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| 鹰嘴豆芽异黄酮对小鼠成骨前体细胞MC3T3-E1增殖及分化的研究 |
| Effects of isoflavones extracted from chickpea sprouts on proliferation and differentiation of mouse osteogenic precursor cells MC3T3-E1 |
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| DOI：10.3969/j.issn.1006-7108.2022.12.002 |
| 中文关键词: 鹰嘴豆芽异黄酮 MC3T3-E1细胞 成骨分化 |
| 英文关键词:isoflavones extracted from chickpea sprouts MC3T3-E1 cells osteogenic differentiation |
| 基金项目:国家自然科学基金项目( 81860746; 82060411); 新疆维吾尔自治区自然科学基金重点项目（2021D01D21） |
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| 中文摘要: |
| 目的 探讨鹰嘴豆芽异黄酮（isoflavones extracted from chickpea sprouts，ICS）对小鼠成骨前体细胞MC3T3-E1增殖、分化的影响及机制。方法 采用CCK-8法检测不同浓度ICS（0、0.1、0.5、1、5、10、50、100 mg/L）对MC3T3-E1细胞在24、48、72 h的增殖作用。成骨诱导培养基中加入不同浓度ICS（0、0.5、1、5 mg/L）观察其对成骨分化不同指标的影响：诱导第7天进行碱性磷酸酶(alkaline phosphatease, ALP)染色观察酶活性；第21天进行茜素红染色观察钙沉积；RT-qPCR检测诱导7 d各组Runt相关转录因子2（runt-related transcription factor 2，RUNX2）、骨钙素（osteocalcin，OCN）和1型胶原蛋白（collagen-1，COL1）mRNA表达水平；通过Western Blot检测诱导7 d RUNX2和COL1的表达水平，同时加入雌激素受体（estrogen receptor，ER）抑制剂ICI182 780（1 μmol/L），检测对成骨分化相关蛋白RUNX2和COL1蛋白表达的影响。结果 ICS在0~10 mg/L浓度范围促进了细胞的增殖（P＜0.05），当浓度大于10 mg/L时对细胞有毒性作用（P＜0.05）；与对照组（0 mg/L）比较，ICS浓度为0.5、1、5 mg/L均能促进ALP活性，具有显著差异，且具有剂量依赖性；诱导21 d后茜素红染色结果显示，ICS能够促进钙沉积，随剂量升高钙结节数量明显增多，具有统计学差异；ICS能够上调成骨分化相关基因RUNX2、OCN和COL1的mRNA转录水平（P＜0.05），促进成骨分化相关蛋白COL1、RUNX2的表达（P＜0.05）；雌激素受体抑制剂ICI 182,780抑制ICS对成骨分化相关蛋白COL1、RUNX2的表达（P＜0.05）。结论 ICS能够促进小鼠成骨前体细胞MC3T3-E1的增殖，并通过上调成骨分化相关基因及蛋白的表达，诱导MC3T3-E1细胞的成骨分化，雌激素受体抑制剂ICI 182,780 能够抑制ICS的成骨诱导分化作用，推测ICS是通过ER相关通路实现促进成骨分化作用的。 |
| 英文摘要: |
| Objective To investigate the effects and its mechanism of isoflavones extracted from chickpea sprouts (ICS) on the proliferation and the differentiation of the osteogenic precursor MC3T3-E1 cells. Methods CCK-8 was used to detect the proliferation of MC3T3-E1 cells at different concentrations of ICS (0, 0.1, 0.5, 1, 5, 10, 50 , 100 mg/L) at 24, 48, 72 h. Different concentrations of ICS (0, 0.5, 1,5 mg/L) were added into the osteogenic induction medium to observe its effects on the osteogenic differentiation: Alkaline phosphatase staining (ALP) assay was performed to observe the enzyme activities after 7-day induction; Alizarin red staining was performed on the 21st day to observe the calcium deposition; RT-qPCR was used to detect the mRNA expression levels of runt-related transcription factor 2 (RUNX2), osteocalcin (OCN) and type 1 collagen (COL1) in each group on the 7th day of induction; Western Blot was used to detect the expression levels of RUNX2 and COL1 on the 7th day of induction, and estrogen receptor (ER) inhibitor ICI182 780 (1 μmol/L) was also added to detect the effect on the protein expression of the osteogenic differentiation-related proteins RUNX2 and COL1.Results ICS promoted cell proliferation in a certain concentration range (1-10 mg/L), and had some toxicity to cells when the concentration was greater than 10 mg/L (P<0.05). Compared with the control group (0 mg/L), ICS at concentrations of 0.5,1,5 mg/L could promote alkaline phosphatase activity, with significant differences in a dose-dependent manner; ICS could promote calcium deposition, with a significant increase in the number of calcium nodules with increasing dose, with statistical differences; ICS could up-regulate the mRNA transcription levels of osteogenic differentiation-related genes RUNX2, OCN and COL1 (P<0.05), and promote the expression of osteogenic differentiation-related proteins COL1 and RUNX2 (P<0.05). The estrogen receptor inhibitor ICI 182,780 inhibited the promoting effect of ICS on osteogenic differentiation-related proteins COL1 and RUNX2 (P<0.05). Conclusion ICS can promote the proliferation of MC3T3-E1 osteogenic precursor cells in mice, and improve the osteogenic differentiation of MC3T3-E1 cells by up-regulating the expression levels of osteogenic differentiation-related genes and proteins. The use of estrogen receptor inhibitor ICI 182,780 can inhibit the osteogenic differentiation of ICS. It is speculated that ICS promotes osteogenic differentiation through ER-related pathway. |
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