胶质瘤细胞和血管内皮细胞的相互作用对侵袭的影响及其与纤维连接蛋白的关系

Influence of interactions of glioma cells and endothelial cells on invasion of glioma cells and its relation with fibronectin

目的 探讨胶质瘤细胞和血管内皮细胞的相互作用对胶质瘤细胞侵袭力的影响及其这种影响与纤维连接蛋白 (FN)表达的关系。方法 在体外利用Transwell共培养系统对人脐静脉内皮细胞和C6胶质瘤细胞进行共培养 ;逆转录 聚合酶链反应 (RT PCR)和免疫细胞化学方法检测经和C6细胞共培养下血管内皮细胞上FN的基因转录及蛋白的表达情况 ;采用体外快速侵袭力测定法检测C6细胞侵袭力在不同状态的血管内皮细胞作用下的变化及FN抗体对这种变化的影响。结果 人脐静脉内皮细胞经和C6细胞共培养后出现FN的基因转录及蛋白的表达 ;在血管内皮细胞 +共培养上清液组 ,C6细胞侵袭Matrigel膜最明显 ,其次是以单纯共培养上清液为诱导剂组 ,两者差异有统计学意义 (P <0 .0 1) ,而在 0 .1%BSA +RPMI 164 0 +血管内皮细胞组 ,孵育 2 4h仍未发现C6细胞侵袭Matrigel膜。而在FN抗体封闭下孵育 2 4h时只有血管内皮细胞 +共培养上清液组的C6细胞出现侵袭 ,但侵袭的细胞数较未封闭的少 (P <0 .0 5 )。结论 胶质瘤细胞和血管内皮细胞相互作用可促进瘤细胞侵袭 ,这种影响与相互作用后内皮细胞表达FN有关。

Objective To investigate the influence of interactions of glioma cells and endothelial cells on invasion of glioma cells and its relation with fibronectin (FN).Methods Human umbilical vein endothelial cells (HUVECs)/C6 glioma cells were co cultured in vitro and the conditioned media were harvested and centrifuged to remove cellular debris,supernatants were placed at 4 ℃ (prepared as chemoattractants for C6 cells and activator for HUVECs),then the gene transcription and expression of FN in HUVECs stimulated by C6 cells were determined by the reverse transcription polymerase chain reaction (RT PCR) and immunocytochemistry respectively.A rapid in vitro quantitative assay for the invasive potential of C6 cells induced by activated HUVECs was carried out.Selected chemoattractants were treated with neutralizing antibody to FN at 1 mg/L final concentration.Results The up regulation of transcription and expression of FN was found in HUVECs after co cultured with C6 cells.The invasive potential of C6 cells in the group with both HUVECs and supernatants from co culture as chemoattractants showed a higher rate of invasion than that in the group with only supernatants from co culture ( P < 0.01). No invasion was found in group with 0.1% BSA and RPMI 1640 and HUVECs after 24 h incubation.However,when selected chemoattractants were treated with neutralizing antibody to FN,invasive potential of C6 cells in the group with both HUVECs and supernatants from co culture as chemoattractants showed a lower rate of invasion than that without treatment with neutralizing antibody to FN ( P < 0.05). No invasion was found in the other groups.Conclusion The invasion of C6 cells might be promoted by interactions of glioma cells and endothelial cells,which was contributed to the expression of FN in endothelial cells.