降钙素基因相关肽通过Hippo通路调控小鼠骨髓间充质干细胞成骨分化的实验研究
目的前期研究发现降钙素基因相关肽（CGRP）能够促进成骨细胞的生物学活性,为了进一步揭示CGRP在骨修复中的作用,检测CGRP对小鼠骨髓间充质干细胞（BMSCs）成骨分化的影响,并对Hippo通路在这个过程中的作用进行了初步探讨。方法在体外诱导培养的BMSCs中加入不同浓度的CGRP,处理48 h后测试碱性磷酸酶（ALP）活性,以筛选的优势浓度;处理7 d后进行茜素红染色,分别检测细胞的分化情况。应用Western blot检测CGRP作用于BMSCs后,Hippo通路核心分子Mst1/2蛋白磷酸化的表达水平;利用Hippo通路抑制剂维替泊芬（Verteporfin）阻断下游Yap信号,逆转录...
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| Published in | 华西口腔医学杂志 Vol. 34; no. 3; pp. 286 - 290 |
|---|---|
| Main Author | |
| Format | Journal Article |
| Language | Chinese |
| Published |
第三军医大学新桥医院口腔科,重庆,400037
2016
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| Subjects | |
| Online Access | Get full text |
| ISSN | 1000-1182 |
| DOI | 10.7518/hxkq.2016.03.014 |
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| Abstract | 目的前期研究发现降钙素基因相关肽（CGRP）能够促进成骨细胞的生物学活性,为了进一步揭示CGRP在骨修复中的作用,检测CGRP对小鼠骨髓间充质干细胞（BMSCs）成骨分化的影响,并对Hippo通路在这个过程中的作用进行了初步探讨。方法在体外诱导培养的BMSCs中加入不同浓度的CGRP,处理48 h后测试碱性磷酸酶（ALP）活性,以筛选的优势浓度;处理7 d后进行茜素红染色,分别检测细胞的分化情况。应用Western blot检测CGRP作用于BMSCs后,Hippo通路核心分子Mst1/2蛋白磷酸化的表达水平;利用Hippo通路抑制剂维替泊芬（Verteporfin）阻断下游Yap信号,逆转录聚合酶链反应检测其对成骨相关因子Ⅰ型胶原蛋白（ColⅠ）、Runt相关转录因子2（Runx2）mRNA的表达影响。结果 ALP活性检测结果显示,与空白对照组相比,10^（-9）、10^（-8）、10^（-7） mol·L^（-1）浓度范围的CGRP都能显著促进小鼠ALP活性的增加（P〈0.05）,以10^（-8） mol·L^（-1）浓度的CGRP为最佳刺激浓度。用10^（-8） mol·L^（-1）浓度的CGRP处理后,茜素红染色显示钙化结节明显增多。CGRP能够显著上调p-Mst1/2蛋白的表达（P〈0.05）;当运用了抑制剂Verteporfin时,显著降低了CGRP诱导的Runx2、ColⅠmRNA的表达（P〈0.05）。结论 CGRP能够促进小鼠BMSCs的成骨分化,且Hippo信号通路介导了CGRP作用于小鼠BMSCs成骨分化的过程。 |
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| AbstractList | 目的前期研究发现降钙素基因相关肽（CGRP）能够促进成骨细胞的生物学活性,为了进一步揭示CGRP在骨修复中的作用,检测CGRP对小鼠骨髓间充质干细胞（BMSCs）成骨分化的影响,并对Hippo通路在这个过程中的作用进行了初步探讨。方法在体外诱导培养的BMSCs中加入不同浓度的CGRP,处理48 h后测试碱性磷酸酶（ALP）活性,以筛选的优势浓度;处理7 d后进行茜素红染色,分别检测细胞的分化情况。应用Western blot检测CGRP作用于BMSCs后,Hippo通路核心分子Mst1/2蛋白磷酸化的表达水平;利用Hippo通路抑制剂维替泊芬（Verteporfin）阻断下游Yap信号,逆转录聚合酶链反应检测其对成骨相关因子Ⅰ型胶原蛋白（ColⅠ）、Runt相关转录因子2（Runx2）mRNA的表达影响。结果 ALP活性检测结果显示,与空白对照组相比,10^（-9）、10^（-8）、10^（-7） mol·L^（-1）浓度范围的CGRP都能显著促进小鼠ALP活性的增加（P〈0.05）,以10^（-8） mol·L^（-1）浓度的CGRP为最佳刺激浓度。用10^（-8） mol·L^（-1）浓度的CGRP处理后,茜素红染色显示钙化结节明显增多。CGRP能够显著上调p-Mst1/2蛋白的表达（P〈0.05）;当运用了抑制剂Verteporfin时,显著降低了CGRP诱导的Runx2、ColⅠmRNA的表达（P〈0.05）。结论 CGRP能够促进小鼠BMSCs的成骨分化,且Hippo信号通路介导了CGRP作用于小鼠BMSCs成骨分化的过程。 Q 254; 目的：前期研究发现降钙素基因相关肽（CGRP）能够促进成骨细胞的生物学活性，为了进一步揭示CGRP在骨修复中的作用，检测CGRP对小鼠骨髓间充质干细胞（BMSCs）成骨分化的影响，并对Hippo通路在这个过程中的作用进行了初步探讨。方法在体外诱导培养的BMSCs中加入不同浓度的CGRP，处理48?h后测试碱性磷酸酶（ALP）活性，以筛选的优势浓度；处理7?d后进行茜素红染色，分别检测细胞的分化情况。应用Western?blot检测CGRP作用于BMSCs后，Hippo通路核心分子Mst1/2蛋白磷酸化的表达水平；利用Hippo通路抑制剂维替泊芬（Verteporfin）阻断下游Yap信号，逆转录聚合酶链反应检测其对成骨相关因子Ⅰ型胶原蛋白（ColⅠ）、Runt相关转录因子2（Runx2） mRNA的表达影响。结果 ALP活性检测结果显示，与空白对照组相比，10-9、10-8、10-7?mol·L-1浓度范围的CGRP都能显著促进小鼠ALP活性的增加（P<0.05），以10-8?mol·L-1浓度的CGRP为最佳刺激浓度。用10-8?mol·L-1浓度的CGRP处理后，茜素红染色显示钙化结节明显增多。CGRP能够显著上调p-Mst1/2蛋白的表达（P<0.05）；当运用了抑制剂Verteporfin时，显著降低了CGRP诱导的Runx2、ColⅠ?mRNA的表达（P<0.05）。结论 CGRP能够促进小鼠BMSCs的成骨分化，且Hippo信号通路介导了CGRP作用于小鼠BMSCs成骨分化的过程。 |
| Abstract_FL | Objective Previous studies have clarified that calcitonin gene-related peptide (CGRP) can promote the biological activity of osteoblasts. To further reveal the role of CGRP in bone repair, we studied its influence on osteogenic differentiation of mouse bone marrow stromal cells (BMSCs) and initially explored the effect of the Hippo signaling pathway with this process. Methods BMSCs were induced to osteogenic differentiate osteoblasts by different concentrations of CGRP for a screening of the optimal concentration. CGRP was added in BMSCs, then the activity of alkaline phosphatase (ALP) and the number of mineralized nodules were examined by specific ALP kits after 48 hours and alizarin red staining fluid after 7 days, respectively. The protein expression of p-Mst1/2 was measured by Western blot. Verteporfin was used to block the downstream Yap signaling. The mRNA expression of collagen typeⅠ(ColⅠ) and runt-related transcription factor 2 (Runx2) were detected by reverse transcription-polymerase chain reaction. Results Compared to the blank group, different concentrations of CGRP (10?9, 10-8, 10?7 mol·L-1), especially 10?8 mol·L-1, significantly increased the ALP activity of BMSCs (P<0.05). Alizarin red staining also showed more mineralized nodules in 10?8 mol·L-1 group. The expression of p-Mst1/2 increased in the CGRP group (P<0.05). Verteporfin treatment effectively decreased the mRNA expression of Runx2 and ColⅠ(P<0.05). Conclusion The Hippo signaling pathway plays a role in CGRP-induced osteogenic differentiation in mouse BMSCs. |
| Author | 王飞 张慧宇 窦予昕 李适廷 张纲 谭颖徽 |
| AuthorAffiliation | 第三军医大学新桥医院口腔科,重庆400037 |
| AuthorAffiliation_xml | – name: 第三军医大学新桥医院口腔科,重庆,400037 |
| Author_FL | Li Shiting Zhang Gang Zhang Huiyu Dou Yuxin Tan Yinghui Wang Fei |
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| Keywords | bone marrow stromal cells 骨髓间充质干细胞 calcitonin gene-related peptide Hippo signaling pathway Hippo信号通路 降钙素基因相关肽 |
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| Notes | 51-1169/R Objective Previous studies have clarified that calcitonin gene-related peptide（CGRP） can promote the biological activity of osteoblasts. To further reveal the role of CGRP in bone repair, we studied its influence on osteogenic differentiation of mouse bone marrow stromal cells（BMSCs） and initially explored the effect of the Hippo signaling pathway with this process. Methods BMSCs were induced to osteogenic differentiate osteoblasts by different concentrations of CGRP for a screening of the optimal concentration. CGRP was added in BMSCs, then the activity of alkaline phosphatase（ALP） and the number of mineralized nodules were examined by specific ALP kits after 48 hours and alizarin red staining fluid after 7 days, respectively. The protein expression of p-Mst1/2 was measured by Western blot. Verteporfin was used to block the downstream Yap signaling. The mRNA expression of collagen typeⅠ（ColⅠ） and runt-related transcription factor 2（Runx2） were detected by reverse transcription-polymerase chain react |
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| Snippet | 目的前期研究发现降钙素基因相关肽（CGRP）能够促进成骨细胞的生物学活性,为了进一步揭示CGRP在骨修复中的作用,检测CGRP对小鼠骨髓间充质干细胞（BMSCs）成骨分化的影响,... Q 254; 目的：前期研究发现降钙素基因相关肽（CGRP）能够促进成骨细胞的生物学活性，为了进一步揭示CGRP在骨修复中的作用，检测CGRP对小鼠骨髓间充质干细胞（BMSCs）成骨... |
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| SubjectTerms | Hippo信号通路 降钙素基因相关肽 骨髓间充质干细胞 |
| Title | 降钙素基因相关肽通过Hippo通路调控小鼠骨髓间充质干细胞成骨分化的实验研究 |
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