沉默CCR7基因后对树突状细胞在体外成熟影响的研究

发布时间：2018-05-13 07:34

本文选题：CCR7 + 树突状细胞（DCs）　；参考：《南昌大学》2012年硕士论文

【摘要】：目的和意义：近年来，诸多研究表明免疫炎症反应参与冠心病的发病机制，而作为免疫炎症反应中最为重要的抗原提呈细胞-树突状细胞（Dendritic Cells，DCs）在免疫反应中发挥着重要的作用。由于趋化因子受体CCR7在未成熟的树突状细胞（Immature dendritic cells，imDCs）表面低表达，而在成熟的树突状细胞表面高表达，推测CCR7可能与DCs的成熟有着密切的关系。另外在免疫反应的过程中imDC主要起着摄取抗原的作用，成熟的DCs将摄取抗原呈递给T淋巴细胞引起免疫炎症反应。CCR7能否调控DCs的成熟目前尚不清楚。本文通过体外原代分离培养大鼠骨髓源性树突状细胞（Rat Bone Marrow-derivedDendritic Cells，BMDCs），进一步通过形态学及其细胞表型进行鉴定。同时构建趋化因子受体CCR7的RNA干扰的慢病毒载体，进一步转染imDCs；转染6天后，通过RT-PCR和Western Blot分别检测CCR7mRNA和蛋白的表达以及ELISA法检测细胞培养上清液中巨噬细胞炎性蛋白1a(MIP-1a)和单核细胞趋化蛋白-1（MCP-1）的含量（由于相关研究表明随着DCs的成熟培养上清液中MIP-1a分泌是逐渐降低的，MCP-1分泌是逐渐增高的）。探讨沉默CCR7基因后对DCs成熟的影响，从而通过抑制DCs的成熟为动脉粥样硬化性心脏病乃至于心血管疾病的治疗提供新的思路。方法:体外分离培养及其鉴定大鼠骨髓源性树突状细胞：取出150-180g（雌雄不限）SD大鼠双下肢股骨和胫骨，用5ml注射器吸取含10%FCS的RPMI-1640培养基冲洗骨髓腔收集骨髓细胞，继而用粒细胞-巨噬细胞集落刺激因子（Granulocyte-macrophage colony stimulating factor, GM-CSF）5ng/ml和白细胞介素-4（Interleukin4，IL-4）5ng/ml诱导分化，培养至第6天收集悬浮细胞（即imDCs），继续用GM-CSF2.5-5ng/ml进行分化培养，一般至12天可获得成熟的树突状细胞。分别收集第6天、9天、12天及14天BMDCs，通过形态学及流式细胞仪检测细胞表型对BMDCs进行鉴定。由吉凯公司完成RNA干扰慢病毒载体构建和慢病毒包装与滴度的检测，获得4个靶点的shRNA，通过RT-PCR检测mRNA和Western Blot检测蛋白筛选最佳的靶点。本研究分为3个组：实验组为转染CCR7-GFP-LV的DCs、阴性对照组为转染NC-GFP-LV的DCs，空白对照组为正常培养的DCs。用筛选出最佳的靶点CCR7-GFP-LV转染第6天的树突状细胞，稳定转染6天后通过RT-PCR和Western Blot检测各组CCR7mRNA和蛋白的表达水平，通过ELISA法检测三个组细胞培养上清液中MIP-1a和MCP-1的含量，通过MTT法检测CCR7沉默后对DCs生长增殖情况的影响。结果:体外培养初期（6-9天）树突状细胞呈半悬浮状态，圆形，无树突；培养至后期（12天左右）呈半悬浮半贴壁状态，圆形或椭圆形，部分细胞可见树突状突起。流式细胞表型的鉴定结果示培养初期低表达CD86，，中度表达MHC-II，至培养后期CD86和MHC-II的表达均增高。ELISA检测结果示随着培养时间的延长MIP-1a含量逐渐降低，而MCP-1含量逐渐增高。由吉凯公司提供4个靶点，经过RT-PCR和Western Blot筛选出最佳靶点为CCR7-RNAi-LV3#,其浓缩病毒滴度为6×108TU/ml.转染CCR7shRNA慢病毒转染后6天（第12天DCs），70-75%左右的BMDCs表达GFP， RT-PCR结果显示CCR7mRNA表达情况实验组（26.85±0.03）%与空白对照组（93.01±0.01）%和阴性对照组（83.69±0.01）%相比P0.05，具有统计学意义；Westeern Blot结果示：CCR7蛋白表达情况实验组（47.86±0.02）%与空白对照组（93.50±0.05）%和阴性对照组（91.35±0.02）%相比P0.05，具有统计学意义；ELISA结果显示MIP-1a含量实验组（30.70±0.50）pg/ml与空白对照组（25.33±1.74）pg/ml和阴性对照组（26.95±0.22）pg/ml相比P0.05,具有统计学意义；MCP-1含量实验组（25.50±0.95）pg/ml与空白对照组（31.06±1.41）pg/ml和阴性对照组（31.44±1.95）pg/ml相比P0.05,具有统计学意义；MTT结果示：CCR7沉默后实验组与空白对照组和阴性对照组相比P0.05,具有统计学意义。结论: CCR7慢病毒载体构建成功，转染CCR7shRNA慢病毒载体后BMDCs的CCR7mRNA和蛋白表达水平明显下调，细胞培养上清液中MIP-1a含量明显增高和MCP-1含量明显下降，同时DCs的生长增殖情况受到明显的抑制；CCR7基因沉默后具有抑制DCs成熟的作用，可以为下一步诱导DCs对相关抗原（如氧化性低密度脂蛋白，ox-LDL）产生免疫耐受做好前期工作，可能为冠心病的治疗提供新的靶点。
[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.

【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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