| Objective:Pelvic organ prolapse?POP?is a group of diseases,which is caused by various reasons,such as degeneration,trauma,leading to the descending of pelvic organs and its surrounding tissues.POP often occurs in elderly women,although it rarely leads to serious illness or death,but it has a great impact on patients‘quality of life.The supporting system of pelvic floor is mainly composed of fascia and ligament composed of connective tissue.Collagen?s the main component which plays a supporting role.Collagen?s mainly synthesized and degraded by fibroblasts.If the function or quantity of fibroblasts is reduced,the content of collagen will be reduced,resulting in the reduction of pelvic floor support,and POP happens.Homobox?Hox?gene is a group of highly conserved gene sequences in mammalian organisms.The proteins encoded by Hox play a role in the regulation of biological bodies by activating or inhibiting a series of downstream genes.HOXA cluster genes are mainly HOXA9,10,11 and 13,which ensure the normal development of female reproductive organs.Animal experiments show that these four genes have spatial specificity in spatial distribution,which are respectively distributed in fallopian tube,uterine body,cervix and upper vagina,upper vagina.This specificity makes the uterus and vagina develop according to their normal morphology.Some studies have shown that there is no difference in uterine histology between HOXA11 knockout mice and wild-type mice in the early stage of development,but in the later stage of development,the stromal cells of mutant mice decrease and the uterosacral ligament disappears.The uterosacral ligament is an important structure to maintain the function of pelvic floor supporting tissue.However,it is still unclear what role HOXA11plays in the occurrence and development of POP and how it works.Therefore,in order to further study the relationship between HOXA11 and POP,the following experiments were designed and completed.Methods:30 cases of vaginal wall tissue of POP group and control group were collected respectively.The expressions of HOXA11,Collagen?,Collagen?,MMP2 and TIMP2in the subepithelial tissues of the vaginal wall of the two groups were detected by Western blot,immunohistochemistry,Masson staining,H&E staining and RT-PCR.The number of fibroblasts in the subepithelial layer of vaginal wall was counted.3 cases of vaginal wall tissue of POP group and control group were collected respectively.The primary culture and purification of fibroblasts were carried out by the adherence method and the differential adherence method.The fibroblasts were identified by cytochemistry,the proliferation of the two groups of cells was detected by MTT method,and the expression of HOXA11 and collagen metabolism related proteins were detected by WB method.Cell transfection technology was used to knockdown and overexpression of HOXA11,collagen metabolism related proteins were detected by WB method,cell proliferation was detected by MTT method,cell apoptosis was detected by flow cytometry.Immunohistochemistry was used to detect the expression of?-catenin in Wnt/?-catenin signaling pathway.After knockdown and overexpression of HOXA11,the changes of key proteins in Wnt/?-catenin signaling pathway,?-catenin,p-?-catenin and p-gsk3?were observed.After adding the Wnt/?-Catenin signal pathway activator lithium chloride and inhibitor XAV-939,the changes of the above proteins and cell proliferation and apoptosis were observed by WB,MTT,and flow cytometry.The interaction of HOXA11with?-Catenin,Tcf4,AXIN1 and GSK3?was detected by immunocoprecipitation.Immunofluorescence and nucleoplasm separation were used to detect the effect of HOXA11 knockdown and overexpression on the nuclear transfer of?-catenin.Different concentrations of 17?-estradiol were added to the two groups of fibroblasts,and MTT was used to detect the proliferation of fibroblasts.After treating the two groups of fibroblasts 48 hours gradient concentration of 17?–estradiol and treating fibroblasts for gradient time with 10-9mM 17?–estradiol,the changes of HOXA11 and collagen metabolism related protein expression in two groups were observed.Finally,17?-estradiol was added and HOXA11 was knocked down at the same time to detect the expression of collagen metabolism related proteins.Results:The expression of HOXA11 in vaginal wall of POP group was significantly lower than that of control group.The expression of Collagen?and Collagen?in the subepithelial tissue of vaginal wall in POP group was low.In the POP group,the fibrils were broken discontinuously,disordered and loose,while in the control group,the collagen fibers were arranged closely and orderly.In pop group,the number of fibroblasts in the subepithelial tissue of vaginal wall was less.The expression of HOXA11,Collagen?and TIMP2 in fibroblasts of POP group was lower than that in the control group,while the expression of MMP2 was higher in POP group than that in the control group.The proliferation rate of fibroblasts of POP group was significantly lower than that of the control group.After knockdown of HOXA11,the proliferation rate and the expression of Collagen?decreased,while the apoptosis of fibroblasts increased.After overexpression of HOXA11,the proliferation and expression of Collagen?increased,while apoptosis of fibroblasts decreased.The expression of?-catenin in the vaginal wall of POP group was lower than that of the control group.After knocking down HOXA11,the expression of?-catenin and p-GSK3?decreased,while the expression of p-?-catenin increased.After adding Wnt/?-catenin signal pathway activator,the expression of?-catenin and Collagen?increased.After overexpression of HOXA11 and addition of Wnt/?-Catenin signal pathway inhibitor,the expression of these proteins was reversed.Immunocoprecipitation showed that HOXA11 interacted with?-catenin and TCF4,but not with AXIN1 and GSK3?.The results of nucleoplasm separation and immunofluorescence showed that?-catenin nuclear transfer decreased after knockdown of HOXA11,but increased after overexpression of HOXA11.When 17?-estradiol was added,the proliferation rate of fibroblasts decreased.With the increase of 17?-estradiol concentration,the proliferation rate decreased gradually,but the cell proliferation rate of POP group was significantly lower than that of the control group.Different concentrations of 17?-estradiol had an effect on the protein expression of the two components.In the control group,HOXA11 and Collagen?secreted the most at the concentration of 10-9mM,while in the POP group,HOXA11 and Collagen?secreted the most at the concentration of 10-8mM.Different treatment time had different effects on the protein expression of the two groups of fibrolasts.The protein expression of POP group and control group was the highest at 48 hours and 24 hours respectively.After17?-estradiol was added as well as knocking down of HOXA11,Collagen?expression decreased.Conclusions:The down-regulation of HOXA11,Collagen?,Collagen?,TIMP2and the up-regulation of MMP2 are closely related to the occurrence and development of POP.The decrease of the number of fibroblasts in the subepithelial tissue of vaginal wall may be related to the occurrence of POP.Decrease of collagen synthesis and increase of collagen decomposition may be the pathophysiological basis of POP.HOXA11 can promote human vaginal fibroblast proliferation,reduce apoptosis and increase Collagen?expression through Wnt/?-catenin signaling pathway.HOXA11can promote the nuclear transfer of?-catenin by interacting with?-catenin,so as to regulate the proliferation and Collagen?synthesis of fibroblasts.However,the sensitivity of the POP fibroblasts to 17?-estradiol was significantly lower than that of the control group.This may be one of the reasons why pop is more common in elderly women.It provides a theoretical basis for explaining the mechanism of POP and hormone replacement therapy of POP. |
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