人蜕膜基质细胞表达的IL-33在诱导早孕母—胎免疫耐受中的作用及其细胞与分子机制

发布时间：2018-05-05 16:09

本文选题：IL-33 + sST2　；参考：《复旦大学》2014年硕士论文

【摘要】：妊娠从免疫学上看类似于器官移植,作为同种移植物的胚胎在母体存活直至分娩,实际上反映母体对胚胎的免疫耐受,母体对胚胎的免疫排斥将导致妊娠失败。揭示母-胎免疫耐受的确切机制,不仅对生殖免疫,而且对移植免疫、肿瘤免疫、自身免疫疾病都有重要意义。母-胎界面主要包括蜕膜基质细胞、蜕膜免疫细胞及滋养细胞,其中蜕膜基质细胞及蜕膜免疫细胞来源于母体,而滋养细胞则来源于胎儿。多年来人们从不同角度研究母-胎界面发生的生物学事件,以阐述母-胎免疫调节的机制,其中包括母-胎界面各组成细胞的生物学功能调节,及母-胎界面Th2型免疫优势的形成。妊娠早期母胎界面的蜕膜被认为是一个免疫特赦组织,妊娠早期大量白细胞在此选择性聚集,其中最主要的是蜕膜NK细胞,约占淋巴细胞总量的70%-80%,另外还有单核巨噬细胞和T细胞等,这些免疫活性细胞在局部组织微环境的调控下能够获得特异性的表型和功能,从而决定母-胎界面免疫反应的取向。IL-33是新近发现的IL-1家族新成员,与受体ST2L及诱饵受体sST2进行相互调控,参与调节Th2型机体免疫,肿瘤进展及转移等过程。此外,研究报道,IL-33/ST2信号调控异常可影响子宫内膜容受性进而导致流产,并且其分泌水平与流产及妊娠期高血压等病理妊娠存在相关性。然而,迄今为止,IL-33在母-胎界面如何发挥作用仍不清楚。第一部分IL-33/ST2在正常妊娠早期及复发性自然流产患者蜕膜基质细胞中的表达目的分析IL-33/ST2在正常妊娠早期和复发性自然流产患者蜕膜基质细胞中的表达水平。方法收集早孕期正常或不明原因复发性自然流产的蜕膜组织,采用免疫组织化学方法检测IL-33/ST2在早孕蜕膜组织中的表达。分离蜕膜基质细胞,采用ELISA、Real-time PCR及Western Blot等技术进一步证实IL-33及ST2在蜕膜基质细胞中的表达,并比较IL-33及ST2表达水平在正常早孕及流产蜕膜基质细胞之间的差别。结果早孕蜕膜组织能检测到IL-33及ST2的表达。ELISA、Real-time PCR及Western Blot进一步证实了IL-33/ST2在蜕膜基质细胞中的表达,并且在转录及翻译水平,复发性自然流产的蜕膜基质细胞IL-33/ST2的表达均明显低于正常妊娠。结论蜕膜组织,尤其是蜕膜基质细胞IL-33及ST2的正常表达对妊娠的维持具有一定的作用。第二部分IL-33/ST2对蜕膜基质细胞生物学行为的调控目的解析IL-33对蜕膜基质细胞增殖及侵袭等生物学行为的调节。方法收集人早孕期正常的蜕膜组织,应用胰酶消化、密度梯度离心法分离、培养正常人早孕期蜕膜基质细胞。不同浓度的IL-33及sST2作用48h后,分别利用BrdU细胞增殖试剂盒、Transwell实验及流式细胞术检测蜕膜基质细胞的增殖能力、侵袭能力及细胞周期。为了验证IL-33对蜕膜基质细胞生物学行为的调节,进一步采用Real-time PCR检测处理后的蜕膜基质细胞中增殖相关分子(PCNA、survivin)及侵袭相关分子(titin, MMP2)的表达。结果BrdU及Transwell实验发现,IL-33以浓度依赖的方式促进蜕膜基质细胞的增殖及侵袭,并且流式细胞术的结果显示,IL-33处理48h后,蜕膜基质细胞的增殖指数升高。此外,Real-time PCR的结果显示IL-33可促进蜕膜基质细胞增殖相关分子(PCNA、survivin)及侵袭相关分子(titin、MMP2)的表达。结论 IL-33可通过ST2L促进蜕膜基质细胞的增殖及侵袭。第三部分 IL-33调控蜕膜基质细胞生物学功能的分子机制目的解析IL-33调控蜕膜基质细胞生物学功能的分子机制。方法分离培养正常人早孕期蜕膜基质细胞,经IL-33处理后,利用ELISA及流式细胞术检测蜕膜基质细胞CCL2及CCR2的表达水平。在培养体系中加入anti-CCL2中和性抗体或CCR2阻滞剂(RS102895)处理后,分别利用BrdU细胞增殖试剂盒、Transwell实验及流式细胞术检测蜕膜基质细胞的增殖能力、侵袭能力及细胞周期。用IL-33处理蜕膜基质细胞,采用Western Blot分析信号分子NF-κB、ERK1/2、JNK及P38的磷酸化水平；在此基础上,用NF-κB抑制剂BAY 11-7082、ERK1/2抑制剂U0126和JNK抑制剂SP600125分别预处理蜕膜基质细胞后再用IL-33处理,采用ELISA及流式细胞术检测蜕膜基质细胞CCL2及CCR2的表达水平。结果经IL-33处理后,蜕膜基质细胞CCL2及CCR2的表达水平增加。拮抗CCL2/CCR2的生物学作用,可下调IL-33的促增殖及促侵袭作用。Western Blot结果显示IL-33可引起NF-κB、ERK1/2及JNK的磷酸化水平升高,NF-κB抑制剂BAY 11-7082. ERK1/2抑制剂U0126预处理细胞后,可下调IL-33引起的CCL2/CCR2升高,然而JNK抑制剂SP600125却对蜕膜基质细胞表达的CCL2及CCR2无上述下调作用。结论 IL-33可通过活化NF-κB和ERKl/2信号分子,进而促进蜕膜基质细胞CCL2和CCR2的表达,并间接促进蜕膜基质细胞的增殖及侵袭等生物学行为。第四部分 IL-33参与调节蜕膜NK细胞的表型和功能目的解析IL-33对蜕膜NK细胞表型及功能的调节作用。方法利用流式细胞术分析蜕膜NK细胞ST2L的表达水平,并比较外周NK细胞与蜕膜NK细胞ST2L的表达水平。用IL-33、sST2或DSCs条件培养液处理蜕膜NK细胞,48h后流式细胞术分析蜕膜NK细胞的表型和细胞因子的表达,细胞毒性检测试剂盒检测NK细胞的细胞毒活性。结果流式细胞术分析发现,蜕膜NK细胞表面表达ST2L,并且ST2L在蜕膜NK的表达水平明显高于外周NK细胞。IL-33和DSCs条件培养液可显著上调蜕膜NK细胞Th2型细胞因子(IL-4、IL-13)、调节性细胞因子(IL-10)及促炎性细胞因子IFN-γ的表达水平,并下调Thl型细胞因子TNF-α的表达。另外,IL-33还可促进NK表面抑制性受体KIR2DL1的表达,并下调其表面杀伤性受体NKP30和NKG2D的表达。细胞毒实验结果表明IL-33可降调节蜕膜NK细胞的细胞毒活性,与此相一致的是经IL-33处理后,NK细胞颗粒酶granzymeA及穿孔素perforin的表达也下降。结论蜕膜基质细胞分泌的IL-33能诱导蜕膜NK细胞的耐受表型及Th2型优势,下调蜕膜NK细胞的细胞毒活性,从而有利于形成母-胎界面免疫耐受微环境。第五部分 IL-33调控蜕膜NK细胞功能和表型的分子机制目的解析IL-33调节蜕膜NK细胞功能和表型的下游信号通路方法分离的蜕膜NK细胞经IL-33处理后,利用Real-time PCR检测NK分化发育相关转录因子的表达。用IL-33处理蜕膜NK细胞,采用Western Blot分析信号分子NF-κB.ERK1/2、JNK及P38的磷酸化水平；在此基础上,用NF-κB抑制剂BAY 11-7082、ERK1/2抑制剂U0126和JNK抑制剂SP600125分别预处理蜕膜基质细胞后再用IL-33处理,流式细胞术分析NK细胞表型、细胞内细胞因子的表达。结果经IL-33处理后,蜕膜NK细胞Nfil3、Id2及Gata3等转录因子表达增高。’Western Blot结果显示IL-33可引起NF-κB、ERK1/2及JNK的磷酸化水平升高,NF-κB抑制剂BAY 11-7082预处理细胞后,可逆转IL-33对蜕膜NK细胞表型及功能的调控作用。结论NF-κB/Nfil3/Id2/Gata3信号通路参与IL-33对蜕膜NK细胞功能的调节作用。
[Abstract]:Pregnancy is immunologically similar to organ transplantation. As an allograft, the embryo survives in the mother's body until delivery, which actually reflects the maternal immune tolerance to the embryo and the maternal immune rejection of the embryo will lead to the failure of pregnancy. The mother fetal interface consists mainly of decidual stromal cells, decidua immune cells and trophoblast cells, in which decidual stromal cells and decidua immune cells originate from the mother, while the trophoblastic cells are derived from the fetus. The mechanism of fetal immune regulation, including the biological function regulation of the constituent cells of the mother fetal interface, and the formation of the Th2 type immune dominance of the mother fetal interface. The decidua of the maternal fetal interface in the early pregnancy is considered as an amnesty of immunization. In the early pregnancy, a large number of leukocytes are selectively gathered here, the most important of which is decidua NK cells. The 70%-80% of the total number of BBA cells, as well as mononuclear macrophages and T cells, can obtain specific phenotypes and functions under the regulation of local microenvironment, thus determining the orientation of the immunoreaction of the mother fetal interface is a new member of the newly discovered family of IL-1 family, with the receptor ST2L and the bait receptor sST2. Mutual regulation, participates in the process of regulating Th2 type body immunity, tumor progression and metastasis. In addition, it is reported that abnormal regulation of IL-33/ST2 signal can affect endometrial receptivity and lead to abortion, and its secretion level is associated with abortion and pregnancy induced hypertension. However, so far, IL-33 is at the maternal fetal interface such as The expression of IL-33/ST2 in the decidua cells of patients with normal pregnancy and recurrent spontaneous abortion analysis of the expression level of IL-33/ST2 in the decidua stromal cells of patients with normal pregnancy and recurrent spontaneous abortion. Methods the normal or unexplained recurrent nature of early pregnancy was collected. The expression of IL-33/ST2 in decidual tissue of early pregnancy was detected by immunohistochemical method. Decidual stromal cells were isolated and the expressions of IL-33 and ST2 in decidua stromal cells were further confirmed by ELISA, Real-time PCR and Western Blot, and the expression levels of IL-33 and ST2 were compared in normal early pregnancy and abortion decidua. The difference between matrix cells. Results early pregnancy decidua tissue can detect the expression of IL-33 and ST2.ELISA, Real-time PCR and Western Blot further confirm the expression of IL-33/ST2 in decidual stromal cells, and the expression of IL-33/ST2 in the decidual stromal cells of recurrent spontaneous abortion is significantly lower than that of normal pregnancy at the level of transcription and translation. Conclusions decidual tissue, especially the normal expression of IL-33 and ST2 in decidual stromal cells, has a certain effect on the maintenance of pregnancy. Second the regulation of biological behavior of decidual stromal cells by the regulation of IL-33/ST2 on the biological behavior of decidual stromal cells. The regulation of IL-33 on the biological behavior of the proliferation and invasion of decidual stromal cells. Methods the normal decidua of human early pregnancy was collected. Membrane tissue, trypsin digestion and density gradient centrifugation were used to isolate the decidual stromal cells of normal human early pregnancy. After different concentrations of IL-33 and sST2 48h, BrdU cell proliferation kits, Transwell experiments and flow cytometry were used to detect the proliferation, invasion and cell cycle of decidual stromal cells. In order to verify IL-33, The biological behavior of decidual stromal cells was regulated, and the expression of PCNA (PCNA, survivin) and invasion related molecules (titin, MMP2) in decidual stromal cells after treatment with Real-time PCR were further used. Results BrdU and Transwell experiments found that IL-33 promoted the proliferation and invasion of decidual stromal cells by the concentration dependent manner. And the results of flow cytometry showed that the proliferation index of decidual matrix cells increased after IL-33 treatment of 48h. In addition, the results of Real-time PCR showed that IL-33 could promote the expression of PCNA (survivin) and invasion related molecules (titin, MMP2) in decidual cells. Conclusion IL-33 can promote the proliferation and invasion of decidual stromal cells by ST2L. Third part of the molecular mechanism of IL-33 regulating the biological function of decidual stromal cells in order to analyze the molecular mechanism of IL-33 regulation of the biological function of decidual stromal cells. Methods to isolate and culture decidual stromal cells from normal human early pregnancy, and to detect the expression of CCL2 and CCR2 in decidual stromal cells by ELISA and flow cytometry after IL-33 treatment. After adding anti-CCL2 neutralization antibody or CCR2 blocker (RS102895) in the culture system, the proliferation ability, invasion ability and cell cycle of decidual stromal cells were detected by BrdU cell proliferation kit, Transwell experiment and flow cytometry. IL-33 was used to treat decidua stromal cells, and Western Blot was used to analyze the signal molecules NF- The phosphorylation level of kappa B, ERK1/2, JNK and P38; on this basis, the decidual stromal cells were treated with NF- kappa B inhibitor BAY 11-7082, ERK1/2 inhibitor U0126 and JNK inhibitor SP600125 respectively, and the expression level of decidual stromal cells was detected by flow cytometry. The expression level of CCL2 and CCR2 in the stromal cells is increased. The biological action of antagonistic CCL2/CCR2 can down regulate the proliferation of IL-33 and promote the invasion of.Western Blot results show that IL-33 can cause NF- kappa B, ERK1/2 and JNK phosphorylation level. CR2 increased, however, JNK inhibitor SP600125 had no down-regulation on CCL2 and CCR2 expressed in decidual stromal cells. Conclusion IL-33 can promote the expression of CCL2 and CCR2 in decidual stromal cells by activating NF- kappa B and ERKl/2 signal molecules, and indirectly promote the proliferation and invasion of decidual stromal cells. Fourth part of the IL-33 ginseng. To regulate the phenotype and function of decidual NK cells and to analyze the regulation of IL-33 on the phenotype and function of decidual NK cells. Methods using flow cytometry to analyze the expression level of ST2L in decidua NK cells and to compare the expression level of ST2L in the peripheral NK cells and decidua NK cells. The decidual NK cells were treated with IL-33, sST2 or DSCs conditioned medium. Flow cytometry was used to analyze the expression of phenotypes and cytokines in decidua NK cells, and cytotoxicity test kit was used to detect cytotoxic activity of NK cells. Results flow cytometry showed that ST2L was expressed on the surface of decidua NK cells, and the expression level of ST2L in decidua NK was significantly higher than that of.IL-33 and DSCs conditioned medium of peripheral NK cells. The expression of Th2 type cytokine (IL-4, IL-13), regulatory cytokine (IL-10) and proinflammatory cytokine IFN- gamma in NK cells, and the expression of TNF- alpha of Thl cytokine. In addition, IL-33 can also promote the expression of KIR2DL1 on the surface inhibitory receptor of NK, and regulate its surface killing receptor NKP30 and expression. The results showed that IL-33 could reduce the cytotoxic activity of decidual NK cells. The expression of NK cell granzyme granzymeA and perforin perforin also decreased after IL-33 treatment. Conclusion IL-33 secreted by decidual stromal cells can induce the tolerance phenotype and Th2 type of decidual NK cells and downregulate the cytotoxicity of decidual NK cells. The fifth part IL-33 regulates the functional and phenotypic mechanism of the decidual NK cells in order to determine the molecular mechanism of the function and phenotype of the decidual NK cells in order to analyze the decidual NK cells of the decidual NK cells that regulate the function and phenotype of the decidua cells by IL-33 treatment and use Real-time PCR to detect the correlation of NK differentiation and development. The decidual NK cells were treated with IL-33, and the phosphorylation level of NF- kappa B.ERK1/2, JNK and P38 was analyzed by Western Blot. On this basis, the NF- kappa B inhibitor BAY 11-7082 was used to treat the thin cell of the decidua matrix and then the cell cell of the decidua matrix was treated respectively. After IL-33 treatment, the expression of Nfil3, Id2, Gata3 and other transcription factors in the decidual NK cells increased. 'Western Blot results showed that IL-33 could cause the increase of NF- kappa B, ERK1/2 and JNK' phosphorylation level. Conclusion NF- kappa B/Nfil3/Id2/Gata3 signaling pathway is involved in the regulation of IL-33 on decidua NK cells.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714

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