Establishment Of Human Immoratalized Endometrial Glandular Cells With Fuctional Characteristics

ObjectiveThe human endometrium is a dynamic tissue, it is important to study endometrial biology .Primary endometrial epithelial cells fall into senescence cultured on plastic dishes, and more complete understanding of endometrial biology has been delayed because of, in part, a lack of an in vitro culture model for endometrial epithelial cells. Our goal was to establish immortalized human endometrial glandular cells induced by HPV16 E6 and E7. Our established cell lines are potentially useful as an experimental model with which to research hormone functions, implantation, and endometrial carcinogenesis.Materials and MethodsMaterials1. SampleHuman endometrial tissue samples were obtained from five patients undergoing hysterectomy as a treatment for uterine myoma. They had regular menstrual cycles and were in late proliferative phase at the time of operation.2.Cell line,plasmid and bacterialPlasmid pcDNA3.1 are offered by Professor Chen and E.coli JM109 are from China Medical University Graduate School. 3. AgentsEndotoxin free ultrapure plasmid DNA purification kit,cell transfection kit , G₄₁₈, mouse anti-CK19 monoclone antibody, mouse anti- HPV16/18 E6 monoclone antibody, mouse anti- HPV16 E7 monoclone antibody, DMSO,fetal calf serum.Methods1 .Establishment and extraction of pcDNA3.1(+)/HPV16E6E7:extracted plasmid according to the direction of cell phect transtection kit.2.Transfection and resistant cell colon screening:EEC were transfected by pcDNA3.1(+)/HPV16E6E7 with cell phect transfection kit.And screened the resistant clone with G₄₁₈.3.RT-PCR detect the expression of HPV16E6/E7 mRNA in cells.4. Western Blot detect the expression of objiective gene in protein level.5.Observation of cell morphology:observed cells with invert microscope.6.Immunocytochemistry:the trial was carried out according to the direction of SABC kit.7.Proliferation experiment-drawn growth curve by MTT and cell counting.8.Analysis of cll cycle by flow cytometry:after PI dying,cells were detected by flow cytometry.9.Soft agar culture:cells were inoculated in double layer agar plate,and two weeks later,counted.Results1 .Extraction of pcDNA3.1(+)/HPV16E6E72.After PCR amplification,agarose gel eletrophoresis detect HPV16 E6,E7 gene fragment about 800bp.3.Western Blot: transformed endometrial glandular epithelium cells express HPV16E6,E7 protein.4.Observation of cell morphology: immortalized cells morphous no obviously change, through contrast phase microscope , the cells adherence tightly,polygon or round,close district to offer mosaicism align.5.Immunocytochemistry: masculine cell focus endochylema are dyied endochylema, nucleus are dyied blue.6.Proliferation experiment: transformed endometrial glandular epithelium cells doubing time about 52.8 hour, after exponential phase of growth,cells arrest accrementition, activity depress, the ability of ingest MTT drop out.7. Analysis of cll cycle by flow cytometry: ransformed endometrial glandular epithelium cells have no apoptotic peak appearance,the proportion of S stage is 33.23%, increase obviously. 8. Soft agar culture: transformed cells have no clone formation.Conclusions1.The primary endometrial glandular cells were transfected with HPV16E6E7 gene,acquired long-term cultures transformed cells over 30rd passage.2.The results strongly suggested that transformed human endometrial glandular cells in long-term culture maintained normal phenotype and growth features on the one hand,and acquired a potentially proliferative ability on the other.However,no tumorigenicity was exhibited,These cells might represent cell populations which were immortalized by HPV16E6E7 gene.