發(fā)布時間：2018-05-05 16:09

  本文選題：IL-33 + sST2　； 參考：《復(fù)旦大學(xué)》2014年碩士論文

【摘要】：妊娠從免疫學(xué)上看類似于器官移植,作為同種移植物的胚胎在母體存活直至分娩,實際上反映母體對胚胎的免疫耐受,母體對胚胎的免疫排斥將導(dǎo)致妊娠失敗。揭示母-胎免疫耐受的確切機制,不僅對生殖免疫,而且對移植免疫、腫瘤免疫、自身免疫疾病都有重要意義。母-胎界面主要包括蛻膜基質(zhì)細胞、蛻膜免疫細胞及滋養(yǎng)細胞,其中蛻膜基質(zhì)細胞及蛻膜免疫細胞來源于母體,而滋養(yǎng)細胞則來源于胎兒。多年來人們從不同角度研究母-胎界面發(fā)生的生物學(xué)事件,以闡述母-胎免疫調(diào)節(jié)的機制,其中包括母-胎界面各組成細胞的生物學(xué)功能調(diào)節(jié),及母-胎界面Th2型免疫優(yōu)勢的形成。妊娠早期母胎界面的蛻膜被認為是一個免疫特赦組織,妊娠早期大量白細胞在此選擇性聚集,其中最主要的是蛻膜NK細胞,約占淋巴細胞總量的70%-80%,另外還有單核巨噬細胞和T細胞等,這些免疫活性細胞在局部組織微環(huán)境的調(diào)控下能夠獲得特異性的表型和功能,從而決定母-胎界面免疫反應(yīng)的取向。IL-33是新近發(fā)現(xiàn)的IL-1家族新成員,與受體ST2L及誘餌受體sST2進行相互調(diào)控,參與調(diào)節(jié)Th2型機體免疫,腫瘤進展及轉(zhuǎn)移等過程。此外,研究報道,IL-33/ST2信號調(diào)控異�？捎绊懽訉m內(nèi)膜容受性進而導(dǎo)致流產(chǎn),并且其分泌水平與流產(chǎn)及妊娠期高血壓等病理妊娠存在相關(guān)性。然而,迄今為止,IL-33在母-胎界面如何發(fā)揮作用仍不清楚。第一部分IL-33/ST2在正常妊娠早期及復(fù)發(fā)性自然流產(chǎn)患者蛻膜基質(zhì)細胞中的表達目的 分析IL-33/ST2在正常妊娠早期和復(fù)發(fā)性自然流產(chǎn)患者蛻膜基質(zhì)細胞中的表達水平。方法 收集早孕期正�；虿幻髟驈�(fù)發(fā)性自然流產(chǎn)的蛻膜組織,采用免疫組織化學(xué)方法檢測IL-33/ST2在早孕蛻膜組織中的表達。分離蛻膜基質(zhì)細胞,采用ELISA、Real-time PCR及Western Blot等技術(shù)進一步證實IL-33及ST2在蛻膜基質(zhì)細胞中的表達,并比較IL-33及ST2表達水平在正常早孕及流產(chǎn)蛻膜基質(zhì)細胞之間的差別。結(jié)果 早孕蛻膜組織能檢測到IL-33及ST2的表達。ELISA、Real-time PCR及Western Blot進一步證實了IL-33/ST2在蛻膜基質(zhì)細胞中的表達,并且在轉(zhuǎn)錄及翻譯水平,復(fù)發(fā)性自然流產(chǎn)的蛻膜基質(zhì)細胞IL-33/ST2的表達均明顯低于正常妊娠。結(jié)論 蛻膜組織,尤其是蛻膜基質(zhì)細胞IL-33及ST2的正常表達對妊娠的維持具有一定的作用。第二部分IL-33/ST2對蛻膜基質(zhì)細胞生物學(xué)行為的調(diào)控目的 解析IL-33對蛻膜基質(zhì)細胞增殖及侵襲等生物學(xué)行為的調(diào)節(jié)。方法 收集人早孕期正常的蛻膜組織,應(yīng)用胰酶消化、密度梯度離心法分離、培養(yǎng)正常人早孕期蛻膜基質(zhì)細胞。不同濃度的IL-33及sST2作用48h后,分別利用BrdU細胞增殖試劑盒、Transwell實驗及流式細胞術(shù)檢測蛻膜基質(zhì)細胞的增殖能力、侵襲能力及細胞周期。為了驗證IL-33對蛻膜基質(zhì)細胞生物學(xué)行為的調(diào)節(jié),進一步采用Real-time PCR檢測處理后的蛻膜基質(zhì)細胞中增殖相關(guān)分子(PCNA、survivin)及侵襲相關(guān)分子(titin, MMP2)的表達。結(jié)果BrdU及Transwell實驗發(fā)現(xiàn),IL-33以濃度依賴的方式促進蛻膜基質(zhì)細胞的增殖及侵襲,并且流式細胞術(shù)的結(jié)果顯示,IL-33處理48h后,蛻膜基質(zhì)細胞的增殖指數(shù)升高。此外,Real-time PCR的結(jié)果顯示IL-33可促進蛻膜基質(zhì)細胞增殖相關(guān)分子(PCNA、survivin)及侵襲相關(guān)分子(titin、MMP2)的表達。結(jié)論 IL-33可通過ST2L促進蛻膜基質(zhì)細胞的增殖及侵襲。第三部分 IL-33調(diào)控蛻膜基質(zhì)細胞生物學(xué)功能的分子機制目的 解析IL-33調(diào)控蛻膜基質(zhì)細胞生物學(xué)功能的分子機制。方法 分離培養(yǎng)正常人早孕期蛻膜基質(zhì)細胞,經(jīng)IL-33處理后,利用ELISA及流式細胞術(shù)檢測蛻膜基質(zhì)細胞CCL2及CCR2的表達水平。在培養(yǎng)體系中加入anti-CCL2中和性抗體或CCR2阻滯劑(RS102895)處理后,分別利用BrdU細胞增殖試劑盒、Transwell實驗及流式細胞術(shù)檢測蛻膜基質(zhì)細胞的增殖能力、侵襲能力及細胞周期。用IL-33處理蛻膜基質(zhì)細胞,采用Western Blot分析信號分子NF-κB、ERK1/2、JNK及P38的磷酸化水平；在此基礎(chǔ)上,用NF-κB抑制劑BAY 11-7082、ERK1/2抑制劑U0126和JNK抑制劑SP600125分別預(yù)處理蛻膜基質(zhì)細胞后再用IL-33處理,采用ELISA及流式細胞術(shù)檢測蛻膜基質(zhì)細胞CCL2及CCR2的表達水平。結(jié)果 經(jīng)IL-33處理后,蛻膜基質(zhì)細胞CCL2及CCR2的表達水平增加。拮抗CCL2/CCR2的生物學(xué)作用,可下調(diào)IL-33的促增殖及促侵襲作用。Western Blot結(jié)果顯示IL-33可引起NF-κB、ERK1/2及JNK的磷酸化水平升高,NF-κB抑制劑BAY 11-7082. ERK1/2抑制劑U0126預(yù)處理細胞后,可下調(diào)IL-33引起的CCL2/CCR2升高,然而JNK抑制劑SP600125卻對蛻膜基質(zhì)細胞表達的CCL2及CCR2無上述下調(diào)作用。結(jié)論 IL-33可通過活化NF-κB和ERKl/2信號分子,進而促進蛻膜基質(zhì)細胞CCL2和CCR2的表達,并間接促進蛻膜基質(zhì)細胞的增殖及侵襲等生物學(xué)行為。第四部分 IL-33參與調(diào)節(jié)蛻膜NK細胞的表型和功能目的 解析IL-33對蛻膜NK細胞表型及功能的調(diào)節(jié)作用。方法 利用流式細胞術(shù)分析蛻膜NK細胞ST2L的表達水平,并比較外周NK細胞與蛻膜NK細胞ST2L的表達水平。用IL-33、sST2或DSCs條件培養(yǎng)液處理蛻膜NK細胞,48h后流式細胞術(shù)分析蛻膜NK細胞的表型和細胞因子的表達,細胞毒性檢測試劑盒檢測NK細胞的細胞毒活性。結(jié)果 流式細胞術(shù)分析發(fā)現(xiàn),蛻膜NK細胞表面表達ST2L,并且ST2L在蛻膜NK的表達水平明顯高于外周NK細胞。IL-33和DSCs條件培養(yǎng)液可顯著上調(diào)蛻膜NK細胞Th2型細胞因子(IL-4、IL-13)、調(diào)節(jié)性細胞因子(IL-10)及促炎性細胞因子IFN-γ的表達水平,并下調(diào)Thl型細胞因子TNF-α的表達。另外,IL-33還可促進NK表面抑制性受體KIR2DL1的表達,并下調(diào)其表面殺傷性受體NKP30和NKG2D的表達。細胞毒實驗結(jié)果表明IL-33可降調(diào)節(jié)蛻膜NK細胞的細胞毒活性,與此相一致的是經(jīng)IL-33處理后,NK細胞顆粒酶granzymeA及穿孔素perforin的表達也下降。結(jié)論 蛻膜基質(zhì)細胞分泌的IL-33能誘導(dǎo)蛻膜NK細胞的耐受表型及Th2型優(yōu)勢,下調(diào)蛻膜NK細胞的細胞毒活性,從而有利于形成母-胎界面免疫耐受微環(huán)境。第五部分 IL-33調(diào)控蛻膜NK細胞功能和表型的分子機制目的 解析IL-33調(diào)節(jié)蛻膜NK細胞功能和表型的下游信號通路方法 分離的蛻膜NK細胞經(jīng)IL-33處理后,利用Real-time PCR檢測NK分化發(fā)育相關(guān)轉(zhuǎn)錄因子的表達。用IL-33處理蛻膜NK細胞,采用Western Blot分析信號分子NF-κB.ERK1/2、JNK及P38的磷酸化水平；在此基礎(chǔ)上,用NF-κB抑制劑BAY 11-7082、ERK1/2抑制劑U0126和JNK抑制劑SP600125分別預(yù)處理蛻膜基質(zhì)細胞后再用IL-33處理,流式細胞術(shù)分析NK細胞表型、細胞內(nèi)細胞因子的表達。結(jié)果 經(jīng)IL-33處理后,蛻膜NK細胞Nfil3、Id2及Gata3等轉(zhuǎn)錄因子表達增高。’Western Blot結(jié)果顯示IL-33可引起NF-κB、ERK1/2及JNK的磷酸化水平升高,NF-κB抑制劑BAY 11-7082預(yù)處理細胞后,可逆轉(zhuǎn)IL-33對蛻膜NK細胞表型及功能的調(diào)控作用。結(jié)論NF-κB/Nfil3/Id2/Gata3信號通路參與IL-33對蛻膜NK細胞功能的調(diào)節(jié)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.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R714

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本文編號：1848356