重组人白细胞介素-1β对人外周血淋巴细胞功能的影响及芍药苷的作用

发布时间：2018-01-31 01:58

本文关键词： 重组人白细胞介素-1β 外周血淋巴细胞 CD25 芍药苷　出处：《安徽医科大学》2009年硕士论文　论文类型：学位论文

【摘要】： 目的: 为阐明白细胞介素-1 (interleukin-1, IL-1)在类风湿关节炎(rheumatoid arthritis, RA)发病机制中的作用及芍药苷(paeoniflorin, Pae)是否通过调节人外周血淋巴细胞(peripheral blood lymphocyte, PBLC)异常的活化、增殖、分化及免疫耐受发挥治疗RA作用,观察重组人IL-1β(recombinant human IL-1β, rhIL-1β)对人PBLC增殖、产生IL-2和IL-10功能的影响及Pae的作用;观察人PBLC经rhIL-1β体外作用后表面CD25表达和其中CD4~+CD25~(high)调节性T细胞(CD4~+CD25~(high) regulatory T cell, CD4~+CD25~(high) Treg)所占比例的变化及Pae的作用。方法: 经知情同意,抽取健康志愿者外周静脉血,采用密度梯度离心法分离人外周血单个核细胞(peripheral blood mononuclear cell, PBMC),采用贴壁法进一步纯化人PBLC进行培养,经rhIL-1β体外作用,检测人PBLC增殖功能,产生IL-2和IL-10水平,表面CD25表达和CD4~+CD25~(high) Treg比例的变化及Pae的作用。 MTT法检测人PBLC的增殖反应;放射性免疫法测定人PBLC培养上清中IL-2的水平;酶联免疫吸附法检测人PBLC培养上清液中IL-10的水平;流式细胞术检测人PBLC表面CD25表达及CD4~+CD25~(high) Treg所占比例。结果: 1. Pae抑制rhIL-1β促人PBLC增殖反应。 rhIL-1β(0.1、1.0、10.0、100.0μg·L~(-1))可增强人PBLC的增殖反应,Pae (10~(-8)、10~(-7)、10~(-6)、10~(-5) mol·L~(-1))体外作用可抑制rhIL-1β诱导的促人PBLC增殖反应。结果表明Pae可恢复rhIL-1β致人PBLC异常的增殖功能至正常水平。 2. Pae降低rhIL-1β体外作用下人PBLC产生炎性细胞因子IL-2的水平,升高抗炎性细胞因子IL-10的水平。 rhIL-1β(0.1、1.0、10.0、100.0μg·L~(-1))可升高人PBLC分泌的IL-2水平,作用24 h产生的IL-2水平显著升高,Pae (10~(-8)、10~(-7)、10~(-6)、10~(-5) mol·L~(-1))可抑制rhIL-1β诱导的人PBLC产生IL-2。rhIL-1β(0.1、1.0、10.0、100.0μg·L~(-1))可抑制人PBLC产生IL-10,rhIL-1β体外作用后产生的IL-10水平随时间延长而升高,作用24 h产生的IL-10水平有显著升高,Pae (10~(-8)、10~(-7)、10~(-6)、10~(-5) mol·L~(-1))体外作用可显著升高rhIL-1β诱导的人PBLC产生IL-10水平。提示Pae可诱导Th0细胞向Th2细胞分化,恢复人PBLC中Th1/ Th2细胞间的平衡。 3. Pae抑制rhIL-1β体外作用下人PBLC的活化,显著降低人PBLC表面CD25的表达水平,升高CD4~+CD25~(high) Treg所占比例。 rhIL-1β(0.1、1.0、10.0、100.0μg·L~(-1))升高人PBLC表面CD25的表达,作用24 h表面CD25表达水平显著升高,Pae (10~(-8)、10~(-7)、10~(-6)、10~(-5) mol·L~(-1))体外作用可显著降低人PBLC表面CD25的表达水平。rhIL-1β体外作用下人PBLC中CD4~+CD25~(high) Treg所占比例无显著性改变;Pae (10~(-7)、10~(-6)、10~(-5) mol·L~(-1))可显著升高CD4~+CD25~(high) Treg在rhIL-1β体外作用下人PBLC中所占比例。结果表明Pae可逆转IL-1引起CD4+CD25- T细胞活化,单一的IL-1体外作用无法影响CD4~+CD25~(high) Treg的表达,Pae可上调CD4~+CD25~(high) Treg在人PBLC中的比例通过诱导免疫耐受发挥调节人PBLC功能的作用。结论: 1. Pae可逆转rhIL-1β的促人PBLC增殖反应。 2. Pae可降低rhIL-1β体外作用下人PBLC分泌IL-2水平,升高IL-10的水平。 3. Pae可抑制rhIL-1β体外作用下人PBLC的活化,下调rhIL-1β体外作用下人PBLC表面CD25表达,升高CD4~+CD25~(high) Treg所占比例。
[Abstract]:Objective:
To understand the interleukin -1 (interleukin-1, IL-1) in rheumatoid arthritis (rheumatoid arthritis, RA) and the effect of paeoniflorin in the pathogenesis of (paeoniflorin, Pae) is regulated by human peripheral blood lymphocytes (peripheral blood, lymphocyte, PBLC) activation, abnormal proliferation, differentiation and immune tolerance play therapy RA effect of recombinant human IL-1 beta (recombinant beta human IL-1, rhIL-1) on the proliferation of PBLC, IL-2 and IL-10 influence function and the role of Pae; the effects of human PBLC by rhIL-1 in vitro after beta surface CD25 expression and CD4~+CD25~ (high) regulatory T cells (CD4~+CD25~ (high) regulatory T cell CD4~+CD25~, (high) Treg) for change and the ratio of Pae function.
Method:
After informed consent, venous blood from healthy volunteers peripheral, isolated from human peripheral blood mononuclear cells by density gradient centrifugation (peripheral blood mononuclear cell, PBMC), using adherent method, further purification of human PBLC were cultured in vitro by rhIL-1 beta, detected the proliferation of PBLC, IL-2 and IL-10 level surface the expression of CD25 and CD4~+CD25~ (high) and Pae change Treg ratio effect.
MTT assay was used to detect the proliferative response of human PBLC. Radioimmunoassay was used to determine the level of IL-2 in human PBLC supernatant. The level of IL-10 in human PBLC supernatant was detected by enzyme-linked immunosorbent assay (ELISA), and CD25 expression and CD4~+CD25~ (high) Treg proportion on PBLC surface were detected by flow cytometry.
Result:
1. Pae inhibited the proliferation of human PBLC by rhIL-1 beta.
RhIL-1 beta (0.1,1.0,10.0100.0 u g - L~ (-1)) can enhance the proliferation of human PBLC, Pae (10~ (-8), 10~ (-7), 10~ (-6), 10~ (-5) mol - L~ (-1)) to promote proliferation of PBLC in vitro can inhibit rhIL-1 induced. The results showed that Pae can restore the function of human PBLC proliferation induced by rhIL-1 abnormal to normal levels.
2. Pae reduces the level of inflammatory cytokines IL-2, which increases the level of inflammatory cytokine IL-2, and increases the level of anti-inflammatory cytokine IL-10 under the effect of rhIL-1 beta in vitro.
RhIL-1 beta (0.1,1.0,10.0100.0 u g - L~ (-1)) can increase the secretion of PBLC high IL-2 level, IL-2 level 24 h significantly increased, Pae (10~ (-8), 10~ (-7), 10~ (-6), 10~ (-5) mol - L~ (-1)) can inhibit rhIL-1 induced by beta the PBLC IL-2.rhIL-1 beta (0.1,1.0,10.0100.0 G - L~ (-1)) can inhibit PBLC production of IL-10, rhIL-1 in vitro after IL-10 levels increased with time, the level of IL-10 24 h have significantly increased Pae (10~ (-8), 10~ (-7), 10~ (-6), 10~ (-5) mol - L~ (-1)) in vitro was significantly increased in rhIL-1 induced human PBLC beta IL-10 level. It suggests that Pae can induce Th0 cells to differentiate into Th2 cells, Th1/ Th2 cells to restore the balance between human PBLC.
3. Pae inhibited the activation of human PBLC under the effect of rhIL-1 beta in vitro, significantly reduced the expression level of CD25 on the human PBLC surface, and increased the proportion of CD4~+CD25~ (high) Treg.
RhIL-1 beta (0.1,1.0,10.0100.0 u g - L~ (-1)) increased expression of CD25 on the surface of expert PBLC, 24 h CD25 surface expression levels were significantly increased, Pae (10~ (-8), 10~ (-7), 10~ (-6), 10~ (-5) mol - L~ (-1)) in vitro can significantly reduce CD4~+CD25~ human PBLC expression level of.RhIL-1 beta in vitro PBLC surface CD25 (high) in the proportion of Treg had no significant change; Pae (10~ (-7), 10~ (-6), 10~ (-5) mol - L~ (-1)) was significantly increased in CD4~+CD25~ (high) Treg in human PBLC beta rhIL-1 in vitro the proportion of cases. The results showed that Pae could reverse IL-1 induced CD4+CD25- activation of T cells in vitro, IL-1 single CD4~+CD25~ (high) could not influence the expression of Treg, Pae CD4~+CD25~ (high) can increase the proportion of Treg in PBLC through the induction of immune tolerance of PBLC play a regulatory function.
Conclusion:
1. Pae can reverse the proliferation of human PBLC in rhIL-1 beta.
2. Pae can reduce the level of IL-2 secreted by human PBLC and increase the level of IL-10 under the effect of rhIL-1 beta in vitro.
3. Pae can inhibit the activation of human PBLC under the effect of rhIL-1 beta in vitro, and reduce the expression of CD25 on the PBLC surface of human rhIL-1 beta in vitro, and increase the proportion of CD4~+CD25~ (high) Treg.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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