催化信号放大系统的制备及其在免疫组化和原位杂？…

目的：应用本实验室制备的催化信号放大（ｃａｔａｌｙｚｅｄ ｓｉｇｎａｌ ａｍｐｌｉｆｉｃａｔｉｏｎ,ＣＳＡ）剂试,提高免疫组化（ＩＨＣ）和原位杂交（ＩＳＨ＿的敏感性。方法：ＣＡＳ法应用于ＩＨＣ检测５０例１０种不同类型的肿瘤切片中４０种不同的抗原和ＩＳＨ检测６种不同的ＲＮＡ成分,并与相应的常规方法进行敏感性比较。同时分析了存在的问题。结果：ＣＡＳ－ＩＨＣ法较传统的ＰＡＯＰ法、ＡＢＣ法和ＬＳＡＢ法敏感     

催化信号放大法的改进

目的:探讨酪胺与过氧化物酶发生酶促反应后,用热缓冲液洗以进一步提高和改善催化信号放大法(CSA)的敏感性和特异性.方法:应用7种不同类型的一抗,2张不同肿瘤的组织芯片,同时进行EnVision法、LsAB法、标准CSA法和改进CSA法染色,比较敏感性和特异性.结果:改进CSA法最为敏感,其次是标准CSA法、EnVision法和LsAB法,改进CSA法的敏感性较标准CSA法高2～3倍.4种方法背景染色基本一致,细胞定位准确.结论:改进后的CSA法的稳定性、敏感性好于标准CSA法.     

应用直接酪胺信号放大方法显示Y染色体荧光原位杂交信号

目的用直接酪胺信号放大(tyramine signal amplification,TSA)方法显示Y染色体荧光原位杂交信号,以提高检测脑组织切片Y染色体荧光原位杂交信号的灵敏度。方法以小鼠Y染色体重复序列pY353/B cDNA为模板标记RNA探针,以雌性小鼠为对照,用原位杂交方法杂交Y染色体特异性基因区域,分别用直接TSA方法和"三步抗体法"显示荧光原位杂交信号。通过计数Y染色体阳性细胞核占所有细胞核的百分率来计算TSA法和"三步抗体法"检测脑组织切片Y染色体荧光原位杂交信号的灵敏度,比较分析两种方法灵敏度的差异。结果"三步抗体法"在脑切片检测Y染色体阳性信号的灵敏度和特异度分别为85.67%和100%;直接TSA法在脑切片检测Y染色体阳性信号的灵敏度和特异度分别为92.82%和100%。结论TSA方法检测脑组织切片Y染色体荧光原位杂交信号的灵敏度比"三步抗体"法有显著的提高,可以用于脑内追踪和分析骨髓干细胞的迁移规律。     

原位杂交及免疫组化法在诊断尖锐湿疣中的应用

原位杂交及免疫组化法在诊断尖锐湿疣中的应用中国第一汽车集团公司医院（１３００１１）潘淑琴张维平北京中日友好医院吴王敬王泰玲在病理活检组织中进行人乳头状瘤病毒（ｈｕ┐ｍａｎｐａｐｉｌｏｍａｖｉｒｕｓ，ＨＰＶ）检测有助于确诊尖锐湿疣。我们对有关病例分别进...     

散发性病毒性脑炎免疫组化和原位杂交研究

２０例临床诊断为散发性病毒性脑炎的尸检和活检脑组织，通过单纯疱疹病毒多克隆抗体免疫组化和生物素标记单纯疱疹病毒ＤＮＡ探针原位杂交研究，５例诊断为单纯疱疹病毒脑炎。结果说明：单纯疱疹病毒是国内散发性病毒性脑炎的重要病原；免疫组化和原位杂交是诊断单纯疱疹脑炎的两种敏感方法。     

大鼠胃促黄体生成素及其受体的免疫组化及原位杂交研究

目的:探讨大鼠胃组织中是否表达促黄体生成素(LH)及其受体(LHR),为进一步研究LH对胃的功能调节提供理论依据.方法:采用免疫组织化学SABC和原位杂交的方法进行研究.结果:在大鼠胃底腺的壁细胞和主细胞呈LH和LHR免疫反应阳性.阳性物质分布于细胞质和细胞膜上.细胞核呈阴性反应.上述细胞同样含有LH和LHR mRNA杂交信号,信号物质亦分布于细胞质内,细胞核呈阴性反应.结论:大鼠胃壁细胞和胃主细胞既能产生LH,又能表达LHR,说明LH对胃壁细胞和胃主细胞功能作用可能是通过旁分泌或自分泌的作用来实现的.     

口腔鳞癌p16／MTS1免疫组化和原位杂交检测

运用免疫组化和原位杂交法对４３例口腔鳞癌的ＭＴＳ１基因，即ｐ１６的表达进行研究，并分析了口腔鳞癌细胞株ｐ１６株的表达。结果４３例口腔癌中２８例为ｐ１６阳性，１５例ｐ１６阴性，ｐ１６基因的改变是口腔鳞癌发展中的频发事件。组化和原杂杂交结合能较好地反映肿瘤组织中ｐ１６基因和蛋白的改变。     

端粒酶催化亚单位在细胞学诊断肺癌中的表达

目的:检测支气管镜涂片中端粒酶催化亚单位(hTERT)的表达,探讨其与肺癌发生的关系,探索细胞学诊断肺癌的新途径。方法:应用免疫组化和原位杂交的方法检测137例支气管镜涂片中hTERT和hTERTmRNA的表达,并与临床病理资料及细胞学诊断进行相关性分析研究。结果:137例病例中,hTERT和hTERTmRNA表达阳性率在细胞学诊断为癌细胞组分别为67.86%(57/84)和79.76%(67/84);疑癌或异型细胞组分别为46.67%(7/15)和60.00%(9/15);未见癌细胞组分别为5.26%(2/38)和39.47%(15/38)。二者的表达在癌细胞组明显高于其他2组(P<0.05)。其中有组织学对照的105例,hTERT和hTERTmRNA阳性表达率与细胞学诊断阳性率相一致。hTERT和hTERTmRNA的阳性表达率呈正相关性(r=0.994,P<0.05),但这二者的表达率与组织病理类型及其他临床特征差异均无显著性(P>0.05)。结论:端粒酶在支气管涂片中的阳性表达与肺癌的发生密切相关。hTERT和hTERTmRNA在癌组织中均存在高表达,且比传统形态学诊断更敏感。而hTERT蛋白的表达在细胞学涂片中更可靠(假阳性率低)。因此检测这二者的表达有助于提高肺癌的细胞学检出率,对早期发现、诊断肺癌有重要意义。     

Preparation of CSA system and its application in IHC and ISH

CatalyZed st~ amplification(CSA) system was successfully ugh in ilnmunoaSSay by BobIDw et alll] and Adams et allzj reported that they aPPlied CSA system to immUnohistOChendstw (IHC ) and in situ hybridization(ISH), ~tively. The core reagent of CSA system is~de crass--linking a kind of ~er molecule such asbiotin, digokin, fluorescein or heaVy mend ion, etc. ThePrinciple of foe system is: twide is catalyZed by horseedsn pemtibe(ar) with HZOz to fonn covatent conjugation siteS in 15 …     

荧光原位杂交和免疫组织化学技术检测乳腺癌HER-2基因扩增和蛋白表达状态对照研究

目的 比较荧光原位杂交(FISH)与免疫组织化学技术(IHC)在检测乳腺癌患者HER-2基因扩增和表达状态方面的应用.方法 采用FISH技术对66例IHC检测结果为HER-2基因过度表达(3+/2+)和低表达或无表达(1+/-)的乳腺癌石蜡切片进行HER-2基因扩增状态检测.结果 用FISH技术检测42例IHC结果显示HER-2基因过度表达(3+/2+)的标本,31例显示HER-2基因扩增,11例无扩增.检测24例IHC检测显示HER-2基因低度表达或无表达(1+/-)的标本,均未显示HER-2基因扩增.两组之间比较,Kappa系数为0.672,P<0.001,显示两项检测技术具有良好的一致性.另外.用FISH技术检测到部分病例显示17号染色体多体性,并且该多体性在HER-2高表达(3+/2+)的病例发生率显著高于低表达或无表达(1+/-)的病例(x~2=4.688,P=0.03).结论 FISH和ICH两项技术具有良好的一致性.IHC可作为HER-2基因扩增和表达状态检测的筛查手段.FISH技术可以作为检测HER-2基因扩增和17号染色体多体性的确诊手段.

Abstract：

Objective To evaluate the application of the immunohistochemistry (IHC) and the fluorescence in situ hybridization (FISH) in detecting the amplification and the expression of HER-2 gene in the breast cancer patients. Methods Sixty-six cases of paraffin-embeded breast cancer samples with overexpression, low or no expression of HER-2 gene as detected by IHC were analyzed for HER-2 gene amplification using FISH. Results Among the 42 samples with HER-2 gene overexpression (3+/2+) detected by IHC, 31 showed positive HER-2 gene amplification and 11 showed negative HER-2 gene amplification in FISH. In the 24 samples with low or no HER-2 gene expression (1+/-) detected by IHC, no HER-2 gene amplification was detected by FISH. The results of the two testing methods showed a good consistency with the kappa coefficient of 0.672 (P<0.001). We also found that the 17 chromosome polysomy in 42% of the samples and the incidence of 17 polysomy was significantly higher in the HER-2 gene overexpression (3+/2+) group than in low or no HER-2 gene expression (1+/-) group (x~2=4.688, P=0.03). Conclusion IHC can be used as a screening method for detecting HER-2 gene amplification, and FISH should be performed in cases of HER-2 gene overexpression (3+/2+) as detected by IHC.     

骨髓活检组织的免疫组织化学和核酸原位杂交研究

目的：建立既适用于常规病理检验又可进行免疫组化及核酸原位杂交研究的骨髓活组织检查方法。方法：活检标本１０例，用ＬｏｗｙＦＭＡ液固定、脱钙，石蜡包埋。切片行常规染色、ＴＧＦ－β１和ＭＩＰ－１α免疫组化及地高辛标记ＤＮＡ探针ｍＲＮＡ原位杂交。结果：ＨＥ染色、鼠源性单抗和兔源性多抗的免疫组化及不同长度探针的ｍＲＮＡ析位杂交均取得理想的效果。结论：ＬｏｗｙＦＭＡ液固定、脱钙后石蜡包埋的骨髓活检组织不仅能满     

Comparison of polymerase chain reaction and catalyzed signal amplification in situ hybridization methods for human papillomavirus detection in paraffin-embedded cervical preneoplastic and neoplastic lesions

BACKGROUND: Two molecular methods for HPV genotyping in formalin-fixed, paraffin-embedded tissue were evaluated: in house polymerase chain reaction assay (PCR) with consensus and type-specific primers and a novel procedure of in situ hybridization-a catalyzed signal amplification system (CSA-ISH, Genpoint, DAKO, Glostrup, Denmark). The number of HPV positive cases and detected viral types were compared in cervical biopsies and cone specimens according to histopathological diagnosis. Primer efficiency in detecting various types of HPV by PCR method was evaluated. METHODS: DNA samples (101) were used as a template to amplify with three pairs of consensus (MY09/11, GP5+/6 +, CPI/IIG) and four type-specific HPV primers (HPV-6/11, 18, 16 and 33). The according histological tissue sections were analyzed with CSA-ISH method, using commercial HPV biotinylated probes HPV-6/11, 16/18 and 31/33/51. RESULTS: The degree of concordance for PCR and CSA-ISH was 64.4%. In 63 of 101 samples (62.4%), HPV was detected by PCR, while only 35 (34.7%) were positive using CSA-ISH. CSA-ISH found lower percentages for all HPV types, except HPV-6/11. A lower percentage of positive results in all high-grade lesions was detected by CSA-ISH. Multiple infections were detected by PCR in only one sample and in three samples by CSA-ISH. Detection with My09/11 primers followed by Gp5+/6+ primers, in nested reaction, gave the highest number of positive results: 58 of 63 (92%). None of the samples diagnosed as condylomata planum or CIN I was positive for HPV-6/11 (low risk type), which was detected exclusively in condylomata acuminatum group. CONCLUSIONS: A significantly higher number of positive samples was detected with PCR than with CSA-ISH method. CSA-ISH method should be improved, especially in detecting HPV in high-grade lesions. CSA-ISH may be more accurate in detection of multiple infections. GP5+/6+ in nested reaction after MY09/11 detected the highest number of positive results. Samples diagnosed as benign lesions positive on HPV-X must be monitored as possible candidates for progression. CIN I lesions, which were HPV negative, probably will not progress. This finding may be important in planning therapy and avoiding unnecessary treatment.     

原位杂交与免疫组化技术用于评价化疗后结直肠癌患者MLH1、MSH2和hMSH6表达水平的对比研究

目的 探讨原位杂交方法和免疫组化方法两种不同方法在检测评价直肠癌术后新辅助化疗治疗后患者三种错配修复蛋白(hMLH1、hMSH2和hMSH6)表达水平的效果。方法选取本院收治的散发性结直肠癌(SCRC)患者260例,取手术切除的肿瘤组织,使用较原位杂交和免疫组化法测定hMLH1、hMSH2和hMSH6表达水平的效果,比较三种指标之间的关联性。结果不同部位的肿瘤各种特征之间未见明显差别。原位杂交法检测hMLH1、hMSH2、hMSH6的阳性率显著高于免疫组化检测的阳性率;免疫组化法检测结果提示hMLH1与hMSH2的表达无明显相关性,hMLH1或hMSH2与hMSH6表达分别呈正相关;原位杂交法检测结果提示hMLH1与hMSH2,hMLH1与hMSH6,hMSH2与hMSH6表达均呈正相关。结论原位杂交法和免疫组化方法两种方法在检测评价直肠癌术后辅助化疗后患者三种错配修复蛋白hMLH1、hMSH2和hMSH6表达水平的效果中原位杂交法更优。     

用原位杂交及免疫组化方法显示人胚胎甲状腺褪黑素受体

目的：在前期工作的基础上进一步观察人胚胎甲状腺组织中腿黑素（Ｍel)受体的存在及其分布。方法：取人胚胎（胎龄６个月）甲状腺,石蜡包埋,切片,以免疫组化ＡＢＣ法及原位杂交方法进行染色。结果：免疫组化方法显示,人胚胎甲状腺细胞的细胞膜、细胞质和细胞核中Ｍel受体mt1和ＭＴ２亚型均呈棕褐色阳性染色;原位杂交方法显示mt１和ＭＴ２分布于甲状腺细胞的细胞质及细胞核内,呈蓝紫色阳性染色。结论：人胚胎甲状腺组织中存在褪黑素受体mt1和ＭＴ２亚型,分布于甲状腺细胞的细胞膜、细胞质、细胞核内。     

荧光原位杂交技术的应用

荧光原位杂交（fluorescence in situ hybridization, FISH））技术是一门新兴的分子细胞遗传学技术,在疾病诊断中发挥了重要作用。本文主要对FISH技术的原理特点以及临床应用作一综述。     

不脱钙骨组织免疫组织化学与原位杂交的方法研究

目的 :探讨在低温塑料包埋制备的不脱钙骨切片上运用免疫组化和原位杂交技术的可行性。方法 :采用改进的甲基丙稀酸甲酯低温塑料包埋法包埋SD大鼠胫骨近端 ,制备不脱钙的骨组织切片。切片行Goldner′s三色法染色观察骨小梁结构 ;采用免疫组化SABC法和原位杂交技术检测骨组织细胞中胰岛素样生长因子 1(IGF 1)mRNA和蛋白表达。结果 :三色法染色示骨小梁结构完整 ,部分骨小梁边缘可见红染的类骨质 ,成骨细胞与破骨细胞清晰可见。免疫组化和原位杂交示IGF 1mRNA和蛋白表达于成骨细胞 骺板软骨细胞及骨髓腔中部分单核细胞的胞浆中。此外 ,在骨小梁基质中也可见IGF 1蛋白的阳性表达。结论 :利用低温塑料包埋制备的不脱钙骨切片可同时进行免疫组化和原位杂交检测观察 ,该方法为骨质疏松等代谢性骨病的分子病理学研究提供了有效的技术平台。     

荧光原位杂交对乳腺癌HER2基因的检测及临床应用

目的探讨荧光原位杂交法(FISH)检测乳腺癌组织中人类表皮生长因子受体2(HER2)表达的临床意义。方法采用FISH和免疫组织化学(IHC)法分别检测28例乳腺癌标本中HER2基因扩增状况和HER2蛋白表达。结果在IHC法检测HER2蛋白表达(++)的22例标本中,FISH检出6例HER2基因扩增,16例无变化。在HER-2蛋白表达(+++)的6例标本中,FISH检出5例HER2基因扩增,1例无扩增。结论对于IHC法初筛为(++)的患者应再进行FISH检测,以确定HER-2基因的状态,从而确定是否应用曲妥珠单抗治疗。