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

1、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         11-02-13 14:47:00     作者：曾蓉蓉，金松    编辑：studa20【摘要】  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

2、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%、4

3、5.6%，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。 【关键词】  荧光原位杂交;宫颈脱落细胞;TERC基因           ABSTRACT Objective: To investigate the optimal methods

4、of fluorescence in situ hybridization on hTERC gene detection. Methods： 140 samples of cervical exfoliated cells were gathered by fluidbase brush sampling and aseptic cotton sampling in the Affiliated Hospital of Hainan Medical College; movies were made by physiological saline infiltration, TCT infi

5、ltration, 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 fl

6、uidbase 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 dige

7、st 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 differ

8、ence(P<0.05). Conclusion： Detecting hTERC gene by fluorescence in situ hybridization, fluidbase brush is better sampling of quantity cells, TCT infiltration is preferable in making movies, bath and hybridization variability have higher success rate.KEY WORDS Fluorescence in situ hybridization; Ce

9、rvical exfoliated cells; TERC gene随着分子生物学的发展，人们运用荧光原位杂交( fluorescence in situ hybridization, FISH)技术检测宫颈脱落细胞中3号染色体长臂26. 3位点端粒酶基因( human telomerase RNA component,hTERC)的扩增,发现它与宫颈病变有着密切的关系1。FISH的原理是以已知的标记单链核酸为探针,按碱基互补原则,与待检材料中未知单链核酸进行结合,形成荧光显微镜下可检测的荧光信号,本实验从取材、制片、消化、变性4个方面,探讨实验方法对FISH杂交率的影响。1 材料与方法1.

10、1 材料来源收集2007年11月2009年3月在海南医学院临床附属医院就诊的140例妇科患者的宫颈脱落细胞标本(排除严重内科疾病及生殖系统炎症)。1. 2 方法FISH检测均以细胞学检查和组织病理学结果为参照标准。1. 2. 1 取材 (1)液基取材刷：患者取膀胱结石位，暴露宫颈，干棉签擦拭分泌物，使用一次性TCT毛刷插入宫颈口,在宫颈外口鳞柱状上皮交界处,以宫颈外口为中心,均匀按顺(逆)时针旋转5周,停留10 s左右，将宫颈刷置于TCT液基中保存;(2)无菌棉签：取材方法同前，棉签放于生理盐水中保存。1. 2. 2 制片 (1)生理盐水低渗：将生理盐水细胞储存液充分震荡10 min，放置12 min后取10 mL细胞储存液 , 1 500 r/min 离心10 min, 去上清，如细胞少，可再取更多液体。加5 mL胶原酶B, 37水浴30 min,间断吹打，离心,加5mL去离子水37水浴20 min,加入2mL固定液(冰乙酸甲醇=13)固定10 min,离心后弃上清,吹打悬浮细胞,重复2次固定，取适量密度细胞悬液10 L用移液器滴片。(2)TCT低渗：胶原酶B 20 min，去离子水40 min，其余