地高辛标记核酸病理形态学检测方法的建立及应用

Establishment and Application of the Pathomorphological Detection Method Using Digoxin for Nucleic Acids Labeling

目的建立地高辛标记原位杂交(ISH)和原位PCR(ISP)方法,探讨其在病理形态学检测中的应用。方法以细胞cDNA或质粒DNA为模板制备地高辛探针,ISH检测肺癌、胃癌或结直肠癌切片上JCV T抗原DNA、beclin 1和SERCA3 mRNA表达水平。在肺癌和胃癌切片上扩增地高辛标记的JCV T抗原DNA。杂交或标记后,进行抗地高辛碱性磷酸酶反应并行NBT/BCIP或Fuchsin显色。结果 PCR扩增获得高纯度地高辛标记DNA探针,甲酰胺杂交液杂交显示JCV T抗原存在于JCI细胞的细胞核中,beclin 1和SERCA3 mRNA阳性信号见于结肠癌和癌旁黏膜上皮细胞质中。ISP扩增了地高辛标记的JCV T抗原DNA,显色后发现JCV T抗原主要定位在JCI细胞、肺泡上皮细胞和肺癌细胞核、胃癌及癌旁黏膜细胞核中。NBT/BCIP显色比Fuchsin显色更为敏感,甲基绿复染使得上述2种显色更加清晰。结论地高辛标记的ISH和ISP操作简便,敏感度高,NBT/BCIP显色和甲基绿复染图像美观。

Objective To introduce the methodology of in situ hybridization（ISH） and PCR（ISP） by digoxin labeling and explore their application in pathomorphological detection and research studies.Methods The digoxin-labeled probes were prepared using cell cDNA or plasmid as template.JC virus（JCV） T antigen DNA,beclin 1 and SERCA3 mRNA were examined on the slides of lung,gastric or colorectal carcinoma by ISH.Additionally,JCV T antigen DNA was amplified and labeled by digoxin on the tissue sections of lung and gastric carcinoma as well as their adjacent normal tissue by ISP.Anti-digoxin alkaline phosphase was reacted and colored by NBT/BCIP or Fuchsin.Results The preparation of digoxin-labeled probes is simple using PCR.ISH showed that JCV T antigen DNA was localized in the nuclei of JCI cells after hybridization in formamide buffer.ISH results also confirmed the distribution of beclin 1 and SERCA3 mRNA in the cytoplasm of colorectal carcinoma cells.JCV T antigen DNA was labeled by digoxin and observed in the nuclei of alveolar cells,gastric epithelial cells,lung and gastric cancer cells by ISP.NBT/BCIP color development was more sensitive than Fuchsin and methyl green staining was beautiful for both NBT/BCIP and Fuchsin.Conclusion Digoxin-labeling ISH and ISP methods were simple and sensitive.Beautiful figures could be obtained using NBT/BCIP color development and methyl green staining.ISH and ISP established in this study were available and applicable for most laboratories.