AQP1在骨肉瘤中的表达和意义

目的探讨骨肉瘤中AQP1的表达和临床意义。方法对39例骨肉瘤和22例骨良性疾病对照组标本采用免疫组化法观察AQP1的表达并用人工计数和自动图像分析两种方法检测其表达差别,同时行CD34抗体染色标记肿瘤微血管,计算所有标本MVD。结果①AQP1表达于肿瘤微血管和小血管内皮细胞及绝大多数骨肉瘤的肿瘤细胞,尤其见于骨肉瘤的肿瘤巨细胞上;②人工计数AQP1阳性细胞百分比（YP）、AQP1人工计数分级、自动图像分析所得平均光密度（MD）,均表明AQP1在骨肉瘤中高表达（P均〈0.01）;③骨肉瘤中AQP1的YP与MVD呈明显正相关（r=0.341,P〈0.05）。结论大多数骨肉瘤肿瘤细胞高表达AQP1的原因不明;AQP1在骨肉瘤生物学行为及肿瘤微血管生成中占有重要作用;提示AQP1有可能作为骨肉瘤治疗的新靶点。     

非小细胞肺癌生物学特性免疫组化图像定量分析

目的探讨非小细胞肺癌(NSCLC)免疫组化图像定量分析中描述光密度参数在免疫组化阳性着色深浅的变化及生物学意义。方法采用S-P法进行肺癌PCNA和BCL-2免疫组化染色,应用计算机病理图像分析系统(MPIAS-500)进行图像分析。结果44例肺癌中PCNA阳性为30例,积分光密度、平均光密度、平均灰度在肺癌肿瘤大小、有无转移、生存时间长短三项指标中两组间均有统计学意义(P<0.01)。BCL-2阳性为37例,其在肺癌肿瘤大小、有无转移、生存时间长短三项指标中积分光密度比较两组间均有统计学意义(P<0.01);平均光密度和平均灰度在前两项两组间有统计学意义(P<0.01和P<0.05)。结论用描述光密度参数指标定量分析肺癌免疫组化图像可以更客观地反应肺癌的发生发展、转移和预后。     

胃黏膜上皮癌变过程中p53蛋白和PCNA的表达及其临床意义

目的探讨肿瘤抑制基因p53蛋白,增殖细胞核抗原(PCNA)在不同胃黏膜上皮病变中的表达状况。方法采用免疫组化ABC法,检测135例不同胃黏膜病变组织和15例正常胃黏膜组织中p53蛋白,PCNA的表达状况。结果胃黏膜上皮各级病变组织中均有不同程度的p53表达,表达率随病变加重而增高,腺癌与萎缩性胃炎比P<0.005。PCNA免疫阳性细胞计数与病变发展有相关性。其定量分析数据除浅表性胃炎与正常组无明显差异外,其余各组间差异显著。结论p53蛋白和PCNA的变化是胃上皮癌变的重要分子学改变。     

雌激素缺乏对兔关节软骨甲状旁腺激素相关肽表达的影响

目的：研究双侧卵巢切除后雌激素缺乏对兔关节软骨甲状旁腺激素相关肽（PTHrP）表达有何影响。方法：手术切除双侧卵巢造成兔体内雌激素水平下降，8周后取实验组和对照组右股骨髁关节标本。经免疫组化方法检测PTHrP的表达。用光镜和图像分析仪测定软骨中PTHrP阳性细胞百分率和表达强度。结果：卵巢切除组关节软骨PTHrP阳性细胞百分率和表达强度均高于正常关节软骨，两间的差异有统计学意义。结论：雌激素水平对关节软骨PTHrP表达具有调节作用。     

肠复康对人大肠癌HT-29细胞增殖的影响及作用机制研究

 目的　研究中药复方肠复康对人大肠癌细胞增殖的影响及探讨其作用机制。方法　建立人大肠癌HT 2 9裸鼠移植瘤模型。设立肠复康低、中、高剂量组和空白组。给药 8周后比较瘤重、抑瘤率及病理变化。免疫组化染色结合图像分析系统半定量检测移植瘤Ki 6 7阳性细胞数和血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)的积分光密度。 结果　肠复康低、中、高剂量组抑瘤率分别为33.13%、4 7.2 6 %、6 6 .0 1%。肠复康各组移植瘤细胞核数量较少 ,细胞及细胞核形态更加规则。肠复康各组Ki 6 7阳性细胞数及VEGF表达均低于空白组。结论　肠复康具有抗人大肠癌HT 2 9裸鼠移植瘤细胞增殖的作用 ,其机理可能与促进癌细胞分化及抑制VEGF的表达有关。     

C-erbB-2和EGFR在大肠癌中过表达及意义

目的 :研究癌基因和表皮生长因子在大肠癌中的表达与临床病理特征及预后的关系。方法 :应用免疫组化 S-P法检测 57例大肠癌组织中 C-erb B-2和 EGFR的表达水平 ,应用计算机图象分析系统对 C-erb B-2、EGFR的阳性细胞数密度进行检测、分析。结果 :Cerb B-2、EGFR在大肠癌中的阳性率分别为 59.6%和 56.1%。 C-erb B-2和 EGFR与瘤体的大小无明显相关性 ,与肿瘤的生长方式、是否有淋巴结转移以及预后有显著相关性 (P<0 .0 5)。结论 :Cerb B-2和 EGFR均有助于评估大肠癌预后 ,C-erb B-2、EGFR均为高表达的患者预后最差     

胃癌组织中VEGF-C与VEGF-D及受体-3的表达及其临床病理意义的研究

目的：探讨胃癌组织中VEGF-C、VEGF—D和VEGFR-3的表达及与临床病理因素的关系。方法：免疫组化方法检测113例胃癌石蜡标本中VEGF-C、VEGF—D及VEGFR-3在蛋白水平的表达，RT—PCR方法检测28例冷冻标本在mRNA水平的表达，分别应用VEGFR-3单抗与CD34单抗利用免疫组化方法检测淋巴管和血管计数。结果：免疫组化结果中，淋巴结转移阳性组VEGF-C／D阳性表达率均高于阴性组，P=0．018，P=0．032。VEGF-C／D阳性组淋巴管记数均高于阴性组，P=0．001，P=0．007。VEGF-C／D的表达均与淋巴管计数相关，RT—PCR结果与免疫组化结果基本吻合，VEGF-C的表达与胃癌患者预后具有相关性。结论：VEGF-C、VEGF—D与胃癌周围淋巴管生成及胃癌淋巴结转移密切相关，VEGF-C有可能成为胃癌的预后因素。     

乳腺癌前病变和乳腺癌中α、γ-tubulin蛋白表达的免疫荧光定量分析及其意义

目的:研究乳腺导管上皮不典型增生(ADH)、导管内癌(DCIS)及浸润性导管癌(IDC)中α-tubulin和Γ-tubulin表达的变化及其意义,进一步探讨中心体异常与乳腺癌发生发展的相关性。方法:研究样本被分为导管上皮不典型增生、导管内癌和浸润性导管癌3组,每组30例。采用流式细胞免疫荧光定量分析技术和免疫组织化学方法,检测上述各组中心体微管蛋白α-tubulin和Γ-tubulin表达水平,与30例正常乳腺组的检测结果比较,并进行统计学分析。结果:在全部样本组中α-tubulin及Γ-tubulin FITC标记的阳性细胞数均数不同,差异有显著性(P=0.000),浸润性导管癌组的过表达最高,正常组织组最低。α-tubulin、Γ-tubulin表达水平随着细胞增殖级别的增加和分化程度的变差呈增高趋势。除不典型增生组和导管内癌组两组间比较以外,其余各组间差异均有显著性(P=0.001)。在每组中α-tubulin和Γ-tubulin的表达程度无统计学差异(P<0.05),显示免疫荧光定量分析和免疫组织化学检测结果趋于一致。结论:乳腺癌发生的早期阶段即存在着中心体蛋白的过度表达,推测这种异常表达在促进细胞恶性转化、形成乳腺癌过程中可能扮演了重要角色。对于中心体蛋白过表达的检测,免疫荧光定量和免疫组化两种技术可相互补充。     

胃癌巨噬细胞浸润和微血管密度与侵袭转移的关系

目的:探讨胃癌组织中浸润巨噬细胞与微血管密度的相关性及其在胃癌侵袭转移中的作用。方法:应用免疫组化SP法,用抗CD68 和抗CD34单克隆抗体对102例胃癌组织进行标记分析,并应用计算机辅助图像分析系统对血管新生的面积进行定量分析。结果:巨噬细胞浸润与胃癌分化程度、浸润深度、淋巴结转移和TNM分期有关,且有显著性差异(p<0.01,p<0.05,p<0.05, p<0.05)。MVD与分化程度、浸润深度、淋巴结转移,TNM分期相关,且有显著性差异(p<0.01)。巨噬细胞计数与MVD呈正相关(r=0.961,p<0.01)。结论:联合检测巨噬细胞浸润度及MVD定量分析可提供胃癌恶性转归更有价值的指标。     

突变型p53蛋白在胃癌和癌旁组织中的表达及形态定量分析

目的　应用图像分析对胃癌、癌旁组织中p5 3蛋白阳性细胞核进行形态测量 ,根据核间差异 ,为胃部良恶性病变的鉴别提供依据。方法　采用SP免疫组化法和图像分析技术分别对 3 3例胃癌和 17例癌旁组织中p5 3阳性细胞核进行检测。结果　免疫组化结果显示 :胃癌中p5 3蛋白阳性表达率和阳性细胞密度均高于癌旁组织 (P <0 .0 5 )。图像分析结果显示 :胃癌和癌旁组织中p5 3阳性细胞核在等效直径、形状因子和异形指数各参数上差异具有显著性 (P <0 .0 1)。结论　以上结果表明 :图像分析能客观对肿瘤标记物进行参数识别 ,可以作为鉴别胃良恶性病变的一个参考指标 ,并有助于胃癌的早期预防。     

A method for quantitative autoradiography over stained sections of tumors exposed in vivo to radiolabeled antibodies.

Quantitative autoradiography can be an important element in calculating dose patterns delivered to tumors and normal tissues of animals injected with radiolabeled antibodies. In this article we develop a computer algorithm that permits quantitative evaluation of grain density over large areas of stained tissue with minimal grain counting. This algorithm, using spectral classification of brightfield and darkfield images, image ratios, and manual counting of small sections of an autoradiograph, yields images defined in terms of grain density, independent of staining artifacts. These quantitative images give a precise indication of intratumor radioimmunoglobulin targeting, and can serve as source functions for dosimetric calculations.     

氨磷汀对顺铂肾毒性损伤的保护作用及其机制的研究

目的 观察顺铂的肾毒性损伤部位、形式与肾功能检查结果的相关性,以了解细胞凋亡的发生机制和氨磷汀的保护机制是否与肾组织Fas和FasL表达改变有关。方法 随机将大鼠分成3组,即对照组（生理盐水）、顺铂组（6mg／kg）和氨磷汀组（顺铂6mg／kg＋氨磷汀200mg／kg）,取其血清标本和肾组织,分别做血清BUN、Cr检测和肾组织病理学检查,并用原位缺口末端标记法（TUNEL）做肾组织凋亡细胞检测、Fas和FasL免疫组化染色,再用图像分析软件对其做阳性细胞计数和染色总灰度值测定。结果 顺铂组动物血清BUN、Cr值均明显高于对照组和氨磷汀保护组,3d时,差异已有统计学意义（P〈0．05）;5d时,两者差异分别为P〈0．01和P〈0．05;10d时,恢复正常。顺铂组肾小管上皮细胞坏死和凋亡均很严重,其凋亡细胞计数明显高于对照组和氨磷汀组（P值均〈0．01）,肾组织Fas和FasL表达的总灰度值,明显高于对照组和氨磷汀组（P值均〈0．01）。结论 氨磷汀对顺铂的肾毒性损伤有保护作用,其机制可能与抑制肾组织Fas和FasL的表达有关。     

Proliferative activity (ki-67 expression) and outcome in high grade osteosarcoma: a study of 27 cases

Purpose. Although pre-operative chemotherapy has improved the prognosis for individuals with osteosarcoma, approximately 40% of patients will die of their disease.The aim of this study was to quantitate proliferative activity in high grade osteosarcomas and to determine whether proliferation is a prognostic factor.Patients. The study consisted of 27 patients with high grade non-metastatic osteosarcoma at various sites for whom pre-operative biopsies and resection specimens were available for review. All patients were treated similarly and had at least 24 months' follow-up from the date of diagnosis.Methods. Proliferative activity (Ki-67 expression) was examined in the diagnostic biopsies immunohistochemically using the MIB-1 antibody. Proliferation was quantitated in two ways; (1) the number of immunopositive cells was counted manually using an ocular grid; or (2) the percentage of immunopositive nuclear area was assessed using morphometric image analysis. Proliferative index was evaluated in relation to patient outcome.Results. Proliferative activity was seen in all biopsies.The median proliferative index as determined by counting cells was 24% (mean of 27%, range of 7-61%) and by image analysis was 2% (mean 3%, range 0.32-8.4).The correlation between MIB-1 proliferation indices determined either by image analysis methodology or manual cell counting was high (Spearman's rho=0.79). Proliferative index did not appear to predict either disease-free or overall survival.Discussion. Tumor proliferation does not appear to be prognostic for high grade osteosarcomas.Whether assessment of this feature in conjunction with other tumor characteristics might be prognostic requires further study.     

一种用流式细胞术检测基因转移效率的新方法

背景与目的：基因转移效率一直以来都是用表达报告基因的细胞（显色或发荧光）占细胞总数的百分比（即转染率）来表达,此百分比是由研究者借助显微镜（或荧光显微镜）计数得出来的。该方法（本文称为人工计数法）存在客观性、准确性不够和工作量大、且不能同时检测基因表达效率等问题。本研究拟寻找一种客观、准确、简便地检测基因转移和表达效率的新方法。方法：以绿色荧光蛋白（GFP）基因为目的基因、重组腺病毒（AdCMV/GFP)为基因载体,用人工计数法测定其对肝癌细胞株HepG2的转染率;用流式细胞术（flow cytometry,FCM)测定其对肝癌细胞株HepG2、Bel7402、Hep3B、SMMC7721和鼻咽癌细胞株CNE－2的转染效率和GFP（绿色荧光蛋白）基因的表达效率（以荧光指数表示）。结果：以FCM测得AdCMV/GFP对HepG2的转染率与载体量呈对数相关（与人工法相似,但前者略高）,荧光指数与载体量呈线性相关（γ＝0．9984）,其它细胞株的结果与HepG2相似。结论：采用FCM法测定基因转移效率克服了主观因素的影响,测定结果客观准确,可以用于测定基因载体对真核细胞的转染效率,或研究转录调控元件的功能。     

心理应激对大鼠胃窦生长抑素mRNA阳性细胞的影响

目的　探讨心理因素对胃窦粘膜生长抑素m RNA 阳性反应细胞的影响。方法　采用心理应激大鼠模型A、B、C三组, 以原位杂交组织化学和微机图像分析技术, 检测大鼠心理应激后胃窦粘膜生长抑素mRNA 阳性反应细胞的表达强度和细胞体积密度(Vv) 、单位体积数密度(Nv) 的变化。结果　在生长抑素m RNA 的表达程度和Vv、Nv 等方面, 三组相比, 闪光间期长短不均加电刺激组(A 组) > 闪光间期恒定加电刺激组(B 组) > 单纯闪光间期长短不均组(C组) 。结论　此结果表明, 心理应激明显增加大鼠胃窦粘膜D 细胞合成和分泌生长抑素     

Morphometric study of tumor angiogenesis as a new prognostic factor in nasopharyngeal carcinoma patients

The aim of this study was to determine the possible prognostic significance of tumor angiogenesis (TA) in nasopharyngeal carcinoma (NPC) patients. Fifty-five NPC patients were evaluated in relation to survival. Endothelial cells were immunohistochemically stained with anti-von Willebrand factor (F-VIII), CD-31 and CD-34 antibodies, and microvessels counted in the most active areas of tumor neovascularization or hotspotsusing both a manual and an automatic method. Overall survival analysis calculated by the Kaplan Meiertest revealed that both methods were correlated with a statistical significance between intratumoral microvessel density (IMD) and overall survival, using either manual (p=0.0141) or automatic counting (p=0.0117). Other angiogenic parameters studied were perimeter, roundness and accumulative area of the microvessels using a morphometric analyzer. Moreover, our results show that cases with high IMD demonstrated a prognostic significance in relation to the accumulative area (p=0.0072).     

全自动显微镜及图像分析系统处理免疫组化图像

目的 :探索全自动显微镜及图像分析系统处理免疫组化图像的新方法。方法 :利用全自动显微镜及图像分析系统采集肩袖撕裂患者肩峰下滑囊(SAB)及正常对照组白细胞介素6(IL-6)免疫组化染色切片图像,建立统一的图像采集、分割及处理标准,并对其分析,采用积分光密度评价阳性表达结果。传统的方法为手动显微镜及早期的图像分析处理软件,取其平均光密度,并对两种方法所测结果进行比较。结果 :新方法与传统方法均能够得出撕裂患者阳性表达数据,但新方法成像质量高,分析结果差异更为显著。结论: 新方法采集的图像质量较高,标准统一,误差较小,人为因素大为减少,结果更为准确,有更好的重复性和可比性。积分光密度与平均光密度相比将面积因素考虑在内,测量物质含量结果更为准确。     

乳腺癌细胞的计算机自动识别—QMSOC的应用研究

应用半自动图像分析仪测量26例乳腺癌(CA组)、5例乳腺纤维腺瘤(FA组)、5例急慢性乳腺炎(IN组)细针针吸穿刺涂片.细胞作几何特征参数,用分步判别法挑选其中7个对识别贡献较大的参数,做计算机自动判别分析:先以18O个随机抽取的细胞作为学习集,推出判别函数组,最后以此函数组对18O个细胞作回代识别,结果显示CA组的专家计算机识到符合率最高,达85%.     

Utility of automated counting to determine absolute neutrophil counts and absolute phagocyte counts for pediatric cancer treatment protocols

BACKGROUND: Absolute neutrophil counts (ANCs) and absolute phagocyte counts (APCs) are used to guide cancer treatment. Although automated counting could replace manual counting, data showing correlations are lacking. By analyzing blood samples from children undergoing cancer treatment, the authors determined whether ANCs and APCs obtained by automated methods correlated positively with ANCs and APCs obtained manually. METHODS: The authors analyzed 3640 consecutive peripheral blood samples. Leukocyte counts determined by Beckman-Coulter Gen-S or HmX analyzers (Beckman-Coulter, Miami, FL) were used to calculate counts obtained by automated or manual methods. Automated differential counts were obtained by automated analyzers and manual differential counts were performed by medical technologists. Counts underwent linear regression analysis. The authors evaluated 5 cutoff values for ANCs and APCs commonly used in decision-making related to cancer treatment: 300/muL, 500/muL, 750/muL, 1000/muL, and 1500/muL. Manually determined ANCs and APCs served as standards to determine the sensitivity, specificity, positive and negative predictive values, and kappa coefficient for automated counting. RESULTS: R(2) values were 0.81 for ANCs determined by manual and automated methods and 0.84 for APCs determined by both methods. The specificity of the automated method was > 90% for all ranges of ANCs and APCs, except one (APCs < 300/muL). There was excellent agreement (kappa > 0.9) between ANCs determined by manual and automated methods and APCs calculated by both methods. CONCLUSIONS: Automated methods of determining ANCs and APCs for children undergoing cancer treatment were reliable and can replace manual counting. Blood smear examination to validate ANCs and APCs determined by automated methods was needed only in selected cases.     

Field template-based design and biological evaluation of new sphingosine kinase 1 inhibitors

Purpose

The Ki67 proliferation index is a prognostic and predictive marker in breast cancer. Manual scoring is prone to inter- and intra-observer variability. The aims of this study were to clinically validate digital image analysis (DIA) of Ki67 using virtual dual staining (VDS) on whole tissue sections and to assess inter-platform agreement between two independent DIA platforms.

Methods

Serial whole tissue sections of 154 consecutive invasive breast carcinomas were stained for Ki67 and cytokeratin 8/18 with immunohistochemistry in a clinical setting. Ki67 proliferation index was determined using two independent DIA platforms, implementing VDS to identify tumor tissue. Manual Ki67 score was determined using a standardized manual counting protocol. Inter-observer agreement between manual and DIA scores and inter-platform agreement between both DIA platforms were determined and calculated using Spearman’s correlation coefficients. Correlations and agreement were assessed with scatterplots and Bland–Altman plots.

Results

Spearman’s correlation coefficients were 0.94 (p < 0.001) for inter-observer agreement between manual counting and platform A, 0.93 (p < 0.001) between manual counting and platform B, and 0.96 (p < 0.001) for inter-platform agreement. Scatterplots and Bland–Altman plots revealed no skewness within specific data ranges. In the few cases with ≥ 10% difference between manual counting and DIA, results by both platforms were similar.

Conclusions

DIA using VDS is an accurate method to determine the Ki67 proliferation index in breast cancer, as an alternative to manual scoring of whole sections in clinical practice. Inter-platform agreement between two different DIA platforms was excellent, suggesting vendor-independent clinical implementability.