【作者】 吕东媛；

【导师】 曹贵方；

【作者基本信息】 内蒙古农业大学 ， 基础兽医学， 2008， 博士

【摘要】 Ghrelin是1999年发现的生长激素促分泌素受体(GHSR)的内源性配体,其最主要功能是通过与GHSR结合刺激生长激素的释放。最近发现生殖器官中有Ghrelin分布,提示其与生殖周期的调节和妊娠有重要关系。雌、孕激素对雌性生殖系统具有重要的调节作用,二者与Ghrelin表达之间是否有关尚无定论。目前未见有关于Ghrelin在乌珠穆沁羊生殖道内的研究报道。本文在获得乌珠穆沁羊Ghrelin (usGhl) cDNA全序列的基础上,对生殖道各组织Ghrelin mRNA进行定位;系统研究了Ghrelin基因在不同发情期和妊娠期的乌珠穆沁羊雌性生殖道各组织内的相对表达量及其与外周血中雌激素和孕酮之间的相互关系;利用体外培养的乌珠穆沁羊输卵管上皮细胞研究雌、孕激素对Ghrelin基因表达量的调节作用;建立usGhl真核表达体系,初步证实构建的重组质粒对动物生长有促进作用。本论文为反刍动物生产性能的提高奠定基础。1.根据GenBank报道的Ghrelin cDNA序列设计特异性引物,运用RT-PCR技术和RACE技术成功克隆了usGhl cDNA全序列。本研究扩增的usGhl cDNA序列全长634bp,包含由116个氨基酸编码的前原乌珠穆沁羊Ghrelin肽。使用Blast网页和DNAStar软件对得到的usGhl cDNA全序列进行分析,无论是cDNA序列还是预测的成熟肽序列都与其它哺乳动物具有较高同源性。本研究结果表明Ghrelin基因在不同动物间表现出较高保守性。2.将乌珠穆沁羊子宫体Ghrelin RT-PCR产物回收并进行地高辛探针标记,用于原位杂交技术检测Ghrelin mRNA在乌珠穆沁羊子宫体、输卵管、宫颈、阴道中的表达位置。结果为:Ghrelin mRNA在内(黏)膜层的上皮细胞和腺体中大量表达,在固有层和肌层少量表达;肌肉组织、结缔组织和外膜层不表达。说明在绵羊雌性生殖道各组织中,Ghrelin mRNA主要表达于内(黏)膜表层细胞。3.运用RT-PCR技术证实Ghrelin基因在乌珠穆沁羊子宫体、输卵管、子宫颈、阴道组织内均有表达且表达量不同:子宫体表达量最高,输卵管次之,子宫颈和阴道内的表达量最少。采用实时荧光定量PCR技术及激素电化学发光测定法研究了乌珠穆沁羊发情及妊娠期生殖道各组织Ghrelin基因表达量与体内雌、孕激素之间的关系。本研究中血清雌二醇和孕酮在发情及妊娠期的浓度变化符合家畜生理周期的一般规律。乌珠穆沁羊生殖道各组织Ghrelin基因的表达量随发情周期及是否妊娠发生规律性改变。其中子宫体、子宫颈和输卵管内Ghrelin基因的表达与雌二醇及孕酮浓度的变化基本呈正相关。阴道内Ghrelin基因的表达与雌二醇及孕酮浓度的变化没有相关性。4.将机械法、细胞沉降及密度梯度离心结合使用,成功获得了原代和传代培养的乌珠穆沁羊输卵管上皮细胞。传一代的输卵管上皮细胞分别添加不同浓度的17-β-雌二醇和孕酮,分别培养24h、48h、72h后用RQ-PCR方法检测Ghrelin基因表达情况。结果显示:17-β-雌二醇和孕酮对体外培养的绵羊输卵管上皮细胞具有促进贴壁和促生长作用;培养时间对Ghrelin基因表达无影响;一定浓度的17-β-雌二醇和孕酮对培养的输卵管上皮细胞内Ghrelin基因的表达有促进作用。5.培养的输卵管上皮细胞添加不同浓度他莫昔芬或米非司酮后再用17-β-雌二醇或孕酮刺激输卵管上皮细胞生长24h、48h、72h。使用RQ-PCR方法检测各组细胞Ghrelin基因表达情况。结果表明:他莫昔芬和米非司酮能够抑制培养的乌珠穆沁羊输卵管上皮细胞的生长;一定浓度他莫昔芬能降低17-β-雌二醇刺激的输卵管上皮细胞内Ghrelin基因的表达,一定浓度米非司酮能降低孕酮刺激的输卵管上皮细胞内Ghrelin基因的表达,且二者的抑制作用均呈现剂量依赖性;培养时间对输卵管上皮细胞内Ghrelin基因的表达量无影响。说明雌、孕激素能够促进培养的绵羊输卵管上皮细胞内Ghrelin基因的表达,雌、孕激素是通过与其受体相结合发挥作用。6.本论文成功构建了Ghrelin真核表达质粒(pcDNA-usGhl),该质粒能在小鼠肌肉组织中特异性表达,并能维持约20天。按1μg质粒/g体重注射pcDNA-usGhl能促进小鼠生长。注射后第10～20天内,pcDNA-usGhl组体重均显着高于空质粒组(P<0.05),PcDNA-usGhl组小鼠的料重比与空质粒组小鼠相比有增加的趋势,但差异不显着。实验中没有发现Ghrelin重组质粒DNA整合到宿主染色体上。观察心脏、肝脏、肾脏、脑及肌肉均没有发现异常肿大及病变现象。更多还原

【Abstract】 Ghrelin was the ligand of GHSR which found in 1999. Its main function was combined with GHSR to stimulate GH releasing. Recently, Ghrelin was found in procreant organs, suggested Ghrelin could regulate procreant cycles and gestation. For the moment, there weren’t any reports about Ghrelin in Ujimqin sheep genital tracts. Estrogen and progesterone could regulate female genital system, but we didn’t know whether they could regulate Ghrelin gene expression or not. Based obtained Ujimqin sheep Ghrelin (usGhl) cDNA complete sequence, this paper deeply researched Ghrelin gene location and expression in genital tracts; studied on relationship between Ghrelin gene and estrogen/progesterone in different oestrum and gestation of female Ujimqin sheep genital tracts; validated Ghrelin gene expression content change in cultured fallopian tube epithelium cells by adding estrogen/progesterone into culture medium. Otherwise, we constructed usGhl eukaryotic expression system (pcDNA-usGhl). Animals experiment confirmed pcDNA-usGhl could accelerate animal growth. The results of this paper could be the foundation to research sheep promoted growth.1. Based on Ghrelin sequence in GenBank, we designed special primers to clone usGhl cDNA complete sequence by RT-PCR and RACE technique. The complete sequence contained 634 bp which included an Ujimqin sheep pre-Ghrelin peptide of 116aa. The relationship of Ghrelin cDNA or forecasted mature peptide sequence between Ujimqin sheep and other mammalians were extremely homologous by NCBI blast function and DNAStar software. The result indicated Ghrelin gene was conservative in different kinds of animals.2. We located Ghrelin mRNA in Ujimqin sheep genital tracts tissues (the body of uterus, the cervix of uterus, fallopian tube and vagina) by in situ hybridization technique (ISH). The result showed: Ghrelin mRNA mainly existed in the epithelium and gland; proper layer and muscle layer were less than epithelium and gland; there weren’t expression in musculature and connective tissue. Indicated Ghrelin mostly expressed in mucous membrane surface layer cells.3. We confirmed Ghrelin gene was expressed in the body of uterus, fallopian tube, cervix of uterus and vagina for the first time by RT-PCR technique. The expressive content of Ghrelin gene in different genital tracts tissues of Ujimqin sheep was different: the highest was the body of uterus, the next was fallopian tube, the cervix of uterus and vagina included its lowest level. Used RQ-PCR and electrochemistry radiation mensuration researched the relationship between Ghrelin and estrogen, progesterone in oestrus and gestation genital tracts tissue. The content chang of estrogen or progesterone in oestrus and gestation Ujimqin sheep serum accorded to currently rule of critter physiological cycle. Ghrelin gene content in Ujimqin sheep genital tracts tissues had disciplinary change by oestrus or gestation concentration: in the body of uterus, the cervix of uterus and fallopian tube was increased or fell with oestrus or gestation content change in serum of body; the content relationship between Ghrelin gene and oestrus or gestation in vagina was not evident.4. We obtained the original generation and the first generation epithelial cells of Ujimqin sheep fallopian tubes epithelium cells by mechanism, cells sedimentation and density grads centrifugal method. 17-β-estradiol and progesterone could accelerate glue and growth of cultured fallopian tubes epithelial cells. We added different concentration 17-β-estrogen and progesterone into the first generation epithelial cells cultured 24h, 48h, 72h. Then examined Ghrelin gene expression content in cells which treated with hormone by RQ-PCR technique. The result showed that culture time didn’t influence Ghrelin gene expression content; certain concentration 17-β-Estradiol or progesterone could promote the expression of Ghrelin gene in cultured fallopian tubes epithelia cell.5. Cultured fallopian tubes epithelia cells were treated with tamoxifen or mifiprestone first, then added estrogen or progesterone corresponding concentration cultured 24h, 48h, 72h. Ghrelin gene expression content of treated cells were detected by RQ-PCR. The results showed: tamoxifen and mifiprestone could restrain growth and intensity increased in cultured fallopian tubes epithelial cells; tamoxifen or mifiprestone could obviously debase Ghrelin gene expression content in cultured fallopian tubes epithelia cells compared with cultured epithelia cells with no tamoxifen or mifiprestone. If increased tamoxifen or mifiprestone concentration in culture medium without FBS, Ghrelin content was gradually debased; culture time didn’t influence Ghrelin gene expression content. Suggested that estrogen and progesterone could promote the expression of Ghrelin. Estrogen and progesterone exerted their function by combining with their corresponding receptors.6. We constructed usGhl eukaryotic expression system (pcDNA-usGhl). Ghrelin gene plasmid could express in mice, and maintained about 20d. Ghrelin recombined plasmid could accelerate mice growth and debased feed and avoirdupois ratio. PcDNA-usGhl DNA didn’t insert mice chromosome in our experiment. There weren’t novel intumescence or pathological changes in mice heart, liver, kidney, brain or muscle. The results indicated pcDNA-usGhl plasmid we constructed was successful.更多还原