發(fā)布時間：2018-06-24 21:26

  本文選題：巨噬細(xì)胞 + F4/80　； 參考：《南方醫(yī)科大學(xué)》2011年碩士論文

【摘要】：[研究背景] 圍著床期子宮內(nèi)膜局部發(fā)生復(fù)雜的神經(jīng)-內(nèi)分泌-免疫調(diào)節(jié),形成特殊的免疫微環(huán)境,在防止病原體感染的同時,調(diào)節(jié)子宮內(nèi)膜的容受性,使之與胚胎發(fā)育同步,以利于胚胎著床及發(fā)育。雖然母胎間免疫耐受的形成機(jī)理至今還尚未研究清楚,但可以肯定的是,免疫細(xì)胞在母胎免疫耐受建立過程中有著非常重要的作用。巨噬細(xì)胞作為體內(nèi)的一種重要免疫細(xì)胞,廣泛參與各種免疫應(yīng)答、免疫效應(yīng)和免疫調(diào)節(jié),對維持正常妊娠有著極為重要的意義。De等報道在動情周期和妊娠不同時期,子宮Mφ的數(shù)量發(fā)生了明顯變化。以往研究表明子宮內(nèi)大部分Mφ匯集于內(nèi)膜表面,主要分布在螺旋動脈和腺體周圍,而這些部位正是胚胎著床和蛻膜形成的始發(fā)點(diǎn),而在非著床區(qū)域Mφ占內(nèi)膜淋巴細(xì)胞的比例明顯降低,推測子宮Mφ在圍著床期間子宮內(nèi)膜上時間-空間分布的特異性和胚胎著床點(diǎn)的富集狀態(tài)可能與內(nèi)膜蛻膜化密切相關(guān),即Mφ不僅參與免疫調(diào)節(jié),而且對蛻膜血管生成有調(diào)控作用。F4/80是小鼠成熟巨噬細(xì)胞表達(dá)的特異性抗原,定位于巨噬細(xì)胞的胞膜和胞質(zhì)；而一氧化氮(NO)作為一種重要的信使分子和活性物質(zhì),對調(diào)節(jié)血管新生和發(fā)育有重要作用,Mφ是體內(nèi)NO重要的合成、儲存與釋放來源。一氧化氮合成酶(nitric oxide synthase, NOS)作為NO合成的唯一限速酶,目前關(guān)于NO的研究多集中于NOS。NOS分離為3種獨(dú)立的基因,分別命名為神經(jīng)型NOS(nerval NOS, nNOS)、誘導(dǎo)型NOS (nducible NOS,iNOS)和內(nèi)皮型NOS(endothelium NOS,eNOS).其中誘導(dǎo)型NOS也稱為巨噬細(xì)胞型,僅在細(xì)胞因子及細(xì)菌脂多糖作用下表達(dá),由巨噬細(xì)胞、膠質(zhì)細(xì)胞等產(chǎn)生,主要作用是參與炎癥反應(yīng)、免疫細(xì)胞對病原體的防御和促進(jìn)子宮內(nèi)膜血管生長作用。而白血病抑制因子(LIF)作為一種被證實(shí)與胚胎著床密切相關(guān)的因子,實(shí)驗(yàn)發(fā)現(xiàn)：胚泡在LIF基因缺失的雌鼠子宮中不能著床,但是給予外源性LIF因子,可誘導(dǎo)部分胚胎著床成功。同時在小鼠實(shí)驗(yàn)中發(fā)現(xiàn),妊娠4-5天的小鼠子宮內(nèi)膜中LIF明顯高表達(dá),提示LIF基因表達(dá)與胚胎著床時間具有一致性,對胚胎著床有啟動效應(yīng)。選擇LIF因子高表達(dá)位點(diǎn)研究Mφ的變化,進(jìn)一步分析Mφ對胚胎著床的影響。 迄今為止,尚未見到國內(nèi)外對于圍著床期不同部位子宮內(nèi)膜Mφ主要功能及其調(diào)節(jié)機(jī)制的連續(xù)動態(tài)觀察和較為全面的研究,因此,本研究選取圍著床期D1.5-D6.5小鼠為研究對象,探討圍著床期小鼠子宮F4/80-Mφ和iNOS的分布范圍變化,以及著床點(diǎn)和非著床點(diǎn)之間LIF.F4/80和iNOS的分布特點(diǎn)。旨在為生殖醫(yī)學(xué)臨床上迫切需要解決的著床障礙、器官移植提供新的思路,也為尋找和開發(fā)主動干預(yù)的免疫學(xué)新途徑提供理論依據(jù)。 [目的] 建立圍著床期小鼠的假孕模型為對照組；分別從組織和細(xì)胞水平上,研究小鼠妊娠早期外周血Mφ隨妊娠時間的變化,胚胎著床前后子宮組織F4/80-Mφ和iNOS的分布特點(diǎn),以及兩者之間的關(guān)聯(lián)性,從而分析子宮組織內(nèi)膜著床點(diǎn)與非著床點(diǎn)Mφ.iNOS和LIF分布的差異,進(jìn)一步探討Mφ在著床過程中的作用。 [方法] 取性發(fā)育成熟的昆明種雌鼠,于動情期以雌雄比1：1的比例合籠,第二天清晨發(fā)現(xiàn)陰道栓者,記錄為妊娠第0.5天(D0.5),依次類推,選取D1.5至D6.5的妊娠小鼠為正常妊娠組(實(shí)驗(yàn)組,N=30)；輸精管結(jié)扎的雄鼠同動情期小鼠交配后小鼠為假孕組(對照組,N=30)。根據(jù)妊娠時間分組,取小鼠外周血和子宮標(biāo)本,利用HE染色和透射電鏡定性觀察子宮組織形態(tài)和Mφ超微結(jié)構(gòu),然后分別用流式細(xì)胞術(shù)(flow cytometry,FCM)和免疫組織化學(xué)方法(SABC法)定量研究圍著床期外周血Mφ、子宮組織Mφ和iNOS表達(dá)量的變化分布,以及著床點(diǎn)和非著床點(diǎn)部位LIF和Mφ、iNOS的分布差異。 [結(jié)果] (1)小鼠子宮解剖形態(tài)觀察以及組織學(xué)表明假孕模型成功建立。 (2)外周血流式細(xì)胞術(shù)檢測顯示：實(shí)驗(yàn)組小鼠F4/80-Mφ數(shù)目顯著高于對照組,差異有統(tǒng)計學(xué)意義(P=0.000)；外周血Mφ與子宮組織Mφ的相關(guān)性分析顯示：兩者存在負(fù)相關(guān)關(guān)系,但相關(guān)關(guān)系不密切。 (3)小鼠子宮F4/80-Mφ免疫組織化學(xué)定位分布：對照組F4/80-Mφ主要分布在子宮內(nèi)膜,肌膜和外膜分布較少；實(shí)驗(yàn)組子宮F4/80-MφD4.5前主要分布于子宮內(nèi)膜和肌膜,D4.5以后分布于外膜。Mφ數(shù)目在圍著床期間不斷發(fā)生變化,著床前D1.5-D3.5Mφ數(shù)目持續(xù)增加,Mφ數(shù)目于D4.5顯著下降到最低值(86.53±27.720,P0.05)；內(nèi)膜Mφ數(shù)目于D2.5達(dá)到高峰(344.6±66.410,P0.05),D4.5以后顯著減少(P0.05)；肌膜Mφ數(shù)目在D2.5達(dá)到高峰(139.8±52.997),D4.5最少(58.8±22.33,P0.05)；外膜Mφ在D3.5達(dá)到峰值(150.2±32.98,P0.05),D4.5以后逐漸恢復(fù)正常水平。 (4)小鼠子宮iNOS免疫組織化學(xué)定位分布：對照組圍著床期小鼠iNOS在子宮內(nèi)膜和肌膜表達(dá)水平較高,外膜中較少見；實(shí)驗(yàn)組小鼠子宮中iNOS陽性細(xì)胞主要分布于子宮內(nèi)膜,D4.5以后外膜表達(dá)量增高。iNOS陽性細(xì)胞數(shù)目隨妊娠時間變化,內(nèi)膜D2.5達(dá)到最高值(83.2±10.498),D3.5下降至D6.5達(dá)最低值(38.0±11.643,P0.05)；肌膜層D3.5為最低值(16.2±6.723,P0.05),D4.5以后恢復(fù)正常；外膜層D3.5達(dá)最高水平(68.8±17.712,P0.05),D4.5天以后iNOS陽性細(xì)胞數(shù)目有所減少,但均高于對照組(P0.05)。兩變量相關(guān)分析顯示：F4/80-Mφ和iNOS表達(dá)量之間存在正相關(guān)關(guān)系,但相關(guān)關(guān)系不密切(r=0.565,P=0.000)。二者在對照組均有較高水平的表達(dá),妊娠后均開始增加,D4.5以后Mφ重新分布,數(shù)量均比著床前有所下降。 (5)小鼠子宮內(nèi)膜F4/80-Mφ和LIF在著床點(diǎn)部位表達(dá)量均顯著高于非著床點(diǎn),差異有統(tǒng)計學(xué)意義(P=0.013,0.040)。iNOS陽性細(xì)胞在著床點(diǎn)的數(shù)目高于非著床點(diǎn),但無顯著性差異。 [結(jié)論] (1)本實(shí)驗(yàn)成功建立圍著床期假孕小鼠模型,為進(jìn)一步研究小鼠子宮巨噬細(xì)胞在圍著床期胚胎植入過程中的作用與機(jī)制奠定了很好的基礎(chǔ)。 (2)圍著床期正常妊娠小鼠外周血巨噬細(xì)胞比例顯著高于假孕小鼠,與子宮局部巨噬細(xì)胞的相關(guān)性不密切,提示圍著床期子宮巨噬細(xì)胞的變化有別于外周血巨噬細(xì)胞的反應(yīng),外周血中巨噬細(xì)胞變化不能完全反映子宮組織巨噬細(xì)胞的數(shù)量變化。 (3)圍著床期間小鼠子宮組織F4/80-Mφ及iNOS陽性細(xì)胞的變化趨勢基本一致,著床前的均主要分布在子宮內(nèi)膜,著床后陽性細(xì)胞數(shù)目均有所下降,分布于外膜,提示巨噬細(xì)胞可能通過iNOS的作用在著床前對子宮內(nèi)膜血管新生有重要作用,著床后可能主要參與免疫耐受的形成。 (4)LIF作為子宮內(nèi)膜著床點(diǎn)的標(biāo)志性分子,與F4/80-Mφ的共同分布于小鼠子宮,二者在著床點(diǎn)的細(xì)胞數(shù)目均顯著高于非著床點(diǎn),這種著床點(diǎn)的高表達(dá)狀態(tài)提示巨噬細(xì)胞可能對胚胎著床位點(diǎn)的選擇有重要意義。
[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.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R321

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