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

病理档案组织中稀有mRNA检测ISH方法的改进及其标准评价方法的建立

基本信息

批准号：
15590315
负责人：
SATO Yuichi
金额：
$ 2.24万
依托单位：
Kitasato University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

项目摘要

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.

免疫染色半定量检测特异性蛋白的表达。目前尚无半定量检测mRNAs表达的原位杂交(ISH)方法。本研究以福尔马林固定和石蜡包埋的正常人体组织为材料，采用ISH方法检测了看家基因β-2-微球蛋白(β-2m)mRNA的表达，以探讨固定条件与蛋白酶K处理的相关性。结果表明，除福尔马林固定一天的心脏组织外，其余所有组织中β-2m的表达都得到了较好的保存。随着注视时间的延长，该基因的表达水平呈下降趋势。此外，即使在相同的条件下，固定的每个组织的最佳蛋白酶K处理也是不同的。我们还发现，在4℃下保存较长时间的组织切片的表达水平低于切开后的组织切片。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，我们能够在3小时内清楚地检测到胰岛素和胰高血糖素的mRNAs。CRNA探针的快速原位杂交在各种研究领域和临床实验室中具有广泛的应用前景。采用高灵敏的ISH法检测福尔马林固定和石蜡包埋的高级别星形细胞瘤组织中自分泌运动因子(AMF)及其受体gp78的表达。间变性星形细胞瘤中AMF mRNA的阳性率显著高于间变性星形细胞瘤。有AMF表达的患者总体生存期明显短于无AMF表达的患者。在间变性星形细胞瘤中，表达AMF的患者的总体生存期也显著短于不表达AMF的患者。这项研究表明，AMF是高级别星形细胞瘤预后较差的因素。