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。成釉蛋白的定位和成釉蛋白mRNA的表达是牙源性肿瘤细胞分化的指标之一。材料与方法：1.正常对照：取孕20天的Wistar大鼠胚胎，用4%多聚甲醛灌流固定标本，观察牙胚中釉原蛋白及其mRNA的分布。2.探针制作：从人丛状成釉细胞瘤中提取总RNA。用引物(正义：5-TCGGTCCTGCCTCAT-CACCATCC-3；反义：5-CCGCTTGGTCTTGCTGTTG-3)进行PCR扩增。将扩增出的基因克隆到载体(Bluescrip)上，用地高辛标记单链RNA探针…通过体外转录获得更多的e。3.杂交：将探针与标本在50℃下杂交16h。用核酸检测试剂盒(Boehringer Mannheim)进行抗原-抗体反应，在光镜下评价杂交效果。结果和讨论：在大鼠和小鼠切牙中，釉原蛋白和mRNA均呈阳性表达，而在未分化的釉质上皮细胞中均为阴性。成釉细胞瘤中的成釉细胞瘤中，成釉蛋白的表达主要集中在滤泡中心区的细胞内，通常在靠近基底膜的柱状细胞胞浆中呈阳性表达。这些发现表明造釉蛋白在成釉细胞瘤中普遍存在。釉原蛋白在腺样体牙源性肿瘤细胞中普遍表达。此外，还观察到釉原蛋白在腺样体牙源性肿瘤中的定位。这些结果从分子水平上证明腺样牙源性肿瘤是一种分化良好的肿瘤。此外，还采用免疫组织化学方法检测成釉细胞瘤的细胞增殖、癌基因产物和抑癌基因产物。这些免疫组织化学研究表明，免疫组织化学检测成釉细胞瘤中的增殖细胞核抗原、c-myc蛋白、ras蛋白和P53蛋白有助于判断成釉细胞瘤的恶性程度。较少

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