Regulation Of Morphology And Synthesis Function By Mechanical Stretch In Human Uterosacral Ligament Fibroblasts

Objective:1 To study regulation of mechanical stretch on the morphology and integrinβ1, talin and extracellular matrix mRNA expression of fibroblasts derived from human uterosacral ligament who are not suffering from pelvic organ prolapse.2 To investigate the relationship between the mechanical stretch and the extracellular matrix remodeling.3 To investigate the effects of mechanical stretch on the pelvic floor dysfunction.Methods :1 The subjects: 15 Patients without pelvic organ prolapse and malignant tumors who underwent hysterectomy surgery in the Obstetric and Gynecological Department of the Second Hospital of Hebei Medical University from October 2009 to September 2010 were recruited in this study. The average age of the patients is 49.6±4.26 years old, the average time of parity is 2.73±1.27,the average body mass index is 24.75±2.44kg/m~2. All the recruited women were not suffer from connective tissue diseases and Pathologically confirmed non-endometriosis, non-estrogen-related ovarian tumors. Excluding acute and chronic complications in patients with pelvic inflammatory disease .Excluding those patients who had the surgery in the uterosacral ligamental site and have a history of estrogen application within the past 3 months.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 were taken from part of the sacral ligament near the cervix, about 5mm or so sacral ligamental specimens were obtained in the hysterectomy of extra fascia, keep fresh in 4℃cold boxes and sent to the laboratory immediately, the whole process is less than 2 hours and requires strict sterile.3 Primary cultured and identification of the fibroblasts: Fibroblasts of sacral ligament derived from old wemen were cultured by collagenase digestion and tissue inoculation method, the cultured cells were identified by morphology with light microscope and the cells'certain antigens were detected with immunocytochemical method.4 Experimental methods for mechanically stimulating the cells in vitro: Using Flexcercell4000 tension system, fibroblasts of 3～5 generations of exponential phase of growth derived from15 cases were stretched for 0,4,15,24 hours, using 0 hours as the control group. The loading process is in the incubator-CO2 . Experimental samples were cultured in a special disposable BioFlex plates which is made of flexible silicone membrane that can be deformed and it is covered by collagen I. The Loading procedure is automatically controlled by the Flexcercell4000 software. Loading parameters: amplitude 10%, sine wave, 0.5Hz. Total RNA was extracted from the fibroblasts immediately after unloading and the expression of mRNA of integrinβ1, talin, CollagenⅠ, CollagenⅢand matrix metalloproteinase-1 were measured by Real-time fluorescence quantitative PCR.Results:1 The fibroblasts show spindle shape mainly and can also show the irregular triangles, polygons and other shapes in Light microscope. The cells connected to each other and form a network structure. The vimentin is 90.4% positive in immunohistochemical staining , smooth muscle actin (α-SMA) is about 8.3% positive and keratin (cytokeratin) is negative.2 In response to mechanical stretch, the morphology and orientation of the uterosacral ligament fibroblasts were changed. The morphology of the fibroblasts is variety from spindle (mainly)to the irregular triangles, polygons and other shapes before stretching. The cells connected to each other and form a network structure, stretched in all directions. After stretched for 4h, 15h, 24h, the shape of the fibroblasts are all spindle and the direction of the fibroblasts perpendicular to the orientation of the maximum strain , the changes of 24 hours'stretching is the most obvious.3 In response to mechanical stretch, the synthetic function of the uterosacral ligament fibroblasts were changed . The integrinβ1 mRNA for 4,15,24 hours'stretch (18.822±0.500, 32.527±2.559,40.357±2.140) were up regulated compared with 0 hour(1.008±0.059), P<0.01. The expressions of talin mRNA for 4,15,24 hours'stretch (109.804±4.861, 106.574±35.477, 863.206±44.298) were up regulated compared with 0 hour(1.009±0.468), except 4h & 15h, the other groups werestatistically different ,p<0.01.collagen I mRNA for 4,15 hours'stretch(0.753±0.075, 0.701±0.023)were down regulated compared with 0 hour (1.000±0.129), P<0.01, but for 24 hour'stretch , the mRNA expression (1.113±0.049) of I-type collagen were restored to the level of 0 hours. The expressions of collagenⅢmRNA for 4,15,24 hours'stretch (4.792±0.036, 6.146±0.594, 5.021±0.594) were up regulated compared with 0 hour(1.008±0.249), except 4h & 24h ,the other groups were statistically different, P<0.01. MMP-1mRNA for 4,15,24 hours'stretch (6.233±0.651, 23.700±0.900, 20.133±0.751)were up regulated compared with 0 hour (1.000±0.100), P<0.01.Conclusions:1 The mechanical stress can change morphology and orientation of the fibroblasts, and promote the synthesis of mRNA of integirnβ1 and talin continuously. At the beginning of Loading, the expression of mRNA of I collagen decreased, then restored to its original level after 24 hours'stretch. The syntheses of CollagenⅢand MMP-1 were increased at first, but decreased after 24 hours'stretch.2 Extracellular matrix remodeling in pelvic floor is an adaptive response to mechanical force.3 Mechanical force maybe play some role in the pathogenic mechanism of POP.