本文選題：膿毒癥 + Siglec-E　； 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究目的:唾液酸結(jié)合免疫球蛋白樣凝集素(sialic-acid-binding-immunoglobulin- like lectins,siglec)最初被認(rèn)定為細(xì)胞表面的跨膜受體,特異性表達(dá)于各種免疫細(xì)胞和造血細(xì)胞表面,能夠抑制TLR信號,因此作為診斷和治療各種疾病的高生物標(biāo)記物引起了人們的關(guān)注。其家族的成員之一,siglec-E (與人同源的是siglec-7和siglec-9),主要表達(dá)于巨噬細(xì)胞和中性粒細(xì)胞表面,是急性炎癥反應(yīng)的重要負(fù)調(diào)節(jié)因子,是膿毒癥治療的潛在靶點。每年有800萬患者因膿毒癥而死亡,膿毒癥仍是大部分住院患者最主要的死因,目前現(xiàn)有的治療方法仍不是很有效。膿毒癥時巨噬細(xì)胞的增殖、活化,功能狀態(tài)對膿毒癥的發(fā)展預(yù)后具有重要的作用。本研究通過LPS對小鼠RAW264.7單核巨噬細(xì)胞進(jìn)行干預(yù),觀察膿毒癥相關(guān)的siglec-E在巨噬細(xì)胞的表達(dá)及釋放規(guī)律,摸索出較為合適的刺激濃度和作用時間,進(jìn)一步探討其與巨噬細(xì)胞分化和吞噬功能改變的關(guān)系,以及膿毒癥時巨噬細(xì)胞吞噬功能障礙的機(jī)制,從而闡明LPS誘導(dǎo)巨噬細(xì)胞表達(dá)和釋放siglec-E的規(guī)律以及其對巨噬細(xì)胞極化的影響,對膿毒癥發(fā)生發(fā)展和預(yù)后的影響,尋找新的有效的治療膿毒癥的方法。研究方法:(1)常規(guī)培養(yǎng)小鼠RAW264.7單核/巨噬細(xì)胞株,選取對數(shù)期細(xì)胞,以2×106個/ml細(xì)胞密度接種,分別設(shè)置對照組和實驗組。對照組不予處理,實驗組予以LPS刺激,在不同的時間點(Oh、6h、12h、24h、36h、48h)分別提取對照組和實驗組細(xì)胞的總RNA和總蛋白。通過實時熒光定量PCR法檢測細(xì)胞裂解液中siglec-E mRNA表達(dá)量;通過Western blot法檢測胞質(zhì)中siglec-E蛋白含量。(2)常規(guī)培養(yǎng)小鼠RAW264.7單核/巨噬細(xì)胞株,選取對數(shù)期細(xì)胞,以2×106個/ml細(xì)胞密度接種,分別設(shè)置對照組和實驗組。對照組不予處理,實驗組細(xì)胞分別加入不同濃度的 LPS (O.O1ug/ml、0.05ug/ml、0.1ug/ml、1ug/ml、2ug/ml)刺激,孵育24h后,分別提取對照組和實驗組細(xì)胞總RNA和總蛋白。通過實時熒光定量PCR法檢測細(xì)胞裂解液中siglec-E mRNA表達(dá);通過Western blot法檢測胞質(zhì)中siglec-E蛋白含量。結(jié)果:(1)在LPS刺激下,不同時間點的siglec-EmRNA表達(dá)量同對照組相比逐漸增加,6h～12h的表達(dá)量增加不明顯,12h～36h的表達(dá)量明顯增加,而36h～48h的表達(dá)量增加緩慢;siglec-E蛋白的表達(dá)量亦是如此。(2)不同劑量的LPS刺激下,siglec-E的mRNA表達(dá)量同對照組相比增高,在給予刺激濃度為O.01ug/ml～O.1ug/ml時的表達(dá)量明顯增多,但增加速度緩慢;而在0.1ug/ml～1ug/ml時表達(dá)量明顯增加,但在1ug/ml～2ug/ml濃度時表達(dá)量卻有所下降;siglec-E的蛋白表達(dá)量與此大致一致。結(jié)論:(1)不同劑量的LPS刺激小鼠RAW264.7單核/巨噬細(xì)胞,可促進(jìn)其表面siglec-E表達(dá)量增加,在0.01ug/ml～1ug/ml范圍內(nèi)表達(dá)量是增加的,但在1ug/ml的濃度時表達(dá)量顯著,而在高于1ug/ml濃度下則產(chǎn)生了抑制,表達(dá)量下降。(2)LPS刺激后,隨干預(yù)時間延長,小鼠單核巨噬細(xì)胞RAW264.7表面siglec-E表達(dá)量逐漸增加,在6h～48h之間表達(dá)量都是增加的,在48h時間點表達(dá)量最為顯著。(3) siglec-E在膿毒癥炎癥時表達(dá)量增加且呈現(xiàn)一定的時間和劑量依賴。
[Abstract]:Research objectives: sialic-acid-binding-immunoglobulin- like lectins (siglec) is initially identified as a transmembrane receptor on the surface of the cell, specifically expressed on the surface of various immune cells and hematopoietic cells, and can inhibit the TLR signal as a high biomarker for the diagnosis and treatment of various diseases. One of the members of the family, siglec-E (homologous to siglec-7 and siglec-9), mainly expressed on the surface of macrophages and neutrophils, is an important negative regulator of acute inflammatory response and a potential target for the treatment of sepsis. 8 million patients die from sepsis and sepsis is still the majority of the year. The main causes of death in hospitalized patients are still not very effective at present. The proliferation, activation and functional status of macrophages in sepsis have an important role in the development of sepsis. This study was conducted through the intervention of RAW264.7 mononuclear macrophages in mice by LPS to observe the siglec-E of sepsis related to the macrophage In order to explore the relationship between macrophage differentiation and phagocytosis, and the mechanism of macrophage phagocytic dysfunction during sepsis, the regulation of LPS induced macrophage expression and release of siglec-E and its polarization to macrophages were elucidated. Influence on the development and prognosis of sepsis to find new effective methods for the treatment of sepsis. Study methods: (1) routine culture of mouse RAW264.7 mononuclear / macrophage strains, selected logarithmic phase cells, 2 x 106 /ml cells density inoculation, the control group and the experimental group were set respectively. The control group was not treated, the experimental group was stimulated by LPS, The total RNA and total protein of the cells in the control group and the experimental group were extracted at different time points (Oh, 6h, 12h, 24h, 36h, 48h). The expression of siglec-E mRNA expression in the cell lysate was detected by real time fluorescence quantitative PCR method, and the content of the protein in the cytoplasm was detected by Western blot. (2) the selected mononuclear / macrophage strains were selected as normal mice. The logarithmic phase cells were inoculated with 2 x 106 /ml cells, and the control group and the experimental group were set respectively. The control group was not treated. The cells of the experimental group were added with different concentrations of LPS (O.O1ug/ml, 0.05ug/ml, 0.1ug/ml, 1ug/ml, 2ug/ml) to stimulate 24h, and then the total RNA and total protein of the cells were extracted from the experimental group and the experimental group. The real-time fluorescence quantification was obtained by real-time fluorescence quantitative analysis. The expression of siglec-E mRNA in cell lysate was detected by PCR and the content of siglec-E protein in cytoplasm was detected by Western blot. Results: (1) the expression of siglec-EmRNA at different time points increased gradually under the stimulation of LPS, and the expression of 6h to 12h increased not obviously, and the expression amount of 12h to 36h was obviously increased. Increase slowly, the expression of siglec-E protein is also the same. (2) under the different doses of LPS stimulation, the expression of mRNA in siglec-E is higher than that of the control group, and the expression amount is significantly increased at the concentration of O.01ug/ml to O.1ug/ml, but the increase rate is slow, while the expression in 0.1ug/ml to 1ug/ml is significantly increased, but in 1ug/ml to 2ug/ml concentration. The expression of siglec-E was in accordance with this. Conclusion: (1) different doses of LPS stimulated the RAW264.7 mononuclear / macrophage in mice, which could increase the expression of siglec-E on the surface of the mouse, and increased in the range of 0.01ug/ml to 1ug/ml, but the expression was significant at the concentration of 1ug/ml, but higher than that of 1ug/ml concentration. (2) after LPS stimulation, the expression of siglec-E in the RAW264.7 surface of mononuclear macrophages increased gradually with the intervention time, and the expression level between 6h and 48h was increased. (3) the expression of siglec-E at the time of sepsis increased and showed a certain time. And dose dependent.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R459.7

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