Study The Mechanism Of Notch Signaling Pathway In The Pathogenesis Of Pelvic Organ Prolapse

Objective:1. To research the expression of collagen ?,collagen ?,notch 1 and hes1in sacral ligament tissues of patients with pelvic organ prolapse and non prolapse.2.To study the effect of Notch signaling pathway activation and inhibition on the secretion of collagen ? and collagen ? in primary cultured sacrum ligament fibroblasts.3.To discuss the influences of different levels of electrical stimulation on the secretion of collagen ? and collagen ? by sacrum ligament fibroblasts and on Notch signaling pathway.4.To investigate the effect of estrogen on the secretion of collagen ? and collagen ? by sacrum ligament fibroblasts and the relationship between estrogen and Notch signaling pathway.Methods:1.Sample collectionThe sacrum ligaments of the patients who underwent hysterectomy and sacral vagina fixation due to pelvic organ prolapse were taken as pop group,and the sacrum ligaments of the non pop patients who underwent hysterectomy due to benign uterine lesions were taken as non pop group,20cases in each group;the pop group was divided into two parts:one was fixed with 4%formaldehyde at room temperature;the other was kept at-80?.The non pop group was divided into three parts:the first two parts were the same as pop group,and the third part was in the aseptic high glucose DMEM medium without serum.2. The expression of collagen ?,collagen ?,notch 1 and nes1 in sacrum ligament fibroblasts of pop group and non pop group were analyzedqualitatively and quantitatively by Imunohistochemistry and Western blot.3. Primary culture and identification of fibroblastsThe primary culture of fibroblasts in sacrum ligament of non pop group was carried out by tissue block method.The cell morphology was observed by inverted phase contrast microscope,and the cell was identified by Immunocytochemistry.All of the cells were fibroblasts from the third to eighth generation of human uterosacral ligament.4. Activation and inhibition of Notch signaling pathwayThe fibroblasts were stimulated with Jagged 1 and DAPT at different concentrations and times.The expressions of collagen ?,collagen ?,Notch 1and Hes1 were detected by Western blot.5. Electrical stimulation of cellsThe fibroblasts were stimulated by Feel Tech function/Army waveform generator and 213 platinum electrode.The parameters of electrical stimulation:frequency?0Hz,10Hz,100Hz,1000Hz,10000Hz?,Voltage 5V,pulse duty cycle:50%,waveform:sine wave,stimulation time:2h.After the stimulation,the expression of collagen ?,collagen ?,notch 1 and hes1 in the cells were detected by Immunocytochemistry and Western blot respectively.6. Estrogen testThe fibroblasts were divided into five groups:C?control?,D?DAPTcollagen ?,notch 1 and hes1 were detected by Western blot after 24 hours of stimulation.7. SPSS 21.0 was used to analyze the data,P<0.05 was considered statistically significant.Results:1.Results of tissue proteins in pop group and non pop groupThe results of immunohistochemistry and Western blot showed that the expression of collagen ?,collagen ?,notch 1 and hes1 in sacrum ligament of pop group was significantly lower than that of non pop group,and the difference was statistically significant.2.Primary culture and identification results of fibroblasts1?On the 5th day of primary culture,scattered cells could be seen around the tissue block with different shapes;on the 10th day,the number of cells around the tissue block were significantly and the shape was irregular,radiating around the tissue block,mainly fusiform cells,with a few triangular and polygonal cells;on the 20th day,the cells around the tissue block were densely arranged,like a fish group or a vortex.The shape is regular,mainly fusiform cells,without obvious arrangement direction.2)Immunocytochemical staining showed that vimentin was positive,cytokeratin and smooth muscle actin were negative.The combined site indicated that the fibroblasts were human uterosacral ligament fibroblasts.3.Experimental results of activation and inhibition of Notch signal pathway.1)In the activated group,with the increase of Jagged1 concentration and time,the expression of hes1 and notch 1 increased,and the expression of Hes1increased more significantly;the expression of collagen ? increased first and then decreased whether in the concentration gradient or time gradient group;the expression of collagen ? increased first and then decreased in the concentration gradient group,and continued to increase in the time gradient group.2)In the inhibition group,with the increase of DAPT concentration and time,the expression of hes1 and notch 1 decreased,the expression of Hes1decreased more significantly.The expression of collagen ? and collagen ? decreased continuously.4.The expression of proteins in fibroblasts after different levels of electrical stimulation.The expression of hes1,notch 1,collagen ? and collagen ? increased first and then decreased with the increase of electrical stimulation frequency.5.Expression of proteins in fibroblasts after inhibition of Notch 1pathway by estrogen stimulation1)The expression of hes1 and notch1 increased only when the estrogen concentration was 10^-8mol/L,but there was no significant change compared with the control group.2)At the concentrations of 10^-8mol/L and 10^-9mol/L,the expression of collagen ? was higher than that of the inhibition group,but there was no significant difference between the two groups and the control group.3)The expression of collagen ? did not change significantly when the concentration of estrogen was 10^-9mol/L,but it increased gradually with the increase of estrogen concentration.Conclusions:1.The inhibition of Notch signaling pathway may be involved in the occurrence and development of POP;2.By interfering with Notch signaling pathway,the metabolism of collagen ? and collagen ? in human sacrum ligament fibroblasts can be affected;properly activating this pathway can promote the production of collagen ? and collagen ?,while over activating can inhibit the production of collagen ? and collagen ?;3.Different levels of electrical stimulation can affect the changes of collagen ? and collagen ? secreted by human sacrum ligament fibroblasts,and its mechanism may be related to Notch signaling pathway;4.A certain concentration of estrogen can alleviate the inhibition of DAPT on the Notch signaling pathway,and can also increase the expression of collagen ? and collagen ? in fibroblasts,but the Notch signaling pathway may not be the main mechanism for estrogen to increase collagen ? and collagen ?.