還原葉酸載體在犬正常組織及淋巴瘤的表現

Abstract

還原葉酸載體(reduced folate carrier, RFC)是細胞輸入葉酸及抗葉酸藥物的最主要運輸物，其在人和齧齒動物各種組織的基因表現已被建立，至今在犬卻少有研究 本研究的主要目的包含 (一) 建構能增幅犬部分 RFC 基因的聚合酶鏈鎖反。應(polymerase chain reaction, PCR)引子及 (二) 偵測犬正常和淋巴瘤組織的 RFC基因表現量。研究樣本包含 5 隻安死術犬而得的 35 種正常組織及 22 個犬淋巴瘤組織。由這些組織萃取 RNA 後反轉錄為 cDNA，再利用 PCR 增幅出 RFC 基因。將電泳下 RFC 條帶亮度與甘油醛-3-磷酸脱氢酶(glyceraldehyde 3-phosphatedehydrogenase, GAPDH)相除，獲得定量的 RFC 基因表現量。 研究結果顯示 RFC在犬正常組織間幾乎都有表現，但表現程度不一，於中樞神經系統及免疫系統可見比平均更高的表現量 犬淋巴瘤組織的 RFC 表現量則遠低於正常淋巴組織(P <。0.001)，且多中心型 B 細胞淋巴瘤、多中心型 T 細胞淋巴瘤及皮膚型 T 細胞淋巴瘤之表現量並無顯著差異。整體而言，RFC 於各種組織間的廣泛分佈支持其在體內葉酸平衡的重要性 而犬淋巴瘤組織的低表現量可能因複雜的機制應組織及，環境的不同做調整，尚須進一步證明。Reduced folate carrier (RFC) is the main carrier which transports folates and antifolates into the cell. RFC gene expression in human and rodent tissues had been established, however, it has not been revealed in dogs yet. The aims of the present study were: (1) to construct specific PCR primers for amplification of canine RFC gene; (2) to assess RFC gene expressions in normal canine and lymphomas tissues. Thirty-five kinds of normal canine tissues were collected from five euthanized dogs and twenty-two lymphomas were enrolled. Total RNA were extracted from normal and lymphoma tissues, and were transcripted reversely to complementary DNA, and then were evaluated for RFC expressions using polymerase chain reaction. RFC expression levels were obtained from quantified bands of RFC divided by glyceraldehyde 3-phosphate dehydrogenase. It was shown that RFC transcripts were expressed ubiquitously but various levels. Higher expressions were observed in central nervous and immune system than the median of all tested tissues. Expressions of RFC in normal lymphoid tissues were significantly higher when compared to B- or T-cell lymphomas (P < 0.001). And there were no significant difference among multicentric B-cell lymphomas, multicentric T-cell lymphomas, and cutaneous T-cell lymphomas. We concluded that the ubiquitous distribution with various levels of RFC in 35 kinds of normal canine tissues and malignant tissues. Higher expressions were observed in central nervous, immune, and urinary systems than others. Stomach had the highest RFC expression, followed by cerebellum and brain stem, with very low levels in duodenum and cardiac muscle. And expressions of RFC were significantly lower in all cases of lymphomas when compared to normal lymphatic organs (thymus, palatine tonsil, lymph node), which composed mainly of lymphocytes.摘要...........i Abstract...........ii Contents...........iii List of Tables...........v List of Figures...........vi Chapter 1 Background and literature review...........1 1.1 Reduced folate carrier (RFC) and antifolates...........1 1.1.1 Folates and folate transporting systems...........1 1.1.2 Functions of RFC...........3 1.1.3 Expression of RFC in normal human and rodent tissues...........4 1.1.4 Prognostic role of RFC...........5 1.1.5 Mechanism and function of antifolates...........6 1.2 Lymphomas...........9 1.2.1 Lymphomas in dogs...........9 1.2.2 Current treatment of lymphomas...........10 Chapter 2 Introduction...........12 Chapter 3 Materials and methods...........14 3.1 Samples collection...........14 3.1.1 Madin-Darby canine kidney (MDCK) cell line...........14 3.1.2 Normal tissues collection...........14 3.1.3 Lymphomas tissues collection...........15 3.2 Analysis of canine RFC gene...........19 3.2.1 Construct specific PCR primers for amplification of canine RFC gene....19 3.2.2 Analyses of canine RFC structure to construct PCR primers for amplification of canine RFC gene...........20 3.2.3 Total RNA extraction...........21 3.2.4 Measurement of RNA concentration...........23 3.2.5 Reverse transcription-polymerase chain reaction (RT-PCR) ...........23 3.2.6 Amplification of glyceraldehydes-3-phosphate dehydrogenase gene (GAPDH...........23 3.2.7 Amplification of RFC gene from MDCK cell lines as a positive control...........24 3.2.8 Sequencing of RFC gene from MDCK cell lines, normal tissues, and lymphomas...........25 3.2.9 Quantification of RFC expression levels...........26 3.3 Statistical analysis...........32 Chapter 4 Results...........33 4.1 Detection of RFC gene expression in normal canine tissues...........33 4.1.1 Animals and samples...........33 4.1.2 Detection of RFC gene expression...........33 4.2 Detection of RFC gene expression in canine lymphomas...........41 4.2.1 Animals and samples...........41 4.2.2 Detection of RFC gene expression...........42 Chapter 5 Discussion...........47 References...........5