二甲双胍通过AMPK通道对界膜中滑膜成纤维细胞的增殖和成骨作用的研究

Effects of metformin on proliferation andosteogenesis of FLSs in interfacial membrane through AMPK pathway

目的研究二甲双胍能否通过AMPK通道抑制界膜中的滑膜成纤维细胞(FLSs)的生长并促进其成骨分化。方法提取原代的FLSs并传4-5代纯化后进行实验,分别加以二甲双胍和促骨生成激动剂(抗坏血酸、β-甘油磷酸、地塞米松)处理。显微镜观察、细胞免疫化学鉴定成纤维细胞;MTT检测药物在不同浓度和时间下对FLSs的生长抑制率;碱性磷酸酶(ALP)和茜素红染色检测药物处理后FLSs随时间变化的ALP活性和成骨情况;Western Blot检测药物作用后通路AMPK及其磷酸化水平情况。结果光镜可见FLSs长梭形,胞体比较大,也可见少量的多角细胞。角质蛋、白波丝蛋白细胞免疫化学染分别为阴性和阳性。MTT测得并计算出二甲双胍对FLSs增殖的抑制率随着浓度增加而增大。二甲双胍组和促骨生成激动剂组中的ALP和茜素红染色分别出现强阳性结果。结论二甲双胍能通过激活AMPK通道抑制界膜中FLSs的生长增值并促进其ALP的合成以及细胞外钙结节的沉积,且具有时间和浓度依赖性。

Objective To investigate whether metformin can inhibit the growth of synovial fibroblasts （FLSs） via loosening interfacial membrane and promoting osteogenic differentiation through AMPK channels. Methods The extraction of primary and FLSs of 4-5 passages after purification used for experiment were treated with Metformin and bone morphogenic agonists （ascorbicacid, betaglycerophosphateand dexamethasone treat- ment）, respectively. The electron microscopy and immunocytochemical staining identified fibroblasts ; MTT detect- ed FLSs growth inhibition rate, which treated with drugs in different concentration and time; Alkaline phosphatase （ALP） staining and alizarin red staining were used to detect the changes of ALP activity and osteogenesis with time after FLSs treated with medicine ; Western Blot was used to detect AMPK and phosphorylation level after drug treat- ment. Results FLSs were with spindle shape, large cell body and small amount of polygonal ceils under electron microscope. The immunocytochemical staining of Keratin and Vimentin was negative and positive, respectively. The inhibition rate of metformin on FLSs proliferation was increased in a concentration dependent manner. Strong positive results were found in ALP and alizarin red staining in the metformin group, which was similar with the bone morphogenetic agonist group. Conclusion Metformin can inhibit FLSs growth and promote the synthesis of alkaline phosphatase and deposition of extracellular calcium nodules through activation of AMPK channel in a time and concentration dependent manner.