核心肽免疫调节抑制类风湿滑膜增生的研究

发布时间：2018-06-17 10:47

本文选题：类风湿关节炎 + 核心肽　；参考：《天津医科大学》2011年硕士论文

【摘要】：目的构建jurkatT淋巴细胞与类风湿关节炎成纤维样滑膜细胞(rheumatoid arthritis fibroblast-like synoviocyte,RAFS)共培养体系,通过观察核心肽(core peptide,CP)对该体系中两种细胞的增殖、凋亡及相关炎性细胞因子表达的影响,探讨CP对RAFS的作用及其机制,为其进一步的临床应用奠定一定的理论基础。方法 1.酶消化法进行RAFS分离培养,电镜及免疫组织化学方法进行鉴定,并观察其生长状态及增殖特性。 2Jurkat T淋巴细胞的培养,观察其生长状态及增殖特性。 3.MTT法观察不同浓度CP对Jurkat T细胞增殖的影响,流式细胞仪检测CP对Jurkat T细胞凋亡的影响,Real-Time PCR法观察CP对Jurkat T细胞炎性相关因子表达的影响。 4.MTT法观察不同浓度CP对共培养体系中Jurkat T细胞及RAFS增殖的影响,流式细胞仪检测CP对共培养体系中Jurkat T细胞及RAFS凋亡的影响,Real-Time PCR法观察CP对共培养体系中Jurkat T细胞及RAFS炎性相关因子表达的影响。结果 1.分离培养得到的RAFS经电镜及免疫组化鉴定以成纤维样滑膜细胞为主,其在体外培养扩增能力较强。 2JurkatT细胞在培养液中悬浮,圆润透亮,随着培养时间的延长,细胞聚集成团生长,细胞体外扩增速度较快,增殖能力强。 3.CP作用于Jurkat T细胞后,第7天高剂量即开始对其体外扩增出现明显抑制作用,中、低剂量分别在第10天、第13天开始表现抑制作用,至第13天三种剂量CP对T细胞的增殖均呈现出抑制作用,且具有剂量依赖性。 4.相对于对照组CP能引起Jurkat T细胞的凋亡增加。作用24h时,中、高剂量CP相对于对照组引起明显的凋亡增加,而低剂量CP则凋亡增加不明显,作用48h时,仅高剂量CP相对于对照组凋亡明显增加。 5.三种剂量CP均抑制了Jurkat T细胞的IL-2mRNA、TNFmRNA的表达,高剂量CP抑制了IL-23p19mRNA的表达。 6.CP作用于混合培养体系后,从第4天开始,高剂量的CP对T淋巴细胞的增殖表现出抑制作用,并一直持续至第13天,中、低剂量在第13天开始表现持抑制作用。高剂量CP在第7天、第10天表现出对RAFS的抑制作用,中、低剂量对RAFS的增殖没有明显的抑制作用。 7.三种浓度CP与空白对照组相比,在24h和48h均能引起共培养体系中JurkatT细胞的凋亡增加。作用24h时,与空白对照组相比,中、高剂量CP能引起共培养体系中RAFS的凋亡增加,作用48h时,只有高剂量CP能引起共培养体系中RAFS的凋亡增加。 8.CP抑制混合培养体系中的Jurkat T的IL-2mRNA及相关炎性因子IL-23p19mRNA、TNFmRNA的表达,高浓度的CP抑制共培养中RAFS的IL23pl9mRNA的表达,中、高浓度CP抑制共培养中RAFS的TNFmRNA的表达。结论 CP能抑制单纯培养及与RAFS混合培养的Jurkat T淋巴细胞的增殖、凋亡 IL-2mRNA及相关炎性细胞因子IL-23p19mRNA、TNFmRNA的表达。高剂量的 CP能抑制与Jurkat T淋巴细胞混合培养的RAFS的增殖、凋亡及相关炎性细胞因子IL-23p19mRNA、TNFmRNA的表达。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329.2

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