目的：探讨荧光原位杂交技术（FISH）检测乳腺癌Her2/neu的扩增情况，比较FISH法与免疫组织化学（IHC）法检测结果的一致性。方法：收集乳腺癌患者手术切除的肿瘤标本50例。经石蜡包埋，组织切片后，应用FISH法检测Her2/neu扩增情况，IHC法检测Her2/neu蛋白表达情况，并对两种检测方法的结果进行统计学分析。结果：50例标本中FISH阳性17例，FISH阴性33例，FISH阳性率34%（17/50）。 50例标本中IHC（~）14例，IHC(0~+)36例，IHC蛋白表达率28%(14/50)。FISH法与IHC法检测结果的总符合率为82%(41/50)(K=0.581)。结论：FISH法具有较高的稳定性与可靠性，临床开展FISH法检测Her2/neu基因扩增情况作为IHC法的补充, 可以更加准确和客观地对乳腺癌患者的治疗和预后进行评价。
目的:β淀粉样蛋白（β-amyloid precursor protein，β-APP）是已知的参与阿尔茨海默病机制的关键因子。β-APP是否参与难治性癫痫中的病理机制并不清楚。这项研究在于了解β-APP的蛋白在难治性癫痫患者术后颞叶皮质和海马组织中的表达是否异常。方法:免疫荧光法半定量测定难治性癫痫患者术后颞叶皮质和海马组织中的β-APP阳性蛋白的荧光值，并应用统计软件对实验数据进行单因素方差分析。结果:免疫荧光强度值分析结果显示β-APP在耐药性癫痫脑组织中表达较对照组明显增高且有统计学意义。结论:β-APP在难治性癫痫脑组织中异常增高，增高的β-APP可能参与了难治性癫痫的病理机制。
目的：机械分离、培养小鼠耳蜗螺旋神经元，并进行免疫荧光细胞学鉴定，为后期进一步的实验研究提供实验材料。方法：采用初出生1～5天以内的昆明小鼠进行解剖、机械分离以获得螺旋神经节组织，进行原代培养后，应用神经微丝蛋白（Neurofilament protein,NFP－H）单克隆抗体进行免疫荧光细胞学鉴定。结果：机械分离后获得的螺旋神经节组织中的螺旋神经元，在体外培养条件下可以存活并进行正常分化。典型的螺旋神经元，其细胞形态呈椭圆形,胞体透明光滑、接近生理形态。荧光染色标记后，胞体和神经突起均显色好，Schwann细胞和成纤维细胞未着色。结论：应用机械分离的方法获得小鼠耳蜗螺旋神经节组织并进行培养，耳蜗螺旋神经元在体外可以稳定地存活生长。培养获得的细胞形态和生存状态接近生理状态，满足电生理、免疫细胞化学、药理学等研究。应用特异性的神经微丝蛋白对培养获得的螺旋神经元进行免疫荧光细胞学鉴定，特异性好，荧光显色好。
Objective To identify micrometastasis in regional lymph nodes of gastric cancer by quantitative real-time reverse transcription-PCR (qRT-PCR) assay and to evaluate the clinical significance of micrometastasis. Methods To study 320 lymph nodes collected from January 2010 to June 2010， 281 of which were from 40 patients with gastric cancer who had undergone a standard gastrectomy with lymphadenectomy， and other 39 of which were from 10 patients with gastroduodenal ulcer. Made CEA， CK-19， and CK-20 as primers， and used qRT-PCR assay in addition to hematoxylin and eosin staining to detect the micrometastasis， and to analyze the clinicopathologic characteristics.Results Totally， micrometastasis were detected by qRT-PCR assay in 31 (15.34%，31/202) lymph nodes of 28 (70.00%， 28/40) patients. Thirty-nine lymph nodes from 10 patients with gastroduodenal ulcer were negative by qRT-PCR and HE staining. The degree of differentiation， depth of gastric mural invasion， and clinical stage had statistically significant correlation with the incidence of lymph node micrometastasis (P＜0.05). Conclusions qRT-PCR assay is a sensitive and specific method to detect lymph node micrometastasis in gastric cancer patients，and it has importantly clinical significance in evaluating clinical staging，prognosis and treatment prescription.
Objective To investigate the gene expression of beta-defensin-4 (mBD-4) and mBD-6 in acute lung injury (ALI) mouse.Methods Sixty adult mice were randomly divided into a control group and a ALI group.ALI was induced by intraperitoneal injection of lipopolysaccharide (LPS) in the ALI group.The control group was treated with same dose of normal saline.The lung tissues were harvested at different time point after stimulation.The expression of mBD-4 and mBD-6 mRNA was measured by real-time quantitative reverse transcription polymerase chain reaction.DNA sequencing was used to confirm the specificity of mBD-4 and mBD-6 cDNA fragment.Results There were no obvious mBD-4 and mBD-6 mRNA expression in mouse lung in the control group at all time points and ALI 6 h group.In the ALI group a marked increasing expression was found on 12 h，1 d and 3 d after LPS stimulation.The mBD-4 mRNA expression was significant higher in the ALI groups of 1 d and 3 d points than that of ALI 12 h group with no obvious difference between each other.There were no significant differences of mBD-6 mRNA expression between ALI groups of 12 h，1 d and 3 d points Conclusion mBD-4 and mBD-6 mRNA is not constitutive expressed in mouse lung and show a up-regulative expression pattern after ALI.
Abstract: Objective To compare the sensitivity and accuracy of autofluorescence bronchoscope (AFB) and white light bronchoscope (WLB) in airway examination for patients with central type lung cancer. Methods From September 2009 to May 2010, 46 patients including 36 males and 10 females with an average age of 62.1 years underwent both AFB and WLB procedures in People’s Hospital of Peking University. Among them, 35 were preliminary diagnostic cases and 11 were postoperative surveillance cases. Local anaesthesia of glottis and airway, and general anaesthesia with continuous intravenous drugs were given before electric bronchoscope was adopted. All patients underwent WLB examination followed by AFB procedure. All suspicious abnormal visual findings were recorded for biopsy and pathological examination. Results All procedures were carried out safely without death or severe complications. We performed bronchoscopy 48 times for all 46 patients and 159 tissues of various sites were taken out for biopsy and pathologic examination which showed 64 malignancies and 95 none malignancies. In 64 malignancies, AFB found all but WLB missed 15 with a missed diagnosis rate of 23.4%. Thirtysix times of examination were performed for the 35 preliminary diagnostic cases and 56 sites of malignancy were found. AFB found all, while WLB missed 12, and 6 sites of malignancy found by AFB were larger in size than those found by WLB. AFB detected 3 cases of multisite malignancy, but WLB missed these diagnoses. The results of AFB and WLB were the same for 26 patients. Twelve times of bronchoscopy were performed for the 11 postoperative surveillance cases and 8 sites of malignancy were found. AFB found them all while WLB missed 3 which were two recurrent cases during the early period after lung cancer surgery. The sensitivity of AFB and WLB was 100.0 % and 76.6%(Plt;0.05) respectively, and the negative predictive value of AFB and WLB was 100.0% and 84.5%(P=0.002) respectively. Conclusion AFB has a better sensitivity and negative predictive value than WLB in detecting mucous canceration lesions in central type lung cancer, and is more accurate in assessment of tumor margins, more sensitive in finding multiple lesions in airway and detecting early cancer recurrence in postoperative surveillance patients.
Objective To detect the expression of forkhead box P3 （FOXP3 ）gene in esophageal squamous cell carcinoma(ESCC) and provide a new basis for immunotherapy of esophageal cancer. Methods Based on fluorescent TaqMan methodology, a realtime quantitative reverse transcription polymerase chain reaction (RT-PCR) for detecting the expression of FOXP3 was set up. In this method, a cloning vector pMD 18-T-FOXP3 was constructed as a standard plasmid. The specific expression of FOXP3 in 42 patients with ESCC and 30 healthy controls were measured by using GeneAmp 7500 Sequence Detection Systems. Results FOXP3 mRNA copy number in ESCC was significantly higher than that in healthy control tissue ［(72.20±23.10)×104copy/μg RNA vs.(0.68±0.34)×104 copy/μg RNA；Plt;0.05］. Conclusion A realtime quantitative RT-PCR method for detecting the expression of FOXP3 gene in ESCC has been successfully established. The expression level of FOXP3 is increased in ESCC compare with healthy controls.
Objective To insure early detection and hence efficient prevention of allograft rejection in transplanted heart, investigate possible applications of NAD(P)H fluorescence components analysis at the level of living cardiac cells to propose new approaches for diagnosis of rejection. Methods NAD(P)H was studied for noninvasive fluorescent probing of the mitochondrial function. Human cardiomyocyte were isolated from one additional endomyocardial biopsy (EMB) of 14 pediatric patients with heart ransplantation. Rat cardiomyocyte (n=5, 13-14 week old) were also isolated by the same approach for human myocytes. Autofluorescence(AF) was recorded in living cardiomyocytes following excitation with 375 nm UVlight and detection by spectrallyresolved time correlated single photon counting (TCSPC), based on the simultaneous measurement of the fluorescence spectra and lifetimes. Rat cardiac cells were divided into four groups: normoxic condition, normoxia with Rotenone, ischemic condition and ischemia with Rotenone. Comparison of cardiomyocyte AF between human and rat; compared kinetics of rat cardiomyocytes AF in normoxic conditions to ischemiamimicking ones, induced at physiological temperatures by reducing cell pH and oxygen content; comparison of cardiomyocyte AF dynamic changes in transplanted pediatric patients presenting either no rejection (R0) or mild rejection (R1). Results We have achieved appropriate isolation of living cardiomyocytes from human biopsies, as well as from rat cardiac tissues and determined their AF. At least a 3-exponential decay with 0.5-0.7ns, 1.9-2.4 ns and 9.0-15.0 ns lifetime pools is necessary to describe human cardiomyocyte AF within 420560 nm spectral range. Rat cardiomyocyte steadystate AF in ischemiamimicking condition was significantly increased when compared normoxic ones (Plt;0.05); application of Rotenone induced a significant increase in AF intensity in ischemic and normoxic condition, however no significant difference between the two groups (Plt;0.05).Human cardiomyocyte AF was found significantly lower in comparison to experimental rat model in the same condition(Plt;0.05). A correlation between changes in steadystate NAD(P)H fluorescence and rejection grades was found when comparison of R1 to R0. R1 showed significantly increased fluorescence intensity (Plt;0.05), without change in the spectra shape, results can be comparable to the effect of ischemiamimic conditions. Conclusion Our studies clearly demonstrated that spectrallyresolved fluorescence spectral analysis coupled to fluorescence lifetime are high sensitive approaches to examine mitochondrial metabolic oxidative state directly in living human cardiomyocytes with good reproducibility. Human cardiomyocytes are more metabolically active than the rat ones, while this activity (and thus ATP production) seems lowered during rejection process. In perspective, the advantage of this method is the possibility of its combination to multiphoton confocal microscopy, which can result in the adaptation of this approach directly to tissue biopsy, as well as in vivo directly via cardiac catheterization without the necessity of cell isolation. This approach provides promising new tool for clinical diagnosis and treatment of allograft rejection, and will enhance our knowledge about cardiomyocyte oxidative metabolism and/or its dysfunction at a cellular level.
Objective To observe preserving effect on myocytes in porcine aortic valve replacement with minimal extracorporeal circulation (MECC). Methods 7 pigs were collected as experimental animals and undertook aortic valve replacement with MECC. Morphological and immunofluorescence intensity changes of right atrial and left ventricular tissues were observed. Results HE staining showed that there were not significant changes and edema or injury of myocytes of right atriums and left ventricles between preoperation and postoperation. Immunofluorescence staining showed complement C3b/c in right atrial myocardial tissues after the operation were a little ber, and innate antibody IgG were a little ber in left ventricular myocardial tissues but similarly weak in right atrial myocardial tissues pre- and post-operation. There was not significant changes in HSPG staining in pre-and post-operative right atrial myocardial tissues, but HSPG were obviously weaker in left ventricular myocardial tissues after the operation. Conclusion MECC is effective on support of porcine aorta valve replacement.