免疫抑制因子MNSFβ在胚胎著床與發(fā)育中的作用機(jī)制的研究
發(fā)布時(shí)間：2018-11-10 11:54
【摘要】：MNSFβ是本實(shí)驗(yàn)室新發(fā)現(xiàn)的一個(gè)著床相關(guān)因子,它是一種由抑制性T細(xì)胞合成的具有非特異性免疫抑制作用的淋巴因子,推測(cè)其可能與母胎間免疫耐受的形成有關(guān)。本課題主要研究MNSFβ的功能及其在胚胎著床中的作用,以進(jìn)一步揭示胚胎著床的分子機(jī)制,從而為不孕、不育機(jī)制的研究、提高人工輔助生育技術(shù)以及開(kāi)發(fā)研制抗著床避孕藥物提供理論基礎(chǔ)；并從中尋找潛在的可實(shí)現(xiàn)生育調(diào)控目的的新環(huán)節(jié)或新的靶分子。本課題主要結(jié)果如下： 1.由于抗MNSFβ抗體尚未市場(chǎng)化,本課題首先制備抗MNSFβ多克隆抗體,通過(guò)ELISA證實(shí)抗體效價(jià)為1：125000,并將之作為本研究的實(shí)驗(yàn)工具之一。 2.應(yīng)用免疫組化,RT-PCR及Western blot技術(shù)證實(shí)MNSFβ表達(dá)于小鼠全身各組織臟器；在小鼠胚胎著床前、后及妊娠期間MNSFβ在子宮組織中的表達(dá)具有動(dòng)態(tài)性變化,即在懷孕4.5天的子宮中表達(dá)減少,之后隨懷孕時(shí)間的延長(zhǎng),表達(dá)逐漸增加,但到懷孕8.5天時(shí),非著床位點(diǎn)的表達(dá)高于著床位點(diǎn)組織。 3.分離小鼠子宮內(nèi)膜細(xì)胞,通過(guò)免疫熒光證實(shí)為基質(zhì)細(xì)胞,并且該細(xì)胞和囊胚均表達(dá)MNSFβ基因；將小鼠子宮內(nèi)膜基質(zhì)細(xì)胞與孕4.5天的囊胚進(jìn)行共培養(yǎng),證實(shí)囊胚可向基質(zhì)細(xì)胞侵襲,粘附,鋪展,最終與其融合,類似著床過(guò)程；用MNSFβ抗血清可明顯抑制小鼠囊胚向子宮內(nèi)膜基質(zhì)細(xì)胞的鋪展與粘附生長(zhǎng),并可誘導(dǎo)胚胎死亡,且呈劑量反應(yīng)關(guān)系。 4.選用人子宮頸鱗癌細(xì)胞系(HCC-94)轉(zhuǎn)染MNSFP表達(dá)或siRNA,用其上清刺激小鼠淋巴細(xì)胞,證實(shí)過(guò)表達(dá)MNSFβ可促進(jìn)淋巴細(xì)胞分泌IL-4,但抑制TNFα的分泌；與之相反,沉默或用抗體中和MNSFβ則抑制IL-4的分泌,卻促進(jìn)TNFα的分泌,提示MNSFβ可能參與母胎免疫耐受。 5.建立MNSFβ條件性基因剔除小鼠,與Cre小鼠交配,獲得雜合型MNSFβ基因敲除小鼠。交配實(shí)驗(yàn)證實(shí)MNSFβ部分基因缺陷導(dǎo)致小鼠生育能力明顯下降,且僅雌鼠受影響；子代雌雄比例失調(diào),并不能得到純合型小鼠。 6.通過(guò)免疫組化、Real-time PCR及WB證實(shí)雜合型小鼠子宮組織的MNSFβmRNA與野生型小鼠相比,無(wú)明顯改變,但其蛋白表達(dá)卻明顯減少；此外,雜合型小鼠子宮腺上皮組織變得疏松,子宮腔腔隙也明顯加寬。 7.選擇胚胎著床與妊娠的不同時(shí)間點(diǎn)(即孕4、5、8、11和14天)觀察子宮中的胚胎數(shù)量和發(fā)育情況,結(jié)果證實(shí)在胚胎著床和胚胎發(fā)育早期子宮中的胚胎數(shù)量正常,但在發(fā)育中期(孕11和14天)胚胎出現(xiàn)明顯異常,即失去胎鼠的形態(tài),并固縮成團(tuán)塊狀。提示MNSFβ參與胚胎發(fā)育。 上述研究結(jié)果提示MNSFβ在小鼠胚胎著床過(guò)程中是必需的,且與免疫抑制相關(guān)。而該基因部分缺陷或表達(dá)減少,在體內(nèi)外均可引起胚胎著床及發(fā)育障礙。因此,MNSFβ有可能成為發(fā)展新型抗生育技術(shù)的潛在靶分子。
[Abstract]:MNSF 尾 is a newly discovered implantation related factor in our laboratory. It is a non-specific immunosuppressive lymphoid factor synthesized by suppressive T cells, which may be related to the formation of maternal and fetal immune tolerance. In this paper, we mainly studied the function of MNSF 尾 and its role in embryo implantation, in order to reveal the molecular mechanism of embryo implantation, so as to study the mechanism of infertility and sterility. To improve artificial assisted fertility technology and develop anti-implantation contraceptive drugs to provide the theoretical basis; And to find out the potential to achieve the purpose of fertility regulation of new links or new target molecules. The main results are as follows: 1. Since the anti MNSF 尾 antibody has not yet been marketized, we first prepared anti MNSF 尾 polyclonal antibody. The titer of anti MNSF 尾 polyclonal antibody was confirmed to be 1: 125 000 by ELISA, and it was used as one of the experimental tools in this study. 2. Immunohistochemical, RT-PCR and Western blot techniques were used to confirm the expression of MNSF 尾 in all tissues of mice. The expression of MNSF 尾 in the uterus was changed dynamically before, after implantation and during pregnancy, that is, the expression of MNSF 尾 decreased in the uterus on the 4.5 day of pregnancy, and then increased gradually with the prolongation of pregnancy time. However, at 8. 5 days after pregnancy, the expression of non-bed site was higher than that of implantation site. 3. Mouse endometrial cells were isolated and identified as stromal cells by immunofluorescence. Both the cells and blastocysts expressed MNSF 尾 gene. Co-culture of mouse endometrial stromal cells with blastocysts on day 4.5 of gestation showed that blastocysts could invade, adhere, spread out to stromal cells and finally fuse with them, similar to implantation process. MNSF 尾 antiserum could significantly inhibit the proliferation and adhesion of mouse blastocyst to endometrial stromal cells, and induce embryo death in a dose-dependent manner. 4. Human cervical squamous cell carcinoma cell line (HCC-94) was used to transfect MNSFP expression or siRNA, supernatant to stimulate mouse lymphocytes. It was proved that overexpression of MNSF 尾 could promote the secretion of IL-4, but inhibit the secretion of TNF 偽 by lymphocytes. On the contrary, silencing or neutralizing MNSF 尾 with antibodies inhibited the secretion of IL-4 and promoted the secretion of TNF 偽, suggesting that MNSF 尾 might participate in maternal and fetal immune tolerance. 5. The MNSF 尾 conditioned gene knockout mice were established and mated with Cre mice to obtain heterozygous MNSF 尾 gene knockout mice. Mating experiments showed that MNSF 尾 partial gene deficiency resulted in a significant decrease in fertility in mice, and only female mice were affected, and homozygous mice could not be obtained due to the imbalance of the ratio of male and female offspring. 6. By immunohistochemistry, Real-time PCR and WB confirmed that the MNSF 尾 mRNA in the uterine tissues of heterozygous mice had no significant change compared with that of wild type mice, but its protein expression was significantly decreased. In addition, the uterine glandular epithelium of the heterozygous mice became loose and the lacunae widened significantly. 7. The number and development of embryos in the uterus were observed at different time points of embryo implantation and pregnancy (that is, 4 days and 14 days of pregnancy). The results showed that the number of embryos in the embryo implantation and early stage of embryo development was normal. But at the middle stage of development (11 and 14 days of gestation), the embryo showed obvious abnormality, that is, it lost the shape of the fetal mouse and pyknosed into a lump. The results suggest that MNSF 尾 is involved in embryonic development. These results suggest that MNSF 尾 is necessary in mouse embryo implantation and is associated with immunosuppression. However, partial defect or decreased expression of the gene can cause embryo implantation and developmental disorders in vivo and in vitro. Therefore, MNSF 尾 may become a potential target molecule for the development of new anti-fertility technologies.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R321
本文編號(hào)：2322405
[Abstract]:MNSF 尾 is a newly discovered implantation related factor in our laboratory. It is a non-specific immunosuppressive lymphoid factor synthesized by suppressive T cells, which may be related to the formation of maternal and fetal immune tolerance. In this paper, we mainly studied the function of MNSF 尾 and its role in embryo implantation, in order to reveal the molecular mechanism of embryo implantation, so as to study the mechanism of infertility and sterility. To improve artificial assisted fertility technology and develop anti-implantation contraceptive drugs to provide the theoretical basis; And to find out the potential to achieve the purpose of fertility regulation of new links or new target molecules. The main results are as follows: 1. Since the anti MNSF 尾 antibody has not yet been marketized, we first prepared anti MNSF 尾 polyclonal antibody. The titer of anti MNSF 尾 polyclonal antibody was confirmed to be 1: 125 000 by ELISA, and it was used as one of the experimental tools in this study. 2. Immunohistochemical, RT-PCR and Western blot techniques were used to confirm the expression of MNSF 尾 in all tissues of mice. The expression of MNSF 尾 in the uterus was changed dynamically before, after implantation and during pregnancy, that is, the expression of MNSF 尾 decreased in the uterus on the 4.5 day of pregnancy, and then increased gradually with the prolongation of pregnancy time. However, at 8. 5 days after pregnancy, the expression of non-bed site was higher than that of implantation site. 3. Mouse endometrial cells were isolated and identified as stromal cells by immunofluorescence. Both the cells and blastocysts expressed MNSF 尾 gene. Co-culture of mouse endometrial stromal cells with blastocysts on day 4.5 of gestation showed that blastocysts could invade, adhere, spread out to stromal cells and finally fuse with them, similar to implantation process. MNSF 尾 antiserum could significantly inhibit the proliferation and adhesion of mouse blastocyst to endometrial stromal cells, and induce embryo death in a dose-dependent manner. 4. Human cervical squamous cell carcinoma cell line (HCC-94) was used to transfect MNSFP expression or siRNA, supernatant to stimulate mouse lymphocytes. It was proved that overexpression of MNSF 尾 could promote the secretion of IL-4, but inhibit the secretion of TNF 偽 by lymphocytes. On the contrary, silencing or neutralizing MNSF 尾 with antibodies inhibited the secretion of IL-4 and promoted the secretion of TNF 偽, suggesting that MNSF 尾 might participate in maternal and fetal immune tolerance. 5. The MNSF 尾 conditioned gene knockout mice were established and mated with Cre mice to obtain heterozygous MNSF 尾 gene knockout mice. Mating experiments showed that MNSF 尾 partial gene deficiency resulted in a significant decrease in fertility in mice, and only female mice were affected, and homozygous mice could not be obtained due to the imbalance of the ratio of male and female offspring. 6. By immunohistochemistry, Real-time PCR and WB confirmed that the MNSF 尾 mRNA in the uterine tissues of heterozygous mice had no significant change compared with that of wild type mice, but its protein expression was significantly decreased. In addition, the uterine glandular epithelium of the heterozygous mice became loose and the lacunae widened significantly. 7. The number and development of embryos in the uterus were observed at different time points of embryo implantation and pregnancy (that is, 4 days and 14 days of pregnancy). The results showed that the number of embryos in the embryo implantation and early stage of embryo development was normal. But at the middle stage of development (11 and 14 days of gestation), the embryo showed obvious abnormality, that is, it lost the shape of the fetal mouse and pyknosed into a lump. The results suggest that MNSF 尾 is involved in embryonic development. These results suggest that MNSF 尾 is necessary in mouse embryo implantation and is associated with immunosuppression. However, partial defect or decreased expression of the gene can cause embryo implantation and developmental disorders in vivo and in vitro. Therefore, MNSF 尾 may become a potential target molecule for the development of new anti-fertility technologies.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R321
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