【摘要】： 人胚胎植入是指從卵子受精到胚泡著床的一系列細胞或分子生物學事件,是一個極其復雜的生理過程,主要包括游離胚泡定位、黏附和侵入以及胎盤形成,是一個連續(xù)的動力生物學現(xiàn)象。研究表明,人早期妊娠過程實際上是在卵巢甾體激素的協(xié)同作用下,通過子宮和胚胎所表達的各種分子,在時間和空間上相互協(xié)同作用下完成的。作為侵入態(tài)的胚胎和接受態(tài)的子宮的同步發(fā)育是成功植入的關(guān)鍵或決定因素,也是胚胎植入調(diào)節(jié)的研究基礎(chǔ)。從目前的動物以及人類的研究資料,胚胎植入受多基因調(diào)控,涉及到數(shù)以百計的分子,形成網(wǎng)絡(luò)級聯(lián)系統(tǒng),這些分子之間可能有相互替代作用。作為侵入態(tài)的胚胎和接受態(tài)的子宮之間細胞因子、生長因子的動態(tài)平衡對于重建基質(zhì)和控制侵入是很關(guān)鍵的,同時也是胚胎植入調(diào)節(jié)的研究基礎(chǔ)。但是關(guān)于母胎界面這些分子的研究多局限于單個因子,而且有關(guān)外源性細胞因子以及激素對胚胎植入的調(diào)節(jié)作用以及細胞因子之間的相互作用的研究尚不多。本研究利用Luminex液相芯片技術(shù),同時測定人測定人早孕絨毛外滋養(yǎng)細胞和蛻膜基質(zhì)細胞兩類細胞培養(yǎng)上清液中l(wèi)eptin、TGF-β1、IL-6、bFGF、IL-1ra、VEGF、EGF、IL-1α的八種蛋白的表達量,探索人體正常情況下的胚胎植入的細胞和分子生物學機理,試圖尋找在人胚胎著床過程中同時起重要作用的多種關(guān)鍵性分子。 IL-1β是胚胎著床必需因子之一,可能是母胎對話的第一啟動因子,而且IL-1β作為一種重要的調(diào)節(jié)因子,通過與相應高親和力受體結(jié)合隨之引起第二波細胞因子的級聯(lián)瀑布效應,還可以影響到人體內(nèi)許多細胞因子的分泌。目前對IL-1β的調(diào)節(jié)作用多數(shù)是針對MMPs及其抑制劑。如何發(fā)揮其對母胎界面的 HCG是胚胎最早產(chǎn)生的分子之一,而且是表明胚胎存在的最特殊的標志。在植入過程中通過旁分泌模式下的局部作用影響妊娠。有研究發(fā)現(xiàn)HCG對新血管發(fā)生的重要細胞因子VEGF的刺激作用具有顯著性,并且HCG使得MMP-9顯著增加,但是不影響TIMP-1,從而有助于滋養(yǎng)細胞的進一步侵入。目前關(guān)于HCG對胚胎植入的影響多數(shù)使用絨癌細胞株進行探討,而用原代培養(yǎng)的滋養(yǎng)細胞和蛻膜細胞對其在早孕期的作用進行研究應該更有價值,但是少見文獻報道。故分別用IL-1β和HCG作為調(diào)節(jié)劑,研究它們對上述母胎兩類細胞分泌的上述八種細胞因子和生長因子的影響,以探索細胞因子和生長因子對滋養(yǎng)細胞侵襲、胚胎植入如何進行調(diào)控,尋找調(diào)控胚胎植入的細胞因子和生長因子網(wǎng)絡(luò)的分子機制,為胚胎植入和妊娠建立提供更多的理論依據(jù)。 第一部分人早孕期滋養(yǎng)細胞和蛻膜細胞表達的細胞因子及生長因子的差異 【目的】 研究人早孕期絨毛外細胞滋養(yǎng)層細胞和蛻膜基質(zhì)細胞分泌的多種細胞因子和生長因子的表達,探討這些細胞因子和生長因子在胚胎植入和妊娠建立過程中的作用。 【方法】 1.按孕周收集人正常早孕(6～9周)的絨毛和蛻膜標本,分別進行離體分離及培養(yǎng)。本實驗分為用0.125%胰蛋白酶和15IU/ml DNA酶,溫和消化絨毛組織;用0.25%胰蛋白酶-0.02%EDTA消化蛻膜組織,用2.5%-1.25%BSA純化絨毛外滋養(yǎng)層細胞和蛻膜基質(zhì)細胞,去除成纖維細胞、巨噬細胞等。得到的絨毛外滋養(yǎng)層細胞(extravillous cytotrophoblasts,EVCT)和蛻膜基質(zhì)細胞(decidualized stromal cells,DSC),按5×10~5/ml接種于分別鋪在24孔培養(yǎng)板中1ml無血清培養(yǎng)液培養(yǎng)24h,鑒定其純度在95%以上后,收集培養(yǎng)上清液。 2.倒置顯微鏡下觀察細胞生長狀態(tài)。 3.用SP免疫組化染色法鑒定細胞純度 將無菌蓋玻片置于培養(yǎng)皿中進行細胞爬片培養(yǎng),待細胞爬片基本長滿時,取出蓋玻片,PBS漂洗5min,3次,4%多聚甲醛4℃固定30 min,第一抗體分別為細胞角蛋白(CK7)和泌乳素(PRL)抗體,然后以鏈霉素抗生物素蛋白-過氧化酶(streptavidin-peroxide,SP)染色法進行染色。加酶底物DAB、H_2O_2顯色,光學顯微鏡觀察,陽性細胞胞質(zhì)呈棕黃色。操作按說明書進行,用PBS代替一抗做陰性對照。 4.利用Luminex xMAP技術(shù)測定EVCT組和DSC組培養(yǎng)上清液中8種細胞因子和生長因子:IL-1α、IL-1ra、IL-6、VEGF、TGF-β1、Leptin、bFGF、EGF的蛋白表達量。比較這些細胞因子在人正常早孕期絨毛外細胞滋養(yǎng)細胞與蛻膜基質(zhì)細胞中表達量的差異。 5.用SPSS10.0軟件進行統(tǒng)計學處理,獨立樣本t檢驗比較EVCT組和DSC組細胞因子和生長因子表達量的差異。顯著水平P＜0.05。 【結(jié)果】 1.DSC及EVCT在24孔培養(yǎng)板正常生長,SP法免疫組化證實DSC的泌乳素(prolactin,PRL)表達陽性,細胞質(zhì)內(nèi)可見大量棕黃色顆粒;EVCT的角蛋白7(cytokeratin7,CK7)表達為陽性,細胞膜可見大量棕黃色顆粒。EVCT和DSC鑒定純度均在95%以上,且生物學特性得以維持,可以用來實驗。 2.EVCT培養(yǎng)上清液中,占優(yōu)勢的細胞因子的含量由高到低依次是Leptin、TGF-β1、IL-6、bFGF、IL-1ra、VEGF、EGF、IL-1α。DSC培養(yǎng)上清液中,占優(yōu)勢的細胞因子的含量由高到低依次是Leptin、TGF-β1、bFGF、IL-1ra、L-6、EGF、IL-1α、VEGF。 3.EVCT和DSC分泌的細胞因子和生長因子的蛋白表達量,兩者之間除EGF外其他因子的表達經(jīng)統(tǒng)計學處理有顯著性差異。獨立t檢驗結(jié)果顯示:除EGF外,IL-1α、TIJ-1ra、IL-6、Leptin、VEGF、TGF-β1、bFGF差異均有統(tǒng)計學差異。 【結(jié)論】 人早孕期絨毛外滋養(yǎng)層細胞和蛻膜基質(zhì)細胞分泌Leptin、IL-6、bFGF、IL-1ra、EGF、VEGF、IL-1α、TGF-β1可能通過自分泌和/或旁分泌的方式參與了早期胚胎植入和妊娠建立過程。母胎兩類細胞中多種細胞因子和生長因子的表達水平不同,推測母胎之間通過這些因子進行對話,有利于細胞因子和生長因子的平衡,并在胚胎植入和妊娠建立過程中發(fā)揮了重要作用。 第二部分重組人白介素1β對人早孕期滋養(yǎng)細胞和蛻膜細胞分泌的細胞因子及生長因子的調(diào)節(jié)作用 【目的】 研究重組人IL-1β對人早孕期絨毛外滋養(yǎng)層細胞和蛻膜基質(zhì)細胞分泌的多種細胞因子和生長因子的影響,探討其對這些因子的調(diào)節(jié)作用,以及對胚胎植入和胎盤形成的影響。 【方法】 1.DSC和EVCT的體外培養(yǎng)方法及鑒定方法同前,于第一次換液時用1ml含10ng/ml IL-1β的無血清培養(yǎng)液培養(yǎng)24h后收集上清液。 2.倒置顯微鏡下觀察細胞生長狀態(tài)。 3.用SP免疫組化染色法進行細胞純度鑒定。(方法同第一部分3) 4.利用Luminex xMAP技術(shù)測定培養(yǎng)上清液中8種細胞因子和生長因子TGF-β1、EGF、IL-1α、IL-1ra、IL-6、bFGF、VEGF、Leptin的蛋白表達量。比較人早孕期絨毛外滋養(yǎng)層細胞與蛻膜基質(zhì)細胞表達的這些細胞因子在加入IL-1β前后蛋白表達量的變化。 5.用SPSS10.0軟件進行統(tǒng)計學處理,配對樣本t檢驗比較EVCT組和DSC組加入IL-1β前后細胞因子和生長因子表達量的差異,顯著水平P＜0.05。 【結(jié)果】 1.EVCT加入含調(diào)節(jié)因子IL-1β的培養(yǎng)液后收集的上清液,與EVCT空白組分泌的細胞因子和生長因子比較:IL-1α、Leptin、EGF表達量增加,而IL-1ra、IL-6、VEGF、TGF-β1、bFGF表達量減少。配對t檢驗結(jié)果顯示:IL-1α、Leptin的增加具有統(tǒng)計學意義,IL-1ra、IL-6、TGF-β1、bFGF、VEGF的減少具有統(tǒng)計學意義。 2.DSC加入含調(diào)節(jié)因子IL-1β培養(yǎng)液后收集的上清液,與DSC空白組分泌的細胞因子和生長因子比較:IL-1ra、Leptin表達量減少,而IL-1α、IL-6、VEGF、TGF-β1、bFGF、EGF表達量增加,其中IL-1ra、IL-6、bFGF、VEGF、Leptin的改變具有統(tǒng)計學意義。配對t檢驗結(jié)果顯示:IL-1ra、Leptin的增加有統(tǒng)計學意義,IL-6、VEGF、TGF-β1、bFGF的減少均有統(tǒng)計學意義。 【結(jié)論】 IL-1β對人早孕期絨毛外細胞滋養(yǎng)細胞和蛻膜基質(zhì)細胞分泌的多種細胞因子和生長因子表達的調(diào)節(jié)存在著差異,不同因子被上調(diào)、下調(diào)或無變化,提示在早孕期間母胎兩類細胞的細胞因子和生長因子網(wǎng)絡(luò)的錯綜復雜相互調(diào)節(jié)變化,可能有助于滋養(yǎng)細胞的浸潤,子宮內(nèi)膜的適度蛻膜化,母胎血管的形成,最終有利于胚胎植入和胎盤形成。 第三部分人絨毛膜促性腺激素對對人早孕期滋養(yǎng)細胞和蛻膜細胞分泌的細胞因子及生長因子的調(diào)節(jié)作用 【目的】 研究人絨毛膜促性腺激素(HCG)對早孕期滋養(yǎng)層細胞和蛻膜基質(zhì)細胞分泌的多種細胞因子和生長因子的影響,探討其對胚胎植入和妊娠維持中的作用。 【方法】 1.收集人正常早孕絨毛和蛻膜標本,分別進行離體培養(yǎng),鑒定其純度在95%以上。(方法同第一部分1)于第一次換液時用1ml含25U/ml人絨毛膜促性腺激素的無血清培養(yǎng)液培養(yǎng)24h后收集上清液。 2.倒置顯微鏡下觀察各細胞生長狀態(tài),并拍照。 3.用SP免疫組化染色法進行細胞純度鑒定。(方法同第一部分3) 4.利用Luminex xMAP技術(shù)測定培養(yǎng)上清液中8種細胞因子和生長因子TGF-β1、EGF、IL-1α、IL-1ra、IL-6、bFGF、VEGF、Leptin的蛋白表達量。比較人早孕期絨毛外細胞滋養(yǎng)層細胞與蛻膜基質(zhì)細胞中這些細胞因子在加入HCG后表達量的變化。 5.用SPSS10.0軟件進行統(tǒng)計學處理,配對樣本t檢驗比較EVCT組和DSC組加入HCG后細胞因子和生長因子表達量的差異,顯著水平P＜0.05。 【結(jié)果】 1.用人絨毛膜促性腺激素處理后,滋養(yǎng)細胞中IL-1α、VEGF、EGF表達量增加,而IL-1ra、IL-6、TGF-β1、FGF、Leptin表達量減少,其中IL-1α、IL-1ra、IL-6、VEGF、TGF-β1、Leptin、FGF的改變具有統(tǒng)計學意義。配對t檢驗結(jié)果顯示:IL-1α、VEGF、EGF的增加具有統(tǒng)計學意義,IL-1ra、IL-6、TGF-β1、Leptin、bFGF的減少具有統(tǒng)計學意義。 2.用人絨毛膜促性腺激素處理后,蛻膜細胞中IL-1α、TGF-β1、Leptin、FGF表達量減少,而IL-1ra、IL-6、VEGF、EGF表達量增加,其中IL-1α、IL-1ra、IL-6、Leptin、FGF的改變具有統(tǒng)計學意義。配對t檢驗結(jié)果顯示:IL-1α、Leptin、,bFGF、TGF-β1的增加具有統(tǒng)計學意義,IL-1ra、IL-6、VEGF、EGF的減少均有統(tǒng)計學意義。 【結(jié)論】 HCG通過旁分泌模式下的作用對母胎兩類細胞分泌的多種細胞因子和生長因子的不同調(diào)節(jié)作用,提示其可能通過對這些因子的影響,從而對母胎血管的發(fā)生,滋養(yǎng)細胞侵入以及胎盤形成等方面起著重要作用,最終有利于胚胎植入和妊娠的維持。
[Abstract]:Human embryo implantation is a series of cellular or molecular biological events from egg fertilization to implantation. It is an extremely complex physiological process, mainly including free blastocyst localization, adhesion and invasion, and placenta formation. It is a continuous dynamic biological phenomenon. The synchronous development of an invasive embryo and a receptive uterus is the key or determinant of successful implantation and the basis for the study of regulation of embryo implantation. Data show that embryo implantation is regulated by multiple genes, involving hundreds of molecules, forming a network cascade system, and these molecules may be substituted for each other. However, studies on these molecules at the maternal-fetal interface are mostly confined to single factors, and there are few studies on the regulation of exogenous cytokines and hormones on embryo implantation and the interaction between cytokines. The expression of leptin, TGF-beta 1, IL-6, bFGF, IL-1ra, VEGF, EGF and IL-1a in the supernatant of extravillous trophoblast and decidual stromal cells of early pregnancy was studied to explore the cellular and molecular biological mechanism of embryo implantation in normal human body, and to find many kinds of proteins that play important roles in embryo implantation. Key molecules.
IL-1beta is one of the essential factors for embryo implantation and may be the first promoter of maternal-fetal dialogue. As an important regulator, IL-1beta can also affect the secretion of many cytokines in the human body by binding to the corresponding high affinity receptors, resulting in cascade cascade effect of the second wave of cytokines. Most of the functions are directed against MMPs and its inhibitors.
HCG is one of the earliest molecules produced by embryos, and is the most specific marker of embryonic existence. It affects pregnancy through local paracrine effects during implantation. The effect of HCG on embryo implantation is mostly studied by choriocarcinoma cell lines, while the effect of primary cultured trophoblasts and decidual cells on early pregnancy should be more valuable, but rarely reported in literature. To investigate the effects of cytokines and growth factors on the above eight cytokines and growth factors secreted by the two types of maternal and fetal cells in order to explore how cytokines and growth factors affect the invasion of trophoblastic cells and how embryo implantation is regulated, and to find the molecular mechanisms regulating the network of cytokines and growth factors for embryo implantation and pregnancy. For more theoretical basis.
Part one differences in cytokines and growth factors expressed in trophoblasts and decidual cells during early pregnancy
[Objective]
To study the expression of cytokines and growth factors secreted by human extravillous trophoblast cells and decidual stromal cells during early pregnancy and to explore the role of these cytokines and growth factors in embryo implantation and pregnancy establishment.
[method]
1. Human chorionic villi and decidua from 6 to 9 weeks of gestation were isolated and cultured in vitro. The chorionic villi were mildly digested by 0.125% trypsin and 15IU/ml DNA enzyme, and the decidua were digested by 0.25% trypsin-0.02% EDTA and purified by 2.5% -1.25% BSA. The extravillous cytotrophoblasts (EVCT) and decidualized stromal cells (DSC) were cultured in a serum-free medium of 1 ml spread in a 24-well plate for 24 hours. The purity of the culture supernatant was over 95%.
2. cell growth was observed under inverted microscope.
3. identification of cell purity by SP immunohistochemical staining.
Cell culture was carried out by placing aseptic cover slides in a culture dish. When the slides were basically full, the cover slides were taken out and rinsed for 5 minutes, 3 times, and fixed for 30 minutes at 4 C with 4% paraformaldehyde. The first antibodies were cytokeratin (CK7) and prolactin (PRL) antibodies, then streptavidin-peroxidase (SP) antibodies. The staining method was used. The enzyme substrate DAB and H_2O_2 were added to stain. The cytoplasm of the positive cells was brown and yellow. The operation was carried out according to the instructions. PBS was used instead of primary antibody as negative control.
4. Luminex xMAP technique was used to determine the expression of 8 cytokines and growth factors: IL-1a, IL-1ra, IL-6, VEGF, TGF-beta 1, Leptin, bFGF and EGF in the supernatants of EVCT and DSC groups.
5. The expression of cytokines and growth factors in EVCT group and DSC group were compared by independent sample t test with SPSS10.0 software. The significant level was P < 0.05.
[results]
1. DSC and EVCT grew normally on the 24-well plate. Proactin (PRL) expression was positive in DSC, and a large number of brown-yellow granules were observed in cytoplasm, cytokeratin 7 (CK7) expression was positive in EVCT, and a large number of brown-yellow granules were found in cell membrane. The purity of both EVCT and DSC were above 95%, and the biological characteristics were obtained. Maintenance can be used for experiments.
2. In the supernatant of EVCT culture, the dominant cytokines were Leptin, TGF-beta 1, IL-6, bFGF, IL-1ra, VEGF, EGF, IL-1a. The dominant cytokines were Leptin, TGF-beta 1, bFGF, IL-1ra, L-6, EGF, IL-1a, VEGF.
3. The expression levels of cytokines and growth factors secreted by EVCT and DSC were significantly different except EGF. Independent t test showed that there were significant differences in the expression levels of IL-1a, TIJ-1ra, IL-6, Leptin, VEGF, TGF-beta 1 and bFGF except EGF.
[Conclusion]
Leptin, IL-6, bFGF, IL-1ra, EGF, VEGF, IL-1a, TGF-beta 1 secreted by human extravillous trophoblast cells and decidual stromal cells during early pregnancy may be involved in early embryo implantation and pregnancy establishment through autocrine and/or paracrine pathways. Dialogue among these factors is beneficial to the balance of cytokines and growth factors, and plays an important role in embryo implantation and pregnancy establishment.
Part II Regulatory effects of recombinant human interleukin-1 beta on cytokines and growth factors secreted by human trophoblasts and decidual cells in early pregnancy
[Objective]
Objective To study the effects of recombinant human IL-1 beta on cytokines and growth factors secreted by human extravillous trophoblast cells and decidual stromal cells in early pregnancy, and to explore its regulatory effects on these factors, as well as on embryo implantation and placenta formation.
[method]
1. The culture method and identification method of DSC and EVCT were the same as before. The supernatant was collected 24 hours after culture with 1 ml serum-free medium containing 10 ng/ml IL-1 beta.
2. cell growth was observed under inverted microscope.
3. the purity of cells was identified by SP immunohistochemical staining. (method is same as part one 3).
4. Protein expression of 8 cytokines and growth factors TGF-beta 1, EGF, IL-1a, IL-1ra, IL-6, bFGF, VEGF and Leptin in culture supernatant were measured by Luminex xMAP technique.
5. The expression of cytokines and growth factors in EVCT group and DSC group before and after adding IL-1 beta were compared by paired t-test with SPSS10.0 software. The significant level was P<0.05.
[results]
1. The expression of IL-1a, Leptin, EGF increased, while the expression of IL-1ra, IL-6, VEGF, TGF-beta 1, bFGF decreased. The results of paired t test showed that the expression of IL-1a, Leptin increased significantly, while IL-1ra, IL-6, TGF-beta 1, bFGF decreased. The decrease of GF and VEGF was statistically significant.
2. The expression of IL-1ra, Leptin decreased, while the expression of IL-1a, IL-6, VEGF, TGF-beta 1, bFGF and EGF increased. The changes of IL-1ra, IL-6, bFGF, VEGF and Leptin in the supernatant of DSC were significant. The increase of Leptin was statistically significant, and the decrease of IL-6, VEGF, TGF- beta 1 and bFGF were statistically significant.
[Conclusion]
Interleukin-1 beta regulates the expression of cytokines and growth factors secreted by human extrachorionic trophoblasts and decidual stromal cells in early pregnancy differently. Different cytokines are up-regulated, down-regulated or unchanged, suggesting that the cytokines and growth factor networks of the two types of cells in early pregnancy are complex and mutually regulated. It is conducive to the infiltration of trophoblast, the moderate decidualization of endometrium, the formation of maternal and fetal blood vessels, and ultimately conducive to embryo implantation and placenta formation.
Part III Regulation of human chorionic gonadotrophin on cytokines and growth factors secreted by trophoblasts and decidual cells in early pregnancy
[Objective]
Objective To study the effects of human chorionic gonadotrophin (HCG) on cytokines and growth factors secreted by trophoblast cells and decidual stromal cells in early pregnancy and its role in embryo implantation and pregnancy maintenance.
[method]
1. Human chorionic villi and decidua of normal early pregnancy were collected and cultured in vitro, the purity of which was more than 95%. (Methods The same as Part I 1) Supernatant was collected 24 hours after culture in serum-free medium containing 25 U/ml human chorionic gonadotrophin at the first fluid exchange.
2. the growth state of each cell was observed under inverted microscope and photographed.
3. the purity of cells was identified by SP immunohistochemical staining. (method is same as part one 3).
4. Protein expression of 8 cytokines and growth factors TGF-beta 1, EGF, IL-1a, IL-1ra, IL-6, bFGF, VEGF and Leptin in culture supernatant were measured by Luminex xMAP technique.
5. The expression of cytokines and growth factors in EVCT group and DSC group after adding HCG was compared by paired t-test with SPSS10.0 software, the significant level was P<0.05.
[results]
1. After treatment with human chorionic gonadotrophin, the expression of IL-1a, VEGF and EGF increased, while the expression of IL-1ra, IL-6, TGF-beta 1, FGF and Leptin decreased. The changes of IL-1a, IL-1ra, IL-6, VEGF, TGF-beta 1, Leptin and FGF were statistically significant. The reduction of TGF- beta 1, Leptin and bFGF was statistically significant.
2. After treatment with human chorionic gonadotrophin, the expression of IL-1a, TGF-beta 1, Leptin and FGF in decidual cells decreased, while the expression of IL-1ra, IL-6, VEGF and EGF increased, including the changes of IL-1a, IL-1ra, IL-6, Leptin and FGF.
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R321

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