本文選題：外周血單個(gè)核細(xì)胞(PBMC) + 反復(fù)種植失敗(RIF)　； 參考：《鄭州大學(xué)》2017年碩士論文

【摘要】：背景及目的由于不孕癥發(fā)病率逐漸升高,輔助生殖技術(shù)(ART)迅速發(fā)展,體外受精-胚胎移植(in vitro fertilization and embryo transfer,IVF-ET)及其衍生技術(shù)使眾多不孕癥患者借助獲得了后代[1]。其中一大部分夫婦在經(jīng)歷過幾次IVF治療后仍然不能成功妊娠,導(dǎo)致了嚴(yán)重的生活質(zhì)量下降,并且每一次失敗都會(huì)帶來巨大的財(cái)務(wù)負(fù)擔(dān)。反復(fù)著床失敗(repeated implantation Failure,RIF)已成為輔助生殖領(lǐng)域研究的熱點(diǎn)和難點(diǎn)[2]。胚胎成功著床的關(guān)鍵因素在于母體和胚胎之間的良好對(duì)話[3],因此如何改善胚胎質(zhì)量、提高子宮內(nèi)膜容受性,促進(jìn)母胎對(duì)話和免疫耐受成為了輔助生殖領(lǐng)域治療RIF的主要研究方向。最近Yoshioka等[4]首先報(bào)道了對(duì)反復(fù)著床失敗患者進(jìn)行宮腔灌注人外周血單個(gè)核細(xì)胞(Peripheral blood mononuclear cell,PBMC),結(jié)果顯示反復(fù)著床失敗患者的臨床妊娠率、著床率和活產(chǎn)率得到了顯著提高。PBMC主要包括T淋巴細(xì)胞、B淋巴細(xì)胞和單核細(xì)胞。PBMC分泌的IL-1α、IL-1β和TNF-α等細(xì)胞因子在胚胎著床中發(fā)揮著重要作用[5]。其機(jī)制可能與PBMC分泌的多種炎性因子在內(nèi)膜容受性上調(diào)和提高胚胎侵襲力有關(guān)。本課題通過體外培養(yǎng)實(shí)驗(yàn)和臨床隨機(jī)對(duì)照試驗(yàn)研究:1.體外培養(yǎng)條件下自體PBMC對(duì)RIF患者子宮內(nèi)膜容受性標(biāo)志物人白血病抑制因子LIF和整合素αVβ3表達(dá)的影響;2.PBMC對(duì)胚胎表面LIF受體表達(dá)的影響;3.研究自體PBMC宮腔灌注對(duì)RIF患者行凍融胚胎復(fù)蘇移植(Frozen-thawed embryo transplantation,FET)助孕妊娠結(jié)局的影響。研究方法1、體外培養(yǎng)條件下自體PBMC對(duì)RIF患者子宮內(nèi)膜容受性標(biāo)志物L(fēng)IF和整合素αVβ3表達(dá)的影響1)選擇2015年5月至2016年5月于鄭州大學(xué)第二附屬醫(yī)院反復(fù)種植失敗患者12例,使用內(nèi)膜取樣器收集患者月經(jīng)第14天子宮內(nèi)膜組織,分離子宮內(nèi)膜腺上皮和間質(zhì)細(xì)胞,體外培養(yǎng)傳至2-3代后冷凍保存。同時(shí)收集其外周血使用Ficoll分離液分離出PBMC細(xì)胞。2)以1×105個(gè)/ml密度將子宮內(nèi)膜細(xì)胞接種于四孔板,37℃,5%CO2培養(yǎng)24h后,更換含雌孕激素培養(yǎng)液1ml,加雌孕激素培養(yǎng)液當(dāng)日記作Day0。3)體外培養(yǎng)至Day2時(shí),實(shí)驗(yàn)組加入以密度為3×106/ml的PBMC細(xì)胞懸液0.5ml。對(duì)照組加入等量培養(yǎng)液。繼續(xù)體外培養(yǎng)至Day6。4)隔日換液,并分別于Day1、3、5收取培養(yǎng)上清液待測(cè)。5)應(yīng)用酶聯(lián)免疫法檢測(cè)培養(yǎng)液中LIF和整合素αVβ3的濃度。2、PBMC對(duì)體外胚胎和子宮內(nèi)膜共培養(yǎng)模型中胚胎表面LIF受體表達(dá)的影響1)收集簽署廢棄胚胎知情同意書的胚胎,其中Day3胚胎共58枚。2)用含雌孕激素的培養(yǎng)液體外培養(yǎng)子宮內(nèi)膜細(xì)胞,模擬人體子宮內(nèi)膜環(huán)境,加雌孕激素培養(yǎng)液當(dāng)日記作Day0。3)A組(實(shí)驗(yàn)組):培養(yǎng)至Day2時(shí),觀察子宮內(nèi)膜貼壁情況,實(shí)驗(yàn)組加入以密度為3×106/ml的PBMC細(xì)胞0.5ml,Day3加入胚胎,共24枚,每孔2枚,養(yǎng)至Day6,觀察囊胚形成情況。4)B組(對(duì)照組):培養(yǎng)至Day2時(shí)添加等量培養(yǎng)液,Day3加入胚胎,共24枚,每孔2枚,養(yǎng)至Day6,觀察囊胚形成情況。5)C組(普通胚胎組):共10枚,不進(jìn)行培養(yǎng),將胚胎復(fù)蘇后直接進(jìn)行檢測(cè)。6)應(yīng)用免疫熒光方法檢測(cè)細(xì)胞期胚胎和囊胚表面LIFR的表達(dá)情況。3、自體PBMC宮腔灌注對(duì)RIF患者FET妊娠結(jié)局的影響1)研究對(duì)象:選取2015.1月至2016.12來我院生殖中心就診的符合反復(fù)種植失敗診斷的患者。2)分組情況:將符合納入條件的患者根據(jù)就診時(shí)間排序并編號(hào)后,應(yīng)用隨機(jī)數(shù)表法進(jìn)行隨機(jī)分組,隨機(jī)分為實(shí)驗(yàn)組和對(duì)照組,截至2016年12月,共入組78例,實(shí)驗(yàn)組A(56例)和對(duì)照組B(22例)。設(shè)置雙盲,研究對(duì)象和主治醫(yī)師均不知情分組情況。3)實(shí)驗(yàn)組于主導(dǎo)卵泡破裂日或服用雌激素內(nèi)膜厚度達(dá)到標(biāo)準(zhǔn)后進(jìn)行孕酮轉(zhuǎn)化當(dāng)日抽取自體外周血4 ml,分離PBMC,方法同第一部分,分別于移植前1天、3天B超引導(dǎo)下用胚胎移植管將200ul PBMC細(xì)胞懸液輕輕注入宮頸內(nèi)口上方。適時(shí)胚胎移植,保胎治療。對(duì)照組宮腔灌注等量培養(yǎng)液,適時(shí)胚胎移植并保胎移植。4)胚胎移植35日后行彩超檢查,超聲下看到孕囊胎芽為臨床妊娠。結(jié)果1、體外培養(yǎng)條件下自體PBMC對(duì)RIF患者子宮內(nèi)膜容受性標(biāo)志物L(fēng)IF和整合素αVβ3表達(dá)的影響1)培養(yǎng)第3天兩組培養(yǎng)液中LIF濃度無明顯差異(P0.05),培養(yǎng)第5天時(shí),與單獨(dú)子宮內(nèi)膜組相比,共培養(yǎng)組培養(yǎng)液中LIF濃度明顯升高(P0.05)。2)培養(yǎng)第3天兩組培養(yǎng)液中整合素αVβ3濃度無明顯差異(P0.05),培養(yǎng)第5天時(shí),與單獨(dú)子宮內(nèi)膜組相比,共培養(yǎng)組整合素培養(yǎng)液中αVβ3濃度明顯升高(P0.05)。2、PBMC對(duì)體外胚胎和子宮內(nèi)膜共培養(yǎng)模型中胚胎表面LIF受體表達(dá)的影響1)本研究共檢測(cè)D3胚胎10枚,為4/II—9/II,對(duì)照組囊胚10枚,級(jí)別為3BB-5BB,實(shí)驗(yàn)組共獲得12枚囊胚,級(jí)別為3BB-5BB。實(shí)驗(yàn)組和對(duì)照組囊胚形成率分別為:50.0%,41.7%,差異無統(tǒng)計(jì)學(xué)意義(P0.05)。2)三組免疫熒光OD值差異顯著P0.05;兩兩比較結(jié)果顯示:LIFR的免疫熒光OD值實(shí)驗(yàn)組囊胚對(duì)照組單獨(dú)胚胎組,P均0.05。3、自體PBMC宮腔灌注對(duì)RIF患者行FET助孕的妊娠結(jié)局1)PBMC治療組和對(duì)照組的平均年齡、不孕年限、不孕類型、既往移植周期數(shù)、既往移植優(yōu)胚數(shù)等相比,均無統(tǒng)計(jì)學(xué)差異(P0.05)。2)PBMC治療組的胚胎種植率和臨床妊娠率均高于對(duì)照組(P0.05)。結(jié)論1.自體PBMC可以提高RIF患者子宮內(nèi)膜細(xì)胞體外培養(yǎng)條件下LIF和整合素αVβ3的濃度,進(jìn)而上調(diào)子宮內(nèi)膜容受性。2.PBMC可以促進(jìn)子宮內(nèi)膜共培養(yǎng)條件下的胚胎表面LIF受體的表達(dá)。3.自體PBMC宮腔灌注可以提高RIF患者行FET助孕的胚胎種植率和臨床妊娠率。
[Abstract]:Background and objective the incidence of infertility is increasing, and assisted reproductive technology (ART) develops rapidly. In vitro fertilization and embryo transfer (in vitro fertilization and embryo transfer, IVF-ET) and its derivatization technology, many infertile patients with the aid of acquired offspring [1]. are still unable to be treated several times after IVF. Successful pregnancy leads to a serious decline in the quality of life and a huge financial burden for every failure. Repeated implantation Failure (RIF) has become a hot and difficult point in the research of assisted reproductive fields. The key factor in the successful implantation of [2]. embryos is the good dialogue between the mother and the embryo [3], so the key factor is the [3] between the mother and the embryo. How to improve the quality of the embryo, improve the receptivity of the endometrium, promote the maternal fetal dialogue and immune tolerance have become the main research direction in the treatment of RIF in the assisted reproductive field. Recently, Yoshioka and other [4] first reported the intrauterine perfusion of human peripheral blood mononuclear cells (Peripheral blood mononuclear cell, PBMC) for patients with repeated implantation failure. The clinical pregnancy rate, implantation rate and survival rate of patients with repeated implantation failure have been significantly improved..PBMC mainly includes T lymphocytes, IL-1 alpha, IL-1 beta and TNF- a secreted by B lymphocytes and mononuclear cells, IL-1 beta and TNF- alpha, which play an important role in embryo implantation and the mechanism may be associated with a variety of inflammatory factors secreted by PBMC. In vitro culture experiment and clinical randomized controlled trial study: 1. the effect of autologous PBMC on the expression of human leukemia inhibitory factor LIF and integrin alpha V beta 3 in endometrium receptive markers in patients with RIF in vitro, and the effect of 2.PBMC on the expression of LIF receptor on the embryo surface; 3 The effect of autologous PBMC intrauterine perfusion on the outcome of pregnancy induced pregnancy in RIF patients (Frozen-thawed embryo transplantation, FET). 1, the effect of autologous PBMC on the endometrial receptive markers LIF and the expression of integrin alpha V beta 3 in RIF patients under in vitro culture, 1) selected from May 2015 to May 2016. 12 cases of recurrent failure in the Second Affiliated Hospital of Zhengzhou University were used to collect endometrium and endometrium for fourteenth days in patients with endometrium, and the endometrial gland epithelium and interstitial cells were isolated and cultured in vitro to the 2-3 generation for cryopreservation. At the same time, the peripheral blood was collected and separated out of PBMC cell.2 by Ficoll separation solution. 1 x 105 /ml densities were collected. After inoculating endometrium cells into four orifice plates, 37 degrees C, 5%CO2 culture for 24h, and replacement of estrogen and progesterone culture solution 1ml, and estrogen and progesterone culture as Day0.3) in vitro culture to Day2, the experimental group added a PBMC cell suspension 0.5ml. control group with a density of 3 x 106/ml, and continued in vitro culture to Day6.4). The concentration of LIF and integrin alpha V beta 3 in the culture medium was detected by enzyme linked immunosorbent assay (.5), respectively,.2, PBMC on the expression of LIF receptor on the embryo surface in the co culture model of the embryo and endometrium 1) to collect the embryos of the informed consent form of the abandoned embryo, of which 58.2 in Day3 embryos were used to contain females. The endometrial cells were cultured outside the liquid of progesterone, the endometrial environment was simulated in the human body, and the estrogen and progesterone culture fluid was used as a diary of Day0.3) A group (experimental group). When cultured to Day2, the wall of the endometrium was observed. The experimental group was added to the PBMC cell 0.5ml of 3 x 106/ml, and Day3 was added to the embryo, 24, 2 of each hole, and observed to Day6. The formation of blastocysts (.4) group B (control group): adding equal amount of culture to Day2, Day3 adding embryos, 24 pieces, 2 holes, Day6,.5 of the blastocyst formation, C group (common embryo group): 10, no culture, and immediately after the embryo resuscitation to detect.6) using immunofluorescence method to detect the LIF cell embryos and blastocyst surface LIF The expression of R.3, the effect of autologous PBMC intrauterine perfusion on the outcome of FET pregnancy in RIF patients 1) the study was to select the cases of.2 in the patients who were diagnosed with repeated implantation failure in the reproductive center of our hospital from 2015.1 months to 2016.12: the random number table was used after the eligible patients were sorted and numbered according to the time of treatment. Groups were divided into experimental group and control group randomly. As of December 2016, 78 cases were enrolled in the group, A (56 cases) and control group B (22 cases) in the experimental group. The experimental group was set double blind, the research subjects and the chief physicians were unaware group.3). The experimental group was taken on the day of the leading follicle rupture day or the estrogen intima thickness reached the standard and took the autologous on the day of progesterone transformation. The peripheral blood was 4 ml, and the PBMC was separated from the first part. 1 days before the transplantation, the 200ul PBMC cell suspension was gently injected into the upper cervix of the cervix under the guidance of the embryo transfer tube on 3 days. The results were 1, the effect of autologous PBMC on the endometrial receptive marker LIF and the expression of integrin alpha V beta 3 in RIF patients was 1). There was no significant difference in LIF concentration in two groups of culture third days (P0.05). The culture medium of co culture group was compared with the individual endometrium group. There was no significant difference in the concentration of integrin alpha V beta 3 in the medium of medium LIF (P0.05).2). The concentration of alpha V beta 3 in the culture fluid of the co culture group was significantly increased (P0.05).2 and PBMC to the LIF receptor on the surface of the embryo and the endometrium culture in the culture medium of the co culture group at fifth days. The concentration of integrin alpha V beta 3 was not significantly different (P0.05) in the culture third days. 1) 10 D3 embryos were detected in this study, 4/II 9/II, 10 blastocysts in the control group and 3BB-5BB, and 12 blastocysts were obtained in the experimental group. The formation rate of the blastocyst in the experimental group and the control group was 50%, 41.7%, the difference was not statistically significant (P0.05).2) and the difference between the three groups of immunofluorescence was significant P0.05; 22 comparative knot. The results showed that the LIFR immunofluorescence o d experimental group had a single embryo group of blastocyst control group, P was 0.05.3, and the autologous PBMC uterine cavity perfusion was 1 for the FET pregnancy outcome of RIF patients. The average age of the PBMC treatment group and the control group, the number of infertility, the type of infertility, the number of previous transplantation cycles, and the number of previously transplanted embryos, were not statistically different (P0.05).2). The embryo implantation rate and clinical pregnancy rate in the PBMC treatment group were higher than those of the control group (P0.05). Conclusion 1. autologous PBMC can improve the concentration of LIF and integrin alpha V beta 3 under the culture conditions of endometrium in RIF patients, and then up regulation of endometrial receptive.2.PBMC can promote the expression of LIF receptor on the surface of endometrium under the condition of co culture. .3. autologous PBMC intrauterine perfusion can improve the embryo implantation rate and clinical pregnancy rate of FET in RIF patients.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R714.8

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