Ion Torrent PGM~(TM)测序仪检测苯丙酮尿症患儿苯丙氨酸羟化酶基因突变

目的评价Ion Torrent PGMTM测序仪检测苯丙酮尿症(PKU)患儿苯丙氨酸羟化酶基因(PAH)突变的可行性。方法提取15例确诊为经典型PKU的患儿及其父母外周血DNA,对PAH全部外显子及外显子-内含子交界区进行PCR反应,用Ion Torrent PGMTM测序仪测序,再对检出突变的样本进行Sanger法验证。结果 Ion Torrent PGMTM平均覆盖深度为1 465倍,平均覆盖率为99.3%;共检出29个突变位点,分属17种突变,其中p.P292L为新发突变,所有检测结果均与Sanger法相一致。结论用Ion Torrent PGMTM测序仪可快速简便检测PKU患儿PAH基因突变。     

Ion Torrent测序技术检测Ⅰ型神经纤维瘤病患者NF1基因突变

摘要：目的：评价Ion Torrent测序技术检测Ⅰ型神经纤维瘤(NF1)基因及其突变类型的可行性。 方法：提取12例NF1患者的外周血DNA,用Ion Torrent个体化基因测序仪(PGM)对患者NF1基因进行测序,用Sanger测序法验证NF1基因相应的突变位点,Ion Torrent PGM检测后覆盖缺失的外显子样本用Sanger测序法重新检测。 结果：检测到10例患者存在NF1基因致病突变,其中2种无义突变,5种小缺失或插入突变,1种错义突变,2种剪接突变。2例患者未检测到致病突变。 结论：Ion Torrent测序技术检测含有大量外显子的NF1基因快速、准确;NF1基因突变可导致氨基酸密码子提前终止。     

白血病NPM1基因突变检测方法的临床适用性比较

目的 分析急性髓细胞白血病(AML)的NPM1(nucleophosmin)基因第12外显子突变,对比3种常用检测方法的临床适用性.方法 随机选择54份AML患者的冻存骨髓细胞标本,提取DNA后PCR扩增NPM1基因第12外显子,分别进行PCR-毛细管电泳、变性高效液相色谱(DHPLC)和直接测序检测.FLT3内部串联重复(ITD)突变的检测采用FLT3 PCR产物分别进行琼脂糖凝胶电泳和PCR-毛细管电泳检测.结果 7例AML患者发现NPM1基因突变,其中5例为常见的A型突变,即960 bp处插入TCTG 4个碱基;1例为D型突变,即960 bp处插入CCTG 4个碱基;另1例为新发现的1种突变,在958 bp处丢失TGGCAGTG 8个碱基,插入GCCCGCGGTTTA 12个碱基.3种基因突变的检测方法检出率均为100%.毛细管电泳检测NPM1基因突变更快速可靠,且可同时检测FLT3-ITD突变.DHPLC的分辨率受实验因素的影响较多.直接测序步骤相对繁琐,而且有杂合子基因序列误读的可能性.结论 AML存在一种NPM1基因的958 bp位点12个碱基置换的基因突变;AML的NPM1基因突变临床检测采用PCR-毛细管电泳法更方便.     

应用新一代半导体靶向测序技术检测枫糖尿症致病基因突变的研究

目的应用Ion Torrent半导体靶向测序技术检测1例枫糖尿症(MSUD)患儿的致病基因突变,明确其致病突变,并探讨该技术用于复杂单基因病检测的可行性。方法采集患儿外周血,提取基因组DNA,经多重PCR扩增富集目的基因片段,构建平均片段大小为300bp左右的文库,经乳液PCR及磁珠颗粒富集,最后采用318半导体测序芯片进行高通量测序,应用Ion Torrent Suite v3.0软件进行数据提取、序列比对及SNPs和Indels提取,再用dbSNP 137数据库过滤得到SNPs和Indels,可疑突变经Sanger法测序验证。结果检出患儿1个新发错义点突变并通过Sanger测序验证,突变是BCKDHB基因第6外显子的NM183050:c.586CT(p.His196Tyr)。结论 Ion Torrent半导体靶向测序技术可对复杂单基因遗传病进行快速、准确地基因诊断。     

急性髓系白血病患儿FLT3-ITD、CEBPA、NPM1、DNMT3A、RAS基因突变特征及临床意义

目的探讨急性髓系白血病(AML)患儿FLT3、CEBPA、NPM1、DNMT3A、NRAS和KRAS基因突变特征及其临床预后意义。方法收集217例初诊AML患儿骨髓标本,采用PCR扩增产物Sanger测序法检测FLT3、CEBPA、NPM1、DNMT3A、NRAS和KRAS基因突变情况,收集临床资料,探究各基因突变的临床特征及预后意义。结果本研究中突变率最高的是NRAS(11.9%),其他依次为CEBPA(10.0%)、FLT3-ITD(5.7%)、KRAS(3.0%)、NPM1(1.4%),未检测出DNMT3A突变。KRAS突变均发生于M5型,NPM1突变均发生于M2型。FLT3-ITD突变组外周血白细胞计数(×109/L)较非突变组明显升高[104.0(19.8,201.0)vs 11.4(3.8,38.7),Z=-3.061,P=0.002];KRAS突变组年龄明显低于非突变组[2.0(1.0,3.3)岁vs 7.0(3.0,10.0)岁,Z=-2.282,P=0.005]。FLT3-ITD突变组患儿总生存率较非突变组明显降低(25.0%vs 52.5%,χ2=4.993,P=0.026);无病生存率呈减低趋势(33.3%vs 60.6%,χ2=3.750,P=0.053)。RAS突变组与非突变组预后差异无统计学意义。结论 FLT3-ITD突变是AML患儿患者的不良预后指标。     

急性髓系白血病FLT3与NPM1基因突变的研究

FMS样酪氨酸激酶3(FLT3)基因内部串联重复(ITD)是近年在急性髓系白血病(AML)患者中发现的常见突变类型,在AML中的发生率为15%～35%,大量研究显示,FLT3突变与外周血高白细胞、骨髓高白血病细胞比例有关,是AML的重要预后影响因素[1-2],核仁磷酸蛋白(NPM1)基因突变也是AML患者最常见的一种基因突变,突变率为25%～35%,在正常核型AML患者中突变率更高,研究显示单独NPM1突变往往预后较好,但当合并FLT3-ITD突变时预后较差[3-6].我们观察了FLT3和NPM1突变在100例AML患者中的发生情况,分析其临床特征、疗效及预后的关系.     

急性髓系白血病中与IDH1/2突变共存的基因突变分析

目的:探讨急性髓系白血病(AML)患者中与IDH1/2突变共存的基因突变及其与部分临床参数的相关性。方法:采用基因组DNA-PCR联合Sanger测序法筛选IDH1/2基因4号外显子突变;采用高通量DNA测序联合Sanger测序法检测IDH1/2突变患者51种肿瘤靶基因突变。结果:358例患者中共检出46例IDH1/2突变,其中35例IDH1突变,11例IDH2突变。97. 8%(45/46) IDH1/2突变患者同时携带其他基因突变,其中双基因突变8例,3个基因突变共存17例,≥4个基因突变共存20例。每例患者平均突变3. 52次。伴随基因突变中最常见的基因为NPM1(n=29,63. 0%),其他依次为:DNMT3A(n=25,54. 3%)、FLT3-ITD(n=7,15. 2%)、TET2 (n=5,10. 9%)及NRAS(n=5,10. 9%)。在IDH1突变组中,伴NPM1突变者的平均外周血白细胞水平高于正常者,伴DNMT3A突变患者的CR率明显低于正常者,差异均有统计学意义(P=0. 034,0. 003);≥4个基因突变患者的白细胞水平明显高于双基因突变者,差异显著(P=0. 037)。结论:95%以上伴IDH1/2突变的AML患者同时存在额外基因突变,基因突变个数及突变类型对患者的临床特征及CR率有一定的影响。     

急性髓系白血病FLT3与NPM1基因突变的研究

FMS样酪氨酸激酶3(FLT3)基因内部串联重复(ITD)是近年在急性髓系白血病(AML)患者中发现的常见突变类型,在AML中的发生率为15%～35%,大量研究显示,FLT3突变与外周血高白细胞、骨髓高白血病细胞比例有关,是AML的重要预后影响因素[1-2],核仁磷酸蛋白(NPM1)基因突变也是AML患者最常见的一种基因突变,突变率为25%～35%,在正常核型AML患者中突变率更高,研究显示单独NPM1突变往往预后较好,但当合并FLT3-ITD突变时预后较差[3-6].我们观察了FLT3和NPM1突变在100例AML患者中的发生情况,分析其临床特征、疗效及预后的关系.     

急性髓系白血病FLT3与NPM1基因突变的研究

FMS样酪氨酸激酶3(FLT3)基因内部串联重复(ITD)是近年在急性髓系白血病(AML)患者中发现的常见突变类型,在AML中的发生率为15%～35%,大量研究显示,FLT3突变与外周血高白细胞、骨髓高白血病细胞比例有关,是AML的重要预后影响因素[1-2],核仁磷酸蛋白(NPM1)基因突变也是AML患者最常见的一种基因突变,突变率为25%～35%,在正常核型AML患者中突变率更高,研究显示单独NPM1突变往往预后较好,但当合并FLT3-ITD突变时预后较差[3-6].我们观察了FLT3和NPM1突变在100例AML患者中的发生情况,分析其临床特征、疗效及预后的关系.     

伴有NPM1基因突变的急性髓细胞白血病患者的临床特征

目的分析急性髓细胞白血病（AML）的NPM1（Nucleophosmin）基因第12外显子突变,探索伴有NPM1基因突变的AML患者的临床特征。方法随机选择98份临床确诊的急性白血病和骨髓增生异常综合征（MDS）患者的冻存骨髓细胞标本,其中78份AML、10份急性淋巴细胞白血病（ALL）和10份MDS提取DNA,行多重PCR,同时扩增NPM1基因第12外显子及FLT3基因第14、15外显子,PCR-毛细管电泳同时检测NPM1和FLT3-ITD基因突变。结果 78例AML患者共检出21例（26.9%）具有NPM1基因突变,10例ALL和10例MDS均未检测出该突变。78例AML中52例核型正常,NPM1阳性19例（36.5%）,26例异常核型AML患者NPM1阳性仅2例（7.7%）,两组间差异有统计学意义（P〈0.05）。AML-M2、M5患者NPM1基因突变发生率高于其他组。NPM1突变型AML患者WBC中位数为42.0（16.3～102.0）×10^9/L,野生型为14.0（3.4～67.2）×10^9/L,两组间差异有统计学意义（P〈0.05）。21例NPM1突变型AML中12例（57.1%）同时伴有FLT3-ITD,57例NPM1野生型患者13例（22.8%）出现FLT3-ITD突变,两组间差异有统计学意义（P〈0.05）。NPM1阳性FLT3阴性患者9例中8例（88.9%）获得CR1,NPM1阳性FLT3阳性12例中3例（25%）获得CR1,NPM1阴性FLT3阴性33例中16例（48.5%）获得CR1,NPM1阴性FLT3阳性13例中2例（15.4%）获得CR1,组间差异有统计学意义（P〈0.05）。结论 NPM1基因突变是AML患者常见的一种突变,尤其是染色体核型正常AML患者发生率较高。伴有NPM1突变的AML患者的临床特征为年龄高,外周血白细胞高,更常见于M2和M5中,伴随FLT3-ITD突变发生率高,CR1率较高。     

Determination of Bradykinin in Blood by a Sensitive Radioimmunoassay

Antibodies of high avidity (maximum Km 1.3 × 10¹⁰ L/M) were produced in rabbits against bradykinin coupled to ovalbumin with toluene-2,4-diisocyanate. Tyrosin⁸ bradykinin was labelled to a specific activity of 530–700 Ci/mmol with ¹²⁵I by means of lactoperoxidase. Sensitivity of the radioimmunoassay was 0.01 μg/1 blood. Specificity studies demonstrated the essential role of the C-terminal arginine of bradykinin for binding to antibody. Mean recovery of [³ PHJbradykinin internal standard after the preparation of 86 blood samples was 39.0%. The major loss occurred during ethanol precipitation. In venous blood collected at random conditions from 32 normal subjects the bradykinin concentration ranged 0.04–0.46 μg/1 and showed no sex difference.     

Na+/Ca2+ exchange inhibitors modulate thapsigargin-induced Ca2+ and Na+ influx in human lymphocytes

Thapsigargin has been shown the elevate intracellular Na(+) concentration in human lymphocytes, but mechanisms underlying thapsigargin-induced Na(+) entry are little understood. In the present study we investigated thapsigargin-induced changes in cytosolic free Na(+) and Ca(2+) concentration in human lymphocytes after inhibition of the Na(+)/Ca(2+) exchange with two structurally unrelated compounds, dimethylthiourea ad bepridil. The intracellular Na(+) increase induced by 5 microM thapsigargin was significantly enhanced in the presence of 5 mM dimethylthiourea or 40 microM bepridil. In contrast, both compounds significantly decreased the thapsigargin-induced intracellular Ca(2+) elevation. No effect of dimethylthiourea or bepridil on thapsigargin-induced Ca(2+) influx was observed in the absence of extracellular Na(+). These observations are consistent with the hypothesis that thapsigargin stimulates Na(+)/Ca(2+ )exchange in human lymphocytes. However, Na(+)/Ca(2+) exchange does not mediate Na(+) influx in human lymphocytes.     

Which has the least immunity depression during postoperative analgesia--morphine, tramadol, or tramadol with lornoxicam?

BACKGROUND: Analgesics are commonly used to provide pain relief after surgery. These drugs produce some extended depression of immunity. A prospective randomized controlled trial was designed to observe expressions of T-lymphocyte subsets (CD3(+), CD3(+)CD4(+) and CD3(+)CD8(+)), natural-killer cells (CD3(-)CD16(+)CD56(+)), and activated T-lymphocytes (CD3(+)CD25(+)) of patients undergoing gastric cancer surgeries and receiving patient-controlled intravenous analgesia (PCIA). METHODS: Forty-five patients undergoing elective gastric cancer surgeries under general anesthesia were randomly allocated into 3 groups. Group I received PCIA using morphine after surgery, group II using tramadol, and group III using tramadol with lornoxicam. The analgesic efficacy was evaluated by visual analog scale (VAS) and Bruggrmann comfort scale (BCS). Expressions of CD3(+), CD3(+)CD4(+), CD3(+)CD8(+), CD3(-)CD16(+)CD56(+), and CD3(+)CD25(+) were measured as percentages of total lymphocytes by flow cytometer at 5 time points. RESULTS: There was no significant difference in analgesic efficacy and the baselines of CD3(+), CD3(+)CD4(+), CD3(+)CD8(+), CD3(-)CD16(+)CD56(+), and CD3(+)CD25(+) in all groups. Compared with the baseline, CD3(+)CD8(+) had no changes in all groups at any time point. Ninety minutes after incision, CD3(+), CD3(+)CD4(+), CD3(-)CD16(+)CD56(+), and CD3(+)CD25(+) were lower in all groups (P<0.05). 24 h after surgery, CD3(+), CD3(+)CD4(+), CD3(-)CD16(+)CD56(+), and CD3(+)CD25(+) were lower in group I and group II (P<0.05); meanwhile CD3(+), CD3(+)CD4(+), and CD3(+)CD25(+) returned to the baseline but CD3(-)CD16(+)CD56(+) was still low (P<0.05) in group III. 48 h after surgery, CD3(+), CD3(+)CD4(+), CD3(-)CD16(+)CD56(+), and CD3(+)CD25(+) returned to the baseline in group II and group III, but not in group I (P<0.05). 72 h after surgery, CD3(+), CD3(+)CD4(+), CD3(+)CD4(+)/CD3(+)CD8(+) returned to the baseline, but CD3(+)CD25(+) and CD3(-)CD16(+)CD56(+) were still low in group I (P<0.05). CONCLUSION: PCIA using lornoxicam with tramadol has the same good analgesic efficacy and less immunity depression than PCIA using morphine or tramadol.     

99Tcm-ECD SPECT非侵入性的定量测定平均CBFl和rCBF理论及其应用研究

目的定量评价大脑及局部的平均脑血流量,应用     

<Superscript>11</Superscript>C-Acetate PET in the Evaluation of Brain Glioma: Comparison with <Superscript>11</Superscript>C-Methionine and <Superscript>18</Superscript>F-FDG-PET

PURPOSE: The aim of the study is to retrospectively investigate the usefulness of (11)C-acetate (ACE)-positron emission tomography (PET) for evaluation of brain glioma, in comparison with (11)C-methionine (MET) and 2-deoxy-2-(18)F-fluoro-D: -glucose (FDG). PROCEDURES: Fifteen patients with brain glioma referred to initial diagnosis were examined with ACE, MET, and FDG-PET. Five patients had low-grade gliomas (grade II), three had anaplastic astrocytomas (grade III), and seven had glioblastomas (grade IV). PET results were evaluated by visual and semiquantitative analysis. For semiquantitative analysis, the standardized uptake value (SUV) and tumor to contralateral normal gray matter (T/N) ratio were calculated. The sensitivity for detection of high-grade gliomas was calculated using visual analysis. RESULTS: Sensitivities of ACE, MET, and FDG were 90%, 100%, and 40%, respectively. ACE and MET T/N ratios were significantly higher than that of FDG. ACE and FDG SUV in high-grade gliomas were significantly higher than that in low-grade gliomas. No significant differences were observed using MET. CONCLUSIONS: ACE PET is a potentially useful radiotracer for detecting brain gliomas and differentiating high-grade gliomas.     

The in vitro Uptake of Vitamin B12 by Diphyllobothrium Latum and its Blockage by Intrinsic Factor

^99mTc-labelling procedures are described. They are standardized and simple enough for technicians without special chemical training to perform, so that smaller hospitals with facilities for isotope scintigraphy can produce their own ^99mTc-labelled compounds. The experimental background for the procedures is described.     

The incidence of discrepant regional myocardial uptake between 201 thallium and 123 I-BMIPP SPECT in patients with coronary heart disease

Myocardial perfusion and fatty acid uptake at rest were assessed by SPECT with ²⁰¹Tl (Tl) and ¹²³I-BMIPP (BMIPP) in 50 consecutive patients with coronary heart disease. Discrepant regional myocardial uptake was observed in 19 patients and classified into the following two groups: mismatch (MM; Tl uptake > BMIPP uptake, n = 14, mean age, 66 years) and paradoxical mismatch (PM; Tl uptake < BMIPP uptake, n = 5, mean age, 68 years). In the MM group, 77% was single- or zero-vessel disease and the artery-perfused region in the mismatched area was almost always ischemia related. Sixty percent of the regions observed with the PM were related to the inferior wall. In the PM group, 80% of cases were associated with multivessel stenoses and 60% of cases was suffered from ischemic attack within a week before scintigraphy. In conclusion, mismatch was related to abnormal fatty acid uptake caused by coronary heart disease. Although the paradoxical mismatch might mainly be related to diaphragmatic attenuation of Tl scans and augmented artifacts of BMIPP scans in the inferior wall, we should not overlook severe coronary heart disease in patients with paradoxical mismatched phenomenon.     

In vivo effects of high phenylalanine blood levels on Na+,K+-ATPase,Mg2+-ATPase activities and biogenic amine concentrations in phenylketonuria

OBJECTIVE: To evaluate the activities of Na+,K+-ATPase and Mg2+-ATPase in erythrocyte membranes from phenylketonuric (PKU) patients and to correlate the enzyme activities with their blood phenylalanine (Phe) levels, biogenic amines as well as with their precursors tyrosine (Tyr) and tryptophan (Try). DESIGN AND METHODS: Twenty three PKU patients were divided into group A (n = 12) on a restricted diet (Phe 1.57 +/- 0.52 mg/dL or 0.10 +/- 0.03 mM) and group B (n = 11) on a "loose" diet (Phe 24.45 +/- 1.50 mg/dL or 1.72 +/- 0.09 mM). The enzyme activities were measured spectrophotometrically, the amino acids with an automatic amino analyser and the biogenic amines with HPLC methods. RESULTS: In group B, plasma amino acids (Tyr, Try), their biogenic amines [adrenaline (A), noradrenaline (NA), dopamine (DA) and serotonin (5HT)], (Na+,K+)-ATPase and Mg2+-ATPase activities were found remarkably decreased (p < 0.001). CONCLUSIONS: High Phe and/or low NA, DA, 5HT plasma levels may indirectly inhibit the erythrocyte membrane Na+,K+-ATPase and Mg2+-ATPase in PKU patients. The observed enzyme inhibitions could be a very informative peripheral marker as regards the neurotoxic Phe brain effects.     

Temporal and spatial characterization of negative regulatory T cells in HIV‐infected/AIDS patients raises new diagnostic markers and therapeutic strategies

BackgroundNegative regulatory T cells (Tregs) not only deplete effector T cells but also inhibit the clearance of HIV during infection, which may allow Tregs to be used as informative diagnostic markers. To facilitate both diagnosis and treatment, a thorough understanding of these regulators by characterizing them on temporal and spatial scales is strongly required.MethodsHundred HIV‐infected/AIDS patients, including 87 males, with an average age of 35.8 years, as well as 20 healthy controls, were enrolled. Flow cytometry was used to analyze CD3+T cells, CD4+T cells, and CD8+T cells to evaluate the immune status of the participants. Then, a group of representative negative regulatory T cells, including CD4+PD‐1+T cells, CD4+PD‐1^highT cells, CD8+PD‐1+T cells, and CD4+CD25^high Tregs was also analyzed to explore their effects on disease progression and intercorrelation.ResultsThe percentages of CD4⁺PD‐1⁺T cells and CD4⁺CD25^highTregs increased in patients with the same ultrahigh significance. Temporally, the patients with both intermediate‐stage and late‐stage disease had higher percentages of CD4⁺PD‐1⁺T cells; however, the percentage of CD4⁺CD25^highTregs only increased in the patients with late‐stage disease. In addition, CD4⁺PD‐1⁺T cells but not CD4⁺CD25^highTregs were negatively correlated with the absolute CD4⁺T cell count. Spatially, no correlations between CD4⁺PD‐1⁺T cells and CD4⁺CD25^highTregs were observed, which suggests these Tregs function differently during immunosuppression.ConclusionsThis study characterized negative regulatory T cells in HIV‐infected/AIDS patients at both temporal and spatial scales and found that CD4⁺CD25⁺Tregs and CD4⁺PD‐1⁺T cells could be used as potential diagnostic markers for identifying different disease stages and monitoring disease progression.     

F applications in drug development and imaging – a review

To control drugs in vivo, new approaches are needed. Considerable progress has been made towards the applications of fluorine (¹⁹F) in pharmacotherapy in this regard. To date, many authors have showed that by using ¹⁹F labelled drugs and non-invasive magnetic resonance imaging (MRI) techniques together, drug biodistribution can be tracked. This review presents methods for ¹⁹F incorporation into pharmaceuticals by forming C–F bonds and drug fluorine oil-water emulsions. Inadequate drug delivery is a major cause of drug resistance, which can be improved using approaches discussed herein aided by ¹⁹F MRI.