摘要
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。
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.
出处
《解剖学报》
CAS
CSCD
北大核心
2013年第2期199-203,共5页
Acta Anatomica Sinica
基金
国家实验细胞资源共享平台资助项目
作者简介
冯海凉（1983-），女（汉族），北京市人，博士，助理研究员。
通讯作者（Towhomcorrespondenceshouldbeaddressed）E-mail：liuyuqin@public．bta．net．cnTel：（olo）69156473
