本文選題：結(jié)核分枝桿菌衍生蛋白 + 細胞凋亡��； 參考：《復旦大學》2011年碩士論文

【摘要】：第一部分 不同毒力結(jié)核桿菌的純化蛋白質(zhì)衍生物對人巨噬細胞凋亡的誘導及其與Toll樣受體-2的相關(guān)性 【目的】了解兩種不同毒力結(jié)核桿菌的純化蛋白衍生物刺激下人單核-巨噬細胞(THP-1)凋亡的差異及其與Toll樣受體-2(TLR-2)的相關(guān)性。 [方法]分別設對照組、高毒力結(jié)核桿菌衍生蛋白(H37Rv-PPD)刺激組和低毒力結(jié)核桿菌衍生蛋白(BCG-PPD)組,分別在3 h、8 h、15 h及24 h刺激分化成熟的THP-1細胞；Hochest染色后觀察細胞凋形態(tài)；流式細胞儀檢測細胞Annexin V蛋白以判定凋亡及TLR-2表達情況；加入TLR-2阻斷劑后,用同樣的方法測定細胞表面TLR-2的表達及凋亡情況。 [結(jié)果]不論用Hochest染色法觀察還是流式細胞儀檢測,都顯示BCG-PPD刺激下細胞以凋亡多見,而H37Rv-PPD刺激下細胞核則多呈壞死狀。從圖中可見,凋亡率隨時間升高,第24小時凋亡比例高達30.2%,而同時間點TLR-2的比例為8.84%；應用TLR-2阻斷劑后,每個時間點TLR-2表達比例均在3%以下,對應時間點凋亡比例下降,第24小時凋亡比例僅為10.5%。H37Rv-PPD可引起TLR-2高表達,第24小時TLR-2表達率為17.2%,該時間點凋亡率僅為7.72%；TLR-2阻斷后,其表達率在對照波動范圍內(nèi),但凋亡率與TLR-2未阻斷前相比變化不大。 [結(jié)論]BCG-PPD主要誘導THP-1的凋亡,且與TLR-2有一定的相關(guān)性；而H37Rv-PPD主要誘導THP-1的壞死。 第二部分 不同毒力結(jié)核桿菌的純化蛋白質(zhì)衍生物誘導人巨噬細胞死亡及TNF-α、IL-1β和IL-10的表達差異 [目的】本實驗對不同毒力結(jié)核桿菌的衍生蛋白(PPD)對人巨噬細胞(THP-1)的影響及其與TNF-α、IL-1β及IL-10的差異性進行研究。 [方法]用H37Rv-PPD和BCG-PPD分別在3.h、8h、15h及24h四個時問點刺激分化成熟的THP-1細胞,再應用Hochest染色染色法,熒光鏡下觀察細胞的轉(zhuǎn)歸差異(凋亡及壞死情況),同時取上清用ELISA法測TNF-α、IL-1β及IL-10的濃度。 [結(jié)果]BCG-PPD刺激下細胞核呈現(xiàn)以橢圓凋亡小體多見,而H37Rv-PPD刺激下細胞核則多呈壞死狀,以壞死多見；H37Rv-PPD干預后上清中TNF-α濃度逐漸升高,至15小時TNF-α濃度達到22000pg/ml,后逐漸下降至基線水平�？傮w而言,BCG-PPD刺激的上清中TNF-α的表達低于H37Rv-PPD刺激的上清中TNF-α的表達。從IL-1β濃度與時間關(guān)系趨勢圖表可以看出,在BCG-PPD干預下上清中IL-1β的濃度在前15小時內(nèi)逐漸升高(達144000pg/ml),24小時下降為9500pg/ml。而在H37Rv-PPD干預下IL-1β則無明顯變化,濃度約在5000pg/ml左右波動。從IL-10濃度與時間關(guān)系圖可以看出,在BCG-PPD干預下,細胞上清中IL-10的濃度從3小時的.3000pg/ml,逐漸下降趨勢,到24小時下降到65.5pg/ml。而在H37Rv-PPD刺激下,3-15小時,IL-10濃度呈上升趨勢,在15小時達到頂峰(濃度達6100pg/ml),24小時下降為2500pg/ml總體而言,BCG-PPD刺激下上清中TNF-a及IL-10的表達量低于H37Rv-PPD刺激組,但BCG-PPD刺激下IL-lβ的表達量卻高于后者。 [結(jié)論]提示高毒力菌株衍生蛋白(H37Rv-PPD)引起THP-1壞死的原因可能與TNF-a的過度表達有關(guān),而凋亡少見可能與IL-10抑制凋亡作用有關(guān),而低毒力菌株衍生蛋白誘導凋亡與IL-1β有關(guān)�？赡芫甓玖Σ町惥痛嬖谟诰甑牡鞍壮煞种�,且與上述幾種細胞因子密切相關(guān)。
[Abstract]:Part one
Apoptosis of human macrophages induced by purified protein derivative of different virulent Mycobacterium tuberculosis and its correlation with Toll like receptor -2
[Objective] to understand the difference of the apoptosis of human mononuclear macrophage (THP-1) stimulated by the purified protein derivatives of two different virulence Mycobacterium tuberculosis and the correlation with the Toll like receptor -2 (TLR-2).
[Methods] the control group, the H37Rv-PPD stimulation group and the low virulence Mycobacterium tuberculosis derived protein (BCG-PPD) group were stimulated to differentiate the mature THP-1 cells in 3 h, 8 h, 15 h and 24 h, and the cell morphology was observed after Hochest, and the flow cytometry was used to detect the Annexin V protein to determine apoptosis and TLR-2. The expression and apoptosis of TLR-2 on the cell surface were measured by the same method after adding TLR-2 blockers.
[results] whether Hochest staining or flow cytometry was used to detect the apoptosis of cells under the stimulation of BCG-PPD, the cell nuclei were mostly dead, and the apoptosis rate increased with time, the percentage of apoptosis was up to 30.2% at twenty-fourth hours, and the proportion of TLR-2 at the same time point was 8.84%; the TLR-2 blocker was used. The ratio of TLR-2 expression at each time point was below 3%, the proportion of apoptosis at the corresponding time point decreased, the percentage of apoptosis was only 10.5%.H37Rv-PPD in twenty-fourth hours and the expression rate of TLR-2 was 17.2%, the rate of apoptosis was only 7.72% at this time point, and the rate of apoptosis was within the range of control after TLR-2 blocking, but the rate of apoptosis and TLR-2 were not. There is little change in comparison before it is blocked.
[conclusion]BCG-PPD mainly induces apoptosis of THP-1, and has a certain correlation with TLR-2, while H37Rv-PPD mainly induces THP-1 necrosis.
The second part
Differential expression of TNF-, IL-1 and IL-10 in human macrophages induced by purified protein derivative of different virulent Mycobacterium tuberculosis
[Objective] to study the effect of PPD on human macrophage (THP-1) and its difference from TNF- alpha, IL-1 beta and IL-10.
[Methods] the mature THP-1 cells were stimulated by H37Rv-PPD and BCG-PPD at four time points of 3.h, 8h, 15h and 24h respectively. Then Hochest staining and staining were used to observe the difference of cell transformation (apoptosis and necrosis) under the fluorescent microscope, and the concentration of TNF- a, IL-1 beta and IL-10 were measured by ELISA method.
[results]BCG-PPD stimulated the nucleus to appear in the ellipsoid apoptotic body more, and the nucleus was mostly bad death with H37Rv-PPD stimulation, and the concentration of TNF- alpha in the supernatant increased gradually after H37Rv-PPD intervention, and the concentration of TNF- alpha reached 22000pg/ml at 15 hours, then gradually decreased to the base line level. In general, TNF- in the supernatant of BCG-PPD stimulation. The expression of alpha was lower than the expression of TNF- alpha in the supernatant of H37Rv-PPD stimulation. From the diagram of the relationship between IL-1 beta concentration and time, the concentration of IL-1 beta in the supernatant under the intervention of BCG-PPD was gradually increased (144000pg/ml) in the first 15 hours, and decreased to 9500pg/ml. in 24 hours, but the IL-1 beta was not obviously changed under the intervention of H37Rv-PPD, and the concentration was about 5000pg/. Ml fluctuation. From the relationship diagram of IL-10 concentration and time, we can see that under the intervention of BCG-PPD, the concentration of IL-10 in the cell supernatant decreased gradually from 3 hours.3000pg/ml to 65.5pg/ml. and under the stimulation of H37Rv-PPD, the concentration of IL-10 showed an upward trend in 3-15 hours, and reached the peak at 15 hours (concentration 6100pg/ml), 24 hours. As a whole, the expression of TNF-a and IL-10 in the supernatant under BCG-PPD stimulation was lower than that in the H37Rv-PPD stimulus group, but the expression of IL-l beta was higher than that of the latter under the BCG-PPD stimulation.
[Conclusion] it is suggested that the cause of THP-1 necrosis caused by high virulent strain derived protein (H37Rv-PPD) may be related to the overexpression of TNF-a, but the rare apoptosis may be related to the inhibition of apoptosis by IL-10, and the induced apoptosis of low virulent strain is related to IL-1 beta. Several cytokines are closely related.
【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R378

【參考文獻】

相關(guān)期刊論文 前1條

1 陳思靜,盧賢瑜;結(jié)核分枝桿菌調(diào)控巨噬細胞凋亡機理的研究進展[J];國外醫(yī)學.臨床生物化學與檢驗學分冊;2005年05期

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