Effect of Inhibiting PPARγ Expression on Osteogenic Differentiation of BMSCs
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摘要:
目的 探究抑制过氧化物酶增殖物激活受体γ（peroxisome proliferator-activated receptor γ，PPARγ）表达对BMSCs成骨分化的影响，为原发性骨质疏松症（primary psteoporosis ，POP）的治疗提供理论基础。 方法 将骨密度正常患者提取的BMSCs分为正常对照组（CON组），POP患者提取的BMSCs分为原发性骨质疏松组（POP组），取POP组细胞加入PPARγ抑制剂，分为抑制剂组（INR组），经成骨诱导分化后，检测各组细胞中骨桥蛋白（osteopontin, OPN）、骨钙素（osteocalcin，OCN）、Osterix、Runt相关转录因子2 （runt-related transcription factor 2 ，Runx2）的表达情况；碱性磷酸酶（alkaline phosphatase，ALP）染色观察各组BMSCs成骨分化情况。 结果 POP组ALP阳性细胞数低于CON组和INR组（P < 0.05）；与CON组相比，POP组中OCN、OPN、Osterix、Runx2表达量降低，差异具有统计学意义（P < 0.05）；与POP组相比，INR组中OCN、OPN、Osterix、Runx2表达量升高，差异具有统计学意义（P < 0.05）。 结论 抑制PPARγ表达后，BMSCs成骨分化特异性基因（ALP、OCN、OPN、Osterix、Runx2）表达增加。 Abstract:Objective To investigate the impact of inhibiting peroxisome proliferator-activated receptor γ （PPARγ） expression on osteogenic differentiation of BMSCs, so as to provide a theoretical basis for the treatment of primary osteoporosis （POP）. Methods BMSCs were extracted from patients with normal bone density and categorized into the normal control group（CON group）. BMSCs extracted from patients with primary osteoporosis were categorized into the primary osteoporosis group （POP group）. The cells from the POP group were treated with a PPARγ inhibitor and divided into the inhibitor group （INR group）. Following osteogenic differentiation, the expression of osteopontin （OPN）, osteocalcin （OCN）, Osterix, and runt-related transcription factor 2 （Runx2） was evaluated in the cells of each group.The alkaline phosphatase （ALP） staining was employed to assess the osteogenic differentiation of BMSCs in each group. Results The quantity of ALP-positive cells in the POP group was lower than that in the CON and INR groups （P < 0.05）. The expression of OCN, OPN, Osterix, and Runx2 was found to be decreased in the POP group when compared to the CON group, with statistically significant differences (P < 0.05）. Conversely, the expression of OCN, OPN, Osterix, and Runx2 was elevated in the INR group when compared to the POP group, with statistically significant differences (P < 0.05）. Conclusion Increased expression of osteogenic differentiation-specific genes （ALP, OCN, OPN, Osterix, Runx2） in BMSCs after inhibition of PPARγ expression. -
Key words:
- POP /
- PPARγ /
- BMSCs /
- Osteogenic differentiation
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图 2 各组PPARγ免疫荧光染色（标尺为50 μm）
A：CON组PPARγ表达的免疫荧光图； B：POP组PPARγ表达的免疫荧光图； C：INR组PPARγ表达的免疫荧光图。
Figure 2. Immunofluorescence staining for PPARγ in each group （scale bar: 50 μm）
图 4 各组ALP染色（中性红染色，标尺为50 μm）
A：CON组ALP染色；B：POP组ALP染色；C：INR组ALP染色。
Figure 4. ALP staining for each group （Neutral red staining，scale bar: 50 μm）
图 5 基因相对表达量
A：BMSCs中OCN基因相对表达量；B： BMSCs中OPN基因相对表达量；C： BMSCs中Osterix基因相对表达量；D： BMSCs中Runx2基因相对表达量。*P < 0.05，**P < 0.01，***P < 0.001。
Figure 5. Relative mRNA expression
图 6 蛋白相对表达量
A：BMSCs中OCN蛋白相对表达量；B：BMSCs中OPN蛋白相对表达量；C：BMSCs中Osterix蛋白相对表达量；D：BMSCs中Runx2蛋白相对表达量。*P < 0.05，**P < 0.01，***P < 0.001。
Figure 6. Relative protein expression
表 1 免疫荧光半定量方差分析（$ \bar x \pm s $，n = 3）
Table 1. The ANOVA for semi-quantification of immunofluorescence （$ \bar x \pm s $，n = 3）
组别 平均荧光强度 CON组 27.65±3.93 POP组 105.98±13.95 INR组 40.72±13.20 F 41.229 P <0.001* *P < 0.05。 
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表 2 各组基因相对表达量（$ \bar x \pm s $，n = 3）
Table 2. Relative expression of genes in each group（$ \bar x \pm s $，n = 3）
组别 OCN OPN Osterix Runx2 CON组 0.95±0.07 1.21±0.20 1.20±0.17 1.23±0.20 POP组 0.40±0.07 0.46±0.03 0.58±0.11 0.48±0.04 INR组 0.66±0.05 0.80±0.03 0.85±0.12 0.86±0.16 F 59.832 31.647 15.947 19.007 P <0.001* 0.001* 0.004* 0.003* *P < 0.05。
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表 3 各组蛋白相对表达量（$ \bar x \pm s $，n = 3）
Table 3. Expression levels of each group of proteins（$ \bar x \pm s $，n = 3）
组别 OCN OPN Osterix Runx2 CON组 0.74±0.07 0.44±0.06 0.55±0.02 0.54±0.16 POP组 0.51±0.11 0.3±0.03 0.22±0.03 0.31±0.08 INR组 0.89±0.28 0.51±0.03 0.38±0.06 0.52±0.02 F 7.189 37.672 93.211 9.690 P 0.006* <0.001* <0.001* 0.002* *P < 0.05。
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