不同代数SD大鼠骨髓间充质干细胞体外增殖和成骨分化能力的实验研究

doi:10.3969/j.issn.1005-4979.2021.01.003

摘要/Abstract

摘要：

目的: 探讨不同代数SD大鼠骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）体外增殖和成骨分化能力的差异。方法: 从SD大鼠骨髓中分离并培养原代BMSCs，免疫荧光染色鉴定BMSCs。传代培养分别获取P1、P3、P5、P7、P9代BMSCs。CCK-8实验检测不同代数BMSCs的增殖能力；显微镜下观察细胞形态学特征；免疫荧光染色检测细胞骨架；流式细胞术检测细胞凋亡情况；碱性磷酸酶（alkaline phosphatase，ALP）染色和茜素红染色比较不同代数BMSCs的体外成骨分化能力；实时聚合酶链式反应（real-time polymerase chain reaction，RT-PCR）和免疫荧光检测不同代数BMSCs成骨分化标志物Ⅰ型胶原A1（collagen type Ⅰ α1，COL1A1）、骨钙素（osteocalcin，OCN）基因表达和蛋白水平。结果: P1、P3、P5代间BMSCs的细胞形态、体外增殖、细胞凋亡及成骨分化能力无显著差别；而P7和P9代BMSCs各方面指标与P1代BMSCs比较，均明显降低（P<0.05）。结论: 5代以后的BMSCs随着传代次数的增加，其细胞增殖和成骨分化能力显著降低。

关键词: 不同代数, 骨髓间充质干细胞, 成骨分化, 组织工程, 细胞骨架

Abstract:

Objective: To investigate the in vitro proliferation and osteogenic differentiation ability of bone marrow mesenchymal stem cells (BMSCs) of SD rats in different generations. Methods: Primary BMSCs were isolated and cultured from rats, and BMSCs were identified using immunofluorescence analysis. The BMSCs were subcultured to obtain P1, P3, P5, P7 and P9 generations of BMSCs. The proliferation of BMSCs in different generations were tested by CCK-8 assay. The cell morphology of BMSCs in different generations were analyzed under microscope and immunofluorescence assay was used for cytoskeleton protein evaluation. Flow cytometry was applied to identify cell apoptosis. Both alkaline phosphatase (ALP) and alizarin red staining tested the osteogenic differentiation ability of BMSCs in different generations in vitro. Real-time polymerase chain reaction (RT-PCR) and immunofluorescence were used to detect the gene expression and protein level of osteogenic marker collagen type Ⅰ α1(COL1A1) and osteocalcin (OCN). Results: There were no significant differences in the morphology, proliferation, cell apoptosis and osteogenic differentiation between P1, P3, P5 generations of BMSCs, but remarkable decrease was observed in P7 and P9 generations of BMSCs more than P1 generation. Conclusion: After 5 generations, the proliferation and osteogenic differentiation ability of BMSCs significantly decreased with aging.

Key words: different generations, bone marrow mesenchymal stem cells, osteogenic differentiation, tissue engineering, cytoskeleton

中图分类号:

R782

王冬佳, 张雄. 不同代数SD大鼠骨髓间充质干细胞体外增殖和成骨分化能力的实验研究[J]. 《口腔颌面外科杂志》, 2021, 31(1): 16-23.

WANG Dongjia, ZHANG Xiong. Comparison of Proliferation and Osteogenic Differentiation Potential in Different Generations of BMSCs Derived from SD Rats[J]. 《Journal of Oral and Maxillofacial Surgery》, 2021, 31(1): 16-23.

https://journal06.magtech.org.cn/Jweb_joms/CN/Y2021/V31/I1/16

图/表 10

表1 引物序列

Table 1 Primer sequence

基因名称	正向引物序列（5′- 3′）	反向引物序列（5′- 3′）
β-actin	F：GGCACCACACCTTCTACAATGAGC	R：GATAGCACAGCCTGGATAGCAACG
COL1A1	F：GGCGAGAGAGGTGAACAAGG	R：GCCAAGGTCTCCAGGAACAC
OCN	F：CAGGCGCTACCTGTATCAATGGC	R：GCCGATGTGGTCAGCCAACTC

图1 免疫荧光染色鉴定大鼠BMSCs（×200） A. CD44在BMSCs细胞膜上呈阳性表达；B. DAPI细胞核染色图；C. CD44和DAPI染色融合结果；D. CD90在BMSCs细胞膜上呈阳性表达；E.DAPI细胞核染色图；F. CD90和DAPI染色融合结果。

Figure 1 Immunofluorescence detection of the cluster of differentiation on rat BMSCs （×200）

图2 光学显微镜下观察不同代数大鼠BMSCs（×100） A. P0代；B. P1代；C. P3代；D. P5代；E. P7代；F. P9代。

Figure 2 Microscopic images of BMSCs in rats with different generations（×100）

图3 荧光显微镜下观察不同代数大鼠BMSCs (×400) A—C. P1代；D—F. P3代；G—I. P5代；J—L. P7代；M—O. P9代。

Figure 3 Immunofluorescence staining of BMSCs in rats with different generations (×400)

图4 P1、P3、P5、P7、P9 代 BMSCs生长曲线 *代表与P1代BMSCs比较，P<0.05，差异有统计学意义。

Figure 4 The growth curve of BMSCs in P1、P3、P5、P7 and P9

图5 P1、P3、P5、P7、P9代 BMSCs细胞凋亡结果 A. 流式细胞术检测细胞凋亡比例；B. 统计学分析结果。*表示P<0.05，与P1代BMSCs比较，差异有统计学意义。

Figure 5 Cell apoptosis of BMSCs in P1, P3, P5, P7 and P9

图6 P1、P3、P5 P7、P9代BMSCs ALP活性表达 A. ALP染色结果；B. ALP半定量结果。*表示P<0.05，与P1代BMSCs比较，差异有统计学意义。

Figure 6 Expression of ALP in P1, P3, P5, P7 and P9 of BMSCs

图7 P1、P3、P5、P7、P9代BMSCs钙结节形成结果 A. 茜素红染色结果；B. 茜素红半定量结果。*表示P<0.05，与P1代BMSCs比较，差异有统计学意义。

Figure 7 Formation of calcium nodules in P1, P3, P5, P7 and P9 of BMSCs

图8 RT-PCR检测不同代数BMSCs COLIA1和OCN mRNA的表达 *表示P<0.05，与P1代BMSCs比较，差异有统计学意义。

Figure 8 The mRNA expression of COL1A1 and OCN in different generations of BMSCs using RT-PCR

图9 免疫荧光检测不同代数BMSCs COL1A1和OCN蛋白表达（×400） A—D. P1代；E—H. P3代；I—L. P5代；M—P. P7代；Q—T. P9代。

Figure 9 Protein levels of COL1A1 and OCN in different generations of BMSCs detected by immunofluorescence staining （×400）

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