IN SITU PCR AND IMMUNOHISTOCHEMICAL STUDIES ON p16 GENE IN PITUITARY ADENOMAS

Objective: To examine the occurrence of p16 gene deletion and to analyze p16 expression on paraffin-embedded human pituitary adenoma specimens. Efforts were made to optimize the technical conditions forin situ PCR. Methods:In situ PCR techniques and inimuno-histochemistry were used. Results: Immunohistochemically, p16-positive tumor cells were observed in all cases with various proportions. The majority of the stromal cells and part of tumor cells was devoid of p16 immunostaining, but signal ofin situ PCR for p16 gene, exon 2, was displayed in these cells. Conclusion: The results implied that p16 gene might not be deleted in these pituitary adenomas. It also indicated thatin situ PCR, both direct and indirect methods, proved feasible and informative to the aim of DNA detection. It is critical to overcome non-specific amplification in directin situ PCR by means of higher annealing temperature, fewer cycle, lower magnesium concentration and stringent washing. A target DNA-deleted sample as the negative control is extremely necessary. For the indirect method, the way to improve the sensitivity is to loosen the conditions for amplification and washing, so that more amplification products are subject to hybridization, and signal detection is facilitated.

原位PCR和免疫组化检测垂体腺癌P16基因

目的研究人垂体腺瘤P16基因的改变，同时探索原位PCR技术的适宜条件和结果确认，以及用于基因缺失检测的可行性。方法原位PCR和免疫组化技术。结界绝大部分间质细胞为P16蛋白免疫组化阴性，而一部分垂体肿瘤细胞呈P16阳性；针对P16基因的原位PCR信号则可见于垂体肿瘤细胞和间质细胞等各种细胞，即P16组化阴性的细胞同样表明有P16基因存在。结论原位PCR可以是P16基因研究的一种有效手段，提示垂体腺癌的P16基因改变形式可能主要是表达过度而不是基因缺失。