Effect Of Estradiol On Female Rat’s Pelvic Myofibroblasts With Mechanical Stress

Objective:To observe the effects of 17-βE2 on matrix metalloproteinase-9,tissue inhibitor of metalloproteinase and β-Catenin of in the specific stressed pelvic skeletal muscle fibroblast of female rats.Methods:1.Research object: 6 female Wistar rats about 2weeks whose average weight is 120±30g are provided by experimental animal center of Shanxi Medical University. They kept healthy after physical examination. The processing method of animals is conformed to the science and technology department of the People’s Republic of China issued "the guidance on treating experimental animals". 2. Fibroblasts primary culture: After collectting skeletal muscle around 3 to 5 mm of female Wistar rats, we using the type II collagenase to digest the original generation lines of fibroblasts in vitro culture, to purificate and to identificate. Choose the 3-5th generations in stable condition of fibroblasts to provide the basis for subsequent strength and 17- beta estradiol intervention experiment. 3. Fibroblast stress Flexcercell 4000 tensile loading system was used to the fibroblasts by 6%, 36 h mechanical stress. And for experiment group,17-βE2 was intervened and cultured for 24 hours. the control group was not given stress or drug. 4.Reverse transcription polymerase chain reaction(RT-PCR):The method of real-time quantitative PCR was used to m RNA extracted from each group of cells. Expressions of each observation target were recorded by electrophoresis photograph.Results:1.Cell culture:After the success of the primitive culture fibroblasts, extend them by the ratio of certain.Identified the fibroblasts with immunohistochemical method. Among the examination vimentin is in positive reaction and cytokeratin is in negative reaction which is regarded as fibroblasts.2.Mechanical stretch: Under the condition of specific strength, under the condition of strength of 6% for 36 hours, traumatic change of fibroblasts is more and more obvious. 3. RT-PCR results: compared with the control group, the relative transcript level of MMP-9m RNA of fibroblast in mechanical stress group is 2.69±0.02, which is higher; the relative transcript level of TIMP-1m RNA and β-catenin m RNA is 0.37±0.01 、0.43±0.01,which is lower; And both differences are statistically significant( P <0.01). Moreover,after intervention of 17-βE2,the relative transcript level of TIMP-1m RNA and β-catenin m RNA is 0.6±0.01、0.63±0.01,lower than the control group,but higher than the mechanical stress group, which is significant statistically(P<0.01). the relative transcript level of MMP-9m RNA is 1.52±0.07, higher than the control group, and lower than the mechanical stress group,there is a statistical significance(P<0.01).Conclusions:This study shows that with specific stress condition of 6%, 36 h, the fibroblasts injury is metabolic. MMP-9 expression of the mechanical stress group, tested by Real-time quantitative PCR, is increased obviously while β-Catenin and TIMP-1expression are decreased greatly which lead to decrease of I type collagen. While the effect of mechanical stress is weakened by 17-βE2, which testifies that estrogen has a protective effect on pelvic floor dysfunction. By studying the biomechanical properties of fibroblasts, it is helpful to reveal the pathogenesis of tissue damage of levator ani muscle in pelvic organ prolapse.