Effects Of17β-estradiol On The Changes Of Fibroblasts Derived From Vaginal Wall In Patients With POP Under Mechanical Stretch

Objective: To investigate the effects of17β-estradiol on the changes ofgene expressions of Collagen type Ⅰ, Collagen type Ⅲ,matrixmetalloproteinase-1and tissue inhibitor of metalloproteinase-1in the vaginalwall fibroblasts in vitro derived from patients with POP exposed tomechanical stretch, and the effects of17β-estradiol on the biochemicalchanges of fibroblasts, and discuss the therapeutic role of estrogenreplacement therapy (ERT) in POP from the cellular perspective.Methods:1. During2009.10-2010.5, twelve patients with POP who underwent surgicaltherapy in the Obstetrics and Gynecology Department of the SecondHospital of Hebei Medical University were recruited in our study. Vaginalwall tissue cut during operations were taken as specimens. All enrolledpatients were postmenopausal women, their average age was（58.3±9.6）years old, the average parity was（2.9±1.2），and theaverage body mass index was (23.78±1.90) kg/m2. According to the PelvicOrgan Prolapse Quantitation (POP-Q), all patients were scored and at leastone part was rated as Ⅲ-Ⅳ degrees. All recruitedpatients had not takensurgical operations and estrogen drugs within the past three months, andwere not suffered with stress urinary incontinence (SUI) and diseasesrelated to collagen metabolism such as rheumatoid arthritis, pulmonaryemphysema and hyperthyroidism et al. Specimens were selected for thepatients’ consent in advance and informed consent forms were signed.2. In vitro culture of fibroblasts: Primary culture of vaginal wall fibroblastswas cultivated by tissue mass culture methods. Fibroblasts wereidentificated by immunocytochemical methods, and then cell growth curve was drawn to understand the cell growth characteristics.3-5generations offibroblasts in logarithmic growth phase were selected for our study.3. Loading mechanical stretch and estrogen intervention: Fibroblasts wereinoculated in the BioFlex culture plates of the Flexcell4000flexiblesubstrates stretched system, and loaded on mechanical stretch with10%elongation, at frequency0.3Hz for12hours, with static culture of cells ascontrol group; after unloaded, cells were treated with17β-estradiol freshlyprepared with ethanol as solvent for48hours, whose final concentrationwas10-8mol/L.Untreated cells were used as blank controls, and cellstreated with the same solvent content were used as solvent controls.4. Reverse transcription polymerase chain reaction (RT-PCR): After unloadedmechanical stretch and the intervention of estrogen, total RNA in the cellswere extracted separately, transcripted reversely to cDNA, andamplificated. The target gene expressions were detected by electrophoresis.All the result data were described as mean±standard deviation. SPSS13.0software was used for statistical analysis. If p﹤0.05, it is believed thatthere is statistically significant.Results:1. Morphology of the cells derived from vaginal wall is long, spindle-shapedand fibroblast-like. The cells grow adhering to the bottom of culture flasksand their boundaries are clear. With the help of immunohistochemicalstaining, the cell are identified as fibroblasts, in which vimentin ispositively expressed, while keratin and smooth muscle actin are negativelyexpressed. In the first to forth days, fibroblasts grow fairly slowly,equivalent to the incubation phase; In the forth to seventh days, cellproliferation becomes active, equivalent to the logarithmic phase; In theseventh to eighth days, cell proliferation tends to smooth, close to thestationary phase.2. Without the mechanical stretch, morphology of fibroblasts is spindle-shaped or polygonal; cells’ boundaries and one of multiple nucleoli areclearly visible. Arrangement of cells is multiple and irregular; Loaded the stretch, morphology of cells become more longer and narrower,referred to as long spindle-shaped, and align of cells is prone to regular,mainly perpendicular to the applied strain field at the periphery of theculture well.3. Compared with the stationary controls, the mRNA expressions ofColⅠand MMP-1increased, while those of Col Ⅲ and TIMP-1decreased;In contrast to the solvent controls,17β-estradiol down-regulated themRNA expressions of MMP-1, meanwhile up-regulated that of ColⅠ、Col Ⅲ、TIMP-1. The differences were statistically significant (P<0.05).Conclusion:1. Mechanical stretch makes the vaginal wall fibroblasts to realign in order toadapt to the mechanical environment, and lead to changes of extracellularmatrix components. So the broken original mechanical equilibrium may beone of mechanisms of POP.2. To some extent17β-estradiol could reverse the biological changes offibroblasts after sustainable mechanical stretching stimulus.