B淋巴細胞在成熟過程當中，於發育後期，VDJ基因重組所引導製造出的免疫球蛋白，將會表現於細胞膜上，成為細胞膜型式的免疫球蛋白基因(membrane immunoglobulin，簡稱mIg)，這些細胞膜上的接受器(receptor)會因抗原的刺激，在免疫球蛋白可變區基因上面，會有體細胞突變(somatic mutation)或點突變(point mutation)的情形發生。依據文獻報告指出，在一些淋巴瘤的案例，如濾泡型淋巴瘤(follicular lymphoma)、黏膜相關淋巴組織B細胞淋巴瘤(mucosa associated lymphoid tissue lymphoma，簡稱MALT lymphoma)等，可能有這種體細胞突變的情況發生。所以在本論文中，針對同樣屬於B細胞腫瘤的慢性淋巴球性白血病(B-Chronic Lymphocytic Leukemia，簡稱B-CLL)，分析其免疫球蛋白重鏈可變區基因，觀察是否也有體細胞突變情形發生，並進一步與濾泡型淋巴瘤比較它們在免疫球蛋白重鍊可變區基因，兩者之間在基因序列的突變差異。首先引用PCR(polymerase chain reaction)方法，針對十個經由實驗室報告及醫師臨床診斷為B細胞慢性淋巴球性白血病的案例，針對免疫球蛋白重鏈可變區基因FR3-FR4區域的放大，以分析慢性淋巴球性白血病的株性來源，確定其中六個案例為單株腫瘤細胞。為了進一步了解B細胞慢性淋巴球性白血病的免疫球蛋白可變區基因，是否有體細胞突變現象，針對其中三個案例，以PCR方法，放大免疫球蛋白重鏈可變區FR1-JH區域，並進行基因選殖及定序。將得到的重鍊可變區基因序列與目前已確認的germline序列做比對，以取代性突變(replacement mutation，簡稱R)和沉默性突變(silent mutation，簡稱S)比值的分佈做結果分析。在案例一，CDR、FR區域都是0.3，顯示沒有突變情形發生。案例二、三的CDR區域是4.0、FR區域是0.25，結果顯示CDR之R/S比值是FR的16倍，因此很可能有抗原刺激存在。D基因部份，全部已比對出的五個案例，有7個氨基酸的改變，整體突變情形看來，並沒有一致性。JH基因部份，所發現的突變固定在codon 99、102、105這幾個好發點上。所以，綜合以上結果顯示，B-CLL可能來自於兩個不同subset，雖然目前無法直接証明某部份的B-CLL與抗原刺激有關，但是未來也許可以利用本實驗所clone出來的免疫球蛋白基因，以In vitro的方式做進一步的分析與探討。 VDJ gene rearrangement occurs during the final stage of B-cell development and membrane immunoglobulin appears on the mature B cells as a result. Subsequently, antigen stimulation results somatic hypermutation in those genes encoding variable region for antigen-binding. Previous findings have shown that somatic hypermutation in some lymphomas including those of follicular lymphoma and mucosa associated lymphoid tissue lymphoma (MALT) are frequently found. In this thesis study, B-cell chronic lymphocytic leukemia (CLL) has been used to examine the changes in its heavy chain gene to determine if the somatic hypermutation occus in this similar lymphocytic disorder. Firstly, the polymerase chain reaction (PCR) method was taken to amplify the FR3-FR4 region of the heavy chain gene of each of the cases to verify their monoclonarity. Among them, six were confirmed as the B-cell malignancy. In order to further determine the possible hypermutation of heavy chain genes within these cases, the full length of the heavy chain genes were PCR amplified in three of these cases. Subsequently, the DNA sequences of these PCR products were determined and used to compare with the published germline sequences. A R/S ratio was used to estimate the presence of hypermutation and significantly high R/S ratio were found in two of the cases in this study. Indicating possible antigen stimulation in these cases. However, no obvious mutation was found in the D gene segments in all cases. Interestingly, mutations were found only in certain nucleotides in JH gene segment of the heavy chain gene. These data suggest that B-CLL might origin from at least two different subsets. As well as some B-CLL might be derivative from antigen stimulation, however, direct evidence would be necessary to draw a more clear picture of the theory. Future study using the heavy chain gene clones prepared from this study would be able to further characterize the involvement of antigen-stimulation among these B-cell proliferative disorders.
