宫颈病变脱落细胞中hTERC基因的扩增及其临床意义

目的:探讨不同宫颈病变和宫颈鳞癌脱落细胞中人端粒酶RNA(hTERC)基因的扩增水平及其临床意义.方法:采用荧光原位杂交技术(HSH)检测10例宫颈鳞癌、68例宫颈上皮内瘤样病变(CIN)及18例宫颈炎患者的宫颈脱落细胞中hTERC基因的扩增情况,并以20例正常宫颈脱落细胞作对照.分析不同宫颈病变和宫颈鳞癌的脱落细胞中hTERC基因的扩增水平与临床特征的相关性.结果:正常宫颈、宫颈炎、CIN Ⅰ-Ⅲ和宫颈鳞癌患者的宫颈脱落细胞中hTERC基因扩增的异常细胞数分别为2.9、6.0、7.4 11.3、17.8和27.7.正常及宫颈炎、CIN与宫颈癌的hTERC基因扩增阳性率分别为10.53%(4/38)、64.23%(43/68)、100%(10/10).细胞学诊断为意义不明的不典型鳞状细胞(ASCUS)的样本中,宫颈癌hTERC基因扩增阳性率明显高于CIN与正常宫颈及炎症(P<0.05),高级别CIN(CINⅡ、CINⅢ)明显高于低级别CIN(CINⅠ)(P<0.005).宫颈鳞癌Ⅱ期以上的hTERC基因扩增率高于Ⅰ期.结论:hTERC基因的异常扩增与CIN和宫颈癌的发生发展相关,hTERC基因的异常扩增可能成为预测宫颈病变恶性进展的有力工具之一.     

新疆维、汉妇女宫颈脱落细胞中hTERC 基因表达

目的:研究hTERC基因在新疆维吾尔族和汉族妇女宫颈脱落细胞中的表达情况及差异性。方法:收集2012年9月至2014年3月在新疆维吾尔自治区人民医院妇科门诊就诊或行宫颈癌机会性筛查的新疆维吾尔族和汉族妇女病例1160例,选择其中HPV感染阳性或TCT阳性或两项同时阳性的465例纳入研究队列,通过F1SH技术检测宫颈脱落细胞中hTERC基因的表达情况。结果:新疆维吾尔族与汉族妇女hTERC基因扩增的总阳性率无显著差异(P=0.620);维吾尔族与汉族妇女正常或慢性炎症组、CIN1组、CIN2组、CIN3组和SCC组hTERC基因扩增的阳性率亦均无显著差异(P=0.918,0.912,0.821,0.922,1.000),hTERC基因的表达率不存在民族差异;维吾尔族与汉族妇女正常或慢性炎症组/CIN1组和CIN2+组基因拷贝数的分布均无显著差异(P=0.925,0.862),且其正常或慢性炎症组/CIN1组和CIN2+组TERC:CEP比例分布亦均无显著差异(P=0.986,P=0.979),hTERC基因的扩增类型不存在民族差异;维吾尔族和汉族妇女hTERC基因扩增阳性率的大小顺序均为:SCC组≈CIN 3组CIN 2组CIN 1组≈正常或慢性炎症组,随着宫颈病变恶性程度的增加,各组中hTERC基因的扩增阳性率有增加的趋势,hTERC基因的表达与宫颈病变的恶性程度呈正相关(相关系数=0.648和0.712,P0.001)。结论:新疆维吾尔族与汉族妇女hTERC基因的表达率不存在民族差异;维吾尔族与汉族妇女hTERC基因的扩增类型不存在民族差异;随着宫颈病变恶性程度的增加,维吾尔族和汉族妇女hTERC基因的扩增阳性率有增加的趋势,hTERC基因的表达与宫颈病变的恶性程度呈正相关。     

高危HPV 感染及hTERC 基因在宫颈癌发生发展中的相关性

目的:探讨高危人乳头瘤病毒及人类染色体端粒酶基因(hTERC)在宫颈癌发生发展中的关系。方法:2010年10月至2011年05月就诊于哈尔滨医科大学附属第一医院妇科患者445例,采用表面等离子体谐振法(SPR)及荧光原位杂交法(FISH)分别检测高危HPV和hTERC的表达情况,对任一结果阳性者行阴道镜下宫颈活检和病理学检查,其中炎症对照组45例,CIN组33例及宫颈癌组6例。结果:单一型HPV29例,多重型26例,最常见为HPV16型合并的二型感染(17例)。在炎症组,CINⅠ组,CINⅡ组、CINⅢ组及宫颈癌组中多重高危HPV比率为17.78%、28.57%、44.44%、50.00%、66.67%,与炎症对照组比较其他组均有统计学意义(P<0.05)。hTERC基因在各组中的阳性表达率分别为2.22%、14.29%、61.11%、75.00%及100%。CIN组及宫颈癌的hTERC基因表达与炎症组比较差异均有统计学意义(P均<0.01),其中CINⅡ及以上各组差异明显。多重HPV感染与hTERC表达呈中等正相关(r=0.462,P<0.01)。结论:随着宫颈病理类型分级升高,HPV感染及hTERC基因表达率升高,hTERC基因在宫颈癌发生发展中起重要作用;高危HPV感染与hTERC基因的异常扩增关系密切,提示二者可能存在因果关系。     

薄层细胞涂片新方法在宫颈细胞学检查中应用的研究

目的：研究一种新的宫颈细胞薄层涂片方法,以提高传统手工涂片方法的制片质量。方法：随机选择的妇科门诊病人100例,随机分成2组,薄层细胞涂片组和手工涂片组,其中薄层细胞涂片组的刮片标本经过我们设计的双层滤网的过滤和分离液离心的步骤去除标本中的黏液、炎细胞和血细胞。这两组得到的涂片根据其细胞分布的均匀度及细胞重叠的多少,把涂片的结果分为满意、基本满意和不满意。结果：薄层细胞涂片组的涂片质量满意率（98％）高于手工涂片组的涂片质量满意程度（32％．P〈0．01）。讨论：薄层细胞涂片法在宫颈细胞学检查中的满意程度明显好于传统手工涂片（P〈0．01）,有统计学意义。效果稳定,适合在大范围内推广。薄层细胞涂片法相对于传统手工涂片有5大优点,大大提高了宫颈细胞学诊断的准确性。     

脂肪组织石蜡切片制作方法探讨

目的探讨脂肪组织石蜡切片制作方法,改善和提高其切片质量。方法将送检的脂肪组织切开固定于中性甲醛液中;取材后将标本放入脱水机中,调整脱水机设置程序,在常规方法的基础上适当增加组织再固定及脱水、透明时间;包埋时采取挤压组织等改进方法。结果通过改进和调整后制作的脂肪组织蜡块平整无塌陷,石蜡切片优良,厚薄均匀,完整,无空洞、无挤压、镜下组织结构完整清晰,细胞无挤压、重叠等现象。细胞形态清晰,着色均匀艳丽。结论通过上述方法的改进,能尽可能多地减少脂肪组织的脂滴,增加脂肪组织的硬度,使组织和石蜡能很好的融合在一起,有利于切出完整的厚薄均匀的石蜡切片,提高制片质量。     

蚊虫连续石蜡切片制备及染色

本研究利用塑化石蜡进行了吸血前后白纹伊蚊整个蚊虫的石蜡包埋、制片及 HE染色 ,获得了染色清晰 ,组织完整的连续石蜡切片 ,并讨论了有关蚊虫石蜡包埋及制片染色的一些注意事项。为进一步利用免疫组化、原位 PCR等技术研究虫媒病毒在蚊虫体内的分布规律提供了可能     

采用不同方法制作胸腹水细胞块的效果比较及临床价值研究

目的:比较不同方法制作胸腹水细胞块的效果以优化胸腹水细胞块制作程序,并对其临床价值进行探讨。方法:以2014年3月至2015年3月我科收集的胸腹水标本120例为研究对象,将每样标本平均分成三组,使用三种不同方法(试管包埋法、直接离心法、细胞块试剂盒法)制作胸腹水细胞块。对不同方法制作细胞块的成功率、完整性及细胞切片恶性细胞的检出率进行考察与比较。结果:细胞块试剂盒法成功率最高,为96.67%,试管包埋法次之,成功率为92.50%,二者相比无显著差异(P0.05)。直接离心法制作成功率为80.83%,远远低于试管包埋法及细胞块试剂盒法,差异有统计学意义(P0.05)。细胞块试剂盒法制作的细胞块完整性最高,完整标本所占比例为96.67%,试管包埋法次之,完整率为94.17%,二者相比无显著差异(P0.05)。直接离心法制作完整率为68.33%,远远低于试管包埋法及细胞块试剂盒法,差异有统计学意义(P0.05)。三种方法恶性细胞检出率以细胞块试剂盒最高,与其他两种方法比较差异有统计学意义(P0.05)。结论:细胞块试剂盒法制作胸腹水细胞块具有最高的成功率及完整性,并且可以显著提高恶性细胞检出率,值得广泛使用。     

内耳整块染色石蜡包埋制片法

内耳制片常用的是火棉胶——石蜡双重包埋H-E片染法。此法制片步骤较多,不易掌握。我采用Bouin氏固定液固定,H-E整块染色、石蜡包埋法制片,操作简便,易于成功,内耳的基本结构(前庭膜、盖膜、毛细胞都完好无损,如取材适当,可在同一切片同时显示壶腹嵴。染色液的配制: 1.苏木精染液的配制: 苏木精 25毫克硫酸铝钾 1.25克碘酸钠5毫克蒸馏水 75毫升由于以上药品称量很小,故需称的较准确。配出来的液体若呈现混浊,则是因为以上药品(主要是碘酸钠)称得不准造成的,这种混浊染色液不能用。 2.复制伊红染色液的配制: 将伊红(Y或B均可)0.5克溶于3毫升蒸馏水中,溶解后将冰醋酸—滴—滴加于其中,边滴边搅动,     

三种不同脂肪染色方法的比较

目的探讨碳蜡(聚乙二醇)包埋技术、冰冻切片及饿酸染色在脂肪染色中的应用,综合比较几种方法的优缺点,以便在病理工作及科研工作中能找到更适合的脂肪染色方法。方法取新鲜脂肪组织及肝组织,每份标本各取3块组织,分为A、B、C三组:A组和B组标本分别进行碳蜡包埋切片和常规冰冻切片,油红O法染色;C组以饿酸浸染组织块后进行石蜡包埋切片及眦复染。结果 A组切片脂肪滴呈红色,细胞核呈蓝色;B组切片脂肪滴呈红色,细胞核呈蓝色;C组切片脂肪滴呈黑色。结论饿酸染色和碳蜡包埋技术在脂肪染色中,具有冰冻切片和石蜡切片的优点,弥补了常规冰冻切片脂肪染色的缺点和局限性,在病理检验及科研中具有一定应用价值。     

一种用于DAPI染色的方法Steedman＇s wax包埋切片法

以植物的胚珠和子房为实验材料,介绍一种用Steedrnan‘s wax包埋对组织切片中的细胞核进行DAPI染色的方法。Steedrnan‘s wax作为一种低熔点多酯蜡,具有与石蜡相似的性质,切片方法同常规石蜡切片,适合于切成厚度大于5μm的连续切片。Steedrnan‘s wax包埋的切片能成功地进行DAPI染色。与用压片法和Technovit 7100或GMA包埋切片法进行的DAPI染色相比,用Steedrnan‘s wax包埋切片法进行的DAPI染色具有廉价、操作简便、可进行连续切片、图象清晰等优点,特别在植物细胞程序化死亡(PCD)的研究中及细胞核DNA含量测定方面,有着较大的应用价值和潜能。     

The detection of hTERC amplification using fluorescence in situ hybridization in the diagnosis and prognosis of cervical intraepithelial neoplasia: a case control study

ABSTRACT: BACKGROUND: Currently the routine non-invasive screening methods for cervical intraepithelial neoplasia (CIN) and cervical cancer are Thinprep cytology test (TCT) and human papillomavirus testing. However, both methods are limited by the high false positive and false negative rates and lack of association with patients' prognosis, especially for the early detection of pro-malignant CIN. The aim of the study was to investigate the role of genomic amplification of human telomerase gene (hTERC) in the diagnosis and prognosis of CIN. METHODS: The study group consisted of specimens of exfoliated cervical cells from 151 patients, including 27 with CIN I, 54 with CIN II/III, 17 with carcinoma in situ, and 28 with invasive squamous carcinoma, as well as 25 patients who were at 2-year follow-up after either Loop Electrosurgical Excision treatment (n = 11) or radical surgery (n = 14). hTERC amplification was detected by dual-color interphase fluorescence in situ hybridization (FISH), and the results were compared with TCT and histologic examination. The final diagnosis was determined by the pathological examination. The control group consisted of specimens of exfoliated cervical cells from 40 normal women. RESULTS: The percentage of cervical exfoliated cells with positive hTERC amplification and incidence rates of hTERC amplification were 9.2% [PLUS-MINUS SIGN] 4.6% and 44.4% (12/27) respectively in patients with CIN I; 16.0% [PLUS-MINUS SIGN] 14.4% and 85.1% (46/54) in patients with CIN II/III; 19.7% [PLUS-MINUS SIGN] 13.3% and 88.3% (15 /17) in patients with carcinoma in situ; 47.0% [PLUS-MINUS SIGN] 25.2% and 100% (28/28)in patients with invasive squamous carcinoma. There was statistically significant difference between the control and study group (P <0.01), and between the patients with various diseases within the study group (P <0.05). CONCLUSION: The detection of genomic amplification of hTERC using FISH is a non-invasive and effective approach for CIN.     

TCT联合HPV检测诊断宫颈病变的临床价值分析

目的:探讨新柏氏液基细胞学技术(TCT)联合人乳头瘤病毒(human papilloma virus,HPV)检测诊断宫颈病变的临床价值。方法:选择哈尔滨医科大学附属第一医院妇科门诊收治的6752例患者,采集其宫颈脱落细胞进行TCT检测,登记患者的年龄及宫颈外观。分别随机选取TCT阳性和阴性结果的患者144例进行HPV检测及阴道镜下活检,分析和比较TCT单独及联合HPV检测诊断宫颈病变的敏感性和特异性。结果:宫颈柱状上皮细胞的外移程度及患者的年龄与宫颈TCT结果均无显著相关性。单独TCT检测诊断宫颈病变的灵敏度为87.04%,特异性为58.55%;单独HPV检测诊断宫颈病变的灵敏度为62.96%,特异性为81.62%;而联合测诊断宫颈病变的灵敏度为100%,特异度为48.29%。结论:TCT联合HPV检测可显著降低宫颈病变的漏诊率。     

The use of fluorescence in situ hybridization (FISH) on paraffin-embedded tissue sections for the study of microchimerism

We describe here a simple and versatile method of fluorescence in situ hybridization (FISH) on paraffin-embedded tissue sections with specific application in the study of microchimerism, that is, the presence of intact foreign cells within an individual. This is accomplished through the use of X and Y chromosome-specific probes to identify the presence of male nuclei within a tissue section from a female, and vice versa. This technique requires only minor modification if at first the hybridization does not yield fluorescent signals of high quality. Analysis of a wide variety of tissue types is possible with this method, and multiple tissue types from one or more individuals can be processed in the same hybridization reaction. This robust FISH method has been used successfully in our laboratory to investigate fetal cell microchimerism in the following paraffin-embedded tissue types: skin, lung, thyroid, adrenal gland, lymph node, heart, spleen, liver, pancreas, kidney, and intestine.     

Telomeres in trisomy 21 amniocytes

Individuals with trisomy 21 have an increased risk of developing leukemia and premature dementia. They also have a higher rate of telomere loss. The aim of the study was to compare telomere length and the hTERC gene copy number, which encodes the telomerase RNA subunit, in amniocytes of trisomy 21 conceptions and normal pregnancies. A quantitative fluorescence-in-situ protocol (Q-FISH) was used to compare telomere length in amniocytes cultured from 11 trisomy 21 conceptions and from 14 normal pregnancies. Quantification was conducted using novel computer software. Fluorescence in situ hybridization (FISH) was used to assess the percentage of cells with additional copies of hTERC. We found that the immunofluorescence intensity, which represents telomere length, was significantly lower in amniocytes from trisomy 21 conceptions compared to the control group. The trisomy 21 group had a higher number of cells with additional copies of hTERC. This observation could be one of the cytogenetic parameters that represent a state of genetic instability and might play a role in the pathomechanism of typical features of Down syndrome, such as dementia and malignancy.     

TCT＋HPVDNA检测＋阴道镜检查对宫颈病变临床价值的研究

目的探讨对宫颈癌的筛查方法及其在防治中的作用。方法2012年3月至2012年8月,在绥化市第一医院妇科门诊行TCT检查的患者,年龄23—75岁,随机分为两组每组52例,一组宫颈液基细胞学检查阳性者行HPV检测,后再行阴道镜宫颈活检。一组宫颈液基细胞学检查阳性者直接行宫颈盲取活检。评价TCT检查联合HPV检测＋阴道镜诊断宫颈病变的作用。结果52例ASC-US患者,行HPV-DNA检测,阳性率为33．33％,HPV-DNA阳性率差异有统计学意义,P〈0.05。结论TCT＋高危型HPV是宫颈癌筛查中的最有效的方式,也是首选方法。若联合阴道镜检查则更能提高宫颈癌的检出率。     

Improved procedure for fluorescence in situ hybridization on tissue microarrays

BACKGROUND: The recently developed tissue microarray (TMA) technology allows the arrangement of up to a thousand tissue specimens on a single microscope slide. This technology enables researchers to perform gene copy number studies on very large series of archival formalin-fixed tissues using fluorescence in situ hybridization (FISH). However, the hybridization properties of individual archival specimens can vary considerably. Therefore a highly optimized protocol is needed to fulfill the task of producing evaluable hybridization signals simultaneously in hundreds of specimens in a TMA. METHODS: The performance of two different FISH protocols, the standard protocol for paraffin embedded tissues and our new optimized protocol, was tested on TMAs using probes for the HER-2 and ZNF217 genes as well as the chromosome 17 centromere. RESULTS: The new protocol resulted in greatly increased signal intensity and an almost 30% increase in the number of tissue samples with evaluable hybridization signals. CONCLUSIONS: Our improved protocol for FISH on TMAs provides standardized hybridization conditions leading to high-quality hybridization signals in the majority of specimens. The increases in the signal intensity and the number of evaluable samples are extremely important for the successful analyses of TMAs by FISH and will allow the utilization of the TMA technology in its full potential.     

两种少量小鼠外周血间期核FISH技术的比较

目的:探讨低渗法与红细胞裂解法应用于小鼠少量外周血间期核荧光原位杂交(fluorescence in situ hybridization,FISH)检测的优缺点,以便于根据实验条件选取最合适的方法。方法:小鼠剪尾巴法获取外周血,采用低渗法或红细胞裂解法破坏红细胞,涂片前先用免疫组化笔在玻片上划好预计涂片区域,重悬沉淀后稍等片刻再吸中间部分用于涂片,涂完后在相差显微镜下观察密度,行常规FISH,比较两种方法处理后的FISH结果。结果:低渗法和红细胞裂解法处理后的片子背景都较干净,密度适中,信号较强。结论:在外周血间期核荧光原位杂交技术中,低渗法和红细胞裂解法都可以获得较满意的FISH结果。低渗法的特点在于试剂便宜且易于获取,但耗时稍长;而红细胞裂解法的特点在于可节省实验时间,但需专用试剂。     

子宫颈癌筛查方法的应用及研究进展

子宫颈癌是妇科常见恶性肿瘤之一,发病率居女性恶性肿瘤第二位。建立可持续、合理、有效的普查方法,早期发现癌前病变是防治宫颈癌的关键。目前宫颈癌筛查方法有宫颈细胞学筛查(巴氏涂片、液基薄层细胞学检测、细胞DNA定量分析技术)、肉眼观察辅以醋酸白和Lugol碘溶液检测法、阴道镜检查、病毒-HPV检测,本文就当前各种筛查方法的应用及研究进展进行概括。     

HPV-DNA 亚型检测联合液基细胞学对宫颈癌 筛查的临床价值

目的:探讨HPV-DNA亚型检测联合液基细胞学对宫颈癌筛查的临床价值.方法:对自愿接受宫颈癌筛查的女性1462例作为研究对象,分别对其进行HPV-DNA亚型检测以及液基细胞学的检查,对于出现阳性的患者进行病理组织学检查.结果:HPV+TCT对宫颈癌早期病变以及癌变的检出率为69.67％明显高于HPV检查的56.28％以及TCT检查的63.89％(P＜0.05);HPV+TCT对GIN Ⅰ、CIN Ⅱ、CINⅢ、癌的检出率分别为91.67％,92.86％、91.67％以及100％.结论:采用HPV-DNA亚型检测联合液基细胞学对宫颈癌筛查,可明显提高其对癌前病变的检出率,是一种高效、简单的检测方法.