Slit3／Robo信号通路在小鼠主动脉平滑肌细胞的表达及其对细胞增殖和迁移的作用

Expression of Slit3/Robo signal pathway in mouse aortic smooth muscle cell and its impact on proliferation and migration

目的观察神经轴突导向分子Slit3及其受体Robo在小鼠主动脉平滑肌细胞（MASMC）中的表达，并探讨外源性Slit3蛋白对MASMC增殖和迁移的作用。方法体外原代培养并采用免疫荧光法鉴定MASMC，采用逆转录-聚合酶链反应和免疫细胞化学法检测Slit3／Robo信号通路在MASMC的表达情况。将细胞分为6组：阴性对照组（DMEM培养基中含牛血清白蛋白86μg/L）、Slit30μg/L组（DMEM培养基中无Slit3）、Slit324μg/L组（DMEM培养基中含Slit324μg/L）、Slit340μg/L组（DMEM培养基中含Slit340μg/L）、Slit380μg/L组（DMEM培养基中含Slit380μg/L）和阳性对照组（DMEM培养基中含血小板源性生长因子10μg/L）。采用CCK．8法分析Slit3对MASMC增殖的作用，采用细胞划痕法和Transwell小室检测Slit3对MASMC迁移的作用。结果（1）MASMC上有Slit2、Slit3、Robol、Rob04mRNA及蛋白的表达，Slit2的mRNA表达水平低于Slit3（P〈0．05），受体Robol与Rob04的mRNA表达水平差异无统计学意义（P〉0．05）。（2）Slit324ug／L组、Slit340μg/L组、Slit380μg/L组的MASMC增殖活性均高于阴性对照组（分别为1．13±0．04、1．19±0．02、1．18±0．08和0．64±0．10，P均〈0．05），Slit340μg/L组的MASMC增殖活性与阳性对照组（1．27±0．05）比较差异无统计学意义（P〉0．05）。（3）细胞划痕实验显示，Slit324μg/L组、Slit340μg/L组、Slit380μg/L组的细胞划痕宽度均小于阴性对照组[分别为（0．40±0．03）cm、（0．32±0．03）cm、（0．30±0．02）cm和（0．49±0．01）cm，P均〈0．05]，Slit380ng／ml组与阳性对照组[（0．22±0．01）cnl]的细胞划痕宽度差异无统计学意义（P〉0．05）。Transwell小室实验显示，Slit324μg/L组、Slit340μg/L组、Slit380μg/L组的MASMC跨膜迁移数量均多于阴性对照组[细胞跨膜迁移数量分别为（46．67±2．23）个、（65．33±3．43）个、（81．67±4．22）个和（39．33±2．03）个，P均〈0．05]，Slit380μg/L组与阳性对照组[（84．00±2．02）个]的MASMC跨膜迁移数量差异无统计学意义（P〉0．05）。结论MASMC上有Slit2、Slit3、Robol和Rob04的表达，外源性Slit3可促进MASMC的增殖和迁移。

Objective To observe the expression of neural axon guidance molecules Slit3 and Robo receptors in mouse aortic smooth muscle cell（MASMC） and investigate the effect of exogenous Slit3 protein on migration and proliferation of MASMC. Methods The primary cultured MASMC were identified by immunofluorescent assay. The expression of Slit3/Robo signal pathway was detected by RT-PCR and immnnocytochemical staining. MASMC were divided into 6 groups： the negative control group （DMEM medium containing bovine serum albumin 86 μg/L）, Slit3 0 μg/L group （DMEM medium without Slit3 ）,Slit3 24 μg/L group （DMEM medium containing Slit3 24 μg/L）, Slit3 40 μg/L group （DMEM medium containing Slit3 40 μg/L）, Slit3 80 μg/L group （DMEM medium containing Slit3 80 μg/L） and the positive control group （DMEM medium containing platelet derived growth factor 10 μg/L）. The effects of exogenous Slit3 on MASMC proliferation and migration were detected by CCK-8 and scratched cells and transwell chambers respectively. Results （ 1 ） The mRNA and protein expressions of Slit2, Slit3, Robol and Robo4 were detected in MASMC. mRNA level of Slit2 was lower than Slit3 （ P 〈 0. 05） and there were no significant difference between mRNA level of Robol and Robo4. （2） The mitogenic responses of MASMC were significantly enhanced in Slit3 24 μg/L group, Slit3 40 μg/L group and Slit3 80 μg/L group compared with negative control group （ 1.13 ± 0. 04, 1.19 ± 0. 02, 1. 18 ± 0. 08 and 0. 64 ± 0. 10 respectively, all P 〈 0. 05 ）. The mitogenic activity of MASMC was the strongest in Slit3 40 μg/L group （ compared with positive control group 1.27 ± 0. 05, P 〉 0. 05 ）. （ 3 ） The autonomous migration activity of MASMC were significantly increased in Slit3 24 μg/L group, Slit3 40 μg/L group, Slit3 80 μg/L group compared with negative control group （ cell scratch width were （0. 40 ± 0. 03 ） cm, （ 0. 32 ± 0. 03 ） cm, （ 0. 30 ± 0.02 ） cm and （ 0. 49 ± 0. 01 ）cm respectively, all P 〈 0. 05 ）. The autonomous migration activity of MASMC was the strongest in Slit3 80 μg/L group （compared with positive control group （0. 22 ± 0.01 ）cm, P〉 0.05 ）. The transmembrane migration activity of MASMC were significantly increased in Slit3 24 μg/L group, Slit3 40 μg/L group, Slit3 80 μg/L group compared with negative control group （the number of cell migration were 46. 67 ± 2. 23, 65.33 ± 3.43, 81.67 ± 4. 22 and 39. 33 ± 2.03 respectively, all P 〈 0. 05 ）. The transmembrane migration activity of MASMC was the strongest in Slit3 80 μg/L group （compared with positive control group 84. 00 ± 2. 02, P 〉 0. 05 ） . Conclusion Slit2, Slit3, Robol and Robo4 were expressed in MASMC, and exogenous Slit3 could promote proliferation and migration of MASMC in vitro.