The Changes Of Cytoskeleton Of Pelvic Floor Function Disorder Patients And Controls Sacral Ligament Fibroblast Cells Before And After Loading

Objective:1To learn the differences in morphology and the levels of proteinexpression of sacral ligament fibroblast cytoskeleton which derive from pelvicfloor dysfunctional(PFD) patients and controls before and after the stress.2To study the relationship of cytoskeleton and pelvic floor dysfunctiondisease on the basis of the change of cytoskeleton before and after the stress.3To explore the role of biomechanics in the diseases pathogenesis ofpelvic floor dysfunction.Methods:1The subjects: Every20cases uterosacral ligament tissue of hospitalizedpatients with pelvic floor dysfunction and benign uterine disease whounderwent hysterectomy surgery in the Gynecological Department of theSecond Hospital of Hebei Medical University from December2011toSeptember2012were recruited in this study. The average age of the PFDgroup patients is57.15±4.59years old, the average time of parity is2.70±1.42,all the PFD patients are in line with uterine prolapse grade Ⅲ-Ⅳin accordance with U.S. pelvic organ prolapse quantitative staging (pelvicorgan prolapse quantitation, POP-Q) scoring criteria; the average age ofcontrol group patients is54.60±3.89years old, the average time of parity is2.60±1.39,the differences on age and production time between the two groupswas not statistically significant. All the recruited patients were confirmed bypostoperative pathology non-endometriosis and non-ovarian tumors whichinfluence the cytoskeleton metabolism. Excluding connective tissue diseasespatients、acute and chronic complications in patients with pelvic inflammatorydisease.Excluding those patients who had the surgery in the uterosacral ligamental site and have a history of estrogen application within the past3months. After obtaining the agreement of the Ethics Committee of The SecondHospital of Hebei Medical University, all patients signed informed consent.2Primary cultured and identification of the fibroblasts: The specimenswere the residual sacral ligament taken from the ablated uterus. The process isaseptic strictly.The organization sent back to the laboratory at4degrees icebox is cultured in the culture bottle with tissue explant.Cells were identifiedwith immunocytochemical staining after culture successfully inorder to beidentified as fibroblasts.3Experimental methods for mechanically stimulating the cells in vitro:Fibroblasts of3～4generations of exponential phase of growth derived fromgroup of15cases were stretched using Flexcercell4000flexible substrateloading system.The loading process is in the incubator-CO2. Experimentalsamples were cultured in a special disposable BioFlex plates which is made offlexible silicone membrane. The Loading procedure is automaticallycontrolled by the Flexcercell4000software.Loading parameters:8%and20%of the amplitude of the sine wave,0.5Hz, loading time:0h,4h,12h,24h.4Confocal laser scanning microscopy and Western Blot experiments:Observe the morphological changes of the cytoskeleton of cells before andafter the stress using the confocal laser scanning microscopy afterimmunofluorescence staining. Total protein was extracted for Western Blotexperiments to assay the protein content of β-actin、vimentin and filamin A.The data were statistically analyzed using SPSS13.0statisticalsoftware.The difference was statistically significant for p <0.05.Results:1There are cells freed from surrounding tissue blocks after cultured in8-10days. The cells covere the whole bottle in about30days. Cells are visiblefor fusiform or polygonal and was arranged for braided or spiral-shaped underthe inverted microscope. The vimentin is positive in immunohistochemicalstaining,while smooth muscle actin (α-SMA) and keratin (cytokeratin) is negative. The cells are confirmed for the sacral ligament fibroblasts accordingto drawn parts.2The cells were irregular polygonal and arranged in different directionsbefore the stress. The microfilament was visible fluorescent strong andScattered in the cytoplasm under the confocal laser scanning microscopy.Cells were gradually stretched to be fusiform and arranged directionconsistently with prolonged load time under strain rate of8%of the force.The microfilament gathered into thicker stress fibers along with thefluorescence brightness enhancement under the confocal laser scanningmicroscopy. It is thus clear that the contours of cells is fuzzy and cellsdisintegrate、 fall off. The cytoskeleton depolymerize、 fracture and thefluorescence is decreased along with stress fibers disintegration thinner underthe confocal laser scanning microscopy.3No significant change compared energizing of the protein content of theβ-actin after loading of8%of the force hours. Protein content of Vimentinshowed decreased and then increased trend compared with afterburner ago.While protein content of filamin A is elevated compared with afterburnerago. The protein content of β-actin and Vimentin in the PFD group is lowerthan that of the control group, however the protein content of filamin A in thePFD group was higher. The protein content of the β-actin、Vimentin andfilamin A is decreased Significantly after loading of20%of the forcecompared with the afterburner before. Moreover the magnitude of PFD groupdecreased is greater than control at the same stress time.Conclusions：1The force of the physiological range make cytoskeleton depolymerizerearrange and change cell morphology and orientation in order to adapt to theforce, while the larger destructive force makes the cytoskeleton fault、disintegrate、apoptosis and fall off.2PFD group whose content of sacral ligament fibroblast cytoskeleton islower compared with the control group is affordability worse on force andeasier to be destroyed than the control group under the same size force. 3The Reaction of cytoskeleton to force is regulated by the related proteinwhich content is changed under the action of the force.4The cytoskeleton plays an important role in the biomechanicsmechanisms of pelvic floor dysfunction disease.