ANGPTL4在调控骨巨细胞瘤细胞增殖、血管生成和破骨分化作用的实验研究

目的探讨人血管生成素样蛋白4(ANGPTL4)基因沉默对骨巨细胞瘤单核基质细胞(GCTSC)增殖、血管生成以及破骨分化的作用。方法体外培养GCTSC细胞系,采用ANGPTL4 shRNA、Scrambled shRNA转染作为ANGPTL4 shRNA组和阴性对照组,另设置无处理的空白对照组。采用实时荧光定量PCR(QPCR)和Western blotting法检测ANGPTL4 mRNA和蛋白表达。MTT法和流式细胞术检测细胞增殖和凋亡。将GCTSC细胞分别与人脐静脉内皮细胞(HUVECs)、小鼠骨髓单核细胞(BMM)共培养,观察HUVECs成管能力和BMM破骨分化能力。结果与空白对照组和阴性对照组比较,ANGPTL4 shRNA组ANGPTL4 mRNA(0.174±0.045)和蛋白表达量(0.098±0.020)均明显降低;而ANGPTL4 shRNA组GCTSC细胞增殖活性降低,凋亡率升高(P<0.05)。与HUVECs共培养后,ANGPTL4 shRNA组闭合管数量(57.35±17.24)%]减少(P<0.05);与BMM共培养后,ANGPTL4 shRNA组TRAP染色阳性的破骨细胞样多核巨细胞数量(48.36±21.79)%]减少(P<0.05)。结论沉默骨巨细胞瘤GCTSC细胞ANGPTL4基因表达后,细胞增殖活性降低,凋亡率增加,并且对肿瘤血管生成和多核巨细胞破骨分化有一定的抑制作用。

Experimental study of the effect of ANGPTL4 on the proliferation,angiogenesis and osteoclastic differentiation of giant cell tumor cells

Objective To investigate the effect of human angiopoietin like protein 4(ANGPTL4)gene silencing on proliferation,angiogenesis and osteoclastic differentiation of stromal-like cells of giant cell tumor(GCTSC).Methods The GCTSC cells was isolated from the GCT tissues and primarily cultured inα-MEM medium.GCTSC cells were transfected with ANGPTL4 shRNA and scrabbled shRNA as ANGPTL4 shRNA group and negative control group,and blank control group without treatment was set up.The expression of ANGPTL4 mRNA and protein was detected by real-time fluorescence quantitative PCR(QPCR)and Western blotting.MTT and flow cytometry were used to detect cell proliferation and apoptosis.GCTSC cells were co-cultured with human umbilical vein endothelial cells(HUVECs)and mouse bone marrow mononuclear cells(BMM)respectively to observe the ability of HUVECs to form tubes and BMM to break bone.Results Compared with blank control group and negative control group,the expression of ANGPTL4 mRNA and protein in ANGPTL4 shRNA group decreased significantly,while the proliferation activity was decreased and apoptosis rate was increased(P<0.05).After co-culture with HUVECs,the number of closed tubes in ANGPTL4 shRNA group decreased(P<0.05);after co-culture with BMM,the number of osteoclast like multinuclear giant cells in ANGPTL4 shRNA group decreased(P<0.05).Conclusion After silencing the expression of ANGPTL4 gene in GCTSC cells,the proliferation activity of GCTSC cells decreased,the apoptosis rate increased,and the angiogenesis and osteoclastic differentiation were inhibited.