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目的:研究肺癌细胞培养上清液对内皮祖细胞(EPCs)功能和活性的影响。方法:密度梯度离心法从大鼠骨髓分离单个核细胞,经差速贴壁后取二次贴壁细胞接种于培养瓶内;收集培养5d的EPCs,用激光共聚焦显微镜鉴定,AC133和vWF双染阳性细胞为正在分化的EPCs;分别用MTT比色法、Transwell小室、Matrigel管腔形成模型检测不同体积分数(0、10%、20%和40%)肺癌细胞培养上清液作用不同时间(6、12、24和48h)对EPCs增殖、迁移及管腔形成能力的影响。结果:肺癌细胞培养上清液作用后,EPCs的增殖活性、迁移的细胞数、管腔形成数逐渐增多,呈剂量及时间依赖效应。作用24h后,对照组EPCs增殖活性OD490值为0.332±0.051,体积分数为10%、20%和40%时OD490值分别为0.411±0.018(P<0.05)、0.446±0.036(P<0.01)和0.467±0.018(P<0.01);对照组迁移细胞数(/×400视野)为37.0±3.937,体积分数为10%、20%和40%时迁移细胞数分别为42.2±3.114(P<0.05)、44.8±3.564(P<0.05)和46.2±4.025(P<0.01);对照组管腔形成数量(/×100视野)为7.0±0.71,体积分数为10%、20%、40%时管腔形成数量分别为8.4±1.14(P<0.05)、9.2±1.30(P<0.05)和10.2±1.30(P<0.01)。结论:在体外,肺癌细胞培养上清液能够促进EPCs的增殖、迁移和管腔形成能力,并在一定范围内呈剂量及时间依赖效应,推测在体内肿瘤细胞可能通过分泌活性物质增强EPCs活性,促进肿瘤内血管生成。
Objective: To investigate the effect of lung cancer cell culture supernatant on the function and activity of endothelial progenitor cells (EPCs). Methods: Mononuclear cells were isolated from bone marrow of rats by density gradient centrifugation and were seeded into culture flasks after secondary adherent cells were differentiated. The EPCs cultured for 5 days were collected and identified by laser confocal microscopy. AC133 and vWF bis The positive staining cells were differentiated into EPCs. MTT assay, Transwell chamber, and Matrigel tube formation assay were used to detect the effect of culture supernatants with different volume fraction (0, 10%, 20% and 40% 6,12,24 and 48h) on EPCs proliferation, migration and lumen formation ability. Results: The proliferative activity of EPCs, the number of migrated cells and the number of lumen formation gradually increased after lung cancer cell culture supernatant, in a dose and time dependent manner. After 24h, the OD490 value of control group was 0.332 ± 0.051, OD490 values were 0.411 ± 0.018 (P <0.05), 0.446 ± 0.036 (P <0.01) and 10% 0.467 ± 0.018 (P <0.01). The numbers of migrated cells in the control group (37 × 3.9) were 37.0 ± 3.937. The numbers of migrated cells were 42.2 ± 3.114 (P <0.05) when the volume fraction was 10%, 20% and 40% , 44.8 ± 3.564 (P <0.05) and 46.2 ± 4.025 (P <0.01). The number of lumen formation in the control group was 7.0 ± 0.71 and the volume fraction was 10%, 20%, 40% The formation numbers were 8.4 ± 1.14 (P <0.05), 9.2 ± 1.30 (P <0.05) and 10.2 ± 1.30 (P <0.01), respectively. CONCLUSION: In vitro, the supernatant of lung cancer cell culture can promote the proliferation, migration and luminal formation of EPCs in a dose-and time-dependent manner within a certain range. It is speculated that in vivo tumor cells may enhance EPCs activity through secretion of active substances, Promote tumor angiogenesis.