大鼠阴茎海绵体肌源性干细胞的分离及鉴定

背景：前期研究在大鼠阴茎海绵体组织中检测到了具有干细胞标志物及肌源性标志物的干细胞。目的：在前期研究基础上,对大鼠阴茎海绵体肌源性干细胞进行分离及表型鉴定。方法：取2月龄SD大鼠阴茎海绵体组织,采用Ⅰ型胶原酶、胰蛋白酶消化分离组织,采用Preplate差速贴壁技术对细胞进行初步纯化,获得PP1~PP6贴壁细胞,并进行流式细胞仪检测、免疫荧光细胞化学鉴定及Western blotting检测。结果与结论：连续6步差速贴壁分离后,PP1~PP6细胞贴附能力逐渐减弱,PP6细胞两三天后开始贴壁形成圆形或梭形。流式细胞仪检测PP6细胞中干细胞抗原1（＋）5.7%、胚胎抗原（＋）2.6%、结蛋白（＋）41.2%。免疫荧光细胞化学显示仅有少量细胞分别表达干细胞抗原1和胚胎抗原,均散在分布,干细胞抗原1主要表达于胞浆,胚胎抗原主要表达于胞核,表达结蛋白的细胞聚集,数量较多,主要表达于胞浆;亦发现有极少量双阳性表达细胞,即干细胞抗原1/胚胎抗原、干细胞抗原1/结蛋白和胚胎抗原/结蛋白。PP1~PP5细胞中未检测到干细胞抗原1及胚胎抗原蛋白明显表达,但在PP6细胞中则检测到干细胞抗原1及胚胎抗原蛋白的表达。结蛋白在PP1~PP6细胞中的表达逐渐增强。提示在大鼠阴茎海绵体中发现具有干细胞及肌源性干细胞表型的细胞。

Isolation and determination of muscle-derived stem cells in the rat penile corpus cavernosum

BACKGROUND：Previous studies have confirmed that stem cells with stem cell markers and muscle-derived stem cell markers were detected in rat penile corpus cavernosum.OBJECTIVE：To perform isolation and phenotype identification of muscle-derived stem cells in rat penile corpus cavernosum on the basis of previous study.METHODS：The penile corpus cavernosum from 2-month-old Sprague-Dawley rats was digested and dissociated by type Ⅰ collagenase and trypsin.Cells were primarily purified by the Preplate differential adhesion technique.PP1-PP6 adhered cells were obtained and the expressions of stem cell markers were detected by flow cytometry,immunofluorescence and western blotting.RESULTS AND CONCLUSION：The adherent capacity of PP1-PP6 cells was gradually weakened after 6-step differential adhesion.After two to three days,PP6 cells began to be adherent and became round or spindle-shaped.Flow cytometry detected stem cell antigen 1（＋） 5.7%,embryonic antigen（＋） 2.6%,and desmin（＋） 41.2% in PP6 cells.Immunofluorescence exhibited that only a few cells expressed stem cell antigen 1 and embryonic antigen,showing diffused distribution.Stem cell antigen 1 was mainly expressed in cytoplasm,and embryonic antigen was mainly expressed in nuclei.Cell aggregation expressing desmin showed a large number and mainly expressed in cytoplasm.Very a few double-positive cells,such as stem cell antigen 1/embryonic antigen,stem cell antigen 1/desmin and embryonic antigen/desmin,were detected.No significant expression of stem cell antigen 1 and embryonic antigen protein was determined in PP1-PP5 cells.However,stem cell antigen 1/embryonic antigen protein was detected in PP6 cells.Desmin expression was gradually enhanced in PP1-PP6 cells.Results indicated that the cells from rat penile corpus cavernosum had the cell phenotype of stem cells and muscle-derived stem cells.