Gene Expression and Development of Odontogenic tumors

牙源性肿瘤的分类与肿瘤细胞的分化程度有关。釉原蛋白的定位和釉原蛋白mRNA的表达是牙源性肿瘤细胞分化的指标之一。本研究制备了釉原蛋白（amelogenin）蛋白和釉原蛋白mRNA（amelogeninmRNA）探针，观察其在牙源性肿瘤中的分布，材料与方法：1.正常对照：取孕20 d Wistar系大鼠胚胎，4%多聚甲醛灌流固定，观察釉原蛋白蛋白（amelogenin）蛋白和釉原蛋白mRNA在牙胚中的分布。2.探针的制备：从人丛状型成釉细胞瘤中提取总RNA。引物（正义：5-TCGGTCCTGCCTCAT-CACCATCC-3 ;反义：5-CCGCTTGGTCTTGTCTGTTG-3）用于使用PCR方法的扩增。将扩增的cDNA加载到载体（Bluescript）上，并用Dig RNA Labeling Kit（Boehringer曼海姆）标记，以制备地高辛标记的单链RNA探针。 ...更多信息 即通过体外转录。3.杂交：探针与标本在50 ℃杂交16小时。使用核酸检测试剂盒（Boehringer曼海姆）进行抗原-抗体反应，以在光学显微镜下评价杂交。结果与讨论：釉原蛋白蛋白和mRNA在大鼠和小鼠切牙中均呈阳性表达，在未分化釉上皮中均呈阴性表达。釉原蛋白mRNA主要表达于滤泡中心区的细胞，多表达于靠近基底膜的柱状细胞胞浆，而釉原蛋白仅在少数成釉细胞瘤中偶见表达。这些发现表明成釉细胞瘤中普遍存在釉原蛋白mRNA。釉原蛋白mRNA在腺样牙源性肿瘤细胞中普遍表达。此外，釉原蛋白在牙源性腺样肿瘤中也有表达。这些结果从分子水平证明了牙源性腺样瘤是一种高分化肿瘤。除上述研究外，还采用免疫组织化学方法检测成釉细胞瘤细胞增殖、癌基因产物和抑癌基因产物。免疫组化结果表明，PCNA、c-myc、ras、p53蛋白的检测有助于评价成釉细胞瘤的恶性程度。少

The classification of odontogenic tumors is related to the differentiation degree of tumor cells. The localization of amelogenin protein and the expression of amelogenin mRNA are among the indexes of odontogenic tumor cell defferentiation. In our study the probes of amelogenin protein and amelogenin mRNA were made and used to observe their distribution in odontogenic tumors.Materials and methods : 1.Normal control : Wistar strain rat embryoes at 20 days after gestation were used for investigation of amelogenin protein and mRNA distribution in tooth germ.The specimens were fixed by 4% paraformaldehyde perfusion. 2.Making of probes : Total RNA was extracted from human plexiform type ameloblastoma. Primers (sense : 5-TCGGTCCTGCCTCAT-CACCATCC-3 ; antisense : 5-CCGCTTGGTCTTGTCTGTTG-3) were used for amplification using PCR method. The amplified cDNA was loaded on Vector (Bluescript) and labeled with Dig RNA Labeling Kit (Boehringer Mannheim) to make Digoxigenin labeled single strand RNA prob … More e by in vitro transcription. 3.Hybridization : The probe was hybridizad with the specimens at 50^0C for 16 hours. Antigen-antibody reaction was performed using Nucleic acid Detection Kit (Boehringer Mannheim) as to evaluate the hybridization under light microscope. In addition, polyclonal anti-amelogenin antibody was used to localize the distribution of amelogenin protein in odontogenic tumors.Results and discussion : Both amelogenin protein and mRNA were positive in rat and mouse incisors but negative in undifferentiated enamel epithelia. Amelogenin mRNA was detected frequently in the cells of the central region of the follicles and usually positive in the cytoplasm of columnar cells near to basement membrane while amelogenin protein was occasionally detected only in a few cases of ameloblastomas. These findins suggest that amelogenin mRNA is generally present in ameloblastomas. Amelogenin mRNA was commonly detected in adenoid ondontogenic tumor cells. In addition the localization of amelogenin protein was also observed in adenoid odontogenic tumors. These results proved at molecular level that adenoid odontogenic tumor is a well differentiated tumor. Apart from the above-mentioned investigations immunohistochemistry was also adopted to evaluate cell proliferation, oncogene product and antioncogene product in ameloblastomas. These immunohistochemical studies indicated that immunodetection of PCNA,c-myc protein, ras protein and p53 protein is helpful in evaluating the malignance of ameloblastomas. Less