PDL2在人胎盘间充质干细胞上的表达调控及生物学意义研究

发布时间：2018-04-04 10:37

  本文选题：间充质干细胞　切入点：胎盘　出处：《滨州医学院》2012年硕士论文

【摘要】：一、PDL2在人胎盘源间充质干细胞上表达的生物学意义 目的：通过沉默PDL2在人胎盘间充质干细胞(human placenta derived mesenchymal stem cells, hPMSCs)上的表达,研究PDL2在hPMSCs上表达对hPMSCs黏附、迁移能力的影响及其在hPMSCs介导的对外周血T细胞的免疫调节中的作用。 方法：(1)应用酶消化法从健康足月产人胎盘组织中分离细胞,培养3代后通过倒置显微镜观察细胞形态、流式细胞术(flow cytometry, FCM)分析细胞表面抗原的表达及体外诱导向成骨细胞、脂肪细胞和神经样细胞分化等方式进行鉴定。(2)逆转录PCR (reverse transcription polymerase chain reaction, RT-PCR)、激光共聚焦显微镜(laser scanning confocal microscopy, LSCM)及FCM检测PDL2在hPMSCs上的表达。(3)通过脂质体途径将化学合成的PDL2siRNA转染入hPMSCs,转染48h后RT-PCR及FCM检测沉默效果。(4)沉默PDL2后,细胞计数法检测hPMSCs的黏附率,苏木精-伊红(hematoxylin-eosin, HE)染色观测沉默PDL2后hPMSCs形态的变化；Transwell细胞培养系统检测hPMSCs在DMEM-LG完全培养基、含SDF-1a的DMEM-LG完全培养基、hPMSCs培养上清3种培养条件下的迁移率。(5) Ficoll密度梯度离心法分离纯化人外周血T细胞,将其与hPMSCs共培养,并用植物血凝素(phytohemagglutinin, PHA)或佛波酯(phorbol12-myristate13-acetate, PMA)刺激。(6)FCM分析T细胞早期活化表型CD69的表达及T细胞周期变化；CFSE标记分析T细胞增殖；细胞胞内染色法检测T细胞对IL-17的分泌。 结果：(1)胎盘组织分离的间充质干细胞贴壁、呈梭形生长,能够定向向成骨细胞、脂肪细胞和神经样细胞分化,其表面抗原CD44、CD105、CD166、CD29呈阳性表达,CD14、CD34和CD45呈阴性表达。(2)基因及蛋白水平检测均显示hPMSCs高表达PDL2；通过脂质体途径能够将PDL2siRNA高效转染入hPMSCs,有效沉默PDL2的表达。(3)沉默PDL2后,细胞接种0.5h后hPMSCs的黏附率较未沉默组未发生明显变化,但在接种1h或3h后,其黏附率明显高于未沉默组细胞(P0.05)；在3种不同的培养条件下,沉默PDL2后,hPMSCs迁移率明显低于未沉默组细胞(P0.01)。(4) hPMSCs能够抑制活化T细胞对CD69的表达,沉默PDL2后,CD69的表达与未沉默组相比无明显变化；沉默PDL2后,T细胞增殖明显增加,但仍低于单纯PMA刺激组；与未沉默组相比,处于G0/G1期的T细胞数量明显减少,处于S期的细胞数量明显增加；在hPMSCs存在条件下,T细胞对IL-17的分泌明显增加,沉默PDL2后,IL-17的分泌被进一步上调。 结论：(1) hPMSCs高表达PDL2;(2) PDL2siRNA能够有效沉默PDL2在hPMSCs上的表达；(3)PDL2不仅在hPMSCs介导的对T细胞的增殖、周期及分泌细胞因子的免疫调节中发挥重要的作用,而且参与了hPMSCs的黏附和迁移。 二、PDL2在人胎盘间充质干细胞上的表达及调控 目的：研究不同代hPMSCs对PDL2的表达变化及IFN-y调节PDL2在hPMSCs上表达的信号调节机制。 方法：(1)实时定量PCR(real-time PCR)及FCM检测不同代hPMSCs对PDL2的表达.(2) RT-PCR及FCM检测IFN-y或／和INCB018424作用后hPMSCs对PDL2mRNA和蛋白水平表达的变化。(3) Western blot检测IFN-y或/和INCBO18424作用后hPMSCs中STAT1和STAT3磷酸化水平变化。 结果：(1)不同代hPMSCs对PDL2在基因水平及蛋白水平的表达均有着显著差异,mRNA水平第20代表达最高,蛋白水平第12代表达最高。(2)IFN-γ作用后,PDL2的mRNA及蛋白表达水平均被明显上调,且STAT1和STAT3的磷酸化水平同样被显著上调。(3) INCB018424处理hPMSCs后,IFN-y刺激及未刺激组PDL2的mRNA、蛋白水平及STAT1和STAT3的磷酸化水平均被明显下调。 结论：不同代hPMSCs对PDL2表达水平不同,IFN-y能够通过JAK-STAT通路上调hPMSCs对PDL2的表达。
[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.

【学位授予单位】：滨州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329.2

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1 陈健琳,郭子宽,徐晨,李欲航,侯春梅,毛宁,陈虎;间充质干细胞分泌TGF-β1抑制异体T细胞反应性[J];中国实验血液学杂志;2002年04期

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