荧光原位杂交检测宫颈脱落细胞hTERC基因实验方法比较观察

Experimental comparison of fluorescence in situ hybridization in cervical exfoliated cell hTERC gene detection

目的:探讨荧光原位杂交技术检测端粒酶基因(hTERC)实验方法的最佳方案。方法:采集海医附院收治的140例患者的宫颈脱落细胞,分别应用液基取材刷、无菌棉签取材法;生理盐水低渗、TCT低渗、直接取液滴片、TCT直接低渗4种制片方法;水浴和直接消化法;甲酰胺法和共变性法,通过检测宫颈脱落细胞hTERC基因表达成功率,对比实验方法的最佳方案。结果:取材方法中液基取材刷与无菌棉签的细胞数量满意率分别为95.5%、14.3%,2种方法差异有统计学意义(P<0.05);生理盐水低渗、TCT低渗、直接取液滴片、TCT直接低渗4种方法杂交成功率分别是80.0%、84.8%、46.2%、82.1%,其中直接取液滴片与其他法差异有统计学意义(P<0.05),其他方法之间比较差异无统计学意义(P>0.05);2种消化法中水浴和直接法杂交成功率分别为88.9%、94.1%,差异无统计学意义(P>0.05),杂信号比例分别为16.7%、45.6%,差异有统计学意义(P<0.05);2种变性法中甲酰胺法和共变性法杂交成功率分别为72.3%、96.0%,差异有统计学意义(P<0.05)。结论:荧光原位杂交技术检测宫颈脱落细胞hTERC基因时,以液基取材刷取材细胞数量多,TCT低渗方法制片效果最好,水浴法和共变性杂交成功率最高。

Objective. To investigate the optimal methods of fluorescence in situ hybridization on hTERC gene detection. Methods： 140 samples of cervical exfoliated cells were gathered by fluid-base brush sampling and aseptic cotton sampling in the Affiliated Hospital of Hainan Medical College; movies were made by physiological saline infiltration, TCT infiltration, direct dropping and direct TCT infiltration. All experimental methods, bath and direct digestion, formamide and denaturation law, were compared by detecting the success expression of cervix castoff cells hTERC gene. Results ： In sampling methods, the satisfactory rate of cell quantity in fluid-base brush and aseptic cotton were 95.5% and 14.3% （ P 〈 0.05 ） ; In hybridization technique, the success rate of 4 methods were 80.0% ,84.8% ,46.2%, 82.1%, respectively, compared to direct dropping method, other three methods were preferable （P 〈 0.05 ） and with no differences among them. In two digest methods, the success rate of hybridization were 88.9% and 94.1%, respectively, and with no difference（P 〉0.05）. But hybridization signal ratio were 16.7% and 45.6%, with significant difference（P 〈0.05）. In two denaturation methods, the success rate were 72.3% and 96.0%, with significant difference （P 〈 0.05）. Conclusion： Detecting hTERC gene by fluorescence in situ hybridization, fluid-base brush is better sampling of quantity cells, TCT infiltration is preferable in making movies, bath and hybridization variability have higher success rate.