右美托咪定调控CCL2-CCR2通路对胆囊癌细胞生物学行为的影响

doi:10.11958/20252587

摘要/Abstract

摘要：

目的探索右美托咪定调控C-C趋化因子配体2（CCL2）-C-C趋化因子受体2（CCR2）通路对胆囊癌细胞增殖、侵袭和细胞周期的影响。方法将GBC-SD细胞分为对照组、右美托咪定低浓度（2 μmol/L）组、右美托咪定高浓度（4 μmol/L）组、右美托咪定高浓度+CCL2组（4 μmol/L右美托咪定和10 μg/L CCL2蛋白）；克隆形成实验和Edu实验测定细胞增殖；Transwell实验测定细胞侵袭和迁移；流式细胞术测定细胞周期和凋亡；Western blot测定增殖细胞核抗原（PCNA）、细胞周期蛋白D1（CyclinD1）、基质金属蛋白酶（MMP）-2、MMP-9、Bcl-2相关X蛋白（Bax）、胱天蛋白酶-3（Caspase-3）、CCL2、CCR2蛋白表达；裸鼠移植瘤实验观察胆囊癌移植瘤生长情况。结果低、高浓度右美托咪定处理后，克隆形成数、Edu阳性率、侵袭及迁移数下降，PCNA、CyclinD1、MMP-2、MMP-9、CCL2及CCR2蛋白表达量降低，G2/M期、S期细胞比例减少，G0/G1期细胞比例、细胞凋亡率及Bax、Caspase-3蛋白表达量升高，且高浓度右美托咪定的效果更明显（P<0.05）；右美托咪定对胆囊癌细胞增殖、侵袭、迁移和细胞周期的抑制作用及对细胞凋亡的促进作用可被CCL2蛋白所逆转（P<0.05）；体内实验显示，右美托咪定可降低裸鼠肿瘤质量、肿瘤体积及肿瘤组织CCL2、CCR2蛋白表达量（P<0.05）。结论右美托咪定通过抑制CCL2-CCR2通路抑制胆囊癌细胞增殖、侵袭，并将细胞周期阻滞在G0/G1期。

关键词: 右美托咪定, 胆囊肿瘤, 细胞系，肿瘤, 趋化因子CCL2, 受体，CCR2, 细胞周期, 细胞增殖, 细胞运动

Abstract:

Objective To explore the effect of dexmedetomidine on the proliferation, invasion and cell cycle of gallbladder cancer cells by regulating the C-C chemokine ligand 2 (CCL2) - C-C chemokine receptor 2 (CCR2) pathway. Methods GBC-SD cells were devided into the control group, the low concentration dexmedetomidine group (2 μmol/L), the high concentration dexmedetomidine group (4 μmol/L) and the high concentration dexmedetomidine+CCL2 group (4 μmol/L dexmedetomidine and 10 μg/L CCL2 protein). The clone formation experiment and Edu experiment were performed to measure cell proliferation. Transwell experiment was performed to measure cell invasion and migration. Flow cytometry was performed to measure cell cycle and apoptosis. Western blot assay was used to measure the proliferating cell nuclear antigen (PCNA), Cyclin D1, matrix metalloproteinase (MMP)-2, MMP-9, Bcl-2 associated X protein (Bax), cysteine-dependent aspartate-specific protease-3 (Caspase-3), CCL2 and CCR2 proteins. The nude mouse transplant tumor experiment was used to determine the growth of gallbladder cancer transplant tumors. Results After treatment with low and high concentrations of dexmedetomidine, the number of cell clone formed, the positive rate of Edu, the numbers of invasions and migrations, the expression levels of PCNA, CyclinD1, MMP-2, MMP-9, CCL2 and CCR2 proteins, the proportions of G2/M and S phase cells decreased, the proportion of G0/G1 phase cells, apoptosis rate and expression levels of Bax and Caspase-3 proteins increased, and the effect of high-concentration dexmedetomidine was more significant (P<0.05). The inhibitory effects of dexmedetomidine on the proliferation, invasion, migration and cell cycle of gallbladder cancer cells, as well as its promoting effect on cell apoptosis could be reversed by CCL2 protein (P<0.05). In vivo experiments showed that dexmedetomidine could reduce tumor mass, tumor volume of nude mice and expression levels of CCL2 and CCR2 proteins (P<0.05). Conclusion Dexmedetomidine inhibits the proliferation and invasion of gallbladder cancer cells, and blocks the cell cycle in the G0/G1 phase by suppressing the CCL2-CCR2 pathway.

Key words: dexmedetomidine, gallbladder neoplasms, cell line, tumor, chemokine CCL2, receptors, CCR2, cell cycle, cell proliferation, cell movement

中图分类号:

R735.8

图/表 16

图1 各组细胞克隆形成实验的结晶紫染色结果 A：对照组；B：右美托咪定低浓度组；C：右美托咪定高浓度组；D：右美托咪定高浓度+CCL2组。图2—8同。

Fig.1 The crystal violet staining results of cell clone formation experiments in each group

图2 各组细胞增殖情况的Edu染色结果（×200）

Fig.2 Results of Edu staining for cell proliferation in each group (×200)

图3 各组细胞PCNA、CyclinD1蛋白表达条带

Fig.3 Protein expression bands of PCNA and CyclinD1 in each group of cells

表1 各组细胞克隆形成数、Edu阳性细胞率及PCNA、CyclinD1蛋白表达比较

Tab.1 Comparison of clone formation numbers, Edu-positive cell rates and protein expressions of PCNA and CyclinD1 between four groups of cells （n=6，$ \bar{x} \pm s$）

组别	克隆形成数/个	Edu阳性细胞率/%
对照组	192.63±17.14	45.18±4.93
右美托咪定低浓度组	141.78±13.52^a	31.84±3.64^a
右美托咪定高浓度组	84.25±9.81^ab	17.76±2.06^ab
右美托咪定高浓度+CCL2组	170.04±15.49^c	39.52±4.38^c
F	64.745^＊＊	55.510^＊＊
组别	PCNA	CyclinD1
对照组	1.31±0.14	1.02±0.11
右美托咪定低浓度组	0.96±0.11^a	0.64±0.07^a
右美托咪定高浓度组	0.43±0.05^ab	0.31±0.03^ab
右美托咪定高浓度+CCL2组	1.18±0.12^c	0.87±0.09^c
F	74.305^＊＊	88.185^＊＊

图4 各组细胞增殖情况的Transwell实验结果（×200）

Fig.4 Results of Transwell assay for cell proliferation in each group (×200)

图5 各组细胞MMP-2、MMP-9蛋白表达条带

Fig.5 Protein expression bands of MMP-2 and MMP-9 in each group of cells

表2 各组细胞侵袭、迁移数及MMP-2、MMP-9蛋白表达比较

Tab.2 Comparison of invasion and migration numbers, expression levels of MMP-2 and MMP-9 proteins between four groups of cells （n=6，$ \bar{x} \pm s$）

组别	侵袭数/个	迁移数/个
对照组	126.83±15.14	142.32±16.38
右美托咪定低浓度组	71.45±8.57^a	87.17±9.84^a
右美托咪定高浓度组	32.72±4.13^ab	41.45±4.66^ab
右美托咪定高浓度+CCL2组	110.24±12.36^c	123.06±14.24^c
F	89.792^＊＊	80.139^＊＊
组别	MMP-2	MMP-9
对照组	1.36±0.13	1.25±0.13
右美托咪定低浓度组	0.91±0.10^a	0.78±0.08^a
右美托咪定高浓度组	0.48±0.05^ab	0.33±0.04^ab
右美托咪定高浓度+CCL2组	1.21±0.13^c	1.04±0.11^c
F	78.065^＊＊	101.924^＊＊

表3 各组细胞G0/G1期、G2/M期、S期细胞比例比较

Tab.3 Comparison of proportions of cells in G0/G1 phase, G2/M phase and S phase between four groups of cells （n=6，%，$ \bar{x} \pm s$）

组别	G0/G1期	G2/M期	S期
对照组	26.18±2.93	24.74±2.81	49.08±5.02
右美托咪定低浓度组	41.42±4.71^a	16.85±1.84^a	41.73±4.32^a
右美托咪定高浓度组	54.83±5.90^ab	11.26±1.42^ab	33.91±3.27^ab
右美托咪定高浓度+CCL2组	33.25±3.67^c	21.57±2.58^c	45.18±5.01^c
F	45.930^＊＊	41.478^＊＊	12.540^＊＊

图6 各组细胞凋亡情况的流式细胞仪测定结果

Fig.6 Results of flow cytometry for the apoptosis status of each group of cells

图7 各组细胞Bax、Caspase-3蛋白表达条带

Fig.7 Protein expression bands of Bax and Caspase-3 in each group of cells

表4 各组细胞凋亡率及Bax、Caspase-3蛋白表达比较

Tab.4 Comparison of apoptosis rates and protein expression of Bax and Caspase-3 between four groups of cells （n=6，$ \bar{x} \pm s$）

组别	细胞凋亡率/%	Bax	Caspase-3
对照组	3.06±0.45	0.74±0.08	0.48±0.05
右美托咪定低浓度组	15.36±2.12^a	1.23±0.16^a	0.85±0.09^a
右美托咪定高浓度组	32.97±3.84^ab	1.68±0.19^ab	1.36±0.15^ab
右美托咪定高浓度+ CCL2组	9.14±1.42^c	0.92±0.10^c	0.61±0.07^c
F	186.359^＊＊	52.044^＊＊	95.179^＊＊

图8 各组细胞CCL2、CCR2蛋白表达条带

Fig.8 Expression bands of CCL2 and CCR2 proteins in each group of cells

表5 各组细胞CCL2、CCR2蛋白表达比较

Tab.5 Comparison of CCL2 and CCR2 protein expression between four groups of cells （n=6，$ \bar{x} \pm s$）

组别	CCL2	CCR2
对照组	1.16±0.12	1.21±0.13
右美托咪定低浓度组	0.81±0.09^a	0.89±0.10^a
右美托咪定高浓度组	0.57±0.06^ab	0.62±0.07^ab
右美托咪定高浓度+CCL2组	0.99±0.10^c	1.08±0.11^c
F	42.432^＊＊	35.900^＊＊

图9 各组裸鼠肿瘤生长情况

Fig.9 Tumor growth status of nude mice in each group

图10 2组裸鼠肿瘤组织CCL2、CCR2蛋白表达条带

Fig.10 Expression bands of CCL2 and CCR2 proteins in tumor tissue of each group of nude mice

表6 2组裸鼠体质量、肿瘤质量、肿瘤体积及CCL2、CCR2蛋白表达比较

Tab.6 Comparison of body weight, tumor mass, tumor volume and expression levels of CCL2 and CCR2 proteins between four groups of nude mice （n=10，$ \bar{x} \pm s$）

组别		体质量/g		肿瘤质量/g
对照组		23.12±2.43		0.61±0.06
右美托咪定组		22.76±2.30		0.34±0.04
t		0.340		11.840^＊＊
组别	肿瘤体积/mm³		CCL2		CCR2
对照组	1 361.85±57.28		0.93±0.10		0.87±0.09
右美托咪定组	967.46±40.12		0.45±0.05		0.41±0.04
t	17.834^＊＊		13.576^＊＊		14.770^＊＊

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