负载肝癌肿瘤抗原的B淋巴细胞诱导特异性抗肿瘤免疫的实验研究

目的:探讨体外经小鼠肝癌细胞不同抗原致敏的CD40配体活化的B淋巴细胞诱导的特异性细胞毒T淋巴细胞(CTL)抗肿瘤免疫的能力.方法:分离、纯化T、B混合淋巴细胞,并在CD40L、rmIL-4联合作用下培养.然后分离T、B淋巴细胞以备用.将凋亡的肝癌细胞及其冻融裂解物作为实验组,1640培养基作为空白对照组分别与B淋巴细胞共同培养,检测培养后各组B细胞表面抗原呈递细胞标记(CD40、CD80、CD86)及主要组织相容性抗原的表达情况.利用混合淋巴细胞增殖实验检测T细胞增殖情况.以负载肿瘤抗原的B淋巴细胞诱导的CTL作为效应细胞,肝癌细胞Hepal-6为靶细胞,LDH释放实验检测杀伤活性.结果:负载肿瘤抗原的B淋巴细胞具有刺激T细胞增殖的能力,实验组的B淋巴细胞,其组织相容性分子及其刺激分子表达明显高于空白对照组,其对靶细胞的杀伤率与空白对照组比较经统计学分析差异有显著性.结论:负载肝癌肿瘤抗原的B淋巴细胞作为抗原呈递细胞诱导的CTL可有效产生特异性抗肝癌免疫作用.     

结节性淋巴细胞为主型霍奇金淋巴瘤3例临床病理观察

目的探讨结节性淋巴细胞为主型霍奇金淋巴瘤(NLPHL)的临床病理特征和病理诊断。方法复习3例NLPHL患者的临床病理资料、免疫表型、EBV原位杂交及抗原受体基因重排PCR检测结果。结果 3例NLPHL中,淋巴结结构破坏,淋巴组织呈结节性或结节-弥漫性增生,可见进行转化的生发中心(PTGC),小淋巴细胞背景中散在大型单个核或多分叶核异型肿瘤细胞。免疫组化:3例肿瘤细胞CD20、PAX5、Oct-2和BOB.1均(+);仅1例CD30弱(+);2例bcl-6(+);CD3、CD15和LMP1(-);CD21(+)显示扩大的不规则滤泡树突细胞网。3例EBV原位杂交均(-)。运用激光显微切割收集肿瘤细胞并对其进行抗原受体基因重排PCR检测,2例IgH单克隆性重排(+)(1例为Fr2a,1例为Fr3a),3例TCRγ多克隆性重排。结论 NLPHL是一种罕见的具有独特临床病理特征的霍奇金淋巴瘤亚型,细致的形态学观察及完善的免疫组化检查有助于诊断和鉴别诊断。     

CD45RO阳性的B细胞性淋巴瘤

目的 研究免疫表型异常淋巴瘤的起源。方法 从48例B细胞淋巴瘤中筛选出3例CD45RO、CD20和CD79a( )，CD3(-)的淋巴瘤。采用免疫组化、PCR和基因扫描技术对其免疫表型，免疫球蛋白重链基因重排(IgH)和T细胞受体基因重排(TCR)进行深入研究。结果 3例淋巴瘤均为结外淋巴瘤。1例为滤泡性淋巴瘤，2例为弥漫性大B细胞性淋巴瘤。PCR和基因扫描显示3例均有IgH克隆性扩增；PCR未显示TCR扩增；高敏感性基因扫描显示低峰值TCR克隆性扩增，提示可能为背景T细胞扩增。结论 CD45RO不是T细胞特异性抗原，CD45RO阳性细胞不能完全等同于T细胞，因此，在研究和临床病理诊断中应选用多种抗体配合使用。     

原发性皮肤边缘区B细胞淋巴瘤临床病理特征

目的 探讨原发皮肤边缘区B细胞淋巴瘤(PCMZL)的临床和病理学特征,诊断和鉴别诊断.方法 对2例PCMZL和1例皮肤淋巴组织增生(CLP)病例进行临床特征、组织形态学、免疫表型及PCR IgH基因重排分析.结果 免疫组化示2例PCMZL的小淋巴细胞CD20和CD79(卅),bcL-2、BOB.1和Oct-2(++),MUM-1(+),CD5、CD10、bel-6和CD23(-);浆细胞CD138、MUM-1(++),限制性表达轻链kappa.1例CLP的小淋巴细胞CD20、CD3和bcl-2(++),MUMl(+);浆细胞同时表达K及λ.IgH扩增2例PCMZL呈单克隆性,1例CLP显示多克隆性.结论 PCMZL属MALT-L家族,形态学上肿瘤细胞由异源性小淋巴细胞组成,免疫学上显示后生发中心标记.浆细胞轻链限制性和B细胞抗原受体基因克隆性重排对诊断有非常重要的帮助.     

蛋白和基因水平探索H/RS细胞的起源

目的：从蛋白、组织和单细胞的基因水平进一步探索H／RS细胞的来源及其克隆性。方法首先对33例经典型霍奇金淋巴瘤(cHL)标本进行B细胞特异性激活蛋白(BSAP)和CD20的检测，然后对33例cHL患的石蜡刮片组织和部分阳性病例的微切割细胞进行免疫球蛋白重链基因克隆性重排检测，并对同一病例的石蜡刮片组织和微切割细胞的扩增产物进行测序分析比较。结果33例中30例的H／RS细胞表达BSAP，10例表达CD20，BSAP和CD20的表达率差异有显性(P=0.000)，对照病例中反应性增生淋巴结的B淋巴细胞和B细胞淋巴瘤肿瘤细胞的BSAP和CD20表达均为100％，T细胞淋巴瘤的肿瘤细胞无表达；33例中的16例石蜡刮片组织IgH基因重排阳性，微切割的19管H／RS细胞有14管出现重排阳性，细胞数目不同的各管阳性率差异无显性(P=0．280)；同一病例的石蜡刮片组织和微切割细胞的PCR产物测序结果均为IgH可变区片段，但是碱基序列并不完全相同。结论：进一步支持cHL中大多数H／RS细胞为B细胞起源，且可能来源于其不同的分化阶段。     

鼻咽部滤泡树突状细胞肉瘤的临床病理观察

目的探讨鼻咽部滤泡树突状细胞肉瘤的临床病理特点、诊断及鉴别诊断要点。方法回顾分析2例鼻咽部滤泡树突状细胞肉瘤的临床表现、影像学、组织病理学特点及免疫组化表型。结果 2例均为女性,年龄分别为53岁和56岁。肿物最大径分别为4.4 cm和4 cm。2例肿瘤均由梭形细胞组成,细胞片状排列,局部略呈车辐状、束状、旋涡状,细胞异型性不明显。免疫组化:vimentin、CD21和CD23均(+),CK(-);例1 EMA(+),CD35(-),EB病毒(+);例2 CD35(+),EMA(-),EBV(-)。例1放弃治疗,随访2个月,目前带瘤生存;例2接受了肿物扩大切除,术后25个月复发,拒绝再次手术,目前带瘤生存32个月。结论鼻咽部滤泡树突状细胞肉瘤非常罕见,明确诊断需结合组织病理学形态、免疫组化表型及EB病毒检测。     

一种新的早幼粒细胞白血病/维甲酸受体α融合基因S亚型变异易位

目的：逆转录聚合酶链反应（RT－PCR）检测早幼粒细胞白血病／维甲酸受体α（PML／RARα）融合基因，筛查变异易位并对变异易位产物测序，以进一步了解变异易位的特点及临床意义。方法：取急性早幼粒细胞白血病（APL）患者骨髓，RT－PCR检测L亚型和S亚型，发现的变异易位经全自动测序仪测定其碱基序列。结果：11例PML／RARα融合基因表达的患者中，S亚型1例、L亚型8例、变异S亚型合并L亚型2例；变异S亚型产物测序得到206bp的碱基序列，检索证实是一种新的变异易位。结论：发现了一种新的S亚型变异易位，证实同一个体可有S和L二种不同亚型并存；提示PML／RARα融合基因的RT－PCR检测中，识别变异易位有重要意义。     

乳腺癌患者外周血中颗粒酶B和穿孔素的表达及意义

目的 分析乳腺癌患者外周血T淋巴细胞及NK细胞中颗粒酶B和穿孔素的表达对乳腺癌的诊断价值，明确颗粒酶B和穿孔素的表达与乳腺癌分子分型、肿瘤分期等临床病理特征的相关性及其对乳腺癌治疗效果的评价。方法 应用流式细胞术检测108例乳腺癌和70例乳腺良性疾病患者样本中CD3⁺T细胞、CD8⁺T细胞及NK细胞中颗粒酶B和穿孔素的表达；采用受试者工作特征(ROC)曲线评估各指标对乳腺癌的诊断效能，比较不同临床病理特征乳腺癌患者颗粒酶B和穿孔素的表达及治疗前后各指标的变化。结果 乳腺癌患者T淋巴细胞亚群与NK细胞中颗粒酶B和穿孔素表达均高于对照组，差异均有统计学意义(P <0.05)。ROC曲线分析结果显示，CD3⁺T细胞、CD8⁺T细胞及NK细胞中颗粒酶B和穿孔素阳性率的曲线下面积(AUC)大于0.5。CD3⁺T细胞中颗粒酶B和穿孔素的表达与乳腺癌肿瘤大小及临床分期呈正相关(P <0.05),CD8⁺T细胞中颗粒酶B也与乳腺癌肿瘤大小及临床分期呈正相关(P...     

多发性骨髓瘤患者IgH-MMSET基因的检测及其意义

目的：检测多发性骨髓瘤(MM)患由于t(4；14)所形成的IgH-MMSET融合基因，探讨其在MM中的临床意义。方法：采用RT-PER法在25例MM患骨髓标本和MM细胞系NCI-H929中检测IgH-MMSET融合基因，并且通过巢式PCR法提高反应的灵敏度。PCR产物纯化后克隆到pGEM-T载体，并用引物M13 Forward测序。将PCR产物的序列与GenBank进行对照，进一步证实发生易位的基因。结果：作为阳性对照的MM细胞系NCI-H929扩增出一明显条带，长度为438bp，经测序后证实为IgH基因与MMSET基因的融合产物。14号染色体及4号染色体上的断裂点分别位于IgH基因的Cμ区及MMSET基因的第3内含子中。25例MM患的骨髓标本扩增后有3例(12．O％)呈现阳性，经分别测序后证实为IgH-MMSET融合基因，扩增产物的长度分别为237bp、239bp和239bp，三第4染色体的断裂方式均与NCI-H929相同，其中l例缺失了MMSET基因的第4外显子的第74位碱基(A)和第75位碱基(T)。结论：MM患IgH-MMSET融合基因是由于t(4；14)所形成，其发生率为12．O％。IgH-MMSET融合基因的出现可能是MM患预后不良的指标之一。     

腮腺黏膜相关淋巴组织型结外边缘区B细胞淋巴瘤1例

患者男性,82岁。主因发现右侧腮腺肿物伴右侧面部疼痛4月余入院。CT扫描显示右侧腮腺实质性肿物突入颞下间隙。临床诊断:右侧腮腺恶性肿物。在全麻下行右侧腮腺深叶及肿物摘除术。病理检查巨检:不规则组织块,5cm×5cm×4cm大小,呈囊性,囊壁完整,表面粗糙,与腮腺组织粘连。剖面呈灰色,有白色液体溢出。镜检:有反应性滤泡出现,瘤细胞为滤泡中心细胞样细胞有亲上皮性,为淋巴上皮病变,伴有浆细胞成分(图1～3)。免疫组化:CD20弥漫( )(图4),背景小淋巴细胞CD5、CD3( ),CD10及AE1/AE3(-)。病理诊断:右侧腮腺黏膜相关淋巴组织型结外边缘区B细…     

222 base pairs in NS5B region and the determination of hepatitis C virus genotype 6

OBJECTIVE: The present study was performed to genotype hepatitis C virus (HCV) by direct sequencing of a 222-bp nucleotide in the NS5B region and comparing the results with those of direct sequencing in the core region. We investigated a new region for HCV genotyping which gave the best performance to discriminate HCV genotype 6a, the unique genotype found in Southeast Asia. METHODS: Plasma samples taken from 57 HCV-infected blood donors were used in this study. RT-PCR products were amplified using primers located in the NS5B region. The 222-bp PCR products were purified and sequenced. The genotype of HCV isolates were obtained by phylogenetic analysis and compared with HCV reference strains stored in the GenBank database. The HCV sequences clustering in the same node were considered to be of the same genotype. RESULTS: Thirty-one, 22 and 4 samples of HCV genotype 3a, 1a and 1b, respectively, were analyzed by this method. Upon comparison with genotyping in the core region, 86 and 14% of the samples yielded concordant and discordant genotype results, respectively. The majority of discordant results (63%; 5 of 8) was observed with HCV genotype 6a which yielded 6a upon core sequencing as opposed to 1a or 3a upon NS5B sequencing. CONCLUSION: HCV genotype 6a obtained by direct sequencing in the core region could not be unequivocally arrived at by sequencing 222 bp in the NS5B region. Hence, sequencing in the core region is preferable for genotyping our specimens, even though longer PCR products are required as this method enables discrimination between genotype 6a and the remaining genotypes.     

Somatic mutation of human immunoglobulin V genes in the X-linked HyperIgM syndrome.

Somatic mutation of Ig variable regions occurs prominently in germinal centers, but it has been debated whether the mutation process initiates in germinal centers or is activated before germinal center entry of B cells. We have analyzed for the presence of somatic mutation in Ig gene rearrangements of the nonpolymorphic human VH6 gene in the X-linked HyperIgM syndrome, which is associated with defective CD40 ligand expression and absence of germinal centers and generation of memory B lymphocytes. IgM and rare IgG VH6 productive rearrangements were isolated from PBL of patients with X-linked HyperIgM syndrome. Although the majority of both the IgM and IgG VH6 rearrangements had a germline VH6 sequence, 7 of 102 VH6 IgM and 1 of 6 IgG rearrangements had a mutated VH6 gene. The mutation frequency (mutations/bp) was 1.4% with a range of 2-9 mutations per clone, a mutation frequency lower, however, than that observed in IgM (3.2%) and IgG (5.4%) VH6 rearrangements of normal individuals. These results suggest that somatic mutation may be initiated in a CD40 ligand-independent pathway before entry of B cells into germinal centers, but fails to achieve the high mutation frequency observed in the presence of germinal centers.     

PCR analysis of the immunoglobulin heavy chain gene in polyclonal processes can yield pseudoclonal bands as an artifact of low B cell number

Polymerase chain reaction (PCR)-based analysis for detecting immunoglobulin heavy chain gene (IgH) rearrangements in lymphoproliferative disorders is well established. The presence of one or two discrete bands is interpreted as a monoclonal proliferation, whereas a smear pattern represents a polyclonal population. Prompted by our observation of discrete bands in histologically reactive processes with a relative paucity of B cells, we sought to determine whether low numbers of B cells in biopsy specimens could artifactually produce pseudomonoclonal bands. We performed IgH PCR analysis on serially diluted DNA samples from 5 B cell non-Hodgkin’s lymphomas (B-NHLs), 5 reactive lymph nodes, 5 reactive tonsils and 10 microdissected germinal centers from a lymph node with follicular hyperplasia. We also assessed multiple aliquots of DNA samples from small biopsy specimens of reactive lymphocytic processes from the stomach (5 cases). PCR products were evaluated using high resolution agarose or polyacrylamide gels, and DNA sequencing was performed on IgH PCR products from two reactive germinal centers, which yielded monoclonal bands of identical size. All 5 B-NHLs harboring monoclonal B cell populations yielded single discrete bands, which were maintained in all dilutions. By contrast, all of the reactive lesions with polyclonal patterns at 50 ng/μl starting template concentration showed strong pseudomonoclonal bands at dilutions of 1:1000 to 1:1500 in placental DNA. Two of the microdissected reactive germinal centers that showed bands of identical size on duplicate reactions were proven to have different IgH sequences by sequencing. We conclude that specimens containing low numbers of polyclonal B cells may produce pseudomonoclonal bands on IgH PCR analysis. IgH PCR analysis should be performed on multiple aliquots of each DNA sample, and only samples that yield reproducible bands of identical size can be reliably interpreted as monoclonal.     

在HeLa细胞中表达人βIVS—Ⅱ—nt—654C→突变基因

OBJECTIVE: To establish a cell model expressing human betaIVS- II -nt 654C --> T allele (beta654 mutant). METHODS: DNA fragment of entire beta-globin gene encompassing the codon region and poly(A) signal of the allele was amplified by long PCR from the genomic DNA of a homozygote with beta65.4 mutation. The amplified fragments were cloned into the Hind III and Xba I sites of the pcDNA3.1 vector. After reidentification of the clone with beta654 mutant by DNA sequencing, the recombinant plasmid was transfected into HeLa cell using liposome method. The expression of the beta654 mutant gene in the transfected cells was detected by RT-PCR. RESULTS: Both the normally processed beta globin mRNA (183bp) and aberrant processed beta globin mRNA (256bp) were identified in the transfected HeLa cells, while no RT-PCR product was detected in the controls. CONCLUSION: The transfected HeLa cells can express human beta654 allele.     

重叠延伸PCR法构建VPS4B基因定点突变真核表达载体

目的 构建含液泡蛋白质分选因子4B(VPS4B)基因定点突变的真核表达载体.方法 用RT-PCR法从HuH-7细胞中扩增VPS4B基因,并克隆到真核载体pXF3H上.采用重叠延伸PCR定点突变技术,构建K180Q和E235Q两种突变质粒,经DNA测序确证定点突变的结果,再将VPS4B及两种突变载体转染HepG2细胞,w...     

Castleman病的临床探讨

目的：旨在提高对Castleman病的诊断和治疗水平,方法：搜集了6个病例,进行分析。结果：本病女性多于男性,中年发病居多,纵膈和腹腔淋巴结是好发部位,临床上局灶型比多中心型多见,病理类型中血管滤泡型和浆细胞型之比为4：2,本病预后较好,结论：该病与人疱疹病毒8（HHV－8）有关,该病患者此病毒抗体阳性率可达90％以上,诊断依靠组织学确定,通过病理本病可和淋巴瘤区分之间的不同,治疗方面,手术切除是首选,应用干扰素类抗病毒药物,有助于控制本病的发展。     

腹部Castleman病影像学诊断

目的：探讨腹部Castleman病的影像学表现。材料与方法：腹部Castleman病4例,女3例,男1例。均行X线、超声、CT检查并经手术及病理证实。结果：病灶为单发,肾形或椭圆形,均有完整的包膜,可见条状、绒毛状或珊瑚状钙化;超声以低回声为主,可见点条状强回声,后方伴声影;CT平扫示软组织肿块,边缘清楚,周围有点条状影,增强肿块与周围点、条状影呈明显均匀强化。结论：Castleman病的影像学表现缺乏特征性;肿块不侵及邻近组织和器官,见到肿块内出现条状、树枝样钙化应想到本病的可能。     

一种人FLT3配基同源异型蛋白关组织中的分布

目的 研究人ＦＬＴ３配基膜外区缺失１３９ｂｐ的同源异型蛋白在组织中的分布特点。方法 采用聚合酶链或逆转录－聚合酶链反应法对ＦＬＴ３配基在人肝脏、脑、骨髓、肌肉、肾脏、正常人外周血及４种白血病细胞系中的分布进行了研究,并利用ＤＮＡ测序法鉴定ＰＣＲ产物。结果 除脑中未见ＦＬＴ３配基表达外,在肝脏、肌肉、骨髓、肾脏、胎肝、正常人外周血细胞及白血病细胞系中均可检测到ＦＬＴ３配基,而膜外区缺失１３９ｂｐ的Ｆ     

Intrinsic constraint on plasmablast growth and extrinsic limits of plasma cell survival

B cells recruited into splenic antibody responses grow exponentially, either in extrafollicular foci as plasmablasts, or in follicles where they form germinal centers. Both responses yield plasma cells. Although many splenic plasma cells survive <3 d, some live much longer. This study shows that early plasma cell death relates to a finite capacity of the spleen to sustain plasma cells rather than a life span endowed by the cell's origin or the quality of antibody it produces. Antibody responses were compared where the peak numbers of plasma cells in spleen sections varied between 100 and 5,000 cells/mm(2). In each response, plasmablast clones divided some five times, with the peak numbers of plasma cells produced relating directly to the number of B cells recruited into the response. The spleen seems to have the capacity to sustain between 20 and 100 plasma cells/mm(2). When this number is exceeded, there is a loss of excess cells. Immunoglobulin variable region gene sequencing, and 5-bromo-2'-deoxyuridine pulse-chase studies indicate that long-lived splenic plasma cells are a mixture of cells derived from the extrafollicular and germinal center responses and cells derived from virgin and memory B cells. Only a proportion has switched immunoglobulin class.