T Cell Receptor Gamma Gene Rearrangements In Mycosis Fougoides And B Cell Lymphoma

IntroductionMycosis fungoides is a low maliganent cutaneous T cell lymphoma which has a variety of manifestation and difficulty of early diagnosis in pathology that u-sually need repeatful biosy and further development of the lesion.Although immunochemistory is an assistantment method to detect the T -cell differentiation antigen, weather the lesion is benign or maliganent is hard to determine . The main character of the maliganent lymphocytes proliferation is clonal , which is manifested by clonal rearrangement of T cell receptor (TCR) in T cell lymphoma. Since the eighties some foreign researchers detected TCR - beta gene rearrangement by Southern blot or TCR gene rearrangement by polymer-ase chain reaction ( PCR). However, Southern blot has been replaced by poly-merase chain reaction because it needs fresh specimen, has lower detection rate and is radiative and time - consuming. PCR is an available and simple method for detecting gene rearrangement because TCR gama (TCR - ) gene rearrangement structure is simple and is almost present in all T lymphoma.Currentlly polymerase chain reaction/agrose ( PCR/agrose ) , polymerase chain reaction/denaturing gradient gel electrophoresis ( PCR/DGGE) , polymerase chain reaction/ temperature gradient gel electrophoresis ( PC R/TGGE) , polymerase chain reaction /sing - strand conformational polymorphism ( PCR/SS-CP) , and polymerase chain reaction/ polyacrylamide gel electrophoresis (PCR/ PAGE) were used. The same research methods were undertaken in nineties in our country. The TCR gama gene rearrangement rates ranged from 52% to 90%. The main difference of these research focused on the number of primers and the design of primers and the method of PCR detecting technique. Although the positive rates of TCR gama gene rearrangement ranged between 80% to 90%by PCR/DGGE, PCR/SSCP, the false positive rate were present. For example, 6% of TCR gama gene rearrangement was noted in nonspecific inflamation. We suggest that the sensitivity and specifity be balanced.At present, these kinds of problem is in further investigation. The one of focuses is to develop a gene diagnosis method with clinically practical prospect. However, the most of the methods used in previous reports were complicated (example, multi - PCR) , time - consuming and dependent upon special devices ( example, PCR/DGGE ). Furthmore, some false positive rates were high which limited clinical use.For further promotion of the clinical practical and scientific value, we undertook sysmetic research by studying the extraction of DNA, the device of primes and the detection rates in different sorts of diseases î–´d samples. We successfully studied: 1. first introduced the " One - step" method to extract DNA from the formalin - fixed paraffin - embeded tissues. As a result, the utilization rates of samples were promoted. At mean time, we used nested - PCR/agrose to detect TCR - y gene rearrangements which is one of the simplest methods with high sensitively and specifity. This method led to simplifized procedures which is avalivable for clinical use and retrospective research. 2. The TCR - y gene rearrangement rates were not high in the peripheral blood of mycosis fougoides ,but the TCR - -y gene rearrangement was present alone in the peripheral blood in the absence of TCR - y gene rearrangement of cutaneous lesions. Up to now, there has been no same report in China.Materials and Methods ,The method of extracting DNA:1. The " one step method" of extracting formalin - fixed paraffin - embeded tissue; the paraffin embedding tissue is cut to 6(xm sedion, every case is cut up to 18 sheet, placed in eppendorf tube, after routine deparaffinization , every tube was added with 100 l degeneration buffer, put in 55 water bath 17hr. After centrifugation, reserved supernatant in 4 refrigerator. At same time, u-sing microsatellize primer to do PCR verification, examing quality.2. Using traditional phenol chloroform method to extract DNA from skin lesion and peripheral blood. Using ultraviolet spectr...