墨旱莲提取物促进小鼠骨髓来源的间充质干细胞迁移和成骨方向分化作用研究

Effects of eclipta extract on migration and osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells

关键词:  墨旱莲  骨髓间充质干细胞  细胞迁移  CXC趋化因子受体4  成骨分化

Key words:  eclipta  bone marrow mesenchymal stem cell  cell migration  CXCR4  osteogenic differentiation

摘要:

[目的] 探究墨旱莲提取物（EHE）对小鼠骨髓来源的间充质干细胞（BMMSCs）迁移和向成骨分化作用的影响。[方法] 提取6周C57BL/6J雌性小鼠后肢股骨和胫骨中的BMMSCs，使用流式细胞术检测细胞表面分子表达。采用血清碱性磷酸酶（ALP）染色、茜素红染色、甲苯胺蓝染色和油红O染色观察BMMSCs向成骨、成脂、软骨细胞多方向的分化情况。通过细胞迁移侵袭（Transwell）实验探究EHE对BMMSCs迁移作用的影响。运用蛋白质印迹法（Western Blot）检测EHE对BMMSCs中CXC趋化因子受体4（CXCR4）表达的影响。通过ALP活性检测，ALP染色和茜素红染色的方法观察EHE对BMMSCs成骨诱导情况影响。[结果] 小鼠原代BMMSCs提取分离后，细胞贴壁生长，形态呈长梭形。流式细胞术结果显示BMMSCs表面分子表达率如下：干细胞抗原1（sca-1）为86%、分化簇（CD）29为99%、CD140α为88.7%、CD90为63.7%、CD45为14%、CD34为1%。经成骨细胞诱导剂诱导，ALP染色后可见被黑染的成骨细胞，茜素红染色后可见红色矿化结节样的成骨细胞；经软骨细胞诱导剂诱导，甲苯胺蓝染色后可见被蓝染的软骨细胞；经成脂细胞诱导剂诱导，油红O染色后可见橘红色脂滴样的脂肪细胞。Transwell实验结果显示，与对照组相比，50 ng/mL基质细胞衍生因子-1（sSDF-1）、0.1 μg/mL和1 μg/mL EHE能显著增加BMMSCs的透膜数量（P<0.05），差异具有统计学意义。Western Blot实验结果显示，与对照组相比，0.1 μg/mL EHE能促进CXCR4蛋白的表达（P<0.05），差异具有统计学意义。ALP活性检测、ALP染色和茜素红染色结果显示，与对照组相比，成骨诱导剂显著诱导BMMSCs向成骨细胞分化（P<0.05），差异具有统计学意义；与成骨诱导剂组相比，1 μg/mL EHE能显著诱导BMMSCs向成骨细胞分化（P<0.05），差异具有统计学意义。[结论] EHE具有促进BMMSCs迁移和向成骨分化作用，其调控BMMSCs迁移的作用机制可能与促进CXCR4蛋白表达相关。

Abstract:

[Objective] To investigate the effects of ecliptaherba (EHE) on the migration and osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) from mice.[Methods] BMMSCs were extracted from the femur and tibia of the hindlimb of 6-week C57BL/6J female mice, and the expression of cell surface molecules was detected by flow cytometry. ALP staining, alizarin red staining, toluidine blue staining and oil red O staining were used to observe the multi-directional differentiation of BMMSCs into osteoblasts, adipocytes and chondrocytes. Transwell experiment was used to explore the effect of EHE on the migration of BMMSCs. Western blot was used to detect the effect of EHE on the expression of CXC chemokine receptor 4(CXCR4) in BMMSCs. The effect of EHE on osteogenic induction of BMMSCs was observed by ALP activity detection, ALP staining and alizarin red staining.[Results] After the extraction and separation of mouse primary BMMSCs, the cells adhered to the wall and grew in a long spindle shape. The results of flow cytometry showed that the surface molecular expression rates of BMMSCs were as follows:Sca-1 was 86%, CD29 was 99%, cd140 α 88.7%, 63.7% for CD90, 14% for CD45 and 1% for CD34. Induced by osteoblast inducer, black stained osteoblasts can be seen after ALP staining, and red mineralized nodular osteoblasts can be seen after alizarin red staining;induced by chondrocyte inducer, blue stained chondrocytes were observed after toluidine blue staining;after being induced by adipocyte inducer, orange fat drop like adipocytes were observed after oil red O staining. The results of Transwell experiment showed that compared with the control group, 50 ng/mL stromal cell derived factor (SDF-1) and 0.1 μg/mL and 1 μg/mL EHE could significantly increase the number of permeable membranes of BMMSCs (P<0.05). The results of Western blot showed that compared with the control group, 0.1 μg/mL EHE could promote the expression of CXCR4 protein (P<0.05). ALP activity test, ALP staining and alizarin red staining showed that compared with the control group, osteogenic inducer significantly induced BMMSCs to differentiate into osteoblasts (P<0.05);compared with osteogenic inducer group, 1μg/mL EHE could significantly induce BMMSCs to differentiate into osteoblasts (P<0.05).[Conclusion] EHE can promote the migration and osteogenic differentiation of BMMSCs, and its mechanism of regulating the migration of BMMSCs may be related to promoting the expression of CXCR4 protein.