Distribution of cytokines in tumor tissues by in situ hybridization

我们建立了ISH技术，使用地高辛标记的cRNA探针在4%多聚甲醛固定和石蜡包埋的组织中检测低表达的mrna如细胞因子。然而，我们的ISH的灵敏度不足以检测肿瘤细胞产生细胞因子mRNA。原位逆转录聚合酶链反应（in situ RT-PCR）方法已被用于检测细胞和组织中mRNA的低水平表达。我们将新开发的原位RT-PCR应用于细胞，并定量比较原位杂交（ISH）的灵敏度。采用体外竞争性RT-PCR检测IL-6 mRNA高、中、低或不表达的人肿瘤细胞系。原位RT-PCR采用Thermus thermophilus DNA聚合酶专用引物组形成高分子量串联物，采用原位地高辛（DIG）直接标记技术。使用dig标记的cRNA探针进行常规ISH。原位RT-PCR和ISH均观察到阳性细胞细胞质中IL-6 mRNA的特异性信号。原位RT-PCR的敏感性是ISH的100倍以上。原位RT-PCR的特异性通过适当的对照试验得到证实。数据表明，我们的原位RT-PCR应用于培养细胞的灵敏度明显高于ISH，并且该技术具有较高的特异性。

We established ISH technique which detect low expression of mRNAs such as cytokines using digoxigenin labeled cRNA probe in 4% paraformaldehyde-fixed and paraffin-embedded tissues. However, the sensitivity of our ISH was not enough to detect the tumor cell producing cytokine mRNA.The in situ reverse transcription-polymerase chain reaction (in situ RT-PCR) method has been used to detect low levels of expression of mRNA in cells an tissues. We applied a newly developed in situ RT-PCR to cells and quantitatively compared the sensitivity of the in situ hybridization (ISH). Human tumor cell lines with either high, moderate, low or no expression of IL-6 mRNA as determined by in vitro competitive RT-PCR were used. In situ RT-PCR was performed using Thermus thermophilus DNA polymerase special primer sets to form high molecular weight concatemers and an in situ digoxigenin (DIG) direct labeling technique. Conventional ISH was performed using DIG-labeled cRNA probe. Specific signals of IL-6 mRNA were observed in the cytoplasm of positive cells by both in situ RT-PCR and ISH. In situ RT-PCR was more than 100-fold more sensitive than ISH. The specific of in situ RT-PCR was confirmed by appropriate control tests. The data indicated that the sensitivity of our in situ RT-PCR applied to culture cells was significantly higher than that of ISH, and that the technique retained high specificity.