Diagnosis and classification of cancers by in situ hybridization using non-radioactive oncogene probes.

我们应用酶免疫组织原位杂交(EIHISH)技术对白血病和癌症进行诊断和分类。用二硝基苯基(DNP)残基化学标记DNA片段。或者DNA片段被紫外线照射，核苷酸序列上相邻的胸苷被二聚化(T-T二聚化)。这些片段被用作非放射性原位杂交的探针。我们寻找了更好的标本固定方式、组织中暴露靶核酸的蛋白酶K浓度、杂交温度、洗涤条件和探针大小。这些条件是在使用不同组织和细胞的原位杂交过程中确定的。实验中使用的组织和细胞包括裸鼠移植的人胃癌、人T细胞白血病细胞、感染乙肝病毒的人肝脏和感染I型疱疹病毒的Vero细胞。DNA探针包括c-myc、T细胞受体β链、HTLV-1、乙肝病毒、HSV-1和甲胎蛋白。冰冻切片、细胞离心和盖印标本比石蜡切片有更强的阳性信号。石蜡切片形态构筑保存最好，冰冻切片形态构筑保存较差。冰冻切片中常可见非特异性染色，尤其是间质组织。这些结果表明，EIHISH适用于细胞生物学实验，但目前用于病理诊断的工具还不成熟。提高EIHISH的敏感性和特异性，将为以癌基因探针为基础的病理诊断提供有用的工具。

We have applied enzyme-immuno-histo-in situ hybridization (EIHISH) technique for the diagnosis and classification of leukemias and cancers. DNA fragments were chemically labeled with dinitrophenyl(DNP) residues. Or DNA fragments were UV- irradiared and adjacent thymidines on the nucleotide sequence were dimerized(T-T dimerization). These fragments were used as probes for non-radioactive in situ hybridization. We searched more better fixation of specimens, concentration of proteinase K for the exposure of target nucleic acids in the tissue, hybridization temperature, washing condition and probe size. These conditions were determined in the course of in situ hybridizations using various tissues and cells. Tissues and cells used in this experiments are as follows: human gastric cancers transplanted in nude mice, human T-cell leukemia cells, human livers infected with hepatitis B virus and Vero cells infected with herpes simplex virus type I. DNA probes used were c-myc, T-cell receptor beta chain, HTLV-1, hepatitis B virus, HSV-1 and alpha-fetoprotein.Frozen-sectioned, cytocentrifuged or stamped specimens gave stronger positive signals than paraffin sections. Morphological architecture was best conserved in paraffin sections but poor in frozen sections. In frozen sections nonspecific staining was often observed especially in stromal tissues. These results indicate that EIHISH is applicable to cell biological experiments but immature tool for pathological diagnosis at present. Improvement of the sensitivity and the specificity in EIHISH will provide useful tool to pathological diagnosis based on oncogene probes.