Abstract:

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.

Key words: Human umbilieal vein endothelial cell , SV40 , RT-PCR , Immunohistochemistry , Transmission electron microscopy