目的: 体外探讨脂多糖(lipopolysaccharide,LPS)刺激产生的炎症条件下,过表达WNT11对成骨细胞成骨能力的影响。方法: 通过慢病毒将WNT11靶向序列转染至成骨细胞后,从功能学角度采用蛋白免疫印迹检测WNT11过表达的效果,并采用CCK-8法检测转染后的细胞增殖活性。体外实验设置3组细胞:LPS-非诱导组、LPS-常规诱导组、LPS-WNT11过表达诱导组,所有细胞体外诱导后经茜素红染色,矿结节及碱性磷酸酶(alkaline phosphatase, ALP)半定量分析,并进行qRT-PCR分析诱导后成骨特异基因ALP、成骨特异性转录因子(runt-related transcription factor 2, Runx2)、骨钙素(osteocalcin, OC)及胶原蛋白I型(collagen I)mRNA的表达水平,综合评价成骨能力的差异。结果: 成骨细胞经慢病毒转杂后,Western blot检测结果显示WNT11表达水平高于非过表达对照组(P＜0.001)。相对于未转染对照细胞,转染组细胞在任何时间点未显示出明显的细胞活性差异(P＞0.05)。成骨细胞在LPS(100 ng/mL)浓度下培养未见明显大量细胞凋亡,细胞经诱导1周和2周后,诱导组可见矿结节生成,且随着时间延长逐渐增多,而对照组无明显矿结节生成。矿结节定量分析和ALP定量分析结果表明诱导1周或2周,WNT11过表达组皆高于常规诱导组(P＜0.000 1);成骨特异基因表达水平亦显示相同的趋势。结论: 过表达WNT11可在体外LPS所致的炎症条件下显著增强成骨细胞的成骨能力。
Objective: To explore the effect of over-expressing WNT11 on osteogenesis of osteoblasts under LPS-incurred inflammatory condition in vitro. Methods: The objective gene sequences targeting for WNT11 were transduced into osteoblasts by lentivirus, then the WNT11 protein level was tested by Western blot assay from the functional perspective. The cells after transduction were evaluated the cell proliferation activity by the CCK-8 method. The experimental cells were enrolled into 3 groups in vitro: non-induced group with LPS, general induced group with LPS and over-expressing WNT11 induced group with LPS. All cells were induced in vitro by alizarin red staining, mineral nodules and alkaline phosphatase (ALP) semi-quantitative analysis, and qRT-PCR was used to analyze the expression levels of osteogenic specific genes ALP, runt-related transcription factor 2 (Runx2), osteocalcin (OC) and collagen I mRNA after induction, and the differences in osteogenic ability were comprehensively evaluated. Results: After transduction using lentivirus, the results of Western blot assay showed the WNT11 protein level was significantly higher than that in control group (P＜0.001). The cell proliferation activity demonstrated no significant differences after transduction, when compared with non-transduction at any time point (P＞0.05). There was no significant apoptosis of osteoblasts cultured in LPS (100 ng/mL) concentration. After 1 week and 2 weeks of induction, mineralized nodules were observed in the induction group and gradually increased with time, while no obvious mineralized nodules were observed in the control group.The quantitatively analysis results of mineralization nodules and ALP activity were significant higher in WNT11 over-expressing group than in general induced group (P＜0.000 1). Moreover, the results of osteogenic relate genes demonstrated similar trends. Conclusion: Over-expressing WNT11 may significantly enhances the osteogenic ability of osteoblasts under LPS-incurred inflammatory condition.
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