PDGF诱导大鼠主动脉平滑肌细胞迁移模型的建立

Establishment of assay for smooth muscle cell migration induced by PDGF in rat aorta

关键词:  平滑肌细胞  Boyden小室  细胞迁移  血小板源性生长因子

Key words:  smooth muscle cell  boyden chamber  cell migration  PDGF

摘要:

［目的］ 建立PDGF诱导的大鼠主动脉平滑肌细胞(A7r5)迁移的模型。［方法］ 采用改良的Boyden小室法，以不同浓度(1、5、10、20、50和100 ng/mL)PDGF-BB为诱导剂，将不同浓度(1×10⁵/mL、2×10⁵/mL、4×10⁵/mL)的A7r5细胞置入 Boyden小室，分别培养5、7、9 h。迁移膜经Giemsa染色后，显微镜下观察迁移到下室的细胞，进行细胞计数。［结果］ 在无PDGF诱导时，迁移细胞数为每视野10个左右；而以10 ng/mL PDGF-BB诱导时，迁移细胞数每视野可达70个左右；当PDGF浓度达到100 ng/mL时，细胞迁移数降至每视野10个左右。随细胞浓度的增高，迁移细胞数目增加，细胞浓度为1×10⁵/mL时，7 h迁移细胞数目为(48±8.944)个；2×10⁵/mL细胞在5 h左右发生迁移的数目为(55±12.864)个，随时间延长而增多，在7 h前后增幅最明显，达(78±15.828)个(P<0.05)；但细胞浓度为4×10⁵/mL时，7 h迁移细胞数为(84±12.213)个(P>0.05)。［结论］ 以10 ng/mL PDGF-BB为诱导剂，以2×10⁵/mL浓度接种，培养7 h建立的迁移模型为最佳。

Abstract:

［Objective］ To establish the model of rat aorta smooth muscle cell(A7r5) migration induced by PDGF.［Methods］ VSMC migration in response to different concentration of PDGF-BB(1, 5,10, 20, 50 and 100 ng/mL) was determined by a modified Boyden chamber method. The various concentration of cell suspension(1×10⁵/mL, 2×10⁵/mL and 4×10⁵/mL) was added into the upper chamber, cells were incubated for 5, 7 and 9 hr respectively. The cells migrated to the lower surface of the filter were stained with giemsa solution, and observed by a light microscope(400). ［Results］ The number of migrated cells (NMC) was about 10/visual field without PDGF. The NMC reached 70/visual field when the cells induced by 10 ng/mL PDGF. The NMC dropped to 10/visual field when the cells induced by 100 ng/mL PDGF. The NMC with 1×10⁵/mL concerntration was 48±8.944 at 7 hr. The NMC with 2×10⁵/mL concerntration was 55±12.864 at 5 hr, and increased dramatically at 7 hr, the NMC reached 78±15.828( P<0.05), but only was 84±12.213( P>0.05) when the cell concentration reached 4×10⁵/mL. ［Conclusion］ The optimal condition is that 2×10⁵/mL cells are added in the upper chamber and the migrated cells are induced for 7 hr. by using 10 ng/mL PDGF-BB.