本文選題：樹突狀細(xì)胞 + miR-200b�。� 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2011年碩士論文

【摘要】：MicroRNA (miRNA)是一種內(nèi)源性、大小約為19~25個堿基的非編碼單鏈小RNA,其作用機(jī)制是通過其反義鏈與靶mRNA分子完全或不完全互補(bǔ)結(jié)合、切割同源性靶mRNA分子或抑制其翻譯,導(dǎo)致特異性靶基因沉默來調(diào)控基因表達(dá)[1-2]。研究表明,miRNA在物種間具有高度的保守性、細(xì)胞或組織特異性、時序性和位相性,即在特定的細(xì)胞或組織、時間、發(fā)育階段才會表達(dá),這決定了組織和細(xì)胞功能的特異性,對組織的發(fā)育起著重要的作用,因此成為目前研究的熱點(diǎn)[3]。樹突狀細(xì)胞(dentritic cells, DCs)是目前發(fā)現(xiàn)的功能最強(qiáng)的抗原提呈細(xì)胞(antigen process cell, APC),從造血干細(xì)胞發(fā)育而來,DC的發(fā)育和分化具有鮮明的時序性和位相性[4-5],因此,非常適合作為靶細(xì)胞研究miRNA對其發(fā)育、增殖、分化與凋亡的調(diào)控。目前有關(guān)miRNA對DC發(fā)育的影響研究甚少,為此我們通過生物信息學(xué)預(yù)測發(fā)現(xiàn)miRNA200b(miR-200b)可能對DC的發(fā)育產(chǎn)生影響。進(jìn)一步檢測外周血單核細(xì)胞分化為DC的不同階段miR-200b的表達(dá),結(jié)果顯示miR-200b在分化過程中變化顯著。結(jié)合我們初步的實(shí)驗(yàn)結(jié)果,有理由推測miR-200b可能對DC的發(fā)育產(chǎn)生影響,為此我們設(shè)計了miR-200b對外周血單核細(xì)胞來源DC發(fā)育影響的研究課題,旨在闡明miR-200b對DC發(fā)育的調(diào)控及其機(jī)制,為深入探討DC的免疫調(diào)控作用提供理論與實(shí)驗(yàn)依據(jù)。 首先,為建立穩(wěn)定的外周血CD14~+單核細(xì)胞來源DC培養(yǎng)體系,通過免疫磁珠法分離外周血CD14~+單核細(xì)胞,比較IMDM和RPMI 1640兩種培養(yǎng)基結(jié)合細(xì)胞因子GM-CSF+IL-4,誘導(dǎo)CD14~+單核細(xì)胞向DC分化是否存在差異,從形態(tài)、表型和功能三個方面進(jìn)行鑒定。研究結(jié)果表明,兩種培養(yǎng)基誘導(dǎo)所得DC在形態(tài)上無顯著差異, iDC與mDC都具備未成熟與成熟DC的典型特征,即未成熟的iDC呈圓球狀,而經(jīng)過LPS誘導(dǎo)刺激后的mDC細(xì)胞表面具有明顯的不規(guī)則突起;流式細(xì)胞儀檢測DC的吞噬能力,結(jié)果顯示, IMDM培養(yǎng)的DC與用RPMI 1640培養(yǎng)的DC具有相似的吞噬能力,在未成熟狀態(tài)下吞噬效率高達(dá)90%以上,而成熟后的吞噬效率則低于50%。進(jìn)一步對誘導(dǎo)獲得DC的細(xì)胞表型進(jìn)行檢測,發(fā)現(xiàn)CD14和CD83的表達(dá)亦無顯著差異,但I(xiàn)MDM培養(yǎng)的DC其CD1a表達(dá)明顯降低,分別為IMDM-iDC(32.34%±9.02%)和IMDM-DC(21.04%±6.33%);RPMI-1640培養(yǎng)體系的CD1a表達(dá)呈高水平,分別為1640-iDC(85.58%±4.26%)和1640-DC(87.68%±5.83%)。經(jīng)過LPS刺激成熟后,作為樹突狀細(xì)胞成熟的表面標(biāo)志CD83表達(dá)亦變化顯著,RPMI 1640-mDC表達(dá)高水平CD83(86.62%±1.37%),而IMDM-mDC則為(64.68%±5.26%);利用MLR體系對誘導(dǎo)所獲DC進(jìn)行功能檢測,即其刺激T細(xì)胞增殖能力,結(jié)果表明,無論是IMDM還是RPMI1640培養(yǎng)的iDC和mDC,它們刺激T細(xì)胞增殖的能力均存在顯著差異。IMDM-iDC和RPMI 1640-iDC的cpm值分別為277.2±25.79和3170±454.12;而IMDM-mDC和RPMI 1640-mDC的cpm值分別為1402.6±128.07和5274.4±426.26。在這兩種培養(yǎng)體系中,iDC刺激T細(xì)胞增殖的能力均顯著低于mDC,而用RPMI 1640-mDC對T細(xì)胞的刺激能力又顯著高于IMDM-mDC,且隨著刺激細(xì)胞濃度梯度的降低, T細(xì)胞增殖能力也逐漸減低。由于不同細(xì)胞因子對DC的發(fā)育與功能起非常重要的調(diào)控作用,為此我們利用懸浮芯片法檢測MLR體系中細(xì)胞因子的表達(dá),結(jié)果表明,IMDM培養(yǎng)條件下的活化的T細(xì)胞表達(dá)較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),但作為DC成熟標(biāo)志的IL-12卻無法檢測到;而RPMI1640培養(yǎng)體系則高表達(dá)IL-12(5658.09±540.46 pg/mL),提示不同培養(yǎng)體系可獲得功能不同的DC。為進(jìn)一步深入研究DC的發(fā)育分化機(jī)制,我們采用RPMI1640培養(yǎng)基進(jìn)行miRNA對DC發(fā)育分化影響的實(shí)驗(yàn)。 其次,通過生物信息學(xué)預(yù)測發(fā)現(xiàn)miR-200b可能對DC的發(fā)育分化產(chǎn)生影響。miR-200家族根據(jù)不同功能區(qū)和“種子區(qū)”序列,分為miR-200a/miR-141和miR-200b/miR-200c/miR-429兩個亞家族;而根據(jù)不同的基因組定位,miR-200家族又分為miR-200a/miR-200b/miR-429和miR-200c/miR-141兩個基因簇[6]。已有研究結(jié)果表明,miR-200家族功能呈多樣性,其中miR-200b在許多腫瘤中存在異常表達(dá)[7-9],對其生物學(xué)功能檢測結(jié)果顯示,在膀胱癌細(xì)胞系T24中過表達(dá)miR-200b可使細(xì)胞形態(tài)發(fā)生顯著改變,細(xì)胞失去偽足樣突起,形狀變得不規(guī)則;進(jìn)一步實(shí)驗(yàn)結(jié)果證明轉(zhuǎn)染miR-200b后的腫瘤細(xì)胞遷移能力也顯著下降。利用生物信息學(xué)軟件預(yù)測miR-200b靶基因,發(fā)現(xiàn)其靶基因Jagged2、WISF1、WASF-3、ZEB1和ZEB2在DC中均有表達(dá),且這些靶基因在3’-UTR均有miR-200b的作用靶點(diǎn)。結(jié)合DC的發(fā)育分化過程和形態(tài)特征與預(yù)測結(jié)果具有相似性,因此我們有理由推測DC可能是miR-200b的作用靶點(diǎn)。為此,我們?nèi)〔煌囵B(yǎng)時間的DC,Real-time PCR檢測miR-200b在DC中的表達(dá)。結(jié)果表明,隨著誘導(dǎo)時間的延長,miR-200b的表達(dá)呈遞減趨勢,提示miR-200b與DC的發(fā)育分化相關(guān)。 第三,依據(jù)上述實(shí)驗(yàn)結(jié)果,進(jìn)一步利用DOTAP轉(zhuǎn)染試劑將過表達(dá)miR-200b和無意義序列NC轉(zhuǎn)染不同發(fā)育階段的DC,即iDC和mDC,通過形態(tài)、表型和功能進(jìn)行相關(guān)的鑒定。結(jié)果顯示,利用DOTAP轉(zhuǎn)染試劑轉(zhuǎn)染小RNA的有效率可達(dá)70%以上;進(jìn)一步對轉(zhuǎn)染miRNA的細(xì)胞進(jìn)行形態(tài)學(xué)觀察,可見轉(zhuǎn)染miR-200b的mDC表面光滑或細(xì)胞突起減少,而轉(zhuǎn)染NC和正常DC表面均可見不規(guī)則突起;流式細(xì)胞術(shù)檢測細(xì)胞表型亦無顯著改變, iDC表型為CD14-CD1a+CD83-,而mDC表型為CD14-CD1a+CD83+ ;吞噬實(shí)驗(yàn)結(jié)果表明,iDC具有很強(qiáng)的吞噬能力,而轉(zhuǎn)染miR-200b的iDC吞噬能力相對較弱,提示miR-200b可能阻礙了iDC的發(fā)育;刺激T細(xì)胞增殖是mDC的主要功能特征,在MLR體系中,轉(zhuǎn)染NC的mDC和正常mDC對T細(xì)胞增殖的刺激能力無顯著差異,而轉(zhuǎn)染miR-200b的mDC對T細(xì)胞增殖的刺激能力則顯著下降;當(dāng)DC與T細(xì)胞的比例為1:10時,mDC、NC-mDC、miR-200b-mDC的cpm值分別為3623±287.7、3366±183.2和2250±136.2; DC與T細(xì)胞的比例為1:50時,mDC、NC-mDC、200b-mDC的cpm值分別為934.8±180.6、882±123和546.8±75.84,上述結(jié)果表明miR-200b可使iDC的吞噬能力降低,mDC刺激T細(xì)胞增殖的能力降低。另外,在MLR反應(yīng)體系中細(xì)胞因子的表達(dá)出現(xiàn)顯著改變,miR-200b-mDC組作為DC成熟標(biāo)志的細(xì)胞因子IL-12分泌量顯著降低,且發(fā)揮刺激T細(xì)胞增殖的細(xì)胞因子IL-2、IL-6也顯著降低,而抑制性因子IL-10雖表達(dá)量很低,卻高于mDC和NC-mDC在MLR體系中的表達(dá)水平,提示miR-200b無論是從形態(tài),還是功能上均對DC的發(fā)育分化起到抑制作用。 上述研究結(jié)果提示, miR-200b對DC的發(fā)育具有抑制作用,但作用靶點(diǎn)尚不清楚,仍需進(jìn)一步研究。本研究為進(jìn)一步系統(tǒng)探討miR-200b調(diào)控DC發(fā)育分化的分子機(jī)制及臨床應(yīng)用提供了理論與實(shí)驗(yàn)依據(jù)。
[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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

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相關(guān)期刊論文 前2條

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本文編號：2044109