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
冯海凉 王春景 顾蓓 杨振丽 刘玉琴
解剖学报 ›› 2013, Vol. 44 ›› Issue (2) : 199-203.
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
Establishment of a simian virus 40 T antigen transfected human umbilical vein endothelial cell line PUMC-HUVEC-T1
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.
人脐静脉内皮 / SV40 / 反转录-聚合酶链反应 / 免疫组织化学 / 透射电镜
Human umbilieal vein endothelial cell / SV40 / RT-PCR / Immunohistochemistry / Transmission electron microscopy
[1] Hayflick L. The limited in vitro lifetime of human diploid cell strains[J]. Exp Cell Res, 1965, 37:614-636.
国家实验细胞资源共享平台;其他(不属于以上基金类别的请自行输入下框)
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