脐带Wharton胶干细胞外基质的制备及其细胞相容性观察

发布时间：2018-11-11 14:32

【摘要】：[目的]建立制备脐带Wharton胶干细胞细胞外基质(extracellular matrix,ECM)的方法并对其生物相容性进行观察。[方法]无菌条件下收集人脐带组织,采用酶消化法分离培养脐带干细胞,使用普通培养基培养7 d后,在原培养基中添加抗坏血酸,再连续培养8 d;加入含有0.5%Triton X-100和20 mM NH 4OH的PBS溶液进行脱细胞处理后,用Hoechst 33258染色观察其DNA残留,采用扫描电镜对其微结构进行观察,通过天狼星红、甲苯胺蓝、番红花O组织学染色和I型胶原、II型胶原、纤维粘连蛋白及层粘连蛋白免疫荧光染色观察微载体的主要成分;将异体脐带干细胞种植在无细胞核DNA残留的ECM上,通过MTT法观察脐带干细胞的增殖生长情况。[结果]通过本实验方法得到的天然脱细胞脐带干细胞ECM,表面无细胞及DNA残留,整体表面有细胞陷窝形成,基质紧密连接。Hoechst 33258荧光染色阴性;天狼星红、甲苯胺蓝和番红花O组织学染色阳性;I型胶原、II型胶原、纤维粘连蛋白和层粘连蛋白免疫荧光染色阳性;在MTT检测细胞增殖中,种植于该ECM上的异体脐带干细胞组的OD值高于单纯平面培养组(P0.05)。[结论]此次实验所得的脱细胞脐带干细胞外基质具有良好的生物相容性,能够为组织工程提供高质量的种子细胞,有望成为组织工程种子细胞的新型培养载体。
[Abstract]:[objective] to establish a method for preparation of umbilical cord Wharton adhesive stem cell extracellular matrix (extracellular matrix,ECM) and to observe its biocompatibility. [methods] Human umbilical cord tissues were collected under aseptic condition. Umbilical cord stem cells were isolated and cultured by enzyme digestion. After 7 days of culture in normal culture medium, ascorbic acid was added to the original medium and cultured for 8 days. After acellular treatment with PBS solution containing 0.5%Triton X-100 and 20 mM NH 4OH, the DNA residue was observed by Hoechst 33258 staining, and its microstructure was observed by scanning electron microscope. The main components of microcarriers were observed by immunohistochemical staining of saffron O, type I collagen, II collagen, fibronectin and laminin. The allogeneic umbilical cord stem cells were implanted on ECM without nuclear DNA residue. The proliferation and growth of cord stem cells were observed by MTT method. [results] the ECM, surface of natural acellular umbilical cord stem cells obtained by this method was free of cells and DNA residues, cell lacunae were formed on the whole surface, matrix was tightly connected. Hoechst 33258 fluorescent staining was negative. Sirius red, toluidine blue and saffron O were positive for histological staining, type I collagen, II collagen, fibronectin and laminin were positive for immunofluorescence staining. In the MTT detection of cell proliferation, the OD value of cord allogeneic stem cells implanted on the ECM group was higher than that of the flat culture group (P0.05). [conclusion] the extracellular matrix of acellular umbilical cord stem cells obtained in this experiment has good biocompatibility and can provide high quality seed cells for tissue engineering, which is expected to be a new culture carrier for tissue engineering seed cells.
【作者单位】：解放军总医院骨科;
【基金】：国家自然科学基金资助项目(编号:31240048) 全军十二五重点项目(编号:BWS11J025)
【分类号】：R318.08

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