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| 人BMSCs自噬水平和成骨分化能力比较 |
| Comparison of autophagic level and osteogenic capability in human BMSCs |
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| DOI：10.3969/j.issn.1006.7108.2018.11.010 |
| 中文关键词: 自噬相关蛋白质类 骨质疏松 骨髓间充质干细胞 成骨分化 |
| 英文关键词:autophagy-related proteins osteoporosis bone marrow mesenchymal stem cell osteogenic differentiation |
| 基金项目:四川省科技厅支撑计划资助项目（2014SZ0235);四川省卫生计生委项目（16PJ550) |
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| 中文摘要: |
| 目的 比较老年骨质疏松（osteoporosis，OP)和健康成年人椎体中骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs)自噬水平和成骨分化能力。方法 取从2组手术患者椎体中培养的BMSCs细胞，Western blot检测自噬相关基因LC3和P62蛋白表达。转染含LC3-GFP质粒的腺病毒后，激光共聚焦下观察代表自噬体的绿色荧光斑点数。细胞经成骨诱导分化培养基刺激后，碱性磷酸酶染色比较早期成骨指标，茜素红染色比较晚期成骨指标钙盐沉积的影响。荧光定量PCR (q-PCR)检测骨形成相关基因I(collagen type I，COL I)、骨钙素（osteocalcin，OCN)、骨桥蛋白（osteopontin, OPN)以及 Runt 相关转录因子2(RUNX2)。结果 Western blot结果显示OP组BMSCs的LC3-II/I表达明显降低，而P62表达明显增髙（P < 0.05);激光共聚焦观察进一步证实OP组中绿色荧光斑点数目显著下降，说明细胞内自噬体数目减少（P <0.05)。两组 BMSCs经成骨诱导后，茜素红和碱性磷酸酶染色发现OP组阳性程度明显弱于正常对照组，而q-PCR结果显示成骨分化的一些关键指标COL I、OCN、OPN和RUNX2在OP组中均显著下降，差异具有统计学意义（P <0. 05)。结论 骨质疏松的BMSCs中自噬水平较正常细胞低，且成骨分化能力更弱。 |
| 英文摘要: |
| Objective To compare the autophagic level and osteogenic capability of human vertebral bone marrow mesenchymal stem cells (BMSCs) from senile osteoporosis (OP) and young non-OP patients. Methods BMSCs was obtained from vertebrae of these two groups of patients and cultured. Western blot was used to detect the expression of autophagy associated proteins, LC3 and P62. The adenovirus containing LC3-GFP fusion protein was used to transfect BMSCs, and autophagy was evaluated by analyzing the number of green fluorescent puncta of autophagosomes under laser confocal microscopy. After stimulation of osteogenic differentiation medium，alkaline phosphatase staining was used to detect the early stage of osteoblast differentiation and alizarin red staining was used to detect the late stage of osteoblast differentiation. Fluorogenic quantitative PCR (q-PCR) was used to detect osteogenic genes， including collagen type I ( COL I), osteocalcin ( OCN), osteopontin ( OPN) and Runt-related transcription factor 2 (RUNX2). Results Western blot analysis showed that the protein expression ratio of LC3 II/I significantly decreased (P < 0. 05)，on the contrary，the protein expression of P62 significantly increased (P < 0. 05). In addition， after adenovirus transfection with LC3-GFP， the laser confocal microscopy observation showed a significant decline of green LC3 puncta that represented the number of autophagosome in BMSCs from OP group (P <0. 05). Furthermore，after osteogenic induction，the optical microscope observation showed that the positive degree of alkaline phosphatase and alizarin red staining was obviously weaker than those in the young control group. The q-PCR result further indicated that osteogenic genes，including COL I，OCN，OPN and RUNX2 all significantly decreased in BMSCs from OP group， and the differences were statistically significant (P <0.05). Conclusion Our study showed that there were low autophagic level and osteogenic capability in the osteoporotic BMSCs. |
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