CAJ | 학술논문

目的：研究受感染宫颈上皮细胞中人乳头瘤病毒整合的检测方法与应用。方法构建串联单拷贝基因重组质粒，用于对经反向斑点杂交法基因检测为16型人乳头瘤病毒感染的宫颈脱落细胞样本进行病毒的早基因分析，通过串联单拷贝基因标准化方法，对采用荧光定量 PCR 技术分析得到的样本中 HBB、病毒早基因 E2与 E6的检测数据分析，计算 HPV 病毒整合参数与平均病毒拷贝数，推断 E2基因完整性及病毒整合发生、病毒拷贝数与已知的病变进程的相关性。结果构建的重组质粒可有效用于标准化同批次荧光定量 PCR 检测 E2基因、E6基因和 HBB 基因的数据。37例16型人乳头瘤病毒阳性宫颈细胞样本中，15例样本结果为 E2基因已破坏，包括10例高度病变组样本及5例正常组样本。不同组 E2基因完整性差异有统计学意义（P ＜0．05）。E2基因被破坏的样本平均病毒拷贝数比 E2基因完整样本显著减少（P ＜0．05）。结论本研究建立的串联单拷贝基因标准化法用于检测16型 HPV 感染的宫颈细胞样本中病毒整合导致的 E2基因破坏有效可行。
목적：연구수감염궁경상피세포중인유두류병독정합적검측방법여응용。방법구건천련단고패기인중조질립，용우대경반향반점잡교법기인검측위16형인유두류병독감염적궁경탈락세포양본진행병독적조기인분석，통과천련단고패기인표준화방법，대채용형광정량 PCR 기술분석득도적양본중 HBB、병독조기인 E2여 E6적검측수거분석，계산 HPV 병독정합삼수여평균병독고패수，추단 E2기인완정성급병독정합발생、병독고패수여이지적병변진정적상관성。결과구건적중조질립가유효용우표준화동비차형광정량 PCR 검측 E2기인、E6기인화 HBB 기인적수거。37례16형인유두류병독양성궁경세포양본중，15례양본결과위 E2기인이파배，포괄10례고도병변조양본급5례정상조양본。불동조 E2기인완정성차이유통계학의의（P ＜0．05）。E2기인피파배적양본평균병독고패수비 E2기인완정양본현저감소（P ＜0．05）。결론본연구건립적천련단고패기인표준화법용우검측16형 HPV 감염적궁경세포양본중병독정합도치적 E2기인파배유효가행。
Objective To establish a method to detect viral integrity of human papillomavirus in women cervical HPV infection. Methods We amplified E6/E7 gene and E2 gene of HPV16,then inserted them into a plasmid containing single copy HBB gene. HPV16 infected cervical epithelium samples were screened out by genotyping with RDB of flow-through hybridization assay.Fluo-rescence quantitive PCR data of HBB,viral E2 gene and viral E6 gene of all samples were standardized by compared with respective parameters of the plasmid.The ratio IHPV and CHPV were calculated to find out E2 gene disruption and viral copies per cell in the cer-vical samples,respectively.Results The plasmid constructed for standardization was proved effective to make the FQ-PCR data of E2 gene,E6 gene and HBB gene comparable.Thirty-seven HPV16 positive cervical epithelium samples included 22 cases from women whose TCT were normal,and 15 cases from women who confirmed HIL/CIN 2-3 or above through colposcopic examina-tion plus biopsy.Fifteen samples were detected E2 gene disruption,including 10 HIL/CIN 2-3 or above samples and 5 TCT normal samples.E2 gene integrity in different groups were statistically significant different(P <0.05).The average viral copies per cell dis-played a significant decline along with E2 gene disruption(P <0.05).Conclusion The tandem single copy gene plasmid standard-ized methord for the detection of E2 gene disruption caused by viral integration in HPV16 infected cervical cells is feasible and effec-tive.