CD82在人早孕期母—胎界面的调节作用
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摘要
母-胎界面滋养细胞具有独特的类似于肿瘤细胞的生物学行为,即高增殖和侵袭能力。滋养细胞增殖与侵袭对于囊胚植入、胎盘发育,并建立恰当的母-胎关系至关重要。滋养细胞增殖与侵袭功能障碍是子痫前期、胎儿宫内生长受限(FGR)、自然流产等妊娠相关疾病的主要原因。但在正常妊娠,滋养细胞很少发生无限制增殖和远处转移,提示可能受到细胞间直接或间接的分子调控,使其促侵袭和抑制侵袭维持生理性动态平衡。母-胎界面微环境细胞与分子间的相互作用可以调节滋养细胞侵入子宫内膜及蜕膜。因此,母-胎界面参与调控滋养细胞侵袭力的关联分子,可以影响胚泡的正常植入和生长。
CD82蛋白是一种广泛表达的Tetraspanin超家族跨膜糖蛋白,是经典的肿瘤细胞侵袭抑制基因。已有研究证实CD82是细胞迁移的一个重要调节蛋白,CD82低表达与肿瘤细胞的侵袭和转移相关,致使多种晚期恶性肿瘤的不良预后。因此我们推测CD82可能参与母-胎界面滋养细胞侵袭能力的调控；解析CD82在母-胎界面的作用机制,及解析调控CD82表达的相关蛋白和基因将有助于阐明生理状态下胚泡植入和病理性滋养细胞疾病的发病机制,对治疗滋养细胞疾病亦具有潜在的临床应用价值。
本研究在前期工作基础上,进一步关注CD82作用于人早孕期滋养细胞的分子机制,拟阐明CD82对滋养细胞功能调控的分子机制；CD82表达调控,及其在母-胎界面促进滋养细胞和蜕膜基质细胞交叉对话,调节滋养细胞侵袭能力的分子机制。
目的分析CD82在正常和早期妊娠失败母-胎界面的表达及其表达调控。
方法收集人早孕期正常或不明原因自然流产的绒毛与蜕膜组织,应用本实验室建立的胰酶消化、密度梯度离心法分离、培养正常人早孕期滋养细胞和蜕膜基质细胞(DSCs)。采用RT-PCR、定量real-time RT-PCR、免疫组织化学、免疫细胞化学和传统免疫印迹杂交法,分别检测母-胎界面CD82的表达,比较分析CD82在正常早孕与早期妊娠失败母-胎界面的表达差异。对原代培养的蜕膜基质细胞进行多种干预,包括妊娠相关激素和炎性介质,用in-cell Western检测蜕膜基质细胞CD82的表达。
结果原代蜕膜基质细胞转录、翻译CD82；而原代滋养细胞不转录和翻译CD82。从mRNA和蛋白水平分析蜕膜组织中CD82的表达水平,发现正常早孕期蜕膜明显低于早期妊娠失败的蜕膜。生理浓度范围hCG能下调蜕膜基质细胞表达CD82(以10kU/L浓度作用最佳)；单独生理浓度孕激素和17β雌二醇对蜕膜基质细胞表达CD82无明显调节作用；但孕激素可以协同hCG进一步下调蜕膜基质细胞表达CD82。LPS促进蜕膜基质细胞表达CD82; IL-1β中和性抗体能拮抗LPS对蜕膜基质细胞表达CD82的促进作用。
结论CD82在人早孕期母-胎界面蜕膜基质细胞表达,明显低于早期妊娠失败蜕膜,提示母-胎界面CD82异常表达可导致妊娠失败。hCG下调蜕膜基质细胞表达CD82,提示CD82与正常妊娠有关,并可能参与控制滋养细胞疾病的发生、发展；LPS通过刺激炎性介质IL-1β的分泌,上调CD82的表达,提示CD82可介导炎症导致的自然流产。
目的解析CD82在人早孕期母-胎界面的生物学功能。
方法原代培养人早孕期蜕膜基质细胞和滋养细胞,利用siRNA干扰技术,成功沉默蜕膜基质细胞CD82的表达,并建立直接或间接共培养体系；利用质粒pCNA3.1(+)-CD82转染滋养细胞肿瘤细胞系BeWo,成功实现BeWo细胞过表达CD82。蜕膜基质细胞经沉默CD82后,与原代滋养细胞共培养,此后采用Matrigel侵袭试验分析滋养细胞的侵袭力。分析BeWo细胞经转染CD82后的侵袭力；分别用定量real-time RT-PCR、in-cell Western和免疫荧光法分析蜕膜基质细胞沉默CD82后,及BeWo细胞过表达CD82后,侵袭相关分子的mRNA和蛋白表达水平。
结果蜕膜基质细胞经CD82沉默后,与原代滋养细胞共培养,使滋养细胞侵袭力明显增强；蜕膜基质细胞表达TIMP1明显下调,integrinβ1和integrinavP3表达明显上调；而MMP2、MMP9、TIMP2和titin表达无明显改变。相反,转染CD82的BeWo细胞侵袭力明显减弱,TIMP1表达明显上调,integrinβ1和integrinαvβ3表达明显下调；同样,MMP2、MMP9、TIMP2和titin表达无明显差异。
结论蜕膜基质细胞表达CD82或滋养细胞过表达CD82均可控制滋养细胞的侵袭力,通过升调节TIMP1表达,实现滋养细胞与蜕膜基质细胞的交叉对话。
目的探讨CD82调节人滋养细胞侵袭性的信号通路。
方法应用siRNA转染蜕膜基质细胞沉默CD82的表达；质粒转染BeWo细胞过表达CD82,然后分析沉默及过表达CD82对侵袭相关的关键信号通路分子表达的调控作用。为了分析CD82抑制滋养细胞侵袭能性的信号通路,建立蜕膜基质细胞和原代滋养细胞共培养体系,及BeWo细胞单独培养体系,分别加入integrinβ1中和性抗体或MAPK/ERK信号通路阻断剂U0126,用Matrigel侵袭试验分析各组滋养细胞的侵袭力,以解析integrinβ1和MAPK/ERK1/2信号通路在CD82调节人早孕期滋养细胞侵袭中的作用；继而用in-cell Western分析各组TIMP1蛋白表达水平。
结果在蜕膜基质细胞与原代滋养细胞共培养系统中,蜕膜基质细胞经沉默CD82后,使滋养细胞侵袭力明显增加,pERK1/2与总ERK1/2的比值明显升高,integrinβ1表达明显增加,TIMP1表达明显下降；而integrinβ1中和性抗体和U0126则明显降低滋养细胞的侵袭力,能逆转CD82沉默对滋养细胞侵袭力的增强作用,解除对磷酸化ERK1/2的升调节作用,提示蜕膜基质细胞表达CD82通过抑制integrinβ1/MAPK/ERK1/2信号通路,控制人早孕期滋养细胞侵袭。integrinβ1中和性抗体和U0126处理也能有效逆转CD82对TIMP1表达的升调作用。同样,转染CD82的BeWo细胞经integrinβ1中和性抗体和U0126处理也呈现类似效果。TIMP1的表达水平与滋养细胞细胞的侵袭力呈直线负相关。
结论因此,CD82主要通过抑制integrinβ1/MAPK/ERK1/2信号通路,促进人早孕期蜕膜基质细胞表达TIMP1,上调的TIMP1依次抑制滋养细胞的侵袭能力。
目的分析人早孕期母-胎界面SDF-1/CXCR4对CD82表达调节作用。
方法收集人早孕期正常蜕膜和绒毛组织,对原代蜕膜基质细胞进行多种干预,包括原代滋养细胞培养上清、重组人SDF-1、重组人CCL2、抗人SDF-1、CXCR4、CCL2中和性抗体或CCR2拮抗剂。用in-cell Western检测蜕膜基质细胞CD82的表达。同时建立蜕膜基质细胞和原代滋养细胞共培养体系,同时引入抗人CXCR4中和性抗体,Matrigel侵袭试验分析共培养体系滋养细胞的侵袭力。
结果滋养细胞培养上清促进蜕膜基质细胞表达CD82；重组人SDF-1亦促进蜕膜基质细胞表达CD82; SDF-1或CXCR4中和性抗体可抑制滋养细胞上清对蜕膜基质细胞表达CD82的升调节作用。CXCR4中和性抗体直接处理滋养细胞明显降低其侵袭力；但CXCR4中和性抗体预处理蜕膜基质细胞后,共培养体系中滋养细胞的侵袭力明显增加；CXCR4中和性抗体分别预处理滋养细胞和蜕膜基质细胞后,共培养体系的滋养细胞侵袭力降低。此外,CXCR4中和性抗体预处理蜕膜基质细胞可以解除CD82对滋养细胞侵袭力的抑制作用。
结论人早孕期母-胎界面滋养细胞分泌SDF-1,通过白分泌方式促进自身侵袭；通过旁分泌升调节蜕膜基质细胞表达CD82,控制滋养细胞的过度侵袭,使其侵袭力维持在正常适度范围,借此介导母-胎界面滋养细胞与蜕膜基质细胞的交叉对话。
目的分析CsA调控母-胎界面CD82表达和滋养细胞侵袭力的分子机制。
方法收集人早孕期正常蜕膜和绒毛组织,用CsA处理原代蜕膜基质细胞,或滋养细胞经CsA预处理后收获培养上清；用抗人SDF-1或CXCR4中和性抗体处理蜕膜基质细胞,然后用in-cell Western检测蜕膜基质细胞CD82的表达。于滋养细胞和蜕膜基质细胞的单独和共培养体系加入CsA, ELISA法分析培养上清中SDF-1的分泌；用抗人CXCR4中和性抗体分别预处理蜕膜基质细胞和/或滋养细胞后,然后进行共培养,在加入CsA后,Matrigel侵袭试验分析共培养体系滋养细胞的侵袭力。
结果CsA对蜕膜基质细胞表达CD82无直接调节作用；但CsA能加强滋养细胞上清对蜕膜基质细胞表达CD82的促进作用；低浓度CsA能促进滋养细胞和共培养体系SDF-1的分泌；抗人SDF-1或CXCR4中和性抗体能抑制CsA通过滋养细胞对蜕膜基质细胞表达CD82的间接促进作用。在加入CsA的共培养体系中,抗人CXCR4中和性抗体预处理蜕膜基质细胞可以部分逆转CD82对滋养细胞侵袭力的抑制作用。
结论CsA促进滋养细胞分泌SDF-1,放大人早孕期母-胎界面滋养细胞对蜕膜基质细胞表达CD82的促进作用；CD82进而抑制滋养细胞侵袭能力。因此,CsA可协调母-胎界面滋养细胞与蜕膜基质细胞之间的交叉对话。
综上所述,本研究发现CD82对人早孕期母-胎界面具有多重调节作用：(1)通过抑制integrinβ1/MAPK/ERK1/2信号通路促进TIMP1表达,抑制滋养细胞侵袭；(2)蜕膜基质细胞通过表达CD82,控制滋养细胞过度侵袭；(3)蜕膜基质细胞CD82过表达可导致早期妊娠失败；(4)滋养细胞通过分泌SDF-1促进蜕膜基质细胞表达CD82,促进人早孕期母-胎界面滋养细胞和蜕膜基质细胞之间的交叉对话,适度控制滋养细胞侵袭能力；(5)CsA通过促进滋养细胞分泌SDF-1,升调节蜕膜基质细胞表达CD82,参与滋养细胞侵袭能力的精密调控。此外,人早孕期母胎界面CD82的表达还受妊娠相关激素的调控。本研究为完善滋养细胞侵袭调控机制,及临床治疗反复自然流产等相关妊娠疾病提供了新策略与新思路。
Implantation of human conceptus involves invasion of trophoblast cells into the uterine epithelium and the underlying stroma that undergoes a complex process of proliferation, migration and differentiation of embryo. A typical feature of placentation in humans is the trophoblasts with high degree invasion to gain access to the maternal circulation in the first trimester pregnancy. An impaired endovascular trophoblast invasion has been confirmed not only associated with pre-eclampsia, fetal intrauterine growth restriction, but also the first-trimester or late miscarriage.
Trophoblast cells display a very unique capability, and physiologically invade into the surrounding tissue that is similar to tumour. Trophoblast and tumor cells share the same biochemical mediators:MMPs and TIMPs. However, as opposed to malignant invasion, trophoblastic invasion during implantation and placentation is stringently controlled both in space and time. The decidua forms a dense cellular matrix that is believed to generate a local cytokine milieu that promotes trophoblast attachment and acts as a physical barrier limiting trophoblast over-invasion.
The CD82 gene (kangai1) encodes a 267 amino acid protein that contains four putative transmembrane domains. It is originally identified based on its function as a metastasis suppressor gene. CD82 plays an important role in inhibiting cancer cell motility, invasion and metastasis, and thus inhibits the formation of tumor metastases without affecting tumor growth. Gellersen et al have found that the expression of CD82 in decidual cell at human maternal-fetal interface is involved in decidual transformation from human endometrial stromal cells (ESCs). It is suggested that CD82 may be involved in the control of trophoblast cell invasion. Therefore, further investigation to elucidate the mechanism of CD82, and to find out how CD82 is regulated might help us to understand the regulation of embryonic implantation and trophoblastic invasion.
In the present study, we focus on the possible molecular mechanism of CD82 on human trophoblast invasiveness in the early pregnancy, and roles of the CD82 in the cross-talking between trophoblast cells and DSCs, which contributes to trophoblast migration and ultimately to successful pregnancy. The identification of these pathways and molecule mechanisms could provide novel targets for the diagnosis and treatment of pathological pregnancies, enabling the translation of basic research discoveries into clinical applications.
The expression of CD82 in tissues and cells was detected by semi-quantified RT-PCR, immunohistochemistry or immunocytochemical staining. We have demonstrated that the decidua and villi in the early pregnancy transcribe CD82 mRNA, but CD82 is only transcribed in primary DSCs, but not in primary trophoblast cells or BeWo cells (a choriocarcinoma cell line). The decidua as well as primary DSCs is stained positive of CD82 on the plasma membrane, but the villi, primary trophoblasts and BeWo cells are negative of CD82. The expression of CD82 in the decidua of normal early pregnancy and the unexplained miscarriage was detected by real-time PCR, immunohistochemistry and traditional western-blot, respectively. We have found that the mRNA level of CD82 in the decidua from the unexplained miscarriage is 97-fold higher than that of the normal early pregnancy (P<0.01). Consistent with transcription level, the decidua from the unexplained miscarriage has a much higher CD82 protein expression than that of the normal early pregnancy by immunohistochemistry and western blot (P<0.01), which suggests that the CD82 over-expression in decidua may restrict appropriate invasion of trophoblasts and lead to early pregnancy wastage.
After isolation and culture of human trophoblast cells and DSCs from the first-trimester pregnancy, the primary DSCs were treated by the pregnancy-associated hormones, estrogen, progesterone and hCG, or proinflammatory cytokines, respectively. Then the CD82 expression of DSCs was determined by in-cell Western. We have found that hCG attenuates the expression of CD82, the maximal inhibition occurs at a concentration of lOkU/L (P<0.05), and progesterone can enhance this effect (P<0.05), but estrogen or progesterone alone cannot modulate the CD82 expression (P>0.05). These results suggest that the syncytiotrophoblast cells secrete hCG that probably participates in regulating the invasion in situ of extravillous trophoblast cells by down-regulating the expression of CD82 in DSCs. Our observation also demonstrates that LPS can promote the CD82 expression through stimulating the IL-1βsecretion.
After isolation, purification and characterization of the first-trimester human trophoblast cells and DSCs, a model of direct and indirect co-culture was set up. The expression of CD82 in DSCs was interfered with siRNA, the BeWo cells were transfected with plasmid pcDNA3.1(+)-CD82, respectively. The effects of CD82 on invasion of trophoblast cells were investigated by Matrigel invasion assay, meanwhile, the invasion-related molecules in the transfected cells were detected by quantified real time RT-PCR, in-cell Western and immunofluorescence, respectively. We have found that the invasive index of human first-trimester trophoblast cells is significantly higher in co-culture with DSCs in CD82 silence than that of the si-negative control (P<0.01), and there is no difference between the direct and indirect co-culture (P>0.05), which suggests that the DSC-expressed CD82 can inhibit the invasion of human trophoblast cells by way of soluble molecules. Our observation also demonstrates that silencing of CD82 in DSCs significantly decreases the mRNA and protein levels of TIMP1 compared to the si-negative control (P<0.01 and P<0.05), and increase the transcription and protein levels of integrinβ1 and integrinαvβ3 (P<0.05). However, the mRNA and protein levels of MMP2, MMP9, TIMP2 and titin shows no statistical difference between the two groups (P>0.05). On the contrast, we have found that invasiveness of the CD82-expressed BeWo cells is significantly lower than that of the control (P<0.01). The TIMP1 mRNA and protein levels are increased, the integrinβ1 and integrinαvβ3 mRNA and protein levels are decreased after trophoblast cells are transfected with CD82 (P<0.05). Therefore, the CD82 inhibits the invasiveness of human trophoblasts in the first-trimester pregnancy through up-regulating the TIMP1 secretion.
After isolation, purification from the first-trimester pregnancy, human DSCs were interfered for CD82, the BeWo cells were transfected with plasmid pcDNA3.1(+)-CD82, respectively. Then the key signal transduction molecules associated with invasion were detected by in-cell Western to screen the possible signal pathway. We have found that integrinβ1 and MAPK/ERK1/2 pathway might play an important role. Thereafter, we treated the CD82-silenced DSCs and CD82-expressed BeWo cells with U0126, MAPK/ERK pathway inhibitor, or anti-integrinβ1 neutralizing antibody, and then the invasiveness of the co-cultured trophoblast cells or BeWo cells was analyzed by Matrigel invasive assay, and the TIMP1 expression in the target cells was analyzed by in-cell Western. It has been found that CD82 in DSCs and BeWo cells significantly suppresses the invasion of human trophoblast cells and promotes protein expression of TIMP1. Compared to the si-negative control, the proportion of phospho-ERK1/2 to total ERK1/2 in the CD82-silenced DSCs was significantly increased (P<0.01). The proportion of phospho-ERK1/2 to total ERK1/2 in the DSCs is decreased after treated with anti-integrinβ1 neutralizing antibody (P<0.01), and anti-integrinβ1 neutralizing antibody can inhibit the up-regulating effect of CD82 silence on phosphorylation of ERK1/2 in DSCs. The inhibitor of MAPK/ERK1/2 signaling pathway, U0126 or anti-integrinβ1 neutralizing antibody can significantly abolish the invasiveness increase induced by CD82 silence. Thereafter, we investigated whether the decreased TIMP1 was involved in the up-regulation of CD82 silence on invasion of trophoblast cells by statistical relationship analysis. It has been also found that the expression of TIMP1 is in negative relation to the invasiveness of trophoblast cells. Hence, it is concluded that the CD82 in DSCs regulates the expression of TIMP1, participates in intercellular communication with human trophoblast cells, and then controls trophoblast invasion by inactivating integrinβ1/MAPK/MAPK3/1 signaling.
After isolation and culture of human trophoblast cells and DSCs from the first-trimester pregnancy, the primary DSCs were treated with the supernatants derive of trophoblasts or/and recombinant human SDF-1, CCL2, anti-SDF-1 neutralizing antibody, anti-CXCR4 neutralizing antibody, anti-CCL2 neutralizing antibody or CCR2 antagonist, respectively. Then the CD82 expression in DSCs was determined by in-cell Western. We pre-treated trophoblasts and/or transfected DSCs with anti-CXCR4 neutralizing antibody, then constructed the co-culture of the two cells, and Matrigel invasion assay was used to detect the trophoblasts invasiveness in the co-culture. We have found that the supernatants from trophoblast cells can up-regulate the CD82 expression in DSCs (P<0.05), recombinant SDF-1 can promote the CD82 expression (P<0.05), and the CD82 expression in DSC is decreased after treated with anti-SDF-1 neutralizing antibody or anti-CXCR4 neutralizing antibody (P<0.05 or P<0.01). In the co-culture, the invasiveness of trophoblast cells was increased when DSCs were pre-treated with anti-CXCR4 neutralizing antibody (P<0.05). However, after trophoblast cells alone or in the co-culture were pre-treated with anti-CXCR4 neutralizing antibody, the trophoblast cells invasion was significantly decreased (P<0.05o r P<0.01).
Therefore, it can be concluded that SDF-1 secreted by trophoblasts can not only promote the invasiveness of them in an autocrine manner, but also control the over-invasiveness of them through up-regulating the CD82 expression in DSCs in a paracrine manner.
After isolation and culture of human trophoblast cells and DSCs from the first-trimester pregnancy, the primary DSCs were treated by CsA or the supernatants derived of the CsA-pretreated trophoblasts, and then treated with or without anti-SDF-1 neutralizing antibody or anti-CXCR4 neutralizing antibody, respectively. Thereafter, in-cell Western was used to evaluate the CD82 expression in DSCs. We constructed the trophoblasts, and DSCs respective culture or co-culture, and then detected the SDF-1 secretion in supernatants by ELISA assay. We also pre-treated trophoblasts and DSCs with anti-CXCR4 neutralizing antibody, then constructed the co-culture of the two cells, and then added by CsA. Matrigel invasion assay was used to detect the trophoblasts invasiveness. We have found that CsA cannot directly affect the KAI/CD82 expression in DSCs (P>0.05), but supernatants derived of the CsA pre-treated trophoblasts can further promote the CD82 expression in DSCs (P<0.01), and the anti-SDF-1 neutralizing antibody or anti-CXCR4 neutralizing antibody inhibits this effect (P<0.05 or P<0.01). CsA can stimulate the SDF-1 secretion in trophoblasts and co-culture unit (P<0.05 or P<0.01). When trophoblast cells alone or in the co-culture were pre-treated with anti-CXCR4 neutralizing antibody, the enhancement of trophoblast cells invasion induced by CsA was decreased (P<0.05 or P<0.01). Moreover, when DSCs were pre-treated with anti-CXCR4 neutralizing antibody, the increase of trophoblasts invasiveness induced by CsA was partly reversed (P<0.05). Therefore, CsA stimulates the SDF-1 secretion of trophoblasts, and enlarges the up-regulation of the trophoblast-derived SDF-1 on the KAI/CD82 expression in DSCs.
In conclusion, CD82 has multiple modulating effects at the maternal-fetal interface:(1) CD82 promotes the TIMP1 expression and in turn suppresses the invasiveness of human first-trimester trophoblasts through inactivating integrinβ1MAPK/ERK1/2 signal pathway; (2) trophoblast cells enhances the CD82 expression in DSCs by secreting SDF-1 in a paracrine manner, and controls the over-invasion of trophoblast. Therefore, CD82 can promote the cross-talk between trophoblast cells and DSCs; (3) CsA stimulates the SDF-1 secretion of trophoblasts, and further up-regulates the CD82 expression in DSCs. Moreover, the expression of CD82 is also regulated by pregnancy-associated hormones hCG. Our results may elucidate human trophoblast invasion regulation mechanism, and provide a new idea for therapeutics of some pregnancy complications, such as miscarriage.
CD82蛋白是一种广泛表达的Tetraspanin超家族跨膜糖蛋白,是经典的肿瘤细胞侵袭抑制基因。已有研究证实CD82是细胞迁移的一个重要调节蛋白,CD82低表达与肿瘤细胞的侵袭和转移相关,致使多种晚期恶性肿瘤的不良预后。因此我们推测CD82可能参与母-胎界面滋养细胞侵袭能力的调控；解析CD82在母-胎界面的作用机制,及解析调控CD82表达的相关蛋白和基因将有助于阐明生理状态下胚泡植入和病理性滋养细胞疾病的发病机制,对治疗滋养细胞疾病亦具有潜在的临床应用价值。
本研究在前期工作基础上,进一步关注CD82作用于人早孕期滋养细胞的分子机制,拟阐明CD82对滋养细胞功能调控的分子机制；CD82表达调控,及其在母-胎界面促进滋养细胞和蜕膜基质细胞交叉对话,调节滋养细胞侵袭能力的分子机制。
目的分析CD82在正常和早期妊娠失败母-胎界面的表达及其表达调控。
方法收集人早孕期正常或不明原因自然流产的绒毛与蜕膜组织,应用本实验室建立的胰酶消化、密度梯度离心法分离、培养正常人早孕期滋养细胞和蜕膜基质细胞(DSCs)。采用RT-PCR、定量real-time RT-PCR、免疫组织化学、免疫细胞化学和传统免疫印迹杂交法,分别检测母-胎界面CD82的表达,比较分析CD82在正常早孕与早期妊娠失败母-胎界面的表达差异。对原代培养的蜕膜基质细胞进行多种干预,包括妊娠相关激素和炎性介质,用in-cell Western检测蜕膜基质细胞CD82的表达。
结果原代蜕膜基质细胞转录、翻译CD82；而原代滋养细胞不转录和翻译CD82。从mRNA和蛋白水平分析蜕膜组织中CD82的表达水平,发现正常早孕期蜕膜明显低于早期妊娠失败的蜕膜。生理浓度范围hCG能下调蜕膜基质细胞表达CD82(以10kU/L浓度作用最佳)；单独生理浓度孕激素和17β雌二醇对蜕膜基质细胞表达CD82无明显调节作用；但孕激素可以协同hCG进一步下调蜕膜基质细胞表达CD82。LPS促进蜕膜基质细胞表达CD82; IL-1β中和性抗体能拮抗LPS对蜕膜基质细胞表达CD82的促进作用。
结论CD82在人早孕期母-胎界面蜕膜基质细胞表达,明显低于早期妊娠失败蜕膜,提示母-胎界面CD82异常表达可导致妊娠失败。hCG下调蜕膜基质细胞表达CD82,提示CD82与正常妊娠有关,并可能参与控制滋养细胞疾病的发生、发展；LPS通过刺激炎性介质IL-1β的分泌,上调CD82的表达,提示CD82可介导炎症导致的自然流产。
目的解析CD82在人早孕期母-胎界面的生物学功能。
方法原代培养人早孕期蜕膜基质细胞和滋养细胞,利用siRNA干扰技术,成功沉默蜕膜基质细胞CD82的表达,并建立直接或间接共培养体系；利用质粒pCNA3.1(+)-CD82转染滋养细胞肿瘤细胞系BeWo,成功实现BeWo细胞过表达CD82。蜕膜基质细胞经沉默CD82后,与原代滋养细胞共培养,此后采用Matrigel侵袭试验分析滋养细胞的侵袭力。分析BeWo细胞经转染CD82后的侵袭力；分别用定量real-time RT-PCR、in-cell Western和免疫荧光法分析蜕膜基质细胞沉默CD82后,及BeWo细胞过表达CD82后,侵袭相关分子的mRNA和蛋白表达水平。
结果蜕膜基质细胞经CD82沉默后,与原代滋养细胞共培养,使滋养细胞侵袭力明显增强；蜕膜基质细胞表达TIMP1明显下调,integrinβ1和integrinavP3表达明显上调；而MMP2、MMP9、TIMP2和titin表达无明显改变。相反,转染CD82的BeWo细胞侵袭力明显减弱,TIMP1表达明显上调,integrinβ1和integrinαvβ3表达明显下调；同样,MMP2、MMP9、TIMP2和titin表达无明显差异。
结论蜕膜基质细胞表达CD82或滋养细胞过表达CD82均可控制滋养细胞的侵袭力,通过升调节TIMP1表达,实现滋养细胞与蜕膜基质细胞的交叉对话。
目的探讨CD82调节人滋养细胞侵袭性的信号通路。
方法应用siRNA转染蜕膜基质细胞沉默CD82的表达；质粒转染BeWo细胞过表达CD82,然后分析沉默及过表达CD82对侵袭相关的关键信号通路分子表达的调控作用。为了分析CD82抑制滋养细胞侵袭能性的信号通路,建立蜕膜基质细胞和原代滋养细胞共培养体系,及BeWo细胞单独培养体系,分别加入integrinβ1中和性抗体或MAPK/ERK信号通路阻断剂U0126,用Matrigel侵袭试验分析各组滋养细胞的侵袭力,以解析integrinβ1和MAPK/ERK1/2信号通路在CD82调节人早孕期滋养细胞侵袭中的作用；继而用in-cell Western分析各组TIMP1蛋白表达水平。
结果在蜕膜基质细胞与原代滋养细胞共培养系统中,蜕膜基质细胞经沉默CD82后,使滋养细胞侵袭力明显增加,pERK1/2与总ERK1/2的比值明显升高,integrinβ1表达明显增加,TIMP1表达明显下降；而integrinβ1中和性抗体和U0126则明显降低滋养细胞的侵袭力,能逆转CD82沉默对滋养细胞侵袭力的增强作用,解除对磷酸化ERK1/2的升调节作用,提示蜕膜基质细胞表达CD82通过抑制integrinβ1/MAPK/ERK1/2信号通路,控制人早孕期滋养细胞侵袭。integrinβ1中和性抗体和U0126处理也能有效逆转CD82对TIMP1表达的升调作用。同样,转染CD82的BeWo细胞经integrinβ1中和性抗体和U0126处理也呈现类似效果。TIMP1的表达水平与滋养细胞细胞的侵袭力呈直线负相关。
结论因此,CD82主要通过抑制integrinβ1/MAPK/ERK1/2信号通路,促进人早孕期蜕膜基质细胞表达TIMP1,上调的TIMP1依次抑制滋养细胞的侵袭能力。
目的分析人早孕期母-胎界面SDF-1/CXCR4对CD82表达调节作用。
方法收集人早孕期正常蜕膜和绒毛组织,对原代蜕膜基质细胞进行多种干预,包括原代滋养细胞培养上清、重组人SDF-1、重组人CCL2、抗人SDF-1、CXCR4、CCL2中和性抗体或CCR2拮抗剂。用in-cell Western检测蜕膜基质细胞CD82的表达。同时建立蜕膜基质细胞和原代滋养细胞共培养体系,同时引入抗人CXCR4中和性抗体,Matrigel侵袭试验分析共培养体系滋养细胞的侵袭力。
结果滋养细胞培养上清促进蜕膜基质细胞表达CD82；重组人SDF-1亦促进蜕膜基质细胞表达CD82; SDF-1或CXCR4中和性抗体可抑制滋养细胞上清对蜕膜基质细胞表达CD82的升调节作用。CXCR4中和性抗体直接处理滋养细胞明显降低其侵袭力；但CXCR4中和性抗体预处理蜕膜基质细胞后,共培养体系中滋养细胞的侵袭力明显增加；CXCR4中和性抗体分别预处理滋养细胞和蜕膜基质细胞后,共培养体系的滋养细胞侵袭力降低。此外,CXCR4中和性抗体预处理蜕膜基质细胞可以解除CD82对滋养细胞侵袭力的抑制作用。
结论人早孕期母-胎界面滋养细胞分泌SDF-1,通过白分泌方式促进自身侵袭；通过旁分泌升调节蜕膜基质细胞表达CD82,控制滋养细胞的过度侵袭,使其侵袭力维持在正常适度范围,借此介导母-胎界面滋养细胞与蜕膜基质细胞的交叉对话。
目的分析CsA调控母-胎界面CD82表达和滋养细胞侵袭力的分子机制。
方法收集人早孕期正常蜕膜和绒毛组织,用CsA处理原代蜕膜基质细胞,或滋养细胞经CsA预处理后收获培养上清；用抗人SDF-1或CXCR4中和性抗体处理蜕膜基质细胞,然后用in-cell Western检测蜕膜基质细胞CD82的表达。于滋养细胞和蜕膜基质细胞的单独和共培养体系加入CsA, ELISA法分析培养上清中SDF-1的分泌；用抗人CXCR4中和性抗体分别预处理蜕膜基质细胞和/或滋养细胞后,然后进行共培养,在加入CsA后,Matrigel侵袭试验分析共培养体系滋养细胞的侵袭力。
结果CsA对蜕膜基质细胞表达CD82无直接调节作用；但CsA能加强滋养细胞上清对蜕膜基质细胞表达CD82的促进作用；低浓度CsA能促进滋养细胞和共培养体系SDF-1的分泌；抗人SDF-1或CXCR4中和性抗体能抑制CsA通过滋养细胞对蜕膜基质细胞表达CD82的间接促进作用。在加入CsA的共培养体系中,抗人CXCR4中和性抗体预处理蜕膜基质细胞可以部分逆转CD82对滋养细胞侵袭力的抑制作用。
结论CsA促进滋养细胞分泌SDF-1,放大人早孕期母-胎界面滋养细胞对蜕膜基质细胞表达CD82的促进作用；CD82进而抑制滋养细胞侵袭能力。因此,CsA可协调母-胎界面滋养细胞与蜕膜基质细胞之间的交叉对话。
综上所述,本研究发现CD82对人早孕期母-胎界面具有多重调节作用：(1)通过抑制integrinβ1/MAPK/ERK1/2信号通路促进TIMP1表达,抑制滋养细胞侵袭；(2)蜕膜基质细胞通过表达CD82,控制滋养细胞过度侵袭；(3)蜕膜基质细胞CD82过表达可导致早期妊娠失败；(4)滋养细胞通过分泌SDF-1促进蜕膜基质细胞表达CD82,促进人早孕期母-胎界面滋养细胞和蜕膜基质细胞之间的交叉对话,适度控制滋养细胞侵袭能力；(5)CsA通过促进滋养细胞分泌SDF-1,升调节蜕膜基质细胞表达CD82,参与滋养细胞侵袭能力的精密调控。此外,人早孕期母胎界面CD82的表达还受妊娠相关激素的调控。本研究为完善滋养细胞侵袭调控机制,及临床治疗反复自然流产等相关妊娠疾病提供了新策略与新思路。
Implantation of human conceptus involves invasion of trophoblast cells into the uterine epithelium and the underlying stroma that undergoes a complex process of proliferation, migration and differentiation of embryo. A typical feature of placentation in humans is the trophoblasts with high degree invasion to gain access to the maternal circulation in the first trimester pregnancy. An impaired endovascular trophoblast invasion has been confirmed not only associated with pre-eclampsia, fetal intrauterine growth restriction, but also the first-trimester or late miscarriage.
Trophoblast cells display a very unique capability, and physiologically invade into the surrounding tissue that is similar to tumour. Trophoblast and tumor cells share the same biochemical mediators:MMPs and TIMPs. However, as opposed to malignant invasion, trophoblastic invasion during implantation and placentation is stringently controlled both in space and time. The decidua forms a dense cellular matrix that is believed to generate a local cytokine milieu that promotes trophoblast attachment and acts as a physical barrier limiting trophoblast over-invasion.
The CD82 gene (kangai1) encodes a 267 amino acid protein that contains four putative transmembrane domains. It is originally identified based on its function as a metastasis suppressor gene. CD82 plays an important role in inhibiting cancer cell motility, invasion and metastasis, and thus inhibits the formation of tumor metastases without affecting tumor growth. Gellersen et al have found that the expression of CD82 in decidual cell at human maternal-fetal interface is involved in decidual transformation from human endometrial stromal cells (ESCs). It is suggested that CD82 may be involved in the control of trophoblast cell invasion. Therefore, further investigation to elucidate the mechanism of CD82, and to find out how CD82 is regulated might help us to understand the regulation of embryonic implantation and trophoblastic invasion.
In the present study, we focus on the possible molecular mechanism of CD82 on human trophoblast invasiveness in the early pregnancy, and roles of the CD82 in the cross-talking between trophoblast cells and DSCs, which contributes to trophoblast migration and ultimately to successful pregnancy. The identification of these pathways and molecule mechanisms could provide novel targets for the diagnosis and treatment of pathological pregnancies, enabling the translation of basic research discoveries into clinical applications.
The expression of CD82 in tissues and cells was detected by semi-quantified RT-PCR, immunohistochemistry or immunocytochemical staining. We have demonstrated that the decidua and villi in the early pregnancy transcribe CD82 mRNA, but CD82 is only transcribed in primary DSCs, but not in primary trophoblast cells or BeWo cells (a choriocarcinoma cell line). The decidua as well as primary DSCs is stained positive of CD82 on the plasma membrane, but the villi, primary trophoblasts and BeWo cells are negative of CD82. The expression of CD82 in the decidua of normal early pregnancy and the unexplained miscarriage was detected by real-time PCR, immunohistochemistry and traditional western-blot, respectively. We have found that the mRNA level of CD82 in the decidua from the unexplained miscarriage is 97-fold higher than that of the normal early pregnancy (P<0.01). Consistent with transcription level, the decidua from the unexplained miscarriage has a much higher CD82 protein expression than that of the normal early pregnancy by immunohistochemistry and western blot (P<0.01), which suggests that the CD82 over-expression in decidua may restrict appropriate invasion of trophoblasts and lead to early pregnancy wastage.
After isolation and culture of human trophoblast cells and DSCs from the first-trimester pregnancy, the primary DSCs were treated by the pregnancy-associated hormones, estrogen, progesterone and hCG, or proinflammatory cytokines, respectively. Then the CD82 expression of DSCs was determined by in-cell Western. We have found that hCG attenuates the expression of CD82, the maximal inhibition occurs at a concentration of lOkU/L (P<0.05), and progesterone can enhance this effect (P<0.05), but estrogen or progesterone alone cannot modulate the CD82 expression (P>0.05). These results suggest that the syncytiotrophoblast cells secrete hCG that probably participates in regulating the invasion in situ of extravillous trophoblast cells by down-regulating the expression of CD82 in DSCs. Our observation also demonstrates that LPS can promote the CD82 expression through stimulating the IL-1βsecretion.
After isolation, purification and characterization of the first-trimester human trophoblast cells and DSCs, a model of direct and indirect co-culture was set up. The expression of CD82 in DSCs was interfered with siRNA, the BeWo cells were transfected with plasmid pcDNA3.1(+)-CD82, respectively. The effects of CD82 on invasion of trophoblast cells were investigated by Matrigel invasion assay, meanwhile, the invasion-related molecules in the transfected cells were detected by quantified real time RT-PCR, in-cell Western and immunofluorescence, respectively. We have found that the invasive index of human first-trimester trophoblast cells is significantly higher in co-culture with DSCs in CD82 silence than that of the si-negative control (P<0.01), and there is no difference between the direct and indirect co-culture (P>0.05), which suggests that the DSC-expressed CD82 can inhibit the invasion of human trophoblast cells by way of soluble molecules. Our observation also demonstrates that silencing of CD82 in DSCs significantly decreases the mRNA and protein levels of TIMP1 compared to the si-negative control (P<0.01 and P<0.05), and increase the transcription and protein levels of integrinβ1 and integrinαvβ3 (P<0.05). However, the mRNA and protein levels of MMP2, MMP9, TIMP2 and titin shows no statistical difference between the two groups (P>0.05). On the contrast, we have found that invasiveness of the CD82-expressed BeWo cells is significantly lower than that of the control (P<0.01). The TIMP1 mRNA and protein levels are increased, the integrinβ1 and integrinαvβ3 mRNA and protein levels are decreased after trophoblast cells are transfected with CD82 (P<0.05). Therefore, the CD82 inhibits the invasiveness of human trophoblasts in the first-trimester pregnancy through up-regulating the TIMP1 secretion.
After isolation, purification from the first-trimester pregnancy, human DSCs were interfered for CD82, the BeWo cells were transfected with plasmid pcDNA3.1(+)-CD82, respectively. Then the key signal transduction molecules associated with invasion were detected by in-cell Western to screen the possible signal pathway. We have found that integrinβ1 and MAPK/ERK1/2 pathway might play an important role. Thereafter, we treated the CD82-silenced DSCs and CD82-expressed BeWo cells with U0126, MAPK/ERK pathway inhibitor, or anti-integrinβ1 neutralizing antibody, and then the invasiveness of the co-cultured trophoblast cells or BeWo cells was analyzed by Matrigel invasive assay, and the TIMP1 expression in the target cells was analyzed by in-cell Western. It has been found that CD82 in DSCs and BeWo cells significantly suppresses the invasion of human trophoblast cells and promotes protein expression of TIMP1. Compared to the si-negative control, the proportion of phospho-ERK1/2 to total ERK1/2 in the CD82-silenced DSCs was significantly increased (P<0.01). The proportion of phospho-ERK1/2 to total ERK1/2 in the DSCs is decreased after treated with anti-integrinβ1 neutralizing antibody (P<0.01), and anti-integrinβ1 neutralizing antibody can inhibit the up-regulating effect of CD82 silence on phosphorylation of ERK1/2 in DSCs. The inhibitor of MAPK/ERK1/2 signaling pathway, U0126 or anti-integrinβ1 neutralizing antibody can significantly abolish the invasiveness increase induced by CD82 silence. Thereafter, we investigated whether the decreased TIMP1 was involved in the up-regulation of CD82 silence on invasion of trophoblast cells by statistical relationship analysis. It has been also found that the expression of TIMP1 is in negative relation to the invasiveness of trophoblast cells. Hence, it is concluded that the CD82 in DSCs regulates the expression of TIMP1, participates in intercellular communication with human trophoblast cells, and then controls trophoblast invasion by inactivating integrinβ1/MAPK/MAPK3/1 signaling.
After isolation and culture of human trophoblast cells and DSCs from the first-trimester pregnancy, the primary DSCs were treated with the supernatants derive of trophoblasts or/and recombinant human SDF-1, CCL2, anti-SDF-1 neutralizing antibody, anti-CXCR4 neutralizing antibody, anti-CCL2 neutralizing antibody or CCR2 antagonist, respectively. Then the CD82 expression in DSCs was determined by in-cell Western. We pre-treated trophoblasts and/or transfected DSCs with anti-CXCR4 neutralizing antibody, then constructed the co-culture of the two cells, and Matrigel invasion assay was used to detect the trophoblasts invasiveness in the co-culture. We have found that the supernatants from trophoblast cells can up-regulate the CD82 expression in DSCs (P<0.05), recombinant SDF-1 can promote the CD82 expression (P<0.05), and the CD82 expression in DSC is decreased after treated with anti-SDF-1 neutralizing antibody or anti-CXCR4 neutralizing antibody (P<0.05 or P<0.01). In the co-culture, the invasiveness of trophoblast cells was increased when DSCs were pre-treated with anti-CXCR4 neutralizing antibody (P<0.05). However, after trophoblast cells alone or in the co-culture were pre-treated with anti-CXCR4 neutralizing antibody, the trophoblast cells invasion was significantly decreased (P<0.05o r P<0.01).
Therefore, it can be concluded that SDF-1 secreted by trophoblasts can not only promote the invasiveness of them in an autocrine manner, but also control the over-invasiveness of them through up-regulating the CD82 expression in DSCs in a paracrine manner.
After isolation and culture of human trophoblast cells and DSCs from the first-trimester pregnancy, the primary DSCs were treated by CsA or the supernatants derived of the CsA-pretreated trophoblasts, and then treated with or without anti-SDF-1 neutralizing antibody or anti-CXCR4 neutralizing antibody, respectively. Thereafter, in-cell Western was used to evaluate the CD82 expression in DSCs. We constructed the trophoblasts, and DSCs respective culture or co-culture, and then detected the SDF-1 secretion in supernatants by ELISA assay. We also pre-treated trophoblasts and DSCs with anti-CXCR4 neutralizing antibody, then constructed the co-culture of the two cells, and then added by CsA. Matrigel invasion assay was used to detect the trophoblasts invasiveness. We have found that CsA cannot directly affect the KAI/CD82 expression in DSCs (P>0.05), but supernatants derived of the CsA pre-treated trophoblasts can further promote the CD82 expression in DSCs (P<0.01), and the anti-SDF-1 neutralizing antibody or anti-CXCR4 neutralizing antibody inhibits this effect (P<0.05 or P<0.01). CsA can stimulate the SDF-1 secretion in trophoblasts and co-culture unit (P<0.05 or P<0.01). When trophoblast cells alone or in the co-culture were pre-treated with anti-CXCR4 neutralizing antibody, the enhancement of trophoblast cells invasion induced by CsA was decreased (P<0.05 or P<0.01). Moreover, when DSCs were pre-treated with anti-CXCR4 neutralizing antibody, the increase of trophoblasts invasiveness induced by CsA was partly reversed (P<0.05). Therefore, CsA stimulates the SDF-1 secretion of trophoblasts, and enlarges the up-regulation of the trophoblast-derived SDF-1 on the KAI/CD82 expression in DSCs.
In conclusion, CD82 has multiple modulating effects at the maternal-fetal interface:(1) CD82 promotes the TIMP1 expression and in turn suppresses the invasiveness of human first-trimester trophoblasts through inactivating integrinβ1MAPK/ERK1/2 signal pathway; (2) trophoblast cells enhances the CD82 expression in DSCs by secreting SDF-1 in a paracrine manner, and controls the over-invasion of trophoblast. Therefore, CD82 can promote the cross-talk between trophoblast cells and DSCs; (3) CsA stimulates the SDF-1 secretion of trophoblasts, and further up-regulates the CD82 expression in DSCs. Moreover, the expression of CD82 is also regulated by pregnancy-associated hormones hCG. Our results may elucidate human trophoblast invasion regulation mechanism, and provide a new idea for therapeutics of some pregnancy complications, such as miscarriage.
引文
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