人胚胎植入过程中细胞因子和生长因子的表达及IL-1β和HCG的调节作用
发布时间：2018-09-11 20:16
【摘要】： 人胚胎植入是指从卵子受精到胚泡着床的一系列细胞或分子生物学事件,是一个极其复杂的生理过程,主要包括游离胚泡定位、黏附和侵入以及胎盘形成,是一个连续的动力生物学现象。研究表明,人早期妊娠过程实际上是在卵巢甾体激素的协同作用下,通过子宫和胚胎所表达的各种分子,在时间和空间上相互协同作用下完成的。作为侵入态的胚胎和接受态的子宫的同步发育是成功植入的关键或决定因素,也是胚胎植入调节的研究基础。从目前的动物以及人类的研究资料,胚胎植入受多基因调控,涉及到数以百计的分子,形成网络级联系统,这些分子之间可能有相互替代作用。作为侵入态的胚胎和接受态的子宫之间细胞因子、生长因子的动态平衡对于重建基质和控制侵入是很关键的,同时也是胚胎植入调节的研究基础。但是关于母胎界面这些分子的研究多局限于单个因子,而且有关外源性细胞因子以及激素对胚胎植入的调节作用以及细胞因子之间的相互作用的研究尚不多。本研究利用Luminex液相芯片技术,同时测定人测定人早孕绒毛外滋养细胞和蜕膜基质细胞两类细胞培养上清液中leptin、TGF-β1、IL-6、bFGF、IL-1ra、VEGF、EGF、IL-1α的八种蛋白的表达量,探索人体正常情况下的胚胎植入的细胞和分子生物学机理,试图寻找在人胚胎着床过程中同时起重要作用的多种关键性分子。 IL-1β是胚胎着床必需因子之一,可能是母胎对话的第一启动因子,而且IL-1β作为一种重要的调节因子,通过与相应高亲和力受体结合随之引起第二波细胞因子的级联瀑布效应,还可以影响到人体内许多细胞因子的分泌。目前对IL-1β的调节作用多数是针对MMPs及其抑制剂。如何发挥其对母胎界面的 HCG是胚胎最早产生的分子之一,而且是表明胚胎存在的最特殊的标志。在植入过程中通过旁分泌模式下的局部作用影响妊娠。有研究发现HCG对新血管发生的重要细胞因子VEGF的刺激作用具有显著性,并且HCG使得MMP-9显著增加,但是不影响TIMP-1,从而有助于滋养细胞的进一步侵入。目前关于HCG对胚胎植入的影响多数使用绒癌细胞株进行探讨,而用原代培养的滋养细胞和蜕膜细胞对其在早孕期的作用进行研究应该更有价值,但是少见文献报道。故分别用IL-1β和HCG作为调节剂,研究它们对上述母胎两类细胞分泌的上述八种细胞因子和生长因子的影响,以探索细胞因子和生长因子对滋养细胞侵袭、胚胎植入如何进行调控,寻找调控胚胎植入的细胞因子和生长因子网络的分子机制,为胚胎植入和妊娠建立提供更多的理论依据。 第一部分人早孕期滋养细胞和蜕膜细胞表达的细胞因子及生长因子的差异 【目的】 研究人早孕期绒毛外细胞滋养层细胞和蜕膜基质细胞分泌的多种细胞因子和生长因子的表达,探讨这些细胞因子和生长因子在胚胎植入和妊娠建立过程中的作用。 【方法】 1.按孕周收集人正常早孕(6～9周)的绒毛和蜕膜标本,分别进行离体分离及培养。本实验分为用0.125%胰蛋白酶和15IU/ml DNA酶,温和消化绒毛组织;用0.25%胰蛋白酶-0.02%EDTA消化蜕膜组织,用2.5%-1.25%BSA纯化绒毛外滋养层细胞和蜕膜基质细胞,去除成纤维细胞、巨噬细胞等。得到的绒毛外滋养层细胞(extravillous cytotrophoblasts,EVCT)和蜕膜基质细胞(decidualized stromal cells,DSC),按5×10~5/ml接种于分别铺在24孔培养板中1ml无血清培养液培养24h,鉴定其纯度在95%以上后,收集培养上清液。 2.倒置显微镜下观察细胞生长状态。 3.用SP免疫组化染色法鉴定细胞纯度 将无菌盖玻片置于培养皿中进行细胞爬片培养,待细胞爬片基本长满时,取出盖玻片,PBS漂洗5min,3次,4%多聚甲醛4℃固定30 min,第一抗体分别为细胞角蛋白(CK7)和泌乳素(PRL)抗体,然后以链霉素抗生物素蛋白-过氧化酶(streptavidin-peroxide,SP)染色法进行染色。加酶底物DAB、H_2O_2显色,光学显微镜观察,阳性细胞胞质呈棕黄色。操作按说明书进行,用PBS代替一抗做阴性对照。 4.利用Luminex xMAP技术测定EVCT组和DSC组培养上清液中8种细胞因子和生长因子:IL-1α、IL-1ra、IL-6、VEGF、TGF-β1、Leptin、bFGF、EGF的蛋白表达量。比较这些细胞因子在人正常早孕期绒毛外细胞滋养细胞与蜕膜基质细胞中表达量的差异。 5.用SPSS10.0软件进行统计学处理,独立样本t检验比较EVCT组和DSC组细胞因子和生长因子表达量的差异。显著水平P＜0.05。 【结果】 1.DSC及EVCT在24孔培养板正常生长,SP法免疫组化证实DSC的泌乳素(prolactin,PRL)表达阳性,细胞质内可见大量棕黄色颗粒;EVCT的角蛋白7(cytokeratin7,CK7)表达为阳性,细胞膜可见大量棕黄色颗粒。EVCT和DSC鉴定纯度均在95%以上,且生物学特性得以维持,可以用来实验。 2.EVCT培养上清液中,占优势的细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培养上清液中,占优势的细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分泌的细胞因子和生长因子的蛋白表达量,两者之间除EGF外其他因子的表达经统计学处理有显著性差异。独立t检验结果显示:除EGF外,IL-1α、TIJ-1ra、IL-6、Leptin、VEGF、TGF-β1、bFGF差异均有统计学差异。 【结论】 人早孕期绒毛外滋养层细胞和蜕膜基质细胞分泌Leptin、IL-6、bFGF、IL-1ra、EGF、VEGF、IL-1α、TGF-β1可能通过自分泌和/或旁分泌的方式参与了早期胚胎植入和妊娠建立过程。母胎两类细胞中多种细胞因子和生长因子的表达水平不同,推测母胎之间通过这些因子进行对话,有利于细胞因子和生长因子的平衡,并在胚胎植入和妊娠建立过程中发挥了重要作用。 第二部分重组人白介素1β对人早孕期滋养细胞和蜕膜细胞分泌的细胞因子及生长因子的调节作用 【目的】 研究重组人IL-1β对人早孕期绒毛外滋养层细胞和蜕膜基质细胞分泌的多种细胞因子和生长因子的影响,探讨其对这些因子的调节作用,以及对胚胎植入和胎盘形成的影响。 【方法】 1.DSC和EVCT的体外培养方法及鉴定方法同前,于第一次换液时用1ml含10ng/ml IL-1β的无血清培养液培养24h后收集上清液。 2.倒置显微镜下观察细胞生长状态。 3.用SP免疫组化染色法进行细胞纯度鉴定。(方法同第一部分3) 4.利用Luminex xMAP技术测定培养上清液中8种细胞因子和生长因子TGF-β1、EGF、IL-1α、IL-1ra、IL-6、bFGF、VEGF、Leptin的蛋白表达量。比较人早孕期绒毛外滋养层细胞与蜕膜基质细胞表达的这些细胞因子在加入IL-1β前后蛋白表达量的变化。 5.用SPSS10.0软件进行统计学处理,配对样本t检验比较EVCT组和DSC组加入IL-1β前后细胞因子和生长因子表达量的差异,显著水平P＜0.05。 【结果】 1.EVCT加入含调节因子IL-1β的培养液后收集的上清液,与EVCT空白组分泌的细胞因子和生长因子比较:IL-1α、Leptin、EGF表达量增加,而IL-1ra、IL-6、VEGF、TGF-β1、bFGF表达量减少。配对t检验结果显示:IL-1α、Leptin的增加具有统计学意义,IL-1ra、IL-6、TGF-β1、bFGF、VEGF的减少具有统计学意义。 2.DSC加入含调节因子IL-1β培养液后收集的上清液,与DSC空白组分泌的细胞因子和生长因子比较:IL-1ra、Leptin表达量减少,而IL-1α、IL-6、VEGF、TGF-β1、bFGF、EGF表达量增加,其中IL-1ra、IL-6、bFGF、VEGF、Leptin的改变具有统计学意义。配对t检验结果显示:IL-1ra、Leptin的增加有统计学意义,IL-6、VEGF、TGF-β1、bFGF的减少均有统计学意义。 【结论】 IL-1β对人早孕期绒毛外细胞滋养细胞和蜕膜基质细胞分泌的多种细胞因子和生长因子表达的调节存在着差异,不同因子被上调、下调或无变化,提示在早孕期间母胎两类细胞的细胞因子和生长因子网络的错综复杂相互调节变化,可能有助于滋养细胞的浸润,子宫内膜的适度蜕膜化,母胎血管的形成,最终有利于胚胎植入和胎盘形成。 第三部分人绒毛膜促性腺激素对对人早孕期滋养细胞和蜕膜细胞分泌的细胞因子及生长因子的调节作用 【目的】 研究人绒毛膜促性腺激素(HCG)对早孕期滋养层细胞和蜕膜基质细胞分泌的多种细胞因子和生长因子的影响,探讨其对胚胎植入和妊娠维持中的作用。 【方法】 1.收集人正常早孕绒毛和蜕膜标本,分别进行离体培养,鉴定其纯度在95%以上。(方法同第一部分1)于第一次换液时用1ml含25U/ml人绒毛膜促性腺激素的无血清培养液培养24h后收集上清液。 2.倒置显微镜下观察各细胞生长状态,并拍照。 3.用SP免疫组化染色法进行细胞纯度鉴定。(方法同第一部分3) 4.利用Luminex xMAP技术测定培养上清液中8种细胞因子和生长因子TGF-β1、EGF、IL-1α、IL-1ra、IL-6、bFGF、VEGF、Leptin的蛋白表达量。比较人早孕期绒毛外细胞滋养层细胞与蜕膜基质细胞中这些细胞因子在加入HCG后表达量的变化。 5.用SPSS10.0软件进行统计学处理,配对样本t检验比较EVCT组和DSC组加入HCG后细胞因子和生长因子表达量的差异,显著水平P＜0.05。 【结果】 1.用人绒毛膜促性腺激素处理后,滋养细胞中IL-1α、VEGF、EGF表达量增加,而IL-1ra、IL-6、TGF-β1、FGF、Leptin表达量减少,其中IL-1α、IL-1ra、IL-6、VEGF、TGF-β1、Leptin、FGF的改变具有统计学意义。配对t检验结果显示:IL-1α、VEGF、EGF的增加具有统计学意义,IL-1ra、IL-6、TGF-β1、Leptin、bFGF的减少具有统计学意义。 2.用人绒毛膜促性腺激素处理后,蜕膜细胞中IL-1α、TGF-β1、Leptin、FGF表达量减少,而IL-1ra、IL-6、VEGF、EGF表达量增加,其中IL-1α、IL-1ra、IL-6、Leptin、FGF的改变具有统计学意义。配对t检验结果显示:IL-1α、Leptin、,bFGF、TGF-β1的增加具有统计学意义,IL-1ra、IL-6、VEGF、EGF的减少均有统计学意义。 【结论】 HCG通过旁分泌模式下的作用对母胎两类细胞分泌的多种细胞因子和生长因子的不同调节作用,提示其可能通过对这些因子的影响,从而对母胎血管的发生,滋养细胞侵入以及胎盘形成等方面起着重要作用,最终有利于胚胎植入和妊娠的维持。
[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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R321
本文编号：2237741
[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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R321
【相似文献】
相关期刊论文 前10条
1 吴霞,李大金,袁敏敏,王明雁;人早孕期绒毛和绒毛外细胞滋养细胞的分离、培养及鉴定[J];生殖与避孕;2004年02期
2 贺银燕;贺晓菊;郭培奋;周雯慧;李大金;;早孕期人蜕膜趋化因子CCL2及其受体CCR2的表达及意义[J];中国免疫学杂志;2008年07期
3 朱晓明;韩涛;王雁玲;;人蜕膜基质细胞条件培养液对滋养细胞浸润功能的影响[J];医学争鸣;2010年05期
4 李蕾,李尚为,曹泽毅,彭芝兰,韩字研;胰岛素样生长因子系统在人早孕蜕膜基质细胞中的基因表达[J];山东医药;2002年01期
5 余秋波;李红梅;董艳玲;王应雄;何俊琳;;自然流产患者蜕膜MMP10的表达及临床意义[J];重庆医科大学学报;2007年07期
6 李蕾;李尚为;曹泽毅;彭芝兰;韩字研;;孕激素对人早孕蜕膜基质细胞IGF-Ⅱ基因表达的影响[J];山东医药;2008年19期
7 李尚为;李蕾;马谊;曹泽毅;彭芝兰;韩字研;;孕激素及胰岛素样生长因子对体外培养的人早孕蜕膜基质细胞增殖活性的影响[J];四川大学学报(医学版);2009年03期
8 史宏,刘伯宁;妊娠期及妊高征滋养细胞E-钙粘附素的表达[J];现代妇产科进展;2002年01期
9 黄煜;李大金;;人早孕蜕膜基质细胞及免疫活性细胞趋化因子受体CXCR6的表达[J];中华微生物学和免疫学杂志;2006年04期
10 徐英萍;王家富;李伟;宋文刚;;小鼠子宫蜕膜基质细胞的分离培养[J];中国组织工程研究与临床康复;2008年25期
相关会议论文 前2条
1 贺银燕;李大金;;早孕蜕膜和蜕膜基质细胞18种趋化因子受体的转录特征及CCR2／CCL2在人蜕膜和蜕膜基质细胞的表达[A];中国免疫学会第五届全国代表大会暨学术会议论文摘要[C];2006年
2 ;移植免疫[A];第七届全国免疫学学术大会论文集[C];2010年
相关博士学位论文 前4条
1 李明清;CD82在人早孕期母—胎界面的调节作用[D];复旦大学;2010年
2 周雯慧;环孢素A促进人早孕期母—胎界面交叉对话的分子机制[D];复旦大学;2007年
3 吴海霞;Th17细胞在人早孕期母—胎界面功能性调节作用[D];复旦大学;2009年
4 邢长英;补肾益气方调控人早孕滋养细胞SOCS3表达改善其生物学功能的研究[D];复旦大学;2006年
相关硕士学位论文 前10条
1 宋华东;人胚胎植入过程中细胞因子谱的变化及米非司酮、环孢素A对其的影响[D];第一军医大学;2007年
2 徐小飞;细菌内毒素脂多糖对人蜕膜基质细胞单核细胞趋化蛋白-1表达影响的研究[D];山东大学;2010年
3 刘晓东;氧浓度对蜕膜基质细胞成骨分化的影响[D];山东大学;2011年
4 何锦霞;人胚胎植入过程中细胞因子和生长因子的表达及IL-1β和HCG的调节作用[D];南方医科大学;2007年
5 张玉杰;TGF-β1对人蜕膜基质细胞趋化因子及其受体表达的影响[D];西北农林科技大学;2012年
6 刘晓宁;GnRH激动剂对人早孕蜕膜基质细胞和绒毛外滋养细胞多种基质金属蛋白酶表达的影响[D];南方医科大学;2007年
7 徐英萍;小鼠子宫蜕膜基质细胞生物学特性及其对树突状细胞增殖影响的研究[D];泰山医学院;2008年
8 魏艳秋;雌、孕激素对人早孕蜕膜基质细胞中BAFFR和Bcl-2/Bax表达的影响[D];山东大学;2009年
9 雷晶;吗啡对体外培养人早孕蜕膜基质细胞ERmRNA、PRmRNA、MORmRNA表达的影响[D];山西医科大学;2011年
10 秦立峗;人胚胎着床过程中细胞因子表达谱的变化及米非司酮对其的影响[D];第一军医大学;2006年
，本文编号：2237741
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2237741.html
最近更新
教材专著
