SPLUNC1在LPS诱导炎症反应中的作用机制研究

发布时间：2018-02-28 19:40

本文关键词： 鼻咽癌 SPLUNC1 TLR4 NF-κB 炎症　出处：《中南大学》2013年博士论文　论文类型：学位论文

【摘要】：目前认为慢性炎症是20-25%的恶性肿瘤发生的主要驱动者,炎-癌分子机制的探讨已成为癌变原理研究领域关注的重要焦点。革兰氏阴性菌是与鼻咽部长期共存的主要病原微生物,是致鼻咽部慢性炎症的常见因素。如果病原微生物长期持续刺激鼻炎上皮则会导致鼻咽局部形成慢性炎症。众所周知,细菌内毒素即脂多糖(lipopoly saccharide, LPS)是革兰氏阴性菌主要毒力因素,它可作用于细胞膜受体CD14和TLR4,从而发挥生物学作用。 SPLUNC1基因作为鼻咽及上呼吸道上皮细胞编码一种分泌性蛋白,是细胞的固有免疫分子。前期研究发现SPLUNC1含有一个BPI结构域,BPI能与革兰氏阴性细菌细胞壁上的LPS特异性地结合。重组的SPLUNC1蛋白能经转染细胞分泌到培养上清液中并具有结合细菌脂多糖,以及杀灭或抑制上呼吸道绿脓杆菌的功能。SPLUNC1在预防病原微生物感染和抑制过度炎症反应方面起到重要作用。本课题将在构建SPLUNC1基因封闭与基因剔除的细胞和动物模型基础上,深入研究LPS诱导的TLR4/NF-κB信号通路介导的炎症反应中SPLUNC1预防或阻断炎-癌反应过程的可能的作用和分子机制,并为鼻咽癌分子诊治提供新靶点及新线索。 LPS可通过TLR4/NF-κB信号通路引起鼻咽癌细胞促炎因子的分泌和鼻咽癌细胞的增殖慢性感染的重要毒力因素被认为是革兰氏阴性细菌细胞壁的主要成分LPS,对LPS的识别与信号传导是宿主细胞抵御革兰氏阴性细菌的关键。同时LPS及其介导的TLR4信号传导途径被认为是慢性感染导致肿瘤发生发展的重要的因素。为了研究LPS引起的慢性炎症对鼻咽癌发生发展的影响,本研究采用合适浓度的LPS (1ug/ml)作用于鼻咽癌细胞系5-8F和6-10细胞系。流式细胞周期分析和MTT结果发现LPS能够促进鼻咽癌细胞系细胞周期进程进而引起细胞增殖。同时LPS刺激24小时后,Real-time PCR和ELISA检测促炎因子IL-6, IL-8, TNF-α和IL-1β的表达发现,LPS能够促进促炎因子的分泌。LPS是TLR4的配体之一。为了w釲PS是否通过TLR4信号通路影响鼻咽癌细胞促炎因子的分泌和细胞增殖,本研究采用Western-blot和Real-time PCR检测了TLR4信号通路相关分子的表达,结果发现LPS能显著提高CD14, TLR4, MyD88,p-p65表达以促进促炎因子的分泌。采用免疫荧光技术和荧光素酶报告基因活性检测发现,在LPS诱导下,鼻咽癌细胞中NF-KB (p65)被活化进入细胞核内。NF/κB (p65)持续活化是慢性炎症发生的主要因素。因此本文认为：在鼻咽癌细胞中,LPS通过TLR4-MyD88-NF-KB (p65)信号通路来促进NF-κB (p65)的活化入核,导致促炎因子的大量分泌进而导致炎症的持续发生和细胞增殖。 SPLUNC1与TLR4在正常和鼻咽癌患者组织中的动态表达前期的研究发现SPLUNC1在正常鼻咽组织中高表达,而在鼻咽癌患者组织中低表达或者不表达；肿瘤细胞表面表达的TLR4能促进肿瘤的免疫逃逸从而促进肿瘤的发生发展。本研究利用鼻咽癌组织芯片采用免疫组化法观察了SPLUNC1与TLR4在正常鼻咽组织和鼻咽癌组织中的表达情况,并分析了两者表达的相关性以及对鼻咽癌患者的预后判断。结果发现在正常鼻咽组织中SPLUNC1的高表达,而炎症组织中SPLUNC1的表达减弱,鼻咽癌组织中SPLUNC1的表达逐渐消失；但是TLR4在正常鼻咽组织中呈弱表达,炎症组织中TLR4表达升高,且在鼻咽癌组织中呈高表达。相关性分析得出SPLUNC1与TLR4的表达呈负相关关系。Kaplan-Meier生存分析显示SPLUNC1表达越低,TLR4表达越高,鼻咽癌患者预后越差。 SPLUNC1通过TLR4/NF-KB信号通路抑制LPS引起的促炎因子的分泌和细胞增殖为了筛选合适的细胞系进行后续实验,本研究对鼻咽癌细胞系进行了SPLUNC1和TLR4的表达筛选,Real-time PCR和WB观察发现鼻咽癌细胞系5-8F和正常鼻咽上皮细胞系NP-69较合适进行后续实验。SPLUNC1含有BPI结构域,能够与细菌LPS结合,为了证实SPLUNC1是制约LPS引起的细胞增殖和促炎因子分泌的关键因素的设想,本研究构建了稳定高表达SPLUNC1的5-8F细胞系和稳定干扰SPLUNC1的NP69细胞系,并使用LPS (1ug/ml)刺激5-8F/ctrl和5-8F/SPLUNC1细胞系以及NP-69/Ctrl和NP69/SPLUNC1si细胞系。通过MTT实验发现稳定转染SPLUNC1的5-8F细胞系明显比空载体转染细胞系增殖慢,同时干扰SPLUNC1的NP69细胞系经LPS刺激后增殖速度比对照组明显加快。通过流式细胞技术也表明SPLUNC1基因能够阻止LPS引起的细胞增殖,导致鼻咽癌细胞的生长阻滞在G0/G1期。同时Real-time PCR和ELISA实验证实,SPLUNC1基因能够在mRNA水平和蛋白水平抑制LPS引起的促炎因子IL-6, IL-8, TNF-α和IL-1β的分泌。以上结果证实SPLUNC1能够抑制LPS引起的促炎因子的分泌和鼻咽癌细胞的增殖。因为LPS可通过TLR4-MyD88-NF-κB (p65)信号通路促进鼻咽癌细胞系促炎因子的分泌和细胞增殖,于是本研究进一步验证了SPLUNC1在这一过程中的作用。使用LPS (1ug/ml)刺激5-8F/ctrl和5-8F/SPLUNC1细胞系以及NP-69/Ctrl和NP69/SPLUNC1si细胞系24小时后,收集蛋白进行Western-blot检测发现,SPLUNC1能够抑制LPS引起的TLR4、 MyD88、p-p65(NF-κB)表达的升高。荧光素酶报告基因检测结果和免疫荧光结果显示,尽管受到LPS的刺激,SPLUNC1也能阻止NF-κB入核,同时也能抑制NF-κB的转录活性。为了在体内证实SPLUNC1抑制LPS引起的炎症反应的功能,本研究构建了Splunc1基因敲除小鼠,取Splunc1敲除小鼠和野生型小鼠肺组织和MEF检测Tlr4/Nf-κb通路关键分子的表达,并对Splunc1敲除小鼠和野生型小鼠进行腹腔注射LPS (5mg/kg)刺激24小时后,取小鼠肺组织进行免疫组化检测,同时收集小鼠外周血进行ELISA检测。Western-blot检测发现Splunc1敲除后,Splunc1敲除小鼠肺组织和MEF (Splunc1-/-)中Cd14, Tlr4、Myd88、p-p65(Nf-κb)表达的升高而Ikba的表达降低。ELISA检测发现Splunc1敲除小鼠LPS刺激后,外周血中促炎因子11-6,11-8表达升高。上述研究结果证明SPLUNC1基因可通过降低TLR4表达和抑制NF-κB活化有效抑制LPS引起的促炎因子的分泌和细胞增殖,这为固有免疫分子SPLUNC1能有效抵抗病原微生物感染引起的炎症反应,维持微环境的稳定提供了有力的证据。
[Abstract]:It is considered that chronic inflammation is the main driver of malignant tumor in 20 - 25 % . The pathogenesis of inflammatory - cancer has become an important focus in the field of carcinogenesis . Gram - negative bacteria are the main pathogenic microorganisms in the chronic inflammation of nasopharyngeal parts . It is well known that bacterial endotoxin , lipopolysaccharide ( LPS ) , is the main virulence factor of Gram - negative bacteria , which can act on the membrane receptor CD14 and the like receptor , thus exerting a biological function . SPLUNC1 encodes a secretory protein as a secretory protein in the nasopharyngeal and upper respiratory epithelial cells , which is an innate immune molecule of the cell . Previous studies have found that SPLUNC1 contains a protein that binds to LPS on the cell wall of the Gram - negative bacterial cell . The recombinant SPLUNC1 protein can be secreted into the culture supernatant by transfected cells and has the function of binding bacterial lipopolysaccharide , and killing or inhibiting the upper respiratory tract pseudomonas aeruginosa . SPLUNC1 plays an important role in the prevention of pathogenic microbial infections and inhibiting excessive inflammation . On the basis of constructing the cell and animal model of SPLUNC1 gene closure and gene knockout , the possible role and molecular mechanism of SPLUNC1 in preventing or blocking inflammatory - cancer response in inflammatory reaction mediated by LPS - induced TLR / NF - 魏B signaling pathway can be further studied , and new targets and new clues are provided for the diagnosis and treatment of nasopharyngeal carcinoma . LPS can induce the secretion of pro - inflammatory cytokines in nasopharyngeal carcinoma cells and the proliferation of nasopharyngeal carcinoma cells through the signaling pathway of NF - 魏B signaling pathway . LPS and IL - 6 , IL - 8 , TNF - 伪 and IL - 1尾 in nasopharyngeal carcinoma cell line were detected by Western - blot and Real - time PCR . Dynamic expression of SPLUNC1 in normal and nasopharyngeal carcinoma patients The expression of SPLUNC1 in normal nasopharyngeal tissues and the expression of SPLUNC1 in nasopharyngeal carcinoma were observed by immunohistochemistry . The expression of SPLUNC1 was decreased in the normal nasopharyngeal tissues , and the expression of SPLUNC1 in the tissues of NPC was decreased . SPLUNC1 inhibits the secretion of pro - inflammatory cytokines and the proliferation of cells induced by LPS via the Toll - like / NF - KB signaling pathway The results showed that SPLUNC1 could inhibit the secretion of IL - 6 , IL - 8 , TNF - 伪 and IL - 1尾 in nasopharyngeal carcinoma cell line by LPS ( 1ug / ml ) . In order to demonstrate the function of SPLUNC1 in inhibiting the inflammatory response induced by LPS in vivo , we constructed a Spluncl knockout mouse . After 24 hours of intraperitoneal injection of LPS ( 5 mg / kg ) by Spluncl knockout mice and wild - type mice , the expression of Cd14 , Tr4 , Myd88 , p - p65 ( Nf - 魏B ) was detected by Western - blot . Western - blot analysis showed that Spluncl knockout mice stimulated inflammatory factors 11 - 6 and 11 - 8 in peripheral blood after LPS stimulation in mice . These results demonstrate that the SPLUNC1 gene can effectively inhibit the secretion of pro - inflammatory factors induced by LPS and inhibit the proliferation of cells by reducing the expression and inhibition of NF - 魏B activation , which provides powerful evidence for the effective resistance of SPLUNC1 to inflammatory response caused by pathogenic microorganism infection and maintaining the stability of the microenvironment .

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R739.63

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