本文選題：細胞外基質金屬蛋白酶誘導因子 + 基質金屬蛋白酶��； 參考：《蚌埠醫(yī)學院》2011年碩士論文

【摘要】：背景：基質金屬蛋白酶(matrix metalloproteinases, MMPs)在動脈粥樣硬化(atherosclerosis, AS)的發(fā)生、發(fā)展過程中扮演重要角色,不僅涉及斑塊局部炎性細胞浸潤、血管平滑肌細胞遷移,還可通過降解細胞外基質導致斑塊破裂,這也是動脈粥樣硬化斑塊不穩(wěn)定甚至破裂,從而引起急性冠脈事件發(fā)生的重要原因之一 研究證實,AS的形成是多因素共同作用的結果。其中,炎性細胞特別是T淋巴細胞和單核細胞在其中發(fā)揮了重要作用。近年研究表明,炎癥反應過程中單核細胞向內膜趨化、遷移、分化成巨噬細胞,后者吞噬脂質形成脂質核心,同時分泌大量MMPs從而導致AS斑塊趨于不穩(wěn)定。細胞外基質金屬蛋白酶誘導因子(extracellular matrix metalloproteinase inducer, EMMPRIN)又稱CD147分子,是一種高度糖基化的單次跨膜蛋白,最早從肺癌細胞系LX-1中被分離出來,為免疫球蛋白超家族(immunogloblin superfamily, IGSF)一員。其在乳腺癌、皮膚癌等腫瘤細胞及腫瘤組織內的成纖維細胞表面高度表達,并可通過旁分泌和自分泌作用誘導成纖維細胞大量表達MMPs。研究證實T細胞表面也有CD147高表達,但在AS中,T淋巴細胞表達的CD147能否誘導單核細胞趨化及分泌MMPs,國內外文獻未見報道。 目的：建立T淋巴細胞和單核細胞的共培養(yǎng)體系以研究細胞與細胞相互作用,通過調控人T淋巴細胞CD147表達,研究其對單核細胞的趨化能力及MMPs分泌的影響,探討AS形成的可能機制和干預靶點,以便為進一步研究AS機制及研發(fā)相關治療藥物提供理論基礎和依據(jù)。 方法：采用RT-PCR、Western Bloting和流式細胞儀等技術,對Jurkat細胞CD147mRNA、蛋白和膜蛋白表達水平進行檢測。從植物血凝素(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)中篩選有效上調Jurkat細胞CD147表達的藥物。采用篩選得到的有效藥物刺激Jurkat細胞，進一步與THP-1細胞共培養(yǎng)，通過趨化實驗：共培養(yǎng)體系中上調CD147表達對單核細胞趨化能力的影響；通過ELISA實驗觀察：親環(huán)素A（CyclophilinA，CyPA）、環(huán)孢素A（CiclosporinA，CsA）預處理T淋巴細胞后和單核細胞共培養(yǎng)24h后，對其上清液中基質金屬蛋白酶-9（matrixmetalloproteinase，MMP-9）蛋白含量的影響。 結果：低濃度組Ara-C刺激Jurkat細胞48h后，與對照組比較細胞CD147mRNA和蛋白表達均明顯升高，其中CD147膜蛋白上調明顯，和對照組比較差異有統(tǒng)計學意義（P0.05）。正常Jurkat細胞與THP-1細胞共培養(yǎng)24h后上清中MMP-9分泌較少；用10ng/ml濃度的Ara-C上調Jurkat細胞CD147表達后，與THP-1細胞共培養(yǎng)24h，上清中MMP-9分泌量明顯升高；同時，單核細胞趨化能力增強（P0.05）。CyPA、CsA分別預處理后JurkatT淋巴細胞與單核細胞THP-1共培養(yǎng)，CyPA可促進上清液中MMP-9分泌，，CsA則抑制上清中MMP-9分泌（P0.05）。 結論： 1.Ara-C可有效上調人T淋巴細胞CD147mRNA和蛋白表達水平。 2.在共培養(yǎng)體系中，上調Jurkat細胞CD147的表達可增強THP-1細胞趨化能力，促進MMP-9分泌。 3.CyPA、CsA分別預處理Jurkat細胞后與THP-1細胞的共培養(yǎng)，CyPA可促進上清中MMP-9分泌，CsA則可抑制上清中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.

【學位授予單位】：蚌埠醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R363

【參考文獻】

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1 范冬梅;楊銘;賈海榮;高瀛岱;王金宏;紀慶;熊冬生;楊純正;;阿糖胞苷上調白血病細胞CD86分子及細胞因子表達的研究[J];細胞與分子免疫學雜志;2008年06期

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