摘要
目的 探讨利用诱导多功能干细胞（induced pluripotent stem cells,iPSc）技术重新编码人皮肤成纤维细胞（human skin fibroblast,HSF）,诱导分化为内皮细胞（endothelial cell,EC）作为种子细胞;以及以脱细胞猪主动脉瓣膜作为支架,体外构建组织工程心脏瓣膜（tissue engineered heart valve,TEHV）的可行性.方法 原代分离培养患者HSF细胞,制备携带OCT4、SOX2、KLF4、c-MYC基因的慢病毒上清感染HSF细胞,观察感染后细胞形态变化.碱性磷酸酶（AP）染色、细胞免疫荧光染色检测鉴定产生的HSF-iPSc.随后用诱导培养液在体外定向分化HSF-iPSc,Western blot检测分化过程中中皮标志物的表达;分化结束后,流式细胞术分选C D31阳性细胞,细胞免疫荧光染色检测HSF-iPSc-EC标志物,吞噬实验和成管实验检测HSF-iPSc-EC的功能.制备脱细胞猪主动脉瓣支架,行HE染色和扫描电镜观察.种植HSF-iPSe-EC于主脉瓣支架构建TEHV,采取静-动-静的模式培养,行HE染色和扫描电镜观察,免疫荧光染色检测α-SMA表达.结果 原代HSF经编码后形态与胚胎干细胞类似,AP染色阳性,并表达多潜能标志物Nanog、SSEA3、SSEA4、Tra-1-60、Tra-1-81.HSF-iPSc分化为EC过程中,内皮标志物CD31、VE-cad、vWF、eNOS不断上调.CD31阳性分选后获得的HSF-iPSc-EC均表达CD31、VE-cad、vWF、eNOS.同时,HSF-iPSc-EC具有明显的乙酰化LDL吞噬和成管能力.猪主动脉瓣膜细胞被完全脱去,且保留完整的纤维网状结构.构建的TEHV表面上内皮样细胞复层生长,排列有序且黏附良好.另外,种植的HSF-iPSc-EC呈空间特异性的表达α-SMA.结论 研究所得HSF-iPSc-EC可作为构建TEHV理想的种子细胞,初步实现了脱细胞主动脉瓣体外再内皮化的预期目标.
Objective To investigate the feasibility of constructing tissue engineered heart valve （TEHV） in deeellularized porcine aortic valve scaffolds by seeding endothelial cells （EC） differentiated from human skin fibroblasts （HSF） derived from pluripotent stem cells （iPSc）.Methods Primary HSF were isolated from the patient and infected by lentiviral supernatant harboring OCT4, SOX2, KLF4, c-MYC genes.Changes in cell morphology were observed after infection.Alkaline phosphatase （AP） staining,immunofluorescence staining were used to identify the derived HSF-iPSc.Then, HSF-iPSc were differentiated directly in induction medium.The markers of endothelial cells were detected by Western Blot during differentiation.CD31-positive cells were sorted by flow cytometry after differentiation.HSF-iPSc-EC markers were detected by immunofluorescence staining, and HSF-iPSc-EC function was analyzed by phagocytosis assay and tube formation.Decellularized porcine aortic valve scaffolds were prepared and observed by HE staining and scanning electron microscopy.TEHV was constructed by seeding HSF-iPSc-EC on the valve scaffolds and cultured by static-rotary-static model.Morphological characteristics of TEHV were observed by HE staining and scanning electron microscopy.α-SMA expression levels were detected by immunofluorescence staining.Results Morphology of the reprogrammed HSF was identical to the embryonic stem cells.AP staining was positive, and pluripotency markers of Nanog, SSEA3, SSEA4, Tra-1-60 and Tra-1-81 were expressed positively.The endothelial markers of CD31, VE-cad, vWF, eNOS were up-regulated continually during the differentiation of HSF-iPSc into EC.CD31, VE-cad, vWF, and eNOS were expressed in sorted HSF-iPSc-EC.Meanwhile, HSF-iPSc-EC had obvious acetylated LDL phagocytosis and tube formation capacity.Cells in porcine aortic valve were stripped completely and the reticular structure of fibers remained intact.Endothelial-like cells displayed stratified growth on the surface of the constructed TEHV with good order and adhesion.In addition,the planted HSF-iPSc-EC had the expression of α-SMA with spatial specificity.Conclusions The obtained HSF-iPSc-EC could be used as ideal seed cells for the construction of TEHV, and the goal of reendothelialization in decellularized aortic valve in vitro was realized preliminarily.
出处
《中华航海医学与高气压医学杂志》
CAS
CSCD
2015年第5期352-355,363,共5页
Chinese Journal of Nautical Medicine and Hyperbaric Medicine
基金
河南省卫生厅资助项目（200903128）
