FGF2调控BMP9诱导的间充质干细胞成骨分化的作用及机制研究

发布时间：2018-11-17 07:14

【摘要】：目的：分析FGF2对BMP9诱导的间充质干细胞（mesenchymal stemcells，MSCS）成骨分化的影响极其相关分子机制。 方法：首先用Western blot检测外源性FGF2或外源性BMP9是否影响MSCs细胞中内源性BMP9或FGF2的蛋白表达；然后以不同滴度的Ad-RFP和AdR-FGF2腺病毒感染细胞，并制备BMP9条件培养基，待Ad-RFP和AdR-FGF2腺病毒作用24h后加入BMP9条件培养基，观察成骨分化早期标志物碱性磷酸酶（Alkaline phosphotase，ALP）的变化；用结晶紫染色分析FGF2和BMP9对间充质干细胞增殖情况的影响；然后用茜素红S染色的方法观察成骨晚期标志物钙盐沉积的变化；通过Western blot检测成骨晚期标志物骨桥蛋白（Osteopotin，OPN）和骨钙蛋白（Osteocalcin，OCN）的变化。 随后，通过PCR技术检测FGF2对BMP9诱导成骨分化相关靶基因ID1、ID2、ID3及成骨关键转录因子Runx2的影响；Western blot检测FGF2对BMP9诱导的Runx2蛋白表达的影响；荧光素酶报告基因实验和Western blot检测FGF2对BMP9活化的经典Smad1/5/8信号途径和非经典MAPKs(p38和ERK1/2)的影响；最后PCR检测FGF2对于BMP9成骨相关Ⅰ型受体ALK1和ALK2的影响。 最后，利用FGF2和BMP9重组腺病毒感染间充质干细胞C3H10T1/2，，利用裸鼠皮下异位成骨实验FGF2对于BMP9观察在裸鼠皮下异位成骨的情况，并用HE、Alcian Blue、 Masson’s Trichrome对骨组织切片进行染色，观察骨组织成熟度情况。 结果：Western blot检测显示外源性FGF2在间充质干细胞株C3H10T1/2细胞中不会影响内源性BMP9蛋白的表达（反之亦然）；而BMP9诱导的ALP活性随着FGF2腺病毒感染滴度的不断增高而逐渐下降；结晶紫染色结果显示FGF2和BMP9对间充质干细胞的增殖都有一定促进作用，两者同时处理细胞时促进作用更明显；在另一种间充质干细胞小鼠胚胎成纤维细胞（Mouse embryonic fibroblasts，MEFs）中，FGF2同样可抑制其BMP9诱导的ALP活性，同时抑制了晚期成骨分化标志物钙盐沉积，以及OPN和OCN的表达。 PCR结果显示FGF2抑制了BMP9相关靶基因ID1、ID2、ID3及成骨关键转录因子Runx2的基因表达，Western blot检测发现FGF2也同时抑制了关键的成骨转录因子Runx2的蛋白表达；FGF2和BMP9本身均可以激活MAPKs中的p38和ERK1/2信号通路，但是FGF2对于BMP9诱导的p38和ERK1/2的活化并无明显影响；值得注意的是，FGF2可以抑制BMP9激活的经典Smad1/5/8信号途径，表现为BMP9诱导的SBE荧光素酶活性下降、及Smad1/5/8磷酸化水平下降；PCR结果显示FGF2抑制了BMP9成骨相关相关Ⅰ型受体ALK1和ALK2的表达。 裸鼠皮下异位成骨实验显示：注射感染了FGF2/GFP和RFP/BMP9腺病毒的间充质干细胞C3H10T1/2后，FGF2明显抑制了BMP9诱导的骨组织形成，而且染色结果显示其骨成熟度也被明显抑制。 结论：FGF2抑制了BMP9诱导的小鼠间充质干细胞的成骨分化及骨形成作用，主要是通过抑制BMP9诱导成骨分化的经典信号通路BMPs-Smad1/5/8而实现的。
[Abstract]:Aim: to investigate the effect of FGF2 on the osteogenic differentiation of mesenchymal stem cells (mesenchymal stemcells,MSCS) induced by BMP9 and its molecular mechanism. Methods: Western blot was used to detect whether exogenous FGF2 or exogenous BMP9 affected the expression of endogenous BMP9 or FGF2 in MSCs cells. Then the cells were infected with different titers of Ad-RFP and AdR-FGF2 adenovirus, and BMP9 conditioned medium was prepared. After 24 hours of treatment with Ad-RFP and AdR-FGF2 adenovirus, BMP9 conditioned medium was added to observe the early osteogenic differentiation marker, alkaline phosphatase (Alkaline phosphotase,. (ALP); The effects of FGF2 and BMP9 on the proliferation of mesenchymal stem cells were analyzed by crystal violet staining, and the changes of calcium salt deposition in late osteogenic stage were observed by alizarin red S staining. The changes of osteopontin (Osteopotin,OPN) and osteocalcin (Osteocalcin,OCN) were detected by Western blot. Then, the effects of FGF2 on BMP9 induced osteogenic differentiation related gene ID1,ID2,ID3 and osteoblast key transcription factor Runx2 were detected by PCR technique.; Western blot was used to detect the effect of FGF2 on BMP9 induced Runx2 protein expression. Luciferase reporter gene experiment and Western blot detection of the effects of FGF2 on the classical Smad1/5/8 signaling pathway activated by BMP9 and non-classical MAPKs (p38 and ERK1/2) were performed. Finally, PCR was used to detect the effect of FGF2 on ALK1 and ALK2 of BMP9 osteoblast related type 鈪

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