论文部分内容阅读
目的研究用人骨髓诱导培养内皮祖细胞(EPCs)并以其作为免疫性基因载体治疗肿瘤的可行性。方法采用梯度离心法从人骨髓中分离单个核细胞,加入人血管内皮生长因子(VEGF165)诱导培养,用流式细胞仪测定细胞表面标志CD133、KDR和CD34的表达情况。以增强型绿色荧光蛋白基因(EGFP)作为报告基因,用流式细胞仪检测携带EGFP基因的腺病毒载体(Ad-EGFP)对EPCs的感染效率。携带人干扰素γ(hγIFN)基因的腺病毒载体(Ad-hγIFN)感染EPCs(EPCs-hγIFN),ELISA法检测细胞EPCs-hγIFN分泌hγIFN的情况。将EPCs-hγIFN与肺癌细胞株H460、胃癌细胞株SGC7901、大肠癌细胞株LoVo混合培养,观察EPCs-hγIFN对肿瘤细胞的作用。结果经过诱导培养2周后,贴壁的单核细胞开始表达CD133、KDR和CD34。Ad-EGFP的感染复数(MOI)为150pfu/EPCs时,感染率达90%以上。EPCs-hγIFN培养上清可以测得浓度稳定且高水平的hγIFN,第1天上清液中的hγIFN浓度为1305.28pg/ml,第14天为1419.10pg/ml。EPCs-hγIFN与H460、SGC7901、LoVo细胞株混合培养4天,肿瘤细胞的生长受到明显的抑制,肿瘤细胞存活率分别为(77.87±6.50)%、(79.36±4.35)%和(69.52±3.78)%,均显著低于对照组(P<0.05)。结论携带hγIFN目的基因的腺病毒转染EPCs后基因表达情况良好,体外实验中表现为抑制肿瘤细胞的生长,可作为免疫性基因载体开展进一步的研究。
Objective To study the feasibility of using human bone marrow to induce endothelial progenitor cells (EPCs) and use them as immune gene carriers to treat tumors. Methods Mononuclear cells were isolated from human bone marrow by gradient centrifugation and cultured with VEGF165. The expression of cell surface markers CD133, KDR and CD34 were detected by flow cytometry. The enhanced green fluorescent protein (EGFP) gene was used as a reporter gene to detect the infection efficiency of EPCs with Ad-EGFP carrying EGFP gene by flow cytometry. The EPCs-hγIFN were transfected with adenovirus vector (Ad-hγIFN) carrying human interferon γ (hγIFN) gene and the hγIFN secreted by EPCs-hγIFN by ELISA. EPCs-hγIFN were mixed with lung cancer cell line H460, gastric cancer cell line SGC7901, and colorectal cancer cell line LoVo to observe the effect of EPCs-hγIFN on tumor cells. Results After two weeks of induced culture, adherent monocytes began to express CD133, KDR and CD34. Ad-EGFP infection multiples (MOI) of 150pfu / EPCs, the infection rate of 90% or more. EPCs-hγIFN culture supernatants showed stable and high levels of hγIFN at day 1, with hγIFN concentrations of 1305.28 pg / ml in day 1 and 1419.10 pg / ml in day 14. EPCs-hγIFN were mixed with H460, SGC7901 and LoVo cell lines for 4 days. The growth of tumor cells was significantly inhibited. The survival rates of tumor cells were (77.87 ± 6.50)%, (79.36 ± 4.35)% and (69.52 ± 3.78) %, Were significantly lower than the control group (P <0.05). Conclusion The adenovirus carrying hγIFN gene has a good gene expression after transfected with EPCs. In vitro experiments showed that it inhibited the growth of tumor cells and could be used as an immunogenic gene carrier for further study.