目的:探讨交感神经递质去甲肾上腺素(NE)对小鼠骨髓间充质干细胞(BMSC)迁移的影响及其机制。方法:(1)取20只3周龄雄性C57BL/6小鼠，处死后取出股骨和胫骨，分离培养BMSC并鉴定。取第2代或3代细胞，分为磷酸盐缓冲液(PBS)组、1 μmol/L NE组、10 μmol/L NE组及100 μmol/L NE组，每组8孔。1 μmol/L NE组、10 μmol/L NE组及100 μmol/L NE组细胞分别在含体积分数1%胎牛血清的低糖DMEM培养基(以下简称低血清培养基)内分别加入终物质的量浓度为1、10、100 μmol/L的NE培养，PBS组细胞在低血清培养基内加入等体积的PBS培养。在刺激前(0 d)及刺激1、3、5 d，采用细胞计数试剂盒8法检测细胞增殖活性(以吸光度值表示)。(2)细胞划痕试验1中，取细胞分为PBS组和单纯NE组，行划痕试验后，单纯NE组细胞采用低血清培养基＋终物质的量浓度为10 μmol/L的NE培养，PBS组细胞采用低血清培养基＋等体积PBS培养。细胞划痕试验2中，取细胞分为PBS组、普萘洛尔＋NE组、酚妥拉明＋NE组，行划痕试验后，普萘洛尔＋NE组细胞每天采用低血清培养基＋终物质的量浓度为1 μmol/L的普萘洛尔、酚妥拉明＋NE组细胞每天采用低血清培养基＋终物质的量浓度为10 μmol/L的酚妥拉明预处理30 min后，均再采用低血清培养基＋终物质的量浓度为10 μmol/L的NE培养；PBS组采用低血清培养基＋等体积PBS培养。细胞划痕试验3中，取细胞分为单纯NE组、单纯(2E，6E)-2，6-二(4-吡啶基亚甲基)环己酮(SC-66)组、SC-66＋NE组，行划痕试验后，单纯NE组采用低血清培养基＋终物质的量浓度为10 μmol/L的NE培养；单纯SC-66组细胞每天采用终物质的量浓度为30 mmol/L的SC-66预处理30 min后，再采用低血清培养基培养；单纯SC-66＋NE组每天采用终物质的量浓度为30 mmol/L的SC-66预处理30 min后，再采用低血清培养基＋终物质的量浓度为10 μmol/L的NE培养。以上3个细胞划痕试验，每组样本数均为6，均计算划痕后24、48、72 h的划痕愈合率。(3)取细胞分为PBS组、单纯NE组、普萘洛尔＋NE组、酚妥拉明＋NE组，每组3孔，PBS组、单纯NE组下室处理分别同细胞划痕试验1相同组，普萘洛尔＋NE组和酚妥拉明＋NE组下室处理分别同细胞划痕试验2相同组，行Transwell实验。常规培养24 h后，计数迁移细胞。(4)取细胞分为PBS组、单纯NE组、普萘洛尔＋NE组、酚妥拉明＋NE组，每组2皿，PBS组、单纯NE组细胞处理分别同细胞划痕试验1相同组，普萘洛尔＋NE组和酚妥拉明＋NE组细胞处理分别同细胞划痕试验2相同组。常规培养24 h后，采用蛋白质印迹法检测细胞蛋白激酶B(Akt)磷酸化水平。对数据行重复测量方差分析、单因素方差分析、独立样本n t检验、LSD-n t检验、Bonferroni校正。n 结果:(1)刺激1 d，100 μmol/L NE组细胞吸光度值明显低于PBS组(n t＝2.986，n P<0.05)；刺激5 d，10 μmol/L NE组细胞吸光度值明显高于PBS组(n t＝3.547，n P<0.01)。(2)细胞划痕试验1中，划痕后24、48、72 h，单纯NE组细胞划痕愈合率分别为(34.4±3.4)%、(52.5±4.7)%、(70.0±3.8)%，明显低于PBS组的(44.1±4.2)%、(80.0±3.6)%、(95.9±2.2)%(n t＝19.320、128.319、221.575，n P<0.01)。细胞划痕试验2中，划痕后24、48、72 h，普萘洛尔＋NE组细胞划痕愈合率均明显低于PBS组(n t＝4.073、9.618、15.272，n P<0.01)。细胞划痕试验3中，划痕后72 h，单纯NE组细胞划痕愈合率明显低于单纯SC-66组(n t＝8.862，n P<0.01)；划痕后24、48、72 h，SC-66＋NE组细胞划痕愈合率均明显低于单纯SC-66组(n t＝3.862、4.290、10.357，n P<0.01)。(3)Transwell实验显示，培养24 h，单纯NE组、普萘洛尔＋NE组及酚妥拉明＋NE组细胞迁移数量均明显少于PBS组(n t＝11.895、10.196、3.222，n P<0.01)。(4)培养24 h后，与PBS组比较，单纯NE组、普萘洛尔＋NE组细胞Akt磷酸化水平明显升高(n t＝8.186、5.996，n P<0.01)。n 结论:NE可抑制小鼠BMSC迁移，Akt信号通路参与了此调控过程。“,”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 n t test, least significant difference n t test, and Bonferroni correction.n 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 ( n t=2.986, n 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 (n t=3.547, n 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 (n t=19.320, 128.319, 221.575, n 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 (n t=4.073, 9.618, 15.272, n 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 (n t=8.862, n 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 (n t=3.862, 4.290, 10.357, n 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 (n t=11.895, 10.196, 3.222, n 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 (n t=8.186, 5.996, n P<0.01).n Conclusions:NE can inhibit the migration of BMSCs in mice, a process in which the signal pathway of Akt is involved in its regulation.