急性混合细胞白血病独特临床生物学特征及预后研究

目的:分析急性混合细胞白血病(HAL)独特的临床生物学特征及预后。方法:采用流式细胞术(FCM)分析白血病细胞的免疫表型,最终确诊56例HAL患者,对其骨髓标本进行细胞形态学及相关细胞化学染色分析,以确定其FAB分型,用聚合酶链反应检测骨髓细胞DNA IgH及TCRγ基因重排,采用兼顾急性髓细胞性白血病(AML)和急性淋巴细胞白血病(ALL)的方案治疗,同时结合多项临床生物学指标分析其转归和预后。结果:确诊的HAL其FAB分型以AML-M1/M2、ALL为主;其免疫分型以B系和髓系混合表达多见。CD34在HAL中呈高表达并且是对患者预后具有影响力的因素(P=0.03)。确诊的56例HAL患者中,28例出现IgH基因单克隆重排阳性(50.76%),22例出现TCRγ基因单克隆重排阳性(39.65%),其中2例IgH和TCRγ基因单克隆重排同时出现阳性。此类患者对治疗反应差、缓解率低为36%。结论:HAL属特殊类型白血病,有其独特的临床生物学特征,对化疗方案不敏感,预后较差。     

N-甲基亚硝基脲诱导小鼠胸腺淋巴瘤TCR基因重排分析

目的探讨N-甲基亚硝基脲（MNU）诱导的小鼠胸腺淋巴瘤的单克隆起源。方法采用巢式PCR方法,对8例MNU诱导的胸腺淋巴瘤组织进行T细胞受体β链（TCRβ）和γ链（TCRγ）克隆性基因重排分析,并对TCRγ基因重排的PCR产物直接测序。结果 8例胸腺淋巴瘤检测TCRβ和TCRγ均呈克隆性基因重排。DNA序列测定证实TCRγ基因PCR扩增产物为基因重排产物。结论巢式PCR TCR基因重排检测及DNA序列分析证实,MNU诱导的小鼠胸腺淋巴瘤是来源于T细胞的肿瘤。     

血清GPAA1、SF、OPN与儿童急性淋巴细胞白血病危险度的关系及对血栓发生风险的评估效能研究

摘要 目的：分析血清糖基磷脂酰肌醇锚附着蛋白1（GPAA1）、铁蛋白（SF）、骨桥蛋白（OPN）与儿童急性淋巴细胞白血病危险度的关系及对血栓发生风险的评估效能。方法：选择我院自2017年1月至2022年12月接诊的112例急性淋巴细胞白血病患儿作为观察组，另选112例性别、年龄与观察组相匹配的健康体检儿童作为对照组。检测两组血清GPAA1、SF、OPN表达水平，分析不同危险度的急性淋巴细胞白血病患儿血清GPAA1、SF、OPN表达水平的差异性，观察急性淋巴细胞白血病患儿的血栓发生情况，通过受试者工作特征曲线（ROC）下面积（AUC）评价血清GPAA1、SF、OPN预测急性淋巴细胞白血病患儿发生血栓的效能。结果：观察组血清GPAA1、SF、OPN表达水平均高于对照组（P＜0.05）；在低危、中危和高危的急性淋巴细胞白血病患儿中，血清GPAA1、SF、OPN表达水平有差异（P＜0.05）；经Spearman相关性分析，血清GPAA1、SF、OPN表达水平与儿童急性淋巴细胞白血病危险度呈正相关（P＜0.05）；在112例急性淋巴细胞白血病患儿中，发生血栓12例，占10.71%；经多因素Logistic回归分析，血清GPAA1、SF、OPN均是急性淋巴细胞白血病患儿发生血栓的独立预测因素（P＜0.05）；经ROC曲线分析，血清GPAA1、SF联合OPN预测急性淋巴细胞白血病患儿发生血栓的AUC为0.901。结论：血清GPAA1、SF、OPN与儿童急性淋巴细胞白血病危险度密切相关，联合预测患儿发生血栓的效能较好，对此病的诊治具有重要指导意义。     

PCR扩增TCRVδ2－Dδ3片段的临床价值

以巢式PCR扩增急性淋巴细胞白血病细胞中TCRVδ2－Dδ3重排片段,同时将生物素掺入初诊期骨髓标本扩增产物中,标记成患者克隆特异性探针,通过点杂交法检测缓解期患者体内残留的白血病细胞．结果提示该法对于临床病情监测有一定的价值．     

DC 与CIK细胞治疗难治复发急性髓细胞白血病的近期临床观察

目的：评估自体DC与CIK 细胞治疗难治复发急性髓细胞白血病的近期疗效与安全性。方法：给予20 例难治复发急性髓细 胞白血病患者树突状细胞(DC)与细胞因子诱导的杀伤细胞(CIK)治疗,20 例难治复发的应用同样化疗方案的急性髓细胞白血病 患者做为对照组;治疗后4 周观察两组患者临床疗效和生存质量(KPS)评分,DC 与CIK 细胞治疗前和治疗后1 周检测T细胞亚 群(CD3+、CD3+CD4+、CD3+CD8+、CD3+CD56+)和细胞因子(IL-12、IL-2、IL-7、IFN-酌及TNF-琢)水平的变化。结果：①DC 与CIK 细胞 治疗组有效率和KPS评分明显高于对照组(P<0.05),所有患者的不良反应轻微,均可耐受。②DC 与CIK 细胞治疗后1 周,患者T 细胞亚群百分比和细胞因子含量较治疗前均明显升高,其中CD3+、CD3+CD56+及IL-12、IL-7 明显升高(P<0.05)。结论：DC与CIK 细胞免疫治疗难治复发急性髓细胞白血病安全有效。     

LFNG在白血病BALL-1细胞中的表达状况研究

O-岩藻糖肽3-β-N-乙酰氨基葡萄糖转移酶(Lunatic Fringe,LFNG)与Notch信号作用密切相关,且LFNG在不同组织细胞中对Notch信号所起的作用不同。为了探讨LFNG在急性B细胞白血病中的表达及对Notch信号的作用,应用real-time PCR和Western-blot在核酸水平及蛋白质水平上检测了LFNG在人急性B淋巴细胞白血病细胞系BALL-1、人正常B淋巴母细胞系HMy2.CIR及正常B细胞中的表达状况,并应用si RNA技术分析了LFNG基因沉默后对白血病BALL-1细胞中Notch信号通路的影响。结果显示:白血病细胞系BALL-1存在LFNG蛋白过度表达,且LFNG基因沉默后抑制白血病B细胞的Notch信号通路。上述结果提示LFNG在白血病B细胞中的异常表达能促进Notch信号。     

DC与CIK细胞治疗难治复发急性髓细胞白血病的近期临床观察

目的：评估自体DC与CIK细胞治疗难治复发急性髓细胞白血病的近期疗效与安全性。方法：给予20例难治复发急性髓细胞白血病患者树突状细胞（DC）与细胞因子诱导的杀伤细胞（CIK）治疗,20例难治复发的应用同样化疗方案的急性髓细胞白血病患者做为对照组;治疗后4周观察两组患者临床疗效和生存质量（KVS）评分,DC与CIK细胞治疗前和治疗后1周检测T细胞亚群（CD3＋、CD3＋CD4＋、CD3＋CD8＋、CD3＋CD56＋）和细胞因子（IL-12、IL-2、IL-7、IFN-γ及TNF—α）水平的变化。结果：（1）DC与CIK细胞治疗组有效率和KPS评分明显高于对照组（P〈0．05）,所有患者的不良反应轻微,均可耐受。（2）DC与CIK细胞治疗后1周,患者T细胞亚群百分比和细胞因子含量较治疗前均明显升高,其中CD3＋、CD3＋CD56＋及IL-12、IL-7明显升高（P〈0．05）。结论：DC与CIK细胞免疫治疗难治复发急性髓细胞白血病安全有效。     

人T细胞急性淋巴细胞白血病小鼠动物模型的建立

目的:通过建立一理想的动物模型来模拟T细胞急性淋巴细胞白血病的发病状态,为探索急性淋巴细胞白血病全新的治疗方法提供平台。方法:采用抗鼠-CD122抗体注射NOD/SCID小鼠进行预处理,通过尾静脉注射9例不同病例的白血病细胞,以及1株T-ALL细胞系。通过检测小鼠体内白血病细胞及脏器白血病细胞浸润情况,观察白血病细胞植入。将白血病细胞进行二次移植,观察移植稳定性。对白血病动物模型进行药物处理,观察小鼠生存期,模拟人体治疗反应。结果:有4例病例的细胞及T-ALL细胞株成功植入。流式细胞检测显示受体小鼠体内较多比例人CD45+细胞表达,免疫组化显示CD45+细胞浸润小鼠不同脏器,系列移植也获得成功。体内药物处理显示地塞米松能延长小鼠的生存期,与临床观察相一致。结论:成功建立经抗鼠CD122单抗预处理的人T细胞急性淋巴细胞白血病NOD/SCID小鼠模型,具有原发疾病的所有主要特征。     

合作小型猪T细胞受体α链的基因结构及其多样性

目的探讨猪TCR基因分子结构的复杂性及其与人类的相似性。方法以公开的猪TCRα链基因为参考序列设计两对特异性引物,用RT-PCR法从合作小型猪外周血、淋巴结和脾脏的淋巴细胞中克隆了93个猪TCRα基因(简称STA)。结果测序分析表明,克隆的猪TCRα链的基因均含有可变的信号肽区和V区、高变的J区和恒定的C区的基因片段,但基因间的核苷酸序列组成都不完全相同,且具有十分复杂的多态性和多样性,基因间的同源性在68.4%~98.7%,这与TCRα链的基本基因结构特征相一致。依据TCRα基因的同源性对其分子结构、遗传演化关系和归类分析发现,在其信号肽区、FR1区和CDR1区、FR2区和CDR2区以及FR3区和CDR3区都存在一些变异集中点和变异热点区。用IMGT/V-QUEST分析方法可将合作小型猪TCRαV区(STAV)、J区(STAJ)基因片段与人类的进行比较分析,发现合作小型猪TCRα与人类的遗传演化关系较近,每个序列都能找到与人类对应的TRAV、TRAJ基因片段,甚至V区的相似性可达92%以上。结论近交培育的合作小型猪在正常状态具有应对外界复杂微生物等环境的TCR遗传多样性分子基础,且适合作为人类免疫学及疾病研究的动物模型。     

小鼠急性髓系白血病模型的构建

MLL基因的异常重排会引发急性淋巴系(ALL)和急性髓系白血病(AML)。该文详细阐述了利用逆转录病毒载体MLL-AF9构建小鼠AML模型的方法。该研究比较了免疫磁珠法与5-氟尿嘧啶(5-FU)方法富集骨髓细胞的效率,以及不同时间点收获的病毒对骨髓Lin-细胞感染效率的影响。通过流式检测发现, 5-氟尿嘧啶富集的骨髓Lin–细能够被48 h收获的病毒高效感染。受体鼠在移植了MLL-AF9感染的骨髓Lin–细胞60天后,外周血、骨髓、脾脏组织中均有大量的白血病细胞浸润, RT-qPCR也验证了白血病靶基因的表达上调,表明小鼠AML模型的成功构建。这项研究为从事白血病研究的科研人员提供了一种有效的小鼠急性髓系白血病模型,为研究白血病发病机理与研发白血病治疗药物提供有用的工具。     

Lack of TdT and immunoglobulin and T-cell receptor gene rearrangements in Hodgkin's disease

To study the pathogenesis of Hodgkin's disease (HD), which today remains obscure, we have undertaken a combined experimental approach: determination of TdT and molecular analysis of rearrangements of immunoglobulin heavy chain (IgH), T-cell receptor (TCR) beta chain and the T-cell rearranging gamma (TRG) genes. TdT determination indicate would the presence of immature cells that are not detected in the normal lymphnode; molecular analysis of the rearrangements of these genes would reveal the presence of even a small monoclonal population of both T and B lineages in the lymphnodes. We believe that the combination of these two types of analysis can indicate whether an expanding lymphoid clone is responsible for this disease. TdT determination was negative in all 41 cases tested. Gene rearrangements were studied in 10 cases for IgH and TCR beta genes and in 5 cases for the TRG gene. No abnormal band beside the germ-line ones was detected in any of our cases, ruling out the presence of a minor neoplastic population. We can explain these results in at least three ways: first, the neoplastic population could represent less than 1% of the total, thus escaping detection by current techniques; second, the neoplastic population is not lymphoid in nature or is composed of mature cells that do not rearrange Ig and TCR genes and therefore belongs to a true non-B, non-T lineage; third, the pathogenesis of HD is completely different from that of non-Hodgkin's lymphomas (NHL) and does not involve the clonal expansion of a cell frozen at a particular maturative stage as is thought to happen in most NHL.     

汉族人群中血管紧张素转换酶抑制剂所致咳嗽与血管紧张素转换酶基因及缓激肽β2受体基因多态性的关系冰

目的：探讨汉族人群中血管紧张素转换酶抑制剂（ACEI）所致咳嗽与血管紧张素转换酶（ACE）基因及缓激肽β2受体（BDK-RB2）基因多态性的关系。方法：应用聚合酶链反应（PCR）方法。检测汉族人群中151例由于服用ACEI引起的咳嗽患者及151例未发生咳嗽的患者的ACEI／D及BDKRB2C／T的多态性,并采用紫外法检测ACE活性。结果：发现ACE基因分布在咳嗽组中II型为47．0％,ID型为42．4％,DD型为10．6％;无咳嗽组分别为39．7％、47．0％、13.3％,两组相比其差异具有统计学意义（P〈0．01）;BDKRB2基因分布在咳嗽组中CC型为21．3％,CT型为50．0％,TT型为28．7％,无咳嗽组分别为22．5％、47．7％、29．8％。两组相比其差异无统计学意义（P〉0．05）;咳嗽组ACE活性水平为[（28．3±10．1）U／L]明显低于无咳嗽组[（40．2±9．4）U／L],两组相比其差异具有统计学意义（P〈0．01）。结论：汉族人群中ACEI所致咳嗽与ACE基因多态性及血清ACE水平有关,BDKRB2C／T与咳嗽间未发现有统计学意义的关联。     

PCR—DGGE法研究葡萄糖-6-磷酸脱氢酶缺乏症基因突变

目的：应用PCR-DGGE法和DNA测序分析云南籍G6PD缺乏症患者基因突变类型和特点、方法应用硝基四氮唑蓝（NBT）纸片法进行G6PD缺乏症定性筛查,G6PD／6PGD比值法验证,应用PCR—DGGE法和DNA测序分析46例云南籍G6PD缺乏症患者基因突变类型和特点。结果：46例云南籍G6PD缺乏症样本中有30例经PCR—DGGE法分析G6PDexon12发现有异常电泳条带,DNA测序证实26例（56、52％）为nt-1388G→A,4例（8.7％）nt-1376G→T．而PCR—DGGE法分析G6PDexon2未发现有异常电泳条带的样本出现。结论：（1）nt-1388G→A（56．52％）、nt-1376G→T（8．7％）是云南省主要的基因突变型也是中国人中最常见的两种突变型,揭示中华民族有着共同的起源;（2）所检样本中未发现nt95A→G。（3）应用PCR—DGGE法结合DNA测序检测G6PD缺乏症患者的基因型,阳性检出率高,方法简便、快捷、灵敏、结果准确可靠。     

Identification of IL-7-dependent bone marrow-derived Thy-1-B220- lymphoid cell clones that rearrange and express both Ig and T cell receptor genes.

Bone marrow stromal cell lines and lymphoid cell lines were co-established from the Whitlock-Witte type of long term liquid cultures of MRL/1 and C57BL/10 (B10) (Thy-1.1) bone marrow cells. The present study investigates the immunologic nature of parental and cloned lymphoid cell lines. Both strains of parental lines and their clones did not grow alone but proliferated on the monolayers of co-established parental stromal cell lines from a syngeneic or alternative strain. When various lymphokines or cytokines were tested for their capacity to support the growth of these lymphoid cell clones, only IL-7 could substitute for the growth-promoting function of stromal cells. These IL-7-dependent clones expressed neither Thy-1 nor B220 Ag. However, all of them from two strains were found to rearrange synchronously H chain of Ig as well as gamma chain of TCR genes. Some of the clones transcribed a mature size of IgH mRNA. Co-expression of mRNA for lambda 5 but not for IgL chain (kappa, lambda) genes resulted in the generation of cell surface mu chain in these clones. Other clones expressed a smaller size of IgH mRNA without exhibiting surface mu chain. Irrespective of the differences in IgH rearrangements and its mRNA expression, a mature size TCR gamma mRNA was detected in all of the clones. Thus, these results demonstrate the existence of untransformed (IL-7-dependent) immature lymphoid cells rearranging both Ig and TCR genes. Their unique features concerning cell surface markers (B220- mu+), specific growth factor requirement, and various modes of Ig/TCR gene rearrangements are discussed in the context of early lymphoid development.     

胰十二指肠切除术后肠黏膜屏障损伤与肠道细菌移位的临床研究（英文）

目的：探讨胰十二指肠切除手术后肠道细菌移位（BT）与术后全身炎症反应综合征（SIRS）关系。方法：40例择期行胰十二指肠切除手术患者,于术前和术后1、3、5天采集外周血,进行血浆D-乳酸,全血细菌DNA检测.全血DNA提取后进行PCR扩增,采用靶基因为大肠杆菌特异性β半乳糖苷酶基因和16SrRNA基因。观察患者术后10天以监测SIRS情况。结果：术前PCR检测全血细菌DNA均为阴性,术后共有13例阳性。术后出现全身炎症反应综合征（SIRS）的患者PCR阳性率为85.7%（12/14）,无SIRS组为3.8%（1/26（）P〈0.01）。PCR阳性组SIRS发生率为93.2%（12/13）,阴性组为7.4%（2/27）（P〈0.01）.PCR阳性的患者外周血血浆D-乳酸浓度较PCR阴性者明显升高（P〈0.01）,有SIRS的患者外周血血浆D-乳酸浓度较无SIRS患者明显升高（P〈0.01）。结论：胰十二指肠切除术后肠黏膜屏障损伤与BT关系密切,术后SIRS和与BT密切相关。PCR技术对术后SIRS有较好的早期预警价值。     

β淀粉样肽单克隆抗体可变区基因的5′RACE扩增及序列分析

目的:克隆并分析抗β淀粉样肽单克隆抗体轻链与重链可变区基因。方法:从分泌抗β淀粉样肽单克隆抗体的杂交瘤细胞株A8中提取总RNA,根据恒定区序列设计基因特异性引物,通过5′RACE法扩增抗体的轻链和重链可变区基因,测定并分析可变区基因序列,并克隆入pMD18-T载体。结果:重链可变区基因序列全长450bp,编码150个氨基酸残基;轻链可变区基因序列全长429bp,编码143个氨基酸残基。在GeneBank中对氨基酸序列进行比对分析,二者均符合小鼠IgG可变区基因的特征。根据Kabat法则对A8抗体轻链和重链可变区氨基酸序列基因进行分析并确定了3个抗原互补决定区(CDR)、4个框架区(FR)和信号肽。结论:通过5′RACE法得到了抗β淀粉样肽单克隆抗体轻链与重链可变区基因,为进一步研究抗体三维结构,以及对该抗体进行人源化改造奠定了基础。     

Rearrangement of IgH genes in normal thymocyte development

IgH chain gene segments are rearranged in 30 to 50% of peripheral T cells. We have analyzed IgH gene rearrangements during normal T cell development, using a well characterized collection of hybridomas derived from fetal, newborn, adult, or aged thymocytes. Our results show that IgH rearrangements occur in the thymus after T cell receptor gene and T cell specific gamma-gene rearrangements but before thymocyte maturation is completed. Therefore IgH gene rearrangements occur at an intermediate stage in thymocyte development. This may be of significance in delineating human lymphoid leukemias. Not all thymocyte hybridomas carried IgH gene rearrangements. Age-related shifts in frequencies of cells with IgH gene rearrangements, probably indicating changes in the composition of thymocyte populations, were found. Finally, a detailed analysis of D to J joins revealed an ordered progression of partial rearrangements at the IgH locus, whereby the most proximal DH-segment, DQ52, is used predominantly at early stages, but that other D to J rearrangements at the same locus may occur subsequently.     

PCR-based clonality assessment in patients with lymphocytic leukaemias: a single-institution experience

PCR-based clonality testing can be performed in all lymphoproliferations by analysing gene rearrangements of antigen receptors, rearrangements that are unique for each kind of lymphocyte. Reactive lymphoproliferations have polyclonally rearranged Ig/TCR genes, whereas malignant proliferations (leukaemias and lymphomas) show clonal rearrangements. The aim of this study was to assess the clinical benefits of clonality testing with previously evaluated consensus primers in leukaemia patients. The study included peripheral blood and bone marrow samples of 67 leukaemia patients (32 B-CLL, 24 B-ALL and 11 T-ALL). Clonality testing was based on PCR amplification of rearranged IgH and TCR genes. During diagnosis, monoclonal pattern was found in all analysed B-CLL and T-ALL samples. Testing in B-ALL patients showed positive results in all bone marrow and one peripheral blood samples. Results of clonality testing in B-CLL patients during follow-up were concordant between peripheral blood and bone marrow. Obtained results corresponded to clinical course in all but one patient. In B-ALL group, results of molecular testing in peripheral blood and bone marrow confirmed remission estimated according to clinical criteria in all except one patient. Before any clinical sign of relapse, monoclonal pattern was found in six/seven patients by bone marrow and in three/seven patients by peripheral blood analysis, respectively. Results of molecular monitoring in T-ALL patients did not confirme clinical evaluation in two patients. Obtained results indicate high accuracy of re-evaluated primers for clonality assessment in ALL and CLL patients at the time of diagnosis. Results of clonality testing in B-ALL patients indicate that bone marrow analysis has higher sensitivity compared to analysis of peripheral blood.