【作者】 韩爱云；

【导师】 张敏红；

【作者基本信息】 中国农业科学院 ， 动物营养与饲料科学， 2010， 博士

【摘要】 本文通过体内、体外试验,系统研究了热应激对肉鸡淋巴细胞钙离子浓度及钙信号转导途径的影响。从分子水平上初步探讨了热应激影响肉鸡免疫功能的机理,为从根本上降低热应激对肉鸡生产的危害提供理论依据。本论文开展了以下三方面的研究:1.热应激对肉鸡淋巴细胞钙离子浓度及免疫功能的影响(1)选择1日龄AA肉公鸡80只,随机分为2个组,每组4个重复,每个重复10只鸡。49日龄时,两个处理组肉鸡分别在温度为22℃和35℃的人工气候舱内处理3 h,研究急性热应激对肉鸡脾淋巴细胞钙离子浓度及细胞免疫功能的影响。结果表明:急性热应激(3 h)可提高肉鸡脾脏淋巴细胞内钙离子浓度及IL-2水平,促进淋巴细胞周期由G1期向S期转变,提高Con A诱导的T淋巴细胞转化率,提高CD4+与CD8+的比值,说明急性热应激可激活肉鸡的细胞免疫机能。(2)选择1日龄AA肉公鸡150只,随机分为3个组,每个组5个重复,每个重复10只。35日龄时,适温组和配对组昼夜维持恒温22℃,高温组温度为27℃—35℃—27℃日循环变化,试验持续14 d,研究持续热应激对肉鸡脾淋巴细胞钙离子浓度及细胞免疫功能的影响。结果表明:日循环高温可使肉鸡淋巴细胞内钙离子浓度升高。高温热应激处理7 d,可显著提高细胞IL-2分泌水平,显著抑制Con A诱导的T淋巴细胞转化率;高温热应激处理14 d,可显著降低淋巴细胞处于S期的比例,提高淋巴细胞处于G1期的比例,对IL-2分泌及T淋巴细胞转化率无显著影响。日循环高温对上述指标的影响与高温降低肉鸡采食量无关。2.高温对体外培养淋巴细胞钙信号转导途径的影响(1)体外分离培养肉鸡脾脏淋巴细胞,研究了高温(44℃)以及高温条件下添加钙通道阻断剂对淋巴细胞内钙离子浓度的影响。结果表明:高温(44℃)可直接影响肉鸡淋巴细胞内钙离子浓度,并且高温使细胞内钙离子浓度升高主要由于细胞内钙库释放起作用引起的;肉鸡淋巴细胞在44℃条件下培养15 min后,撤除高温继续培养1.5 h,细胞内钙离子浓度可以一定程度上得到恢复,但并不能恢复到起始水平;高温导致的肉鸡脾淋巴细胞[Ca2+]i升高能被IP3受体拮抗剂2-APB抑制,而不受钙释放激活通道(CRAC)抑制剂SKF-96365和Ryanodine受体(RYR)阻断剂钌红的影响,说明高温升高肉鸡淋巴细胞钙离子浓度,主要由于内质网上的IP3受体介导的钙释放通道起作用引起的。(2)体外培养肉鸡脾脏淋巴细胞,研究了细胞培养液中添加钙离子载体A23187和钙离子螯合剂BAPTA-AM对细胞IL-2基因表达及IL-2蛋白分泌的影响。结果表明:钙离子载体A23187使肉鸡淋巴细胞内钙离子浓度升高时,IL-2mRNA基因表达及IL-2蛋白分泌显著升高;钙离子螯合剂BAPTA-AM使肉鸡淋巴细胞钙离子浓度显著降低时,IL-2mRNA表达及IL-2蛋白分泌显著降低。说明淋巴细胞钙离子浓度变化对IL-2mRNA基因表达和蛋白分泌具有直接关系。(3)体外培养肉鸡脾脏淋巴细胞,研究了高温(44℃)条件下细胞培养液中添加受体抑制剂对淋巴细胞内钙离子及IL-2基因表达的影响。结果表明:高温可使肉鸡淋巴细胞[Ca2+]i及IL-2mRNA表达量升高,这一作用能被磷脂酶C(PLC)抑制剂U73122,IP3受体抑制剂2-APB及钙调素激酶(CaM-PK)抑制剂W7所抑制,说明IP3/Ca2+信号转导通路在高温导致细胞[Ca2+]i及IL-2 mRNA表达量升高的过程中起重要调节作用。3.吡啶甲酸铬对热应激肉鸡淋巴细胞钙离子浓度及免疫功能的影响(1)选择1日龄AA肉公鸡300只,随机分为5个组(Ⅰ–Ⅴ),每组5个重复,每个重复12只鸡。在15日龄时,试验Ⅰ–Ⅳ组肉鸡分别饲喂含0,400,800,1200μg /kg Cr的日粮2周后,再进行日循环高温(27℃—35℃—27℃)处理2周,试验Ⅴ组肉鸡则维持恒温22℃作为适温对照组。研究吡啶甲酸铬对热应激肉鸡脾淋巴细胞钙离子浓度及免疫功能的影响。结果表明:日粮补铬(800-1200μg/kg)可显著降低热应激肉鸡的血浆中胰岛素水平,抑制淋巴细胞内钙离子浓度和IL-2浓度升高,提高淋巴细胞转化率。表明高温条件下,日粮添加CrPic可缓解热应激对肉鸡细胞免疫功能造成的负面影响。(2)体外培养肉鸡脾淋巴细胞,研究了高温条件下在细胞培养液中分别添加胰岛素、吡啶甲酸铬、胰岛素+吡啶甲酸铬对肉鸡脾淋巴细胞内钙稳态的影响。结果表明:同时添加CrPic和胰岛素可以明显加快钙离子的恢复速度,说明CrPic可通过增强胰岛素功能,进而调节肉鸡淋巴细胞钙离子浓度。综合上述研究结果,可以得出,热应激可通过影响肉鸡淋巴细胞钙离子浓度及IP3/Ca2+信号转导途径,进而影响肉鸡免疫功能;日粮补充CrPic可通过增强胰岛素功能调节肉鸡淋巴细胞钙离子浓度,进而缓解热应激对肉鸡免疫功能的负面影响。更多还原

【Abstract】 In vivo and in vitro experiments were conducted to investigate the effects of heat stress on calcium signal transduction of lymphocytes from broilers chickens. We explored the mechanism of heat stress modulate immune response at molecular level, in order to provide a basis for research into the nutritional strategies of reducing the negative effects of heat stress on broilers. This thesis was devoted to makes the research from three respects:1. Effect of heat stress on Ca2+ concentration of lymphocyte and immune response from broiler chickens(1) We investigated the effect of acute heat stress on Ca2+ concentration and cellular immunity in splenic lymphocytes from broiler chickens. Eighty 1-d-old Arbor Acres male broiler chickens were randomly allotted to 2 treatments, each treatment represented by 4 replicates of 10 birds each. At 49 d, the chickens of 2 treatments were housed in two controlled environment chambers, which temperature were kept 22℃and 35℃for 3 h, respectively. The result shows that acute heat stress caused a significant increase the [Ca2+]i and the secretion of interleukin-2 (IL-2) in lymphocytes,enhanced the ConA-stimulated lymphocyte proliferation significantly, increased the ratio of CD4+ to CD8+ in lymphocytes, and promoted the transition of activated T cells from the G1 to the S phase . These results suggest that acute heat stress could enhance immune function.(2) The experiment was conducted to investigate the effect of chronic heat stress on Ca2+ concentration and cellular immunity in splenic lymphocytes from broiler chickens. One hundred and fifty 1-d-old Arbor Acres male broiler chickens were randomly allotted to 3 treatments, each treatment represented by 5 replicates of 10 birds each. At 35 d, the normal thermal groups (NT) and paired-feeding group (PF) were housed in controlled environment chambers at 22℃.The high-temperature group (HT) was housed in a controlled environment chamber with daily cyclic high temperature (27℃—35℃—27℃), the experiment last two weeks. The result shows that chronic heat stress increased the [Ca2+]i of lymphocyte. After 7-d heat exposure,the secretion of IL-2 by splenic lymphocytes was increased significantly, and ConA-stimulated lymphocyte proliferation was suppressed significantly. The 14-d heat stress decrease the S phase proportion, and increase the G1 phase proportion of lymphocyte. In addition, the indicators mentioned above were not associated with feed intake of heat stress broiler chickens.2.Effect of high temperature on calcium signal transduction of lymphocyte from broiler chickens(1) We investigated effect of high temperature (44℃) and calcium channel blockers on the [Ca2+]i of lymphocyte . The rusult shows that expousure to high temperature induced an increase in the [Ca2+]i of lymphocyte. And the increase in [Ca2+]i was dependent mainly on the intracellular Ca2+ stores; Incubation at 40℃for 1.5 h, following heating for 15min at 44℃, resulted in partial recovery of the lymphocytes [Ca2+]i, but it did not return to the basal level; The increase in [Ca2+]i was induced by high temperature could be inhibited by Inositol 1,4,5-trisphosphate receptor (IP3R) blocker 2-APB, but not be influenced by calcium release activated channel blocker SKF-96365 and ryanodine receptor antagonists ruthenium red. The rusults suggest that the heat-induced increase in [Ca2+]i was dependent mainly on the IP3R-mediated calcium release channel on the endoplasmic reticulum(2) It was investigated that the effect of the Ca2+ ionophore and the membrane-permeant Ca2+ chelator on the IL-2 mRNA expression and IL-2 protein secretion. The rusult shows that when the Ca2+ ionophore A23187 increased the lymphocytes [Ca2+]i, the IL-2mRNA expression and IL-2 protein secretion of lymphocyte were increased; when the membrane-permeant Ca2+ chelator BAPTA-AM decreased the lymphocytes [Ca2+]i, the IL-2mRNA expression and IL-2 protein secretion of lymphocyte were decreased. The results suggest that the lymphocytes [Ca2+]i directly related with IL-2 mRNA expression and IL-2 protein secretion.(3) We investigated effect of the receptor blockers under high temperature on [Ca2+]i and the IL-2 mRNA expression in lymphocyte. The result shows that high temperature induce an increase in [Ca2+]i and the IL-2 mRNA expression of lymphocytes, and the increasement could be inhibited by the phosphatidylinositol-specific phospholipase C inhibitor U73122, IP3 receptor blocker 2-APB and CaM-dependent protein kinase (CaM-PK) antagonist W7. It suggests that the IP3/Ca2+ signal transduction plays an important role in the process of Ca2+ to IL-2 mRNA expression.3. Effect of Chromium Picolinate on Ca2+ concentration of lymphocyte and immune response from heat stress broiler chickens(1) In order to investigate the effect of chromium picolinate (CrPic) on Ca2+ concentration of lymphocyte and immune response from heat stress broiler chickens, three hundred 1-d-old Arbor Acres male broiler chickens were randomly allotted to 5 treatments (Ⅰ-Ⅴ), each treatment represented by 5 replicates of 12 birds each. At 15 d,the broilers in treatmentⅠ-Ⅳwere pre-fed 0, 400,800,1200μg /kg Cr of diet for 2 weeks, then they were housed in 3 controlled environment chambers with daily high cyclic temperature (27℃—35℃—27℃) for 14 d, the broilers in treatmentⅤwere kept in 22℃as the control group. The result shows that supplemental chromium (800-1200μg/kg Cr) decreased serum insulin concentration, supressed the increasement of the lymphocytes [Ca2+]i and IL-2 concentration, increased the lymphocyte proliferation. The results suggets that the CrPic could alleviate the negative effect on the cellular immune response of heat stress broiler chickens.(2) In vitro experiments were conducted to investigate the effects insulin, CrPic and insulin+CrPic on calcium homostasis of lymphocyte.The result shows that insulin+CrPic treatment caused more significant increase in [Ca2+]i recovery rate than CrPic or insulin treatment. It suggest that the CrPic could modulate the lymphocytes [Ca2+]i in potentiating insulin action.更多还原