调控T淋巴细胞CD147表达对单核细胞的趋化及MMP-9分泌的影响

发布时间：2018-04-28 18:05

本文选题：细胞外基质金属蛋白酶诱导因子 + 基质金属蛋白酶　；参考：《蚌埠医学院》2011年硕士论文

【摘要】：背景：基质金属蛋白酶(matrix metalloproteinases, MMPs)在动脉粥样硬化(atherosclerosis, AS)的发生、发展过程中扮演重要角色,不仅涉及斑块局部炎性细胞浸润、血管平滑肌细胞迁移,还可通过降解细胞外基质导致斑块破裂,这也是动脉粥样硬化斑块不稳定甚至破裂,从而引起急性冠脉事件发生的重要原因之一研究证实,AS的形成是多因素共同作用的结果。其中,炎性细胞特别是T淋巴细胞和单核细胞在其中发挥了重要作用。近年研究表明,炎症反应过程中单核细胞向内膜趋化、迁移、分化成巨噬细胞,后者吞噬脂质形成脂质核心,同时分泌大量MMPs从而导致AS斑块趋于不稳定。细胞外基质金属蛋白酶诱导因子(extracellular matrix metalloproteinase inducer, EMMPRIN)又称CD147分子,是一种高度糖基化的单次跨膜蛋白,最早从肺癌细胞系LX-1中被分离出来,为免疫球蛋白超家族(immunogloblin superfamily, IGSF)一员。其在乳腺癌、皮肤癌等肿瘤细胞及肿瘤组织内的成纤维细胞表面高度表达,并可通过旁分泌和自分泌作用诱导成纤维细胞大量表达MMPs。研究证实T细胞表面也有CD147高表达,但在AS中,T淋巴细胞表达的CD147能否诱导单核细胞趋化及分泌MMPs,国内外文献未见报道。目的：建立T淋巴细胞和单核细胞的共培养体系以研究细胞与细胞相互作用,通过调控人T淋巴细胞CD147表达,研究其对单核细胞的趋化能力及MMPs分泌的影响,探讨AS形成的可能机制和干预靶点,以便为进一步研究AS机制及研发相关治疗药物提供理论基础和依据。方法：采用RT-PCR、Western Bloting和流式细胞仪等技术,对Jurkat细胞CD147mRNA、蛋白和膜蛋白表达水平进行检测。从植物血凝素(Phytohaem agglutinin,PHA),乙酸肉豆蔻佛波醇(phorbol myristate acetate, PMA),阿糖胞苷(cy to sine arabinoside, Ara-C)中筛选有效上调Jurkat细胞CD147表达的药物。采用筛选得到的有效药物刺激Jurkat细胞，进一步与THP-1细胞共培养，通过趋化实验：共培养体系中上调CD147表达对单核细胞趋化能力的影响；通过ELISA实验观察：亲环素A（CyclophilinA，CyPA）、环孢素A（CiclosporinA，CsA）预处理T淋巴细胞后和单核细胞共培养24h后，对其上清液中基质金属蛋白酶-9（matrixmetalloproteinase，MMP-9）蛋白含量的影响。结果：低浓度组Ara-C刺激Jurkat细胞48h后，与对照组比较细胞CD147mRNA和蛋白表达均明显升高，其中CD147膜蛋白上调明显，和对照组比较差异有统计学意义（P0.05）。正常Jurkat细胞与THP-1细胞共培养24h后上清中MMP-9分泌较少；用10ng/ml浓度的Ara-C上调Jurkat细胞CD147表达后，与THP-1细胞共培养24h，上清中MMP-9分泌量明显升高；同时，单核细胞趋化能力增强（P0.05）。CyPA、CsA分别预处理后JurkatT淋巴细胞与单核细胞THP-1共培养，CyPA可促进上清液中MMP-9分泌，，CsA则抑制上清中MMP-9分泌（P0.05）。结论： 1.Ara-C可有效上调人T淋巴细胞CD147mRNA和蛋白表达水平。 2.在共培养体系中，上调Jurkat细胞CD147的表达可增强THP-1细胞趋化能力，促进MMP-9分泌。 3.CyPA、CsA分别预处理Jurkat细胞后与THP-1细胞的共培养，CyPA可促进上清中MMP-9分泌，CsA则可抑制上清中MMP-9分泌。
[Abstract]:Background: matrix metalloproteinases (MMPs) plays an important role in the development of atherosclerosis (atherosclerosis, AS), which involves not only local inflammatory cell infiltration, vascular smooth muscle cell migration, but also the disruption of plaque by reducing the extracellular matrix, which is also atherosclerosis. One of the important causes of acute coronary events is unstable or even ruptured plaques.
Studies have shown that the formation of AS is the result of multiple factors. Among them, inflammatory cells, especially T and monocytes, play an important role. In recent years, the study showed that monocyte chemotaxis, migrated and differentiated into macrophages during the process of inflammation, and the latter phagocyted lipid to form the lipid core and secreted a large number of MM. Ps leads to the instability of AS plaques. The extracellular matrix metalloproteinase inducer (extracellular matrix metalloproteinase inducer, EMMPRIN), also known as CD147, is a highly glycosylated single transmembrane protein, which was first isolated from the lung cancer cell line LX-1, as the immunoglobulin superfamily (Immunogloblin superfami). Ly, IGSF). It is highly expressed on the surface of fibroblasts in tumor cells, such as breast cancer, skin cancer, and tumor tissue, and can be induced by paracrine and autocrine induced fibroblasts to express a large number of MMPs. studies to confirm the high expression of CD147 on the surface of T cells, but in AS, the CD147 expressed by T lymphocytes can induce mononuclear cells. The chemotaxis and secretion of MMPs have not been reported in the literature at home and abroad.
Objective: to establish a co culture system of T lymphocyte and monocyte to study the interaction between cell and cell, and to study the effect of T lymphocyte CD147 expression on the chemotactic ability of mononuclear cells and the effect of MMPs secretion, and to explore the possible mechanism of AS formation and the target of intervention in order to further study the mechanism of AS and to develop the related treatment. Drugs provide theoretical basis and basis.
Methods: RT-PCR, Western Bloting and flow cytometry were used to detect the expression level of CD147mRNA, protein and membrane protein in Jurkat cells. It was screened effectively from plant hemagglutinin (Phytohaem agglutinin, PHA), nutmeg phorbol (phorbol myristate acetate, PMA) and cytarabine. The drugs expressed by CD147 in Jurkat cells were used to stimulate Jurkat cells by screening effective drugs and co culture with THP-1 cells. Through chemotactic experiments, the effect of CD147 expression on the chemotactic capacity of monocytes was up-regulated by co culture system; and by ELISA experiment, cyclophilin A (CyclophilinA, CyPA), cyclosporin A (CiclosporinA, CsA) were observed. After pretreatment with T lymphocytes, the effects of 24h co cultured with monocytes on the content of matrix metalloproteinase -9 (matrixmetalloproteinase, MMP-9) in the supernatant were studied.
Results: after the low concentration group Ara-C stimulated the Jurkat cell 48h, the expression of CD147mRNA and protein in the cell was significantly higher than the control group, and the CD147 membrane protein up regulation was obviously up, and there was a significant difference between the control group and the control group (P0.05). The MMP-9 secreted less in the normal Jurkat cells and THP-1 cells after the co culture of 24h; 10ng/ml concentration Ara-C. After the expression of CD147 in Jurkat cells was up-regulated, 24h was co cultured with THP-1 cells, and the secretion of MMP-9 in the supernatant increased significantly. At the same time, the chemotactic capacity of monocyte increased (P0.05).CyPA, CsA was pre treated with CsA, and CyPA could promote the secretion of MMP-9 in the supernatant.
Conclusion:
1.Ara-C can effectively up regulate the expression of CD147mRNA and protein in human T lymphocytes.
2. in co culture system, upregulation of CD147 expression in Jurkat cells can enhance the chemotaxis ability of THP-1 cells and promote MMP-9 secretion.
3.CyPA and CsA co cultured with THP-1 cells after pretreatment of Jurkat cells, CyPA promoted the secretion of MMP-9 in the supernatant, while CsA inhibited the secretion of MMP-9 in the supernatant.

【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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