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Flow cytometric analysis of CD45, CD11b and NSA expression in leukocytes of healthy and tumor bearing dogs and MDSCs changes in canine mammary carcinoma cell line bearing NOD/SCID mice
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骨髓來源抑制細胞(myeloid derived suppressor cells, MDSCs)為一群異型性的不成熟骨髓細胞組成，在腫瘤、感染、敗血症等病理狀況下增多並活化，對於身體的先天及後天免疫均具有抑制作用，為腫瘤細胞躲避免疫反應的機制之一。目前獸醫學無MDSCs的相關發表研究，但有報告指出在轉移腫瘤的犬隻的抗腫瘤免疫反應較弱。我們假設腫瘤會改變MDSCs的表現，因此本研究利用流式細胞儀分析比較健康犬、良性腫瘤及惡性腫瘤犬隻白血球表現人及小鼠MDSCs的標記物CD11b、共同白血球標記物CD45及嗜中性球特異性抗體NSA的差異。並建立犬腫瘤異種移植於NOD/SCID小鼠，評估以gemcitabine治療後能否藉由控制MDSCs影響腫瘤形成。結果呈現在惡性腫瘤患犬，中度至高度CD45表現、細胞大、顆粒性高的細胞群比例顯著少於健康犬。腫瘤患犬表現CD11b的比例顯著少於健康犬。在惡性腫瘤患犬，表現中等強度CD11b的細胞百分比、表現中等強度NSA細胞百分比顯著上升。我們推測目標細胞表現中等或低強度的CD45、中等程度的CD11b及NSA。此外我們建立犬混合性乳腺癌細胞株DE-SF異種移植於NOD/SCID小鼠，顯示NOD/SCID小鼠為良好異種移植的平台。在轉殖DE-SF一個月後，小鼠脾臟及骨髓的MDSCs較無腫瘤小鼠高。研究指出gemcitabine可減少荷腫瘤小鼠脾臟MDSCs。體外試驗中此藥物對DE-SF細胞一半致死濃度近200 μM，不具強細胞殺滅毒性。以60 mg gemcitabine/kg BW腹腔注射於荷DE-SF小鼠後，脾臟及骨髓MDSCs均無下降。推測其可能原因為藥物劑量不足以殺滅NOD/SCID小鼠脾臟MDSCs亦或NOD/SCID小鼠MDSCs對gemcitabine不具感受性。總結本研究，在腫瘤患犬確實與健康犬隻呈現不同的白血球標記物表現，未來可循此路徑進一步找出影響腫瘤生長的MDSCs。NOD/SCID小鼠是否為評估減少MDSCs藥物效果平台需再研究。
Myeloid derived suppressor cells (MDSCs) compromise of a group of heterogeneous immature myeloid cells, which are expanded and activated under pathological conditions, such as cancer, infection, sepsis. MDSCs have suppressive activities on both innate and acquired immunity, and are recognized as one of the mechanisms how tumor escape immune response. At present, although there is no published research regarding to MDSCs in veterinary medicine, studies revealed that there is a decrease in antitumor immune response among dogs with metastatic cancer. We hypothesize that tumor will change circulating MDSCs, therefore, by using flow cytometry, this study compares differences in leukocyte expressions of CD11b, a positive marker for MDSCs in mice and human, common leukocyte marker CD45 and neutrophil specific antibody (NSA) expressions between healthy dogs and benign and malignant tumor bearing dogs. Results revealed that there is a significant decrease in the percentage of large and high granularity cells with medium or increased CD45 fluorescence intensity in dog with malignant tumor comparing with healthy controls. The percentage of CD11b positive leukocytes significantly decreased in tumor bearing dogs. In dogs with malignant tumor, leukocytes expressing medium intensity in CD11b and NSA increased significantly. Taken together, we suppose that our target cells express medium or decreased intensity in CD45, medium intensity in CD11b and NSA. We establish DE-SF, a canine complex mammary carcinoma cell line, xenograft in NOD/SCID mice, which proved that NOD/SCID mice is a good host for xenografts. The MDSCs percentage in both spleen and bone marrow increased comparing with naive control. Researches revealed that gemcitabine reduces the number of MDSCs in the spleen of tumor bearing mice. This drug doesn''t have a strong cytotoxic effect on DE-SF in vitro since the half lethal dose is nearly 200 μM. One day after 60 mg/kg gemcitacine IP injection in DE-SF bearing mice, MDSCs percentage in spleen and bone marrow did not reduce. We suppose that this result might be due to an insufficient dose to eliminate MDSCs in NOD/SCID mice or insensitivity of NOD/SCID mice MDSCs to gemcitabine. In conclusion, tumor bearing dogs shows different leukocyte marker expressions comparing with healthy controls, by which we could make a closer approach to find MDSCs. Whether NOD/SCID mice can be used to evaluate the effect of MDSCs eliminating drugs needs further research.
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