| Objective:1 To study the expression and significance of integrin ?1 and nesprin-2 in the sacral ligament of patients with pelvic organ prolapse(POP)and control.2 The effects of mechanical stress on integrin ?1 and nesprin-2 in primary cultured fibroblasts of the sacral ligament.3 To study its role in the pathogenesis of pop.Methods:1 The subjects: 12 Patients with pelvic organ prolapsed(POP)who underwent surgical treatment in the Obstetric and Gynecological Department of the Second Hospital of Hebei Medical University from March 2016 to September 2016 were recruited in the case group.Over the same period 12 patients without POP who had total hysterectomy for uterine benign diseases were recruited in the control group.There were no endometriosis,diabetes and other diseases of respiration system,digestive system and cardiovascular system in both two groups.All those patients who underwent the surgery had no history of estrogen application within the past 3 months.There was no statistically significant difference in Age,pregnant times,productive times and body mass index between the case group and the control group..2 Specimen: After obtaining the agreement of the Ethics Committee of The Second Hospital of Hebei Medical University,all patients signed informed consent.The specimens of two groups were taken from part of the sacral ligament near the cervix,one fresh tissue was put in the cold box with-20? to the laboratory for tissue protein extraction,the other piece of specimens preserved 4% fixed in paraformaldehyde solution 24 h was used for immunohistochemical staining.take one piece from the sacral ligament near the cervix,and placed in a culture medium containing with anti-penicillinand anti-streptomycin 4 ? cold box and sent to the laboratory for cell culture,laboratory immediately.The whole process is less than 2 hours and required strictly sterile.3 The expressions of integrin?1 and nesprin-2 in pop and non pop sacral ligament tissues were studied qualitatively and quantitatively by immunohistochemistry and Western blot.4 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 immunocytochemical method.5 Experimental methods for mechanically stimulating in the cells:Sacral ligament fibroblasts of 3-5 generations in exponential phase of growth from non-pop were stretched by Flexcercell 4000 tension system for 0,6,12,24 hours.The loading process is in the incubator-C02.Loading parameters: amplitude15%,sine wave,0.5Hz.In the setting time will be removed immediately in the cell culture plate after different times of mechanical stretching.then,observe the morphology of cultured cells,finally,extract fibroblast protein.The change in the expression of Integrin?1 and nesprin-2 before and after stretch was determined by western bolt simultaneously.The collected data are analyzed with SPSS21.0 software.Two-tailed test with P<0.05 was considered significant.Results:1 The results of immunehistochemical staining show that the expression of integrin ?1 are detected in the cell membrane of fibroblast,the expression of nesprin-2 located in the cytoplasm and nucleus.The different expression of integrin?1 and nesprin-2 in non-pop and pop sacral ligament was determined by western bolt simultaneously.The result is that the ratio of reference and integrin beta 1 expression is(2.09 ± 0.12)in non-pop group and(1.29 ± 0.04)in pop group,P<0.05.The result is that the ratio of reference and nesprin-2expression is(1.09 ± 0.06)in non-pop group and(0.49 ± 0.02)in pop group,P<0.05.Compared with the normal group,the expression levels of integrin-1and nesprin-2 in the case group were obviously lower than that of the normal group,and the difference was statistically significant,P<0.05.2 In the fifth day after inoculation,the primary sacral ligament fibroblasts began to appear free nuclei,seventh days to Tenth days began to have the cell from the tissue block edge free,adherent growth,the cell is bigger,the majority fibroblast cell is the shuttle shape or the irregular triangle,the boundary is clear.Aboat fifth days to twenty days or so,when the monolayer cells reach 80%-90%,it is a long spindle shape,radial,braided arrangement.After subculture,the full extension of the sacral ligament fibroblasts showed a flat spindle shaped.3 The expression of vimentin in cultured cells comes from mesenchymal origin cells,keratin expression is in epithelial cells.the inverted microscope.The cultured cells were stained by immunocytochemistry.:The vimentin is positive with brown located in cytoplasm.while the keratin(cytokeratin)is negative.The cells are confirmed for the sacral ligament fibroblasts according to drawn parts.4 Before stretching the morphology of fibroblasts in the sacral ligament is varied,which is long spindle shaped or irregular triangle,and its arrangement is also multi direction,without obvious law.Under the 15% strain,with the extension of stress time,the cells showed a change in the force field,which was longer than the primary cells,and showed a long spindle shape.5 Under the 15% strain stimulous,the primary culture of pelvic ligament fibroblasts of integrin beta 1 expression and internal reference,in Oh,6h,12 h,24h respectively is(1.40 ± 0.02,1.35 ± 0.02,1.64 ± 0.05,3.11 ± 0.04),There was no significant difference between 0h and 6h,P>0.05.0h,12 h,24h compared with each other,the result is the difference was statistically significant,P<0.05.we can kown that with the extension of time the expression of integrin beta 1 up regulation.,which is up to 24 h to do more.under the 15% strain stimulous,the primary culture of pelvic ligament fibroblasts of nesprin-2 expression and internal reference,in Oh,6h,12 h,24hrespectively is(0.40 ± 0.06,0.64 ± 0.02,0.60 ± 0.04,2.45 ± 0.06),There was no significant difference between 6h and 12 h,P>0.05.0h compared with6 h,12h,24 h,the difference was statistically significant,P<0.05.we can kown that 0h as the control,with the extension of time the expression of nesprin-2up regulation.,which is up to 24 h to do more.Conclusions:1 Mchanical force can affect the expression levels of cytoskeletal protein integrin beta 1 and nesprin-2,can change the morphology of the cells and make the fibroblasts of sacral cytoskeleton adapt to the new enviroment,to control cell fate regulation,signal transduction,gene transcription and expression.2 Non pop patients had a more stable cytoskeleton system,integrin beta 1and nesprin-2 expression decreased than non-pop patients,and played an important role in the pathogenesis of pop.3 Cytoskeleton plays an important role in the mechanism of pelvic floor dysfunction. |
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