脂肪干细胞和血管内皮细胞共培养复合脱细胞基质材料修复兔尿道缺损的实验研究

Repair of urethral defects in rabbits using adipose stem cells and vascular endothelial cells co-cultured with acellular matrix materials

目的本实验拟探讨应用脂肪干细胞(adipose-derived stem cells,ADSCs)和血管内皮细胞(vascular endothelial cells,VEC)体外共培养,联合脱细胞基质材料构建人工尿道修复兔尿道缺损的效果。方法体外分离培养、扩增获得兔脂肪干细胞和血管内皮细胞,分别将ADSCs、VEC以及二者共培养细胞群与脱细胞基质结合。将48只雄性成年新西兰兔阴茎腹侧尿道制作成缺损长度为2.0 cm的动物模型,然后利用随机数字表法分为4组。无细胞对照组:单纯应用异种脱细胞基质修复组。ADSCs组:脂肪干细胞复合异种脱细胞基质手术修复组;VEC组:血管内皮细胞复合脱细胞基质手术修复组;ADSCs与VEC共培养组:ADSCs与VEC共培养后复合异种脱细胞基质手术修复组。分别于术后第1周、2周、4周、8周取各组实验动物的重建尿道组织,进行组织学及免疫组化观察分析。术后第8周观察各组动物排尿情况,记录有无尿瘘和尿道狭窄。结果体外培养扩增的ADSCs呈梭形生长,VEC形态多样,呈铺路石样;两者共培养7 d时部分细胞仍保持原有的梭形,另外一部分细胞呈多角形,巢样分布,14 d时观察可见细胞间存在许多相互连接的突触,并且部分细胞可融合成团块状。术后4周、8周分别对各组兔子尿道行免疫组织化学染色分析,可见ADSCs与VEC共培养组上皮血管内皮细胞因子(VEGF)、平滑肌肌动蛋白(a-SMA)明显多于无细胞对照组、ADSCs组、VEC组。术后8周无细胞对照组并发症发生率为75%(9/12),ADSCs组为41.7%(5/12),VEC组为33.3%(4/12),ADSCs与VEC共培养组为16.7%(2/12),四组间差异有统计学意义(χ2=8.914,P<0.05)。进一步两两比较发现,无细胞对照组与ADSCs与VEC共培养组相比差异有统计学意义(P<0.0083)。无细胞对照组与ADSCs组、无细胞对照组与VEC组、ADSCs组与VEC组、ADSCs组与ADSCs和VEC共培养组及VEC组与ADSCs和VEC共培养组比较,差异均无统计学意义(P>0.0083)。结论ADSCs和VEC经体外共培养后可作为种子细胞应用于组织工程尿道的构建和尿道缺损的修复。VEC可诱导ADSCs向上皮细胞方向转变,并增加尿道组织血管的发生。

Objective To co-culture adipose derived stem cells(ADSCs)and vascular endothelial cells(VEC)in vitro and add acellular matrix materials for reconstructing artificial urinary urethral defect.Methods Isolation,culture and amplification of rabbit ADSCs and VEC were performed.Respectively ADSCs,VEC and both were seeded to acellular matrix.A total of 48 male adult New Zealand rabbits were randomly divided into 4 groups with a length of urethral defect at 2.0 cm.Ccell-free control group was a simple application of acellular matrix;ADSCs:acellular matrix composite surgical repair;VEC:VEC acellular matrix surgical repair;ADSCs&VEC co-culturing group:ADSCs plus VEC co-cultured with acellular matrix composite.Urethral tissues were examined by histology and immunohistochemistry at 1,2,4 and 8 weeks.Urethral fistula and stricture were recorded at 8 weeks.Results ADSCs were fusiform shapes in vitro and cellular morphology of VEC was diverse and slabstone.Both were co-cultured for 7 days,some cells still retained original spindle and other cells were polygonal and nest-like.At 14 days,there were a lot of interconnected synapses and some of them fused into a mass.Urethra of each rabbit was observed at 4 and 8 weeks post-operation.The number of capillary and smooth muscle was higher in ADSCs&VEC co-culturing than that in Cell-free control/ADSCs/VEC group.Vascular endothelial growth factor(VEGF)and smooth muscle actin(SMA)were positive in ADSCs vs.VEC.After 8 weeks,the complications occurred in 9/12 cases(75%)in Cell-free,5/12(41.7%)in ADSCs,4/12(33.3%)in VEC and 2/12(16.7%)in ADSCs&VEC.And the differences were statistically significant(χ2=8.914,P<0.05).Conclusion Cell-free and ADSCs&VEC,the difference was statistically significant(P<0.0083);comparing Cell-free&ADSCs,Cell-free&VEC,ADSCs&VEC,ADSCs&ADSCs&VEC and VEC&ADSCs&VEC,the differences were not statistically significant(P>0.0083).Conclusion After in vitro co-culturing,ADSCs and VEC may be used as seeds for tissue engineering urethra and repairing urethral defect.And VEC transforms ADSCs into epithelial cells and increases the occurrence of blood vessels in urethra.