| Objective:1To explore the role of mechanical force in pelvic floor dysfunctiondisease.2To investigate the influence on fibroblasts cell morphology andcytoskeleton of mechanical force in human sacral ligament.3To probe the contribution of the functional changes of cytoskeletonmorphology to the pathogenesis of pelvic floor dysfunction.Methods:1Object: To select20patients with Non POP (except for malignanttumor) who underwent hysterectomy surgery in the Obstetric andGynecological Department of the Second Hospital of Hebei MedicalUniversity from October2011to September2012, mean age49.8±2.89,parity2.56±1.07, BMI25.01±2.89kg/m2.There were no connective tissuedisease of all subjects, postoperative pathology confirmed non ovarianendocrine tumor, non endometriosis estrogen-related diseases, patients withacute and chronic pelvic inflammatory disease and sequelae of pelvicinflammatory disease were all removed, and also get rid of the patients withThe site operation history and recent history of estrogen. All patients signedinformed consent.2Materials: All patients signed an informed consent of the SecondHospital of Hebei Medical Ethics Committee. The specimen was the part ofuterosacral ligament near cervix, extrafascial hysterectomy collected about5mm of the sacral ligament,4℃ice box sent to the laboratory, the transportprocess was less than2hours and required strict aseptic.3Primary culture and identification of fibroblasts: Using tissue blockmethod and collagenase digestion method to primary culture the fibroblasts of uterosacral ligament, inverted phase contrast microscope for cellmorphological observation, take notes of the growth curve and cells wereidentified by immunocytochemical staining.4Study of cell mechanics: Using Flexcercell4000tension system,fibroblasts of3～5generations of exponential phase of growth derived from20cases were stretched for0,4,12,24hours, using0hour as the controlgroup. The loading process was in the incubator-CO2. Experimental sampleswere cultured in the special disposable BioFlex plates which were made offlexible silicone membrane that can be deformed and it was covered bycollagen I. The Loading procedure was automatically controlled by theFlexcercell4000software. Loading parameters: amplitude12%and20%,sine wave,0.5Hz. Total protein was extracted from the fibroblastsimmediately after unloading. Laser scanning confocal microscope forfibroblast cytoskeletal morphological observation before and after loading.Western blot immunoblotting for fibroblast cytoskeletal protein expressionbefore and after loading.Results：1Under the light microscope, the fibroblasts were mainly composed ofspindle, irregular triangle or polygonal shapes, and connected to each otherinto a network. The vimentin is90.4%positive in immunohistochemicalstaining, smooth muscle actin (α-SMA) is about8.3%positive and keratin(cytokeratin) is negative.2Under stress, there were changes in cell morphology and extensiondirection. The morphology of the fibroblasts was variety from spindle(mainly) to the irregular triangles, polygons and other shapes beforestretching. The cells connected to each other into a network, stretched in alldirections.12%mechanical force4hours, cells and cell processes are pulledperpendicular to the maximum stress direction.20%mechanical force4hours, the cells began to fall and collapse, with the extension of the time ofthe force, cells gradually lose the adherent state, shedding significantly todeath. 3The results of confocal laser scanning microscope staining: Cellcytoplasmic filaments showed green fluorescence, actin fiber diameter anduniform distribution,12%mechanical force, the shape begin irregular andperpendicular to the maximum stress direction, actin fiber is thin and unevendistribution, actin fluorescent staining intensity decreased with time ofloading. After12hours loading, actin staining intensity gradually restored tothe morphology before loading, actin staining intensity of cells from normalto decreased to normal.20%mechanical force, Cytoplasmic actindepolymerize and disappear, actin fluorescent staining intensity decreasedwith time of loading. After12hours loading, the intensity of staining did notreturn to the morphology before loading, but gradually weaken or evendisappear.4Western-Blot results: The loading range12%, expression of actinprotein in4h(0.6128±0.0002),12h(0.6128±0.0002),24h(0.6129±0.0002)have no significant change with0h(0.6130±0.0001), no statisticallysignificant differences between groups, P>0.01; Expression of α-tubulinprotein content4h(0.5709±0.0002),12h(0.5710±0.0002),24h(0.5712±0.0002) have no significant change with0h(0.5712±0.0001), nostatistically significant differences between groups, P>0.01; Expression ofβ-tubulin protein content4h(0.5709±0.0001),12h(0.5711±0.0001),24h(0.5711±0.0001) have no significant change with0h(0.5712±0.0004), nostatistically significant differences between groups, P>0.01; Expression ofvimentin protein in4h(0.5880±0.0002) was lower than0h(0.8394±0.0002),there was significant difference between the group, P=0.000<0.01, theexpression level of12h(0.5995±0.0001)was higher than4h, there wassignificant difference between the groups, P=0.000<0.01, the expressionlevel of24h(0.7436±0.0001) higher than12h, there was significantdifference between the group, P=0.000<0.01. The loading range20%,expression of actin protein in4h(0.5375±0.0001),12h(0.3659±0.0002),24h(0.2716±0.0000) have significant changes with0h(0.6131±0.0001),statistically significant differences between groups, P=0.000<0.01; Expression of α-tubulin protein content4h(0.5087±0.0003),12h(0.3903±0.0002),24h(0.1943±0.0002) have significant changes with0h(0.5712±0.0001), statistically significant differences between groups,P=0.000<0.01; Expression of β-tubulin protein content4h(0.5057±0.0001),12h(0.3901±0.0001),24h(0.1886±0.0003)have significant changes with0h(0.5712±0.0004), statistically significant differences between groups,P=0.000<0.01; Expression of vimentin protein content4h(0.4616±0.0003),12h(0.3120±0.0002),24h(0.2716±0.0001)have significant changes with0h(0.8394±0.0002), statistically significant differences between groups,P=0.000<0.01.Conclusions：1A certain range of mechanical force can change the morphology ofthe cells and the stretch direction, lead to rearrangement of the cytoskeleton,and the mechanical signals can be transformed by the second messenger,regulate gene expression and protein content.2The cytoskeleton plays a supporting role mainly within cells,cytoskeleton morphology, structure and change of protein content can leadto some degree of changes in morphological and biological cell function.3Connection between the cytoskeleton, integrins and extracellularmatrix is the main pathway of cell signal transduction, morphological andfunctional alterations of the cytoskeleton can be directly affect signaltransduction, eventually lead to the change of cell biology function.4Mechanical forces play a role in the pathogenesis of pelvic floordysfunction. |
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