| Objections:1. To isolate and purify the adipose-derived stem cells and fibroblasts in vitro.2. To establish the animal model of stress urinary incontinence. Urodynamic indices and morphological changes of the urethra and surrounding tissues were observed after different numbers of two kinds of cells combined and transplanted to the middle of periurethral. And compare the stress urinary incontinence﹐s treatment effects of each groups.Methods:1. Adopting collagenase I to acquire rat adipose-derived stem cells, which was identified by flow cytometry according to different cell surface markers. And applying the improved two-steps enzymatic digestion to obtain fibroblasts, which was identified via immunohistochemistry. The cell populations were purified by the pre-plating technique according to different cellular adherent ability. The cell proliferation and morphological study were conducted under inverted phase contrast microscope for subsequent ideal source of cells.2. 36 non-pregnant female SD rats were randomly divided into two groups:normal group, which were not received any treatment; the experimental group which were performed vaginal dilation and bilateral ovariectomy. 2 weeks later,maximum bladder capacity and abdominal leak point pressure were measured by BL-420 biological function tester in all rats. The model were successfully established.3. After A month, all successful models in experimental groups were randomlydivided into five groups(6/group). The mixture of ADSCs and fibroblasts were classified 1×104,1×105 and 1×106group. Then, under the microscope, all cells were implanted to the middle of periurethral at 3:00 and 9:00, respectively. At the same time, to establish a control group of saline and a blank group. Urodynamic indices were measured again after a month in order to assess the treatment effects for stress urinary incontinence4. After a month all rats sacrificed. Evaluating the morphologic changes of the urethra and surrounding tissues by HE staining after cell transplantation, which were obtained from the urethra and vaginal wall.5. Statistical analysis: Analysis was performed with SPSS 13.0 for Windows(SPSS Inc). If the data was consistented with a normal distribution and homogeneity of variance, Statistical significance was assessed by comparing mean(±SD) values with Student’s t-test for independent groups.; ANOVA was adopted among groups,pairwise comparisons was used LSD-t; Otherwise, Wilcoxon test was used. The results were considered statistically significant when P value was less than 0.05.Results:1. ADSCs were mostly being a swirling colony of polygonal and spindle. The cell morphology was consistent. The cell were expressing CD29、CD44. But CD45 was not. The positive rate was 96.63%, 78.53% and 0.75%, respectively. Fibroblasts were morphologically similar to ADSCs, identified by immunohistochemical. The vimentin was brownish yellow granules, positively located in the cytoplasm. However,the keratin was negatively expressed. Observed by inverted phase contrast microscope,the cell morphology of fibroblasts and ADSCs after third passages had no significant changes. And the cell still remains strong proliferation activity.2. Vaginal dilation and bilateral ovariectomy can successfully establish the animal model of SUI. Maximum bladder capacity(ml) in experimental group and normal group were 1.83 ± 0.54, 3.08 ± 0.28, and abdominal leak point pressure(cm H2O) were 76.02 ± 2.56, 24.91 ± 2.94. The experimental group was significantly lower than the normal group(P <0.001).3. 1 month later, a different quantity of ADSCs and fibroblasts were injected.According to urodynamic testing, the MBC were 3.08 ± 0.28, 1.89 ± 0.36, 1.93 ± 0.60,2.10 ± 0.55, 2.23 ± 0.66 and 2.54 ± 0.38 in normal group, SUI group, saline group,1×104 group, 1×105 group and 1×106 group, respectively. Between groups showed:normal group and other groups had a statistical difference(P <0.001), 1×106group and saline group, SUI group were of great significance. Between other groups showed no difference(P> 0.05); ALPP were 76.02 ± 2.56, 26.26 ± 3.01, 29.17 ± 4.09, 32.96 ±4.99, 36.82 ± 4.22 and 41.16 ± 3.57, individually. Between groups showed: normal group and other groups were statistically different(P <0.001), but the SUI group and saline group had no significance(P>0.05). However,1×104 group and SUI group;1×105group and SUI group, saline group;1×106group and SUI group, saline group,1×104 group had a significantly difference,respectively.4. The morphologic changes of the urethra and surrounding tissues after a different quantity of ADSCs and fibroblasts transplantation: the urethra has a complete muscular structure in normal group. All bundles of muscle fibers were packed closely. The urethra of SUI group was impaired seriously. For example,muscular became thin and disordered, muscle fibers appeared loose, broken and atrophy. The saline group, had no significant changes, but urethral scar formation and fibrosis was obvious. Cell injection group, the injection local of urethral muscular was thickened(P<0.01). But the arrangement of muscle fiber is still relatively loose and disordered. The surrounding connective tissue became close. However, there had not significant difference between each number of cell groups(P>0.05).The anterior vaginal wall showed that the mucous, muscular and the outer layers were clearly visible in normal group. The bundles of smooth muscle were thick and braided. The collagen was abundance and dense. In SUI group, muscular became thinner and shorter. The connection structure was disappeared between normal muscle fibers. The gap widen between all bundles of collagen fiber and appeared scattering and breakage.The saline group and cell injection groups had no significant changes observed in the anterior vaginal wall(P>0.05).Conclusions:1. By using the method of collagenase digestion and pre-plating technique can obtain ADSCs, which had relative higher purity and better biological activity; and through two-step enzymatic digestion and pre-plating technique can acquire a higher purity of fibroblasts.2. As for the selection of transplantation concentration, 1×106may be a better choice for Fibroblasts and Adipose-derived Stem Cells combined transplantation for treatment of stress urinary incontinence in rat models. |
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