 or 2000 mg/kg chitosan in a single factorial arrangement. The trial lasted for 42 days. The objective of the experiment 1 was to elucidate the effects of chitosan on humoral immune functions and cellular immune functions, and further to demonstrate the promotion of chitosan on immune functions of broiler chicks. The experiment 2 was conducted to investigate the effects of chitosan on endocrine secretion in broiler chicks. The experiment 3 was conducted to investigate the effects of chitosan on relative expression of iNOS mRNA and cPLA_2 mRNA in small intestine, and to elucidate the molecular mechanisms for chitosan to affect immune functions. The experiment 4 was conducted in vitro to demonstrate the effects of different levels of chitosan(0, 40, 80, 160 and 320μg/ml) on secretion functions and relative expression of iNOS mRNA and cPLA_2 mRNA of peritoneal macrophage, and further to investigate the molecular mechanisms by which chitosan modulated immune functions .
Under the present experimental conditions, the results showed as follows:
①The dietary chitosan improved humoral immune functions and cellular immune functions in broiler chicks in a dose-dependent relationship. The supplementation of 500 - 1000 mg/kg chitosan improved the New castle disease antibody titer, T cell group contents and the concentrations of IgG, IgM and IgA, and then strengthen the immune functions of broiler chicks. However, the addition of 2000 mg/kg chitosan to diet was less effective.
②Chitosan influenced concentrations of immunocyte factors(IL-1 and IL-2) and correlated hormone in serum of broilers in a quadratic dose-dependent manner(P<0.05). The supplementation of 500 - 1000 mg/kg chitosan improved the concentrations of IL-1, IL-2 and TNF-α, and suppressed the ACTH and COR contents. However, the addition of 2000 mg/kg chitosan to diet was less effective. This suggested that the effects of chitosan on immune functions was related to the levels of immunocyte factors and correlated hormones in serum of broiler chicks.
③The experiment in vivo was conducted to study the molecular mechanisms for chitosan to modulate immune functions. The results showed that dietary chitosan supplementation enhanced NO content and iNOS activity in serum as well as iNOS mRNA relative expression in small intestine of broiler chickens in a quadratic dose-dependent manner(P<0.05), and the supplementation of 500-1000 mg/kg chitosan in the diet showed the better effects. The supplementation of 500-1000 mg/kg chitosan improved the concentrations of PLA_2,AA and PGE_2 in serum as well as cPLA_2 mRNA relative expression in small intestine of broiler chickens in a quadratic dose-dependent manner(P<0.05). But positive effects tended to be suppressed when the addition of chitosan in diet was increased to 2000 mg/kg.
④The experiment in vitro showed that chitosan could enhanced iNOS mRNA relative expression of peritoneal macrophage in broiler chicks(P<0.05), and promote the secretion of NO and iNOS in peritoneal macrophage. The addition of chitosan would also promote cPLA_2 mRNA relative expression and cPLA_2 activity of peritoneal macrophage(P<0.05), and increase the concentrations of PLA_2, AA, PGE_2 and LTB_4 in culture liquid. This suggested that the chitosan would promote the secretion functions of peritoneal macrophage in broiler chicks.
⑤The present results implied that the mechanisms for chitosan to affect immune functions of broiler chickens were probably that chitosan activated expression of iNOS mRNA and improved iNOS activity and NO secretion; or that chitosan activated cPLA_2 mRNA expression and cPLA_2 activity, and promoted AA production which is synthesized from membrane phospholipid by cPLA_2.
⑥In conclusion, the broiler chicks fed a diet containing 500-1000 mg/kg chitosan had higher immune functions compared with birds given a control diet.
引文
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