本文選題：GnIH + 顆粒細(xì)胞��； 參考：《華中農(nóng)業(yè)大學(xué)》2015年博士論文

【摘要】：促性腺激素抑制激素(GnIH)是近期發(fā)現(xiàn)含12個(gè)氨基酸的激素,在哺乳動(dòng)物上又稱(chēng)為RF酰胺相關(guān)肽(RFRP)。據(jù)推測(cè)GnIH與抑制素生理功能相似,能夠抑制促性腺激素的合成和釋放,并能以旁分泌或自分泌方式直接參與調(diào)控卵巢功能。本研究先用不同濃度的GnIH注射雄性小鼠,再用不同濃度的GnIH對(duì)小鼠卵泡顆粒細(xì)胞進(jìn)行體外培養(yǎng),從下丘腦、垂體、睪丸和卵泡顆粒細(xì)胞角度分析GnIH的作用及其機(jī)制。為了證明研究結(jié)果的可靠性,在本實(shí)驗(yàn)室現(xiàn)有抑制素基因疫苗基礎(chǔ)上成功構(gòu)建了三種新型基因疫苗,分析了這些疫苗在HeLa細(xì)胞中的表達(dá)情況。最后采用電穿孔肌肉注射方法將這些疫苗免疫小鼠,分析免疫反應(yīng)及對(duì)小鼠產(chǎn)仔數(shù)的影響;同時(shí)用這些疫苗免疫灘羊,從免疫反應(yīng)、血清激素濃度變化及產(chǎn)羔數(shù)等方面分析基因免疫對(duì)繁殖的影響,為進(jìn)一步優(yōu)化提高繁殖力的基因疫苗奠定基礎(chǔ)。主要研究?jī)?nèi)容與結(jié)果如下:1.GnIH對(duì)雄鼠生殖與生殖內(nèi)分泌的調(diào)控及分子機(jī)制研究挑選32只小鼠,依據(jù)年齡和體重在各組均衡的原則,分為4組,每組8只。試驗(yàn)組小鼠每天兩次,分別皮下注射150μl含GnIH 0(對(duì)照)、1、3和6μg的生理鹽水液,連續(xù)11天,然后收集血樣,用ELISA方法檢測(cè)LH、T和INH B的濃度;提取下丘腦、垂體、睪丸組織,提取組織中RNA及蛋白后,用qPCR分別檢測(cè)GnRH I、Kiss-1、FSHβ、LHβ、GnRHR和HSF-2的mRNA表達(dá);用western bolt檢測(cè)P450scc、StAR、3β-HSD、LHR和AR蛋白表達(dá);用H.E染色檢測(cè)睪丸組織形態(tài)變化;用TUNEL方法檢測(cè)生殖細(xì)胞凋亡。結(jié)果顯示:GnIH處理能夠明顯降低血漿中LH濃度和下丘腦組織中GnRH I mRNA、Kiss-1 mRNA的表達(dá);下調(diào)FSHβ、LHβ、GnRH受體、P450scc、StAR和3β-HSD基因的表達(dá),進(jìn)而降低血中睪酮水平。此外,GnIH處理還能下調(diào)精子發(fā)生相關(guān)基因(LHR、AR、HSF-2)的表達(dá),降低血中INHB的濃度和睪丸組織中INHβb mRNA的表達(dá)量,導(dǎo)致睪丸生殖細(xì)胞形態(tài)異常,誘發(fā)凋亡。這些結(jié)果表明:GnIH抑制小鼠睪丸類(lèi)固醇生成及精子發(fā)生的機(jī)制可以解釋為(1)GnIH在下丘腦通過(guò)直接抑制GnRH I、Kiss-1mRNA的表達(dá)來(lái)抑制GnRH釋放,或者在垂體抑制GnRHR mRNA的表達(dá),從而減少腺垂體LH的分泌;(2)直接作用于睪丸組織,降低p450scc、star和β-hsd基因的表達(dá)來(lái)抑制睪酮生成;(3)通過(guò)直接下調(diào)睪丸中l(wèi)hr、ar和hsf-2的表達(dá)來(lái)抑制精子發(fā)生。2.gnih對(duì)小鼠卵巢顆粒細(xì)胞類(lèi)固醇生成的作用及分子機(jī)制首先分離pmsg注射后48h的小鼠卵巢顆粒細(xì)胞,體外培養(yǎng)24h、48h、72h、96h后,提取顆粒細(xì)胞總rna和蛋白,用qpcr和westernblot方法檢測(cè)了gnih的受體(gpr147)在小鼠卵巢顆粒細(xì)胞上的表達(dá)情況。然后用不同濃度(10ng/ml、100ng/ml、1000ng/ml)的小鼠gnih(rfrp-3)培養(yǎng)原代顆粒細(xì)胞24h后,收集上清液,提取顆粒細(xì)胞總rna和蛋白。用elisa方法檢測(cè)雌二醇(e2)、孕酮(p)的水平;用qpcr和westernblot方法檢測(cè)顆粒細(xì)胞類(lèi)固醇生成相關(guān)酶基因(p450scc、3β-hsd、star)、fshrmrna和p-erk1/2蛋白的表達(dá);用流式細(xì)胞技術(shù)檢測(cè)顆粒細(xì)胞的凋亡。結(jié)果顯示:小鼠顆粒細(xì)胞上確有g(shù)pr147表達(dá);中劑量(100ng/ml)gnih能夠明顯降低p450scc、3β-hsd和star、fshrmrna的表達(dá),并能誘導(dǎo)顆粒細(xì)胞發(fā)生明顯的凋亡(p0.05);低劑量和中劑量gnih能夠顯著降低顆粒細(xì)胞孕酮的合成(p0.05),還能下調(diào)p-erk1/2蛋白的表達(dá)。這些結(jié)果表明:小鼠顆粒細(xì)胞上確有g(shù)pr147的表達(dá);gnih能夠在體外直接作用于小鼠卵巢顆粒細(xì)胞,通過(guò)降低顆粒細(xì)胞中fhsrmrna和p-erk1/2蛋白的表達(dá)來(lái)降低顆粒細(xì)胞孕酮的合成及存活率,從而間接影響卵泡發(fā)育。3.gnih與抑制素共表達(dá)新型基因疫苗的構(gòu)建、鑒定及其對(duì)小鼠繁殖力的影響先將豬源inhα(1-32)連接到乙肝表面抗原s基因c末端,合成sinh;將牛源gnih(rfrp-3)基因連接到乙肝表面抗原s基因c末端,合成srfrp。將sinh和srfrp基因片段分別插入到pires真核表達(dá)載體兩個(gè)多克隆位點(diǎn),構(gòu)建抑制素和促性腺激素抑制激素共表達(dá)質(zhì)粒,即p-sinh/srfrp(簡(jiǎn)稱(chēng)“共表達(dá)質(zhì)粒”)。然后將組織纖溶酶原信號(hào)肽序列(tpa)信號(hào)肽序列分別插入到p-sinh/srfrp疫苗目的基因的n-端,構(gòu)建帶有信號(hào)肽的抑制素和促性腺激素抑制激素共表達(dá)疫苗,即p-tpa-sinh/tpa-sfrfp(簡(jiǎn)稱(chēng)“信號(hào)肽共表達(dá)質(zhì)粒”);將sinh插入到pires載體上游多克隆位點(diǎn),構(gòu)建成抑制素單表達(dá)質(zhì)粒,即p-sinh(簡(jiǎn)稱(chēng)“單表達(dá)質(zhì)粒,抑制素疫苗”),用作陽(yáng)性對(duì)照。應(yīng)用雙酶切和測(cè)序分析驗(yàn)證這些疫苗是否構(gòu)建成功,同時(shí)將其轉(zhuǎn)染真核細(xì)胞后檢測(cè)在細(xì)胞中的表達(dá)情況。然后,選取6周齡體重相似的40只昆明小鼠,分為四組,應(yīng)用電穿孔法將上述三種疫苗分別免疫小鼠,間隔2周加強(qiáng)免疫2次,并以生理鹽水作為陰性對(duì)照。在初次免疫后第0 d、14d、28d和42d收集血樣,用間接ELISA方法檢測(cè)其中抗抑制素和抗RFRP水平。采血后母鼠與公鼠合籠,連續(xù)記錄三個(gè)胎次的產(chǎn)仔數(shù)。雙酶切和測(cè)序結(jié)果表明,三種質(zhì)粒中目的基因的插入位點(diǎn)、方向和順序完全正確,且都能在真核細(xì)胞中表達(dá);免疫結(jié)果表明,在第三次免疫后2周,p-TPA-SINH/TPA-SFRFP疫苗免疫小鼠后血漿抗INH和抗RFRP-3抗體陽(yáng)性值(P/N值)明顯高于p-SINH和p-SINH/SRFRP疫苗產(chǎn)生的(p0.05),而且陽(yáng)性小鼠比率分別是100%(抗INH抗體)和90%(抗RFRP抗體);繁殖結(jié)果表明,三種質(zhì)粒免疫小鼠后其產(chǎn)仔數(shù)顯著高于對(duì)照組(生理鹽水組),并且p-TPA-SINH/TPA-SRFRP疫苗免疫后小鼠的產(chǎn)仔數(shù)明顯高于p-SINH疫苗的(p0.05)。這些結(jié)果表明,p-SINH、p-SINH/SRFRP和p-TPA-SINH/TPA-SRFRP質(zhì)粒被成功構(gòu)建;三種質(zhì)粒免疫小鼠后,均能刺激機(jī)體產(chǎn)生抗INH和/或GnIH(RFRP-3)抗體,并可提高產(chǎn)仔數(shù)。其中,信號(hào)肽雙表達(dá)質(zhì)粒的免疫和提高產(chǎn)仔數(shù)效果最好。4.GnIH與抑制素共表達(dá)新型基因疫苗免疫綿羊?qū)ζ渖臣に丶胺敝沉Φ挠绊懱暨x32只健康母灘羊,按照年齡和體重相似的原則分為四組,每組8只,分別肌肉注射信號(hào)肽共表達(dá)疫苗(A組)、共表達(dá)疫苗(B組)和抑制素苗(C組)各0.6mg及0.4 ml生理鹽水(D組)。加強(qiáng)免疫2次,間隔20 d。在第3次免疫后當(dāng)天所有綿羊進(jìn)行同期發(fā)情處理。分別在初次免疫后20、30、40、60 d后頸靜脈收集血清,并在最后一次采血之后引入公羊,直至所有綿羊妊娠,最后分別統(tǒng)計(jì)分娩之后各組綿羊的雙羔率。用間接ELISA方法檢測(cè)血清中抗INH和抗RFRP-3抗體水平;用RIA方法檢測(cè)同情發(fā)情后綿羊外周血中FSH、LH、E2和P的水平。結(jié)果表明,在初次免疫后20 d,三種疫苗都能誘發(fā)明顯的免疫反應(yīng),血中抗INH和抗RFRP-3抗體的陽(yáng)性值(P/N值)顯著高于對(duì)照組(p0.05);信號(hào)肽共表達(dá)疫苗能夠誘發(fā)綿羊產(chǎn)生更高抗INH和抗RFRP-3抗體水平;在第3次免疫后20 d,A組綿羊外周血中FSH和LH水平顯著高于C組(p0.05);雙羔率在A、B、C和D組中分別是37.5%,37.5%,12.5%,0。這些結(jié)果表明,表達(dá)INH和RFRP-3雙基因的新型基因疫苗能夠誘發(fā)綿羊體液免疫反應(yīng),增加外周血中促性腺激素水平,并且在一定程度上能夠提高母羊的雙羔率。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：S814

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