| Background and Objective Detrusor instability (DI) is a common micturation dysfunction in urology. At present, the pathogenesis of DI has not been elucidated and the treatment of DI are not satisfactory. Gap junctions (GJ) are unique channels that transmit ions and small molecular metabolites between adjacent cells. They play an important role in maintaining excitatory communication among adjacent cells. Recent studies of the detrusor have suggested a new viewpoint for the pathogenesis of DI. Detrusor is a kind of excitable tissue, the excitability of detrusor may play a key role in the occurring of DI, but there were no reports about the functional changes of gap junction intercellular communication (GJIC) in DI. We deduce that the excitation via GJIC served as one of the important reasons for the pathogenesis of DI. In this study, we used RT-PCR techniques, western blot, fluorescence redistribution after photobleaching (FRAP) techniques and laser scanning confocal microscope (LSCM) to detect the levels of connexin mRNA expression and connexin43 protein and the functional changes of gap junctional mediated intercellular communication in DI respectively. We hope to study the relationship between the expression of gap junction gene connexin and its protein in bladder detrusor, and find out the pathogenesis of DI to provide a new direction in clinical treatment.To study the functional changes of gap junctional mediated intercellular communication in DI of bladder in rats so as to demonstrate the feasibility of resisting excitatory communication as the target of therapy for DI. Material and Methods: A total of 43 Wistar female rats of 2 months, weighing 180-210gm were used. Proximal urethra were partial ligated to produce the rat bladder outlet obstruction (BOO) models. Intravesical pressure was measured 6 weeks later. 25 rats were chosen as DI group.15 rats of normal bladder tissue were used as control group. For transmission electron microscopy studied the ultrastructure in the samples of detrusor. RT-PCR techniques, in transcript levels, were employed to detect the expression of connexin in DI. Western blot techniques, in protein levels, were employed to detect the levels of Cx43 protein in DI. Primary cell cultures were set up from fragments of rat bladder for BOO models and normal rat bladder. The fresh bladder tissues were excised under sterile condition and rinsed with sterile saline. After removal of the serosa and mucosa, small tissue fragments were digested with collagenase Ⅳ at 4℃ for 12h. The cells were cultured in DMEM supplemented with 15% fetal bovine serum at 37℃ and 5% CO2. Using indirect immunofluorescence method we examined whether the expression pattern of connexin43 protein in the detrusor cells. Cultured detrusor cells were loaded with the fluorescent dye 6-CFDA. The function of GJIC in the cultured bladder detrusor cells were detected by FRAP techniques. After the samples were loaded, the functions of GJIC in the detrusor cells were detected by Leica laser scanning confocal microscope. Three types of cells were selected for fluorescense microscope: (i ) bleached cells, in contact with adjacent cells that need to be bleached by the laser; (ii) single cells, not connected with other cells that also need to be bleached by the laser; (iii) unbleached cells, in contact with adjacent cells that need not to be bleached by the laser. All FRAP procedures, consisting of loading, washing, selecting, bleaching and scanning had to be finished within 30-40 minutes.After the three types of cells were identified, they were scanned. In accordance with the Leica TCS NT instruction, parameters for the bleach/time series, the fluorescence recovery rates were recorded for a period of 4 minutes after bleaching. The mean fluorescence recovery rates was calculated according to the formula. The effect of 18β-GA at various concentrations on cultured rat bladder detrusor cells of DI group were observed by FRAP techniques under LSCM in this study. Meanwhile, the effect of 18β-GA of the same concentrations at...
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