Fernandes Patrícia Dias, Guerra Fabiana S, Sales Natália M, Sardella Thais B, Jancar Sonia, Neves Josiane S
Universidade Federal do Rio de Janeiro, Instituto de Ciências Biomédicas, Laboratório de Farmacologia da Dor e da Inflamação, Brazil.
Universidade Federal do Rio de Janeiro, Instituto de Ciências Biomédicas, Laboratório de Farmacologia da Dor e da Inflamação, Brazil.
J Pharmacol Toxicol Methods. 2015 Jan-Feb;71:83-9. doi: 10.1016/j.vascn.2014.09.001. Epub 2014 Sep 6.
Ehrlich tumor is a mammary adenocarcinoma with aggressive behavior. Inoculated in mice peritoneal cavity, the Ehrlich tumor grows in ascitic form (EAT). Since inflammation modulates tumor progression we further investigated the inflammatory response during EAT growth.
Balb/C mice were intraperitoneal inoculated with 5×10(5) Ehrlich cells and after every 2days, blood samples were collected for hemoglobin, hematocrit, platelets and leukocytes counts. The ascitic fluid was collected for protein concentration and cell count. Phenotype analysis of the peritoneal cells was made by FACS, prostaglandin E2 (PGE2) and cytokines by ELISA, nitric oxide (NO) by nitrate conversion protocol, and cyclooxygenase-1 (COX1), COX2 and inducible nitric oxide synthase (iNOS) by immunoblotting.
Following EAT inoculation into the peritoneal cavity there was a rapid increase in ascitis volume and protein concentration. The cell number in ascitis remained stable until day 8 (lag phase) followed by a sharp increase. As tumor progressed, blood leukocytes increased and erythrocyte decreased. Phenotypic analysis showed that during the lag phase the percentage of F4/80(+) cells remained similar to control levels and around 7% of this population was also positive for the GR1 marker. These double-positive cells (probably inflammatory monocytes) markedly increased at day 6. The percentage of F4/80-GR1(+)cells (probably neutrophils) was low and did not significantly vary during tumor progression. CD4(+) and CD8(+) cells were not detected in the time points analyzed. iNOS and COX1 expression increased after day 2 reaching peak levels on day 10. COX2 enzyme expression did not change significantly over time. Sustained increase in PGE2 and NO levels was observed. IL-10 and MCP-1 peaked at day 14 and IL-1β increased progressively till day 10. IFN-γ levels were low till day 10, increasing progressively after that.
These data extended the characterization of the inflammatory response during Ehrlich ascitis tumor growth, further validating it as a useful model for antitumor drugs screening.
艾氏瘤是一种具有侵袭性的乳腺腺癌。接种于小鼠腹腔后，艾氏瘤以腹水形式生长（EAT）。由于炎症可调节肿瘤进展，我们进一步研究了EAT生长过程中的炎症反应。
将5×10⁵个艾氏细胞腹腔接种到Balb/C小鼠体内，每2天采集一次血样，检测血红蛋白、血细胞比容、血小板和白细胞计数。收集腹水检测蛋白浓度和细胞计数。通过流式细胞术对腹腔细胞进行表型分析，通过酶联免疫吸附测定法检测前列腺素E2（PGE2）和细胞因子，通过硝酸盐转化法检测一氧化氮（NO），通过免疫印迹法检测环氧合酶-1（COX1）、COX2和诱导型一氧化氮合酶（iNOS）。
将EAT接种到腹腔后，腹水体积和蛋白浓度迅速增加。腹水细胞数量在第8天（潜伏期）前保持稳定，随后急剧增加。随着肿瘤进展，血白细胞增加而红细胞减少。表型分析显示，在潜伏期，F4/80⁺细胞百分比与对照水平相似，该群体中约7%的细胞GR1标记也呈阳性。这些双阳性细胞（可能是炎性单核细胞）在第6天显著增加。F4/80-GR1⁺细胞（可能是中性粒细胞）百分比很低，在肿瘤进展过程中无明显变化。在所分析的时间点未检测到CD4⁺和CD8⁺细胞。iNOS和COX-1表达在第2天后增加，在第10天达到峰值水平。COX2酶表达随时间无明显变化。观察到PGE2和NO水平持续升高。IL-10和MCP-1在第14天达到峰值，IL-1β在第10天前逐渐增加。IFN-γ水平在第10天前较低，之后逐渐升高。
这些数据扩展了艾氏腹水瘤生长过程中炎症反应的特征，进一步验证了其作为抗肿瘤药物筛选有用模型的价值。
