miR-200b对外周血单核细胞来源树突状细胞发育的影响

发布时间：2018-06-20 11:30

本文选题：树突状细胞 + miR-200b　；参考：《中国人民解放军军事医学科学院》2011年硕士论文

【摘要】：MicroRNA (miRNA)是一种内源性、大小约为19~25个碱基的非编码单链小RNA,其作用机制是通过其反义链与靶mRNA分子完全或不完全互补结合、切割同源性靶mRNA分子或抑制其翻译,导致特异性靶基因沉默来调控基因表达[1-2]。研究表明,miRNA在物种间具有高度的保守性、细胞或组织特异性、时序性和位相性,即在特定的细胞或组织、时间、发育阶段才会表达,这决定了组织和细胞功能的特异性,对组织的发育起着重要的作用,因此成为目前研究的热点[3]。树突状细胞(dentritic cells, DCs)是目前发现的功能最强的抗原提呈细胞(antigen process cell, APC),从造血干细胞发育而来,DC的发育和分化具有鲜明的时序性和位相性[4-5],因此,非常适合作为靶细胞研究miRNA对其发育、增殖、分化与凋亡的调控。目前有关miRNA对DC发育的影响研究甚少,为此我们通过生物信息学预测发现miRNA200b(miR-200b)可能对DC的发育产生影响。进一步检测外周血单核细胞分化为DC的不同阶段miR-200b的表达,结果显示miR-200b在分化过程中变化显著。结合我们初步的实验结果,有理由推测miR-200b可能对DC的发育产生影响,为此我们设计了miR-200b对外周血单核细胞来源DC发育影响的研究课题,旨在阐明miR-200b对DC发育的调控及其机制,为深入探讨DC的免疫调控作用提供理论与实验依据。首先,为建立稳定的外周血CD14~+单核细胞来源DC培养体系,通过免疫磁珠法分离外周血CD14~+单核细胞,比较IMDM和RPMI 1640两种培养基结合细胞因子GM-CSF+IL-4,诱导CD14~+单核细胞向DC分化是否存在差异,从形态、表型和功能三个方面进行鉴定。研究结果表明,两种培养基诱导所得DC在形态上无显著差异, iDC与mDC都具备未成熟与成熟DC的典型特征,即未成熟的iDC呈圆球状,而经过LPS诱导刺激后的mDC细胞表面具有明显的不规则突起;流式细胞仪检测DC的吞噬能力,结果显示, IMDM培养的DC与用RPMI 1640培养的DC具有相似的吞噬能力,在未成熟状态下吞噬效率高达90%以上,而成熟后的吞噬效率则低于50%。进一步对诱导获得DC的细胞表型进行检测,发现CD14和CD83的表达亦无显著差异,但IMDM培养的DC其CD1a表达明显降低,分别为IMDM-iDC(32.34%±9.02%)和IMDM-DC(21.04%±6.33%);RPMI-1640培养体系的CD1a表达呈高水平,分别为1640-iDC(85.58%±4.26%)和1640-DC(87.68%±5.83%)。经过LPS刺激成熟后,作为树突状细胞成熟的表面标志CD83表达亦变化显著,RPMI 1640-mDC表达高水平CD83(86.62%±1.37%),而IMDM-mDC则为(64.68%±5.26%);利用MLR体系对诱导所获DC进行功能检测,即其刺激T细胞增殖能力,结果表明,无论是IMDM还是RPMI1640培养的iDC和mDC,它们刺激T细胞增殖的能力均存在显著差异。IMDM-iDC和RPMI 1640-iDC的cpm值分别为277.2±25.79和3170±454.12;而IMDM-mDC和RPMI 1640-mDC的cpm值分别为1402.6±128.07和5274.4±426.26。在这两种培养体系中,iDC刺激T细胞增殖的能力均显著低于mDC,而用RPMI 1640-mDC对T细胞的刺激能力又显著高于IMDM-mDC,且随着刺激细胞浓度梯度的降低, T细胞增殖能力也逐渐减低。由于不同细胞因子对DC的发育与功能起非常重要的调控作用,为此我们利用悬浮芯片法检测MLR体系中细胞因子的表达,结果表明,IMDM培养条件下的活化的T细胞表达较高的免疫抑制性因子IL-10(56.67±33.41 pg/mL),以及IL-6(315.48±15.43 pg/mL)和IL-8(17300.76±2273.89 pg/mL),但作为DC成熟标志的IL-12却无法检测到;而RPMI1640培养体系则高表达IL-12(5658.09±540.46 pg/mL),提示不同培养体系可获得功能不同的DC。为进一步深入研究DC的发育分化机制,我们采用RPMI1640培养基进行miRNA对DC发育分化影响的实验。其次,通过生物信息学预测发现miR-200b可能对DC的发育分化产生影响。miR-200家族根据不同功能区和“种子区”序列,分为miR-200a/miR-141和miR-200b/miR-200c/miR-429两个亚家族;而根据不同的基因组定位,miR-200家族又分为miR-200a/miR-200b/miR-429和miR-200c/miR-141两个基因簇[6]。已有研究结果表明,miR-200家族功能呈多样性,其中miR-200b在许多肿瘤中存在异常表达[7-9],对其生物学功能检测结果显示,在膀胱癌细胞系T24中过表达miR-200b可使细胞形态发生显著改变,细胞失去伪足样突起,形状变得不规则;进一步实验结果证明转染miR-200b后的肿瘤细胞迁移能力也显著下降。利用生物信息学软件预测miR-200b靶基因,发现其靶基因Jagged2、WISF1、WASF-3、ZEB1和ZEB2在DC中均有表达,且这些靶基因在3’-UTR均有miR-200b的作用靶点。结合DC的发育分化过程和形态特征与预测结果具有相似性,因此我们有理由推测DC可能是miR-200b的作用靶点。为此,我们取不同培养时间的DC,Real-time PCR检测miR-200b在DC中的表达。结果表明,随着诱导时间的延长,miR-200b的表达呈递减趋势,提示miR-200b与DC的发育分化相关。第三,依据上述实验结果,进一步利用DOTAP转染试剂将过表达miR-200b和无意义序列NC转染不同发育阶段的DC,即iDC和mDC,通过形态、表型和功能进行相关的鉴定。结果显示,利用DOTAP转染试剂转染小RNA的有效率可达70%以上;进一步对转染miRNA的细胞进行形态学观察,可见转染miR-200b的mDC表面光滑或细胞突起减少,而转染NC和正常DC表面均可见不规则突起;流式细胞术检测细胞表型亦无显著改变, iDC表型为CD14-CD1a+CD83-,而mDC表型为CD14-CD1a+CD83+ ;吞噬实验结果表明,iDC具有很强的吞噬能力,而转染miR-200b的iDC吞噬能力相对较弱,提示miR-200b可能阻碍了iDC的发育;刺激T细胞增殖是mDC的主要功能特征,在MLR体系中,转染NC的mDC和正常mDC对T细胞增殖的刺激能力无显著差异,而转染miR-200b的mDC对T细胞增殖的刺激能力则显著下降;当DC与T细胞的比例为1:10时,mDC、NC-mDC、miR-200b-mDC的cpm值分别为3623±287.7、3366±183.2和2250±136.2; DC与T细胞的比例为1:50时,mDC、NC-mDC、200b-mDC的cpm值分别为934.8±180.6、882±123和546.8±75.84,上述结果表明miR-200b可使iDC的吞噬能力降低,mDC刺激T细胞增殖的能力降低。另外,在MLR反应体系中细胞因子的表达出现显著改变,miR-200b-mDC组作为DC成熟标志的细胞因子IL-12分泌量显著降低,且发挥刺激T细胞增殖的细胞因子IL-2、IL-6也显著降低,而抑制性因子IL-10虽表达量很低,却高于mDC和NC-mDC在MLR体系中的表达水平,提示miR-200b无论是从形态,还是功能上均对DC的发育分化起到抑制作用。上述研究结果提示, miR-200b对DC的发育具有抑制作用,但作用靶点尚不清楚,仍需进一步研究。本研究为进一步系统探讨miR-200b调控DC发育分化的分子机制及临床应用提供了理论与实验依据。
[Abstract]:MicroRNA (miRNA) is an endogenous, non coded single strand RNA of about 19~25 base, and its mechanism is to cut the homologous target mRNA molecule or inhibit its translation through its antisense chain and the target mRNA molecule completely or completely, and inhibit its translation, which leads to the specific target gene silence to regulate gene expression [1-2]. research. MiRNA is in substance. Interspecies have high conservatism, cell or tissue specificity, timing and phase, which are expressed in a specific cell or tissue, time, and developmental stage. This determines the specificity of the tissue and cell function and plays an important role in the development of tissue. Therefore, it has become a hot spot of [3]. dendritic cells (dentritic cells, DC). S) is the most powerful antigen presenting cell (antigen process cell, APC) found at present. From the development of hematopoietic stem cells, the development and differentiation of DC have a distinct timing and phase [4-5]. Therefore, it is very suitable as a target cell to study the regulation of miRNA on its development, proliferation, differentiation and apoptosis. At present, miRNA has a shadow on the development of DC. There was little research, so we found that miRNA200b (miR-200b) may have an impact on the development of DC by bioinformatics. Further detection of the expression of miR-200b at different stages of DC in peripheral blood mononuclear cells showed that miR-200b was significantly changed during the process of differentiation. 0b may have an effect on the development of DC. Therefore, we have designed the research subject of the effect of miR-200b on the development of DC in peripheral blood mononuclear cells, aiming at clarifying the regulation and mechanism of miR-200b on the development of DC, and providing theoretical and experimental basis for the in-depth study of the immune regulation and control of DC.
First, in order to establish a stable peripheral blood CD14~+ monocyte derived DC culture system, CD14~+ mononuclear cells from peripheral blood were separated by immunomagnetic beads, and two cultures of IMDM and RPMI 1640 were compared with the cytokine GM-CSF+IL-4 to induce the differentiation of CD14~+ mononuclear cells to DC, and the morphological, phenotypic and functional three aspects were identified. The results showed that there was no significant difference in morphology between the two culture medium induced DC. Both iDC and mDC had the typical characteristics of immature and mature DC, that is, the immature iDC was round spheroid, and the mDC cell surface after LPS induced stimulation had obvious irregular protruding, and the flow cytometry detected the phagocytosis of DC, and the result showed IMDM culture. The DC cultured with RPMI 1640 had similar phagocytosis ability, and the phagocytic efficiency was more than 90% in the immature state, while the phagocytic efficiency after maturity was lower than that of 50%., and the expression of CD14 and CD83 was not significantly different, but the expression of CD14 and CD83 was also not significantly different, but the DC CD1a expression of IMDM culture was significantly reduced. Not IMDM-iDC (32.34% + 9.02%) and IMDM-DC (21.04% + 6.33%); CD1a expression in RPMI-1640 culture system was high level, 1640-iDC (85.58% + 4.26%) and 1640-DC (87.68% + 5.83%). After LPS stimulation, the expression of CD83 as the surface marker of dendritic cells was also significant, RPMI 1640-mDC expressed high CD83 (86.62% + 1.37%). While IMDM-mDC was (64.68% + 5.26%), using the MLR system to detect the induced DC, that is, it stimulated the proliferation of T cells. The results showed that both IMDM and RPMI1640 cultured iDC and mDC had significant differences in the ability to stimulate T cell proliferation by.IMDM-iDC and RPMI 1640-iDC, respectively 277.2 + 25.79 and 3170 + 454, respectively. .12, while the CPM values of IMDM-mDC and RPMI 1640-mDC were 1402.6 + 128.07 and 5274.4 + 426.26. respectively in these two cultures. The ability of iDC to stimulate T cell proliferation was significantly lower than mDC, while RPMI 1640-mDC on T cells was significantly higher than that of RPMI 1640-mDC. Since different cytokines play an important role in regulating the development and function of DC, we use the suspension chip method to detect the expression of cytokines in the MLR system. The results show that the activated T cells in the IMDM culture conditions express a higher immunosuppressive factor IL-10 (56.67 + 33.41 pg/mL), and IL-6 (315.48 + 15.43 pg/). ML) and IL-8 (17300.76 + 2273.89 pg/mL), but the IL-12 as a symbol of DC maturity can not be detected, while RPMI1640 culture system is highly expressed IL-12 (5658.09 + 540.46 pg/mL), suggesting that different cultures can obtain different functional DC. to further study the development and differentiation mechanism of DC. Experiments on the effect of differentiation.
Secondly, through bioinformatics prediction, it is found that miR-200b may affect the development and differentiation of DC, and the.MiR-200 family is divided into two subfamilies of miR-200a/miR-141 and miR-200b/miR-200c/miR-429 according to the sequence of different functional areas and "seed region", and the miR-200 family is divided into miR-200a/miR-200b/miR-429 and miR according to the location of different genomes. The results of -200c/miR-141 two gene cluster [6]. show that the function of miR-200 family is diverse, of which miR-200b has abnormal expression of [7-9] in many tumors. Its biological function detection results show that the overexpression of miR-200b in the bladder cancer cell line T24 can cause significant changes in cell morphology, and the cells lose the form of pseudo foot like protrusions. Further experimental results showed that the migration ability of tumor cells after transfection of miR-200b was also significantly decreased. MiR-200b target gene was predicted by bioinformatics software, and its target genes, Jagged2, WISF1, WASF-3, ZEB1 and ZEB2 were expressed in DC, and these targets were targeted at miR-200b in 3 '-UTR. The developmental differentiation and morphological characteristics are similar to the predicted results. Therefore, we have reason to speculate that DC may be the target of miR-200b. Therefore, we take DC of different culture time and Real-time PCR to detect the expression of miR-200b in DC. The results show that the expression of miR-200b is decreasing with the prolongation of induction time, suggesting miR-200b. It is related to the development and differentiation of DC.
Third, according to the results mentioned above, DOTAP transfection reagent will be further used to transfect miR-200b and nonsense sequence NC to different developmental stages of DC, namely, iDC and mDC, through morphological, phenotypic and functional identification. The results show that the efficiency of transfection of small RNA by DOTAP transfection reagent can be more than 70%, and the transfection of miRNA is further improved. Morphological observation showed that the mDC surface of the transfected miR-200b was smooth or the cell protuberance decreased, while the transfected NC and the normal DC surface showed irregular protuberance, and the cell phenotype was not significantly changed by flow cytometry, the iDC phenotype was CD14-CD1a+CD83-, and the mDC phenotype was CD14-CD1a+CD83+. The phagocytic experiment showed that iDC was very strong. The phagocytosis of iDC transfected with miR-200b is relatively weak, suggesting that miR-200b may impede the development of iDC, and the proliferation of T cells is the main functional feature of mDC. In the MLR system, there is no significant difference in the stimulating ability of NC's mDC and normal mDC to the proliferation of T cells. When the ratio of DC to T cells is 1:10, the CPM values of mDC, NC-mDC and miR-200b-mDC are 3623 + 287.73366 + 183.2 and 2250 + 136.2 respectively. When the proportion of DC and T cells is 1:50, mDC, NC-mDC, 200b-mDC CPM values are 934.8 + 180.6882 + 123 and 546.8 + 75.84 respectively. In addition, the expression of cytokines in the MLR reaction system was significantly changed. The secretion of cytokine IL-12 in the miR-200b-mDC group was significantly reduced as a marker of DC maturation, and the cytokine IL-2, which stimulated the proliferation of T cells, was also significantly reduced, while the inhibitory factor IL-10, although low in expression, was higher than mDC and N. The expression level of C-mDC in MLR system suggests that miR-200b can inhibit the development and differentiation of DC both in morphology and function.
These results suggest that miR-200b has an inhibitory effect on the development of DC, but the target points are still unclear. This study provides a theoretical and experimental basis for further systematic study of the molecular mechanism and clinical application of miR-200b to regulate and regulate the development and differentiation of DC.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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