本文選題：CCR7 + 樹突狀細(xì)胞（DCs）��； 參考：《南昌大學(xué)》2012年碩士論文

【摘要】：目的和意義：近年來，諸多研究表明免疫炎癥反應(yīng)參與冠心病的發(fā)病機(jī)制，而作為免疫炎癥反應(yīng)中最為重要的抗原提呈細(xì)胞-樹突狀細(xì)胞（Dendritic Cells，DCs）在免疫反應(yīng)中發(fā)揮著重要的作用。由于趨化因子受體CCR7在未成熟的樹突狀細(xì)胞（Immature dendritic cells，imDCs）表面低表達(dá)，而在成熟的樹突狀細(xì)胞表面高表達(dá)，推測(cè)CCR7可能與DCs的成熟有著密切的關(guān)系。另外在免疫反應(yīng)的過程中imDC主要起著攝取抗原的作用，成熟的DCs將攝取抗原呈遞給T淋巴細(xì)胞引起免疫炎癥反應(yīng)。CCR7能否調(diào)控DCs的成熟目前尚不清楚。本文通過體外原代分離培養(yǎng)大鼠骨髓源性樹突狀細(xì)胞（Rat Bone Marrow-derivedDendritic Cells，BMDCs），進(jìn)一步通過形態(tài)學(xué)及其細(xì)胞表型進(jìn)行鑒定。同時(shí)構(gòu)建趨化因子受體CCR7的RNA干擾的慢病毒載體，進(jìn)一步轉(zhuǎn)染imDCs；轉(zhuǎn)染6天后，通過RT-PCR和Western Blot分別檢測(cè)CCR7mRNA和蛋白的表達(dá)以及ELISA法檢測(cè)細(xì)胞培養(yǎng)上清液中巨噬細(xì)胞炎性蛋白1a(MIP-1a)和單核細(xì)胞趨化蛋白-1（MCP-1）的含量（由于相關(guān)研究表明隨著DCs的成熟培養(yǎng)上清液中MIP-1a分泌是逐漸降低的，MCP-1分泌是逐漸增高的）。探討沉默CCR7基因后對(duì)DCs成熟的影響，從而通過抑制DCs的成熟為動(dòng)脈粥樣硬化性心臟病乃至于心血管疾病的治療提供新的思路。 方法:體外分離培養(yǎng)及其鑒定大鼠骨髓源性樹突狀細(xì)胞：取出150-180g（雌雄不限）SD大鼠雙下肢股骨和脛骨，用5ml注射器吸取含10%FCS的RPMI-1640培養(yǎng)基沖洗骨髓腔收集骨髓細(xì)胞，繼而用粒細(xì)胞-巨噬細(xì)胞集落刺激因子（Granulocyte-macrophage colony stimulating factor, GM-CSF）5ng/ml和白細(xì)胞介素-4（Interleukin4，IL-4）5ng/ml誘導(dǎo)分化，培養(yǎng)至第6天收集懸浮細(xì)胞（即imDCs），繼續(xù)用GM-CSF2.5-5ng/ml進(jìn)行分化培養(yǎng)，一般至12天可獲得成熟的樹突狀細(xì)胞。分別收集第6天、9天、12天及14天BMDCs，通過形態(tài)學(xué)及流式細(xì)胞儀檢測(cè)細(xì)胞表型對(duì)BMDCs進(jìn)行鑒定。由吉?jiǎng)P公司完成RNA干擾慢病毒載體構(gòu)建和慢病毒包裝與滴度的檢測(cè)，獲得4個(gè)靶點(diǎn)的shRNA，通過RT-PCR檢測(cè)mRNA和Western Blot檢測(cè)蛋白篩選最佳的靶點(diǎn)。本研究分為3個(gè)組：實(shí)驗(yàn)組為轉(zhuǎn)染CCR7-GFP-LV的DCs、陰性對(duì)照組為轉(zhuǎn)染NC-GFP-LV的DCs，空白對(duì)照組為正常培養(yǎng)的DCs。用篩選出最佳的靶點(diǎn)CCR7-GFP-LV轉(zhuǎn)染第6天的樹突狀細(xì)胞，穩(wěn)定轉(zhuǎn)染6天后通過RT-PCR和Western Blot檢測(cè)各組CCR7mRNA和蛋白的表達(dá)水平，通過ELISA法檢測(cè)三個(gè)組細(xì)胞培養(yǎng)上清液中MIP-1a和MCP-1的含量，通過MTT法檢測(cè)CCR7沉默后對(duì)DCs生長(zhǎng)增殖情況的影響。 結(jié)果:體外培養(yǎng)初期（6-9天）樹突狀細(xì)胞呈半懸浮狀態(tài)，圓形，無(wú)樹突；培養(yǎng)至后期（12天左右）呈半懸浮半貼壁狀態(tài)，圓形或橢圓形，部分細(xì)胞可見樹突狀突起。流式細(xì)胞表型的鑒定結(jié)果示培養(yǎng)初期低表達(dá)CD86，，中度表達(dá)MHC-II，至培養(yǎng)后期CD86和MHC-II的表達(dá)均增高。ELISA檢測(cè)結(jié)果示隨著培養(yǎng)時(shí)間的延長(zhǎng)MIP-1a含量逐漸降低，而MCP-1含量逐漸增高。由吉?jiǎng)P公司提供4個(gè)靶點(diǎn)，經(jīng)過RT-PCR和Western Blot篩選出最佳靶點(diǎn)為CCR7-RNAi-LV3#,其濃縮病毒滴度為6×108TU/ml.轉(zhuǎn)染CCR7shRNA慢病毒轉(zhuǎn)染后6天（第12天DCs），70-75%左右的BMDCs表達(dá)GFP， RT-PCR結(jié)果顯示CCR7mRNA表達(dá)情況實(shí)驗(yàn)組（26.85±0.03）%與空白對(duì)照組（93.01±0.01）%和陰性對(duì)照組（83.69±0.01）%相比P0.05，具有統(tǒng)計(jì)學(xué)意義；Westeern Blot結(jié)果示：CCR7蛋白表達(dá)情況實(shí)驗(yàn)組（47.86±0.02）%與空白對(duì)照組（93.50±0.05）%和陰性對(duì)照組（91.35±0.02）%相比P0.05，具有統(tǒng)計(jì)學(xué)意義；ELISA結(jié)果顯示MIP-1a含量實(shí)驗(yàn)組（30.70±0.50）pg/ml與空白對(duì)照組（25.33±1.74）pg/ml和陰性對(duì)照組（26.95±0.22）pg/ml相比P0.05,具有統(tǒng)計(jì)學(xué)意義；MCP-1含量實(shí)驗(yàn)組（25.50±0.95）pg/ml與空白對(duì)照組（31.06±1.41）pg/ml和陰性對(duì)照組（31.44±1.95）pg/ml相比P0.05,具有統(tǒng)計(jì)學(xué)意義；MTT結(jié)果示：CCR7沉默后實(shí)驗(yàn)組與空白對(duì)照組和陰性對(duì)照組相比P0.05,具有統(tǒng)計(jì)學(xué)意義。 結(jié)論: CCR7慢病毒載體構(gòu)建成功，轉(zhuǎn)染CCR7shRNA慢病毒載體后BMDCs的CCR7mRNA和蛋白表達(dá)水平明顯下調(diào)，細(xì)胞培養(yǎng)上清液中MIP-1a含量明顯增高和MCP-1含量明顯下降，同時(shí)DCs的生長(zhǎng)增殖情況受到明顯的抑制；CCR7基因沉默后具有抑制DCs成熟的作用，可以為下一步誘導(dǎo)DCs對(duì)相關(guān)抗原（如氧化性低密度脂蛋白，ox-LDL）產(chǎn)生免疫耐受做好前期工作，可能為冠心病的治療提供新的靶點(diǎn)。
[Abstract]:Objective and significance: in recent years, many studies have shown that immune inflammatory response is involved in the pathogenesis of coronary heart disease, and Dendritic Cells (DCs), the most important antigen presenting cell in the immune response, plays an important role in the immune response. Because chemokine receptor CCR7 is in the immature dendritic cells (Im The surface of mature dendritic cells, imDCs) is low expression and is highly expressed on the surface of mature dendritic cells. It is presumed that CCR7 may have a close relationship with the maturation of DCs. In addition, in the process of immune response, imDC plays an important role in the uptake of antigen, and the mature DCs will take the antigen to the T lymphocyte to cause the immune inflammation reaction to.CCR7. It is not clear to regulate the maturity of DCs. In this paper, Rat Bone Marrow-derivedDendritic Cells (BMDCs) in vitro was isolated and cultured in vitro. It was further identified by morphology and cell phenotype. At the same time, the lentivirus vector with the RNA interference of chemokine receptor CCR7 was constructed, and the transfection of imDCs was further transfected. After 6 days, the expression of CCR7mRNA and protein was detected by RT-PCR and Western Blot, and the content of macrophage inflammatory protein 1a (MIP-1a) and monocyte chemoattractant protein -1 (MCP-1) in cell culture supernatant was detected by ELISA method. (the correlation study showed that MIP-1a secretion in the mature culture supernatant of DCs was gradually reduced. The effect of the silent CCR7 gene on the maturation of DCs is explored, thus providing a new idea for the treatment of atherosclerotic heart disease and even on the treatment of cardiovascular diseases by inhibiting the maturity of DCs.
Methods: the rat bone marrow derived dendritic cells were isolated and cultured in vitro: the femur and tibia of 150-180g (male and female and male) SD rats were removed and the bone marrow cells were washed with the RPMI-1640 culture medium containing 10%FCS in the 5ml syringe, and then the granulocyte macrophage colony stimulating factor (Granulocyte-macrophage colony) was used. Stimulating factor, GM-CSF) 5ng/ml and interleukin -4 (Interleukin4, IL-4) 5ng/ml induced differentiation, culture to sixth days to collect suspension cells (imDCs), continue to use GM-CSF2.5-5ng/ml for differentiation and culture, generally to 12 days to obtain mature dendritic cells. Collect sixth days, 9 days, 12 days and 14 days BMDCs, through morphology and flow fineness. BMDCs was identified by cell phenotype. The RNA interfering lentivirus carrier was constructed by Jikai Corporation and the detection of the package and titer of lentivirus was completed. The shRNA of 4 targets was obtained. The best targets were screened by RT-PCR detection of mRNA and Western Blot detection protein. This study was divided into 3 groups: the experimental group was the DCs and negative of CCR7-GFP-LV transfected. The group was DCs transfected with NC-GFP-LV, and the blank control group was used for the normal culture of DCs. to transfect the best target CCR7-GFP-LV to sixth days of dendritic cells. The expression level of CCR7mRNA and protein in each group was detected by RT-PCR and Western Blot after 6 days of stable transfection, and MIP-1a and MCP-1 in the supernatant of three groups of cells were detected by ELISA method. The effects of CCR7 silencing on the growth and proliferation of DCs were detected by MTT.
Results: the dendritic cells were semi suspended, round and non dendrite in the early stage of culture (6-9 days). The cells were semi suspended and half adherent in the later period (about 12 days), round or oval, and some cells showed dendritic protuberance. The identification results of the phenotype of flow cytometry showed that the low expression of CD86 in the early stage of culture was MHC-II, to CD86 and M in the later period of culture. The expression of HC-II increased with.ELISA detection, and the content of MIP-1a gradually decreased with the prolongation of culture time, while the content of MCP-1 increased gradually. The best target was CCR7-RNAi-LV3# with RT-PCR and Western Blot, and the concentration of the concentration of the virus was 6 days after the transfection of 6 x 108TU/ml. transfected CCR7shRNA lentivirus (first). 2 days DCs), 70-75% BMDCs expressed GFP, RT-PCR results showed that CCR7mRNA expression in the experimental group (26.85 + 0.03)% compared with the blank control group (93.01 + 0.01)% and negative control group (83.69 + 0.01)% compared to P0.05, with statistical significance; Westeern Blot results showed that CCR7 egg white expression in the experimental group (47.86 + 0.02)% and the blank control group (93.50 + 0. 05)% compared with the negative control group (91.35 + 0.02)% (91.35 + 0.02)% compared with P0.05, with statistical significance; ELISA results showed that the MIP-1a content experimental group (30.70 + 0.50) pg/ml compared with the blank control group (25.33 + 1.74) pg/ml and the negative control group (26.95 + 0.22) pg/ml compared to P0.05, with statistical significance; MCP-1 content experimental group (25.50 + 0.95) pg/ml and blank control group (31.06) + 1.41) pg/ml and negative control group (31.44 + 1.95) pg/ml compared with P0.05, with statistical significance, MTT results showed that after CCR7 silencing, the experimental group was statistically significant compared with the blank control group and the negative control group P0.05.
Conclusion: the construction of CCR7 lentivirus vector was successful. The expression level of CCR7mRNA and protein in BMDCs was obviously reduced after transfection of CCR7shRNA lentivirus vector. The content of MIP-1a in the cell culture supernatant was obviously increased and the content of MCP-1 decreased obviously. Meanwhile, the growth and proliferation of DCs was obviously suppressed, and the CCR7 gene was silenced to inhibit the maturity of DCs. It can be used for the next step to induce DCs to produce immune tolerance to related antigens, such as oxidative low density lipoprotein (ox-LDL), and to provide new targets for the treatment of coronary heart disease.

【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王絢卉;于曉風(fēng);曲紹春;徐華麗;韓冬;睢大{|;;洋參二醇皂苷注射液對(duì)大鼠急性心肌梗死的影響及其機(jī)制研究[J];中國(guó)中藥雜志;2009年24期

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