不同毒力结核杆菌的纯化蛋白质衍生物（PPD）对人巨噬细胞凋亡的诱导及其细胞内信号通路的初步研究

发布时间：2018-05-31 00:31

本文选题：结核分枝杆菌衍生蛋白 + 细胞凋亡　；参考：《复旦大学》2011年硕士论文

【摘要】：第一部分不同毒力结核杆菌的纯化蛋白质衍生物对人巨噬细胞凋亡的诱导及其与Toll样受体-2的相关性【目的】了解两种不同毒力结核杆菌的纯化蛋白衍生物刺激下人单核-巨噬细胞(THP-1)凋亡的差异及其与Toll样受体-2(TLR-2)的相关性。 [方法]分别设对照组、高毒力结核杆菌衍生蛋白(H37Rv-PPD)刺激组和低毒力结核杆菌衍生蛋白(BCG-PPD)组,分别在3 h、8 h、15 h及24 h刺激分化成熟的THP-1细胞；Hochest染色后观察细胞凋形态；流式细胞仪检测细胞Annexin V蛋白以判定凋亡及TLR-2表达情况；加入TLR-2阻断剂后,用同样的方法测定细胞表面TLR-2的表达及凋亡情况。 [结果]不论用Hochest染色法观察还是流式细胞仪检测,都显示BCG-PPD刺激下细胞以凋亡多见,而H37Rv-PPD刺激下细胞核则多呈坏死状。从图中可见,凋亡率随时间升高,第24小时凋亡比例高达30.2%,而同时间点TLR-2的比例为8.84%；应用TLR-2阻断剂后,每个时间点TLR-2表达比例均在3%以下,对应时间点凋亡比例下降,第24小时凋亡比例仅为10.5%。H37Rv-PPD可引起TLR-2高表达,第24小时TLR-2表达率为17.2%,该时间点凋亡率仅为7.72%；TLR-2阻断后,其表达率在对照波动范围内,但凋亡率与TLR-2未阻断前相比变化不大。 [结论]BCG-PPD主要诱导THP-1的凋亡,且与TLR-2有一定的相关性；而H37Rv-PPD主要诱导THP-1的坏死。第二部分不同毒力结核杆菌的纯化蛋白质衍生物诱导人巨噬细胞死亡及TNF-α、IL-1β和IL-10的表达差异 [目的】本实验对不同毒力结核杆菌的衍生蛋白(PPD)对人巨噬细胞(THP-1)的影响及其与TNF-α、IL-1β及IL-10的差异性进行研究。 [方法]用H37Rv-PPD和BCG-PPD分别在3.h、8h、15h及24h四个时问点刺激分化成熟的THP-1细胞,再应用Hochest染色染色法,荧光镜下观察细胞的转归差异(凋亡及坏死情况),同时取上清用ELISA法测TNF-α、IL-1β及IL-10的浓度。 [结果]BCG-PPD刺激下细胞核呈现以椭圆凋亡小体多见,而H37Rv-PPD刺激下细胞核则多呈坏死状,以坏死多见；H37Rv-PPD干预后上清中TNF-α浓度逐渐升高,至15小时TNF-α浓度达到22000pg/ml,后逐渐下降至基线水平。总体而言,BCG-PPD刺激的上清中TNF-α的表达低于H37Rv-PPD刺激的上清中TNF-α的表达。从IL-1β浓度与时间关系趋势图表可以看出,在BCG-PPD干预下上清中IL-1β的浓度在前15小时内逐渐升高(达144000pg/ml),24小时下降为9500pg/ml。而在H37Rv-PPD干预下IL-1β则无明显变化,浓度约在5000pg/ml左右波动。从IL-10浓度与时间关系图可以看出,在BCG-PPD干预下,细胞上清中IL-10的浓度从3小时的.3000pg/ml,逐渐下降趋势,到24小时下降到65.5pg/ml。而在H37Rv-PPD刺激下,3-15小时,IL-10浓度呈上升趋势,在15小时达到顶峰(浓度达6100pg/ml),24小时下降为2500pg/ml总体而言,BCG-PPD刺激下上清中TNF-a及IL-10的表达量低于H37Rv-PPD刺激组,但BCG-PPD刺激下IL-lβ的表达量却高于后者。 [结论]提示高毒力菌株衍生蛋白(H37Rv-PPD)引起THP-1坏死的原因可能与TNF-a的过度表达有关,而凋亡少见可能与IL-10抑制凋亡作用有关,而低毒力菌株衍生蛋白诱导凋亡与IL-1β有关。可能菌株毒力差异就存在于菌株的蛋白成分之中,且与上述几种细胞因子密切相关。
[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.
【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R378

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