인간 내 항문괄약근을 이용한 내외괄약근의 전구세포 분리 동정에 대한 연구

Abstract

Introduction: We investigated internal and external sphincter progenitor cells as potential tools for tailored cell therapies for fecal incontinence. Methods: Sphincter progenitor cells were isolated with the preplate technique from normal internal and external anal sphincters from 10 patients with rectal cancer who underwent abdominoperineal resection. The isolated cells and differentiated muscle fibers were identified with an immunofluorescence assay, western blotting, and RT–PCR. The proliferation of the progenitor cells with or without radiotherapy was compared with quantitative clonogenic and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Results: Immunofluorescence assay before differentiation confirmed that the internal anal sphincter progenitor cells expressed CD34 and neural-glial antigen 2 (NG2), whereas the external anal sphincter progenitor cells expressed CD34, PAX7, and NG2. After differentiation, the internal anal sphincter progenitor cells expressed α-SMA, calponin, and desmin, whereas the external anal sphincter progenitor cells expressed MYC, MYOG, and desmin. Differential expression profiles were observed for the two cell types with western blotting and RT–PCR, which were identical to those observed with immunofluorescence. In MTT assays of the internal anal sphincter progenitor cells, cell viability was lower in the radiotherapy group than in the nonradiotherapy group at 24 h (14·1% vs 11·5%, respectively, p = 0·033), 48 h (14·7% vs 11·9%, respectively, p = 0·022), 72 h (15·3% vs 12·5%, respectively, p = 0·012), 96 h (16·2% vs 13·1%, respectively, p = 0·012), and 120 h (14·8% vs 13·6%, respectively, p = 0·536), as confirmed in the external anal sphincter progenitor cells and with clonogenic assays. Conclusions: This is the first study to differentially harvest internal and external sphincter muscle progenitor cells from humans. We suggest that internal- or external-anal-sphincter-muscle-derived cells can be used for tailored cell therapies for fecal incontinence.