TLR4基因单核苷酸多态性与军团菌感染的体外实验研究
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摘要
目的从体外实验探讨TLR4基因单核苷酸多态性TLR4(2244G→A)、(2299A→G)和(2242T→C)与军团菌感染的关系。方法用军团菌刺激健康人(n=54)外周血单个核细胞(peripheral blood mononuclear cell,PBMC),real-time RT-PCR检测PBMC中TLR4及My D88的表达水平,ELISA法检测培养上清液中IL-1β、IL-6及TNF-α的水平,采用SNa Pshot技术对TLR4多态性(2244G→A)、(2299A→G)和(2242T→C)进行分型检测。结果体外实验显示TLR4(2244G→A)AA型TNF-α表达显著高于GG/GA型(P=0.036 7),然而GG/GA型My D88、IL-6的水平显著高于AA型(P=0.035 2,P=0.031 7)。但是TLR4(A2299G)及TLR4(T2242C)各种基因型之间TNF-α、IL-6、IL-1β及TLR4、My D88的表达均无差异(P>0.05)。结论 TLR4基因单核苷酸多态性与军团菌感染的宿主易感性可能无关。
Objective To explore the association between Toll-like receptor 4 single nucleotide polymorphism( SNP) 2244 G → A,2299 A→G,2242 T→C and the susceptibility of Legionella infection in vitro experimental research. Methods The peripheral blood mononuclear cells( PBMC) from healthy subjects( n = 54) were stimulated with L. pneumophila. Real-time RT-PCR was used to detect the mRNA expression level of TLR4 and My D88. The levels of TNF-α,IL-1 b and IL-6 were measured by enzyme-linked immunosorbent assay( ELISA). TLR4 genotyping was performed with SNa Pshot technology. Results In vitro assay results showed that the TNF-αexpression was significantly higher in TLR4( G2244 A) AA than in GG/GA( P = 0. 036 7). The mRNA expression of My D88 and the level of IL-6 were significantly lower in TLR4( G2244 A) AA than in GG/GA( P = 0. 035 2,0. 031 7). Whereas the expression of TNF-α,IL-6,IL-1β,TLR4 and My D88 showed no statistical difference among different genotypes of TLR4 A2299 G and TLR4 T2242 C polymorphism( P > 0. 05). Conclusion The findings indicate that there is no association about the TLR4(2244 G→A),(2299 A→G),(2242 T→C) polymorphism with susceptibility to L. pneumophila infection.
Objective To explore the association between Toll-like receptor 4 single nucleotide polymorphism( SNP) 2244 G → A,2299 A→G,2242 T→C and the susceptibility of Legionella infection in vitro experimental research. Methods The peripheral blood mononuclear cells( PBMC) from healthy subjects( n = 54) were stimulated with L. pneumophila. Real-time RT-PCR was used to detect the mRNA expression level of TLR4 and My D88. The levels of TNF-α,IL-1 b and IL-6 were measured by enzyme-linked immunosorbent assay( ELISA). TLR4 genotyping was performed with SNa Pshot technology. Results In vitro assay results showed that the TNF-αexpression was significantly higher in TLR4( G2244 A) AA than in GG/GA( P = 0. 036 7). The mRNA expression of My D88 and the level of IL-6 were significantly lower in TLR4( G2244 A) AA than in GG/GA( P = 0. 035 2,0. 031 7). Whereas the expression of TNF-α,IL-6,IL-1β,TLR4 and My D88 showed no statistical difference among different genotypes of TLR4 A2299 G and TLR4 T2242 C polymorphism( P > 0. 05). Conclusion The findings indicate that there is no association about the TLR4(2244 G→A),(2299 A→G),(2242 T→C) polymorphism with susceptibility to L. pneumophila infection.
引文
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[10]Hang J,Zhou W,Zhang H,et al.TLR4 Asp299Gly and Thr399Ile polymorphisms are very rare in the Chinese population[J].J Endotoxin Res,2004,10(4):238-240.
[2]Plantinga TS,Ioana M,Alonso S,et al.The evolutionary history of TLR4 polymorphisms in Europe[J].J Innate Immun,2012,4(2):168-175.
[3]Mansur A,von Gruben L,Popov AF,et al.The regulatory tolllike receptor 4 genetic polymorphism rs11536889 is associated with renal,coagulation and hepatic organ failure in sepsis patients[J].J Transl Med,2014,12:177.
[4]Tellería-Orriols JJ,García-Salido A,Varillas D,et al.TLR2-TLR4/CD14 polymorphisms and predisposition to severe invasive infections by Neisseria meningitidis and Streptococcus pneumoniae[J].Med Intensiva,2014,38(6):356-362.
[5]Hawn TR,Verbon A,Janer M,et al.Toll-like receptor 4 polymorphisms are associated with resistance to Legionnaires’disease[J].Pro Natl Acad Sci USA,2005,102(7):2487-2489.
[6]冯凯,周钢桥,翟芸,等.TLR4基因多态性在中国人群中的初步研究[J].中华医学遗传学杂志,2005,22(1):99-101.
[7]王永堂.TLR4基因启动子区SNPs及其与严重创伤病人预后的关联研究[D].重庆:第三军医大学,2009.
[8]Michel O,Levan TD,Stern D,et al.Systemic responsiveness to lipopolysaccharide and polymorphi ms in the toll-like receptor 4gene in human beings[J].J Allergy Clin Immunol,2003,112(5):923-929.
[9]吕欣,愈卫锋,黄盛东,等.内毒索受体TLR4基因多态性“Asp299Gly”的分型检测[J].第二军医大学学报,2004,25(11):1195-1198.
[10]Hang J,Zhou W,Zhang H,et al.TLR4 Asp299Gly and Thr399Ile polymorphisms are very rare in the Chinese population[J].J Endotoxin Res,2004,10(4):238-240.