Preliminary Experimental Study On Autologous Muscle Satellite Cell Injection For Female Stress Urinary Incontinence

Part â…  Isolation and purification of rat primary muscle satellite cell and characterization of its muscle differentiation in vitro cultureObjective: to establish a method of isolation and purification of rat primary muscle satellite cells. Characterization of myotube cell generated from muscle satellite cells in vitro culture was also observed.Method: (1) Single nuclear cells of muscle tissues were isolated by the way of improved two-step enzymatic digestion. Majority of fibroblasts, endothelia and other mixed heterogeneous cell population were removed through pre-plating technique according to different cellular adherent ability and thus muscle satellite cells of relative high purity were obtained. (2) Morphological study was conducted under inverted phase contrast microscope. Marker of skeletal muscle tissues, desmin, was identified on the harvested cells by histocytochemistry. (4) MTT assay was used to measure growth curve of rat primary muscle satellite cells. (5) Muscle differentiation of satellite cells was also observed under different culture conditions, including changed culture medium or prolonged culture time.Results: (1) High purity muscle satellite cells were obtained through combination of improved two-step enzymatic digestion and pre-plating technique, which is confirmed by desmin histocytochemistry. (2) It is necessary to use poly-lysine or collagen-coated dishes to enable adhesion of muscle satellite cells. Majority of muscle satellite cells completes adhesion course within 24 hours after inoculation. Morphology of muscle satellite cells are similar to that of fibroblasts with the fusiform shape, both belonging to fibroblast cell type. (3) When cultured in vitro,the latent phase of muscle satellite cell is at the 1st to 2nd days. The platform phase is at the 5th to 6th day. (4) Myotube cell gradually formed when cell confluence is more than 60% to 70% or differential medium with lower fetal bovine serum and horse serum is used in vitro culture. Sometimes spontaneous contraction of myotube cells could be observed under better culture condition.Conclusion: Method combining improved two-step enzymatic digestion with pre-plating technique is a kind of easy and practicable in some degree to obtain relative high purity of muscle satellite cells. Muscle satellite cells could form myotube cell with multiple nuclei without any special induction. Myotube cell has characterization of spontaneous contraction.Prat â…¡ Effects of IGF-1 on muscle satellite cell proliferation and changes of PI3K cell transduction pathway involvedObjective: To research proliferative effects of IGF-1 on the rat primary muscle satellite cells and mechanisms involved.Method: (1) MTT cell proliferation assay measures proliferative status of satellite cells under IGF-1 of different concentration (2ng/ml, 20ng/ml, 50ng/ml and 100ng/ml) at different culture phases (24 and 48 hours). (2) To measure changes of components of PI3K cell signaling transduction pathway under IGF-1 stimulation. 3 experimental groups of muscle satellite cells of second passage from the same rat were established and all the groups were ensured at the same cell cycle with at least 16 hours serum-free medium treatment when the cells grow to 30% to 40% confluence. group 1 was treated with 5% FBS as basal stimulation;group 2 was treated with additional 20ng/ml IGF-1 stimulation; group 3 was treated with IGF-1 and PI3K signaling pathway inhibitor, LY294002 (20uM), which was pre-incubated with cells 1 hour just before experiment. 2 hours later under different growth conditions, cellular protein and total RNA were extracted. Then Expression of FOXO1, phospho-FOXO1 and P27Kip1 protein were analyzed by western blotting assay. (3) RT-PCR assay measured expression of P27Kip1 mRNA at the level of transcription.Results: (1) IGF-1 of 2ng/ml display slightly proliferative effects, while IGF-1 of 20ng/ml is enough to activate and stimulate rapid proliferation of muscle satellite cells. Effect of IGF-1 of 50ng/ml or 100ng/ml is just a little higher than IGF-1 of 20ng/ml. (2) Muscle satellite cells proliferate rapidly and are apt to fuse mutually to form myotube cells because of IGF-1 stimulation. Thus it's necessary to observe status of cell proliferation and cell passage or culture medium replacement should be done in time. (3) IGF-1 elevates the levels of phospho-FOXO1 protein and could inhibit expression of P27Kip1 at the level of transcription.Conclusion: (1) IGF-1 of 20ng/ml is enough to stimulate rapid proliferation of muscle satellite cells. This growth factor is thus helpful to get large numbers of muscle satellite cells from limit tissues in a short time for tissue engineering medicine. (2) It is necessary to observe status of cell proliferation closely to avoid myotube cells formation when IGF-1 was utilized to stimulate muscle satellite proliferation in vitro culture. (3) IGF-1 stimulated cell proliferation through elevating level of phospho-FOXO1 protein, resulting in inhibited expression of P27Kip1 at the level of transcription.Part â…¢ Experimental study of Effects of injection of autologous muscle satellite cells for female stress urinary incontinenceObjective: (1) To establish effective animal model of female stress urinary incontinence for further experimental study. (2) To evaluate urodynamic outcomes (abdominal leak point pressure, ALPP) of muscle satellite cells injection around urethral and morphological changes of urethral after cells injection 1 month later.Method: (1) Establishment of female animal stress urinary incontinence model: total of 30 female Spague-Dawley rats were utilized to do this study. Among them, 20 rats were used to mimic female stress urinary incontinence. Therefore, there were 3 groups of female rats. Group 1 was normal control (n=10); group 2 was treated with bilateral ovarectomy and repeated vaginal dilation as female stress urinary incontinence model control(n=10); group 3 was treated with the same management as group 2 initially and 2 months later autograft musclel satellite cells were injected around proximal urethral. 3 month later all the animals of each group received urodynamic measurements to evaluate changes of abdominal leak point pressure. (2) At the end of the second month, autograft muscle satellite cells of rats in group 2 were isolated and amplified by IGF-1. Then these cells were injected around the proximal urethral with Hamilton micro-injection needles. (3) At the mean time when urodynamic measurements were conducted, proximal urethral tissue of each rat was harvested and fixed by 4% paraform, the injected cites with satellite cells were most important. Thereafter, the samples were hematoxylin/eosin stained, examined microscopically and photographed.Results: (1) The abdominal leak point pressure of group 2 and group 3 were significantly lower than that of group 1. Abdominal leak point pressure of group 3, which was treated with cell therapy, was higher than that of group 2. However, the leak point pressure of group 3 was indeed lowerthan that of group 1. the difference has statistical significance. (2) The muscle satellite cells could form irregular muscle fiber at the injected cites and enhance the muscle of the urethral.Conclusion: (1) It is reliable for bilateral ovarectomy and repeated vaginal dilation to establish female animal model of stress urinary incontinence for experimental study. (2) Cell therapy mediated by muscle satellite cells could enhance the abdominal leak point pressure of stress urinary incontinence animal significantly. It has promise to be a new kind of bulking agent for female stress urinary incontinence in the future. The bulking effects may be one of causes of improvement of ALPP. However, enhancement of urethral contraction can not be excluded because of intensification of urethral muscle. (3) More study on the numbers of injected cells and long-term influence on the bladder function should be done before its clinical application.