LPS对巨噬细胞氧依赖性杀伤结核分枝杆菌的促进作用及其机制

发布时间：2018-05-31 09:40

本文选题：LPS + 结核分枝杆菌(M.tb)　；参考：《蚌埠医学院》2013年硕士论文

【摘要】：研究背景：结核病是由结核分枝杆菌(Mycobacteriumtuberculosis，M.tb)感染引起的，目前仍是发病率和病死率最高的传染病之一。它被认为是一种慢性炎症性疾病，伴有巨噬细胞吞噬、杀伤结核菌过程贯穿其发生、发展的始终[1]。以往的研究表明[2]，机体感染M.tb后，主要通过补体受体进入巨噬细胞，并通过干扰巨噬细胞吞噬和溶酶体成熟而长期寄生于宿主巨噬细胞内并造成潜伏感染。Toll样受体（Toll-likereceptors,TLRs）介导天然免疫，与抗TB免疫反应相关。研究发现[3]，Toll样受体4（Toll-likereceptor4,TLR4）在巨噬细胞抗M.tb的发生、发展中发挥着重要作用。有资料显示[4]，非M.tb来源的TLR4受体激动剂LPS可通过TLR4途径和NOX2NADPH氧化酶及ROS信号通路促进THP-1巨噬细胞吞噬和杀伤M.tb。探讨LPS促进巨噬细胞吞噬及杀伤M.tb的机制，对于其作为免疫佐剂或潜在的临床抗结核预防用药，具有重要价值。目的：研究LPS对巨噬细胞氧依赖性杀伤结核分枝杆菌的促进作用，初步阐明其机制。方法：（1）结核分枝杆菌减毒株M.tbH37Ra用苏通氏液体培养基于细菌培养箱中培养1个月后接种至罗琴氏固体培养基，37°C培养1个月，无菌接种环收集M.tb菌体于1mL匀浆器中，加入生理盐水反复研磨，取细菌悬液，用生理盐水经10000rpm×2min洗涤3次，抗酸染色阳性且镜下观察细菌分散。分光光度计检测菌液浓度，用生理盐水配成浓度为5×106/mL的M.tb悬液，4°C备用。（2）10-100μg/mL浓度异硫氰酸荧光素(FITC)，在磷酸盐缓冲液（PBS，pH7.2）中标记M.tb，流式细胞术检测标记率和平均荧光强度（MFI）。FITC标记的M.tb以10∶1的比例感染巨噬细胞THP-1(A)18h-24h，以流式细胞术检测分析感染模型的感染率。（3）取FITC标记的M.tb与佛波醇酯(PMA)诱导分化的THP-1巨噬细胞(THP-1(A))共孵育1h-6h后，观察PMA活化的THP-1(A)细胞吞噬FITC标记M.tb的时间动力学变化。（4）0，100，1000ng/mL不同浓度LPS刺激THP-1(A)细胞24h后，与M.tb按不同比例（1:1-1:100）共培养30min，流式细胞术检测LPS对THP-1(A)细胞吞噬M.tb吞噬率的促进作用。（5）0，10，100，1000，10000ng/mL不同浓度LPS分别刺激PMA分化前后的THP-1细胞24h后，再与M.tb以1:50浓度比共孵育30min，流式细胞术检测LPS在THP-1细胞吞噬M.tb功能中的促进作用。（6）分别以抗人TLR4抗体HTA125阻断TLR4，以DPI(diphenyleneiodoniumchloride)阻断NOX2NADPH氧化酶后，再以LPS（1000ng/mL）作用THP-1(A)细胞24h，1:50浓度比例加入M.tb作用30min，流式细胞术分析TLR4及NOX2NADPH氧化酶信号途径在LPS促进THP-1(A)细胞吞噬M.tb中的作用。（7）不同浓度荧光素二醋酸酯(FDA（0.1-1.0μg/mL）)，在磷酸盐缓冲液（PBS，pH7.2）中标记M.tb。取佛波醇酯(PMA)诱导分化的THP-1(A)细胞与FDA标记M.tb于37℃孵育30min，洗涤细胞，加入小牛血清，置37℃孵育0～60min后，三蒸水裂解细胞，流式细胞术检测裂解细胞释放的M.tb荧光强度变化，分析计算THP-1(A)细胞对吞噬M.tb的杀伤功能。同时观察刺激剂LPS及阻断剂HTA125和DPI对杀伤功能的影响。（8）流式细胞术检测LPS（100ng/mL）对M.tb感染前后THP-1(A)细胞表面TLR4表达水平、胞内ROS产量的促进作用；Westernblot检测THP-1(A)细胞NOX2蛋白表达水平；荧光定量PCR检测THP-1(A)细胞胞内TLR4mRNA，NOX2mRNA基因表达水平。结果:（1）100μg/mL的FITC浓度标记M.tb的标记率可达(99.02±0.74)%，用于后续实验需要。（2）PMA诱导分化的THP-1(A)细胞在4h对M.tb-FITC的吞噬率未加台盼蓝淬灭组为(78.82±3.21)%与加台盼蓝淬灭组(68.88±0.46)%相比，差异具有统计学意义(P0.05)。至6h时吞噬率(93.65±1.47)%达饱和。（3）THP-1(A)与M.tb比例从1∶1增加至1∶100，其吞噬率从(2.17±0.15)%增加至(35.62±2.49)%。100ng/mL的LPS在THP-1(A)：M.tb-FITC达1:50时，吞噬率达(65.94±2.25)%，差异有统计学意义(P0.05)。（4）10ng/mL浓度的LPS即可促进巨噬细胞对M.tb的吞噬率，且随浓度增加吞噬率逐渐增高，10000ng/mL的LPS浓度其吞噬率达(94.07±3.55)%较对照组(23.41±3.22)%增加，差异有统计学意义(P0.05)。（5）10μMDPI处理的巨噬细胞对M.tb的吞噬率降低至(75.93±1.06)%，20μM时降低更显著，吞噬率达(58.16±0.98)%(P0.05)。HTA125预处理的巨噬细胞吞噬M.tb吞噬百分率(38.57±1.35)%和LPS刺激组(83.25±2.53)%相比较，差异有统计学意义(P0.05)。（6）1.0μg/mLFDA对M.tb的标记率可达(98.21±0.92)%，可满足杀伤实验的需要。THP-1(A)细胞对M.tb0~60min杀伤率随时间增加而增加，60min的杀伤率可达(54.16±2.17)%，与0min杀伤率(15.21±0.63)%相比较，差异具有显著性。（7）100ng/mLLPS对THP-1(A)细胞30min杀伤M.tb的MFI值可达365.23±1.58与对照组608.35±2.16比较，，差异有显著性（P0.01）。且随浓度增加杀伤能力增强，10000ng/mL的MFI值降低至216.59±4.21。（8）HTA125及DPI预处理的THP-1(A)细胞对M.tb-FDA的杀伤能力降低至385.92±25.98和302.35±25.47，较LPS刺激组268.54±32.73，差异有统计学意义（P0.01）。（9）LPS可增强被M.tb抑制的巨噬细胞表面TLR4受体的表达率。（10）Westernblot结果显示LPS有助于上调被M.tb抑制的THP-1(A)巨噬细胞NOX2蛋白的表达量。（11）LPS可增强被M.tb抑制的THP-1(A)细胞ROS的产量。（12）荧光定量PCR检测基因表达量结果显示，经LPS刺激后，被M.tb抑制的THP-1(A)胞内的TLR4mRNA，NOX2mRNA基因表达量均明显升高。结论：LPS能通过上调TLR4及NOX2的表达水平和功能，促进巨噬细胞对M.tb的吞噬和氧依赖性杀伤。
[Abstract]:Background: tuberculosis is caused by Mycobacteriumtuberculosis (M.tb) infection. It is still one of the highest morbidity and mortality. It is considered to be a chronic inflammatory disease with macrophage phagocytosis and the process of killing Mycobacterium tuberculosis. The development of the previous [1]. study showed that [2], after M.tb infection, mainly through the complement receptor into macrophages, and by interfering with macrophages phagocytosis and lysosome maturation and long parasitic in host macrophages and causing latent infection of.Toll like receptor (Toll-likereceptors, TLRs) mediated natural immunity, related to the anti TB immune response. The study found [3], Toll like receptor 4 (Toll-). Likereceptor4, TLR4) plays an important role in the development of macrophage against M.tb. Some data show that [4], a non M.tb source of TLR4 receptor agonist LPS can promote the phagocytosis and killing of THP-1 macrophages by TLR4 pathway and NOX2NADPH oxidase and ROS signaling pathway, and the mechanism of macrophage phagocytosis and killing of macrophages is promoted. It is of great value as an adjuvant or potential antituberculosis drug.
Objective: To study the effect of LPS on macrophage oxygen dependent killing of Mycobacterium tuberculosis, and elucidate its mechanism.
Methods: (1) the strain M.tbH37Ra of Mycobacterium tuberculosis was inoculated with the culture medium of Santong's liquid for 1 months and inoculated to the ropey's solid medium for 1 months and 37 [37] C for 1 months. The sterile ring collected the M.tb bacteria in the 1mL homogenizer. The bacterial suspension was repeated by adding physiological saline to the bacterial suspension, and the saline was washed by 10000rpm x 2min. 3 times, the anti acid staining was positive and the bacterial dispersion was observed under the microscope. The spectrophotometer was used to detect the concentration of bacterial liquid, and the concentration of M.tb suspension with a concentration of 5 x 106/mL was mixed with physiological saline, and 4 degree C was used for reserve.
(2) 10-100 mu g/mL concentration of fluorescein isothiocyanate (FITC), labeled M.tb in phosphate buffer solution (PBS, pH7.2), the detection marking rate of flow cytometry and the average fluorescence intensity (MFI).FITC labeled M.tb were infected with THP-1 (A) 18h-24h in the ratio of 10 to 1, and the infection rate of the infection model was detected by flow cytometry.
(3) the time dynamics of THP-1 (A) cells activated by PMA activated THP-1 (A) cells phagocytic FITC labelled M.tb was observed after the FITC labeled M.tb was incubated with THP-1 macrophages (THP-1 (A)) induced by phorbol ester (PMA).
(4) 01001000ng/mL stimulated THP-1 (A) cell 24h with different concentrations of LPS, and co culture 30min with M.tb in a different proportion (1:1-1:100). Flow cytometry was used to detect the effect of LPS on the phagocytosis of THP-1 (A) cells.
(5) 0,10100100010000ng/mL with different concentrations of LPS stimulated 24h of THP-1 cells before and after PMA differentiation, and then incubated 30min with M.tb at 1:50 concentration ratio, and flow cytometry was used to detect the role of LPS in THP-1 cell phagocytosis of M.tb.
(6) TLR4 was blocked by anti human TLR4 antibody HTA125, NOX2NADPH oxidase was blocked by DPI (diphenyleneiodoniumchloride), and THP-1 (A) cell 24h by LPS (1000ng/mL), and M.tb action was added at the 1:50 concentration ratio.
(7) at different concentrations of fluorescein two acetate (FDA (0.1-1.0) g/mL), M.tb. in phosphate buffer solution (PBS, pH7.2) was labeled by M.tb. to induce THP-1 (A) cells and FDA markers to incubate 30min, washing cells, adding calf serum, incubated at 37 centigrade for 0 ~ three Lysic cells, and flow cytometry to detect cleavage The changes in the fluorescence intensity of M.tb released by the cell were analyzed. The killing function of THP-1 (A) cells to the phagocytosis of M.tb was analyzed and the effects of stimulant LPS and blockers HTA125 and DPI on the killing function were also observed.
(8) flow cytometry was used to detect the level of TLR4 expression on the surface of THP-1 (A) cells before and after M.tb infection by flow cytometry, the promotion of intracellular ROS production, and Westernblot to detect the expression level of NOX2 protein in THP-1 (A) cells, and fluorescence quantitative PCR to detect the expression level of THP-1 (A) cells.
Results: (1) the labeling rate of M.tb labeled with 100 FITC g/mL could reach (99.02 + 0.74)%, which is necessary for subsequent experiments.
(2) PMA induced differentiation of THP-1 (A) cells in the 4H phagocytosis rate of M.tb-FITC was (78.82 + 3.21)% and (68.88 + 0.46)% with trypan quenching (68.88 + 0.46)%, and the difference was statistically significant (P0.05). The phagocytosis rate (93.65 + 1.47)% reached saturation to 6h.
(3) the ratio of THP-1 (A) and M.tb increased from 1 to 1 to 1: 100, and the phagocytosis rate increased from (2.17 + 0.15)% to (35.62 + 2.49)%.100ng/mL LPS at 1:50 in THP-1 (A):M.tb-FITC, and the phagocytosis rate was (65.94 + 2.25)%, and the difference was statistically significant (P0.05).
(4) the LPS of 10ng/mL concentration could promote macrophage phagocytosis rate of M.tb, and the phagocytosis rate increased with concentration, and the LPS concentration of 10000ng/mL was (94.07 + 3.55)% higher than that of the control group (23.41 + 3.22)%, the difference was statistically significant (P0.05).
(5) the phagocytosis rate of M.tb in 10 MDPI treated macrophages decreased to (75.93 + 1.06)%, and decreased more significantly at 20 u M. The phagocytosis rate was (58.16 + 0.98)% (P0.05).HTA125 pretreated macrophages phagocytic percentage of M.tb phagocytosis (38.57 + 1.35)% and LPS stimulation group (83.25 + 2.53)%, the difference was statistically significant (P0.05).
(6) the labeling rate of 1 mu g/mLFDA to M.tb could reach (98.21 + 0.92)%. The killing rate of.THP-1 (A) cells increased with time, and the killing rate of 60min was up to (54.16 + 2.17)%, compared with 0min killing rate (15.21 + 0.63)%, the difference was significant.
(7) the MFI value of 30min killing M.tb in THP-1 (A) cells was 365.23 + 1.58 compared with that of the control group (608.35 + 2.16). The difference was significant (P0.01). The MFI value of 10000ng/mL decreased to 216.59 + 4.21. with the increase of killing ability with the increase of concentration.
(8) the killing ability of THP-1 (A) cells pretreated with HTA125 and DPI decreased to 385.92 + 25.98 and 302.35 + 25.47, compared with LPS stimulation group (268.54 + 32.73), and the difference was statistically significant (P0.01).
(9) LPS can enhance the expression rate of TLR4 receptor on macrophages inhibited by M.tb.
(10) Westernblot results showed that LPS could help upregulate the expression of NOX2 protein inhibited by M.tb in THP-1 (A) macrophages.
(11) LPS can enhance the yield of ROS inhibited by M.tb (THP-1) (A) cells.
(12) the fluorescence quantitative PCR detection gene expression results showed that after LPS stimulation, the TLR4mRNA and NOX2mRNA gene expression in THP-1 (A) cells were significantly increased by M.tb inhibition. Conclusion: LPS can promote the macrophage to M.tb phagocytosis and oxygen dependent killing by up regulation of the expression level and function of TLR4 and NOX2.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R392

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