【摘要】：非結(jié)核分枝桿菌(Nontuberculous mycobacteria,NTM)一直被認(rèn)為是僅限于與臨床相關(guān)的環(huán)境性細(xì)菌,且因?yàn)槿藗儗?duì)結(jié)核分枝桿菌(Mycobacterium tuberculosis,MTB)的高度關(guān)注而更加忽視了它的存在,直到艾滋病的大肆流行,NTM才被醫(yī)療衛(wèi)生界所重視。NTM是機(jī)會(huì)致病菌,且具有多種可變的致病潛力,大部分NTM是腐物寄生菌,其在自然環(huán)境中分布廣泛。迄今為止已發(fā)現(xiàn)170多種,其中約1/3與人類疾病有關(guān)。巨噬細(xì)胞的吞噬作用可以有效抑制MTB在體內(nèi)的增殖,當(dāng)然MTB亦可干擾巨噬細(xì)胞的凋亡,以逃逸免疫系統(tǒng)對(duì)MTB的清除作用。近年研究表明NTM的臨床表現(xiàn)和致病機(jī)制與MTB十分相似,鑒于國內(nèi)外關(guān)于NTM研究的報(bào)道相對(duì)較少,所以本文主要參考了一些MTB的相關(guān)研究。偶發(fā)分枝桿菌(M.fortuitum)為一種快速生長(zhǎng)型的NTM,多分離自土壤及水體中,且常造成創(chuàng)傷部位和軟組織感染,當(dāng)宿主免疫力較低時(shí)即可導(dǎo)致發(fā)病。為探討M.fortuitum對(duì)巨噬細(xì)胞凋亡的具體影響,進(jìn)行了以下試驗(yàn)研究:首先,將分離自牛腸系膜淋巴結(jié)中的偶發(fā)分枝桿菌重新進(jìn)行培養(yǎng)鑒定,構(gòu)建偶發(fā)分枝桿菌感染THP-1細(xì)胞的感染模型,利用Hoechst 33258染色和透射電鏡對(duì)M.fortuitum感染后THP-1細(xì)胞的凋亡情況進(jìn)行觀察,發(fā)現(xiàn)M.fortuitum感染可以造成THP-1細(xì)胞的凋亡,主要是在感染后6h至24h這個(gè)時(shí)間段細(xì)胞的凋亡變化較為明顯。利用熒光定量PCR方法、Western-blot檢測(cè)M.fortuitum感染后凋亡相關(guān)基因caspase-3、caspase-8及bcl-2在mRNA轉(zhuǎn)錄水平以及蛋白水平的變化,結(jié)果顯示:偶發(fā)分枝桿菌感染后caspase-3、caspase-8的表達(dá)量在mRNA轉(zhuǎn)錄水平以及蛋白水平會(huì)隨著作用時(shí)間延長(zhǎng)而不斷升高,具有一定的時(shí)間依賴性,不同的是caspase-3的mRNA轉(zhuǎn)錄水平于感染后12h時(shí)最高,而其蛋白水平則于24h時(shí)達(dá)到最高,這可能是因?yàn)榈鞍姿降谋磉_(dá)與mRNA轉(zhuǎn)錄水平相比,具有一定的時(shí)間延后性;Bcl-2的表達(dá)量則與caspase-3、caspase-8不同,從mRNA轉(zhuǎn)錄水平來看,在感染的起始階段,其Bcl-2的表達(dá)量較高,從4h起逐漸下降,12h時(shí)最低,尤以分離株的下降最為明顯,并且與對(duì)照組差異顯著,蛋白水平上,偶發(fā)分枝桿菌感染THP-1細(xì)胞后,其Bcl-2的表達(dá)低于對(duì)照組。上述結(jié)果說明M.fortuitum感染后能引起THP-1細(xì)胞的凋亡,使得caspase-3、caspase-8的表達(dá)量升高,而減少了抗凋亡基因Bcl-2的表達(dá)。采用ELISA方法檢測(cè)M.fortuitum感染后,與THP-1凋亡有關(guān)的細(xì)胞因子TNF-α、IL-6和IL-10分泌量的變化,結(jié)果顯示TNF-α的分泌量隨著作用時(shí)間加長(zhǎng)而不斷升高,12h時(shí)達(dá)到最高,呈現(xiàn)一定的時(shí)間依賴性;IL-6在0h-4h表達(dá)量較高,隨后開始下降;在感染的初始階段,IL-10的表達(dá)量較高,從感染后的6h開始則出現(xiàn)明顯的下降趨勢(shì)。這一結(jié)果說明M.fortuitum感染THP-1后能使促凋亡細(xì)胞因子TNF-α大量分泌,有助于細(xì)胞內(nèi)凋亡過程的繼續(xù)進(jìn)行,而IL-6和IL-10表達(dá)量的降低可能是偶發(fā)分枝桿菌并沒能抵抗巨噬細(xì)胞的清除作用。本試驗(yàn)初步研究了M.fortuitum感染對(duì)THP-1凋亡的影響。結(jié)果發(fā)現(xiàn),M.fortuitum感染THP-1后經(jīng)上調(diào)凋亡相關(guān)基因caspase-3、caspase-8的表達(dá),減少抗凋亡基因Bcl-2的表達(dá),調(diào)控相關(guān)細(xì)胞因子TNF-α、IL-6及IL-10的分泌,最終引起巨噬細(xì)胞的凋亡。這些試驗(yàn)結(jié)果將為揭示NTM對(duì)巨噬細(xì)胞凋亡影響的機(jī)制奠定基礎(chǔ),并為進(jìn)一步研究NTM病免疫機(jī)制提供一定理論依據(jù)。
[Abstract]:Nontuberculous mycobacteria (NTM) has been considered to be limited to clinical related environmental bacteria, and because of the high attention to Mycobacterium tuberculosis (Mycobacterium tuberculosis, MTB), the existence of it is ignored. Until the epidemic of AIDS, NTM is valued by the health community as a.NTM. It is an opportunistic pathogen and has a variety of variable pathogenicity potential. Most NTM is a parasitic fungus, which is widely distributed in the natural environment. Up to now, more than 170 are found, about which about 1/3 are related to human disease. Macrophage phagocytosis can effectively inhibit the proliferation of MTB in the body, and of course MTB can also interfere with the apoptosis of macrophages. The scavenging effect of escaping immune system on MTB. Recent studies have shown that the clinical and pathogenic mechanisms of NTM are very similar to that of MTB. In view of the relatively few reports about the NTM study at home and abroad, this paper mainly refers to some related studies of MTB. Mycobacterium sporogenes (M.fortuitum) is a fast growing NTM, which is separated from the soil and is more isolated from the soil. In water, it often causes the site of trauma and the infection of soft tissue. When the host immunity is low, the specific effects of M.fortuitum on the apoptosis of macrophages are discussed. The following experimental studies have been carried out. First, the isolated mycobacteria isolated from the mesenteric lymph nodes were recultured and identified to construct the Mycobacterium even. The infection model of THP-1 cells was observed by Hoechst 33258 staining and transmission electron microscopy. The apoptosis of THP-1 cells after M.fortuitum infection was observed. It was found that M.fortuitum infection could cause the apoptosis of THP-1 cells, mainly in the period of 6h to 24h after infection, the apoptosis of cells was more obvious. The fluorescence quantitative PCR method, We, was used. Stern-blot detected the changes of Caspase-3, Caspase-8 and Bcl-2 at mRNA transcriptional level and protein level after M.fortuitum infection. The results showed that the expression of Caspase-3, Caspase-8, at mRNA transcriptional level and protein level, increased with the time of action. The difference is that the mRNA transcriptional level of Caspase-3 is highest at 12h after infection, and the protein level reaches the highest at 24h, which may be because the expression of the protein level has a certain time delay compared to the mRNA transcriptional level, and the expression of Bcl-2 is different from that of Caspase-3, Caspase-8, from the mRNA transcriptional level, in the sense of sense. The expression of Bcl-2 was higher in the initial stage of dyeing, decreased gradually from 4h, and the lowest in 12h, especially with the drop of isolated strains, and the difference was significant with the control group. On the protein level, the expression of Bcl-2 was lower than that of the control group after infection of THP-1 cells. The results indicated that M.fortuitum infection could cause THP-1 cells after infection. Apoptosis, which increased the expression of Caspase-3 and caspase-8, reduced the expression of the anti apoptotic gene Bcl-2. The changes in the cytokine TNF- a, IL-6 and IL-10 secretion related to THP-1 apoptosis were detected by ELISA method. The results showed that the secretion of TNF- a increased with the longer time of action, and reached the highest in 12h. IL-6 expressed a certain time dependence; the expression of 0h-4h was higher and then began to decline; in the initial stage of infection, the expression of IL-10 was higher, and a significant decline from the beginning of 6h after infection showed that the M.fortuitum infection of THP-1 could make the apoptotic cell factor TNF- alpha secreted in large quantities and contribute to the apoptosis in the cells. The decrease in the expression of IL-6 and IL-10 may be the decrease in the expression of Mycobacterium tumefaciens and did not resist the scavenging effect of macrophages. The effect of M.fortuitum infection on the apoptosis of THP-1 was preliminarily studied. The results showed that the expression of Caspase-3, Caspase-8 was reduced by M.fortuitum infection after THP-1 infection, and the anti apoptotic basis was reduced. The expression of Bcl-2 regulates the secretion of related cytokines, TNF- a, IL-6 and IL-10, and eventually induces apoptosis of macrophages. These results will provide a basis for revealing the mechanism of NTM on the apoptosis of macrophages and provide a theoretical basis for further study of the immune mechanism of NTM disease.
【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S852.61

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