向尿路上皮细胞定向诱导分化的脂肪干细胞与小肠黏膜下层支架的生物相容性研究

A biocompatibility research of urothelium-induced adipose stem cells and small intestinal submucosa

目的分离、扩增兔脂肪干细胞,诱导脂肪干细胞向尿路上皮细胞分化,并将诱导后细胞与小肠黏膜下层复合,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 6只8周龄新西兰兔腹股沟脂肪组织,通过胶原酶消化法分离脂肪干细胞,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,并进行分化后细胞的鉴定。随后将分化后细胞种植在小肠黏膜下层上,通过扫描电镜、组织学切片观察诱导后细胞在小肠黏膜下层上的生长情况。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%)、CD45(1.6%)。通过间接共培养诱导分化后,细胞免疫荧光、Western blot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小肠黏膜下层上生长,粘附较好。组织学检查可见小肠黏膜下层表面细胞平铺生长。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。小肠黏膜下层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 assess the biocompatibility of cells and scaffold materials in order to provide experimental basis for construction of tissue-engineered tissues.Methods A total of 8-week-old male New Zealand white rabbits were chosen to obtain adipose stem cells(ADSCs)and the groin fat tissues.ADSCs were separated with collagenase digestion method,then cultured and amplified in vitro.The third passage of ADSCs were used to confirm the phenotype with flow cytometry.After that,the third passage of ADSCs and urothelial cells were indirectly cultured for 2 weeks with Transwell system.Induced ADSCs were differentiated into urothelial cells,and the expression of UCs markers was detected with cell immunofluorescence and Western blot.After induction,the ADSCs were cultured on small intestinal submucosa(SIS),the material characterization and the adhesion of the cells on the material surface was observed with HE and scanning electron microscope(SEM).Results ADSCs were successfully cultured and proliferated in vitro.Surface antigen CD molecules were detected with flow cytometry.CD29(99.6%),CD44(98.2%),CD31(1.9%),and CD45(1.6%)were highly expressed.The epithelial cell marker(UP1a)expression increased in the induced cells.Immunofluorescence staining demonstrated that the induced cells rather than ADSCs were positive for specific urothelial marker,UP1a.Western blot indicated that the UP1a expression of the induced cells was increased.SEM observed that the prepared SIS scaffold materials had three-dimensional spatial structure with good porosity.The cells could be spread on the surface of the scaffold material.Conclusion Induced ADSCs 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 SIS 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.