喉鳞癌中HPV的免疫组化,原位杂交,PCR和间接原位PCR检测

目的 探讨ＨＰＶ与喉癌发生的关系。方法 采用免疫组化,原位杂交,ＰＣＲ和间接原位ＰＣＲ技术检测５０例喉鳞状细胞癌中的ＨＰＶ感染情况。结果 免疫组化衣壳抗原阳性６２例（１２％）,原位杂交阳性１３例（２６％）,ＰＣＲ阳性１０例（２０％）,原位ＰＣＲ阳性１７例（３４％）,综合上述方法阳性率为４２％（阳性２１例）。结论 ＨＰＶ感染与喉癌有着明显的关系,间接原位ＰＣＲ在检测ＨＰＶ感染时具有较高的敏感性,特异     

原发性中枢神经系统淋巴瘤的临床病理及与EB病毒的关系

目的　探讨原发性中枢神经系统淋巴瘤 (primarycentralnervoussystemlymphoma ,PCNSL)的临床病理特点及与EB病毒 (EBV)的关系。方法　对 6例PCNSL进行临床病理观察 ,采用ABC法行LCA、CD2 0、CD79a、CD4 5RO、CD3免疫组化标记 ,并以生物素标记的EBER 1寡核苷酸为探针进行原位杂交检测。结果　 6例均无HIV感染病史 ,其中 5例为B细胞来源淋巴瘤 ,1例为T细胞来源淋巴瘤 ,EB病毒原位杂交检测均为 (- )。结论　PCNSL多数为B细胞性淋巴瘤 ,少数为T细胞性淋巴瘤 ,非HIV感染的PCNSL的发生可能与EB病毒感染关系不密切。     

Comparative study with in situ hybridization and immunocytochemistry in detection of HIV-1 in formalin-fixed paraffin-embedded cell cultures

The sensitivities of three immunohistological techniques were compared in this study for detecting human immunodeficiency virus (HIV-1) in infected cultured human lymphocytes that had been formalin-fixed and paraffin-embedded. The techniques included in situ hybridization (ISH) with HIV-1 cDNA; immunocytochemistry with HIV-1 p24 monoclonal antibody (ICC-m); and immunocytochemistry with HIV-1 polyclonal antibody from a patient with acquired immunodeficiency syndrome (AIDS) (ICC-p). Procedures were optimized for enzyme digestion and for antibody reaction conditions. HIV-1--infected cells and noninfected control cells were tested. Noninfected controls were uniformally negative by all three methods. Infected cells had the highest positivity rate by the ISH method (p less than or equal to 0.0001), and the ICC-p method was more positive than the ICC-m (p less than or equal to 0.0001). Both the ICC-p and the ICC-m techniques were more positive with the cocultivated cell cultures than the ISH, which was more sensitive with the infected continuous cell line (P less than or equal to 0.0001). The ICC-p method had a lower standard deviation on positive results than either the ICC-m or ISH method. The variability observed with these test procedures, reagents, and specimens suggests that these are important technological parameters in detecting p24, with implications for detecting other HIV-1 markers in infected tissues.     

应用荧光原位杂交技术筛选bcr基因探针及分析微小残留病

目的　定量检测慢性髓细胞白血病（ Ｃ Ｍ Ｌ） 造血干细胞移植（ Ｈ Ｓ Ｃ Ｔ） 后ｂｃｒ 基因重排。方法　应用荧光原位杂交（ Ｆ Ｉ Ｓ Ｈ） 技术,从 Ｃ Ｅ Ｐ Ｈ 酵母人工染色体（ Ｙ Ａ Ｃ） 文库的３ 个ｂｃｒ 相关候选克隆中筛选探针,并对７ 例造血干细胞移植后病人进行检测,同时进行细胞遗传学和逆转录聚合酶链反应（ Ｒ Ｔ Ｐ Ｃ Ｒ） 分析。结果　确定了定位于２２ｑ１１ ｂｃｒ 基因区域且可在 Ｐｈ 染色体阳性细胞中易位至９ｑ３４的 Ｙ Ａ Ｃ 克隆７６５ Ｅ３ ,应用该 Ｙ Ａ Ｃ Ｄ Ｎ Ａ 探针进行荧光原位杂交,发现７ 例病人中５ 例存在ｂｃｒ 易位阳性细胞（６ ％ ～９８ ％ ） ,常规 Ｇ 显带检出３ 例, Ｐ Ｃ Ｒ 检出６ 例。结论　 Ｆ Ｉ Ｓ Ｈ 可能成为白血病造血干细胞移植后疗效观察和微小残留病定量监测的重要手段。     

连续R显带和荧光原位杂交技术在白血病诊断中的应用

目的探讨连续R显带和荧光原位杂交技术(FISH)在鉴定自血病染色体复杂变异易位中的应用。方法对15例核型分析有疑问的标本在R显带基础上再进行荧光原位杂交。对比分析同一中期分裂相的显带和FISH结果。结果4例应用BCR／ABL探针；4例PML／RARa探针；4例AML1／ETO探针；2例IgH探针；1例MLL探针。经显带结果与FISH结果对比分析，9例疑似的复杂易位得到确证，并精确定位。6例核型结果得到修正。结论连续R显带和FISH方法在复杂变异易位的鉴定方面与传统的核型分析和直接FISH检测相比具有明显的优势。在自血病的诊断方面．对于未知异常染色体的检出将具有更广泛的应用前景。     

组织芯片制备与原位分析技术

目的 建立组织芯片制备技术并观察在各种原位分析中的应用。方法 1．在传统病理学技术的基础上制备各种组织蜡块作为组织供体，再用组织阵列仪按照预先设计制作各种组织阵列蜡块，经切片和烤片后即成为各种组织芯片。2．用各种组织芯片按照常规方法进行HE染色、免疫组化(IHC)、原位杂交(ISH)和原位PCR(IS-PCR)实验分析。结果 (1)用本方法可分别制备低、中和高密度组织芯片，而且组织芯片排列整齐，外形美观，组织点大小一致且不易脱落。(2)HE染色结果均一，核浆对比明显，便于进行诊断和比较诊断；IHC和免疫荧光染色清晰，便于观察和诊断；ISH和IS-PCR实验效果较好，可用于各种基因扩增分析。结论 (1)本方法是一种比较成熟的组织芯片制备技术。(2)用本方法制备的组织芯片可以进行HE染色和各种原位分析。     

原位杂交与免疫组化双染法在淋巴瘤诊断中的应用

目的在淋巴瘤病例中建立EB病毒编码小RNA(EBER)原位杂交与免疫组化双重染色法。方法使用双染试剂盒在石蜡切片上先做原位杂交再做免疫组化。结果双染成功后细胞和组织结构完整。原位杂交阳性细胞的胞核呈蓝黑色,免疫组化阳性细胞的胞膜呈红色,双阳性细胞的核与膜蓝红对比鲜明,背景清晰。结论此方法可直观地判断感染EB病毒的细胞属于B细胞还是T细胞,有助于淋巴瘤的诊断。     

糖尿病患者颈动脉、眼动脉及视网膜中央动脉的改变

目的应用高频超声检测糖尿病患者颈动脉、眼动脉及视网膜中央动脉病变,为临床提供有参考价值的数据.方法检测32例糖尿病患者(合并或者无高血压)和14例正常对照组颈动脉内中膜复合体厚度(IMT),弹性指数(Ep),僵硬指数(β),眼动脉(OA)、视网膜中央动脉(CRA)的收缩期峰值血流速度(Vs)、舒张末期流速(Vd)、血管阻力指数(RI).结果与对照组比较,糖尿病无高血压及合并高血压患者颈动脉IMT增厚,Ep、β增高(P<0.01),眼动脉、视网膜中央动脉的Vd下降(P<0.01),RI增高(P<0.01).结论糖尿病患者颈动脉内中膜复合体增厚,弹性度降低,视网膜血液供应不良.高频超声在糖尿病血管病变的诊断中具有重要的实用价值.     

荧光原位杂交技术检测骨髓增生异常综合征患者5、7、8号染色体异常及临床意义

目的用荧光原位杂交（FISH）技术分析骨髓增生异常综合征（MDS）患者的染色体改变及预后。方法用常规细胞遗传学分析和FISH法分析37例MDS患者8、5、7号染色体的异常变化。用SPSS11．5统计软件，对患者的遗传学异常与疾病转归、预后之间关系进行相关性检验。结果检出染色体异常21例（56．8％），其中复杂异常6例（16．2％），8号染色体异常9例（24．3％），-5／5q-异常2例（5．4％），-7／7q-异常2例（5．4％）。平均随访12个月，1例失访，22例存活，14例死亡，12例转变为急性白血病。复杂核型与MDS的急性白血病转化及死亡密切相关；8号染色体三体和-7／7q-与死亡相关。结论FISH能敏感地检测出小克隆的异常，应用多种探针并结合染色体检测能较准确判断MDS患者的预后，异常核型比例高提示预后差。     

原位杂交法检测MDR1基因在食管癌中的表达及与p53的关系

目的:了解食管癌组织MDR1基因表达的临床意义及其与p53的关系。方法:用原位杂交法检测了57例食管癌组织标本中MDR1基因的表达,免疫组化法检测了其中44例p53蛋白的表达并进行临床病理分析及相关性分析。结果:57例食管癌组织中MDR1mRNA阳性表达率为35%(20/57),其中食管癌无淋巴结转移的患者阳性表达为29%(8/29),有淋巴结转移患者阳性表达分别为41%(12/28),高于无淋巴结转移患者的表达。临床Ⅱ期食管癌患者阳性表达分别为27%(8/30),临床Ⅲ-Ⅳ期阳性表达为44%(12/27),高于临床Ⅱ期患者的表达。食管癌患者MDR1mRNA表达与肿瘤大小、部位、分化程度、肿瘤浸润的程度无明显相关。44例食管癌组织中p53蛋白阳性表达率为73%(32/44),其中食管癌浸润至肌层,p53表达率为45%(5/11),肿瘤浸润至外膜,p53表达率为82%(27/33),差异有显著性,P<0.01。食管癌患者p53表达与肿瘤大小、部位、分化程度、有无淋巴结转移、临床分期无明显相关。32例p53蛋白表达阳性的组织中有10例MDR1mRNA表达阳性,它们之间的表达在统计学上无明显相关。结论:MDR1基因...     

神经母细胞瘤原位荷瘤与转移瘤裸鼠模型建立的实验研究

目的用自建的细胞系QDDQ建立起原位荷瘤与转移瘤裸鼠模型,探讨神经母细胞瘤(Neuroblastoma,NB)的转移特性。方法取NB新鲜瘤组织,利用酶消化法,建立起体外细胞系,以(3～7)×107/ml浓度的单细胞悬液0.5 ml接种于裸鼠,建立荷瘤鼠模型。结果 NB原位荷瘤与转移瘤裸鼠模型成功建立,实验鼠共30只,致瘤25只,致瘤率为83.3%。10只原位荷瘤,15只转移荷瘤。结论建立NB细胞动物模型,对于进一步研究其转移特性、分子生物学特征,提供了良好的平台。     

荧光原位杂交法快速鉴定金黄色葡萄球菌和凝固酶阴性葡萄球菌

目的 建立一种新的荧光原位杂交法,快速鉴定血培养中的金黄色葡萄球菌(金葡菌)和凝固酶阴性葡萄球菌。方法 将荧光同位素标记的针对金葡菌和凝固酶阴性葡萄球菌的核糖体16SrRNA序列的互补的DNA寡核苷酸探针,在同一张玻片上50℃杂交1 h,用杂交缓冲液50℃洗涤10min,干燥后置荧光显微镜下观察,全过程大约2 h。结果 探针的特异性经标准菌株和临床分离菌株证实。将259份临床标本测试与培养法比较,金葡菌的特异性和敏感性均为100％,CoNs的特异性和敏感性分别为100％和95.5％,荧光原位杂交法的最低检出限为103CFU/ml。结论 本方法适用于血培养中的金葡菌和凝固酶阴性葡萄球菌的快速鉴定。     

Comparison of polymerase chain reaction methods, in situ hybridization, and enzyme immunoassay for detection of Chlamydia pneumoniae in atherosclerotic carotid plaques

Chlamydia pneumoniae has been associated with cardiovascular diseases and has been shown by different methods to be present in atherosclerotic lesions. However, not all studies have found C. pneumoniae in atherosclerotic tissues. We compared polymerase chain reaction (PCR) methods, in situ hybridization (ISH), and measurement of chlamydial lipopolysaccharide (cLPS) by enzyme immunoassay (EIA) from homogenized atherosclerotic tissue in the detection of C. pneumoniae. In a study population of 110 patients with carotid artery disease, cLPS was found in 22.2%, and DNA by PCR was found in 34.3% and by ISH in 39.4% of the samples. Poor repeatability was shown to complicate PCR, and the technical problems inherent in ISH were not insignificant. In contrast, the cLPS EIA test was fast and easy to perform. If the sensitivity could be increased, for example, by testing multiple tissue pieces, cLPS EIA might provide a standardized commercial method for the detection of chlamydia in tissue samples, and it, thus, merits further characterization and validation in different patient populations.     

Effect of X‐ray irradiation on pharmacokinetics of irinotecan hydrochloride and expression of CES1 and CYP3A1 in rats

Concurrent chemoradiation with irinotecan hydrochloride (CPT‐11) is accepted for cancer treatment. However, the effects of X‐ray irradiation on chemotherapeutics in the plasma remain unclear. We evaluated the pharmacokinetics of CPT‐11 in rats after exposure to X‐ray irradiation and examined the changes of protein and mRNA expression of CES1 and CYP3A1. The X‐ray irradiation with 1 Gy and 5 Gy was delivered to the whole body of rats. CPT‐11 at 30 and 60 mg/kg, respectively, was intravenously infused 24 h after irradiation. CPT‐11 was determined by RP‐HPLC in plasma. ELISA and PCR were used to analyze the protein and mRNA expression of CES1 and CYP3A1, respectively. Compared with control rats, the X‐ray irradiation decreased the AUC of CPT‐11 (30 mg/kg) by 15.6% at 1 Gy and 39.0% at 5 Gy and increased the CL by 60.0% at 5 Gy. The X‐ray irradiation could also decrease the AUC of CPT‐11 (60 mg/kg) and increase the CL. In addition, the protein and mRNA expression of CES1 and CYP3A1 were increased significantly in rats after irradiation. This study found significant changes in the pharmacokinetics of CPT‐11 in rats after exposure to X‐ray irradiation, and they might be due to significant increases in the expressions of CYP3A1 and CES1. The pharmacokinetics of CPT‐11 should be rechecked, and the optimal CPT‐11 dose should be reevaluated during concurrent chemoradiation therapy.     

联合间期和中期荧光原位杂交确定成人急性白血病患者11q23/MLL异常

目的 探讨快速、敏感、有效揭示11q23／MLL基因重排的方法，确定11q23／MLL异常存成人急性白血病（AL）中的发生情况及其临床特征，指导AL风险治疗。方法112例成人AL患者骨髓细胞经24h短期培养按常规方法制备染色体标本，R显带行核型分析；LSIMLL双色分离信号DNA探针行间期荧光原位杂交（FISH）筛选异常信号，有异常信号者行中期FISH确定11q23／MLL基因重排。结果 112例AL患者FISH揭示9例11q23／MLL易位（检出率8．0％），其中常规细胞遗传学分析（CCA）只检出4例（检出率3．6％）。3例CCA示del（11）（q23）者FISH揭示2例为11q23／MLL易位，1例为11号染色体长臂末端缺失。在1例正常核型、1例11q＋和1例无11q23明显异常者，FISH揭示为11q23／MLL易位。除9例易何外，FISH揭示8例存在MLL基因扩增，包括多倍体、均匀染色区（hsr）和双微染色体（dmin）。AL伴11q23／MLL异常者多诊断为B系祖细胞急性淋巴细胞白血病（pro-B ALL）、急性单核细胞白血病（AMoL）或急性双表型白血病（BAL）。结论 使用MLL双色分离信号DNA探针行FISH确定11q23／MLL异常是快速敏感的方法，其检出率高于CCA，有效揭示11q23／MLL易位和扩增。临床诊断pro-B ALL、AMoL或BAL，尤其正常核型者应行FISH以确定11q23／MLL异常。     

Safety,tolerability, and pharmacokinetics of single and multiple topical ophthalmic administration of imatinib mesylate in healthy subjects

For the long‐term efficacy of dry eye disease treatment, relieving underlying inflammation is necessary. Imatinib mesylate is a novel ophthalmic formulation of imatinib mesylate, which is expected to alleviate inflammation by inhibiting the discoidin domain receptor 1 activity. This study aims to evaluate the safety and pharmacokinetics of imatinib mesylate in healthy subjects. A randomized, double‐blind, placebo‐controlled study was conducted. In a single ascending dose, 16 subjects received a single eye drop of imatinib mesylate 0.1%, 0.3%, or matching placebo. In the multiple ascending dose (MAD), subjects received multiple eye drops of imatinib mesylate 0.1%, 0.3%, or matching placebo once daily for 7 days. Safety and tolerability were assessed by ophthalmic examination, including the visual analog scale (VAS) to monitor the burning sensation in the eyes. A total of four treatment‐emergent adverse events (TEAEs) occurred during the study. All TEAEs were mildly severe with no serious cases. VAS results in the 0.1% MAD group exhibited highest score of two points, whereas it was less than one point in others. Insignificant difference between the imatinib mesylate and placebo groups in the VAS results was seen. After a single dose administration of imatinib mesylate 0.1%, all plasma concentrations were below the lower limit of quantification. The peak plasma concentrations of imatinib were less than 0.54 µg/L in all groups. In conclusion, a single and multiple topical ophthalmic administration of imatinib mesylate was well‐tolerated in healthy subjects. Because there was minimal systemic exposure to imatinib, the adverse effect in the body seems to be insignificant.     

Magnetic resonance spectroscopy for measuring the biodistribution and in situ in vivo pharmacokinetics of fluorinated compounds: validation using an investigation of liver and heart disposition of tecastemizole

BACKGROUND AND OBJECTIVE: The study of biodistribution and in situ pharmacokinetics is a challenging, but sometimes very important, aspect of premarketing characterization of drugs. We aimed to develop a non-invasive fluorine magnetic resonance (MR) spectroscopic method for the absolute quantitation of a mono-fluorinated compound and of its metabolites in the heart and liver of healthy subjects for this purpose. METHOD: We used fluorine MR spectroscopy (MRS) at 4 T (Tesla) and external standardization in an open label multiple-dose study. Twenty-three healthy adult subjects were enrolled in the study. The surface coil localized fluorine MR spectrum was monitored in the heart and liver at baseline and after oral administration of multiple doses of tecastemizole. Steady-state measurements were made at set time points that depended upon dose, and washout measurements were made only on subjects in which in vivo fluorine signal was observed. RESULTS AND DISCUSSION: At 4 T, under the given experimental conditions, the method had a lower limit of quantitation (LLOQ) of about 2.6 microm and a limit of detection (LOD) of about 0.3 microm for solution state samples (linewidth approximately 15 Hz). The measurement reproducibility was 6.4% using a 50 microm phantom. The effect of MR operator and spectral analyst on the calculated calibration curve slope was small, with inter-rater correlation coefficients of 0.999 and 0.998 respectively. MR signal from fluorine-containing tecastemizole-related moieties was observed in situ only at day 8 in the liver of three of five subjects dosed at 270 mg/day. The average in situ concentration was estimated to be 58+/-22 microm, with an average test-retest reproducibility of 216%. Extrapolating the in vitro results to human measurements, with an approximate linewidth of 250 Hz, predicts in situ LOD and LLOQ values of approximately 6 and 44 microm respectively. However, the human study had a fluorine MRS LOD of approximately 20 microm. The decrease in sensitivity and the increase in variability of the in vivo, in situ measurements compared with the validation study most likely arose from coil placement and incomplete rephasing of the MR signal by the respiratory phase compensation method. CONCLUSION: The measured concentrations were the lowest ever recorded for a multi-dose exogenous mono-fluorinated compound in the human liver using a validated fluorine MR quantitation method. The proposed non-invasive MR method for studying the biodistribution and in situ pharmacokinetics of mono-fluorinated compounds in the liver and heart should have broader application to the development of non-invasive biomarkers.     

Design,fabrication and in vitro evaluation of a novel polymer‐hydrogel hybrid scaffold for bone tissue engineering

The development of a bone mechanically‐compatible and osteoinductive scaffold is important for bone tissue engineering applications, particularly for the repair and regeneration of large area critically‐sized bone defects. Although previous studies with weight‐bearing scaffolds have shown promising results, there is a clear need to develop better osteoinductive strategies for effective scaffold‐based bone regeneration. In this study, we designed and fabricated a novel polymer‐hydrogel hybrid scaffold system in which a load‐bearing polymer matrix and a peptide hydrogel allowed for the synergistic combination of mechanical strength and great potential for osteoinductivity in a single scaffold. The hybrid scaffold system promoted increased pre‐osteoblastic cell proliferation. Further, we biotinylated human recombinant bone morphogenetic protein 2 (rhBMP2), and characterized the biotin addition and its effect on rhBMP2 biological activity. The biotinylated rhBMP2 was tethered to the hybrid scaffold using biotin‐streptavidin complexation. Controlled release studies demonstrated increased rhBMP2 retention with the tethered rhBMP2 hybrid scaffold group. In vitro evaluation of the hybrid scaffold was performed with rat bone marrow stromal cells and mouse pre‐osteoblast cell line MC3T3‐E1 cells. Gene expression of alkaline phosphatase (ALP), collagen I (Col I), osteopontin (OPN), bone sialoprotein (BSP), Runx‐2 and osteocalcin (OC) increased in MC3T3‐E1 cells seeded on the rhBMP2 tethered hybrid scaffolds over the untethered counterparts, demonstrating osteoinductive potential of the hybrid graft. These findings suggest the possibility of developing a novel polymer‐hydrogel hybrid system that is weight bearing and osteoinductive for effective bone tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.     

探讨白血病患者骨髓中VEGF、bFGF、sICAM-1及CD105的表达对髓血屏障调节机制的影响

目的探讨白血病患者骨髓中VEGF、bFGF、sICAM-1及CD105的表达对髓血屏障调节机制的影响。方法采用原位杂交方法,用CD105标记骨髓活化的血管内皮细胞;用ELISA方法检测骨髓液VEGF、bFGF、sICAM-1的表达水平。结果 CD105在白血病的表达以急性髓系白血病(AML-M5)最高,急性髓系白血病(AML)AML-M3表达最低。AML-M5的阳性表达率为(52.32±15.34)%,明显高于正常对照组[(2.32±0.87)%](P<0.05);AML-M3的阳性表达率为[(3.56±1.21)%]与正常对照组差异无统计学意义(P>0.05);CLL阳性表达率与正常对照组差异无统计学意义(P>0.05);其他白血病(T-ALL、B-ALL、M1、M2、M4、CML)的阳性表达率均明显高于正常对照组(P<0.05);CD105在高白细胞白血病组的表达明显高于在低白细胞白血病组的表达(P<0.05);骨髓液VEGF、sI-CAM含量在CML表达最高,bFGF在B-ALL表达最高,VEGF、sICAM含量在T-ALL明显高于与正常对照组(P<0.05),而bFGF含量在T-ALL与正常对照组差异无统计学意义(P>0.05);CLL表达VEGF、bFGF与正常对照组差异无统计学意义(P>0.05);CLL表达sICAM明显高于正常对照组;其他白血病(B-ALL、M1、M2、M3、M4、M5、CML)VEGF、bFGF、sICAM均明显高于正常对照组(P<0.05);骨髓液CD105在AML-3的表达与VEGF、bFGF、sI-CAM的表达呈负相关。在其他类型白血病与VEGF、bFGF、sICAM的表达呈正相关。VEGF、sICAM在高白细胞白血病组的表达明显高于在低白细胞白血病组的表达(P<0.05);而bFGF在高白细胞白血病组与低白细胞白血病组的表达无明显差异(P>0.05)。结论白血病时骨髓CD105、VEGF、bFGF及sICAM的表达在不同类型白血病高低不同,由骨髓入血的白血病细胞数量也有所不同,说明这些血管生成因子,参与构成了骨髓内的诱导微环境,影响了髓血屏障的正常调节机制,导致髓血屏障的结构和功能紊乱。