GnRH主动免疫对雄性藏绵羊甲状腺功能的影响

发布时间：2018-01-30 08:50

本文关键词： GnRH 主动免疫雄性藏绵羊甲状腺功能　出处：《四川农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：20世纪60年代末期,激素免疫学的产生为动物生殖调控提供了新思路。用外源性GnRH主动免疫动物,会刺激动物机体产生大量GnRH特异性抗体,引起生殖内分泌系统平衡的失调,改变下丘脑-垂体-性腺轴间的正常反馈调节关系,以减少生殖激素的合成和分泌及终止配子的发生,从而达到免疫去势的目的。GnRH主动免疫可避免外科阉割引起的动物高度应激、外科伤口容易感染,增加动物发病率甚至导致动物死亡等诸多不足,已成为最有望替代外科阉割的安全友好方法。本试验用蛋白结构修饰后的GnRH并列体二聚物(G6k-GnRH-Tandem-Dimer)与卵清蛋白偶联,以Specol为佐剂,主动免疫雄性藏绵羊,以研究其对雄性藏绵羊甲状腺功能的影响。试验选取24周龄、体重17-19 Kg的雄性藏绵羊30只,随机分为三组,每组10只。其中一组于25周龄时各只绵羊颈部皮下注射2 mLGnRH并列体二聚物卵清蛋白复合物(TDK-OVA)的Specol乳化剂(含TDK 100 μg),8周后加强免疫一次,注射剂量及方法同初次免疫,为免疫去势组。一组在试验开始前一周进行外科手术去势,为手术去势组。剩余一组不做任何处理,为完整对照组。初次免疫当天记为0 wpv。初免当天及初免后第4、8、12、16 wpv(即每4周1次,直至屠宰)颈静脉采集血样,酶联免疫法(ELISA)测定血清抗体滴度及LH、FSH、T、TSH、T3、T4含量变化。加强免疫后8周(即16 wpv),屠宰所有试验绵羊,分离下丘脑、垂体及两侧睾丸。Real-Time quantitative PCR分析下丘脑、垂体生殖相关基因(GnRH-R、TSH-β、TRH、TSH-R、MT1、MT2、DIO2、DIO3) mRNA表达变化。结果显示,免疫去势组产生了良好的抗体反应,初免后机体产生抗体,加免后血清抗GnRH抗体滴度快速升高,由加免当天的18.67%升高到12周(12 wpv)的最高峰(55.62%),后保持在较高水平,直至屠宰(48.85%)。与完整对照组相比,免疫去势组血清T含量显著下降(p0.05),屠宰时仍维持在较低水平(4.27ng/mL).与完整对照组相比,手术去势组和免疫去势组血清T3和T4无显著下降(p0.05),GnRH主动免疫显著下调绵羊垂体GnRH-R、TSH-β及下丘脑TRH、TSH-R、DI02mRNA表达水平(p0.05),而手术去势组绵羊垂体GnRH-R mRNA表达水平显著高于免疫去势组和完整对照组(p0.05)。屠宰时,完整对照组睾丸平均重量为51.71±7.58 g,平均体积为49.04±6.66 cm3,免疫去势组睾丸平均重量为41.58±5.77 g,平均体积为39.91±6.02 cm3。与完整对照组相比,免疫去势组藏绵羊睾丸发生萎缩,其重量及体积均下降到对照组睾丸的80%(p0.05)上述研究结果表明：GnRH主动免疫尽管使下丘脑TRH、TSH-R、DI02、DI03及垂体TSH-β mRNA表达水平下调,但对血清中TSH、T3及T4浓度无明显影响。总之,GnRH主动免疫对雄性藏绵羊甲状腺功能无不利影响。
[Abstract]:In the end of 1960s, the production of hormone immunology provided a new idea for animal reproductive regulation. Active immunization with exogenous GnRH can stimulate animal body to produce a large number of GnRH specific antibodies. It causes the imbalance of reproductive endocrine system and changes the normal feedback regulation relationship between hypothalamus-pituitary-gonad axis in order to reduce the synthesis and secretion of reproductive hormones and the termination of gametes. In order to achieve the purpose of immune castration. GnRH active immunization can avoid high stress caused by surgical castration, surgical wounds are prone to infection, increase animal morbidity and even lead to animal death and many other deficiencies. It has become the most promising safe and friendly method for surgical castration. In this study, the GnRH paratactic dimer (G6k-GnRH-Tandem-Dimer) was modified with protein structure. Coupled with ovalbumin. Male Tibetan sheep were immunized with Specol as adjuvant to study its effect on thyroid function of male Tibetan sheep. They were randomly divided into three groups. At 25 weeks of age, one sheep was subcutaneously injected with Specol emulsifier (TDK-OVA) for 2 mLGnRH paratactic dimer ovalbumin complex (TDK-OVA). TDK 100 渭 g). Once after 8 weeks, the dose and method of immunization were the same as the primary immunization group. One group underwent surgical castration one week before the trial, and the remaining group did not do any treatment. The first immunization day was 0 wpv.The blood samples were collected from jugular vein on the day of the first immunization and after the first immunization at 81216 wpv (that is once every 4 weeks until slaughtering). Elisa was used to determine the titer of serum antibody and the content of TSHT3T4 in serum. All the tested sheep were slaughtered 8 weeks after immunization. The hypothalamus, pituitary and bilateral testis. Real-time quantitative PCR were isolated to analyze the hypothalamus and pituitary gene-related gene GnRH-RnTSH- 尾. The results showed that the immune ovariectomized group had a good antibody response, and the body produced antibody after the first immunization. The titer of serum anti GnRH antibody increased rapidly from 18.67% on the day of administration to the highest peak of 55.62% at 12 weeks and remained at a higher level. Compared with the intact control group, the serum T content in the ovariectomized group decreased significantly (p 0.05). The serum T3 and T4 levels in ovariectomized group and immune castrated group were not significantly decreased compared with the control group (P 0.05). The expression of GnRH-RnTSH- 尾 and TRH-TSH-RnDI02 mRNA in hypothalamus was significantly down-regulated by GnRH (p0.05). However, the expression of GnRH-R mRNA in the pituitary gland of the ovariectomized sheep was significantly higher than that of the ovariectomized group and the intact control group. The average weight and volume of testis were 51.71 卤7.58 g and 49.04 卤6.66 cm ~ (3) in the intact control group and 41.58 卤5.77 g in the castration group respectively. The average volume was 39.91 卤6.02 cm ~ (3). Compared with the control group, the testis of Tibetan sheep in the immune castrated group were atrophied. The weight and volume decreased to 80% of the testis of the control group (p 0.05). The results showed that the hypothalamus TRH TSH-RH DI02 was induced by the active immunization of 1% GnRH. The expression of TSH- 尾 mRNA in DI03 and pituitary gland was down-regulated, but there was no significant effect on serum TSH-T _ 3 and T _ 4 levels. GnRH active immunization had no adverse effect on thyroid function of male Tibetan sheep.
【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S826

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