原位杂交和免疫组化研究人类胰腺癌的Ki-67基因表达

Study of gene expression of Ki-67 in human pancreatic carcinoma using in situ hybridazation and immunochemistry

目的应用地高辛标记的 Ki-67 cRNA(against exon 13 of the gene)进行 mRNA 原位杂交和免疫组化分析胰腺癌中 Ki-67基因的 mRNA 转录和蛋白翻译,研究 Ki-67基因结构和功能表达的关系。方法40例胰腺癌(20例高分化腺癌,20例低分化腺癌),9例非胰腺肿瘤(5例正常胰腺,4例慢性胰腺炎),Ki-67基因第13外显子 cDNA 片段(435 bp)由 PCR 扩增,然后 PCR 产物克隆到质粒(3.9kb PCR^(TM)Ⅱvector)进行扩增,以此模板合成地高辛标记的 cDNA 探针,应用 mRNA 原位杂交结合免疫组化研究 Ki-67基因在胰腺癌的转录和蛋白翻译。结果应用地高辛标记的 Ki-67 mRNA原位杂交和 MIB-1免疫组化成功地检测胰腺癌巢中明显高于正常的 Ki-67 mRNA 转录和蛋白翻译,胰腺癌 Ki-67表达明显高于非胰腺癌,并与胰腺癌组织学分级密切相关。结论本研究首次证明:人类胰腺癌存在 Ki-67基因的过度转录和翻译;且此基因的表达强度与胰腺癌的组织学分级亦密切相关,此基因的 exon 13的转录在最终 Ki-67蛋白的过度表达中起极重要的作用。

Objective To investigate,at transcriptional and protein translational levels,using in situ hybridization and immunochemistry,the gene expression of the Ki-67 protein in pancreatic carcino- ma so as to get insight of the gene structure and function.Methods Forty pancreatic cancer,5 normal pancreatic and 4 chronic pancreatitis tissue specimens were collected.A 435bp cDNA fragment located in codon 2,exon 13 of the Ki-67 antigen gene was amplified by the polymerase chain reaction(PCR). The DIG-labeled cRNA probes were transcribed using a commercialy DIG RNA labeling kit.Localiza- tion of the Ki-67 protein and the specific mRNA,by combining immunohistochemistry(ICH)with DIG-labeled in situ hybridization(ISH),was performed in this experiment.Results Successful local- ization of the Ki-67 protein mRNA in pancreatic tissue sections,prepared in routinely formalin-fixed and paraffin-embeded way,was first accomplished in this experiment.Analyzing the Ki-67 mRNA tran- scription in 17 pancreatic cancer specimens with Ki-67 ICH labeling index>20%,we found that stronger mRNA signals were also observed in poorly differentiated specimens with Ki-67 index>50% than in those of well differentiated cases with the ICH labeling index of 20-50%.A high expression of both the mRNA and the protein was observed in pancreatic adenocarcinomas with poor differentiation. Conclusion This is the first report that abnormal overexpression of the gene encoding Ki-67 protein has been detected not only at the protein level,but also at the mRNA level in pancreatic carcinoma.The abnormal overexpression of the Ki-67 protein might be correlated with the transcription of exon 13 of the gene.