【作者】 何亚萍；

【导师】 李卓娅； 王健；

【作者基本信息】 华中科技大学 ， 免疫学， 2012， 博士

【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。更多还原

【Abstract】 MNSFβ, a novel implantation-related factor newly identified in our lab, is a nonspecific immune inhibitory lymphokine produced by suppressor T cell.It was presumed that MNSFP could be related with the participation in the formation of immune tolerance between the embryo and maternal endometrial tissues. The subject mainly studied the function of MNSFβgene and explored the roles the gene played in the embryo implantation so as to further reveal the molecular mechanism of embryo implantation, provide the theoretical basis with the research of infertility mechanism, improvement of the artificial assisted reproductive technology and development of anti-implantation contraceptive drugs and probe for the new potential link or new target molecules to achieve the purposes of fertility regulation. The main results are as follows: 1. As the anti-MNSFβpolyclonal antibody was not commercial, it was prepared at first in the lab. The antibody titers were confirmed as 1:125000 by ELISA. The antibody was used as one of the experimental tools in this study.2. The immunohistochemistry(IHC), RT-PCR and Western blot techniques were applied to confirm that the MNSFβwas expressed in most tissues and organs of the mouse. In the periods of the mouse embryo preimplantation, implantation and pregnancy, the MNSFβexpression had its dynamic changes in its uterine tissues. The results showed that the MNSFβwas lowly expressed on day 4.5 of the pregnancy, the MNSFβexpression gradually increased with the prolongation of the pregnancy time, and the expression of the MNSFβwas much higher in the interimplantation sites than in the implantation sites on day 8.5.3. The primary mouse uterine endometrial cells were isolated by random cycling on day 4.5 pregnant mouse uterine from which blastocysts were flushed. The isolated cells were stromal cells identified by immunofluorescence assays. And the MNSFP was expressed in mouse blastocysts and endometrial stromal cells by the IHC method assay. The mouse endometrial stromal cells were co-cultured with the blastocysts of the day 4.5 pregnancy with the results of the spread, invasion, attachment and fusion of the mouse blastocysts to the endometrial stromal cells similar to implantation process in vivo, the process of which was observed daily under an inverted microscope. MNSFβantiserum was used to inhibit the spreading and adhesive growth of the mouse blastocysts to endometrial stromal cells and induce the death of the embryo, which presented a dose-dependent relationship.4. The transfection of MNSFβexpression plasmid or siRNA into HCC-94 cells and re-suspense of mouse lymphocytes in the culture medium from the different transfected HCC-94 cell supernatants showed that MNSFβcould promote the secretion of IL-4 and inhibit the secretion of TNFa from mouse lymphocytes.In contrast, the secretion of TNFa from lymphocytes sharply increased with the siRNA and MNSFP antibody blocking. The results suggested that MNSFβmight be involved in maternal-fetal immune tolerance.5. The heterozygous MNSFβknockout mice were obtained by mating of the MNSFβconditional gene knockout mice with Cre-mice. The mating experiments confirmed that part of the MNSFβgene defects led to the significant fertility decrease in mice, but only the fertility of the female mice was affected. The male and female ratio of their offspring mice was imbalanced and the homozygous mice could not be obtained.6. The methods of the IHC, Real-time PCR and Western blot also confirmed that the expression level of MNSFβmRNA in the heterozygous mice uterine tissue had no more significant change than that of wild-type mice, but the expression of MNSFP protein level was significantly reduced. In addition, the structure of the heterozygous mice uterine gland-epithelial tissues became very loose and the uterine cavities were significantly widened.7. The observation of the numbers of embryos in the uterus and their develop ment by selecting different times of the embryo implantation and pregnancy (i. e. day 4,5,8,11 and 14 of pregnancy) demonstrated the normality of the num bers of embryos in the uterus of embryo implantation and early embryonic develop ment, but some obvious abnormalities of the embryos appeared:loss of the shap e of fetal mice and formation of clumps shape in the period of the medium-ter m embryo development (day 11 and 14 of pregnancy). The results suggested th e MNSF P was involved in the embryonic development.To sum up, it could be concluded that the MNSFβwas required in th e process of embryo implantation in mice and related with immunosuppressio n. Part of the defects or reduction of the gene expression could cause its dis orders in the implantation of the embryo and development in vitro and in vi vo; therefore, it is possible that the MNSFβbecome the potential target m olecule for the development of a novel contraceptive technology.更多还原