巨噬细胞在围着床期小鼠子宫中的变化及作用

发布时间：2018-06-24 21:26

本文选题：巨噬细胞 + F4/80　；参考：《南方医科大学》2011年硕士论文

【摘要】：[研究背景] 围着床期子宫内膜局部发生复杂的神经-内分泌-免疫调节,形成特殊的免疫微环境,在防止病原体感染的同时,调节子宫内膜的容受性,使之与胚胎发育同步,以利于胚胎着床及发育。虽然母胎间免疫耐受的形成机理至今还尚未研究清楚,但可以肯定的是,免疫细胞在母胎免疫耐受建立过程中有着非常重要的作用。巨噬细胞作为体内的一种重要免疫细胞,广泛参与各种免疫应答、免疫效应和免疫调节,对维持正常妊娠有着极为重要的意义。De等报道在动情周期和妊娠不同时期,子宫Mφ的数量发生了明显变化。以往研究表明子宫内大部分Mφ汇集于内膜表面,主要分布在螺旋动脉和腺体周围,而这些部位正是胚胎着床和蜕膜形成的始发点,而在非着床区域Mφ占内膜淋巴细胞的比例明显降低,推测子宫Mφ在围着床期间子宫内膜上时间-空间分布的特异性和胚胎着床点的富集状态可能与内膜蜕膜化密切相关,即Mφ不仅参与免疫调节,而且对蜕膜血管生成有调控作用。F4/80是小鼠成熟巨噬细胞表达的特异性抗原,定位于巨噬细胞的胞膜和胞质；而一氧化氮(NO)作为一种重要的信使分子和活性物质,对调节血管新生和发育有重要作用,Mφ是体内NO重要的合成、储存与释放来源。一氧化氮合成酶(nitric oxide synthase, NOS)作为NO合成的唯一限速酶,目前关于NO的研究多集中于NOS。NOS分离为3种独立的基因,分别命名为神经型NOS(nerval NOS, nNOS)、诱导型NOS (nducible NOS,iNOS)和内皮型NOS(endothelium NOS,eNOS).其中诱导型NOS也称为巨噬细胞型,仅在细胞因子及细菌脂多糖作用下表达,由巨噬细胞、胶质细胞等产生,主要作用是参与炎症反应、免疫细胞对病原体的防御和促进子宫内膜血管生长作用。而白血病抑制因子(LIF)作为一种被证实与胚胎着床密切相关的因子,实验发现：胚泡在LIF基因缺失的雌鼠子宫中不能着床,但是给予外源性LIF因子,可诱导部分胚胎着床成功。同时在小鼠实验中发现,妊娠4-5天的小鼠子宫内膜中LIF明显高表达,提示LIF基因表达与胚胎着床时间具有一致性,对胚胎着床有启动效应。选择LIF因子高表达位点研究Mφ的变化,进一步分析Mφ对胚胎着床的影响。迄今为止,尚未见到国内外对于围着床期不同部位子宫内膜Mφ主要功能及其调节机制的连续动态观察和较为全面的研究,因此,本研究选取围着床期D1.5-D6.5小鼠为研究对象,探讨围着床期小鼠子宫F4/80-Mφ和iNOS的分布范围变化,以及着床点和非着床点之间LIF.F4/80和iNOS的分布特点。旨在为生殖医学临床上迫切需要解决的着床障碍、器官移植提供新的思路,也为寻找和开发主动干预的免疫学新途径提供理论依据。 [目的] 建立围着床期小鼠的假孕模型为对照组；分别从组织和细胞水平上,研究小鼠妊娠早期外周血Mφ随妊娠时间的变化,胚胎着床前后子宫组织F4/80-Mφ和iNOS的分布特点,以及两者之间的关联性,从而分析子宫组织内膜着床点与非着床点Mφ.iNOS和LIF分布的差异,进一步探讨Mφ在着床过程中的作用。 [方法] 取性发育成熟的昆明种雌鼠,于动情期以雌雄比1：1的比例合笼,第二天清晨发现阴道栓者,记录为妊娠第0.5天(D0.5),依次类推,选取D1.5至D6.5的妊娠小鼠为正常妊娠组(实验组,N=30)；输精管结扎的雄鼠同动情期小鼠交配后小鼠为假孕组(对照组,N=30)。根据妊娠时间分组,取小鼠外周血和子宫标本,利用HE染色和透射电镜定性观察子宫组织形态和Mφ超微结构,然后分别用流式细胞术(flow cytometry,FCM)和免疫组织化学方法(SABC法)定量研究围着床期外周血Mφ、子宫组织Mφ和iNOS表达量的变化分布,以及着床点和非着床点部位LIF和Mφ、iNOS的分布差异。 [结果] (1)小鼠子宫解剖形态观察以及组织学表明假孕模型成功建立。 (2)外周血流式细胞术检测显示：实验组小鼠F4/80-Mφ数目显著高于对照组,差异有统计学意义(P=0.000)；外周血Mφ与子宫组织Mφ的相关性分析显示：两者存在负相关关系,但相关关系不密切。 (3)小鼠子宫F4/80-Mφ免疫组织化学定位分布：对照组F4/80-Mφ主要分布在子宫内膜,肌膜和外膜分布较少；实验组子宫F4/80-MφD4.5前主要分布于子宫内膜和肌膜,D4.5以后分布于外膜。Mφ数目在围着床期间不断发生变化,着床前D1.5-D3.5Mφ数目持续增加,Mφ数目于D4.5显著下降到最低值(86.53±27.720,P0.05)；内膜Mφ数目于D2.5达到高峰(344.6±66.410,P0.05),D4.5以后显著减少(P0.05)；肌膜Mφ数目在D2.5达到高峰(139.8±52.997),D4.5最少(58.8±22.33,P0.05)；外膜Mφ在D3.5达到峰值(150.2±32.98,P0.05),D4.5以后逐渐恢复正常水平。 (4)小鼠子宫iNOS免疫组织化学定位分布：对照组围着床期小鼠iNOS在子宫内膜和肌膜表达水平较高,外膜中较少见；实验组小鼠子宫中iNOS阳性细胞主要分布于子宫内膜,D4.5以后外膜表达量增高。iNOS阳性细胞数目随妊娠时间变化,内膜D2.5达到最高值(83.2±10.498),D3.5下降至D6.5达最低值(38.0±11.643,P0.05)；肌膜层D3.5为最低值(16.2±6.723,P0.05),D4.5以后恢复正常；外膜层D3.5达最高水平(68.8±17.712,P0.05),D4.5天以后iNOS阳性细胞数目有所减少,但均高于对照组(P0.05)。两变量相关分析显示：F4/80-Mφ和iNOS表达量之间存在正相关关系,但相关关系不密切(r=0.565,P=0.000)。二者在对照组均有较高水平的表达,妊娠后均开始增加,D4.5以后Mφ重新分布,数量均比着床前有所下降。 (5)小鼠子宫内膜F4/80-Mφ和LIF在着床点部位表达量均显著高于非着床点,差异有统计学意义(P=0.013,0.040)。iNOS阳性细胞在着床点的数目高于非着床点,但无显著性差异。 [结论] (1)本实验成功建立围着床期假孕小鼠模型,为进一步研究小鼠子宫巨噬细胞在围着床期胚胎植入过程中的作用与机制奠定了很好的基础。 (2)围着床期正常妊娠小鼠外周血巨噬细胞比例显著高于假孕小鼠,与子宫局部巨噬细胞的相关性不密切,提示围着床期子宫巨噬细胞的变化有别于外周血巨噬细胞的反应,外周血中巨噬细胞变化不能完全反映子宫组织巨噬细胞的数量变化。 (3)围着床期间小鼠子宫组织F4/80-Mφ及iNOS阳性细胞的变化趋势基本一致,着床前的均主要分布在子宫内膜,着床后阳性细胞数目均有所下降,分布于外膜,提示巨噬细胞可能通过iNOS的作用在着床前对子宫内膜血管新生有重要作用,着床后可能主要参与免疫耐受的形成。 (4)LIF作为子宫内膜着床点的标志性分子,与F4/80-Mφ的共同分布于小鼠子宫,二者在着床点的细胞数目均显著高于非着床点,这种着床点的高表达状态提示巨噬细胞可能对胚胎着床位点的选择有重要意义。
[Abstract]:[research background]
There is a complex neuroendocrine immunoregulation in the endometrium in the surrounding bed, which forms a special immune microenvironment. It regulates the receptivity of the endometrium while preventing the infection of the pathogen and synchronizes the embryonic development with the embryo to facilitate the implantation and development of the embryo, although the mechanism of the immune tolerance between the mother fetus has not yet been studied yet. But it is certain that immune cells play a very important role in the establishment of maternal fetal immune tolerance. Macrophages, as an important immune cell in the body, are widely involved in various immune responses, immune responses and immunomodulatory, and are very important for the maintenance of normal pregnancy, such as.De and other reports in the estrous cycle and pregnancy. At the same time, the number of M pHi in the uterus changed significantly. Previous studies showed that most of the M pHi in the uterus was gathered on the surface of the endometrium, mainly in the spiral arteries and glands, which were the starting points of the embryo implantation and decidua formation, while the proportion of M Phi in the non implantation area was significantly reduced, and M Phi was speculated. The specificity of the time space distribution on the endometrium and the preconcentration of the embryo implantation point during the implantation of the endometrium may be closely related to the endometriosis. That is, M Phi not only participates in the immunoregulation, but also regulates the formation of the decidua vascularization, and the.F4/80 is the specific antigen expressed in the mature macrophages of mice, located in the membrane of the macrophage and in the membrane of macrophages. Nitric oxide (NO), as an important messenger and active substance, plays an important role in regulating angiogenesis and development. M phi is an important synthesis of NO in the body and the source of storage and release. Nitric oxide synthase (NOS) is the only speed limiting enzyme synthesized by NO. Currently, the research on NO is mostly concentrated on NOS.NOS. 3 separate genes, named NOS (nerval NOS, nNOS), inducible NOS (nducible NOS, iNOS) and endothelial NOS (endothelium NOS, eNOS), are also called macrophage type, which are expressed only under the action of cytokines and bacterial lipopolysaccharide, and are produced by macrophages, glial cells and so on. The main role is to participate. Inflammation, the defense of the immune cells to the pathogen and the promotion of the growth of the endometrium, and the leukemia inhibitory factor (LIF) is a factor that is closely related to the implantation of the embryo. The experiment found that the blastocyst can not catch the bed in the uterus of the female rat with the deletion of LIF gene, but the exogenous LIF factor can induce some embryos to be induced. At the same time, it was found in mice that the LIF was highly expressed in the endometrium of the mouse endometrium on the 4-5 day of pregnancy, suggesting that the expression of LIF gene was consistent with the implantation time of the embryo and had a starting effect on the implantation of the embryo. The changes of M pHi in the high expression site of LIF factor were selected to further analyze the effect of M Phi on the implantation of embryos.
Up to now, there has not been a continuous dynamic observation and a more comprehensive study on the main function and regulation mechanism of endometrium M Phi at the different parts of the surrounding bed. Therefore, this study selects the D1.5-D6.5 mice in the surrounding bed period as the research object, and discusses the distribution of F4/80-M phi and iNOS in the substage of the surrounding bed, as well as the changes in the distribution of the F4/80-M phi and iNOS in the surrounding bed. The distribution of LIF.F4/80 and iNOS between the bed point and the non implantation point is designed to provide a new way of thinking for the implantation barrier which is urgently needed in the clinical practice of reproductive medicine, and to provide a theoretical basis for the search and development of a new immunological approach for active intervention.
[Objective]
The pseudo pregnancy model of the mice in the surrounding bed was established as the control group, and the changes of M Phi with the pregnancy time in the early pregnancy of the pregnant mice, the distribution of F4/80-M and iNOS in the uterus tissue before and after the implantation of the embryo, and the relationship between the two were studied on the level of tissue and cell, respectively, and the correlation between the implantation sites of the endometrium and the non implantation site M Phi was analyzed. The difference between the distribution of.INOS and LIF further explores the role of M in implantation.
[method]
In the estrous stage of Kunming female mice, the female mice were caged in the estrus period with the ratio of male to male to 1:1. The vaginal suppositories were found on the morning of second days, recorded as 0.5 days of pregnancy (D0.5), and the pregnant mice from D1.5 to D6.5 were selected as normal pregnancy group (experimental group, N=30), and the male mice with spermatodefered tube ligated with estrus mice were the pseudo pregnancy group. Control group, N=30). According to the time of pregnancy group, take the peripheral blood and uterus specimens of mice, use HE staining and transmission electron microscope to observe the morphology of the uterus and the ultrastructure of M phi, and then use flow cytometry (flow cytometry, FCM) and immunohistochemical method (SABC method) to study the peripheral blood M phi, M and iNOS of the uterus tissue, respectively. The distribution of expression, and the distribution difference of LIF and M, iNOS in implantation site and non implantation point.
[results]
(1) morphological observation of uterus and histology showed that the model of pregnancy was successfully established.
(2) peripheral blood flow cytometry showed that the number of F4/80-M pHi in the experimental group was significantly higher than that of the control group, and the difference was statistically significant (P=0.000). The correlation analysis between the peripheral blood M phi and the uterine tissue M Phi showed that there was a negative correlation between the two groups, but the correlation was not close.
(3) the distribution of immunohistochemical localization of F4/80-M pHi in mice uterus: F4/80-M pHi in the control group was mainly distributed in the endometrium, and the distribution of the musculi and epicardium was less; the experimental group was mainly distributed in the endometrium and the musculus membrane before F4/80-M [D4.5] in the experimental group. After D4.5, the number of.M pHi in the outer membrane was constantly changed during the surrounding bed, and the number of D1.5-D3.5M Phi before the implantation was measured. The number of M Phi decreased to the lowest value (86.53 + 27.720, P0.05), and the number of M pHi in the intima reached the peak (344.6 + 66.410, P0.05) and decreased significantly after D4.5 (P0.05). The number of M in the musculi membrane reached a peak in D2.5 (139.8 + 52.997), D4.5 (58.8 + 22.33, P0.05), and the outer membrane reached its peak (150.2 + 32.98,). After D4.5, the normal level was gradually restored.
(4) the immunohistochemical localization of iNOS in mice uterus: the expression level of iNOS in the endometrium and muscularis of the mice in the control group was higher, and the outer membrane was rare. The iNOS positive cells in the mice uterus were mainly distributed in the endometrium in the experimental group, and the number of.INOS positive cells in the outer membrane after D4.5 changed with the time of pregnancy, and the intima was D2. .5 reached the highest value (83.2 + 10.498), D3.5 decreased to the minimum value of D6.5 (38 + 11.643, P0.05), D3.5 of muscle membrane was the lowest (16.2 + 6.723, P0.05), and D4.5 returned to normal after D4.5, and the highest level of D3.5 in outer membrane (68.8 + 17.712, P0.05). The number of iNOS positive cells decreased after D4.5, but higher than that of the control group (P0.05). Two variable related points. The analysis showed that there was a positive correlation between F4/80-M phi and iNOS expression, but the correlation was not close (r=0.565, P=0.000). Two in the control group had a high level of expression, after pregnancy all began to increase, after D4.5 M Phi redistribution, the number was lower than before the implantation.
(5) the expression of F4/80-M phi and LIF in the implantation site of the endometrium of mice was significantly higher than that in the non implantation site, and the difference was statistically significant (P=0.013,0.040) the number of.INOS positive cells at the implantation point was higher than that of the non implantation point, but there was no significant difference.
[Conclusion]
(1) this experiment successfully established a model of pseudo pregnancy mice in the peri bed period, which laid a good foundation for the further study of the role and mechanism of the mouse uterine macrophages during the implantation of the surrounding bed embryos.
(2) the proportion of macrophages in peripheral blood of normal pregnant mice in the surrounding bed period was significantly higher than that of the pseudo pregnancy mice, and the correlation between the macrophages and macrophages in the uterus was not closely related, suggesting that the changes in the macrophages in the bed period were different from those of the peripheral blood macrophages. The change of macrophages in the peripheral blood could not fully reflect the number of macrophages in the uterus. Quantity changes.
(3) the change trend of F4/80-M phi and iNOS positive cells in the mouse uterus during the surrounding bed was basically the same. The pre implantation was mainly distributed in the endometrium. The number of positive cells after the implantation decreased and distributed in the outer membrane. It suggested that the macrophage may play an important role in the neovascularization of the endometrium before the bed by the role of iNOS. It may be mainly involved in the formation of immune tolerance later.
(4) as a marker of the implantation site of the endometrium, LIF is distributed in the mouse uterus with F4/80-M phi, and the number of cells at the implantation point of the two is significantly higher than that of the non implantation point. The high expression of this implantation point suggests that the macrophage may be of great significance to the selection of the bed point of the embryo.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R321

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