摘要
目的评价碱性成纤维细胞生长因子(bFGF)联合复合细胞膜片注射治疗雌性大鼠压力性尿失禁(SUI)的效果。方法体外实验:提取原代脂肪干细胞(ADSC),将其培养成细胞膜片并进行表征,获取大鼠心脏微血管内皮细胞,与ADSC膜片构建复合细胞膜片。采用Transwell实验评估血管内皮细胞的迁移能力,采用CCK-8实验评估ADSC、血管内皮细胞的增殖能力。动物实验:选取雌性Sprague Dawley(SD)大鼠24只(体重约为200 g),分为对照组(未进行任何处理)、SUI组(仅采用阴部神经离断法进行造模)、ADSC膜片组(SUI造模的基础上注射ADSC膜片进行治疗)、复合细胞膜片+bFGF组(SUI造模的基础上注射搭载有大鼠心脏微血管内皮细胞的ADSC膜片联合bFGF注射治疗)共4组,每组各6只。完成注射4周后,对以上4组大鼠进行尿动力学检测,并对尿道取材,采用Masson染色及结蛋白(Desmin)、α-平滑肌肌动蛋白(α-SMA)、CD31免疫荧光染色评估建模和修复效果。结果ADSC膜片和复合细胞膜片+bFGF均构建成功。Transwell实验结果显示,单纯ADSC、ADSC膜片、ADSC膜片+bFGF培养基中迁移细胞数量分别为(35±3)、(78±4)、(186±7)个/视野,ADSC膜片+bFGF培养基中迁移细胞数量显著多于ADSC膜片培养基和单纯ADSC培养基(P值均<0.05),ADSC膜片培养基显著多于单纯ADSC培养基(P<0.05)。CCK-8实验结果显示,培养第3、5、7天,添加bFGF的培养基ADSC的A 450值分别为0.51±0.02、1.01±0.04、1.23±0.07,不添加bFGF的培养基分别为0.40±0.01、0.78±0.03、0.90±0.05,均显著高于培养第1天(基线)的0.20±0.01、0.20±0.02(P值均<0.05);添加bFGF的培养基血管内皮细胞的A 450值分别为0.88±0.01、1.51±0.02、1.80±0.02,不添加bFGF的培养基分别为0.72±0.01、1.20±0.02、1.60±0.02,均显著高于培养第1天(基线)的0.41±0.01、0.40±0.02(P值均<0.05);添加bFGF的培养基培养第3、5、7天ADSC和血管内皮细胞的A 450值均显著高于不添加bFGF的培养基同时间(P值均<0.05)。尿动力检测结果显示,SUI组的膀胱漏尿点压(LPP)和膀胱最大容量(MBC)均显著小于对照组(P值均<0.05),验证大鼠SUI模型建模成功。ADSC膜片组大鼠LLP和MBC均显著大于SUI组(P值均<0.05),复合细胞膜片+bFGF组大鼠LLP和MBC均显著大于ADSC膜片组和SUI组(P值均<0.05)。尿道组织学Masson染色显示,与SUI组相比,ADSC膜片组和复合细胞膜片+bFGF组的平滑肌和横纹肌肌层明显增厚,肌细胞数量增多;复合细胞膜片+bFGF组的平滑肌和横纹肌肌层较ADSC膜片组更厚,肌细胞数量更多。SUI组的尿道括约肌Desmin、α-SMA、CD31阳性细胞比例均显著低于对照组(P值均<0.05),ADSC膜片组和复合细胞膜片+bFGF组Desmin、α-SMA、CD31阳性细胞比例均显著高于SUI组(P值均<0.05),复合细胞膜片+bFGF组Desmin、α-SMA、CD31阳性细胞比例均显著高于ADSC膜片组(P值均<0.05)。结论bFGF联合复合细胞膜片注射可以有效治疗雌性大鼠的SUI,且在功能学和组织学方面的效果均优于ADSC膜片。
Objective To evaluate the effect of basic fibroblast growth factor(bFGF)combined with composite cell sheet injection on the stress urinary incontinence(SUI)in female rats.Methods In vitro experiments:primary adipose-derived stem cells(ADSC)were extracted,cultured into cell sheets and characterised;rat cardiac microvascular endothelial cells were obtained and used to construct the composite cell sheets with ADSC sheets.Transwell assay was used to assess the migration of vascular endothelial cells,and cell counting kit-8(CCK-8)assay was used to assess the cell proliferation of ADSC and vascular endothelial cells.Animal experiments:24 female Sprague-Dawley(SD)rats(weighing about 200 g)were selected and divided into control group(without any treatment),SUI group(modelling by pubic nerve dissection),ADSC cell sheet group(injected with ADSC cell sheet after SUI modeling),and composite cell sheet+bFGF group(injected with ADSC cell sheet carrying rat cardiac microvascular endothelial cells after SUI modeling),with 6 rats in each group.Four weeks after intervention,urodynamic tests were performed on all the rats,and the urethra was sampled.Masson staining and immunofluorescence staining with Desmin,α-smooth muscle actin(SMA),and CD31 were used to evaluate the modelling and repair effects.Results Both ADSC cell sheet and composite cell sheet were successfully constructed.Transwell assay showed that the number of migrated cells in ADSC group,ADSC cell sheet group,and ADSC cell sheet+bFGF group were 35±3,78±4,and 186±7 cells per field of view,respectively;the number of migrated cells in ADSC cell sheet+bFGF group was significantly more than that in ADSC cell sheet group and ADSC group(both P<0.05),and the number of migrated cells in ADSC cell sheet group was significantly more than that in ADSC group(P<0.05).The results of CCK-8 experiments showed that on the 3rd,5th and 7th days of culture,the A 450 values of ADSC were 0.51±0.02,1.01±0.04,and 1.23±0.07 in the bFGF group,and 0.40±0.01,0.78±0.03,0.90±0.05 in the control group,which were all significantly higher than the baseline on day 1 of culture(0.20±0.01,0.20±0.02)(all P<0.05).The A 450 values of vascular endothelial cells were 0.88±0.01,1.51±0.02,and 1.80±0.02 in the bFGF group on the 3rd,5th and 7th days of culture,and 0.72±0.01,1.20±0.02,1.60±0.02 in the control group,which were significantly higher than the baseline on day 1 of culture(0.41±0.01,0.40±0.02)(all P<0.05).The A 450 values of ADSC and vascular endothelial cells in the bFGF group were significantly higher than those in the control group on days 3,5,and 7 of culture(all P<0.05).The results of urodynamic test showed that the leak point pressure(LPP)and maximal bladder capcity(MBC)of the SUI group were significantly smaller than those of the control group(both P<0.05),which verified the success of SUI modeling.The LLP and MBC in the ADSC cell sheet group were significantly larger than those of the SUI group(both P<0.05).The LLP and MBC in the composite cell sheet+bFGF group were significantly larger than those in the ADSC group and the SUI group(all P<0.05).Masson staining showed that ADSC cell sheet group and composite cell sheet+bFGF group had thicker muscle layer and more smooth muscle and striated muscle cells than SUI group,and composite cell sheet+bFGF group had thicker striated muscle layer and more smooth muscle and striated muscle cells than ADSC cell sheet group.The proportions of urethral sphincter Desmin,α-SMA,and CD31-positive cells in the SUI group were all significantly lower than those in the control group(all P<0.05).The proportions of Desmin,α-SMA,and CD31-positive cells in the ADSC cell sheet group and the composite cell sheet+bFGF group were significantly higher than those in the SUI group(all P<0.05).The proportions of Desmin,α-SMA,and CD31-positive cells in the composite cell sheet+bFGF group were significantly higher than those in the ADSC cell sheet group(all P<0.05).Conclusion bFGF combined with composite cell sheet injection can effectively treat SUI in female rats,and its effect is better than that of adipose stem cell sheets both functionally and histologically.
作者
王宇晖
刘猛
杨铭
方文卓
金扬旺
傅强
WANG Yuhui;LIU Meng;YANG Ming;FANG Wenzhuo;JIN Yangwang;FU Qiang(Department of Urinary Surgery,Shanghai Sixth People’s Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200233,China)
出处
《上海医学》
2025年第5期259-268,共10页
Shanghai Medical Journal
基金
国家自然科学基金(82170694)
上海市科学技术委员会科技计划项目(20ZR1442100)。
