發(fā)布時(shí)間：2018-04-04 10:37

  本文選題：間充質(zhì)干細(xì)胞　切入點(diǎn)：胎盤(pán)　出處：《濱州醫(yī)學(xué)院》2012年碩士論文

【摘要】：一、PDL2在人胎盤(pán)源間充質(zhì)干細(xì)胞上表達(dá)的生物學(xué)意義 目的：通過(guò)沉默PDL2在人胎盤(pán)間充質(zhì)干細(xì)胞(human placenta derived mesenchymal stem cells, hPMSCs)上的表達(dá),研究PDL2在hPMSCs上表達(dá)對(duì)hPMSCs黏附、遷移能力的影響及其在hPMSCs介導(dǎo)的對(duì)外周血T細(xì)胞的免疫調(diào)節(jié)中的作用。 方法：(1)應(yīng)用酶消化法從健康足月產(chǎn)人胎盤(pán)組織中分離細(xì)胞,培養(yǎng)3代后通過(guò)倒置顯微鏡觀察細(xì)胞形態(tài)、流式細(xì)胞術(shù)(flow cytometry, FCM)分析細(xì)胞表面抗原的表達(dá)及體外誘導(dǎo)向成骨細(xì)胞、脂肪細(xì)胞和神經(jīng)樣細(xì)胞分化等方式進(jìn)行鑒定。(2)逆轉(zhuǎn)錄PCR (reverse transcription polymerase chain reaction, RT-PCR)、激光共聚焦顯微鏡(laser scanning confocal microscopy, LSCM)及FCM檢測(cè)PDL2在hPMSCs上的表達(dá)。(3)通過(guò)脂質(zhì)體途徑將化學(xué)合成的PDL2siRNA轉(zhuǎn)染入hPMSCs,轉(zhuǎn)染48h后RT-PCR及FCM檢測(cè)沉默效果。(4)沉默PDL2后,細(xì)胞計(jì)數(shù)法檢測(cè)hPMSCs的黏附率,蘇木精-伊紅(hematoxylin-eosin, HE)染色觀測(cè)沉默PDL2后hPMSCs形態(tài)的變化；Transwell細(xì)胞培養(yǎng)系統(tǒng)檢測(cè)hPMSCs在DMEM-LG完全培養(yǎng)基、含SDF-1a的DMEM-LG完全培養(yǎng)基、hPMSCs培養(yǎng)上清3種培養(yǎng)條件下的遷移率。(5) Ficoll密度梯度離心法分離純化人外周血T細(xì)胞,將其與hPMSCs共培養(yǎng),并用植物血凝素(phytohemagglutinin, PHA)或佛波酯(phorbol12-myristate13-acetate, PMA)刺激。(6)FCM分析T細(xì)胞早期活化表型CD69的表達(dá)及T細(xì)胞周期變化；CFSE標(biāo)記分析T細(xì)胞增殖；細(xì)胞胞內(nèi)染色法檢測(cè)T細(xì)胞對(duì)IL-17的分泌。 結(jié)果：(1)胎盤(pán)組織分離的間充質(zhì)干細(xì)胞貼壁、呈梭形生長(zhǎng),能夠定向向成骨細(xì)胞、脂肪細(xì)胞和神經(jīng)樣細(xì)胞分化,其表面抗原CD44、CD105、CD166、CD29呈陽(yáng)性表達(dá),CD14、CD34和CD45呈陰性表達(dá)。(2)基因及蛋白水平檢測(cè)均顯示hPMSCs高表達(dá)PDL2；通過(guò)脂質(zhì)體途徑能夠?qū)DL2siRNA高效轉(zhuǎn)染入hPMSCs,有效沉默PDL2的表達(dá)。(3)沉默PDL2后,細(xì)胞接種0.5h后hPMSCs的黏附率較未沉默組未發(fā)生明顯變化,但在接種1h或3h后,其黏附率明顯高于未沉默組細(xì)胞(P0.05)；在3種不同的培養(yǎng)條件下,沉默PDL2后,hPMSCs遷移率明顯低于未沉默組細(xì)胞(P0.01)。(4) hPMSCs能夠抑制活化T細(xì)胞對(duì)CD69的表達(dá),沉默PDL2后,CD69的表達(dá)與未沉默組相比無(wú)明顯變化；沉默PDL2后,T細(xì)胞增殖明顯增加,但仍低于單純PMA刺激組；與未沉默組相比,處于G0/G1期的T細(xì)胞數(shù)量明顯減少,處于S期的細(xì)胞數(shù)量明顯增加；在hPMSCs存在條件下,T細(xì)胞對(duì)IL-17的分泌明顯增加,沉默PDL2后,IL-17的分泌被進(jìn)一步上調(diào)。 結(jié)論：(1) hPMSCs高表達(dá)PDL2;(2) PDL2siRNA能夠有效沉默PDL2在hPMSCs上的表達(dá)；(3)PDL2不僅在hPMSCs介導(dǎo)的對(duì)T細(xì)胞的增殖、周期及分泌細(xì)胞因子的免疫調(diào)節(jié)中發(fā)揮重要的作用,而且參與了hPMSCs的黏附和遷移。 二、PDL2在人胎盤(pán)間充質(zhì)干細(xì)胞上的表達(dá)及調(diào)控 目的：研究不同代hPMSCs對(duì)PDL2的表達(dá)變化及IFN-y調(diào)節(jié)PDL2在hPMSCs上表達(dá)的信號(hào)調(diào)節(jié)機(jī)制。 方法：(1)實(shí)時(shí)定量PCR(real-time PCR)及FCM檢測(cè)不同代hPMSCs對(duì)PDL2的表達(dá).(2) RT-PCR及FCM檢測(cè)IFN-y或／和INCB018424作用后hPMSCs對(duì)PDL2mRNA和蛋白水平表達(dá)的變化。(3) Western blot檢測(cè)IFN-y或/和INCBO18424作用后hPMSCs中STAT1和STAT3磷酸化水平變化。 結(jié)果：(1)不同代hPMSCs對(duì)PDL2在基因水平及蛋白水平的表達(dá)均有著顯著差異,mRNA水平第20代表達(dá)最高,蛋白水平第12代表達(dá)最高。(2)IFN-γ作用后,PDL2的mRNA及蛋白表達(dá)水平均被明顯上調(diào),且STAT1和STAT3的磷酸化水平同樣被顯著上調(diào)。(3) INCB018424處理hPMSCs后,IFN-y刺激及未刺激組PDL2的mRNA、蛋白水平及STAT1和STAT3的磷酸化水平均被明顯下調(diào)。 結(jié)論：不同代hPMSCs對(duì)PDL2表達(dá)水平不同,IFN-y能夠通過(guò)JAK-STAT通路上調(diào)hPMSCs對(duì)PDL2的表達(dá)。
[Abstract]:Biological significance of PDL2 expression on human placental mesenchymal stem cells
Objective: through silencing PDL2 in human placenta derived mesenchymal stem cells (human placenta derived mesenchymal stem cells, hPMSCs) on the expression of PDL2 on the adhesion of hPMSCs expression in hPMSCs, peripheral blood T cells migration and hPMSCs mediated immune regulation effect.
Methods: (1) cells were isolated from healthy full-term placental tissue by enzyme digestion method, cultured after 3 generations of cell morphology was observed by inverted microscope, flow cytometry (flow cytometry, FCM) of the cells were induced to osteoblasts in vitro and expression of cell surface antigen, fat cell and nerve cell differentiation etc. identification. (2) PCR (reverse transcription polymerase chain RT reaction, RT-PCR), laser scanning confocal microscope (laser scanning confocal microscopy, LSCM) and the expression of FCM PDL2 was detected in hPMSCs. (3) PDL2siRNA was transfected into hPMSCs by chemical synthesis of liposome, transfection of 48h RT-PCR and FCM detection of silence results. (4) after silencing PDL2, adhesion detection of hPMSCs cell counting rate, hematoxylin eosin staining (hematoxylin-eosin, HE) changes of hPMSCs morphology observation of PDL2 silencing; Transwell cells The detection of hPMSCs in DMEM-LG medium containing SDF-1a DMEM-LG culture medium, hPMSCs culture supernatant of 3 culture conditions. The migration rate (5) separation and purification of T cells from human peripheral blood by Ficoll density gradient centrifugation and the co cultured with hPMSCs, and with phytohemagglutinin (phytohemagglutinin, PHA) or by ester (phorbol12-myristate13-acetate, PMA) stimulation. (6) analysis of FCM T expression and cell cycle changes of T cells in the early activation of CD69 phenotype; analysis of T cell proliferation marker CFSE; intracellular staining of T cells was assayed by IL-17.
Results: (1) the separation of placenta mesenchymal stem cells adherent, spindle shaped growth can be directed into osteoblasts, adipocytes and neuron like cell differentiation, its surface antigen CD44, CD105, CD166, CD29 showed positive expression of CD14, CD34 and CD45 negative expression (2) detection. Gene and protein level showed high expression of hPMSCs PDL2; by liposome can be efficiently transfected into PDL2siRNA pathway hPMSCs, effectively inhibit the expression of PDL2. (3) after silencing of PDL2 cells after inoculation with 0.5h hPMSCs, the adhesion rate is silent group did not change significantly, but in 1H or 3h after inoculation, the adhesion the rate was significantly higher than that in non silent cells (P0.05); in 3 different culture conditions, PDL2 silencing, hPMSCs migration rate was significantly lower than that of group (P0.01). The silence cells (4) hPMSCs can inhibit the activation of T expression on CD69 cells, PDL2 silencing, CD69 expression and silencing group obvious change Silence; after PDL2, the proliferation of T cells was significantly increased, but still lower than that of PMA group; compared with non silencing group in G0/G1 phase T cells decreased significantly, in the number of cells in S phase was significantly increased; in the condition of hPMSCs, T cells on the secretion of IL-17 increased significantly after PDL2 was silenced, the secretion of IL-17 was further increased.
Conclusion: (1) the high expression of hPMSCs PDL2; (2) PDL2siRNA can effectively inhibit the expression of PDL2 in hPMSCs; (3) PDL2 not only in hPMSCs mediated proliferation of T cells, immune cycle and secretion of cytokines play an important role in the regulation of, and participate in hPMSCs adhesion and migration.
Expression and regulation of two, PDL2 on human placental mesenchymal stem cells
Objective: To study the changes in the expression of PDL2 in different generations of hPMSCs and the modulation mechanism of IFN-y to regulate the expression of PDL2 on hPMSCs.
Methods: (1) real time quantitative PCR (real-time PCR) FCM expression and detection of different generation hPMSCs to PDL2. (2) change on the level of PDL2mRNA and protein expression of hPMSCs RT-PCR and FCM detection of IFN-y or / and INCB018424 after the action. (3) Western blot detection of IFN-y or / and INCBO18424 after hPMSCs and STAT1 the phosphorylation level of STAT3 change.
Results: (1) the different generation of hPMSCs on the expression of PDL2 in gene and protein levels were significantly different, mRNA reached the highest level of twentieth representatives, Twelfth representatives of the highest protein level. (2) IFN- gamma function, mRNA PDL2 and protein expression level were significantly increased, and STAT1 and STAT3 phosphorylation the level was also significantly increased. (3) INCB018424 after hPMSCs treatment, IFN-y stimulation and non stimulation mRNA group PDL2, STAT1 and STAT3 protein levels and phosphorylation levels were significantly reduced.
Conclusion: the expression level of PDL2 in different generations of hPMSCs is different, and IFN-y can increase the expression of hPMSCs to PDL2 through the JAK-STAT pathway.

【學(xué)位授予單位】：濱州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R329.2

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本文編號(hào)：1709586