發(fā)布時(shí)間：2018-04-28 18:05

  本文選題：細(xì)胞外基質(zhì)金屬蛋白酶誘導(dǎo)因子 + 基質(zhì)金屬蛋白酶　； 參考：《蚌埠醫(yī)學(xué)院》2011年碩士論文

【摘要】：背景：基質(zhì)金屬蛋白酶(matrix metalloproteinases, MMPs)在動(dòng)脈粥樣硬化(atherosclerosis, AS)的發(fā)生、發(fā)展過(guò)程中扮演重要角色,不僅涉及斑塊局部炎性細(xì)胞浸潤(rùn)、血管平滑肌細(xì)胞遷移,還可通過(guò)降解細(xì)胞外基質(zhì)導(dǎo)致斑塊破裂,這也是動(dòng)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(shí),AS的形成是多因素共同作用的結(jié)果。其中,炎性細(xì)胞特別是T淋巴細(xì)胞和單核細(xì)胞在其中發(fā)揮了重要作用。近年研究表明,炎癥反應(yīng)過(guò)程中單核細(xì)胞向內(nèi)膜趨化、遷移、分化成巨噬細(xì)胞,后者吞噬脂質(zhì)形成脂質(zhì)核心,同時(shí)分泌大量MMPs從而導(dǎo)致AS斑塊趨于不穩(wěn)定。細(xì)胞外基質(zhì)金屬蛋白酶誘導(dǎo)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(xì)胞系LX-1中被分離出來(lái),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(xì)胞及腫瘤組織內(nèi)的成纖維細(xì)胞表面高度表達(dá),并可通過(guò)旁分泌和自分泌作用誘導(dǎo)成纖維細(xì)胞大量表達(dá)MMPs。研究證實(shí)T細(xì)胞表面也有CD147高表達(dá),但在AS中,T淋巴細(xì)胞表達(dá)的CD147能否誘導(dǎo)單核細(xì)胞趨化及分泌MMPs,國(guó)內(nèi)外文獻(xiàn)未見(jiàn)報(bào)道。 目的：建立T淋巴細(xì)胞和單核細(xì)胞的共培養(yǎng)體系以研究細(xì)胞與細(xì)胞相互作用,通過(guò)調(diào)控人T淋巴細(xì)胞CD147表達(dá),研究其對(duì)單核細(xì)胞的趨化能力及MMPs分泌的影響,探討AS形成的可能機(jī)制和干預(yù)靶點(diǎn),以便為進(jìn)一步研究AS機(jī)制及研發(fā)相關(guān)治療藥物提供理論基礎(chǔ)和依據(jù)。 方法：采用RT-PCR、Western Bloting和流式細(xì)胞儀等技術(shù),對(duì)Jurkat細(xì)胞CD147mRNA、蛋白和膜蛋白表達(dá)水平進(jìn)行檢測(cè)。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(diào)Jurkat細(xì)胞CD147表達(dá)的藥物。采用篩選得到的有效藥物刺激Jurkat細(xì)胞，進(jìn)一步與THP-1細(xì)胞共培養(yǎng)，通過(guò)趨化實(shí)驗(yàn)：共培養(yǎng)體系中上調(diào)CD147表達(dá)對(duì)單核細(xì)胞趨化能力的影響；通過(guò)ELISA實(shí)驗(yàn)觀察：親環(huán)素A（CyclophilinA，CyPA）、環(huán)孢素A（CiclosporinA，CsA）預(yù)處理T淋巴細(xì)胞后和單核細(xì)胞共培養(yǎng)24h后，對(duì)其上清液中基質(zhì)金屬蛋白酶-9（matrixmetalloproteinase，MMP-9）蛋白含量的影響。 結(jié)果：低濃度組Ara-C刺激Jurkat細(xì)胞48h后，與對(duì)照組比較細(xì)胞CD147mRNA和蛋白表達(dá)均明顯升高，其中CD147膜蛋白上調(diào)明顯，和對(duì)照組比較差異有統(tǒng)計(jì)學(xué)意義（P0.05）。正常Jurkat細(xì)胞與THP-1細(xì)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(diào)Jurkat細(xì)胞CD147表達(dá)后，與THP-1細(xì)胞共培養(yǎng)24h，上清中MMP-9分泌量明顯升高；同時(shí)，單核細(xì)胞趨化能力增強(qiáng)（P0.05）。CyPA、CsA分別預(yù)處理后JurkatT淋巴細(xì)胞與單核細(xì)胞THP-1共培養(yǎng)，CyPA可促進(jìn)上清液中MMP-9分泌，，CsA則抑制上清中MMP-9分泌（P0.05）。 結(jié)論： 1.Ara-C可有效上調(diào)人T淋巴細(xì)胞CD147mRNA和蛋白表達(dá)水平。 2.在共培養(yǎng)體系中，上調(diào)Jurkat細(xì)胞CD147的表達(dá)可增強(qiáng)THP-1細(xì)胞趨化能力，促進(jìn)MMP-9分泌。 3.CyPA、CsA分別預(yù)處理Jurkat細(xì)胞后與THP-1細(xì)胞的共培養(yǎng)，CyPA可促進(jìn)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(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 范冬梅;楊銘;賈海榮;高瀛岱;王金宏;紀(jì)慶;熊冬生;楊純正;;阿糖胞苷上調(diào)白血病細(xì)胞CD86分子及細(xì)胞因子表達(dá)的研究[J];細(xì)胞與分子免疫學(xué)雜志;2008年06期

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