Improvement of ISH method for the detection of rare mRNAs in pathology archival tissues and establishment of its standard evaluation method

免疫染色半定量检测特异性蛋白表达。目前尚无半定量检测mrna表达的原位杂交（ISH）方法的报道。为了检验固定条件与蛋白酶K处理的相关性，本研究采用ISH法检测了1 ~ 16 d福尔马林固定和石蜡包埋的正常人体组织中β -2-微球蛋白（β - 2m） mRNA的保藏基因。结果表明，除心脏组织外，经福尔马林固定1天后β - 2m的表达保存最多。随着固定时间的延长，表达量有降低的趋势。此外，即使在相同条件下，每个固定组织的最佳蛋白酶K处理也是不同的。我们还发现，长时间保存在4℃的组织切片的表达水平比切割后的组织切片低。为了提高检测灵敏度，对使用cRNA探针的快速ISH方法进行了改进。使用这种改进的方法，我们可以在3小时内清楚地检测到胰岛素和胰高血糖素mrna。基于cRNA探针的快速ISH在各个研究领域和临床实验室都有广泛的应用前景。采用高灵敏的ISH法检测福尔马林固定和石蜡包埋病理档案高级别星形细胞瘤组织中自分泌运动因子（AMF）及其受体gp78 mRNA的表达。AMF mRNA的阳性率明显高于间变性星形细胞瘤。AMF表达患者的总生存期明显短于无AMF表达患者。在间变性星形细胞瘤中，表达AMF的患者的总生存期也明显短于不表达AMF的患者。本研究表明，AMF是高级别星形细胞瘤的不良预后因素。

The specific protein expression has been semi-quantitatively examined by immunostaining. No in situ hybridization (ISH) method to detect the expression of mRNAs semi-quantitatively has been reported. In this study, to examine the correlation of the fixation conditions and proteinase K treatment, house keeping gene such as beta-2-microglobulin (beta-2M) mRNA was detected by ISH method using one day to 16 days formalin-fixed and paraffin-embedded normal human tissues. As a result, the expression of beta-2M was most preserved in all but heart tissue fixed by formalin for one day. And the expression levels tend to be reduced during elongation of fixation time. Furthermore, optimal proteinase K treatments were different in each tissue that was fixed even if in the same conditions. We also demonstrate that the expression levels of tissue sections that stored at 4 C for a long time were decreased than those in tissue sections just after cutting. To raise detection sensitivity, the rapid ISH method using cRNA probes has been improved. Using this modified method, we were able detect insulin and glucagon mRNAs clearly within three hours. The rapid ISH with cRNA probes could have many applications in various research fields and clinical laboratories. The expressions of autocrine motility factor (AMF) and its receptor gp78 mRNA were detected in formalin-fixed and paraffin-embedded pathology archival high-grade astrocytoma tissues by the highly sensitive ISH method. The positive rate of AMF mRNA was significantly higher than in anaplastic astrocytomas. The overall survival of patients with AMF expression was significantly shorter than patients without AMF expression. In anaplastic astrocytomas, the overall survival of patients with AMF expression was also significantly shorter than patients without AMF expression. This study demonstrated that AMF is a poor prognostic factor in high-grade astrocytomas.