肌源干细胞联合纤维蛋白胶支架移植对压力性尿失禁大鼠的治疗研究

Therapeutic effect and mechanism of muscle derived stem cells combined with fibrin glue scaffold on stress urinary incontinence in rats

目的探讨肌源干细胞(MDSCs)联合纤维蛋白胶支架对压力性尿失禁大鼠的治疗效应及相关机制。方法建立压力性尿失禁的大鼠模型,并根据注射介质的不同分为5组,其中FM组为MDSCs联合可注射性纤维蛋白胶组;M组为单纯MDSCs注射组;F组为单纯可注射性纤维蛋白胶组;D组为溶剂对照组;S组为假手术组。在注射后1、2周以及4周通过实时荧光定量PCR和免疫组织化学法检测注射部位近端尿道组织胰岛素样生长因子-1(IGF-1)、血管内皮细胞生长因子(VEGF)的mRNA和蛋白表达情况。通过第八因子相关抗原(FVⅢR Ag)表达检测毛细血管生长情况。通过HE染色观察注射部位的近端尿道组织病理变化。结果注射后1周,FM组的VEGF和IGF-1 mRNA表达均高于D和S组(均P<0.05),VEGF mRNA和蛋白表达均高于M组(均P<0.05),而VEGF和IGF-1蛋白表达均高于F、D、S组(均P<0.05);注射后2周,FM组的VEGF、IGF-1 mRNA和蛋白表达均高于D组(均P<0.05),而VEGF蛋白表达均高于F、D组(均P<0.05);注射后1周,M组的VEGF和IGF-1 mRNA和蛋白均高于D组,VEGF mRNA高于S组,差异均有统计学意义(均P<0.05)。注射后4周,FM组的VEGF和IGF-1 mRNA的表达降至正常水平,而VEGF和IGF-1的蛋白表达明显降低,两两比较差异均无统计学差别(均P>0.05)。注射后4周,FM组的近端尿道毛细血管密度均高于其他组(均P<0.01),M组的近端尿道毛细血管密度均高于D组(均P<0.01)。与D组比较,FM组可见尿道外括约肌层增厚,注射部位有新生毛细血管及新鲜肌纤维形成,肌纤维密度增大;M组可见尿道外括约肌层增厚,注射部位可见部分新生肌纤维和少许新生毛细血管;F组肌层仍较疏松,注射部位可见新生毛细血管。结论MDSCs联合纤维蛋白胶作为组织工程支架可促进模型大鼠近端尿道的IGF-1、VEGF表达,促进新生毛细血管的形成和尿道肌层的再生修复。

Objective To investigate the therapeutic effect and mechanism of muscle derived stem cells(MDSCs)combined with fibrin glue scaffold on stress urinary incontinence in rats.Methods The rat models of stress urinary incontinence were established and divided into 5 groups according to the different injection media,FM group was MDSCs combined with injectable fibrin glue group;M group was simple MDSCs injection group;F group was simple injectable fibrin glue group;D group was solvent control group;S group was sham operation group,only related operations were performed,but no media were injected.The expressions of insulin-like growth factor-1(IGF-1)and vascular endothelial growth factor(VEGF)were detected by RT-PCR and immunohistochemistry at 1,2 and 4 weeks after injection.Microvascular growth was detected by FVⅢR Ag expression.HE staining was used to observe the histopathological changes of the urethra near the injection site.Results One week after injection,the mRNA expressions of VEGF and IGF-1 in FM group were higher than those in D and S groups(all P<0.05),the mRNA and protein expressions of VEGF were higher than those in M group(all P<0.05),and the protein expressions of VEGF and IGF-1 in FM group were higher than those in F,D and S groups(all P<0.05).Two weeks after injection,the expressions of VEGF mRNA,IGF-1 mRNA and protein in FM group were higher than those in D group(all P<0.05),and the expression of VEGF protein in FM group was higher than that in F and D groups(all P<0.05).One week after injection,the mRNA and protein levels of VEGF and IGF-1 in group M were higher than those in group D,and VEGF mRNA was higher than that in group S,and the differences were statistically significant(all P<0.05).Four weeks after injection,the mRNA expressions of VEGF and IGF-1 decreased to the normal level,while the protein expressions of VEGF and IGF-1 decreased significantly in the FM group.There was no significant difference between the two groups(all P>0.05).At 4 weeks after injection,the proximal urethral capillary density in FM group was higher than that in other groups(all P<0.01),and the proximal urethral capillary density in M group was higher than that in D groups(all P<0.01).Compared with group D,the external urethral sphincter layer was thickened,new capillaries and fresh muscle fibers were formed at the injection site,and the density of muscle fibers increased in group FM.In group M,thickening of external urethral sphincter was observed,and some new muscle fibers and a few new capillaries were observed at the injection site.In group F,the muscle layer was still loose,and new capillaries were seen at the injection site.Conclusions MDSCs combined with fibrin glue as tissue engineering scaffold can promote the expression of IGF-1 and VEGF in the proximal urethra of model rats,increase the formation of neovascularization,and promote the regeneration and repair of urethral muscle layer.