本文選題：LPS + 結(jié)核分枝桿菌(M.tb)　； 參考：《蚌埠醫(yī)學(xué)院》2013年碩士論文

【摘要】：研究背景：結(jié)核病是由結(jié)核分枝桿菌(Mycobacteriumtuberculosis，M.tb)感染引起的，目前仍是發(fā)病率和病死率最高的傳染病之一。它被認(rèn)為是一種慢性炎癥性疾病，伴有巨噬細(xì)胞吞噬、殺傷結(jié)核菌過(guò)程貫穿其發(fā)生、發(fā)展的始終[1]。以往的研究表明[2]，機(jī)體感染M.tb后，主要通過(guò)補(bǔ)體受體進(jìn)入巨噬細(xì)胞，并通過(guò)干擾巨噬細(xì)胞吞噬和溶酶體成熟而長(zhǎng)期寄生于宿主巨噬細(xì)胞內(nèi)并造成潛伏感染。Toll樣受體（Toll-likereceptors,TLRs）介導(dǎo)天然免疫，與抗TB免疫反應(yīng)相關(guān)。研究發(fā)現(xiàn)[3]，Toll樣受體4（Toll-likereceptor4,TLR4）在巨噬細(xì)胞抗M.tb的發(fā)生、發(fā)展中發(fā)揮著重要作用。有資料顯示[4]，非M.tb來(lái)源的TLR4受體激動(dòng)劑LPS可通過(guò)TLR4途徑和NOX2NADPH氧化酶及ROS信號(hào)通路促進(jìn)THP-1巨噬細(xì)胞吞噬和殺傷M.tb。探討LPS促進(jìn)巨噬細(xì)胞吞噬及殺傷M.tb的機(jī)制，對(duì)于其作為免疫佐劑或潛在的臨床抗結(jié)核預(yù)防用藥，具有重要價(jià)值。 目的：研究LPS對(duì)巨噬細(xì)胞氧依賴(lài)性殺傷結(jié)核分枝桿菌的促進(jìn)作用，初步闡明其機(jī)制。 方法：（1）結(jié)核分枝桿菌減毒株M.tbH37Ra用蘇通氏液體培養(yǎng)基于細(xì)菌培養(yǎng)箱中培養(yǎng)1個(gè)月后接種至羅琴氏固體培養(yǎng)基，37°C培養(yǎng)1個(gè)月，無(wú)菌接種環(huán)收集M.tb菌體于1mL勻漿器中，加入生理鹽水反復(fù)研磨，取細(xì)菌懸液，用生理鹽水經(jīng)10000rpm×2min洗滌3次，抗酸染色陽(yáng)性且鏡下觀察細(xì)菌分散。分光光度計(jì)檢測(cè)菌液濃度，用生理鹽水配成濃度為5×106/mL的M.tb懸液，4°C備用。 （2）10-100μg/mL濃度異硫氰酸熒光素(FITC)，在磷酸鹽緩沖液（PBS，pH7.2）中標(biāo)記M.tb，流式細(xì)胞術(shù)檢測(cè)標(biāo)記率和平均熒光強(qiáng)度（MFI）。FITC標(biāo)記的M.tb以10∶1的比例感染巨噬細(xì)胞THP-1(A)18h-24h，以流式細(xì)胞術(shù)檢測(cè)分析感染模型的感染率。 （3）取FITC標(biāo)記的M.tb與佛波醇酯(PMA)誘導(dǎo)分化的THP-1巨噬細(xì)胞(THP-1(A))共孵育1h-6h后，觀察PMA活化的THP-1(A)細(xì)胞吞噬FITC標(biāo)記M.tb的時(shí)間動(dòng)力學(xué)變化。 （4）0，100，1000ng/mL不同濃度LPS刺激THP-1(A)細(xì)胞24h后，與M.tb按不同比例（1:1-1:100）共培養(yǎng)30min，流式細(xì)胞術(shù)檢測(cè)LPS對(duì)THP-1(A)細(xì)胞吞噬M.tb吞噬率的促進(jìn)作用。 （5）0，10，100，1000，10000ng/mL不同濃度LPS分別刺激PMA分化前后的THP-1細(xì)胞24h后，再與M.tb以1:50濃度比共孵育30min，流式細(xì)胞術(shù)檢測(cè)LPS在THP-1細(xì)胞吞噬M.tb功能中的促進(jìn)作用。 （6）分別以抗人TLR4抗體HTA125阻斷TLR4，以DPI(diphenyleneiodoniumchloride)阻斷NOX2NADPH氧化酶后，再以LPS（1000ng/mL）作用THP-1(A)細(xì)胞24h，1:50濃度比例加入M.tb作用30min，流式細(xì)胞術(shù)分析TLR4及NOX2NADPH氧化酶信號(hào)途徑在LPS促進(jìn)THP-1(A)細(xì)胞吞噬M.tb中的作用。 （7）不同濃度熒光素二醋酸酯(FDA（0.1-1.0μg/mL）)，在磷酸鹽緩沖液（PBS，pH7.2）中標(biāo)記M.tb。取佛波醇酯(PMA)誘導(dǎo)分化的THP-1(A)細(xì)胞與FDA標(biāo)記M.tb于37℃孵育30min，洗滌細(xì)胞，加入小牛血清，置37℃孵育0～60min后，三蒸水裂解細(xì)胞，流式細(xì)胞術(shù)檢測(cè)裂解細(xì)胞釋放的M.tb熒光強(qiáng)度變化，分析計(jì)算THP-1(A)細(xì)胞對(duì)吞噬M.tb的殺傷功能。同時(shí)觀察刺激劑LPS及阻斷劑HTA125和DPI對(duì)殺傷功能的影響。 （8）流式細(xì)胞術(shù)檢測(cè)LPS（100ng/mL）對(duì)M.tb感染前后THP-1(A)細(xì)胞表面TLR4表達(dá)水平、胞內(nèi)ROS產(chǎn)量的促進(jìn)作用；Westernblot檢測(cè)THP-1(A)細(xì)胞NOX2蛋白表達(dá)水平；熒光定量PCR檢測(cè)THP-1(A)細(xì)胞胞內(nèi)TLR4mRNA，NOX2mRNA基因表達(dá)水平。 結(jié)果:（1）100μg/mL的FITC濃度標(biāo)記M.tb的標(biāo)記率可達(dá)(99.02±0.74)%，用于后續(xù)實(shí)驗(yàn)需要。 （2）PMA誘導(dǎo)分化的THP-1(A)細(xì)胞在4h對(duì)M.tb-FITC的吞噬率未加臺(tái)盼藍(lán)淬滅組為(78.82±3.21)%與加臺(tái)盼藍(lán)淬滅組(68.88±0.46)%相比，差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。至6h時(shí)吞噬率(93.65±1.47)%達(dá)飽和。 （3）THP-1(A)與M.tb比例從1∶1增加至1∶100，其吞噬率從(2.17±0.15)%增加至(35.62±2.49)%。100ng/mL的LPS在THP-1(A)：M.tb-FITC達(dá)1:50時(shí)，吞噬率達(dá)(65.94±2.25)%，差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 （4）10ng/mL濃度的LPS即可促進(jìn)巨噬細(xì)胞對(duì)M.tb的吞噬率，且隨濃度增加吞噬率逐漸增高，10000ng/mL的LPS濃度其吞噬率達(dá)(94.07±3.55)%較對(duì)照組(23.41±3.22)%增加，差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 （5）10μMDPI處理的巨噬細(xì)胞對(duì)M.tb的吞噬率降低至(75.93±1.06)%，20μM時(shí)降低更顯著，吞噬率達(dá)(58.16±0.98)%(P0.05)。HTA125預(yù)處理的巨噬細(xì)胞吞噬M.tb吞噬百分率(38.57±1.35)%和LPS刺激組(83.25±2.53)%相比較，差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 （6）1.0μg/mLFDA對(duì)M.tb的標(biāo)記率可達(dá)(98.21±0.92)%，可滿(mǎn)足殺傷實(shí)驗(yàn)的需要。THP-1(A)細(xì)胞對(duì)M.tb0~60min殺傷率隨時(shí)間增加而增加，60min的殺傷率可達(dá)(54.16±2.17)%，與0min殺傷率(15.21±0.63)%相比較，差異具有顯著性。 （7）100ng/mLLPS對(duì)THP-1(A)細(xì)胞30min殺傷M.tb的MFI值可達(dá)365.23±1.58與對(duì)照組608.35±2.16比較，，差異有顯著性（P0.01）。且隨濃度增加殺傷能力增強(qiáng)，10000ng/mL的MFI值降低至216.59±4.21。 （8）HTA125及DPI預(yù)處理的THP-1(A)細(xì)胞對(duì)M.tb-FDA的殺傷能力降低至385.92±25.98和302.35±25.47，較LPS刺激組268.54±32.73，差異有統(tǒng)計(jì)學(xué)意義（P0.01）。 （9）LPS可增強(qiáng)被M.tb抑制的巨噬細(xì)胞表面TLR4受體的表達(dá)率。 （10）Westernblot結(jié)果顯示LPS有助于上調(diào)被M.tb抑制的THP-1(A)巨噬細(xì)胞NOX2蛋白的表達(dá)量。 （11）LPS可增強(qiáng)被M.tb抑制的THP-1(A)細(xì)胞ROS的產(chǎn)量。 （12）熒光定量PCR檢測(cè)基因表達(dá)量結(jié)果顯示，經(jīng)LPS刺激后，被M.tb抑制的THP-1(A)胞內(nèi)的TLR4mRNA，NOX2mRNA基因表達(dá)量均明顯升高。結(jié)論：LPS能通過(guò)上調(diào)TLR4及NOX2的表達(dá)水平和功能，促進(jìn)巨噬細(xì)胞對(duì)M.tb的吞噬和氧依賴(lài)性殺傷。
[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.
【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R392

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 吳淑燕;李瓊;儲(chǔ)元元;李Z腦

本文編號(hào)：1959179