本文關(guān)鍵詞： 重組人白細胞介素-1β 外周血淋巴細胞 CD25 芍藥苷　出處：《安徽醫(yī)科大學(xué)》2009年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的: 為闡明白細胞介素-1 (interleukin-1, IL-1)在類風濕關(guān)節(jié)炎(rheumatoid arthritis, RA)發(fā)病機制中的作用及芍藥苷(paeoniflorin, Pae)是否通過調(diào)節(jié)人外周血淋巴細胞(peripheral blood lymphocyte, PBLC)異常的活化、增殖、分化及免疫耐受發(fā)揮治療RA作用,觀察重組人IL-1β(recombinant human IL-1β, rhIL-1β)對人PBLC增殖、產(chǎn)生IL-2和IL-10功能的影響及Pae的作用;觀察人PBLC經(jīng)rhIL-1β體外作用后表面CD25表達和其中CD4~+CD25~(high)調(diào)節(jié)性T細胞(CD4~+CD25~(high) regulatory T cell, CD4~+CD25~(high) Treg)所占比例的變化及Pae的作用。 方法: 經(jīng)知情同意,抽取健康志愿者外周靜脈血,采用密度梯度離心法分離人外周血單個核細胞(peripheral blood mononuclear cell, PBMC),采用貼壁法進一步純化人PBLC進行培養(yǎng),經(jīng)rhIL-1β體外作用,檢測人PBLC增殖功能,產(chǎn)生IL-2和IL-10水平,表面CD25表達和CD4~+CD25~(high) Treg比例的變化及Pae的作用。 MTT法檢測人PBLC的增殖反應(yīng);放射性免疫法測定人PBLC培養(yǎng)上清中IL-2的水平;酶聯(lián)免疫吸附法檢測人PBLC培養(yǎng)上清液中IL-10的水平;流式細胞術(shù)檢測人PBLC表面CD25表達及CD4~+CD25~(high) Treg所占比例。 結(jié)果: 1. Pae抑制rhIL-1β促人PBLC增殖反應(yīng)。 rhIL-1β(0.1、1.0、10.0、100.0μg·L~(-1))可增強人PBLC的增殖反應(yīng),Pae (10~(-8)、10~(-7)、10~(-6)、10~(-5) mol·L~(-1))體外作用可抑制rhIL-1β誘導(dǎo)的促人PBLC增殖反應(yīng)。結(jié)果表明Pae可恢復(fù)rhIL-1β致人PBLC異常的增殖功能至正常水平。 2. Pae降低rhIL-1β體外作用下人PBLC產(chǎn)生炎性細胞因子IL-2的水平,升高抗炎性細胞因子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產(chǎn)生的IL-2水平顯著升高,Pae (10~(-8)、10~(-7)、10~(-6)、10~(-5) mol·L~(-1))可抑制rhIL-1β誘導(dǎo)的人PBLC產(chǎn)生IL-2。rhIL-1β(0.1、1.0、10.0、100.0μg·L~(-1))可抑制人PBLC產(chǎn)生IL-10,rhIL-1β體外作用后產(chǎn)生的IL-10水平隨時間延長而升高,作用24 h產(chǎn)生的IL-10水平有顯著升高,Pae (10~(-8)、10~(-7)、10~(-6)、10~(-5) mol·L~(-1))體外作用可顯著升高rhIL-1β誘導(dǎo)的人PBLC產(chǎn)生IL-10水平。提示Pae可誘導(dǎo)Th0細胞向Th2細胞分化,恢復(fù)人PBLC中Th1/ Th2細胞間的平衡。 3. Pae抑制rhIL-1β體外作用下人PBLC的活化,顯著降低人PBLC表面CD25的表達水平,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的表達,作用24 h表面CD25表達水平顯著升高,Pae (10~(-8)、10~(-7)、10~(-6)、10~(-5) mol·L~(-1))體外作用可顯著降低人PBLC表面CD25的表達水平。rhIL-1β體外作用下人PBLC中CD4~+CD25~(high) Treg所占比例無顯著性改變;Pae (10~(-7)、10~(-6)、10~(-5) mol·L~(-1))可顯著升高CD4~+CD25~(high) Treg在rhIL-1β體外作用下人PBLC中所占比例。結(jié)果表明Pae可逆轉(zhuǎn)IL-1引起CD4+CD25- T細胞活化,單一的IL-1體外作用無法影響CD4~+CD25~(high) Treg的表達,Pae可上調(diào)CD4~+CD25~(high) Treg在人PBLC中的比例通過誘導(dǎo)免疫耐受發(fā)揮調(diào)節(jié)人PBLC功能的作用。 結(jié)論: 1. Pae可逆轉(zhuǎn)rhIL-1β的促人PBLC增殖反應(yīng)。 2. Pae可降低rhIL-1β體外作用下人PBLC分泌IL-2水平,升高IL-10的水平。 3. Pae可抑制rhIL-1β體外作用下人PBLC的活化,下調(diào)rhIL-1β體外作用下人PBLC表面CD25表達,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.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392

【參考文獻】

相關(guān)期刊論文 前9條

1 李景怡,吳玉章,方勇飛,王勇,張靜波,李陽;CD28/CTLA-4在類風濕關(guān)節(jié)炎患者外周血和關(guān)節(jié)積液中的表達及意義[J];第三軍醫(yī)大學(xué)學(xué)報;2004年06期

2 毛立群,馮玲,閆燕華;類風濕性關(guān)節(jié)炎患者外周血T_(H1)/T_(H2)細胞的研究[J];免疫學(xué)雜志;2002年S1期

3 張浩,董書魁,李雪麗,王清娟,田京霞;類風濕性關(guān)節(jié)炎患者外周血細胞內(nèi)IFN-γ和IL-4的三色流式分析[J];免疫學(xué)雜志;2004年02期

4 孫嫵弋,張玲玲,吳麗,汪倪萍,王華,岳莉,張運芳,魏偉;重組人白細胞介素-1受體拮抗劑對大鼠膠原性關(guān)節(jié)炎的治療作用[J];中國藥理學(xué)通報;2005年01期

5 常艷;魏偉;張磊;徐紅梅;;白芍總苷對IL-1α誘導(dǎo)膠原性關(guān)節(jié)炎大鼠成纖維樣滑膜細胞功能的影響[J];中國藥理學(xué)通報;2007年02期

6 王志堅，萬軍梅，，陳敏珠，徐叔云;白芍總甙對正常人與類風濕性關(guān)節(jié)炎患者單核細胞和淋巴細胞功能的影響[J];中國藥理學(xué)通報;1994年03期

7 汪慶童;張玲玲;孫嫵弋;吳麗;魏偉;;重組人白細胞介素1受體拮抗劑對大鼠佐劑性關(guān)節(jié)炎的治療作用[J];安徽醫(yī)科大學(xué)學(xué)報;2006年06期

8 王曉玉;魏偉;唐麗琴;吳虹;楊宇清;陳尹;;芍藥苷對佐劑性關(guān)節(jié)炎大鼠腹腔巨噬細胞吞噬功能及其產(chǎn)生細胞因子的影響[J];安徽醫(yī)科大學(xué)學(xué)報;2007年02期

9 李萍;畢黎琦;栗占國;;表達Foxp3的調(diào)節(jié)性T細胞在類風濕關(guān)節(jié)炎發(fā)病中的意義[J];中國免疫學(xué)雜志;2006年04期

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