犬自然發生腫瘤主要組織相容複合物 及細胞激素表現概述之特徵

Abstract

犬自然發生腫瘤之主要組織相容複合物(Major histocompatibility complex, MHC)及細胞激素模版(profile)的詳盡資料非常有限，然而，此資料對於基因治療之應用相當重要。本研究乃自台北的動物醫院蒐集62隻患有腫瘤的犬隻。將腫瘤塊研磨至單一細胞懸浮液(single cell suspension)，使用流式細胞儀分析下述各項資料：1)腫瘤細胞之MHC I, II含量，2)腫瘤浸潤淋巴球(Tumor infiltrating lymphocyte, TIL)之CD3, 4, 8, 21，及3)週邊血液淋巴球(Peripheral blood lymphocyte, PBL)之MHC I, II,及CD3, 4, 8, 21。並使用Real-time RT PCR偵測IL-2, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, TNF-α, TGF-β, IFN-γ之mRNA含量。將上述資料配合病歷及動物之預後，使用Biomarker Patterns Software分析，得到MHC I 表現量72.58%之數值，若低於此數值以乳房腫瘤(Mammary gland tumor, MGT)居多，若高於此數值則以其他腫瘤為主。MGT中，MHC I表現低於72.58%者超過91%(33/36)。3個良性MGT中，2個(67%)MHC I表現量低；而33個惡性MGT中，高達31個(94%)MHC I低表現。進一步比較，健康乳腺細胞(Healthy mammary gland, HMG)與腫瘤細胞細胞激素之表現，發現MGT之IL-8顯著較高 (p<0.05)。將MGT依組織病理學細分為良性、惡性，並將惡性MGT細分為simple、complex、tubulopapillary惡性上皮性腫瘤。雖然IL-8在MGT表現量顯著高於HMG，但惡性MGT中只有simple carcinoma之IL-8有此顯著性差異，其他類別之MGT無此現象。使用免疫組織化學染色後發現，IL-8表現似乎與血管新生成正相關。除此之外，同樣與HMG比較，良性MGT之IL-10、IL-12表現顯著較低(p<0.05)，IL-15則極顯著較低(p<0.01)。進一步將惡性腫瘤細分後，僅tubulopapillary carcinoma之IL-12顯著低於HMG。另外，比較惡性及良性MGT時發現，惡性MGT之IL-8及IL-13表現顯著較良性MGT為高。其次，MGT病畜之PBL似乎含有較健康犬隻高比例的非T非B細胞。如將細胞表面分子配合預後資料，得到下述預後較佳的profile且具有統計學上顯著性差異(p<0.01)，包括腫瘤細胞低MHC II，TIL之高CD4、CD21，及PBL之高MHC II、非CD4,8,21及低CD21。 由以上之結果可見，本實驗所調查的自然發生腫瘤中，MGT有較高比例MHC I表現量較低，而MHC I 低表現可能和腫瘤惡性有關。自PBL及腫瘤細胞之細胞表面分子與細胞激素基因profile得到的結果，可能有助於了解腫瘤的致病機制、導致病畜預後及日後犬腫瘤免疫治療之應用。 關鍵字：自然發生腫瘤、主要組織相容複合物、腫瘤浸潤淋巴球、週邊血液淋巴球、細胞激素。

There is lack of detailed MHC (Major histocompatibility complex) and cytokine profile data available in canine spontaneous tumors. However, this data is essential in a gene therapy application. 62 cancer dog patients were mostly collected from animal hospital in Taipei. Single cell suspensions were prepared. The flow cytometry was used to analyze the MHC I, II of tumor cells, subpopulations of TIL (Tumor infiltrating lymphocyte), PBL (Peripheral blood lymphocyte) including MHC I, II, and CD3, 4, 8, 21. In addition, tumor cells cytokines, such as IL-2, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, TNF-α, TGF-β, IFN-γ were studied by real-time RT PCR. These data were compared with the case history and their prognosis by Biomarker Patterns Software and we found that the cutoff value of MHC I expression in MGT (Mammary gland tumor) and non-MGT was 72.58%. Lower than 72.58% was MGT, and higher would be non-MGT. The results from 36 MGT and 15 non-MGT were collected. For MGT, 33 of 36 cases (91%) the MHC I were low in density (lower than 72.58%). 2 of 3 (67%) benign tumor and 31/33 (94%) malignant tumors expressed low MHC I in density. Therefore, among most spontaneous tumors, MGT tent to expresses low MHC I and low MHC I expression may have correlation with tumor malignancy. Data below are cytokine mRNA from MGT, benign MGT, and tubulopapillary carcinoma in malignant MGT compared with healthy mammary gland (HMG). IL-8 in MGT was significantly higher, while IL-10, IL-12, IL-15 in benign MGT and IL-12 in tubulopapillary carcinoma are significantly lower than HMG. Moreover, malignant MGT express significantly higher IL-8 and IL-13 mRNA level than benign MGT. Although IL-8 is significantly higher in MGT than HMG, only malignant MGT and especially simple carcinoma is significantly higher than mammary gland. Other subtypes of MGT has no significance compared to HMG. IL-8 may have positive correlation with angiogenesis. In addition, MGT tended to have more non-T, non-B cells than healthy dogs in the PBL. In cell surface marker, low MHC II in tumor cell, high CD4 and CD21 in TIL, high MHC II, non-CD4,8,21 and low CD21 in PBL has statistically significance in prognosis. From data above, MGT tend to express lower percentage of MHC I among spontaneous tumors collected in this study. In addition, low MHC I may be correlated with tumor malignancy. In addition, cell surface marker and mRNA expression profile in PBL and tumor cell may help us understand tumorigenesis mechanism, and the correlations with prognosis, which is useful for the study of canine cancer immunotherapy.