大鼠阴茎海绵体肌源性干细胞多向分化潜能的鉴定

发布时间：2018-11-02 06:52

【摘要】：目的：应用大鼠阴茎海绵体分离、纯化肌源性干细胞（muscle derived stem cells,MDSCs）,检测干细胞标志物的表达，并将分离得到的细胞进行传代培养；检测分离细胞在特定条件下向成脂细胞及成骨细胞分化的潜能；检测分离细胞在特定条件下向内皮细胞分化的潜能。 方法：取2月龄SD雄性大鼠为实验对象，用酶消化法结合percoll密度梯度离心法及改良的Preplate差速贴壁法，分离、纯化大鼠阴茎海绵体肌源性干细胞。利用免疫荧光细胞化学法检测肌源性干细胞标记物干细胞抗原1（Sca-1）和结蛋白（Desmin）在PP6细胞中的表达，并对PP6细胞进行传代培养。将第3代PP6细胞在成脂及成骨诱导分化培养基中培养21d，同时对照组采用干细胞培养基培养；分别于诱导0d、7d、14d、21d通过油红“O”染色，观察是否有脂滴形成；通过茜素红染色，观察是否有成骨细胞形成的钙结节。将第3代PP6细胞在血管内皮细胞生长因子（vascularendothelial growth factor, VEGF）和碱性成纤维细胞生长因子（Basic fibroblast growthfactor, bFGF）共同诱导21d后，免疫荧光细胞化学法鉴定内皮细胞标志物CD31及胎肝激酶-1（Flk-1）的表达；分别在诱导0d、5d、10d、15d、21d，通过逆转录-聚合酶链反应（RT-PCR）检测干细胞标志物（Oct-4）和内皮细胞标志物CD31、内皮型一氧化氮合酶（eNOS）、Flk-1的mRNA表达量；通过Dil-Ac-LDL摄取实验检测细胞吞噬低密度脂蛋白的能力；划痕24h后观察划痕空白处的距离，评估细胞的迁移能力。 结果：分离、纯化的PP6细胞表达肌源性干细胞标志物Sca-1和Desmin。在成脂诱导分化过程中，诱导前3d可见细胞的形状由长梭形变为圆形或者三角形，排列紧密，呈叠瓦样，细胞核清晰，细胞分裂增殖明显减慢，油红O染色诱导组与对照组均未见细胞红染；7d左右在相差显微镜下可观察到胞质内有少量的高折光性小脂滴出现，大小均匀，主要集中在细胞核周围，油红O染成红色；14d左右细胞内脂滴逐渐增多并融合成大脂滴，细胞核被挤到一边或者消失；诱导21d细胞分化达高峰，，细胞体积达到最大，胞质内脂滴数量最多，继续诱导，细胞内未见空泡增多。而对照组细胞形态未发生变化，且油红O染色阴性。在成骨诱导分化过程中，诱导前7d可见细胞的形状由长梭形变为圆形或者三角形，排列紧密，细胞核清晰，细胞分裂增殖明显减慢，茜素红染色两组均未见钙结节形成；诱导14d后，细胞出现层叠、聚集生长态势，融合的细胞呈现“叠瓦样”改变，细胞体积变大、形态呈多边形，核大，核仁清晰，细胞内颗粒增多，细胞外有少量基质分泌，茜素红染色可见散在分布的钙结节形成；诱导21d时镜下可见细胞内颗粒增多，细胞外基质分泌旺盛，茜素红染色显示细胞间红色矿化基质沉淀，钙结节形成。在内皮细胞诱导分化过程中，PP6诱导21d后表达内皮细胞标志物（CD31和Flk-1）；诱导21d后与诱导0d比较，干细胞标志物Oct-4的mRNA表达量下降（下降倍数为0.445±0.025），而内皮细胞标志物mRNA表达量升高（CD31、eNOS、Flk-1升高倍数分别为2.541±0.146、2.364±0.175、2.538±0.100），差异均有统计学意义（P0.05）；Dil-Ac-LDL摄取实验提示诱导后的细胞具有吞噬低密度脂蛋白的能力；划痕实验观察到诱导组空白处距离为282.01±3.84，未诱导组空白处距离为450.35±1.86，差异有统计学意义（P0.05）。 结论：大鼠阴茎海绵体中分离纯化的细胞表达肌源性干细胞标记物，并且在特定条件下可分化为成脂细胞、成骨细胞及内皮细胞，进一步证实了阴茎海绵体中存在肌源性干细胞。
[Abstract]:Objective: To detect the expression of stem cell marker in rat corpus cavernosum, purify muscle source stem cells (MDSCs), to detect the expression of stem cell markers, and to detect the potential of isolated cells to differentiate into adipocytes and osteoblasts under specific conditions; Detection of the potential of isolated cells to differentiate into endothelial cells under specific conditions. Methods: Two-month-old SD male rats were taken as experimental subjects, and percoll density gradient centrifugation and modified Preplate differential attachment method were combined with enzyme digestion to separate and purify the muscular source of corpus cavernosum of rats. The expression of Sa-1 and Desmin in PP6 cells was detected by immunofluorescence cytochemical method, and the P6 cells were passaged. culturing the 3rd generation PP6 cells in lipoid and bone inducing differentiation medium, and culturing in the control group by using stem cell culture medium; respectively inducing 0d, 7d, 14d, 21d; over-oiled red "O" staining to see if lipid droplets were formed; by staining with Congo red, observe whether osteoblasts were formed or not. The expression of the endothelial cell marker CD31 and fetal liver kinase-1 (Flk-1) was identified by immunofluorescence cytochemical method after 21days induction by vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). 5d, 10d, 15d, 21d, detecting stem cell markers (Oct-4) and endothelial cell markers CD31, endothelial nitric oxide synthase (VEGF) and Flk-1 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR); Ability to evaluate cell migration by observing the distance in the blank space after scratch 24h Ability. Results: Isolated, purified PP6 cells express muscle-derived stem cell markers Sca-1 and D In the process of lipoid induction and differentiation, the shape of the visible cells induced by the induction of lipoid induction was circular or triangular in shape of long shuttle, closely aligned, stacked tiles, clear cell nucleus, obvious decrease in cell division and proliferation, and neither the oil red O staining induction group nor the control group It can be seen that a small amount of high-refractive small lipid droplets appear in the cytoplasm and the size is even, mainly in the periphery of the nucleus, the oil red O is dyed red, and the fat drops in the left and right cells are gradually increased and fused into big fat drops, and the nuclei are squeezed to one. The differentiation of 21d cells reached the peak, the volume of cells reached the maximum, the number of intracellular lipid droplets was the most, continued to induce, and the cells were not In the control group, the morphology of the cells did not change and the oil was red. O staining was negative. In the process of bone induction and differentiation, the shape of the visible cells was circular or triangular in shape of long shuttle, the arrangement was tight, the cell nucleus was clear, cell division and proliferation were obviously slowed down, and no calcium nodules were found in the two groups. The cells were laminated, aggregated and grown, the fused cells showed 鈥渟tacked tile鈥

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