Study The Mechanism Of Mechanical Force In The Pathogenesis Of Pelvic Organ Prolapse

Objective:1 To Research the expression of CollagenI and Wnt/β-catenin signaling pathway in ligament tissue of patients with pelvic organ prolapse.2 To study the influence on Wnt/β-catenin signaling pathway and secreting Collagen I ability after mechanical force stretching the original generation of sacral ligament fibroblasts.3 To study the influence on secreting MMP-1 ability after mechanical force stretching the original generation of sacral ligament fibroblasts.4 From the level of signaling pathways study mechanical force role in the pathogenesis of pelvic organ prolapse.Methods:1 The subjectsTo select patients with pelvic organ proplase who underwent surgical treatment in the Obstetric and Gynecological Department of June 2016 to September 2016 as the case group.Over the same period,patients without POP who had total hysterectomy for uterine benign diseases were recruited in the control group.The two groups of patients were no Endometrium Endometriosis,chronic pulmonary emphyse ma,associated with connective tissue diseases such as rheumatoid arthritis disease.There was no statistically significant difference in Age,pregnant times,productive times,body mass index.2 SpecimenAfter obtaining the agreement of the Ethics Committee of The Second Hospital of Hebei Medical University,all patients signed informed consent.The specimens were taken from the sacral ligament tissue near the cervix by a deputy director or above physicians under laparoscopy.Which is part of thenon-POP patients’ sacral ligament tissue placed into serum-free DMEM high glucose sterile medium,sent by 4°C cold boxes to the laboratory immediately,performed primary cell culture,The entire drawing and transportation process is less than 1 hour and requires strict sterile;One part were placed in the 4%formalin fixative,for embedding,slicing,and another part were placed in the specimen bag stored at-80 ° C for the extraction of tissue proteins.A portion of POP patients’ sacral ligament tissue were placed in the 4% formalin fixative for embedding,slicing,and the other placed in the specimen bag stored at-80 ° C for the extraction of tissue proteins.3 ImmunohistochemistryThe sections were immunohistochemically stained with SP three steps,to observe the expression of CollagenI,Wnt16 and β-catenin in case and control groups.4 Using Western blot method to detect the expression of CollagenI,Wnt16 and β-catenin in the sacral ligament of POP and non-POP groups.5 Primary cultured and identification of the fibroblasts:Fibroblasts of Sacral ligament derived from old women were cultured by tissue inoculation method.The morphology of cultured cells was observed by inverted phase contrast microscope.The cultured cells were identified with immuno cytochemical method.6 Experimental method for mechanically stimulating in the cells:Sacral ligament fibroblasts of 3-5 generations in exponential phase of growth were stretched by Flexcercell4000 tension system for 0,6,12 hours.Loading parameters:amplitude 15%,sine wave,0.5Hz.The loading process is in the incubator-CO2.The morphological changes of fibroblasts after mechanical stimulating were observed under a laser confocal microscope.At the same time,the change in the expression of Wnt16,β-catenin and CollagenI before and after stretching were determined by Western blot simultaneously.The collected data are analyzed with 21.0 software.Two-tailed test with P<0.05 was considered significant.7 Elisa assayThe change on the secretion of MMP-1 unstretched and stretched fibroblasts were determined by Elisa assay.Results:1 The immunohistochemical staining showed that the expression of CollagenI,Wnt16,β-catenin in the patients’ sacral ligament tissue was significantly lower than that of the control group.2 The result of Western blot showed that the expression of CollagenI,Wnt16,β-catenin in the case group was lower than that in the control group,and the difference was statistically.3 On the 5th day of the primary culture,a small number of cells can be observed around the tissue mass,and the cells’ morphology were different,but mainly were spindle cells;On the 10 th day,the cells around the tissue block were significantly increased,radially arounding the tissue mass,we can visible triangular and multi-angle cells,but mainly were spindle cells;On the 20 th day,the cells were more and more dense than before,and the vast majority were spindle cells,but they have no obvious direction.The vimentin was positive in immunocytochemical staining,keratin and smooth muscle actin were negative,suggesting that fibroblasts.4 In response to mechanical stretch,the morphology and arrangement of the uterosacral ligament fibroblasts were changed.Before stretching,the cells were polygonal and spindle-shaped,and have no obvious direction.After stretching,cells elongated,long spindle shape,the same direction of gowth,prpendicular to the direction of maximum stress,and the gap of intercellular were increased.5 The Western blot results showed that the expression of Wnt16,β-catenin,CollagenI was significantly reduced after 6 hours of mechanical stimulating,and compared with 0 hour,the difference was statistically;When the mechanical force was loaded for 12 hours,the expression levels of the three were increased,but with 0 hour is still showing a downward trend,and the difference is statistically.The rise and fall of the three were strongly correlated.6 Elisa assay results showed that after mechanical loading of fibroblasts6、12 hours,the amount of MMP-1 secreted by fibroblasts was significantly increased,compared with the unattributed group,the difference is statistically.The rate of MMP-1 secretion of fibroblasts after mechanical loading was significantly accelerated.Conclusions:1 The inhibition of Wnt / β-catenin signaling pathway may be involved in the development and progression of POP.2 A certain degree of mechanical force can inhibite the Wnt / β-catenin signaling pathway,the degree of inhibition of the mechanical force on the Wnt/ β-catenin signaling pathway may be related to the time of mechanical force.3 The Wnt/β-catenin signaling pathway can regulate the ability of fibroblasts to secrete of CollagenI.4 A certain degree of mechanical force can promote the secretion of MMP-1 by fibroblasts.