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  本文選題：類風濕關(guān)節(jié)炎 + 核心肽��； 參考：《天津醫(yī)科大學》2011年碩士論文

【摘要】：目的 構(gòu)建jurkatT淋巴細胞與類風濕關(guān)節(jié)炎成纖維樣滑膜細胞(rheumatoid arthritis fibroblast-like synoviocyte,RAFS)共培養(yǎng)體系,通過觀察核心肽(core peptide,CP)對該體系中兩種細胞的增殖、凋亡及相關(guān)炎性細胞因子表達的影響,探討CP對RAFS的作用及其機制,為其進一步的臨床應用奠定一定的理論基礎(chǔ)。方法 1.酶消化法進行RAFS分離培養(yǎng),電鏡及免疫組織化學方法進行鑒定,并觀察其生長狀態(tài)及增殖特性。 2Jurkat T淋巴細胞的培養(yǎng),觀察其生長狀態(tài)及增殖特性。 3.MTT法觀察不同濃度CP對Jurkat T細胞增殖的影響,流式細胞儀檢測CP對Jurkat T細胞凋亡的影響,Real-Time PCR法觀察CP對Jurkat T細胞炎性相關(guān)因子表達的影響。 4.MTT法觀察不同濃度CP對共培養(yǎng)體系中Jurkat T細胞及RAFS增殖的影響,流式細胞儀檢測CP對共培養(yǎng)體系中Jurkat T細胞及RAFS凋亡的影響,Real-Time PCR法觀察CP對共培養(yǎng)體系中Jurkat T細胞及RAFS炎性相關(guān)因子表達的影響。 結(jié)果 1.分離培養(yǎng)得到的RAFS經(jīng)電鏡及免疫組化鑒定以成纖維樣滑膜細胞為主,其在體外培養(yǎng)擴增能力較強。 2JurkatT細胞在培養(yǎng)液中懸浮,圓潤透亮,隨著培養(yǎng)時間的延長,細胞聚集成團生長,細胞體外擴增速度較快,增殖能力強。 3.CP作用于Jurkat T細胞后,第7天高劑量即開始對其體外擴增出現(xiàn)明顯抑制作用,中、低劑量分別在第10天、第13天開始表現(xiàn)抑制作用,至第13天三種劑量CP對T細胞的增殖均呈現(xiàn)出抑制作用,且具有劑量依賴性。 4.相對于對照組CP能引起Jurkat T細胞的凋亡增加。作用24h時,中、高劑量CP相對于對照組引起明顯的凋亡增加,而低劑量CP則凋亡增加不明顯,作用48h時,僅高劑量CP相對于對照組凋亡明顯增加。 5.三種劑量CP均抑制了Jurkat T細胞的IL-2mRNA、TNFmRNA的表達,高劑量CP抑制了IL-23p19mRNA的表達。 6.CP作用于混合培養(yǎng)體系后,從第4天開始,高劑量的CP對T淋巴細胞的增殖表現(xiàn)出抑制作用,并一直持續(xù)至第13天,中、低劑量在第13天開始表現(xiàn)持抑制作用。高劑量CP在第7天、第10天表現(xiàn)出對RAFS的抑制作用,中、低劑量對RAFS的增殖沒有明顯的抑制作用。 7.三種濃度CP與空白對照組相比,在24h和48h均能引起共培養(yǎng)體系中JurkatT細胞的凋亡增加。作用24h時,與空白對照組相比,中、高劑量CP能引起共培養(yǎng)體系中RAFS的凋亡增加,作用48h時,只有高劑量CP能引起共培養(yǎng)體系中RAFS的凋亡增加。 8.CP抑制混合培養(yǎng)體系中的Jurkat T的IL-2mRNA及相關(guān)炎性因子IL-23p19mRNA、TNFmRNA的表達,高濃度的CP抑制共培養(yǎng)中RAFS的IL23pl9mRNA的表達,中、高濃度CP抑制共培養(yǎng)中RAFS的TNFmRNA的表達。 結(jié)論 CP能抑制單純培養(yǎng)及與RAFS混合培養(yǎng)的Jurkat T淋巴細胞的增殖、凋亡 IL-2mRNA及相關(guān)炎性細胞因子IL-23p19mRNA、TNFmRNA的表達。高劑量的 CP能抑制與Jurkat T淋巴細胞混合培養(yǎng)的RAFS的增殖、凋亡及相關(guān)炎性細胞 因子IL-23p19mRNA、TNFmRNA的表達。
[Abstract]:Objective to construct a co-culture system of jurkat T lymphocytes and rheumatoid arthritis fibroblast-like synoviocytein (Raffs), and to observe the proliferation of two kinds of cells in this system by core peptide (CPP). To investigate the effect of CP on RAFS and its mechanism, and to lay a theoretical foundation for its further clinical application. Method 1. RAFS was isolated and cultured by enzyme digestion, identified by electron microscope and immunohistochemistry, and its growth state and proliferation characteristics were observed. 2 Jurkat T lymphocyte culture. 3. MTT assay was used to observe the effect of CP on the proliferation of Jurkat T cells. Effects of CP on apoptosis of Jurkat T cells were detected by flow cytometry. The effects of CP on the expression of inflammatory related factors in Jurkat T cells were observed by Real-Time PCR. 4. MTT assay was used to observe the effects of CP at different concentrations on the proliferation of Jurkat T cells and RAFS in co-culture system. The effect of CP on Jurkat T cells and RAFS apoptosis was detected by flow cytometry. The effect of CP on the expression of Jurkat T cells and RAFS inflammatory related factors in co-culture system was observed by Real-Time PCR. Result 1. The RAFS isolated and cultured were mainly fibroid synovial cells by electron microscope and immunohistochemistry. 2Jurkat T cells were suspended in culture medium, smooth and bright, with the extension of culture time. After treated with CP on Jurkat T cells, the proliferation of Jurkat T cells began to be inhibited by high dose on the 7th day, and the medium and low doses on the 10th day, respectively. On the 13th day, the growth of T cells was inhibited by the three doses of CP in a dose-dependent manner. Compared with the control group, CP increased the apoptosis of Jurkat T cells. At 24h exposure, the apoptosis of medium and high dose CP was significantly increased compared with that of control group, while that of low dose CP was not obvious. At 48 h, the apoptosis of high dose CP was significantly increased compared with that of control group, and that of low dose CP was significantly higher than that of control group (P < 0.05). All three doses of CP inhibited the expression of IL-2mRNA-TNFmRNA in Jurkat T cells, and high-dose CP inhibited the expression of IL-23p19mRNA. 6. After treated with CP in mixed culture system, the proliferation of T lymphocytes was inhibited by high-dose CP from the 4th day. And continued until the 13 th day, the low dose on the 13 th day began to show inhibitory effect. High dose CP showed inhibitory effect on RAFS on the 7th day and 10th day, while low dose had no obvious inhibitory effect on RAFS proliferation. Compared with the control group, the apoptosis of Jurkat T cells in co-culture system was increased at 24 h and 48 h after CP treatment. Compared with the control group, the apoptosis of RAFS in co-culture system was increased by high dose CP at 24 h, and increased at 48 h after treatment. Only high dose CP could increase the apoptosis of RAFS in co-culture system. 8. CP inhibited the expression of IL-2mRNA and IL-23p19mRNA-TNFmRNA in Jurkat T and IL-23p19mRNA-TNFmRNA in co-culture system, and high concentration of CP inhibited the expression of IL23pl9 mRNA in co-culture. High concentration CP inhibited the expression of TNF mRNA in RAFS. Conclusion CP can inhibit the proliferation of Jurkat T lymphocytes and the expression of apoptotic IL-2mRNA and IL-23p19mRNA-TNFmRNA in Jurkat T lymphocytes cultured solely or co-cultured with RAFS. High dose CP could inhibit the proliferation, apoptosis and the expression of IL-23p19mRNA-TNFmRNA in RAFS co-cultured with Jurkat T lymphocytes.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329.2

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