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目的研究体外内皮祖细胞对C6胶质瘤细胞增殖能力的影响。方法取SD大鼠脾脏采用密度梯度离心法获取内皮祖细胞,通过观察其分化过程中的形态特征,激光共聚焦检测其吞噬乙酰化低密度酯蛋白(DiI-acLDL)和结合荆豆凝集素-1(FITC-UEA-1)的特异性,免疫荧光检测细胞表面抗原CD34和CD31的表达,对EPCs进行鉴定。按0、10%、20%、30%、40%、50%6个浓度将内皮祖细胞条件培养基加至C6胶质瘤细胞的常规培养基中观测其对C6胶质瘤细胞增殖的影响;分别采用内皮祖细胞条件培养基和常规培养基孵育C6胶质瘤细胞24h,流式细胞仪检测2组的细胞周期。结果激光共聚焦鉴定DiI-acLDL和FITC-UEA-1双染色阳性细胞即为正在分化的内皮祖细胞;免疫荧光检测内皮祖细胞一致性表达CD34和CD31。随着内皮祖细胞条件培养基含量的增加,36h和48h各浓度组的光密度值也随着增加,且50%内皮祖细胞条件培养基组D(490)值(2.018±0.220)、(2.388±0.448)均高于对照组(1.163±0.103)、(1.106±0.174),且差别有统计学意义(P<0.01)。50%含有内皮祖细胞条件培养基培养的C6胶质瘤细胞的增殖率(即S+G2/M)明显高于对照组[(34.650±1.492)%vs(29.587±2.504)%,P<0.05]。结论体外内皮祖细胞条件培养基能促进C6胶质瘤细胞的增殖,其机制可能与其分泌的各种血管生成类细胞因子有关。
Objective To investigate the effect of endothelial progenitor cells (EPCs) on the proliferation of C6 glioma cells in vitro. Methods The spleen of SD rats were obtained by density gradient centrifugation and the morphological characteristics of the cells were observed by laser scanning confocal microscopy. DiI-acLDL and phage lectin- 1 (FITC-UEA-1). Immunofluorescence was used to detect the expression of cell surface antigen CD34 and CD31. EPCs were identified. Endothelial progenitor cells conditioned medium was added to conventional culture medium of C6 glioma cells at 0, 10, 20, 30, 40, 50% concentration to observe its effect on the proliferation of C6 glioma cells The C6 glioma cells were incubated with endothelial progenitor cells conditioned medium and conventional medium for 24 h respectively. The cell cycle of the two groups was detected by flow cytometry. Results Confocal laser scanning confocal laser microscopy showed that the differentiated endothelial progenitor cells were positive for DiI-acLDL and FITC-UEA-1 double staining positive cells. The expression of CD34 and CD31 in endothelial progenitor cells was detected by immunofluorescence. With the increase of the conditioned media of EPCs, the optical density values increased with the increasing of 36h and 48h, and the values of D (490) (2.018 ± 0.220), (2.388 ± 0.448) were higher than that of the control group (1.163 ± 0.103) and (1.106 ± 0.174), respectively, and the difference was statistically significant (P <0.01). The proliferation rate (ie S + G2 / M) of C6 glioma cells cultured in conditioned media containing 50% endothelial progenitor cells was significantly higher than that in the control group [(34.650 ± 1.492)% vs (29.587 ± 2.504)%, P <0.05 ]. Conclusion Conditioned media of endothelial progenitor cells in vitro can promote the proliferation of C6 glioma cells, which may be related to the secretion of various angiogenic cytokines.