免疫抑制因子MNSFβ在胚胎着床与发育中的作用机制的研究
发布时间：2018-11-10 11:54
【摘要】：MNSFβ是本实验室新发现的一个着床相关因子,它是一种由抑制性T细胞合成的具有非特异性免疫抑制作用的淋巴因子,推测其可能与母胎间免疫耐受的形成有关。本课题主要研究MNSFβ的功能及其在胚胎着床中的作用,以进一步揭示胚胎着床的分子机制,从而为不孕、不育机制的研究、提高人工辅助生育技术以及开发研制抗着床避孕药物提供理论基础；并从中寻找潜在的可实现生育调控目的的新环节或新的靶分子。本课题主要结果如下： 1.由于抗MNSFβ抗体尚未市场化,本课题首先制备抗MNSFβ多克隆抗体,通过ELISA证实抗体效价为1：125000,并将之作为本研究的实验工具之一。 2.应用免疫组化,RT-PCR及Western blot技术证实MNSFβ表达于小鼠全身各组织脏器；在小鼠胚胎着床前、后及妊娠期间MNSFβ在子宫组织中的表达具有动态性变化,即在怀孕4.5天的子宫中表达减少,之后随怀孕时间的延长,表达逐渐增加,但到怀孕8.5天时,非着床位点的表达高于着床位点组织。 3.分离小鼠子宫内膜细胞,通过免疫荧光证实为基质细胞,并且该细胞和囊胚均表达MNSFβ基因；将小鼠子宫内膜基质细胞与孕4.5天的囊胚进行共培养,证实囊胚可向基质细胞侵袭,粘附,铺展,最终与其融合,类似着床过程；用MNSFβ抗血清可明显抑制小鼠囊胚向子宫内膜基质细胞的铺展与粘附生长,并可诱导胚胎死亡,且呈剂量反应关系。 4.选用人子宫颈鳞癌细胞系(HCC-94)转染MNSFP表达或siRNA,用其上清刺激小鼠淋巴细胞,证实过表达MNSFβ可促进淋巴细胞分泌IL-4,但抑制TNFα的分泌；与之相反,沉默或用抗体中和MNSFβ则抑制IL-4的分泌,却促进TNFα的分泌,提示MNSFβ可能参与母胎免疫耐受。 5.建立MNSFβ条件性基因剔除小鼠,与Cre小鼠交配,获得杂合型MNSFβ基因敲除小鼠。交配实验证实MNSFβ部分基因缺陷导致小鼠生育能力明显下降,且仅雌鼠受影响；子代雌雄比例失调,并不能得到纯合型小鼠。 6.通过免疫组化、Real-time PCR及WB证实杂合型小鼠子宫组织的MNSFβmRNA与野生型小鼠相比,无明显改变,但其蛋白表达却明显减少；此外,杂合型小鼠子宫腺上皮组织变得疏松,子宫腔腔隙也明显加宽。 7.选择胚胎着床与妊娠的不同时间点(即孕4、5、8、11和14天)观察子宫中的胚胎数量和发育情况,结果证实在胚胎着床和胚胎发育早期子宫中的胚胎数量正常,但在发育中期(孕11和14天)胚胎出现明显异常,即失去胎鼠的形态,并固缩成团块状。提示MNSFβ参与胚胎发育。 上述研究结果提示MNSFβ在小鼠胚胎着床过程中是必需的,且与免疫抑制相关。而该基因部分缺陷或表达减少,在体内外均可引起胚胎着床及发育障碍。因此,MNSFβ有可能成为发展新型抗生育技术的潜在靶分子。
[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 伪 by lymphocytes. On the contrary, silencing or neutralizing MNSF 尾 with antibodies inhibited the secretion of IL-4 and promoted the secretion of TNF 伪, 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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R321
本文编号：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 伪 by lymphocytes. On the contrary, silencing or neutralizing MNSF 尾 with antibodies inhibited the secretion of IL-4 and promoted the secretion of TNF 伪, 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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R321
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