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首页> 外文期刊>American Journal of Physiology >Novel and quantitative DNA dot-blotting method for assessment of in vivo proliferation.
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Novel and quantitative DNA dot-blotting method for assessment of in vivo proliferation.

机译：用于评估体内增殖的新型定量DNA点印迹法。

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摘要

Immunohistochemical assessment of 5-bromo-2-deoxyuridine (BrdU) in tissue sections is a widely used method to evaluate cell proliferation in vivo. However, this method requires time-consuming preparation of paraffin sections and laborious counting of BrdU-labeled nuclei on multiple sections. Here, we report the development of a rapid and reliable method to quantitate BrdU incorporation in intestinal and liver tissues using a dot-blot method. In vivo models of colon or liver proliferation were used to analyze the usefulness and reliability of this new method. Mice were killed after BrdU injection, and genomic DNA was isolated from the tissues, denatured, and dot-blotted onto a nitrocellulose membrane. The incorporated BrdU was detected with a BrdU monoclonal antibody, and the signal intensity was densitometrically quantified. Results were compared with BrdU index determined by conventional immunohistochemistry on the same tissue samples. The patterns of colonic BrdU incorporation during fasting and refeeding, measured by the dot-blotting method and the immunohistochemical method, were similar. The BrdU incorporation in the regenerating liver after partial hepatectomy, evaluated by these two different methods, showed a strong correlation (R(2) = 0.91, P < 0.01). In addition, the inhibition of colon proliferation by the phosphoinositol 3-kinase inhibitor wortmannin was demonstrated by this dot-blotting method. In conclusion, the dot-blotting technique described in this report provides an accurate, more efficient, and possibly more reliable method for the assessment of in vivo proliferation compared with conventional immunohistochemical determination of BrdU-labeling index.
机译：免疫组织化学评估组织切片中的5-溴-2-脱氧尿苷（BrdU）是一种广泛用于评估体内细胞增殖的方法。但是，此方法需要耗时的石蜡切片制备以及费力的在多个切片上计数BrdU标记的核。在这里，我们报告了一种快速可靠的方法的发展，该方法可以使用点印迹法对肠道和肝脏组织中的BrdU掺入进行定量。结肠或肝脏增殖的体内模型用于分析这种新方法的有用性和可靠性。 BrdU注射后杀死小鼠，并从组织中分离基因组DNA，变性并点印迹到硝酸纤维素膜上。用BrdU单克隆抗体检测掺入的BrdU，并通过光密度定量定量信号强度。将结果与相同组织样品上通过常规免疫组织化学测定的BrdU指数进行比较。通过斑点印迹法和免疫组织化学法测量的禁食和再喂养期间结肠BrdU掺入的模式相似。通过这两种不同的方法评估，部分肝切除术后再生肝中的BrdU掺入显示出很强的相关性（R（2）= 0.91，P <0.01）。另外，通过这种斑点印迹法证明了磷酸肌醇3-激酶抑制剂渥曼青霉素对结肠增殖的抑制。总之，与传统的免疫组织化学方法测定BrdU标记指数相比，本报告中描述的斑点印迹技术提供了一种准确，更有效且可能更可靠的方法来评估体内增殖。

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