猿猴病毒40 T抗原转化的人脐静脉内皮细胞系PUMC-HUVEC-T1的建立

目的 建立猿猴病毒40（SV40）T抗原转化的人脐静脉内皮细胞（HVUEC）模型，为内皮研究提供可利用资源。方法 分离人脐静脉内皮细胞，原代培养。感染含有SV40大T、小T抗原的慢病毒，连续传代培养。对感染后的HUVEC，RT-PCR检测大T的表达，免疫细胞化学检测vWF、CD31、CD34的表达及结合凝集素能力，透射电镜观察内皮细胞的超微结构，Matrigel检测管状成型，进行染色体核型分析，皮下接种BABL/c-nu裸鼠检测致瘤性，PCR法进行种属鉴定及支原体检测，短串联重复序列（STR）检测鉴定细胞身份。结果 转化后的人脐静脉内皮细胞命名为PUMC-HUVEC-T1，扁平多角状，汇合时典型铺路石排列，体外传代40代以上（1∶3～4）；细胞中有SV40LT mRNA的表达；vWF、CD31、CD34表达均阳性，可结合荆豆凝集素-1（UEA-1）；电镜可见WP小体；不同代数细胞核型正常稳定，裸鼠体内接种不成瘤。PUMC-HUVEC-T1种属鉴定为人源性，STR结果与原代HUVEC一致，无支原体的污染，国家实验细胞资源共享平台收藏。结论 建立了易获得、背景清楚、质量可靠的SV40 T抗原转化的HUVEC细胞系PUMC-HUVEC-T1。

Establishment of a simian virus 40 T antigen transfected human umbilical vein endothelial cell line PUMC-HUVEC-T1

Objective To establish a simian virus 40(SV40)T antigen transfected human umbilical vein endothelial cells (HVUEC) model for endothelial research.Methods Primary human umbilical vein endothelial cells were separated and infected with SV40 large T and small T antigen, and then continuously sub-cultured in vitro. The infected HUVEC of large T expression was detected by RT-PCR. vWF, CD31, CD34 expression and lectin binding were determined by immuno-cytochemistry. Ultra-structure was observed by transmission electron microscopy and the formation of endothelial tubes was accessed by Matrigel.The karyotype was analyzed and tumorigenicity was detected by subcutaneous inoculation in BABL/c nude mice. Mycoplasma and species were checked by PCR. Short tandem repeat(STR) profiling was employed for cell identity.Results The SV40 T antigen transfected cell line was designated as PUMC-HUVEC-T1 and had SV40 LT mRNA expression. The cells were passaged (1∶3-4) for more than 40 times in vitro. Morphologically PUMC-HUVEC-T1 arrayed like pitching stone when reaching confluency. PUMC-HUVEC-T1 showed positive expression of vWF, CD31, CD34, and could bind lectin in vitro. WP corpuscles were identified by electron microscopy. The cells formed vascular network-like structures when planted and cultured on Matrigel. The karyotype was nomal and stable between different passages. No tumor formed in BABL/c-nude mice. PUMC-HUVEC-T1 was conformed of its human origin and its STR was consistent with that of the original HUVEC. No mycoplasma was detected. Conclusion A SV40 T antigen transformed HUVEC cell line PUMC-HUVEC-T1 was successfully established which is accessible with clear background and reliable quality. It would provide a solid base for the endothelial research. It is deposited by cell resource center and is available for distribution.