骨髓细胞形态学联合原位免疫组化在原发骨髓淋巴瘤患者中的诊断价值

目的:探讨骨髓细胞形态学联合原位免疫组化在原发骨髓淋巴瘤患者中的诊断价值。方法:收集西安交通大学医学院第一附属医院2010年1月-2019年12月23例确诊的原发骨髓淋巴瘤患者的临床资料,回顾性分析骨髓细胞形态学、原位免疫组化和免疫分型等的特点,明确骨髓细胞形态学联合原位免疫组化在原发骨髓淋巴瘤中的诊断价值。结果:原发骨髓淋巴瘤以B细胞淋巴瘤为主,其中弥漫大B细胞淋巴瘤为最常见的病理类型;所有患者骨髓均可见典型的淋巴瘤细胞,联合原位免疫组化78.26%的患者能够被诊断为原发骨髓淋巴瘤,91.30%的患者确诊为淋巴瘤。结论:骨髓细胞形态学联合原位免疫组化在原发骨髓淋巴瘤患者中具有重要诊断价值。

生长抑素mRNA与神经肽Y，催产素在大鼠前脑内的分布及其共存现象──地高辛标记c－RNA探针原位杂交与免疫组化联合法

本实验应用地高辛标记ｃＲＮＡ探针原位杂交组化和免疫组化联合法在同一切片上先后显示了生长抑素ｍＲＮＡ神经元和催产素神经元，生长抑素ｍＲＮＡ神经元和神经肽Ｙ神经元。结果表明生长抑素ｍＲＮＡ及神经肽Ｙ广泛地共存于大鼠的大脑新皮质，尾壳核，以及海马等处的神经元中。所有位于大脑新皮质，尾壳核处的神经肽Ｙ神经元均含有生长抑素ｍＲＮＡ，部分位于海马的神经肽Ｙ神经元含有生长抑素ｍＲＮＡ，而所有位于下丘脑弓状核，室周核的神经肽Ｙ神经元均不含有生长抑素ｍＲＮＡ；生长抑素ｍＲＮＡ与催产素虽然共同分布于下丘脑许多核区，但未见共存于同一神经元。本文对地高辛标记ｃＲＮＡ探针原位杂交组化以及它与免疫组化联合法的技术问题进行了讨论。

全自动免疫组化和原位杂交染色机染色失败解决方案

该文分析总结了Leica Bond Max全自动免疫组化和原位杂交染色机染色失败的原因,以及对常见人员操作失误与机器自身故障进行了总结,并提供了对应的解决方案。

甲状腺自身免疫病原位浸润细胞与DR抗原异常表达及意义系列研究(简介)

甲状腺自身免疫病,因其发病机理未完全明了,临床上尚无有效治疗方法。本项目首家应用甲状腺粗针穿刺技术及免疫组化、免疫电镜方法进行了一系列原位研究,取得了重要成果。主要有以下四部分内容:

应用组织芯片研究CD44v9 mRNA在乳腺癌中的表达及与Ki-67相关性

目的:探讨CD44v9 mRNA和Ki-67在乳腺癌中表达的关系。方法:应用组织芯片分别结合原位杂交和免疫组化方法检测乳腺癌中CD44v9 mRNA和Ki-67的表达。结果:乳腺癌组织CD44v9 mRNA和Ki-67表达水平明显高于正常乳腺组织(P<0.05)。乳腺癌组织中CD44v9 mRNA的表达与淋巴结转移、远处转移具有显著相关性,Ki-67的表达与组织病理分级、淋巴结转移均有显著相关性。它们的表达与肿瘤大小、年龄等无关(P>0.05)。两者表达成正相关(r=0.367,P<0.01)。结论:CD44v9 mRNA和Ki-67与乳腺癌的恶性生物学行为密切相关,可作为预测肿瘤转移潜能的指标。组织芯片技术是病理学研究的一种新方法,具有高效、简便及标准化的特点。

Survivin mRNA及其蛋白和bcl-2在子宫内膜癌中的表达及意义

目的:探讨凋亡抑制基因SurvivinmRNA及其蛋白和bcl-2在子宫内膜癌中的表达及与临床病理学特征的关系。方法:采用原位杂交和免疫组化法检测53例子宫内膜癌,33例非典型增生子宫内膜和30例正常子宫内膜中Survivin基因的表达,同时采用免疫组化法检测子宫内膜癌中bcl-2蛋白的表达。结果:SurvivinmRNA及其蛋白从正常子宫内膜、非典型增生到子宫内膜癌,阳性表达率逐渐增加(P<0.01)。53例子宫内膜癌中SurvivinmRNA与其蛋白阳性表达一致,两者与组织分化呈正相关(P<0.05),而与临床分期无关。bcl-2与子宫内膜癌组织分化呈负相关,而与临床分期无关。Survivin与bcl-2无关。结论:Survivin基因在子宫内膜癌中表达上调,提示其在子宫内膜癌的发生发展中起作用,它的检测对子宫内膜癌早期诊断提供依据。Survivin与bcl-2基因通过不同的机制参与子宫内膜癌的发生。

Clinicopathologic significance of HER-2/neu protein expression and gene amplification in gastric carcinoma

AIM:To study the HER-2/neu protein expression and gene amplification in gastric carcinoma and their relation.METHODS:One hundred and forty-five formalin-fixed and paraffin-embedded tumor tissue samples from Chinese gastric carcinoma patients were studied with immunohistochemistry(IHC)and fluorescence in situ hybridization(FISH)methods.Clinicopathologic data about all patients were collected.RESULTS:The levels of HER-2 3+,HER-2 2+and HER21+were measurable in 6.9%,8.3%and 17.2%of the samples,respectively.No HER-2 was stained in 67.6% of the samples.FISH showed that HER-2 gene was amplified in 18 samples,10 HER-2 3+samples,5 HER-2 2+samples,and 3 HER-2 1+samples with IHC staining.HER-2 status was not correlated with the sex and age of patients,and tumor size,location or differentiation,but with the depth of invasion,TNM stage,lymph node and distant metastasis as well as histopathological classification of gastric cancer(P<0.05).CONCLUSION:All samples with IHC as HER-2 expression should be analyzed with FISH.Detection of HER-2 gene amplification can assess the malignant biological behaviors and prognosis of gastric cancer.

Detection of Mycoplasma Hyorhinis Infection in Ovarian Cancer with in situ Hybridization and Immunohistochemistry

OBJECTIVE To detect Mycoplasma hyorhinis in ovarian cancer tissues and the relationship between mycoplasma infection and ovarian cancer. METHODS All specimens obtained from 109 cases with ovarian cancer were fixed in freshly prepared 10% neutral buffered formalin, embedded in paraffin, and cut into 4-μm sections for insitu hybridization （ISH） and then detected with immunohistochemistry （IHC）. The expressions of 16S rRNA and P37 protein from mycoplasma hyorhinis were detected respectively using ISH and IHC. SPSS 13.0 software was employed to analyze the relationship between the results of the study and clinical pathological materials. RESULTS The expression rate of mycoplasma hyorhinis 16S rRNA gene and P37 protein was 20.2% （22/109） and 43.1% （47/109 cases） in ovarian cancer tissues, respectively, but it was 0 （0/30 cases） in the normal ovarian tissues. The difference in mycoplasma infection ratio between ovarian cancer tissues and normal tissues was extremely significant （P 〈 0.001）. Anyhow, we didn＇t found any association between the mycoplasma infection and clinical pathological characters. CONCLUSION There was a mycoplasma infection in ovarian cancer tissues, which may play a role in oncogenesis of ovarian cancer.

Characterization of gastric cancer models from different cell lines orthotopically constructed using improved implantation techniques

AIM:To develop orthotopic gastric cancer mouse models from different cell lines and characterize the tumor features to assist further in preclinical trials and clinical treatment strategies.METHODS:Human gastric cancer SGC-7901 and BGC823 cell suspensions were injected subcutaneously into nude mice to develop solid tumors,and tumor tissue pieces were then implanted under the serous coat of the stomach.An autopsy was performed on all animals of the SGC-7901 and BGC-823 models to observe the primary tumor growth and metastases using pathological and immunohistochemical methods.RESULTS:Both models showed large tumors in situ resulting in pressure and infiltration of the adjacent organs.The gastric cavity became smaller,along with stenosis of the cardia or pylorus.There were biological and statistical differences between the two models.The metastasis rate in involved organs (lymph nodes,kidney,spleen,testis) was significantly higher in the BGC-823 model compared to the SGC-7901 model (P < 0.05 or P < 0.01).The median survival of the BGC-823 model was shorter than that of SGC-7901 (23 d vs 84 d,P < 0.05).Histopathologically,the primary tumor and metastatic lesions of the two models showed obvious atypia and mucus in the cytoplasm.Compared with the SGC-7901 model,BGC-823 appeared more poorly differentiated (absence of adenoid structure),had a smaller volume,and richer capillary structure.Immunohistochemical staining revealed cytokeratin 20 and epithelial membrane antigen expression was positive in the SGC-7901 tumors,while negative in BGC-823 ones.CONCLUSION:Models using the SGC-7901 and BGC-823 cell lines were established which could function in gastric cancer research on carcinogenesis mechanism and drug discovery.The two models showed different tumor behavior and the latter was more malignant than the former.

Clinicopathologic features of 112 cases with mantle cell lymphoma

Objective： This study aims to explore the clinicopathologic features of 112 patients with mantle cell lymphoma （MCL）. Methods： Data from 112 MCL cases were collected, and immunohistochemical assay was conducted. Fluorescence in situ hybridization （FISH） detected a break in the CCND 1 gene. The t-test was used in the statistical analysis. Results： All tumor cells in the 112 cases expressed B cell-related antigen, including 1 blastoid subtype and 1 polymorphic subtype. Among all cases, 106 expressed CD5 and 104 expressed cyclin D1. A break in the CCND1 gene was not found in 3 cases with CDS-MCL. IgH/CCND 1 polyploid was observed in 2 classic cases. Conclusion： MCL is a type of special immunophenotypic B-cell lymphoma, The prognoses ofblastoid and polymorphic subtypes are poor. Special subtypes should be classified during diagnosis.

Expression of Bmi-1 and EZH2 in tissues adjacent to human epithelial ovarian cancer cells of orthotopic implantation in nude mice

Objective This study investigated the feasibility of screening residual normal ovarian tissues based on the expression of Bmi-1 and EZH2 in tissues adjacent to orthotopic ovarian carcinomas in nude mice. Methods The human epithelial ovarian cancer cell line OVCAR3 was grown in subcutaneous tissues and the tumor tissues were orthotopically implanted. The expression levels of Bmi-1 and EZH2 were detected by immunohistochemical staining and RT-PCR in cancer tissues, proximal and remote tissues with respect to the cancer tissues, and normal ovarian tissues of nude mice.Results Thirty-five ovarian tissue samples with normal biopsy results were obtained from 40 cases of human epithelial ovarian cancer in the nude mice in which the tumor tissues were orthotopically implanted. Bmi-1 and EZH2 expression levels were lower in proximal paraneoplastic tissue samples than in cancer tissue samples(P < 0.05) and higher than in remote paraneoplastic tissue samples(P < 0.01). No significant difference was found in the expression levels of Bmi-1 and EZH2 using immunohistochemistry among residual normal ovarian tissues obtained from orthotopically implanted models that differed in severity. The expression of Bmi-1 and EZH2 was negative in 20 normal ovarian tissue samples.Conclusion The expression levels of Bmi-1 and EZH2 were reduced with increasing distance from the cancer tissues. Negative expression of these tumor-associated genes can be used as a standard for the screening of normal ovarian tissues adjacent to tumor tissues. Normal ovarian tissues can be obtained from the tissues adjacent to tumors.

Study on the protein expression and amplification of HER2 gene in gastric cancer

Objective: The aim of the study was to investigate the human epidermal growth factor receptor 2(HER2) gene amplification and protein expression and interpretation points in the stomach mixed carcinomas. Methods: Immunohistochemistry(IHC) and fluorescence in situ hybridization(FISH) technique were used to detect HER2 gene amplification and expression of HER2 protein in 442 cases of gastric mixed carcinoma. Results: The expression rate of HER2 protein was 41.2%(182/442): the HER2 protein expression IHC 3+ extensive type in 18 cases, partial type in 21 cases, focal type in 8 cases, accounting for 10.6%(47/442); the HER2 protein expression IHC 2+ extensive type in 23 cases, partial type in 28 cases, focal type in 11 cases, accounting for 14.0%(62/442); the HER2 protein expression IHC 1+ extensive type in 27 cases, partial type in 31 cases, focal type in 15 cases, accounting for 16.5%(73/442). HER2 gene amplification rate of 442 cases was 16.1%(71/442). In 182 cases of HER2 protein positive expression, the HER2 gene cluster amplification rate was 14.8%(27/182), large granular amplification rate 11.0%(20/182), punctate amplification rate 6.0%(11/182) and high polysomy 7.1%(13/182). In 71 cases of HER2 gene amplification, there was 42 cases of HER2 protein expression IHC 3+, 22 cases of HER2 protein expression IHC 2+, and 7 cases of IHC 1+. Conclusion: HER2 detection of gastric mixed carcinoma has great heterogeneity, HER2 protein positive expression is divided into extensive type, partial type and focal type, and HER2 gene positive amplification is divided into cluster amplification, large granular amplification, punctate amplification and high polysomy. These typing of HER2 protein expression and HER2 gene amplification provide reference index to quantify for targeted therapeutic effect of anticancer drugs.

Expression of ER protein from DCIS to IDC in ductal breast cancer

Objective: We studied the alterations in the expression of estrogen receptor alpha (ER) in the progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinomas (IDC). Methods: The mastectomy specimens of 120 cases containing both DCIS and IDC were examined. The expression of ER proteins were examined by immunohistochemistry. The difference of the expression of ER proteins between DCIS and IDC were compared. Results: There were 58.33% of the cases with DCIS expressing ER proteins, and 40.00% of the cases IDC expressing ER proteins. There was a significant decrease of ER expression in IDC compared to DCIS (χ2 = 4.034, P = 0.045). Conclusion: These findings substantiate the notion that breast cancer progression is often associated with alterations in expressions of ER. The underlying mechanisms of these alterations need further investigation.

脑组织切片技术中防脱片制作及注意事项

目的:老鼠大脑组织所含成分和结构特殊,其组织与玻片粘附性差,在免疫组化染色和原位杂交实验中脱片率很高,故应用一种新型粘片剂APES(氨醛三己氧基硅烷)制作防脱片。方法:载玻片经酸浸泡、清洗、烤干、浸入1∶50丙酮稀释的APES中,浸泡20 s,再放入100%丙酮反复3次浸泡清洗,制作防脱片。结果:APES制作防脱片应用在免疫组化和原位杂交实验中防脱片效果稳定、可靠。结论:APES制作防脱片效果特好,方法简单,脑组织及其他组织实验都可应用,可在临床和科研实验室中推广使用。

Increased expression of neuropeptide Y and its mRNA in STZ-diabetic rats

OBJECTIVE: To study the relationship between neuropeptide Y (NPY) and diabetes by examining the content and distribution of NPY and its mRNA expression in the hypothalamus and pancreas of STZ-diabetic rats. METHODS: Thirty Wistar rats were randomly divided into 3 groups (diabetic group, diabetic insulin treatment group, and control group). After feeding for 24 weeks, the rats were sacrificed. The expression of NPY in the hypothalamus and pancreas was detected with immunohistochemistry and in situ hybridization. RESULTS: (1) The hypothalamic content of NPY and its mRNA were significantly increased in STZ-diabetic rats in comparison with normal controls. Increased expression of NPY mRNA was found only in the arcuate nucleus and not in the paraventricular nucleus in diabetic rats, suggesting that NPY was produced in the arcuate nucleus. (2) The hypothalamic content of NPY and its mRNA in STZ-diabetic rats were visibly reduced after insulin treatment compared with that in untreated diabetic rats. This supports the hypothesis that insulin deficiency in the brain may be responsible for increased hypothalamic NPY gene expression in diabetic rats. (3) The increase of hypothalamic NPY in STZ diabetic rats associated with hyperphagia and polydipsia could be reversed by insulin replacement, suggesting that increased hypothalamic NPY contributes to the pathophysiological progress of the diabetic state. (4) The present study demonstrated for the first time that the content of NPY and its mRNA in the pancreas was increased in STZ-diabetic rats, and that the distribution of NPY-positive cell in islets was changed from the periphery to the whole islet. The content and distribution of NPY and its mRNA in islets were not changed by insulin treatment. CONCLUSION: Increased NPY in the hypothalamus results in hypophagia and polydipsia, while the implication of increased NPY in the pancreas of diabetic rats is not clear.