大鼠脂肪间充质干细胞的分离、培养及其向少突胶质前体细胞的诱导分化

doi:10.16098/j.issn.0529-1356.2022.05.003

摘要/Abstract

摘要：

目的改良酶消化法提取脂肪间充质干细胞(ADSCs)，并探讨诱导其分化为少突胶质前体细胞(OPCs)的可行性，为脱髓鞘疾病治疗种子细胞来源提供有效途径。方法取SD大鼠腹股沟区脂肪，Ⅰ型胶原酶和胰蛋白酶联合消化法分离培养ADSCs，Ⅰ型胶原酶组作为对照，细胞计数，观察细胞形态。取第3代细胞，流式细胞术检测细胞的表面标志物CD29、CD90和CD45；成脂诱导，油红O染色；利用干细胞分化培养液及OPCs培养液将ADSCs诱导为OPCs，免疫荧光检测α-N-乙酰基神经氨酸 α-2、8-唾液酸转移酶1（A2B5）、NG2；利用OPCs培养液继续培养OPCs分化为少突胶质细胞(OLs)。结果 Ⅰ型胶原酶组分离的ADSCs为（9.143±0.5084）×10⁷/L，联合酶组为（13.43±0.4809）×10⁷/L，联合酶组较Ⅰ型胶原酶组ADSCs数量多，差异有统计学意义（P＜0.05，n=7）；细胞形态观察显示，ADSCs贴壁生长，呈长梭形；流式细胞术检测CD29 阳性率为99.8%，CD90阳性率为 99.4%，CD45 阳性率为0.35%；成脂诱导后倒置光学显微镜下可见脂滴，油红O染色脂滴呈红染；ADSCs向OPCs诱导第16天，A2B5和NG2表达阳性，A2B5阳性率为（87.03±0.94）%，NG2阳性率为（90.07±0.96）%;继续培养OPCs 3 d，其分化为OLs，髓鞘碱性蛋白（MBP）表达阳性。结论联合酶消化较单独胶原酶消化获取的细胞数量多，分离培养的细胞符合ADSCs的特征，体外可诱导分化为OPCs。

关键词: 脂肪间充质干细胞, 分离, 培养, 诱导分化, 少突胶质前体细胞, 神经脱髓鞘疾病, 流式细胞术, 大鼠

Abstract:

Objective To explore the possibility of rat adiposederived stem cells (ADSCs) to differentiate into oligodendrocyte precursor cells(OPCs) and find an effective way to treat demyelinating disease. Methods ADSCs from the inguinal region of SD rats were isolated, digested with collagenase type Ⅰ and trypsin, collagenase type Ⅰ digestion method as control, counted and compared; Cultured in vitro and observed the growth characteristics. After ADSCs subcultured 3 times of passages, CD29, CD90 and CD45 were detected by flow cytometry; After differentiation into adipocyte, the cells were identified by the staining of oil red O; After differentiation into OPCs by stem cell differentiation medium and OPCs induced differentiation medium, the expression of α-N-acetylneuraminic acid α-2, 8-sialyltransferase Ⅰ(A2B5) and NG2 was detected by immunofluorescent staining. Results The number of ADSCs in the combined enzyme group was higher than the collagenase type 1 group (P<0.05, n=7); ADSCs grew in a long shuttle type and their morphology tended to be stable after passage. The surface marker CD29,CD90 were positive, and CD45 was negative. After adipogenic induction, oil red O staining showed red lipid droplets of varying sizes in the cells. After OPCs induction, immunofluorescence detection showed that positive reaction of cell surface fluorescence was seen with antibody to A2B5 and NG2,(87.03±0.94)% expressed A2B5, (90.07±0.96)% expressed NG2. After cultured for 3 days, immunofluorescence detection showed that positive reaction of cell surface fluorescence was seen with antibody to myelin basic protein (MBP). Conclusion ADSCs are obtained by combined enzyme digestion and the cells are much more than collagenase alone and can be induced to OPCs in vitro.

Key words: Adipose-derived stem cell, Isolation, Cultivation, Induced differentiation, Oligodendrocyte Precursor cell, Demyelinating disease, Flow cytometry, Rat

中图分类号:

张雅群付丽任译延乔妍梓赵冬梅. 大鼠脂肪间充质干细胞的分离、培养及其向少突胶质前体细胞的诱导分化[J]. 解剖学报, 2022, 53(5): 557-562.

ZHANG Ya-qun FU Li REN Yi-yan QIAO Yan-zi ZHAO Dong-mei. Isolation and culture of rat adipose-derived stem cells and differentiation into oligodendrocyte precursor cells[J]. Acta Anatomica Sinica, 2022, 53(5): 557-562.

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