HPV-18型晚期基因L1在宫颈脱落细胞中的表达与突变研究

Expression and mutation of HPV18 late gene L1 in exfoliated cervical cells

目的:研究人乳头瘤病毒HPV-18型晚期基因L1在不同宫颈上皮病变患者脱落细胞中表达分布与基因突变特征。方法:收集单纯感染人乳头瘤病毒HPV-18型宫颈上皮病变组织脱落细胞,免疫细胞化学分析其中L1基因的蛋白表达情况。提取总DNA,特异性引物针对HPV-18 L1基因进行PCR扩增检测,所得产物进行Sanger基因测序分析。结果:HPV-18 L1基因在宫颈炎、CINⅠ+Ⅱ级、CINⅢ级和宫颈癌组织中蛋白表达阳性率分别为100%、81.8%、34.6%和12.2%。在不同病理分级宫颈上皮病变患者脱落细胞标本中,HPV-18 L1蛋白表达阳性率差异有统计学意义(P<0.01)。Sanger法基因测序显示L1基因序列存在突变,突变率9.2%,共发现11类核苷酸变异,其中4类属错义突变。结论:HPV-18型晚期基因L1蛋白表达量与宫颈组织病理病变严重程度呈负相关趋势。HPV-18 L1基因突变较为突出,存在地区流行型突变株。L1基因突变与宫颈恶性病变程度可能存在一定关系。

Objective:To analyze the expression and variation of HPV-18 late protein L1 in cervical exfoliated cells at different stages.Methods:Exfoliated cells were obtained from cervical epithelia infected with human papillomavirus strain 18(HPV-18),and subjected to detection of L1 protein expression by immunocytochemistry.Total DNA was extracted,and amplified by polymerase chain reaction(PCR) using primers specific to HPV-18 L1 gene.Then the amplified products were analyzed by Sanger gene sequencing.Results:L1 protein was positively expressed in cervical inflammation of HPV-18,CIN Ⅰ+Ⅱ,CIN Ⅲ and cervical cancer tissues,with expression rate of 100%,81.8%,34.6% and 12.2%,respectively.The positive rate of L1 protein expression was statistically different in exfoliated cervical cells in CIN with different grade(P<0.01).Sanger sequencing revealed mutation in L1 gene sequence,with a mutation rate of 9.2%.A total of 11 nucleotide variants were found,among which 4 were missense mutations.Conclusion:Expression level of HPV-18 L1 protein is negatively correlated with the severity of cervical lesion by histopathology.Mutation of HPV-18 L1 gene is relatively prominent,and characterized by endemic mutant strains,which indicates that L1 gene mutation may be related to the malignant degree of cervical lesions.