免疫组织化学实验技术教学实践与体会

免疫组织化学实验技术课程在医学研究生教学中具有重要地位,如何在限定的学时内让选修该课程的学生掌握其基本实验操作技能,对培养和提高研究生基本科研素质具有重要意义。通过精简理论教学内容,加强实验操作教学,运用现代教育技术优化教学过程,重视实验带教前的准备工作,督促每位学生自己动手进行实验操作,改革课程考核评定体系,取得了满意的教学效果。

过氧化物还原酶4在口腔鳞癌中的表达及临床意义

目的研究过氧化物还原酶4(peroxiredoxin 4,PRDX4)在口腔鳞癌组织中的表达及临床意义。方法通过苏木素-伊红染色方法明确口腔鳞癌患者手术中所取组织的病理类型,荧光定量PCR实验检测30例口腔鳞癌患者正常组织和肿瘤组织中PRDX4的表达,通过免疫组织化学实验检测101例正常和口腔鳞癌组织中PRDX4的表达,并分析其与患者临床病理特征的关系。结果荧光定量PCR实验结果显示,PRDX4在正常组织中的2^(-△CT)值为2.064±0.2023,在口腔鳞癌组织中的2^(-△CT)值为4.170±0.2866(P<0.01)。免疫组织化学实验结果显示,PRDX4表达强度与口腔鳞癌患者生存率(P=0.0132)、淋巴结转移(P=0.0033)和组织病理分级(P=0.0353)显著相关。结论PRDX4在口腔鳞癌组织中高表达,且与患者不良预后存在密切关系。

Diagnosis of intestinal tuberculosis using a monoclonal antibody to Mycobacterium tuberculosis

AIM:To investigate the utility of immunohistochemical(IHC) staining with an antibody to Mycobacterium tuberculosis(M.tuberculosis) for the diagnosis of intestinal tuberculosis(TB).METHODS:We retrospectively identified 10 patients(4 males and 6 females;mean age = 65.1 ± 13.6 years) with intestinal TB.Clinical characteristics,including age,gender,underlying disease,and symptoms were obtained.Chest radiograph and laboratory tests,including sputum Ziehl-Neelsen(ZN) staining,M.tuberculosis culture,and sputum polymerase chain reaction(PCR) for tubercle bacilli DNA,as well as Tuberculin skin test(TST) and QuantiFERON-TB gold test(QFT),were examined.Colonoscopic records recorded on the basis of Sato's classification were also reviewed,in addition to data from intestinal biopsies examined for histopathological findings,including hematoxylin and eosin staining,and ZN staining,as well as M.tuberculosis culture,and PCR for tubercle bacilli DNA.For the present study,archived formalin-fixed paraffin-embedded(FFPE) intestinal tissue samples were immunohistochemically stained using a commercially available species-specific monoclonal antibody to the 38-kDa antigen of the M.tuberculosis complex.These sections were also stained with the pan-macrophage marker CD68 antibody.RESULTS:From the clinical data,we found that no patients were immunocompromised,and that the main symptoms were diarrhea and weight loss.Three patients displayed active pulmonary TB,six patients(60%) had a positive TST,and 4 patients(40%) had a positive QFT.Colonoscopic findings revealed that all patients had type 1 findings(linear ulcers in a circumferential arrangement or linear ulcers arranged circumferentially with mucosa showing multiple nodules),all of which were located in the right hemicolon and/or terminal ileum.Seven patients(70%) had concomitant healed lesions in the ileocecal area.No acid-fast bacilli were detected with ZN staining of the intestinal tissue samples,and both M.tuberculosis culture and PCR for tubercle bacilli DNA were negative in all samples.The histopathological data revealed that tuberculous granulomas were present in 4 cases(40%).IHC staining in archived FFPE samples with anti-M.tuberculosis monoclonal antibody revealed positive findings in 4 patients(40%);the same patients in which granulomas were detected by hematoxylin and eosin staining.M.tuberculosis antigens were found to be mostly intracellular,granular in pattern,and primarily located in the CD68 + macrophages of the granulomas.CONCLUSION:IHC staining with a monoclonal antibody to M.tuberculosis may be an efficient and simple diagnostic tool in addition to classic examination methods for the diagnosis of intestinal TB.

Effects of different fixatives on the TrkB-immunoreactivity in rat brain

Objective: To find out an effective fixative in immunohistochemistry for high-affinity neurotrophin receptor-tyrosine kinase (Trk) B. Methods: Comparing the results from four groups of adult rats which were fixed by different fixatives before the brain sections were processed for TrkB immunohistochemistry. Results: In the four groups, TrkB immunoreactive cells were observed throughout the whole brain, but the intensity of immunoreactive cells and the background staining exhibited a marked difference among the groups. Conclusion: Using 0.3%-0.5% paraformaldehyde in 75% saturated picric acid 0.1 mol/L di-sodium hydrogen phosphate buffer as the fixative may yield the best quality of TrkB immunoreactivity.

Changes of the gastric endocrine cells in the C57BL/6 mouse after implantation of murine lung carcinoma: An immunohistochemical quantitative study

AIM: The regional distributions and relative frequencies of some gastric endocrine cells of C57BL/6 mice were studied by immunohistochemical method using seven types of specific antisera against chromogranin A (CGA), serotonin, somatostatin, gastrin, cholecystokinin (CCK)-8, glucagon and human pancreatic polypeptide (HPP) after subcutaneous implantation of murine lung carcinoma (3LL) cells. METHODS: The experimental animals were divided into two groups, one is non-implanted sham and the other is 3LL-implanted group. Samples were collected from the two regions of stomach (fundus and pylorus) at 28 d after implantation of 3LL cells (1x105 cell/mouse). RESULTS: In this study, all the seven types of immunoreactive (IR) cells were identified except for HPP. Most of these IR cells in the gastric portion were generally spherical or spindle in shape (open-type cell) while cells showing round in shape (closed-type cell) were found occasionally. The regional distributions of gastric endocrine cells in the 3LL-implanted group were similar to those of non-implanted sham. However, significant decreases of some types of IR cells were detected in 3LL-implanted group compared to those of non-implanted sham. In addition, the IR cells showing degranulation were numerously detected in 3LL-implanted group. CGA-, serotonin- and somatostatin-IR cells in the fundus and pylorus regions, and gastrin-IR cells in the pylorus regions of 3LL-implanted groups significantly decreased compared to those of non-implanted sham. However, no changes on frequencies of CCK-8- and glucagon-IR cells were demonstrated between 3LL-implanted and non-implanted groups. CONCLUSION: Endocrine cells are the anatomical units responsible for the production of gut hormones, and the change in their density would reflect a change in the capacity of producing these hormones. Implantation of tumor cell mass (3LL) induced severe quantitative changes of gastric endocrine cell density, and the abnormality in density of gastric endocrine cells may contribute to the development of gastrointestinal symptoms such as anorexia and indigestion, frequently encountered in patients with cancer.

Changes of the intestinal endocrine cells in the C57BL/6 mouse after implantation of murine lung carcinoma (3LL): An immunohistochemical quantitative study

AIM: To study the distributions and frequencies of intestinal endocrine cells in the C57BL/6 mouse with immunohistochemical method using seven types of specific antisera against chromogranin A (CGA), serotonin,somatostatin, glucagons, gastrin, cholecystokinin (CCK)-8 and human pancreatic polypeptide (hPP) after abdominal subcutaneous implantation of murine lung carcinoma (3LL).METHODS: The experimental animals were divided into two groups, one is non-implanted Sham and the other is 3LL-implanted group. Samples were collected from six regions of intestinal tract at 28th d after implantation of 3LL cells (1×105 cell/mouse).RESULTS: In this study, five types of immunoreactive (IR) cells were identified except for gastrin and hPP. The regional distributions of the intestinal endocrine cells in the 3LL-implanted group were similar to those of the non-implanted Sham. However, significant decreases of IR cells were detected in 3LL-implanted group compared to those of non-implanted Sham. CGA- and serotonin-IR cells significantly decreased in 3LL-implanted groups compared to that of non-implanted Sham. Somatostatin-IR cells in the jejunum and ileum and CCK-8-IR cells in the jejunum of 3LL-implanted groups significantly decreased compared to that of non-implanted Sham. In addition,glucagon-IR cells were restricted to the ileum and colon of non-implanted Sham.CONCLUSION: Implantation of tumor cell mass (3LL)induced severe quantifiable changes of intestinal endocrine cell density and the abnormality in density of intestinal endocrine cells may contribute to the development of gastrointestinal symptoms such as anorexia and indigestion, frequently encountered in patients with cancer.

趋化因子CXCL5在肝癌中的表达及其临床意义

目的探讨趋化因子配体5(CXCL5)在原发性肝细胞肝癌(HCC)中的表达,分析其在肝癌发生、发展、侵袭及转移中的作用。方法收集218例HCC组织标本及其临床病理资料,采用免疫组化(IHC)Elvision检测肝癌组织、癌旁组织、周围正常组织中CXCL5的表达,染色后阅片、评分、对比其在各组之间的表达差异;采用相关分析、非参数检验、Log-Rank、Cox回归分析和Logistic回归分析进行统计学分析,P<0.05认为有统计学意义。结果 IHC结果显示CXCL5在癌组织中的阳性率高于癌旁组织(P<0.01)和周围正常组织(P<0.01);癌组织CXCL5中高表达与门静脉癌栓形成、BCLC分期、TNM分期、肝癌并静脉侵犯正相关,与病理分级负相关;CXCL5是肝癌患者的独立预后因素之一(HR1.734;P=0.001)。结论 CXCL5在HCC中表达增高,与肝癌的病理分级、恶性程度、门静脉癌栓、临床分期呈正相关,其高表达是HCC的独立预后危险因子之一。