Isolation And Culture Of Human Endometrial Glandular Epithelial Cells By Differential Centrifugation And OPN/MMP-9Expression And Significance In The Cells

Part OneThe evaluation of endometrial epithelial cells useing the hydrolysis atroom temperature combined with differential centrifugationObjectives Explore the feasibility of room temperature enzymolysis combinedwith improved differential centrifugation on the isolation and culture of humanendometrial glandular epithelial cell and provide the basis for further explorationchoose endometrial drug intervention timing.Methods1.We collected endometrium specimens from patients with endometriosis.?Eachsample was divided in two for cell isolation via room temperature enzymolysiscombined with improved differential centrifugation (improved group) or37?water bath digestion combined with mesh method (control group)?We thencompared the number,and purity of the isolated and cultured using two methodendometrial glandular epithelial cell by immunocytochemical staining and lightmicroscopy.2.Changes in cell morphology was observed by light microscopy? OD valuesmeasured at different points in time cell by MTT assay and drawing growth curve.Results1.The cell isolation and culture was successful for all specimens. We obtained (9±1.7) ×107gland epithelial cells per1g endometrial tissuein the improved group, ascompared with (3.9±0.78)×107cells in the control group. The immunohistochemistryrevealed that the expression of cytokeratin was positive for both groups and thepurity for the improved and control group was respectively (97.8±0.002)%and (88.6±0.006)%, which had statistically significant difference (P <0.05).2. Exterior features of epithelial cells are similar but cell growth cycle has asignificant difference in light microscopy of two groups?Modified cells began todecline at15days, while the control cells began to decline in8-10days.3. MTT assay showed that the cells in the OD450value of the two different points intime increases with time in the3~7d increases. Cultured cells were seeded in twogroups adherent completely in the third day, and OD3values between the twogroups was no significant difference?OD450of improved group was significantlyhigher OD450of the control group from the sixth day (P <0.01), The growth rate andthe activity of the epithelial cells which isolated by the improvement group wassignificantly higher than the control group(P <0.01), there was a significant differencein differences.Conclusion1.2. OPN/MMP9epithelial cells isolated and cultured in the first6,7,8,9days werehighly expressed than isolated and cultured3,12days, is further confirmed epithelialcells in primary culture activity optimum time is7-8day, the best time to carry outdrug intervention. Part Two The expression and significiance of OPN/MMP9in endometrialepithelial cellsObjectivesAnalysis of differences between OPN and MMP9epithelial cells in the mRNA inadenomyosis eutopic and normal endometrium, Discussion of OPN and MMP9expression in ectopic endometrium features between OPN expression and MMP9inadenomyosis.Methods?1.Select the10patients with uterine adenomyosis as the experimental group,6patients with uterine fibroids as a control group, through in vitro culture ofendometrial epithelial cells using real-time quantitative reverse transcription-polymerase chain reaction (Real-time RT-PCR) analysis OPN/MMP-9mRNA in theexperimental group and the control group endometrial glandular epithelial cellsexpress.2.Real-time quantitative reverse transcription poly-merase chainreaction (Real-time RT-PCR) were used to measure the levels of OPN/MMP9mRNAof AMT eutopic endometrial epithelial cells at different timesResults1.OPNmRNA expression in cultured endometrial epithelial cells: epithelial cells in theexperimental group than in the control group OPNmRNA expression increasedsignificantly ((P <0.05)); while the same group proliferating epithelial cellsOPNmRNA of expression and secretion compared with the menstrual cycle hasnothing to do, the difference was not statistically significant (P>0.05)2. Expression of MMP-9mRNA in cultured endometrial epithelial cells: epithelial cells in theexperimental group than in the control group OPNmRNA expression increased significantly ((P<0.05)); while the same group of glandular proliferation compared significantly increasedsecretory epithelial expression of MMP-9mRNA secretory phase, the difference was statisticallysignificant ((P <0.05)). 3.OPN/MMP-9mRNA differentially expressed in normal endometrial glandularepithelial cells and endometrial epithelial expression of the same reign:OPN/MMP-9mRNA expression in the normal menstrual cycle, the endometriumdifferent isolated and cultured epithelial cells: OPN/MMP9mRNA separated innormal human endometrial epithelial cells in different menstrual cycle low expressionin the proliferative phase, while in the menstrual cycle secretory phase was highlyexpressed, there was a significant difference (P <0.05) differences; OPN/MMP9mRNA in adenomyosis isolated and cultured endometrial epithelial cellscompared with normal endometrial epithelial cell proliferation resulting separationperiod, significant differences in the secretion of both (P <0.05)Conclusion?1. OPN in adenomyosis endometrial epithelial cells highly expressed, but nothing todo with the separation of the different endometrial menstrual cycle, no menstrualcycle, MMP-9in the eutopic endometrium of adenomyosis high expression ofepithelial secretory cells.2. OPN/MMP9epithelial cells isolated and cultured in the first7,8,9days were highlyexpressed than the3.12days, is further confirmed epithelial cells in primary cultureactivity optimum time is the7-8day, the best time to carry out drug intervention.