【摘要】：目的：分析FGF2對BMP9誘導的間充質(zhì)干細胞（mesenchymal stemcells，MSCS）成骨分化的影響極其相關(guān)分子機制。 方法：首先用Western blot檢測外源性FGF2或外源性BMP9是否影響MSCs細胞中內(nèi)源性BMP9或FGF2的蛋白表達；然后以不同滴度的Ad-RFP和AdR-FGF2腺病毒感染細胞，并制備BMP9條件培養(yǎng)基，待Ad-RFP和AdR-FGF2腺病毒作用24h后加入BMP9條件培養(yǎng)基，觀察成骨分化早期標志物堿性磷酸酶（Alkaline phosphotase，ALP）的變化；用結(jié)晶紫染色分析FGF2和BMP9對間充質(zhì)干細胞增殖情況的影響；然后用茜素紅S染色的方法觀察成骨晚期標志物鈣鹽沉積的變化；通過Western blot檢測成骨晚期標志物骨橋蛋白（Osteopotin，OPN）和骨鈣蛋白（Osteocalcin，OCN）的變化。 隨后，通過PCR技術(shù)檢測FGF2對BMP9誘導成骨分化相關(guān)靶基因ID1、ID2、ID3及成骨關(guān)鍵轉(zhuǎn)錄因子Runx2的影響；Western blot檢測FGF2對BMP9誘導的Runx2蛋白表達的影響；熒光素酶報告基因?qū)嶒灪蚖estern blot檢測FGF2對BMP9活化的經(jīng)典Smad1/5/8信號途徑和非經(jīng)典MAPKs(p38和ERK1/2)的影響；最后PCR檢測FGF2對于BMP9成骨相關(guān)Ⅰ型受體ALK1和ALK2的影響。 最后，利用FGF2和BMP9重組腺病毒感染間充質(zhì)干細胞C3H10T1/2，，利用裸鼠皮下異位成骨實驗FGF2對于BMP9觀察在裸鼠皮下異位成骨的情況，并用HE、Alcian Blue、 Masson’s Trichrome對骨組織切片進行染色，觀察骨組織成熟度情況。 結(jié)果：Western blot檢測顯示外源性FGF2在間充質(zhì)干細胞株C3H10T1/2細胞中不會影響內(nèi)源性BMP9蛋白的表達（反之亦然）；而BMP9誘導的ALP活性隨著FGF2腺病毒感染滴度的不斷增高而逐漸下降；結(jié)晶紫染色結(jié)果顯示FGF2和BMP9對間充質(zhì)干細胞的增殖都有一定促進作用，兩者同時處理細胞時促進作用更明顯；在另一種間充質(zhì)干細胞小鼠胚胎成纖維細胞（Mouse embryonic fibroblasts，MEFs）中，F(xiàn)GF2同樣可抑制其BMP9誘導的ALP活性，同時抑制了晚期成骨分化標志物鈣鹽沉積，以及OPN和OCN的表達。 PCR結(jié)果顯示FGF2抑制了BMP9相關(guān)靶基因ID1、ID2、ID3及成骨關(guān)鍵轉(zhuǎn)錄因子Runx2的基因表達，Western blot檢測發(fā)現(xiàn)FGF2也同時抑制了關(guān)鍵的成骨轉(zhuǎn)錄因子Runx2的蛋白表達；FGF2和BMP9本身均可以激活MAPKs中的p38和ERK1/2信號通路，但是FGF2對于BMP9誘導的p38和ERK1/2的活化并無明顯影響；值得注意的是，F(xiàn)GF2可以抑制BMP9激活的經(jīng)典Smad1/5/8信號途徑，表現(xiàn)為BMP9誘導的SBE熒光素酶活性下降、及Smad1/5/8磷酸化水平下降；PCR結(jié)果顯示FGF2抑制了BMP9成骨相關(guān)相關(guān)Ⅰ型受體ALK1和ALK2的表達。 裸鼠皮下異位成骨實驗顯示：注射感染了FGF2/GFP和RFP/BMP9腺病毒的間充質(zhì)干細胞C3H10T1/2后，F(xiàn)GF2明顯抑制了BMP9誘導的骨組織形成，而且染色結(jié)果顯示其骨成熟度也被明顯抑制。 結(jié)論：FGF2抑制了BMP9誘導的小鼠間充質(zhì)干細胞的成骨分化及骨形成作用，主要是通過抑制BMP9誘導成骨分化的經(jīng)典信號通路BMPs-Smad1/5/8而實現(xiàn)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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