Toll样受体4基因3’未翻译区多态性对其蛋白表达的影响

Influence of single nucleotide polymorphism of toll like receptor 4 gene at 3’untranslated region on its protein expression

目的探讨TLR4基因3’未翻译区(3’UTR)基因多态性对全血培养后TLR4蛋白表达的影响。方法分别采用单管双向等位基因特异性扩增和限制性片段长度多态法检测269名汉族健康献血者Toll样受体4(TLR4)基因3’UTR 11 367和编码区896位点的基因多态性。其中89例全血标本,用全血培养模型检测内毒素刺激前后TLR4蛋白表达的变化,并测定培养上清中肿瘤坏死因子(TNF)-α水平。结果89例健康献血者中,11 367位点等位基因G和C的频率分别为82．58%和17．42%；63例是G等位基因纯合子(GG),21例为杂合子(GC),5例是C等位基因纯合子(CC)。基因型GC与CC内毒素刺激后TLR4表达增加程度显著低于GG纯合子表达增加程度(P＜0．05)。并且GG纯合子TNF-α诱生水平显著高于GC和CC基因型。在269例健康献血者中未发现896位点的G等位基因。结论TLR4基因3’UTR 11 367G/C基因多态性对全血培养TLR4蛋白表达产生显著影响,并与LPS刺激后TNF-α分泌变化有关。

Objective To investigate whether single nucleotide polymorphism （SNP） of toll like receptor 4 （TLR4） at 3＇ untranslated region （3＇ UTR） influences TLR4 protein expression in whole blood culture. Methods A total of 89 samples out of 269 whole blood samples taken from healthy persons were used for cell culture model with/without lipopolysaccharide （LPS） stimulation to measure TLR4 expression level by flow cytometry. SNP of TLR4 gene at 3＇ UTR 11367G/C and coding region 896A/G was genotyped with the method of single-tube bi-directional allele specific amplification （SB-ASA） and restriction fragment length polymorphism （RFLP） technique, respectively. TNF-α production in the whole blood culture after stimulated by LPS was also measured. Results Frequency of TLR4 11367 SNP was 17.42%. Genotype frequencies of 11 367 loci were 70.79% （ GG）, 23.60% （GC） and 5.61% （ CC）, respectively. Among 89 individuals were 63 with homozygate and 21 with heterozygote （GC） for the G allele （GG） as well as five with genotype CC. The TLR4 expression in leukocytes was higher in GG homozygate after LPS stimulation compared with individuals with heterozygous GC and homozygous CC （ P 〈 0.05 ）. In addition, the highest values of TNF-α production was found in GG homozygate compared with individuals with heterozygous GC and homozygous CC （ P 〈 0.05 ）. No G allele was detected in 896 loci among 269 blood donors. Conclusions SNP of TLR4 gene at 3＇ UTR exerts significant influence on TLR4 expression, which may be associated with secretion of TNF-α posterior to LPS stimulation.