AG490对成骨细胞和破骨细胞之间相互作用的影响

doi:10.3877/cma.j.issn.1674-1366.2017.02.002

目的

体外建立MC3T3-E1细胞与RAW 264.7细胞间接共培养体系，观察AG490对共培养体系中破骨细胞形成的影响。

方法

MC3T3-E1细胞经矿化诱导培养后，与RAW 264.7细胞进行间接共培养，经一定浓度AG490（1、5、10 μmol/L）分别处理后，细胞计数试剂盒（CCK-8）检测细胞增殖，氮偶联法检测成骨细胞碱性磷酸酶（ALP）活性，实时荧光定量聚合酶链反应（PCR）检测成骨细胞相关基因骨保护素（OPG）、Ⅰ型胶原（COL1）、ALP、骨钙素（OCN）的表达，抗酒石酸酸性磷酸酶（TRAP）和苏木素染色检测破骨细胞的形成，蛋白印迹法（Western blot）检测破骨细胞中RANK的表达。采用单因素方差分析和双因素方差分析对数据进行统计分析。

结果

AG490下调MC3T3-E1细胞矿化过程中OPG、COL1、ALP、OCN mRNA的表达（F_OPG=30.120，P_{OPG，AG490 1 μmol/L}=0.021、P_{OPG，AG490 5 μmol/L}= 0.221、P_{OPG，AG490 10 μmol/L}= 0.003；F_ALP= 67.352，P_{ALP，AG490 1 μmol/L}= 0.034、P_{ALP，AG490 5 μmol/L}= 0.001、P_{ALP，AG490 10 μmol/L}= 0.156；F_COL1= 28.158，P_{COL1，AG490 1 μmol/L}= 0.054、P_{COL1，AG490 5 μmol/L}= 0.002、P_{COL1，AG490 10 μmol/L}= 0.4；F_OCN= 125.375，P_{OCN，AG490 1 μmol/L}<0.001、P_{OCN，AG490 5 μmol/L}<0.001、P_{OCN，AG490 10 μmol/L}<0.001）。与RAW264.7细胞单独培养作为对照组相比，MC3T3-E1细胞与RAW 264.7细胞间接共培养明显促进成熟破骨细胞的形成，AG490在这个过程中对破骨细胞的形成起到抑制作用（F= 85.391，P_{共培养组-对照组}<0.001，P_{共培养+AG490组-共培养组}= 0.035），同时与对照组相比，明显抑制破骨细胞中RANK蛋白的表达（F_RANK= 376.25，P_{RANK，AG490 1 μmol/L}= 0.468、P_{RANK，AG490 5 μmol/L}= 0.003、P_{RANK，AG490 10 μmol/L}<0.001）。

结论

AG490抑制MC3T3-E1细胞成骨向分化，并通过影响OPG/RANKL/RANK信号轴的传递抑制成骨细胞与破骨前体细胞间接共培养过程中破骨细胞的形成。

关键词: 共同培养技术, 核因子κB受体活化因子, AG490, 成骨向分化, 破骨向分化

Objective

To establish a co-culture system of MC3T3-E1 cells and RAW 264.7 cells in vitro, and then explore the effect of AG490 on osteoclastogenesis in this system.

Methods

The mineralized MC3T3-E1 cells and RAW 264.7 cells were co-cultured after the addition of different concentrations of AG490 (1 μmol/L, 5 μmol/L, 10 μmol/L) . The cell proliferation was analyzed by CCK-8 assay, ALP activity was analyzed by a simultaneous-coupling azo dye method, and osteoblast-related genes OPG, COL1, ALP, OCN were analyzed by RT-PCR in mineralized MC3T3-E1 cells. TRAP and hematoxylin staining were performed to observe the osteoclastogenesis, and Western blot was adopted to analyze the RANK expression in osteoclasts. Data were evaluated by One-Way ANOVA and Two-Way ANOVA.

Results

AG490 inhibited the ALP activity and the expression of osteoblast-related genes OPG, COL1, ALP, OCN in mineralized MC3T3-E1 cells (F_OPG= 30.120, P_{OPG, AG490 1 μmol/L}= 0.021, P_{OPG, AG490 5 μmol/L}= 0.221, P_{OPG, AG490 10 μmol/L}= 0.003; F_ALP= 67.352, P_{ALP, AG490 1 μmol/L}= 0.034, P_{ALP, AG490 5 μmol/L}= 0.001, P_{ALP, AG490 10 μmol/L}= 0.156; F_COL1= 28.158, P_{COL1, AG490 1 μmol/L}= 0.054, P_{COL1, AG490 5 μmol/L}= 0.002, P_{COL1, AG490 10 μmol/L}= 0.4; F_OCN= 125.375, P_{OCN, AG490 1 μmol/L}<0.001, P_{OCN, AG490 5 μmol/L}<0.001, P_{OCN, AG490 10 μmol/L}<0.001) . Compared with RAW264.7 cells alone group (control group) , the indirect co-culture of MC3T3-E1 cells and RAW264.7 cells successfully promoted osteoclast formation, which could be significantly inhibited by AG490 (F= 85.391, P_{co-culture-control}<0.001, P_{co-culture+AG490-co-culture}= 0.035) , meanwhile the addition of AG490 down-regulated the RANK in the osteoclasts in protein level (F_RANK= 376.25, P_{RANK, AG490 1 μmol/L}= 0.468, P_{RANK, AG490 5 μmol/L}=0.003, P_{RANK, AG490 10 μmol/L}<0.001) .

Conclusions

AG490 can inhibit osteoblastic differentiation in MC3T3-E1 cells, and might play an important role in osteoclast formation inhibition in co-culture of MC3T3-E1 cells and RAW 264.7 cells by blockade of OPG/RANKL/RANK signal axis.

Key words: Culture techniques, Receptor activator of nuclear factor-kappa B, AG490, Osteoblastic differentiation, Osteoclastic differentiation

表1 实时荧光定量PCR引物序列

基因	引物序列
OPG	F:5′-CTGGAGTGCACGATGCCAGCTA-3′
?	R:5′-TCCGTGCTCGGCGATGGACGTC-3′
RANKL	F:5′-GAGACAGACATCGGGAGGGAT-3′
?	R:5′-GTGGGATGTGAACTCGGAAGCT-3′
ALP	F:5′-ACGGAGGCATTGACTCTGAACG-3′
?	R:5′-GGAAGCACCAACGAGAGGAGCT-3′
COL1	F:5′-GAGATCGTGCTGAACGGTAATT-3′
?	R:5′-GATTGACATGCATGTTGAATGA-3′
OCN	F:5′-AGCGAAGACCTAGGAGACAAGT-3′
?	R:5′-TGAGGATCGTAACTCGCGAGAG-3′
GAPDH	F:5′-AAGGTCGGTGTGAACGGATTTA-3′
?	R:5′-TGTAGACCATGTAGTTGAGGTA-3′

表1 实时荧光定量PCR引物序列

基因	引物序列
OPG	F:5′-CTGGAGTGCACGATGCCAGCTA-3′
?	R:5′-TCCGTGCTCGGCGATGGACGTC-3′
RANKL	F:5′-GAGACAGACATCGGGAGGGAT-3′
?	R:5′-GTGGGATGTGAACTCGGAAGCT-3′
ALP	F:5′-ACGGAGGCATTGACTCTGAACG-3′
?	R:5′-GGAAGCACCAACGAGAGGAGCT-3′
COL1	F:5′-GAGATCGTGCTGAACGGTAATT-3′
?	R:5′-GATTGACATGCATGTTGAATGA-3′
OCN	F:5′-AGCGAAGACCTAGGAGACAAGT-3′
?	R:5′-TGAGGATCGTAACTCGCGAGAG-3′
GAPDH	F:5′-AAGGTCGGTGTGAACGGATTTA-3′
?	R:5′-TGTAGACCATGTAGTTGAGGTA-3′

图1 CCK-8检测AG490对MC3T3-E1细胞和RAW 264.7细胞增殖的影响

表2 不同浓度AG490对MC3T3-E1细胞矿化过程中成骨细胞相关基因表达影响

AG490浓度(μmol/L)	基因相对表达量					F值					P值
AG490浓度(μmol/L)	OPG	ALP	COL1	OCN	RANKL	OPG	ALP	COL1	OCN	RANKL	OPG	ALP	COL1	OCN	RANKL
0	2.35	1.67	5.62	1.02	1.43	30.120	67.352	28.158	125.375	8.164	-	-	-	-	-
1	1.21	1.17	3.75	0.51	1.51	30.120	67.352	28.158	125.375	8.164	0.021	0.034	0.054	0.000	0.557
5	1.01	0.78	1.47	0.19	1.39	30.120	67.352	28.158	125.375	8.164	0.221	0.001	0.002	0.000	0.924
10	0.58	0.63	0.93	0.08	1.31	30.120	67.352	28.158	125.375	8.164	0.003	0.156	0.400	0.000	0.376

表2 不同浓度AG490对MC3T3-E1细胞矿化过程中成骨细胞相关基因表达影响

AG490浓度(μmol/L)	基因相对表达量					F值					P值
AG490浓度(μmol/L)	OPG	ALP	COL1	OCN	RANKL	OPG	ALP	COL1	OCN	RANKL	OPG	ALP	COL1	OCN	RANKL
0	2.35	1.67	5.62	1.02	1.43	30.120	67.352	28.158	125.375	8.164	-	-	-	-	-
1	1.21	1.17	3.75	0.51	1.51	30.120	67.352	28.158	125.375	8.164	0.021	0.034	0.054	0.000	0.557
5	1.01	0.78	1.47	0.19	1.39	30.120	67.352	28.158	125.375	8.164	0.221	0.001	0.002	0.000	0.924
10	0.58	0.63	0.93	0.08	1.31	30.120	67.352	28.158	125.375	8.164	0.003	0.156	0.400	0.000	0.376

图2 ALP染色检测AG490对MC3T3-E1细胞成骨向分化的影响

图3 实时荧光定量PCR检测MC3T3-E1细胞中OPG、ALP、COL1、OCN、RANKL mRNA表达水平

图4 TRAP和苏木素染色检测MC3T3-E1细胞和RAW 264.7细胞间接共培养体系中成熟破骨细胞的形成

表3 不同浓度AG490对MC3T3-E1细胞和RAW 264.7细胞共培养过程中RANK蛋白表达影响

AG490浓度(μmol/L)	基因相对表达量		F值		P值
AG490浓度(μmol/L)	对照组	共培养组	对照组	共培养组	对照组	共培养组
0	3.27	3.18	376.25	376.25	-	0.723
1	3.25	2.94	376.25	376.25	-	0.468
5	3.39	2.16	376.25	376.25	-	0.003
10	3.13	0.12	376.25	376.25	-	0.000

表3 不同浓度AG490对MC3T3-E1细胞和RAW 264.7细胞共培养过程中RANK蛋白表达影响

AG490浓度(μmol/L)	基因相对表达量		F值		P值
AG490浓度(μmol/L)	对照组	共培养组	对照组	共培养组	对照组	共培养组
0	3.27	3.18	376.25	376.25	-	0.723
1	3.25	2.94	376.25	376.25	-	0.468
5	3.39	2.16	376.25	376.25	-	0.003
10	3.13	0.12	376.25	376.25	-	0.000

图5 Western blot检测RANK蛋白水平的表达

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The effect of AG490 on the crosstalk between osteoblasts and osteoclasts in vitro

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The effect of AG490 on the crosstalk between osteoblasts and osteoclasts in vitro