The Study Of Inhibition Of Notchl On Stem-like Cells Obtained By Endometrial Biopsy

Objective:1. The aim of this study was to search for the presence of cells with plastic adherence, clonogenicity, and differentiation in human endometrium.2. Construct lentivirus-Notch1-shRNA particles, and high titer lentivirus particles were determined and collected.3. The effectiveness and security of lentivirus-Notch1-shRN A to inhibit the development and growth of stem-like cells were studied.Methods:1. The exploration of the isolation and culture of eutopic endometrial glandular epithelial cell and its stromal cell as an in vitro model of endometriosis. Clonogenicity of the endometrial stem-like cells. The gene levels of epithelial markers (EMA, CK, CD49f) and stromal markers(THY-1(CD90),collagen type I,5B5, vimentin) were detected by real-time quantitative PCR and western blot.2. Construct lentivirus-Notch1-shRNA particles. High titer lentivirus particles wer determined and collected.3. Study the effect of lentivirus-Notch1-shRNA particles on the expression of Notchl mRNA and protein and its influence on the change of stem cell makers in stem-like cells.The cellular morphology was observed by optical microscope.Endometrial stem-like cells were identified by IHC. LV-Notch1-shRNA particles was transfected into stem-like cells,the empty vector as control.The Notchl mRNA and protein expression were detected by real time PCR and western blot.. The gene levels of BMI1, c-Myc,CD133,Oct4,Sox2 and Nanog in the stem-like cells, were detected by real-time quantitative PCR. The cell migration and invasion function was evaluated.Results:1. The strongest positive immunostaining of Nothcl was observed in stem-like cells.Notch 1 protein were significantly downregulated by LV-Notchl-shRNA than control group(P<0.05).2. The protein of Notchl is high expression in endometrial epithelial stem-like cells than control group(P< 0.05). The protein of Notch1 is high expression in endometrial stromal stem-like cells than control group (P< 0.05). After treated with LV-Notchl-shRNA,the expression of Nothcl were inhibited by 70% than control group(p<0.05).3. After transfected with LV-Notch1-shRNA for 72h,the expression of stem cell makers (BMI1, c-Myc, Sox2, Oct4, Nanog,CD133) were decreased with different degrees (P<0.05).4. After transfected with LV-Notch1-shRNA for 72h,the cell clone-forming rat were decreased with different degrees in in endometrial epithelial stem-like cells and endometrial stromal stem-like cells (P<0.05).5. After transfected with LV-Notch1-shRNA for 72h,the migration rate of endometrial epithelial stem-like cells and endometrial stromal stem-like cells were significant decreased,24h migration rate 48% vs 75%(P<0.05),48% vs 78%(P<0.01), respectively.6. The cell invasion function were evaluated by transwell assay,showed the invasion of endometrial epithelial stem-like cells and endometrial stromal stem-like cells were significant decreased,24h after the train crossed the transwell cell per low magnification field of vision respectively 33.37±3.3,46±5.4,difference was significant(P<0.05).Conclusion:1. Endometrial cell clones derived from in vitro cultured purified stromal cells obtained by endometrial biopsy display characteristic stem cell features, including clonality; long-term culturing properties; multilineage differentiation potential; expression of EMA,CK,CD49f,THY-1,collagen typeⅠ,5B5,vimentin. We conclude that adult stem cells are present in endometrial biopsies.2. The LV-Notch1-shRNA have been constructe successfully.The recombinant lentiviral vector is safe,effective and convenient,and can be packaged high-titer,high-purity lenti virus.1. LV-Notch1-shRNA can infect endometrial stem like cells effectively in vitro and can inhibit the expression of Notchl gene, which resulted in suppressing the cloning efficiency, migration rate and invasion function.