New method for sensitive in situ telomerase assay and it's application.

端粒重复序列扩增检测方法的引入意味着可以在几乎所有类型肿瘤的小样本中检测到端粒酶。然而，目前还不知道是否所有或只有一小部分肿瘤细胞具有端粒酶活性。正常组织中活化的淋巴细胞可能会产生假阳性结果。采用细胞端粒重复序列合成法和原位PCR结合原位杂交技术检测端粒酶活性。用单引物扩增端粒序列，用地高辛标记的寡核苷酸探针进行原位杂交检测扩增产物。（1）在没有TS引物的情况下，没有检测到信号。（2）不加Taq DNA聚合酶时，检测不到信号。（3）TRAP阴性标本无信号。（4）特异性间接原位PCR。（5）HL 60细胞经VD3诱导分化后端粒酶活性下降，PHA刺激的淋巴细胞端粒酶活性上升。(6)In结果表明，原位PCR检测的阳性细胞数与预期阳性细胞数基本一致，这种间接方法大大提高了原位PCR的特异性，可同时观察细胞形态和端粒酶活性，可用于微小残留病的检测。

The introduction of the telomeric repeat amplification protocol assay means telomerase can be detected in small samples from almost all types of tumors. However it is not known whether all or only a small subset of tumor cells have telomerase activity. Activated lymphocytes in normal tissue may give false positive results. We used in cell telomeric repeat synthesis and PCR in situ with in situ hybridization to document telomerase activity. The intracellular amplification of telomere sequences was achieved with single primer pairs and the PCR products were detected by subsequent in situ hybridization using digoxigenin-labeled oligonucleotide probes specific for the amplification products.The validity and specificity of our methods were confirmed as follows. (1)Without TS primer, no signal was detected. (2)Without Taq DNA polymerase, no signal was detected. (3)TRAP negative samples gave no signals. (4)Specific indirect in-situ PCR was done. (5)HL60 cells showed down-regulation of telomerase activity after differentiation by VD3 and PHA-stimulated lymphocytes showed up-regulation of telomerase activity. (6)In artificial cell mixtures, there was an approximate correlation between the expected and observed number of positive cells.This indirect method greatly increases the specificity of in situ PCR and enables the simultaneous observation of cell morphology and telomerase activity and could be used to detect minimal residual diseases.