| Objective: Bladder outlet obstruction is the most common lower urinary system disease in children,and children with congenital bladder outlet obstruction are mostly secondary to urethral abnormalities.Posterior urethral valve is the most common cause of bladder outlet obstruction in children.However,the current treatments of the disease cannot fundamentally improve the bladder function and long-term outcomes of end-stage renal disease,which still affect the quality of life and even the health of children.Therefore,clarifying the timing of bladder pathogenesis changes,looking for effective methods to treat the disease and choosing a suitable node for intervention are of great significance for the repair of bladder injury with congenital bladder outlet obstruction.With the deepening of stem cell research,dedifferentiated fat(DFAT)cells may be able to improve bladder injury caused by congenital bladder outlet obstruction and open up a new therapeutic direction for this disease.This study aimed to clarify the pathological changes of the bladder after partial bladder outlet obstruction(PBOO)in neonatal rats and to explore the ability of DFAT cells cultured by insert culture to induce differentiation into smooth muscle cells in vitro.Methods: 1.Forty-five newborn SD rats were randomly divided into two groups,30 in PBOO group and 15 in control group.The rats in PBOO group were divided into 5groups at 2d,4d,6d,8d,and 10 d after PBOO surgery,with 6 rats in each group.The rats in control group were divided into 5 groups at 2d,4d,6d,8d and 10 d after sham operation,with 3 rats in each group.HE staining were used to observe bladder tissue morphology,and tissue immunofluorescence were used to detect the expression of calponin 1,vimentin,SMMHC and collagen Ⅲ and observe the pathological changes of bladder tissue after PBOO.2.DFAT cells were isolated and cultured in vitro using insert culture,cell morphology and growth condition were observed by inverted phase contrast microscope,the identify and purity of the cells was detected by flow analysis of the surface antigen phenotype.The DFAT cells were induced to differentiate into smooth muscle cells in vitro,and the expression of αSMA was detected by cellular immunofluorescence.Results: 1.After PBOO,the bladder wall thickened and the bladder tissue was compensatory hyperplasia.With the obstruction time increased,the bladder volume increased and the bladder wall gradually became thinner.HE staining showed that inflammatory cell infiltration on the second day after PBOO,the arrangement of myocytes disordered,the submucosa and muscular layer thickened,and the muscle gap increased on the 4th day after PBOO.Tissue immunofluorescence showed that the expressions of calponin 1,vimentin,SMMHC and collagen Ⅲ increased first and then decreased.The highest expression of calponin 1,vimentin and SMMHC was on the 4th day after PBOO,and the highest expression of collagen Ⅲ was on the 8th day after PBOO.2.The DFAT cells cultured by insert culture were fibroblast-like,spindle-shaped and valley-like arrayed.The expressions of surface antigen markers in flow cytometry analysis of DFAT cells were CD29+CD90+CD31-CD45-,and the percentage of positive cells were CD29+(96.93 ± 3.61)%,CD90+(98.65 ± 0.84)%,CD31+(0.16 ± 0.13)%,CD45+(0.16 ± 0.10)%.After inducted in vitro,αSMA staining were positive,the rate ofαSMA positive cells was(73.56 ± 8.44)%.Conclusion: 1.PBOO caused bladder damage,smooth muscle hyperplasia,epithelial-mesenchymal transition and collagen deposition occurred in the bladder tissue on the second day after PBOO in newborn rats.Smooth muscle hyperplasia and epithelial-mesenchymal transition of the bladder tissue were most significant on the 4th day after PBOO,and the collagen deposition of the bladder tissue was most significant on the 8th day after PBOO.2.The DFAT cells cultured by insert culture can be induced to differentiate into smooth muscle cells in vitro,which provides a possibility for its application in bladder tissue engineering. |
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