【摘要】： 目的 DC-SIGN位于樹(shù)突狀細(xì)胞表面,是近年發(fā)現(xiàn)的與結(jié)核病發(fā)生密切相關(guān)的一個(gè)免疫分子,近年體外細(xì)胞水平研究結(jié)果顯示結(jié)核菌可通過(guò)其胞壁成分ManLAM與DC-SIGN分子結(jié)合,開(kāi)通下述幾種機(jī)制抑制宿主抗結(jié)核免疫應(yīng)答:抑制未成熟樹(shù)突狀細(xì)胞成熟;誘導(dǎo)周?chē)幢桓腥镜臉?shù)突狀細(xì)胞處于半成熟狀態(tài);上調(diào)樹(shù)突狀細(xì)胞表達(dá)免疫抑制因子IL-10等。本研究通過(guò)探討DC-SIGN分子配體ManLAM對(duì)肺結(jié)核患者樹(shù)突狀細(xì)胞黏附BCG能力的影響,進(jìn)一步研究DC-SIGN分子在結(jié)核病發(fā)生過(guò)程中的作用。 方法 1.含有EGFP基因的大腸桿菌-分枝桿菌穿梭表達(dá)載體的構(gòu)建用KpnⅠ和XbaⅠ對(duì)質(zhì)粒pEGFP和pHSP70-LA73進(jìn)行雙酶切,以制備目的基因和載體大片段,用T4連接酶連接,然后轉(zhuǎn)化已制備好的大腸桿菌感受態(tài)細(xì)胞DH5α,用含卡那霉素的固體LB培養(yǎng)基篩選重組子。 2.綠色熒光蛋白標(biāo)記BCG 應(yīng)用eppendorf電轉(zhuǎn)化儀將重組質(zhì)粒轉(zhuǎn)化入制備好的BCG感受態(tài)細(xì)胞,培養(yǎng)至對(duì)數(shù)生長(zhǎng)期后,用熱誘導(dǎo)的方法使綠色熒光蛋白表達(dá),從而標(biāo)記BCG。 3.ManLAM對(duì)肺結(jié)核患者樹(shù)突狀細(xì)胞黏附BCG能力影響的研究分離研究對(duì)象外周血單個(gè)核細(xì)胞,誘導(dǎo)樹(shù)突狀細(xì)胞生長(zhǎng),用流式細(xì)胞術(shù)檢測(cè)其表面DC-SIGN表達(dá)率,并在肺結(jié)核患者樹(shù)突狀細(xì)胞與不同濃度ManLAM預(yù)孵育后,再以5:1的比例加入標(biāo)記BCG共孵育,流式細(xì)胞儀檢測(cè)樹(shù)突狀細(xì)胞表面綠色熒光,測(cè)定黏附率。 結(jié)果 1.酶切鑒定條帶正確,分枝桿菌穿梭質(zhì)粒構(gòu)建成功,命名為pHSP70-EGFP。 2.倒置熒光顯微鏡觀察細(xì)菌涂片,可見(jiàn)細(xì)菌發(fā)出較強(qiáng)的綠色熒光,證明綠色熒光蛋白在BCG中表達(dá),標(biāo)記BCG成功。 3.肺結(jié)核患者外周血樹(shù)突狀細(xì)胞表面DC-SIGN表達(dá)率為(86.69±3.66)%,明顯高于健康對(duì)照組。肺結(jié)核患者外周血樹(shù)突狀細(xì)胞和標(biāo)記BCG的黏附率為(89.84±1.57)%,而伴隨著ManLAM濃度的不斷增加,樹(shù)突狀細(xì)胞和標(biāo)記BCG的黏附率不斷下降。 結(jié)論 1.可以用穿梭質(zhì)粒pHSP70-EGFP在BCG中表達(dá)綠色熒光蛋白,從而標(biāo)記BCG。 2.樹(shù)突狀細(xì)胞主要通過(guò)與ManLAM配體結(jié)合而黏附BCG,同時(shí)也證明DC-SIGN是肺結(jié)核患者樹(shù)突狀細(xì)胞表面結(jié)核分枝桿菌的主要受體。
[Abstract]:Objective DC-SIGN, located on the surface of dendritic cells, is an immune molecule closely related to the occurrence of tuberculosis in recent years. In recent years, the results of cell level studies in vitro showed that tuberculous bacillus could bind to DC-SIGN molecule through its cell wall component ManLAM, which could inhibit host anti-TB immune response by inhibiting immature dendritic cell maturation. The peripheral uninfected dendritic cells were induced to be semi-mature, and the expression of immunosuppressive factor IL-10 was up-regulated by dendritic cells. The purpose of this study was to investigate the effect of DC-SIGN ligand ManLAM on the ability of dendritic cells to adhere to BCG in pulmonary tuberculosis patients, and to further study the role of DC-SIGN molecule in the pathogenesis of tuberculosis. Method 1. Construction of Escherichia coli -Mycobacterium Shuttle expression Vector containing EGFP Gene the plasmids pEGFP and pHSP70-LA73 were digested with Kpn 鈪，

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