【作者】 李云；

【导师】 周杰；

【作者基本信息】 安徽农业大学 ， 基础兽医学， 2013， 硕士

【摘要】 皖南花猪是安徽省黄山市的地方优良猪种之一,具有繁殖率高、产仔多、肉质好等优良特性。脂联素是主要由脂肪组织细胞分泌的一种生物活性物质,与动物的生殖性能有着十分密切的关系。本试验通过测定分析皖南花猪血清中脂联素(Adiponectin, Adp), GnRH和GnIH含量；下丘脑组织中脂联素、脂联素受体(Adiponectin receptor, AdpR)、GnRH和GnIH基因表达量的发育性变化规律及相关性,进而探讨Adp与皖南花猪生殖作用的联系。通过重组脂联素(recombinant adiponectin, rAdp)对小鼠下丘脑GnRH细胞和GnIH细胞的GnRH和GnIH基因表达的影响,探索Adp对下丘脑生殖激素合成的影响。1体内试验：脂联素与GnRH和GnIH在皖南花猪下丘脑组织中的发育性变化及其相关性研究随机选取0d、30d、45d、90d、180d的皖南花猪各10头(公、母各5头)进行试验。ELISA法测定血清中Adp、GnRH和GnIH激素的水平；用FQ-PCR法检测下丘脑组织中Adp、AdpR1、AdpR2、GnRH和GnIHmRNA的表达。结果显示：(1)皖南花猪血清脂联素与GnRH和GnIH水平均有极显著的发育性变化,在30d时脂联素极显著低于其他日龄,而GnRH和GnIH显著或极显著高于其他日龄(P<0.05或P<0.01)。(2)皖南花猪下丘脑组织脂联素mRNA表达量很低,AdpR1、AdpR2mRNA在不同发育阶段的表达量相对稳定(P>0.05),AdpR1mRNA表达量在各个日龄均显著高于AdpR2(P<0.05)。皖南花猪下丘脑组织GnRH、GnIHmRNA发育性变化总体呈下降趋势,GnRH、GnIHmRNA在下丘脑中的发育性变化总体呈下降趋势,GnRH和GnIHmRNA表达量分别在45d和90d最低。刚出生的母猪下丘脑GnRHmRNA表达量显著高于其他日龄(P<0.05)(3)性别间比较发现,公猪AdpR1mRNA在Od, AdpR2和GnRH mRNA在30d显著高于母猪(P<0.05)(4)相关性分析结果：皖南花猪下丘脑组织中AdpR1与GnRH表达量显著负相关(P<0.05)：血清中Adp与GnRH水平显著负相关(P<0.05),母猪Adp与GnRH显著负相关(P<0.05)。GnRH与GnIH含量在公、母猪和总体上均极显著正相关(P<0.01)。以上结果提示：在皖南花猪发育过程中脂联素可能通过内分泌并由AdpR1介导从而抑制GnRH的分泌,而这种抑制主要表现在母猪中。2体外试验：脂联素对小鼠下丘脑细胞生殖激素基因表达的影响选取怀孕18d的小鼠在无菌条件下取胎鼠下丘脑组织分离培养下丘脑细胞。培养5d后用重组脂联素处理,试验设置4h和24h两个时间点,每个时间点设置2μg/ml和10μg/ml两个浓度。FQ-PCR方法检测下丘脑细胞中AdpR1、AdpR2、GnRH和GnIH mRNA表达量。结果显示：(1)重组脂联素对脂联素受体基因表达的影响：2μg/ml的重组脂联素处理4h后AdpR2mRNA表达量极显著下降(P<0.01),其余均无显著变化。此外,下丘脑细胞中AdpR1mRNA表达量显著高于AdpR2的mRNA表达量(P<0.01)：(2)重组脂联素对下丘脑GnRH细胞和GnIH细胞瓶中GnRH和GnIH基因表达的影响：下丘脑细胞加入重组脂联素24h后,10μg/ml组的GnRH和GnIHmRNA的表达量受到极显著的抑制(P<0.01)。加入4h后,两个浓度组对GnRH和GnIHmRNA表达量均无显著的影响(P>0.05)。以上结果提示：脂联素可能由AdpR1介导抑制GnRH和GnIH基因的表达,且脂联素对GnRH和GnIH的调节有一定的剂量效应和时间效应。更多还原

【Abstract】 Wannanhua pig is the local breed in Huangshan, China, which has the characteristics of early maturity, high reproduction rate, disease resistance and high-quality pork. Adiponectin (Adp) is one of the most abundant adipokines mainly secreted by adipose tissues and had closely relationship with the reproduction particularly. In order to understand the interrelationship between the Adp and the hypothalamo-pituitary-gonadal (HPG) axis in Wannanhua pigs, we measured serum levels of Adp and reproductive related hormones and the mRNA expression of these genes. In addition, the effects of recombinant adiponectin (rAdp) on cultured hypothalamic cells of mice were also studied in this paper in order to discover the influence of adiponectin on hypothalamus reproductive endocrine.1The developmental patterns and correlation of Adiponectin, Adiponectin receptors, GnRH and GnⅢ in the hypothalamus of Wannanhua pigsFive Wannanhua boars and five Wannanhua gilts at birth,30,45,90and180days of age were selected. The concentration of serum adiponectin, GnRH and GnIH at different days of age were detected by ELISA, while the developmental patterns and gender differences of Adp, AdpR1, AdpR2, GnRH and GnIH/mRNA expression in hypothalamus were detected by Real-time fluorescent quantitative PCR(FQ-PCR). The results showed as follows:(1) The Adp, GnRH and GnIH level in serum were significantly changed with age increasing. At30days of age, the Adp content was lower than other days (P<0.01) while the GnRH and GnIH content were higher than other days (P<0.05or P<0.01).(2) In hypothalamus, the mRNA expression of Adp was very low, while AdpR1, AdpR2mRNA had relatively steady expression levels at different stages of growth (P>0.05) and the mRNA expression of AdpR1were significantly higher than AdpR2at any days of age. The GnRH and GnIH mRNA expression in hypothalamus declined generally with age increasing, which at the bottom at45d or90d respectively. While the GnRH mRNA expression in hypothalamus of the gilts at birth was significantly higher than other days (P<0.05),(3) With the comparison between different genders, we can find that AdpR1mRNA expression at birth and AdpR2and GnRH mRNA expression at30days of boars were significantly higher than those of gilts (P<0.05).(4) The analysis of correlation: There was significant negative correlation between the AdpR1and GnRH mRNA in hypothalamus (P<0.05). The Adp and GnRH level in serum were inversely correlated (P<0.05) in gilts; The serum GnRH and GnIH content were significant correlation (P<0.01) both in boars and in gilts.The results indicated that the adiponectin may suppress production of GnRH in hypothalamus by endocrine pathway and mediated by AdpRl during the development of Wannanhua pigs and the suppression was mainly found in gilts.2Effect of rAdp on GnRH and GnIH gene expressions in cultured hypothalamic cells of mice.Hypothalamic cells were isolated abacterially from hypothalamus at18d fetal mice. Hypothalamic cells were cultured after some treatment. Cells treated with rAdp five days after subculture. FQ-PCR methods were used to detect the expression of AdpRl, AdpR2, GnRH and GniH mRNA in cells after4h and24h rAdp treatment. The results showed as follows:(1) Effect of rAdp on AdpR mRNA expression in cultured hypothalamic cells of mice:AdpR2mRNA levels decreased significantly after4h treatment of2μg/ml rAdp, while others gene expressions had no significant difference with rAdp treatment in hypothalamic cells. In addition, AdpRl mRNA levels were significantly higher than AdpR2mRNA expression (P<0.01).(2) Effect of rAdp on GnRH and GnIH mRNA expression in cultured hypothalamic cells of mice:In hypothalamic cells, GnRH and GnIH mRNA levels decreased significantly after24h treatment of10μg/ml rAdp (P<0.01). The rAdp had no effects on the mRNA expression of GnRHand GnIH after4h treatment.In hypothalamic cells, rAdp may inhibit the GnRHand GnIH mRNA expression, and its action is mainly mediated by AdpR1. Regulation of adiponectin on GnRH and GnIH has a certain dose-effect and time-effect.更多还原