CAJ | 학술논문

目的：建立人乳头瘤病毒6型（HPV6）全基因体外组织工程皮片培养模型，为进一步研究 HPV病毒周期奠定基础。方法用电转的方法，将 HPV6全长线性基因和质粒 pEGFP-▲EGFP 共转染 hTERT 细胞，G418抗性筛选，Southern 印迹法检测细胞内 HPV 病毒含量；3T3 J2滋养层细胞、I 型鼠尾胶原与含 HPV6基因的 hTERT 细胞（HPV6.hTERT 细胞）混合后，在金属网格上共同培养，逐渐形成皮片样结构。 HE 染色和免疫组化检测皮片的组织结构和 HPV6 L1蛋白表达，电镜检查皮片病毒颗粒。结果 HPV6全长线性基因成功转入 hTERT 细胞，Southern 印迹法检测细胞内含 HPV6 DNA；与3T3 J2细胞、I 型鼠尾胶原共同培养的 HPV6. hTERT 细胞随时间而增殖分化，逐渐形成具有疣状增生外观的皮片。皮片 HE 染色显示，培养7 d 即出现典型的皮肤分层结构；培养21 d 皮片可见明显乳头瘤样增生、空泡细胞、角化过度、角化不全等 HPV 感染组织病理表现。免疫组化显示皮片上部有 HPV6 L1蛋白表达。电镜检测发现皮片中存在 HPV6病毒颗粒。结论 HPV6全基因组织工程皮片培养模型为 HPV 的生物学研究提供了一个平台，但在应用上有一定的局限性。
목적：건립인유두류병독6형（HPV6）전기인체외조직공정피편배양모형，위진일보연구 HPV병독주기전정기출。방법용전전적방법，장 HPV6전장선성기인화질립 pEGFP-▲EGFP 공전염 hTERT 세포，G418항성사선，Southern 인적법검측세포내 HPV 병독함량；3T3 J2자양층세포、I 형서미효원여함 HPV6기인적 hTERT 세포（HPV6.hTERT 세포）혼합후，재금속망격상공동배양，축점형성피편양결구。 HE 염색화면역조화검측피편적조직결구화 HPV6 L1단백표체，전경검사피편병독과립。결과 HPV6전장선성기인성공전입 hTERT 세포，Southern 인적법검측세포내함 HPV6 DNA；여3T3 J2세포、I 형서미효원공동배양적 HPV6. hTERT 세포수시간이증식분화，축점형성구유우상증생외관적피편。피편 HE 염색현시，배양7 d 즉출현전형적피부분층결구；배양21 d 피편가견명현유두류양증생、공포세포、각화과도、각화불전등 HPV 감염조직병리표현。면역조화현시피편상부유 HPV6 L1단백표체。전경검측발현피편중존재 HPV6병독과립。결론 HPV6전기인조직공정피편배양모형위 HPV 적생물학연구제공료일개평태，단재응용상유일정적국한성。
Objective To establish a model for preparation of tissue-engineered skin grafts with hTERT cells carrying human papillomavirus type 6 (HPV 6) genome in vitro, so as to lay a foundation for studying HPV life cycle. Methods The full-length linear HPV6 genome and plasmid pEGFP-▲EGFP were electrophoretically cotransferred into hTERT cells. After selection using G418 resistance, Southern blotting was performed to determine the viral load of HPV6 in transfected cells. 3T3 J2 trophoblastic cells, type I rat-tail collagen and hTERT cells containing the full-length HPV6 genes (HPV6.hTERT cells)were mixed and cocultured on metal meshes to form skin graft-like structures. Hematoxylin and eosin (HE)staining was performed to observe the structure of formed skin grafts, an immunohistochemical assay to measure the expression of HPV6 L1 protein, and electron microscopy to observe virus particles in the skin grafts. Results The linear HPV6 gene was successfully transferred into hTERT cells, and Southern blotting showed the presence of HPV6 DNA in the transferred hTERT cells. The HPV6.hTERT cells, which were cocultured with 3T3 J2 trophoblastic cells and type I rat-tail collagen, proliferated and differentiated over time, and gradually formed skin grafts giving the appearance of verrucous hyperplasia. HE staining showed that the cocultured HPV6.hTERT cells could form typical stratified structure of skin after 7 days of cultivation, and histopathologic features of HPV infection, including obvious papillomatous hyperplasia, presence of vesicular cells, hyperkeratosis and parakeratosis, could be observed after 21 days. The immunohistochemical assay showed the expression of HPV6 L1 protein in the upper portion of skin grafts, and electron microscopy revealed the presence of HPV6 virus particles in skin grafts. Conclusions The established model for preparation of tissue-engineered skin grafts using HPV 6 genome-carrying cells provides a basis for biological studies of HPV, but its application is limited to some degree.