Effects and mechanism of norepinephrine on the migration of bone marrow mesenchymal stem cells in mice
10.3760/cma.j.cn501120-20200325-00194
- VernacularTitle:去甲肾上腺素对小鼠骨髓间充质干细胞迁移的影响及其机制
- Author:
Yanan KONG
1
;
Jing JIN
;
Biao CHENG
Author Information
1. 安徽医科大学第一附属医院整形外科，合肥 230022
- Keywords:
Norepinephrine;
Mesenchymal stem cells;
Cell migration assays
- From:
Chinese Journal of Burns
2020;36(12):1173-1182
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the effects and mechanism of sympathetic neurotransmitter norepinephrine (NE) on the migration of bone marrow mesenchymal stem cells (BMSCs) in mice.Methods:(1) Twenty 3-week-old male C57BL/6 mice were sacrificed for isolating, culturing, and identifying BMSCs from the femur and tibia. Cells of the second or third passages were divided into phosphate buffer solution (PBS) group, 1 μmol/L NE group, 10 μmol/L NE group, and 100 μmol/L NE group, with 8 wells in each group. Cells in 1 μmol/L NE group, 10 μmol/L NE group, and 100 μmol/L NE group were cultured in low-sugar Dulbecco′s modified eagle medium containing 1% volume fraction of fetal bovine serum (hereinafter referred to as low-serum medium) added with NE in final molarity of 1 μmol/L, 10 μmol/L, and 100 μmol/L, respectively. Cells in PBS group were cultured in low-serum medium added with the same volume of PBS. Before stimulation (0 d) and on stimulation day 1, 3, 5, cell counting kit 8 method was used to detect cell proliferation activity (expressed as the absorbance value). (2) In cell scratch test 1, cells were divided into PBS group and simple NE group. After the scratch test, cells in simple NE group were cultured with low-serum medium+ NE in final molarity of 10 μmol/L, and cells in PBS group were cultured with low-serum medium+ the same volume of PBS. In cell scratch test 2, cells were divided into PBS group, propranolol+ NE group, and phentolamine+ NE group. After the scratch test, cells in propranolol+ NE group were pretreated with low-serum medium+ propranolol in final molarity of 1 μmol/L for 30 minutes each day, cells in phentolamine+ NE group were pretreated with low-serum medium+ phentolamine in final molarity of 10 μmol/L for 30 minutes each day, and then they were cultured with low-serum medium+ NE in final molarity of 10 μmol/L. Cells in PBS group were cultured with low-serum medium+ the same volume of PBS. In cell scratch test 3, cells were divided into simple NE group, simple (2E, 6E)-2, 6-bis (4-pyridylmethylene) cyclohexanone (SC-66) group, and SC-66+ NE group. After the scratch test, cells in simple NE group was cultured with low-serum medium+ NE in final molarity of 10 μmol/L, cells in simple SC-66 group were cultured with low-serum medium after being pretreated with SC-66 in final molarity of 30 mmol/L for 30 minutes every day, cells in SC-66+ NE group were cultured with low-serum medium+ NE in final molarity of 10 μmol/L after being pretreated with SC-66 in final molarity of 30 mmol/L for 30 minutes every day. In the above 3 cell scratch tests, the sample numbers in each group were all 6, and the scratch healing rates at post scratch hour (PSH) 24, 48, and 72 were all calculated. (3) Cells were divided into PBS group, simple NE group, propranolol+ NE group, and phentolamine+ NE group, with 3 wells in each group. The lower chamber treatment methods of PBS group and simple NE group were the same as those of the same groups in cell scratch test 1. The lower chamber treatment of propranolol+ NE group and phentolamine+ NE group were the same as those of the same groups in cell scratch test 2. After the Transwell experiment was performed and the cells were routinely cultured for 24 hours, the migrated cells were counted. (4) Cells were divided into PBS group, simple NE group, propranolol+ NE group, and phentolamine+ NE group, with 2 dishes in each group. The cell treatment of PBS group and simple NE group were the same as those of the same groups in cell scratch test 1. The cell treatment of propranolol+ NE group and phentolamine+ NE group were the same as those of the same groups in cell scratch test 2. After 24 hours of routine culture, the phosphorylation level of protein kinase B (Akt) of cells was detected by Western blotting. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, independent sample t test, least significant difference t test, and Bonferroni correction. Results:(1) After 1 day of stimulation, the absorbance value of cells in 100 μmol/L NE group was significantly lower than that in PBS group ( t=2.986, P<0.05). After 5 days of stimulation, the absorbance value of cells in 10 μmol/L NE group was significantly higher than that in PBS group ( t=3.547, P<0.01). (2) In cell scratch test 1, at PSH 24, 48, and 72, the scratch healing rates of cells in simple NE group were (34.4±3.4)%, (52.5±4.7)%, and (70.0±3.8)%, which were significantly lower than (44.1±4.2)%, (80.0±3.6)%, and (95.9±2.2)% in PBS group ( t=19.320, 128.319, 221.575, P<0.01). In cell scratch test 2, at PSH 24, 48, and 72, the scratch healing rates of cells in propranolol+ NE group were significantly lower than those in PBS group ( t=4.073, 9.618, 15.272, P<0.01). In cell scratch test 3, at PSH 72, the scratch healing rates of cells in NE group was significantly lower than that in simple SC-66 group ( t=8.862, P<0.01). At PSH 24, 48, and 72, the scratch healing rates of cells in SC-66+ NE group were significantly lower than those in simple SC-66 group ( t=3.862, 4.290, 10.357, P<0.01). (3) The Transwell experiment showed that after 24 hours of culture, the numbers of migrated cells in simple NE group, propranolol+ NE group, and phentolamine+ NE group were significantly less than the number in PBS group ( t=11.895, 10.196, 3.222, P<0.01). (4) After 24 hours of culture, the phosphorylation levels of Akt of cells in simple NE group and propranolol+ NE group were significantly higher than the level in PBS group ( t=8.186, 5.996, P<0.01). Conclusions:NE can inhibit the migration of BMSCs in mice, a process in which the signal pathway of Akt is involved in its regulation.
