制备人脐静脉内皮细胞和人肺腺癌细胞融合细胞的新方法.
制备人脐静脉内皮细胞和人肺腺癌细胞融合细胞的新方法.
机构信息
Department of Neonatology, Chengdu Women's and Children's Central Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, China.
出版信息
Technol Cancer Res Treat. 2021 Jan-Dec;20:15330338211034260. doi: 10.1177/15330338211034260.
PURPOSE
Human umbilical endothelial cells (HUVECs) have been proved to be an effective whole-cell vaccine inhibiting tumor angiogenesis. In this study, we fused HUVECs with human lung adenocarcinoma cells A549 s, aiming at preparing lung cancer vaccine to achieve dual effects of anti-tumor angiogenesis and specific immunity to tumor cells.
METHODS
A549 cells were induced by ethyl methane sulfonate (EMS) and 8-azaguanine (8-AG) to get hypoxanthine guanine phosphoribosyl transferase (HGPRT) auxotrophic A549 cells. Then Fused HGPRT auxotrophic A549 cells with primary HUVEC cells by combining electrofusion with polyethylene glycol (PEG). Afterward the fusion cells were screened by HAT and HT selective medium and sorted by flow cell sorter to obtain high-purity HUVEC-A549 cells. Finally, HUVEC-A549 cells were identified by karyotype analysis and western blotting.
RESULTS
The fusion efficiency of HUVEC-A549 cells prepared by combining electrofusion with polyethylene glycol (PEG) was significantly higher than that of electrofusion and PEG (43.0% vs 17.60% vs 2.71%, < 0.05). After screened by HAT and HT selective medium and sorted by flow cell sorter, the proportion of HUVEC-A549 cells can count for 71.2% ± 3.2%. The mode of chromosomes in HUVEC-A549 cells was 68, and the chromosome was triploid. VE-cadherin and platelet endothelial cell adhesion molecule-1 (CD31) were highly expressed in HUVECs and HUVEC-A549 cells, but not in A549 cells.
CONCLUSIONS
These results indicate that HUVEC-A549 cells retain the biological characteristics of human umbilical vein endothelial cells and A549 cells. It can be used in the experimental study of lung cancer cell vaccine.
目的
人脐静脉内皮细胞（HUVEC）已被证明是一种有效的抑制肿瘤血管生成的全细胞疫苗。在本研究中，我们将 HUVEC 与人类肺腺癌细胞 A549 融合，旨在制备肺癌疫苗，以达到抗肿瘤血管生成和肿瘤细胞特异性免疫的双重效果。
方法
用乙基甲烷磺酸（EMS）和 8-氮鸟嘌呤（8-AG）诱导 A549 细胞，获得次黄嘌呤鸟嘌呤磷酸核糖转移酶（HGPRT）缺陷型 A549 细胞。然后通过电融合与聚乙二醇（PEG）将 HGPRT 缺陷型 A549 细胞与原代 HUVEC 细胞融合。接着通过 HAT 和 HT 选择性培养基筛选融合细胞，并通过流式细胞分选对其进行纯化，获得高纯度的 HUVEC-A549 细胞。最后，通过染色体核型分析和 Western blot 鉴定 HUVEC-A549 细胞。
结果
电融合与聚乙二醇（PEG）联合法制备的 HUVEC-A549 细胞融合效率明显高于电融合法和 PEG 法（43.0% vs 17.60% vs 2.71%， < 0.05）。经过 HAT 和 HT 选择性培养基筛选和流式细胞分选后，HUVEC-A549 细胞的比例可占 71.2% ± 3.2%。HUVEC-A549 细胞的染色体模式为 68 条，染色体为三倍体。HUVEC 和 HUVEC-A549 细胞中高表达血管内皮钙黏蛋白（VE-cadherin）和血小板内皮细胞黏附分子-1（CD31），而 A549 细胞中不表达。
结论
这些结果表明 HUVEC-A549 细胞保留了人脐静脉内皮细胞和 A549 细胞的生物学特性，可用于肺癌细胞疫苗的实验研究。
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