嚴(yán)重創(chuàng)傷對小鼠骨髓來源的樹突狀細(xì)胞誘導(dǎo)T細(xì)胞應(yīng)答能力的影響
發(fā)布時(shí)間：2018-08-01 11:49
【摘要】： 目的:創(chuàng)傷是對人類生命健康與社會(huì)生產(chǎn)的嚴(yán)重威脅之一,在我國已成為繼心腦血管疾病和惡性腫瘤之外最大的死亡原因。嚴(yán)重創(chuàng)傷后機(jī)體免疫功能紊亂可導(dǎo)致創(chuàng)傷后感染和器官功能障礙等并發(fā)癥,深入研究有助于我們進(jìn)一步了解創(chuàng)傷及其并發(fā)癥的機(jī)制,并指導(dǎo)臨床治療。長期以來,針對創(chuàng)傷后免疫功能紊亂的研究涉及了中性粒細(xì)胞、單核-巨噬細(xì)胞和T、B淋巴細(xì)胞等諸多方面,但作為免疫系統(tǒng)重要的成員之一——樹突狀細(xì)胞(dendritic cell, DC)在創(chuàng)傷后免疫功能紊亂中的作用和地位迄今未被闡明。DC是聯(lián)系先天性免疫和適應(yīng)性免疫的橋梁,作為專職的抗原提呈細(xì)胞(APC),DC在啟動(dòng)和調(diào)節(jié)先天性及適應(yīng)性免疫應(yīng)答中具有關(guān)鍵性的作用。已有研究證實(shí),機(jī)體遭遇創(chuàng)傷后,DC在其分化發(fā)育過程、抗原提呈功能和細(xì)胞因子產(chǎn)生等方面均發(fā)生了障礙。但作為中樞免疫器官的骨髓,在創(chuàng)傷后向DC分化的過程及骨髓來源DC(BMDC)的功能是否會(huì)發(fā)生改變,尚未見報(bào)道。本研究著重探討了創(chuàng)傷對小鼠BMDC刺激T細(xì)胞增殖能力的影響。 方法:①制備動(dòng)物創(chuàng)傷模型,致傷后24 h分離小鼠骨髓細(xì)胞,體外應(yīng)用重組小鼠粒細(xì)胞/巨噬細(xì)胞—集落刺激因子(rmGM-CSF)誘導(dǎo)樹突狀細(xì)胞;②通過混合淋巴細(xì)胞反應(yīng)(MLR)檢測未成熟、成熟DC誘導(dǎo)異源T細(xì)胞的應(yīng)答能力;③應(yīng)用流式細(xì)胞術(shù)檢測BMDC表面主要組織相容性復(fù)合物II類分子(MHC II)及共刺激分子CD40、CD80、CD86表達(dá);④應(yīng)用酶聯(lián)免疫吸附試驗(yàn)(ELISA)檢測脂多糖(LPS)刺激的BMDC培養(yǎng)上清中白細(xì)胞介素-12(IL-12)p40、IL-12 p70以及白細(xì)胞介素-10(IL-10)水平的變化。 結(jié)果:①在同等誘導(dǎo)條件下,創(chuàng)傷組小鼠骨髓細(xì)胞在體外誘導(dǎo)的DC(CD11c陽性細(xì)胞)百分率與正常對照組相比無明顯差異,但其總數(shù)量明顯低于正常對照組(骨髓細(xì)胞誘導(dǎo)DC的擴(kuò)增倍數(shù)分別為3.9±0.5 vs 5.4±0.6,P 0.05);②創(chuàng)傷組小鼠BMDC,在經(jīng)過LPS誘導(dǎo)成熟前后,其介導(dǎo)的MLR值均明顯低于相應(yīng)的對照組值(P 0.05);③以CD11c陽性細(xì)胞圈門,創(chuàng)傷小鼠BMDC在LPS刺激前后的CD40表達(dá)均明顯低于正常對照組(4±1.0% vs 22±3.5%,P 0.01;56±7.5% vs 91±8.0%,P 0.01),但MHC II、CD80和CD86表達(dá)在兩組間無統(tǒng)計(jì)學(xué)差異(P 0.05);④創(chuàng)傷組小鼠BMDC在體外經(jīng)LPS誘導(dǎo)成熟后,其IL-12 p40、IL-12 p70分泌水平均明顯低于正常對照組(45±6.5 ng/L vs 78±6.8 ng/L,P 0.05;9±1.0 ng/L vs 18±1.9 ng/L,P 0.05),但分泌IL-10的能力與正常對照組相比無統(tǒng)計(jì)學(xué)差異(P 0.05)。兩組未成熟DC分泌上述細(xì)胞因子水平均無統(tǒng)計(jì)學(xué)差異(P 0.05) 結(jié)論:①創(chuàng)傷小鼠骨髓細(xì)胞對GM-CSF刺激增殖的敏感性降低;②創(chuàng)傷小鼠的BMDC功能發(fā)生障礙,其介導(dǎo)的混合淋巴細(xì)胞反應(yīng)顯著降低,對T細(xì)胞增殖的激發(fā)能力受到抑制;③創(chuàng)傷小鼠BMDC誘導(dǎo)T細(xì)胞應(yīng)答的能力降低可能與其CD40表達(dá)及IL-12分泌減少有關(guān)。
[Abstract]:Objective: trauma is one of the serious threats to human life, health and social production. In our country, it has become the biggest cause of death outside the cardiovascular and cerebrovascular diseases and malignant tumors. The immune dysfunction of the body after severe trauma can lead to complications such as post traumatic infection and organ dysfunction. Further research will help us to further understand the creation of the disease. The mechanism of injury and its complications and guiding clinical treatment. For a long time, the study of post-traumatic immune dysfunction involves neutrophils, mononuclear macrophages, T, B lymphocytes and many other aspects, but as one of the important members of the immune system, the dendritic cell (DC) is in the post-traumatic immune dysfunction. The role and status have not been elucidated so far that.DC is a bridge linking innate and adaptive immunity. As a full-time antigen presenting cell (APC), DC plays a key role in the initiation and regulation of congenital and adaptive immune responses. It has been proved that after the body suffers injury, the DC is in its differentiation and development and the antigen presentation function However, there have been obstacles in the production of cytokine, but it has not been reported whether the bone marrow, as a central immune organ, can differentiate into DC after trauma and the function of bone marrow DC (BMDC). This study focuses on the effect of trauma on the proliferation of T cells stimulated by BMDC in mice.
Methods: (1) the animal model of trauma was prepared. The bone marrow cells of mice were separated by 24 h after injury. The recombinant mouse granulocyte / macrophage colony stimulating factor (rmGM-CSF) was used to induce dendritic cells in vitro. (2) the response ability of immature T cells induced by mature DC was detected by mixed lymphocyte reaction (MLR); (3) flow cytometry was used to detect the cells. The main histocompatibility complex II molecules (MHC II) and co stimulator CD40, CD80, CD86 expression on the BMDC surface; (4) the enzyme linked immunosorbent assay (ELISA) was used to detect the levels of interleukin -12 (IL-12) and interleukin (IL) in the culture supernatant of BMDC culture of LPS (LPS).
Results: (1) there was no significant difference in the percentage of DC (CD11c positive cells) induced by bone marrow cells in the trauma group in vitro compared with the normal control group, but the total number of bone marrow cells was significantly lower than that of the normal control group (3.9 + 0.5 vs 5.4 + 0.6, P 0.05) of the bone marrow cells induced by bone marrow cells (P 0.05). (2) the BMDC in the trauma group was in L The MLR value of PS was significantly lower than that of the corresponding control group (P 0.05) before and after the induction of maturation. (3) the expression of CD40 in the traumatic mice BMDC before and after LPS stimulation was significantly lower than that of the normal control group (4 + 1% vs 22 + 3.5%, P 0.01, 56 + 7.5% vs 91 + 8%, P 0.01), but there was no statistical difference between the two groups. Difference (P 0.05); (4) the level of IL-12 P40 and IL-12 p70 secretion in the IL-12 P40 and IL-12 p70 in the trauma group was significantly lower than that in the normal control group (45 + 6.5 ng/L vs 78 + 6.8 ng/L, P 0.05, 9 + 1 ng/L 18 + 1.9), but there was no statistical difference between the normal control group and the normal control group. There was no significant difference in secretion of cytokines mentioned above (P 0.05).
Conclusion: (1) the sensitivity of bone marrow cells in traumatized mice decreased to the proliferation of GM-CSF, and the BMDC function in traumatic mice was impaired, the mixed lymphocyte reaction was significantly reduced and the ability to stimulate the proliferation of T cells was inhibited. The ability to respond to BMDC induced T cells in traumatized mice may be associated with the expression of CD40 and the secretion of IL-12. The reduction is related.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392
本文編號：2157482
[Abstract]:Objective: trauma is one of the serious threats to human life, health and social production. In our country, it has become the biggest cause of death outside the cardiovascular and cerebrovascular diseases and malignant tumors. The immune dysfunction of the body after severe trauma can lead to complications such as post traumatic infection and organ dysfunction. Further research will help us to further understand the creation of the disease. The mechanism of injury and its complications and guiding clinical treatment. For a long time, the study of post-traumatic immune dysfunction involves neutrophils, mononuclear macrophages, T, B lymphocytes and many other aspects, but as one of the important members of the immune system, the dendritic cell (DC) is in the post-traumatic immune dysfunction. The role and status have not been elucidated so far that.DC is a bridge linking innate and adaptive immunity. As a full-time antigen presenting cell (APC), DC plays a key role in the initiation and regulation of congenital and adaptive immune responses. It has been proved that after the body suffers injury, the DC is in its differentiation and development and the antigen presentation function However, there have been obstacles in the production of cytokine, but it has not been reported whether the bone marrow, as a central immune organ, can differentiate into DC after trauma and the function of bone marrow DC (BMDC). This study focuses on the effect of trauma on the proliferation of T cells stimulated by BMDC in mice.
Methods: (1) the animal model of trauma was prepared. The bone marrow cells of mice were separated by 24 h after injury. The recombinant mouse granulocyte / macrophage colony stimulating factor (rmGM-CSF) was used to induce dendritic cells in vitro. (2) the response ability of immature T cells induced by mature DC was detected by mixed lymphocyte reaction (MLR); (3) flow cytometry was used to detect the cells. The main histocompatibility complex II molecules (MHC II) and co stimulator CD40, CD80, CD86 expression on the BMDC surface; (4) the enzyme linked immunosorbent assay (ELISA) was used to detect the levels of interleukin -12 (IL-12) and interleukin (IL) in the culture supernatant of BMDC culture of LPS (LPS).
Results: (1) there was no significant difference in the percentage of DC (CD11c positive cells) induced by bone marrow cells in the trauma group in vitro compared with the normal control group, but the total number of bone marrow cells was significantly lower than that of the normal control group (3.9 + 0.5 vs 5.4 + 0.6, P 0.05) of the bone marrow cells induced by bone marrow cells (P 0.05). (2) the BMDC in the trauma group was in L The MLR value of PS was significantly lower than that of the corresponding control group (P 0.05) before and after the induction of maturation. (3) the expression of CD40 in the traumatic mice BMDC before and after LPS stimulation was significantly lower than that of the normal control group (4 + 1% vs 22 + 3.5%, P 0.01, 56 + 7.5% vs 91 + 8%, P 0.01), but there was no statistical difference between the two groups. Difference (P 0.05); (4) the level of IL-12 P40 and IL-12 p70 secretion in the IL-12 P40 and IL-12 p70 in the trauma group was significantly lower than that in the normal control group (45 + 6.5 ng/L vs 78 + 6.8 ng/L, P 0.05, 9 + 1 ng/L 18 + 1.9), but there was no statistical difference between the normal control group and the normal control group. There was no significant difference in secretion of cytokines mentioned above (P 0.05).
Conclusion: (1) the sensitivity of bone marrow cells in traumatized mice decreased to the proliferation of GM-CSF, and the BMDC function in traumatic mice was impaired, the mixed lymphocyte reaction was significantly reduced and the ability to stimulate the proliferation of T cells was inhibited. The ability to respond to BMDC induced T cells in traumatized mice may be associated with the expression of CD40 and the secretion of IL-12. The reduction is related.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R392
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相關(guān)期刊論文 前4條
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