| BackgroundHuman pelvic floor supporting tissues basically play the role of bearing pelvic organs. The female pelvic floor supporting tissues mainly comprise the pelvic outlet closure of the multi-layer muscle, fascia and the uterine ligaments. These tissues, for various reasons (such as:pregnancy, childbirth, constipation, chronic cough, etc.), are so damaged or torn that their supporting function is weakened, namely the occurrence of displacement and dysfunction of uterine and adjacent rectum, bladder and vagina wall. That is clinically termed pelvic floor dysfunction (PFD), which mainly referring to the stress urinary incontinence (SUI) and pelvic organ prolapse (POP). Present treatments of PFD mainly include surgery, conservative drugs and rehabilitation of pelvic floor muscles, but their deficiencies are obvious, especially for the injured ligament tissue. Scars are left behind during the repairing process, which is not the true meaning of regeneration. And the structure and function of uterine uterosacral ligament-main ligament complex, which takes the main supporting force to the pelvic floor, are closely related to the occurrence and development of PFD. Therefore, the real problem that needs to solve treating this disease is how to restore its normal structure and function after the injury of the uterine ligament. The birth of tissue engineering provides a new means for the repair of the uterine ligaments. Recent studies have demonstrated that during ligaments injury repair process, some stem cells originated from bone marrow mesenchymal, that are similar to the surrounding ligament cells, accumulate in the injury area. Injecting into the damaged knee tendon, the patellar tendon with bone marrow mesenchymal stem cells or fibroblasts fibronectin matrix can affect the expression of its histologic features, ultrastructure and the necessary extracellular matrix protein mRNA, which makes the damaged knee tendon and ligaments more easier to recover. In addition, co-culture of bone marrow BMSCs and tendon or ligament fibroblasts could increase the expression of related genes of rabbit tendon and ligament. Bone marrow mesenchymal stem cells (BMSCs) is a kind of primitive cells with super self-reproduction and directional differentiation, which can be induced directionally into various phenotypes of progeny cells externally or internally.The present study aims to study the effect of normal cells in vitro and mechanical stretch-induced and injured cells with stretch stimulation on BMSCs differentiating into uterine ligament fibroblasts in a co-culture system. In the experiment, mechanical stretch is used to stimulate and injure rat uterine ligament fibroblasts cultured in vitro, resulting in PFD cell injury model, and the BMSCs are co-cultured (non-contact) in vitro with normal uterine ligament fibroblasts and mechanical stretch-induced and injured uterine ligament fibroblasts. At the same time, detection of expression of elastin, lysyloxidase (LOX), the Fibulin-5 of co-cultured and induced BMSCs, will make further understanding of the differentiation potential and biological characteristics of BMSCs. This dissertation studies the non-contact co-cultured and induced BMSCs in vitro differentiating into uterine ligament fibroblasts, provides possible research ideas and develops a new therapy from the root for PFD from the perspective of tissue engineering.Methodology1. Isolation, culture and passage of rat BMSCs in vitro:Rat BMSCs were isolated and separated by Percoll using intensity gradient centrifugation and passage adherence screening method. BMSCs were identified through cell morphologic observation, cell-cycle detection and by some characteristic phenotype molecule. Biological function of rat BMSCs is tested through differentiation of rat BMSCs into Osteoblasts and Lipoblasts in vitro.2. Isolated culture and identification of rat pelvic ligament fibroblasts in vitro: Rat pelvic ligament tissue is primarily cultured to obtain uterus ligament fibroblasts. Expression of collagen I and collagen III in the pelvic ligament fibroblasts are detected through morphological methods and immunohistochemistry.3. Constructing analogue of mechanic stimulated rat pelvic ligament fibroblasts in vitro:A cyclic 10% uniaxia strain at 1HZ is applied on rat pelvic ligament fibroblasts for continuous 3,6,12,24,36 hours mechanical stretched injury, and the change of organelles is detected by transmission electron microscope (TEM). Laser scanning confocal microscope is used to identify the change of cytoskeletal filament which has been colored by phalloidine. Apoptosis is observed through using trypan blue. The mRNA expressions of collagen I and collagen III in the mechanical stretched injury pelvic ligament fibroblasts are measured by real time RT-PCR.4. Establishment a model of co-culture rat BMSCs with pelvic ligament fibroblasts:To non-contact co-culture rat BMSCs with the mechanic stretched (10% deformation with 1 Hz,12h) rat pelvic ligament fibroblasts for 3 days,6days and 12 days separately. The mRNA expressions of elastin, LOX, and Fibulin-5 in the BMSCs are measured by real time RT-PCR, and the synthesis of elastin, LOX, and Fibulin-5 in BMSCs is detected by western-blot.Data were presented as mean±standard error. Statistical significance was analyzed by one-way ANOVA using the SPSS 16.0 software. P<0.05 was considered significant.Results1. The obtained rat BMSCs appear spindle-shape or platypelloid shape, but when they are enhanced to cover the entire bottle wall, they show swirl and radiated arrangement. By means of flow cytometry (FCM), the fourth generation of BMSCs has the phenotype of positive expression of CD90 (over 99.0% of the cell), CD44 (over 98.8% of the cell), and negative expression of CD34 (1.8%), CD45 (3.7%), suggesting that 80% of the forth generation BMSCs are in G0/G1 period which indicate that 80% of it are in stationary phase.. The cell morphologic feature showed that the cell we got could be induced into osteoblasts and adipogenic cells which appear characteristics of stem cells.2. Pelvic ligament fibroblasts was elongated and reshaped like spindle or stellate, and cell morphology was homogeneous and presented in order, immunohis tochemistry staining showed that the 4th passage fibroblast cells had positive expression of type I and III collagen which is in line with the characteristics of fibroblasts.3. In the experiment, the cell body is elongated by 10% Load and 1 Hz. The F-action and nucleus of the stretched cell bodies are labeled by fluorescent, phalloidine and DAPI. Under laser confocal scanning microscope, the F-action are disaggregated and reset. The shorter mechanic stimulated rat pelvic ligament fibroblasts had no obvious changed, longer stimulated begins to die locally, and cells become vacuoles gradually; part of the organelles crack; nucleus move to the margin; little amount of the rest rough endoplasmic reticulum and mitochondrial still can be identified.4. With mechanical stretch stimulation after 3h,6h,12h, the mRNA expressions of collagens I and collagen III in the pelvic ligament fibroblasts increased, but decreased after 24h,36h. The mechanical stretch stimulation after 12h, the mRNA expressions of collagens I and collagen III is at best. Compared to the control group, the mRNA expressions increased, but decreased after 36h.5. After being co-cultured indirectly3,6,12d, through real-time quantitative RT-PCR and Weston-Blot detection, the expressions of BMSCs elastic protein, LOX, Fibulin-5 mRNA and protein are as follows:(1) The mRNAI of the 3d indirectly stretched co-cultured group are 2.01±0.12, 1.92±0.07 and 2.02±0.09 respectively, indirectly co-cultured group are 1.41±0.06, 1.26±0.06 and 1.16±0.11 respectively, control group are 1.00±0.03,1.00±0.04 and 1.00±0.06 respectively. The protein of the 3d indirectly stretched co-cultured group are 0.30±0.11,0.45±0.07,0.29±0.14 respectively, indirectly co-cultured group are 0.21±0.15,0.39±0.08,0.20±0.15 respectively, control group are 0.17±0.07,0.35±0.10, 0.14±0.07respectively, but were not significantly different(P> 0.05).(2) The mRNAI of the 6d of indirectly stretched co-cultured group are 3.81±0.13,2.11±0.07 and 3.90±0.11 respectively, of the 6d indirectly co-cultured group are 2.69±0.08,1.62±0.11 and 2.59±0.09 respectively, of the 3d control group are 1.29±0.05,1.11±0.04 and 1.21±0.07 respectively. The protein of the 3d indirectly stretched co-cultured group are 0.45±0.09,0.61±0.14,0.37±0.07 respectively, indirectly co-cultured group are 0.39±0.06,0.45±0.14,0.23±0.04 respectively, control group are 0.19±0.12,0.36±0.06,0.15±0.04 respectively. Through single factor analysis of variance, there is significant statistical difference (P< 0.05). By further comparison, it is found there is statistical difference (P< 0.05) when the BMSCs elastic protein, mRNAI of the 6d indirectly co-cultured group increased strikingly in comparison with the control group, the difference is statistically significant (P< 0.05); the BMSCs elastic protein, mRNAI of the 6d indirectly stretched co-cultured group increased strikingly in comparison with that of the indirectly co-cultured group, the difference is statistically significant (P< 0.05).(3) The mRNAI of the 12d indirectly stretched co-cultured group are 4.54±0.16, 2.50±0.09 and 4.21±0.15respectively, of the 12d indirectly co-cultured group are 2.93±0.14,1.71±0.09 and 2.89±0.08 respectively, of the 12d control group are 1.50±0.04,1.19±0.18and 1.60±0.07respectively. The protein of the 12d indirectly stretched co-cultured group are 0.42±0.14,0.49±0.11,0.24±0.06 respectively, of the 12d indirectly co-cultured group are 0.42±0.14,0.49±0.110.24±0.06 respectively, of the 12d control group are 0.20±0.13,0.38±0.15,0.18±0.06 respectively. Through single factor analysis of variance, there is significant statistical difference (P<0.05). By further comparison, it is found there is statistical difference (P< 0.05) when the BMSCs elastic protein, mRNAI of the 12d indirectly co-cultured group increased strikingly in comparison with that of the control group, the difference is statistically significant (P< 0.05); the BMSCs elastic protein, mRNAI of the 12d indirectly stretched co-cultured group increased strikingly in comparison with that of the indirectly co-cultured group, the difference is statistically significant (P< 0.05). Conclusions1. After 12h with 1Hz and 10% mechanical stretch stimulation, the injured cells are similar with the ligament fibroblasts ultrastructural changes of POP patients reported in the literature, while avoiding a large number of cell apoptosis. So 12h with 1Hz can be used as a reasonable uterine ligament fibroblast stretch of time and frequency.2. Non-contact co-culture rat BMSCs with normal cells or with mechanical stretched pelvic ligament fibroblasts can induce BMSCs differentiating into uterine ligament fibroblasts, yet the injured cells with stretch stimulation have a stronger effect. After induced differentiation, the rat BMSCs can synthesize and secrete elastin, LOX, Fibulin-5, with part of the secretory function of uterine ligament fibroblasts.
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