The Preliminary Research On Change In The Cytoskeleton System By Mechanical Stretch In Human Uterosacral Ligament Fibroblasts

Objective:1To study influence on the morphology of cytoskeleton and regulation ofthe expression of β-actin, β-tubulin and vinculin in fibroblasts derived fromhuman uterosacral ligament of POP and non-POP patients after two kinds ofmechanical stretching.2To investigate the relationship between the mechanical stretch and thecytoskeleton remodeling.3To investigate the effects of mechanical stretch on the pelvic organprolapse from the level of cytoskeleton.Methods:1The subjects:15Patients with pelvic organ prolapse who underwentsurgical treatment in the Obstetric and Gynecological Department of theSecond Hospital of Hebei Medical University from October2011toSeptember2012were recruited in the case group. Over the same period15patients without POP who had total hysterectomy for uterine benign diseaseswere recruited in the control group. There were no endometriosis, diabetes andother diseases of respiration system, digestive system and cardiovascularsystem which influencing cytoskeleton metabolism in both two groups. Allthose patients who underwent the surgery had no history of estrogenapplication within the past3months. There was no statistically significantdifference in Age, pregnant times, productive times and body mass indexbetween the case group and the control group.2Specimen: After obtaining the agreement of the Ethics Committee ofThe Second Hospital of Hebei Medical University, all patients signedinformed consent. The specimens of two groups were taken from part of thesacral ligament near the cervix, kept fresh in4℃cold boxes and sent to the laboratory immediately. The whole process is less than2hours and requiredstrictly sterile.3Primary cultured and identification of the fibroblasts: Fibroblasts ofsacral ligament derived from old women were cultured by tissue inoculationmethod. The morphology of cultured cells was observed by inverted phasecontrast microscope, HE staining and Wright-Giemsa staining.The culturedcells were identified with immunocytochemical and immunofluorescentmethod.4Experimental methods for mechanically stimulating in the cells: Sacralligament fibroblasts of3～5generations in exponential phase of growth fromtwo groups were stretched by Flexcercell4000tension system for0,4,12,24hours. The loading process is in the incubator-CO2. Loading parameters:amplitude10%and20%, sine wave,0.5Hz. After removing cell culture platesof the case group and control group in the setting time, the cells wereimmediately operated by immunofluorescent staining.The influence on F-actinin sacral ligament fibroblasts cytoskeleton from two groups was observed byconfocal laser scanning microscopy and immunofluorescence microscopyafter two different kinds of mechanical stretching. The change in theexpression of β-actin, β-tubulin and vinculin before and after stretch wasdetermined by western bolt simultaneously. The collected data are analyzedwith SPSS13.0software. Two-tailed test with P<0.05was consideredsignificant.Results:1The cultured cells showed spindle shape or irregular triangles with clearboundary. The monolayer cells of80%-90%confluence were arranged inradial pattern or swirling pattern after30days. The vimentin was positive inimmunohistochemical staining and immunofluorescent staining, keratin andsmooth muscle actin were negative. Combined with the position of drawingmaterials, the cultured cells were confirmed as fibroblasts derived from humanuterosacral ligament.2In response to mechanical stretch, the morphology and arrangement of the uterosacral ligament fibroblasts were changed. Under10%stretch, themorphology of the fibroblasts was variety from pleomorphism to the singlespindle over time, the direction of the fibroblasts was perpendicular to theorientation of the maximum strain, and the change was the most obvious after24hours’ stretching. However, damage and obvious crimple were observedin some cells under10%stretch, causing lost of the original morphology.3Cytoskeletal filaments marked by FITC-phalloidin showed greenfluorescence, and cell nucleus marked by PI showed red fluorescence underconfocal laser scanning microscopy and immunofluorescence microscopy.Strong fluorescent strength and clear stress fibers were observed before stretchin sacral ligament fibroblasts from two groups. Under10%stretch, There waschange in force field compliance of filaments over time in both groups, but thefluorescent strength was down regulated with the lowest value at4h, but for24hour’ stretch, the fluorescent strength was restored to the level of staticstrength group. Under20%stretch, the fluorescent strength was continuouslydown regulated over time, the damage of microfilament structure was clearlydiscovered after24hour’ stretch, breaking up into smaller clumps sectionally.Moreover, the fluorescent strength with different stretch of cytoskeletonfilaments in control group was generally higher than that in case group at eachstage.4The results of western blot showed that the expression of β-actin andβ-tubulin in sacral ligament fibroblasts cytoskeleton from two groups had noobvious change with10%stretch(P>0.05). However, the expression ofvinculin was up regulated over time(P<0.05). With20%stretch, theexpressions of β-actin, β-tubulin and vinculin were continuously downregulated in sacral ligament fibroblasts cytoskeleton from two groups(P<0.05).Moreover, the expressions of above proteins in cytoskeleton in control groupwere generally higher than that in case group with different stretch at eachstage(P<0.05).Conclusions：1Two different kinds of mechanical force can change morphology and orientation of the fibroblasts and have an adaptive or destructive effect on themorphology of cytoskeleton. The expressions of β-actin, β-tubulin andvinculin in the cytoskeleton are also influenced.2Cytoskeleton remodeling under mechanical force plays an importantrole in the occurrence and development of POP. Patients without POP havemore stable cytoskeleton system than the ones with POP.3Mechanical force plays an important role in the pathogenic mechanismof POP.