复发性呼吸道乳头状瘤细胞原代培养中人乳头状瘤病毒含量的变化  

Changes of HPV content in primary culture of recurrent respiratory papilloma cells

作　　者：肖洋[1] 周思含 王军[1] XIAO Yang;ZHOU Sihan;WANG Jun(Department of Laryngology,Beijing Tongren Hospital,Capital Medical University,Key Laboratory of Otolaryngology Head and Neck Surgery(Capital Medical University),Ministry of Education,Beijing,100730,China)

机构地区：[1]首都医科大学附属北京同仁医院咽喉科,耳鼻咽喉头颈科学教育部重点实验室(首都医科大学),北京100730

出　　处：《中国耳鼻咽喉头颈外科》2021年第12期770-773,共4页Chinese Archives of Otolaryngology-Head and Neck Surgery

基　　金：北京市医院管理局儿科学科协同发展中心儿科专项创新推广项目(XTCX201823)。

摘　　要：目的优化复发性呼吸道乳头状瘤(recurrent respiratory papilloma,RRP)细胞在体外原代培养的条件,并检测培养不同时间点单位细胞人乳头状瘤病毒(HPV)的含量。方法采用组织块培养法培养7例喉乳头状瘤组织,利用低速离心法和过筛网去除细胞碎片来优化培养系统,并于培养的第2、7、14天采用荧光半定量PCR法检测HPV分型及病毒的相对含量变化。结果 原代细胞培养第2天,显微镜下可见"凹空状"HPV感染的原代上皮细胞。培养第10天大部分HPV感染的原代上皮细胞出现了明显的凋亡和坏死,未被感染的、直径较小的上皮细胞开始增殖。在传代培养的第30~45天后,原代细胞逐渐衰老死亡。HPV6在离体后拷贝数迅速降低,最后完全检测不到;而HPV11虽然在离体后拷贝数出现迅速降低,但随着培养时间延长,即使细胞的状态不佳,HPV11仍然能维持着较低的拷贝数。结论 RRP原代细胞的体外培养虽可以存活30 d以上,但随着培养时间的延长,HPV含量不能随细胞增殖而稳定扩增,HPV拷贝数骤降甚至消失,尚不能作为HPV感染性肿瘤的体外研究模型。OBJECTIVE To optimize the culture conditions of primary human recurrent respiratory papillomatosis(RRP) cells in vitro and to detect the content of HPV per unit cell at different time points.METHODSIn this study,7 cases of laryngeal papilloma tissues were cultured by tissue block culture method.The culture system was optimized by removing cell fragments using low-speed centrifugation and sieving.On the 2nd,7th and 14th days of culture,the HPV typing and the relative content of HPV were detected by quantitative Real-Time PCR.RESULTS On the second day of primary cell culture,the ’concave’ HPV infected primary epithelial cells were observed under the microscope.On the 10th day of culture,most HPV infected primary epithelial cells showed obvious apoptosis and necrosis,and uninfected epithelial cells with small diameter began to proliferate.After 30-45 days of subculture,the primary cells gradually aged and died.The copy number of HPV6 decreased rapidly in vitro and could not be detected at last.Although the copy number of HPV11 also decreased rapidly in vitro,with the extension of culture time,HPV11 can still maintain a low copy number even if the cell state is poor.CONCLUSION Although theprimary cells of recurrent respiratory papilloma can survive for more than 30 days in vitro,with the extension of culture time,the HPV content can not expand stably with cell proliferation,and the HPV copy number drops or even disappears.It can not be used as an in vitro research model of HPV infectious tumor.

关 键 词：乳头状瘤病毒科 乳头状瘤病毒感染 复发性呼吸道乳头状瘤 原代细胞培养 体外技术 

分 类 号：R739.65[医药卫生—肿瘤]