GnIH对小鼠繁殖的调控及其分子机制和基因免疫技术研究

发布时间：2018-06-18 05:08

本文选题：GnIH + 颗粒细胞　；参考：《华中农业大学》2015年博士论文

【摘要】：促性腺激素抑制激素(GnIH)是近期发现含12个氨基酸的激素,在哺乳动物上又称为RF酰胺相关肽(RFRP)。据推测GnIH与抑制素生理功能相似,能够抑制促性腺激素的合成和释放,并能以旁分泌或自分泌方式直接参与调控卵巢功能。本研究先用不同浓度的GnIH注射雄性小鼠,再用不同浓度的GnIH对小鼠卵泡颗粒细胞进行体外培养,从下丘脑、垂体、睾丸和卵泡颗粒细胞角度分析GnIH的作用及其机制。为了证明研究结果的可靠性,在本实验室现有抑制素基因疫苗基础上成功构建了三种新型基因疫苗,分析了这些疫苗在HeLa细胞中的表达情况。最后采用电穿孔肌肉注射方法将这些疫苗免疫小鼠,分析免疫反应及对小鼠产仔数的影响;同时用这些疫苗免疫滩羊,从免疫反应、血清激素浓度变化及产羔数等方面分析基因免疫对繁殖的影响,为进一步优化提高繁殖力的基因疫苗奠定基础。主要研究内容与结果如下:1.GnIH对雄鼠生殖与生殖内分泌的调控及分子机制研究挑选32只小鼠,依据年龄和体重在各组均衡的原则,分为4组,每组8只。试验组小鼠每天两次,分别皮下注射150μl含GnIH 0(对照)、1、3和6μg的生理盐水液,连续11天,然后收集血样,用ELISA方法检测LH、T和INH B的浓度;提取下丘脑、垂体、睾丸组织,提取组织中RNA及蛋白后,用qPCR分别检测GnRH I、Kiss-1、FSHβ、LHβ、GnRHR和HSF-2的mRNA表达;用western bolt检测P450scc、StAR、3β-HSD、LHR和AR蛋白表达;用H.E染色检测睾丸组织形态变化;用TUNEL方法检测生殖细胞凋亡。结果显示:GnIH处理能够明显降低血浆中LH浓度和下丘脑组织中GnRH I mRNA、Kiss-1 mRNA的表达;下调FSHβ、LHβ、GnRH受体、P450scc、StAR和3β-HSD基因的表达,进而降低血中睾酮水平。此外,GnIH处理还能下调精子发生相关基因(LHR、AR、HSF-2)的表达,降低血中INHB的浓度和睾丸组织中INHβb mRNA的表达量,导致睾丸生殖细胞形态异常,诱发凋亡。这些结果表明:GnIH抑制小鼠睾丸类固醇生成及精子发生的机制可以解释为(1)GnIH在下丘脑通过直接抑制GnRH I、Kiss-1mRNA的表达来抑制GnRH释放,或者在垂体抑制GnRHR mRNA的表达,从而减少腺垂体LH的分泌;(2)直接作用于睾丸组织,降低p450scc、star和β-hsd基因的表达来抑制睾酮生成;(3)通过直接下调睾丸中lhr、ar和hsf-2的表达来抑制精子发生。2.gnih对小鼠卵巢颗粒细胞类固醇生成的作用及分子机制首先分离pmsg注射后48h的小鼠卵巢颗粒细胞,体外培养24h、48h、72h、96h后,提取颗粒细胞总rna和蛋白,用qpcr和westernblot方法检测了gnih的受体(gpr147)在小鼠卵巢颗粒细胞上的表达情况。然后用不同浓度(10ng/ml、100ng/ml、1000ng/ml)的小鼠gnih(rfrp-3)培养原代颗粒细胞24h后,收集上清液,提取颗粒细胞总rna和蛋白。用elisa方法检测雌二醇(e2)、孕酮(p)的水平;用qpcr和westernblot方法检测颗粒细胞类固醇生成相关酶基因(p450scc、3β-hsd、star)、fshrmrna和p-erk1/2蛋白的表达;用流式细胞技术检测颗粒细胞的凋亡。结果显示:小鼠颗粒细胞上确有gpr147表达;中剂量(100ng/ml)gnih能够明显降低p450scc、3β-hsd和star、fshrmrna的表达,并能诱导颗粒细胞发生明显的凋亡(p0.05);低剂量和中剂量gnih能够显著降低颗粒细胞孕酮的合成(p0.05),还能下调p-erk1/2蛋白的表达。这些结果表明:小鼠颗粒细胞上确有gpr147的表达;gnih能够在体外直接作用于小鼠卵巢颗粒细胞,通过降低颗粒细胞中fhsrmrna和p-erk1/2蛋白的表达来降低颗粒细胞孕酮的合成及存活率,从而间接影响卵泡发育。3.gnih与抑制素共表达新型基因疫苗的构建、鉴定及其对小鼠繁殖力的影响先将猪源inhα(1-32)连接到乙肝表面抗原s基因c末端,合成sinh;将牛源gnih(rfrp-3)基因连接到乙肝表面抗原s基因c末端,合成srfrp。将sinh和srfrp基因片段分别插入到pires真核表达载体两个多克隆位点,构建抑制素和促性腺激素抑制激素共表达质粒,即p-sinh/srfrp(简称“共表达质粒”)。然后将组织纤溶酶原信号肽序列(tpa)信号肽序列分别插入到p-sinh/srfrp疫苗目的基因的n-端,构建带有信号肽的抑制素和促性腺激素抑制激素共表达疫苗,即p-tpa-sinh/tpa-sfrfp(简称“信号肽共表达质粒”);将sinh插入到pires载体上游多克隆位点,构建成抑制素单表达质粒,即p-sinh(简称“单表达质粒,抑制素疫苗”),用作阳性对照。应用双酶切和测序分析验证这些疫苗是否构建成功,同时将其转染真核细胞后检测在细胞中的表达情况。然后,选取6周龄体重相似的40只昆明小鼠,分为四组,应用电穿孔法将上述三种疫苗分别免疫小鼠,间隔2周加强免疫2次,并以生理盐水作为阴性对照。在初次免疫后第0 d、14d、28d和42d收集血样,用间接ELISA方法检测其中抗抑制素和抗RFRP水平。采血后母鼠与公鼠合笼,连续记录三个胎次的产仔数。双酶切和测序结果表明,三种质粒中目的基因的插入位点、方向和顺序完全正确,且都能在真核细胞中表达;免疫结果表明,在第三次免疫后2周,p-TPA-SINH/TPA-SFRFP疫苗免疫小鼠后血浆抗INH和抗RFRP-3抗体阳性值(P/N值)明显高于p-SINH和p-SINH/SRFRP疫苗产生的(p0.05),而且阳性小鼠比率分别是100%(抗INH抗体)和90%(抗RFRP抗体);繁殖结果表明,三种质粒免疫小鼠后其产仔数显著高于对照组(生理盐水组),并且p-TPA-SINH/TPA-SRFRP疫苗免疫后小鼠的产仔数明显高于p-SINH疫苗的(p0.05)。这些结果表明,p-SINH、p-SINH/SRFRP和p-TPA-SINH/TPA-SRFRP质粒被成功构建;三种质粒免疫小鼠后,均能刺激机体产生抗INH和/或GnIH(RFRP-3)抗体,并可提高产仔数。其中,信号肽双表达质粒的免疫和提高产仔数效果最好。4.GnIH与抑制素共表达新型基因疫苗免疫绵羊对其生殖激素及繁殖力的影响挑选32只健康母滩羊,按照年龄和体重相似的原则分为四组,每组8只,分别肌肉注射信号肽共表达疫苗(A组)、共表达疫苗(B组)和抑制素苗(C组)各0.6mg及0.4 ml生理盐水(D组)。加强免疫2次,间隔20 d。在第3次免疫后当天所有绵羊进行同期发情处理。分别在初次免疫后20、30、40、60 d后颈静脉收集血清,并在最后一次采血之后引入公羊,直至所有绵羊妊娠,最后分别统计分娩之后各组绵羊的双羔率。用间接ELISA方法检测血清中抗INH和抗RFRP-3抗体水平;用RIA方法检测同情发情后绵羊外周血中FSH、LH、E2和P的水平。结果表明,在初次免疫后20 d,三种疫苗都能诱发明显的免疫反应,血中抗INH和抗RFRP-3抗体的阳性值(P/N值)显著高于对照组(p0.05);信号肽共表达疫苗能够诱发绵羊产生更高抗INH和抗RFRP-3抗体水平;在第3次免疫后20 d,A组绵羊外周血中FSH和LH水平显著高于C组(p0.05);双羔率在A、B、C和D组中分别是37.5%,37.5%,12.5%,0。这些结果表明,表达INH和RFRP-3双基因的新型基因疫苗能够诱发绵羊体液免疫反应,增加外周血中促性腺激素水平,并且在一定程度上能够提高母羊的双羔率。
[Abstract]:Gonadotropin suppressive hormone (GnIH) is a recent discovery of a hormone containing 12 amino acids and is also known as RF amide related peptide (RFRP) in mammals. It is presumed that GnIH and inhibin are similar in physiological function, can inhibit the synthesis and release of gonadotropin, and can directly regulate ovarian function by paracrine or autocrine methods. This study was first used in this study. The male mice were injected with different concentrations of GnIH and cultured in vitro with different concentrations of GnIH. The effect and mechanism of GnIH were analyzed from the angle of hypothalamus, pituitary, testis and follicle granulosa cells. In order to prove the reliability of the results, it was successfully constructed on the basis of the existing inhibin gene vaccine in this laboratory. Three new genetic vaccines were used to analyze the expression of these vaccines in HeLa cells. Finally, these vaccines were used to immunize these vaccines to analyze the immune response and the effect on the number of offspring in mice. At the same time, these vaccines were used to immunize the beach sheep and analyze the basis of the immune response, the change of serum hormone concentration and the number of lambs. The main research contents and results are as follows: 32 mice were selected by 1.GnIH on the regulation of reproductive and reproductive endocrinology and molecular mechanism of male rats. According to the principle of age and weight balance in each group, there were 4 groups of 8 mice in each group. Two times, subcutaneous injection of 150 mu l containing GnIH 0 (control), 1,3 and 6 mu g saline solution for 11 days, and then collecting blood samples, and using ELISA to detect the concentration of LH, T and INH B; extract the hypothalamus, pituitary, and testis tissue, extract RNA and protein in the tissue. Bolt was used to detect the expression of P450scc, StAR, 3 beta -HSD, LHR and AR. The morphological changes of the testis were detected by H.E staining, and the apoptosis of germ cells was detected by TUNEL method. The results showed that GnIH treatment could significantly reduce the LH concentration in the plasma and the GnRH I in the hypothalamus. In addition, GnIH treatment can also reduce the expression of LHR, AR, HSF-2, the concentration of INHB in the blood and the expression of INH beta B mRNA in the testis, causing the abnormal morphology of the testicular germ cells and inducing the death of the testis. These results suggest that GnIH inhibits the steroid formation of testis in mice. And the mechanism of spermatogenesis can be explained as (1) GnIH in the hypothalamus can inhibit GnRH release by directly inhibiting the expression of GnRH I, Kiss-1mRNA, or inhibiting the expression of GnRHR mRNA in the pituitary, thus reducing the secretion of LH in the adenohypophysis; (2) it directly acts on the testis tissue and reduces the expression of P450scc, star and beta -hsd genes to inhibit testosterone production; (3) (3) The expression of LHR, AR and hsf-2 in testis was directly regulated to inhibit the effect of.2.gnih on steroid formation of mouse ovarian granulosa cells and the molecular mechanism was first isolated from the mouse ovarian granulosa cells of 48h after PMSG injection. After the culture of 24h, 48h, 72h, 96h, the granule cell total RNA and protein were extracted. The expression of NIH receptor (gpr147) on the mouse ovarian granulosa cells and then using GnIH (rfrp-3) in mice with different concentrations (10ng/ml, 100ng/ml, 1000ng/ml) to cultivate the original granulosa cells 24h, collect the supernatant and extract the total RNA and protein in granular cells. The level of estradiol (E2) and progesterone (P) is detected by ELISA. The expression of P450scc, 3 beta -hsd, star, fshrmrna and p-erk1/2 protein were detected in granulosa cells. The apoptosis of granulosa cells was detected by flow cytometry. The results showed that the expression of gpr147 was found on the mouse granulosa cells, and the medium dose (100ng/ml) GnIH could significantly reduce P450scc, 3 beta -hsd and star, the expression of fshrmrna. The granulosa cells have obvious apoptosis (P0.05), and low and medium dose GnIH can significantly reduce the synthesis of progesterone (P0.05) in granulosa cells, but also reduce the expression of p-erk1/2 protein. These results show that the expression of gpr147 in the mouse granulosa cells is true; GnIH can directly act on mouse ovarian granulosa cells in vitro and reduce the particles by reducing the particles in vitro. The expression of fhsrmrna and p-erk1/2 protein in cells to reduce the synthesis and survival of progesterone in granulosa cells, thereby indirectly affecting the construction of a novel gene vaccine that CO expressed.3.gnih and inhibin in the follicle development. The identification and its impact on mice fertility first connected pig source INH alpha (1-32) to the C terminal of the hepatitis B surface antigen S gene, and synthesized Sinh; cattle The source GnIH (rfrp-3) gene was connected to the C terminal of the hepatitis B surface antigen S gene, and srfrp. was synthesized by srfrp. to insert Sinh and srfrp fragments into the two polyclonal sites of the eukaryotic expression vector of Pires, respectively, to construct inhibin and gonadotropin inhibiting hormone co expression plasmids, namely, p-sinh/srfrp (simply called "co expression plasmid"). Then the fibrinolytic enzyme was expressed as a fibrinolytic enzyme. The sequence of signal peptide sequence (TPA) was inserted into the n- end of the p-sinh/srfrp vaccine target gene, and a costatin and gonadotropin suppressive hormone co expression vaccine with a signal peptide was constructed, that is, p-tpa-sinh/tpa-sfrfp ("signal peptide co expression plasmid"), and Sinh was inserted into the upstream polyclonal site of the Pires carrier and constructed as a inhibin single. The expression plasmid, p-sinh ("single expression plasmid, inhibin vaccine"), was used as a positive control. Double enzyme cutting and sequencing analysis were used to verify whether these vaccines were successfully constructed and then transfected into eukaryotic cells to detect the expression in the cells. Then, 40 Kunming mice with similar weight at the age of 6 weeks were selected and divided into four groups. The three kinds of vaccines were immunized by hole method, and the immunization was strengthened 2 times at intervals of 2 weeks, and the normal saline was used as negative control. The blood samples were collected at zeroth D, 14d, 28d and 42d after the initial immunization. The anti inhibin and anti RFRP levels were detected by indirect ELISA. The number of baby mice and male rats were kept in continuous recording of the number of birth numbers of three fetal times. And sequencing results showed that the insertion sites, direction and order of the target genes in the three plasmids were completely correct and could all be expressed in the eukaryotic cells, and the immune results showed that the positive values of anti INH and anti RFRP-3 antibody (P/N value) of the plasma in the p-TPA-SINH/TPA-SFRFP vaccinated mice were significantly higher than those of the p-SINH and p-SINH/SRFRP vaccines after the third immunization. The incidence of (P0.05) and positive mice were 100% (anti INH antibody) and 90% (anti RFRP antibody), and the reproduction results showed that the number of offspring of the three plasmids was significantly higher than that of the control group (normal saline group), and the litter size of the mice after the p-TPA-SINH/TPA-SRFRP vaccine was significantly higher than that of the p-SINH vaccine (P0.05). These results showed that the results showed that the number of the mice was significantly higher than that of the p-SINH vaccine (P0.05). P-SINH, p-SINH/SRFRP and p-TPA-SINH/TPA-SRFRP plasmids were successfully constructed. After three plasmids were immunized with mice, the body could stimulate the body to produce anti INH and / or GnIH (RFRP-3) antibodies and increase the number of offspring. Among them, the immunization of the double expression plasmid of the signal peptide and the improvement of the number of offspring were best.4.GnIH and inhibin co expressed a new gene vaccine to immunize sheep. The effects of the reproductive hormone and fecundity were selected in 32 healthy female flat sheep. According to the principle of age and weight similarity, four groups were divided into 8 rats, each of which was injected into the A group by intramuscular injection of signal peptide (group B) and inhibin vaccine (group C), each 0.6mg and 0.4 ml physiological saline (group D). The immunization was strengthened 2 times and the interval 20 D. was third times of immunization. All sheep were treated with estrus on the same day after the first immunization. After the first 20,30,40,60 D, the blood serum was collected in the neck vein. After the last blood collection, the sheep was introduced to all the sheep gestation. Finally, the two lambs rate of all the sheep after the delivery was calculated. The level of anti INH and anti RFRP-3 antibody in the serum was detected by indirect ELISA method. The RIA method was used to detect the levels of FSH, LH, E2 and P in the peripheral blood of sheep after sympathetic estrus. The results showed that the three vaccines could induce obvious immune responses after the first 20 D, and the positive value of anti INH and anti RFRP-3 antibody (P/N value) in the blood was significantly higher than that of the control group (P0.05). The level of anti RFRP-3 antibody and the level of FSH and LH in peripheral blood of group A were significantly higher than that in group C (P0.05) after third times of immunization, and the rate of double lambs in A, B, C and D were 37.5%, 37.5%, 12.5%, 0., respectively. The results showed that the new gene vaccine expressing INH and double genes could induce the humoral immune response of sheep and increase gonadotropin in peripheral blood. Level, and to a certain extent, can increase the double lamb rate of ewes.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S814

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