基于iTRAQ标记结合2D nano HPLC-ESI-OrbiTrap MS/MS技术筛选卵巢癌血清标记物

  目的  利用相对和绝对同位素标记（iTRAQ）和纳升级两维高效液相色谱-电喷雾-OrbiTrap质谱（2D nano HPLC-ESI-OrbiTrap MS/MS）蛋白组学方法探寻卵巢癌血清标记物,以提高早期卵巢癌诊断率。  方法  收集卵巢癌患者血清20例（卵巢癌组）,CA125异常的卵巢良性囊肿16例（良性囊肿组）,健康者20例（正常对照组）,每组取10例组内等量混合后去除高丰度蛋白,iTRAQ试剂标记,进行强离子交换柱分离多肽、nanoLC分离并在线连接电喷雾串联OrbiTrap质谱分析,筛选出重要的差异蛋白。随后采用Western blot方法,将筛选出的重要差异蛋白进行56例临床血清样本逐例验证。  结果  共鉴定出差异蛋白326个。其中重点筛选出了8个重要的差异蛋白:蛋白PARD3、SRP1-alpha、LAMA4、LRP16、IgSF2、NRAMP1、NF-E2-related factor 1和APOA4;验证了3个重要差异蛋白:PARD3、NRAMP1、APOA4。  结论  应用iTRAQ标记的定量蛋白质组学技术筛选出了8个重要的差异蛋白,可能是潜在的肿瘤标记物。其中APOA4蛋白有可能成为区分恶性与良性卵巢疾病的生物标志物。      

UPLC/Q-TOF MS/MS技术评价防腐剂硼酸和叠氮化钠对尿液代谢物的影响

目的：在基于超高效液相色谱-四级杆-飞行时间串联质谱（UPLC/Q-TOF MS/MS）的尿液代谢组学研究中,确定硼酸和叠氮化钠两种防腐剂是否适用于尿液样品的处理,并确定其适宜的浓度范围。方法：收集6名健康志愿者的晨尿,等量混匀后分装,根据处理方式不同,分为对照组、叠氮化钠组和硼酸组。对照组样品不添加任何防腐剂,叠氮化钠组在尿液中分别添加终浓度为0.1、1和10 mmol/L的叠氮化钠,硼酸组在尿液中分别添加终浓度为2、20和200 mmol/L的硼酸。应用UPLC/Q-TOF MS/MS技术对样品进行检测,采用主成分分析、偏最小二乘判别分析和正交偏最小二乘判别分析等化学计量学方法进行数据处理,比较不同处理组间的代谢图谱。结果：偏最小二乘判别分析得分图显示,3个浓度的叠氮化钠处理组以及2 mmol/L硼酸处理组与对照组间无明显分离趋势;200 mmol/L硼酸处理组与对照组分离趋势明显,两组尿液代谢物存在差异。结论：0.1~10 mmol/L叠氮化钠和2 mmol/L硼酸适用于基于UPLC/Q-TOF MS/MS技术的尿液代谢组学研究。     

TACE Combined with PSE in Treating Hepatocelluar Carcinoma with Hypersplenism

OBJECTIVE To observe the effectiveness and safety of transcatheter arterial chemoembolization (TACE) combined with partial splenic embolization (PSE) in treating primary hepatocelluar carcinoma (HCC) with hypersplenism. METHODS Thirty HCC patients with liver cirrhosis, portal hypertension and hypersplenism were treated with TACE and PSE. The degree of tumor volume reduction and remission of hypersplenism were observed. RESULTS The tumor reduction rate of the HCC was 73.3%. Twenty-eight patients had hypersplenic remission with a rate of 93.3%. There were no severe complications such as hepatic abscesses. CONCLOUSION TACE combined with PSE is a safe and effective method to treat HCC with liver cirrhosis, portal hypertension and hypersplenism.     

MB-WCX联合MALDI-TOF MS建立结直肠癌血清诊断模型

 目的 运用弱阳离子磁珠（magnetic beads based weak cation exchange, MB-WCX）联合基质辅助激光解吸离子飞行时间质谱（matrix assisted laser desorption ionization time of flight mass spectrometry, MALDI-TOF MS）建立结直肠癌血清蛋白组学诊断模型。方法 收集我院正常对照（健康体检者）、结直肠癌术前及术后患者血清标本各72例,弱阳离子磁珠分离血清多肽,MALDI-TOF MS建立正常对照、结直肠癌术前及术后患者血清蛋白表达谱,ClinProt Tools 2.0软件分析差异表达峰并建立诊断模型,液相色谱-电喷雾离子化质谱（liquid chromatography-eletronic spray ionization mass/mass, LC-ESIMS/MS）鉴定差异表达蛋白。结果 对比分析正常对照、结直肠癌术前及术后血清蛋白图谱,共发现80个差异表达峰,12个峰差异具有统计学意义（均P<0.01）,与对照组相比,其中9个差异峰在结直肠癌术前的血清蛋白图谱中显示升高,术后显示降低,3个峰在结肠癌术前的血清蛋白图谱中显示降低,术后显示升高。遗传算法（genetic algorithm, GA）模型诊断结直肠癌的敏感度和特异性分别为99.31%和96.49%。GA模型中m/z: 2663.36、m/z: 4793.17和m/z: 5343.48的差异表达峰经鉴定分别为纤维蛋白原α前体亚型1（isoform 1 of Fibrinogen alpha chain precursor, FGA）、组蛋白赖氨酸甲基转移酶SETD7（histone-lysine N-methyltransferase SETD7, SETD7）和黏蛋白5AC（Mucin-5AC precursor, MUC5AC）。结论 血清蛋白质谱模型能够准确区分正常对照与结直肠癌患者,但尚需更进一步研究证实。     

应用MALDI-TOF MS技术建立贲门癌血清蛋白指纹图谱诊断模型

背景与目的:贲门癌是常见的消化道肿瘤,预后差,5年生存率低.早期诊断是改善其预后的关键因素之一.近年来,蛋白质组学的迅速发展推进了肿瘤标志物研究的进程,其中基质辅助激光解析离子化飞行时间质谱(matrix assisted laser desorption ionization time-of-flight mass spectrometry,MALDI-TOF MS)作为常用的蛋白质组学技术,成为检测和验证血液等体液肿瘤标志物的新技术及平台.本研究利用MALDI-TOF MS技术检测贵门癌患者血清蛋白指纹图谱,建立贲门癌诊断模型,探讨其临床应用价值.方法:收集河北医科大学第四医院2009年3月-2010年5月胸外科贲门癌患者血清63例和健康志愿者血清54例,采用弱阳离子蛋白芯片(WCX磁珠)对血清进行分析前处理,MALDI-TOF MS技术进行血清蛋白图谱检测,所得结果用ZUCI-蛋白芯片数据分析系统进行处理.运用遗传算法(genetic arithmetic,CA)结合支持向量机(support vector machine,SVM)运算建立贲门癌蛋白指纹图谱诊断模型:将117例标本随机分为训练组和盲法测试组,经训练后验证诊断模型的特异度和灵敏度.结果:采集贵门癌患者和健康对照者的血清蛋白指纹图谱,经数据对比分析找到69个有显著性差异的质荷比峰(P＜0.01);从中筛选出差异最显著的10个蛋白质荷比峰建立诊断模型(m/z分别为2 863、4 655、3 883、3 241、3 952、2 295、2 741、1 546、4 054和2 671).通过验证,该诊断模型的灵敏度为96.83%、特异度为90.74%.结论:应用MALDI-TOF MS技术能够检测贲门癌患者血清蛋白指纹图谱,建立贲门癌诊断模型.该模型在贲门痛诊断中具有较高的灵敏度和特异度,有一定的临床应用价值.     

Intravesical chemotherapy with 4′-epi-Adriamycin in patients with superficial bladder tumors

Summary We evaluated the effects of 4-epi-Adriamycin (EPI), a derivative of Adriamycin (ADR), in intravesical instillation chemotherapy. The patients received two courses of three daily instillations of 50–80 mg EPI dissolved in 30 ml physiological saline on 3 consecutive days, with an interval of 4 days between courses. Full evaluation was possible in 33 of 35 patients with superficial bladder tumors treated with EPI. Complete response was observed in 4 cases and partial response in 14 cases, giving a response rate of 55%. Side effects such as pollakiuria and pain on micturition occurred in 9 cases. EPI appears to be an effective agent for intravesical instillation chemotherapy in patients with superficial bladder tumors.     

应用SELDI-TOF MS技术分析肝癌血清差异表达蛋白

目的应用蛋白质组学技术分析肝癌血清中差异表达蛋白的临床意义。方法应用表面加强激光解析电离飞行时间质谱(SELDI-TOF-MS)技术，对肝癌、肝硬化、慢性乙型肝炎及健康对照组血清蛋白质进行检测，应用Ciphergen公司软件对蛋白质波谱进行分析。结果质荷比(m／z)为11492．92的蛋白质波峰强度仅肝癌组明显增强，与患者的肝功能、AFP等均无相关性。以波峰的信号噪声比(S/N)大于5为标准，该蛋白质诊断肝癌的灵敏度为50.8％，特异性为95．6％。结论质荷比(m／z)为11492．92的蛋白质可能是肝癌的生物学标志物，对AFP阴性患者的实验室诊断可能具有辅助作用。     

Genistein抑制鸟嘌呤氧化损伤的CGC/MS研究

为了探讨genistein抗肿瘤与抗氧化之间的关系，用Fenton反应（H2O2＋Fe2+→·OH＋OH－＋Fe3+）造成鸟嘌呤的氧化损伤，用毛细管柱气相色谱／质谱分析法（CGC／MS）研究了genistein对这一过程的影响。结果表明genstein具有抑制8-经基鸟嘌呤生成的作用，60μmol·L－1genistein可使8-经基鸟嘌呤的生成量降低50％，提示genistein的抗肿瘤效果可能与其保护遗传物质的氧化损伤有关，这种作用可能是genistein通过清除H2O2造成的。     

Chemoradiotherapy with concurrent gemcitabine and cisplatin with or without sequential chemotherapy with gemcitabine/cisplatin vs chemoradiotherapy with concurrent 5-fluorouracil in patients with locally advanced pancreatic cancer �C a multi-centre randomised phase II study

Background:

No standard treatment for locally advanced pancreatic cancer (LAPC) is defined.

Patients and methods

Within a multi-centre, randomised phase II trial, 95 patients with LAPC were assigned to three different chemoradiotherapy (CRT) regimens: patients received conventionally fractionated radiotherapy of 50 Gy and were randomised to concurrent 5-fluorouracil (350 mg m⁻² per day on each day of radiotherapy, RT-5-FU arm), concurrent gemcitabine (300 mg m⁻²), and cisplatin (30 mg m⁻²) on days 1, 8, 22, and 29 (RT-GC arm), or the same concurrent treatment followed by sequential full-dose gemcitabine (1000 mg m⁻²) and cisplatin (50 mg m⁻²) every 2 weeks (RT-GC+GC arm). Primary end point was the overall survival (OS) rate after 9 months.

Results:

The 9-month OS rate was 58% in the RT-5-FU arm, 52% in the RT-GC arm, and 45% in the RT-GC+GC arm. Corresponding median survival times were 9.6, 9.3, and 7.3 months (P=0.61) respectively. The intent-to-treat response rate was 19, 22, and 13% respectively. Median progression-free survival was estimated with 4.0, 5.6, and 6.0 months (P=0.21). Grade 3/4 haematological toxicities were more frequent in the two GC-containing arms, no grade 3/4 febrile neutropaenia was observed.

Conclusion:

None of the three CRT regimens tested met the investigators'' definition for efficacy; the median OS was similar to those previously reported with gemcitabine alone in LAPC.     

Myelodysplastic syndrome with complex karyotypic abnormality in a patient with Waldenström's macroglobulinemia after sequential treatment with chlorambucil and fludarabine

A therapy-related myelodysplastic syndrome (t-MDS) during the course of Waldenstr?m's macroglobulinemia (WM) has been observed in rare patients. In most of them, the condition developed after treatment with alkylating agents. We experienced a 65-year-old male patient who was diagnosed as WM. He was treated with intermittent oral chlorambucil for 12 months and three cycles of fludarabine, and complete response was achieved after fludarabine treatment. During routine outpatient follow-up, severe anemia occurred. His bone marrow aspirate showed dysplastic hemopoiesis with ringed sideroblasts and siderocytes, which is consistent with MDS (refractory anemia with ringed sideroblasts). Cytogenetic analysis showed complex chromosomal abnormalities including 5q deletion, 12p deletion and monosomy 18. When decision is made to treat WM with chlorambucil and/or fludarabine, a potential risk for t-MDS or therapy-related acute myeloid leukemia should be considered and a close hematologic monitoring is needed.     

运用MALDI-TOF MS方法建立食管癌患者血清蛋白指纹图谱诊断模型

 目的 本研究利用基质辅助激光解析离子化飞行时间质谱（matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF MS）技术检测食管癌患者血清蛋白指纹图谱,建立食管癌诊断模型,探讨其临床应用价值。方法采用弱阳离子蛋白芯片（WCX磁珠）对血清进行分析前处理,运用MALDI-TOF MS技术检测119例标本（75例食管癌和44例健康对照）血清蛋白质谱图,通过蛋白芯片数据分析系统进行数据处理,以遗传算法结合支持向量机运算建立食管癌与健康对照组、早期食管癌与中晚期食管癌组诊断模型,随机抽取79例建模标本（50例食管癌和29例健康对照）进行训练与交叉验证,并选择新病例（30例食管癌和23例健康对照）血清标本进行测试。结果采集食管癌患者和健康对照者的血清蛋白质纹图谱,经数据分析找到75个有显著性差异的质荷比峰（P<0.05）和71个有非常显著性差异的质荷比峰（P<0.01）;软件包运算后,建立两个诊断模型：模型1：区分食管癌与健康对照组,由11个蛋白质峰（2 087,2 210,3 258,3 973,4 283,4 645,4 092,4 210,1 985,2 818和2 046 Da）组成,该诊断模型检测食管癌的敏感度为92.4%,特异性为87.4%;模型2：区分早期食管癌与中晚期食管癌组,由8个蛋白质峰（4 195,4 074,4 268,2 106,4 905,5 965,2 863 和 3 953 Da）组成,该诊断模型检测食管癌的敏感度为87.5%,特异性为89.7%。结论运用MALDI-TOF MS技术结合磁珠分选的方法可检测食管癌血清质谱图,建立具有较高的敏感度和特异性食管癌诊断模型。     

MS—SSCA法在检测基因异常甲基化中的应用

目的：介绍一种简单,敏感的筛查多个标本,多个CG二核苷酸甲基化状况的方法,即甲基化敏感的单链构象分析法（methylation-sensitive,Single-strand confomation Analysis,MS-SSCA)。方法：单链DNA的胞嘧啶可被亚硫酸氢盐修饰转变成尿啶啶,而5’甲基胞嘧啶仍保持不变,这种修饰处理可在甲基化和非甲基化基因组DNA间产生DNA序列差异,而这种DNA序列差异可通过PCR－SSCP法较为灵敏地筛查出,利用该原理检测乳腺癌细胞BRCA1基因启动子区13个CG二核苷酸甲基化状况。结果：在癌细胞中发现两种异常甲基化,即高度甲基化及部分甲基化,结论：MS－SSCA法在基因甲基化检测中将有很大的应用潜能,为探讨肿瘤发生机制提供了新的分子分析方向。     

Successful treatment with a low-dose cisplatin–etoposide regimen for patients with diencephalic syndrome

Diencephalic syndrome (DS) is a rare but rapidly fatal condition, usually occurring during the first year of life, as a result of a hypothalamic/chiasmatic tumor. The purpose of this study was to induce an objective tumor response and to achieve rapid weight recovery by using ten three-day courses of reduced-dose cisplatin-etoposide. Between 2004 and 2009, eight pediatric patients with DS as a result of an hypothalamic tumor and with a median age at diagnosis of 6.5?months (range 4-60?months) received 10 monthly courses of cisplatin (25?mg/m(2)/day on days 1-3) and etoposide (100?mg/m(2)/day on days 1-3). Under chemotherapy, rapid weight recovery was observed for all patients; tumor response was observed for six (75?%; partial response in four and minimum response in two). The other two had stable disease at completion of treatment. Mean time to weight recovery was 6?months (range 5-7?months) for pilomyxoid astrocytoma patients, and 3.3?months (range 3-4?months) for those with pilocytic astrocytoma. For DS patients who received nutritional support (enteral or parenteral nutrition) the mean time for weight recovery was 5?months (range 3-7?months) whereas children who were able to orally ingest a high-energy diet had a mean time for weight recovery of 8.66?months (range 3-19?months). After follow-up ranging from 22 to 89?months (median 38?months) all patients are alive. A low-dose cisplatin-etoposide regimen is highly effective regarding tumor response and treatment of DS symptoms/cachexia without causing significant side-effects.