诱导向尿路上皮分化的脂肪干细胞与膀胱脱细胞基质支架的生物相容性研究

The biocompatibility research of adipose stem cells induced to differentiate into urothelium and bladder acellular matrix scaffolds

目的探讨脂肪干细胞向尿路上皮细胞分化的可行性,并将诱导后细胞与膀胱脱细胞基质复合培养,评价细胞与支架材料的生物相容性,为进一步构建组织工程化组织提供实验基础。方法取6只8周龄雄性新西兰大白兔的腹股沟脂肪组织和膀胱组织,通过胶原酶消化法分离脂肪干细胞(adipose-derived stem cells,ADSCs),同时通过胰蛋白酶消化的方法获取尿路上皮细胞,流式细胞鉴定后将第3代脂肪干细胞与尿路上皮细胞间接共培养2周,并对分化后细胞进行表型鉴定。随后将分化后细胞种植在膀胱脱细胞基质上,通过扫描电镜、组织学切片观察诱导后细胞在膀胱脱细胞基质上的生长情况。结果脂肪干细胞培养成功并在体外扩增,流式术检测表面抗原CD分子,细胞高表达CD29(99.6%)、CD44(98.2%);低表达CD31(1.9%)及CD45(1.6%)。通过间接共培养诱导分化后,细胞免疫荧光、Western blotting检测到尿路上皮标志物UPIa的表达,而未诱导的脂肪干细胞无表达。扫描电镜提示诱导后细胞在膀胱脱细胞基质上生长,黏附较好。组织学检查可见膀胱脱细胞基质表面细胞平铺生长。结论脂肪干细胞向尿路上皮诱导分化后可作为泌尿系组织工程的种子细胞,可能是尿路上皮细胞之外的又一新选择。膀胱脱细胞基质能支持诱导后的脂肪干细胞良好的生长,该支架可作为载体材料用于泌尿系组织工程的修复与重建。

Objective To explore the feasibility of differentiation of adipose stem cells into urothelial cells, evaluate the biocompatibility of cells and scaffolds, and provide experimental basis for further construction of tissue-engineered tissue. Methods The adipose derived stem cells(ADSCs) were isolated from the inguinal adipose tissue and bladder tissue of six 8-week-old male New Zealand white rabbits by collagenase digestion, and the urothelial cells were obtained by trypsin digestion. After flow cytometry identification, the third generation adipose stem cells and urothelial cells were co-cultured indirectly for 2 weeks, and the differentiated cells were phenotyped. Then the differentiated cells were implanted on the acellular matrix of bladder. The growth of the cells on the acellular matrix of bladder was observed by scanning electron microscopy and histological section. The surface antigen CD molecules were detected by flow cytometry. Results The adipose stem cells were cultured successfully and expanded in vitro. The cells expressed CD29(99.6%), CD44(98.2%), CD31(1.9%) and CD45(1.6%). After inducing differentiation by indirect co-culture, the expression of epithelial cell marker(UP1 a) was detected by immunofluorescence and Western blotting, but the uninduced adipose stem cells did not. Scanning electron microscopy(SEM) showed that the cells grew well and adhered well on the acellular matrix of bladder after induction. Histological examination showed that the cells on the surface of acellular matrix of bladder grew horizontally. Conclusion Induced adipose stem cells can differentiate into urothelial cells that can be used as seeding cells for urinary tissue engineering, which may be another choice out of urothelial cells. The bladder acellular matrix can support the good growth of the induced stem cells, which can be used as the carrier material for the repair and reconstruction of the urinary tissue engineering.