猪膀胱细胞外基质对人脐带Wharton胶间充质干细胞生物相容性实验

Biocompatibility of Wharton's jelly mesenchymal stem cells with porcine bladder accellular matrix

  • 摘要: 目的 探索化学法制备的猪膀胱细胞外基质(porcine bladder accellular matrix,PBAM)对人脐带Wharton胶间充质干细胞(Wharton's jelly mesenchymal stem cells,WJ-MSCs)的生物相容性,为组织工程膀胱的研究提供新的种子细胞。 方法 采用化学脱细胞法制备PBAM,行HE、甲苯胺蓝、Hoechst33258荧光染色检测脱细胞效果,扫描电镜观察PBAM形态,无水乙醇法检测其孔隙率。组织块法培养WJ-MSCs,观察形态并及行成骨、成脂诱导。比较不同浓度的PBAM浸提液对WJ-MSCs增殖的影响。实验组将WJ-MSCs复合至PBAM进行培养,对照组以DMEM培养,第3、5、7天收集细胞并计数,对细胞-支架复合物行Hoechst33258及AO染色,扫描电镜观察WJ-MSCs在PBAM上的生长状态。 结果 化学脱细胞法成功制备的PBAM。HE、甲苯胺蓝和Hoechst33258染色证实PBAM上无细胞残留,扫描电镜(SEM)观察PBAM表面呈多孔结构,孔隙率为93%。组织块法培养WJ-MSCs第6天可见WJ-MSCs呈鱼群样排列,行成脂、成骨诱导后,油红“O”和茜素红染色阳性。不同浓度PBAM浸提液对WJ-MSCs的增殖效果与DMEM培养液无差异。两组增殖高峰均在第5天,实验组细胞增加率高于对照组(P<0.05)。将WJ-MSCs复合至PBAM培养第5天后,Hoechst33258染色见大量WJ-MSCs生长于PBAM表面,AO染色提示为活细胞。电镜扫描可见大量人脐带WJ-MSCs黏附生长。 结论 PBAM对WJ-MSCs具有较好的相容性,可为进一步研究组织工程膀胱提供实验基础。

     

    Abstract: Objective To explore the biocompatibility of porcine bladder accellular matrix (PBAM) prepared by chemical method and human umbilical cord Wharton mesenchymal stem cells (WJ-MSCs), and provide new seed cells for further research of tissueengineered bladder. Methods PBAM was prepared by chemical decellular method, and HE, toluidine blue and Hoechst33258 fluorescent staining were used to detect the effect of the decellularization. The appearance of PBAM was observed by scanning electron microscope and the porosity was detected by anhydrous ethanol. WJ-MSCs were cultured by tissue block method, and its morphology was observed, then osteogenic and adipogenic differentiation were induced. The effects of different concentrations of PBAM leaching liquor on the proliferation of WJ-MSCs were compared. The experimental group combined WJ-MSCs to PBAM for culture, and the control group was cultured by the DMEM. At 3 days, 5 days, 7 days of cultivation, the WJ-MSCs were collected and counted. The cell-scaffold was observed by Hoechst33258 and AO staining. And the growing of WJ-MSCs on PBAM was observed by scanning electron microscope (SEM). Results PBAM was successfully prepared by chemical acellular method. HE, toluidine blue and Hoechst33258 staining confirmed no cell residue on PBAM. SEM showed a porous structure on the surface of PBAM, and the porosity of PBAM was 93%. At 6 days of cultivation, the WJ-MSCs were fibroblast-like, and after osteogenic and adipogenic induction, the results of oil red “O” and Alizarin Red stain were positive. The effect of different concentrations of PBAM leaching liquor on the proliferation of WJ-MSCs was similar to that of DMEM. The proliferation peaked at the fifth day in both two groups, and the proliferation rate of WJ-MSCs in the experimental group was higher than that of the control group (P< 0.05). At the fifth day after WJ-MSCs combined to the PBAM, Hoechst33258 staining showed that a large number of WJ-MSCs grew on the surface of PBAM, and AO staining suggested that they were living cells. SEM showed that a large number of WJ-MSCs adhered and grown. Conclusion PBAM shows good compatibility with WJ-MSCs, which provides an experimental basis for further study of tissue-engineered bladder.

     

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