STR—PCR定量检测供受者嵌合体方法的建立及临床应用

利用STRs的高度多态性等特性,用竞争性聚合酶链反应（PCR）结合聚丙烯凝胶电泳、染色和凝胶成像分析等技术,建立定量检测异基因造血干细胞移植后供受者嵌合体的方法,用无关个体的DNA混合实验进行可行性和准确性研究,并同时做染色体分带进行对照,以此基础上对2例非清髓异基因造血干细胞移植病人的植入情况进行动态检测。结果显示,扩增产物中供受者DNA含量的百分比同扩增前混合样本的比例呈显著直线相关,STR－PCR检测嵌合体方法简单、快速、可靠、敏感性高,所需标本量少且不受性别限制,是检测NAST供受者嵌合体的新方法。     

吻合血管同种异体骨移植术后微嵌合现象的研究

目的 研究吻合血管同种异体骨移植术后受体微嵌合现象（microchimerism）的存在及其与免疫耐受的相关性。方法 吻合血管同种异体大段股骨干移植术后不同时期进行X线和组织学切片检查，并对不同组织器官进行性别决定因子Y（Sry）半定量聚合酶链反应（PCR）分析。结果 实验组在术后不同时期的X线及组织学表现为典型的骨折愈合模式；对照组X线及组织学表现为密度增高的移植骨被大量骨痂包绕，以及畸形愈合等排斥反应征象。Sry半定量PCR分析显示，实验组术后器官组织的微嵌合发生率及嵌合率明显高于对照组，且随术后期的延长而增高。结论 吻合血管同种异体骨移植术后，受体的一些器官组织存在着微嵌合现象，且微嵌合发生率与受体对移植骨组织相容性呈正相关。     

非清髓异基因外周血造血干细胞移植后供体细胞植入证据的研究

采用染色体荧光原位杂交（FISH）并结合常规染色体和染色体分带等方法,观察并比较了4例血液病人非清髓异基因外周血干细胞移植（NAPBSCT）后的植入情况。结果4例病人均有不同程度的植入,全部形成混合性嵌合体,其中2例转化为完全植入。统计结果分析表明,FISH分析中期分裂相与间期细胞的结果无统计学差异。与常规染色体检测结果相比较,FISH分析中期分裂相或间期细胞的植入率高,但无统计学差异。但FISH具有操作简单、实验周期短、结果敏感可靠等优点,适合用于性别不合的NAPBSCT后植入证据检测。     

异基因外周血造血干细胞移植41例临床分析

为分析异基因外周血造血干细胞移植（allo-PBSCT)治疗恶性血液病的效果和并发症的防治，用allo-PBSCT治疗恶性血液病41例，急性移植物抗宿主病（GVHD）预防采用环孢素A（CsA）＋短程氨甲蝶呤（MTX）、霉酚酸酯（MMF）联合CsA＋短程MTX两种方案。结果显示，全部患者均顺利造血重建。HLA完全相合移植急性GVHDⅡ度以上者8例（21．05％）。慢性GVHD发生率76％。急性白血病CR1和慢性髓细胞白血病－慢性期（CML－CP）、HLA完全相合移植34例，复发3例（8．82％）；随访2年以上者存活率71．43％（10／14）。表明allo－PBSCT急性GVHD发生率并不高于骨髓移植，但慢性GVHD发生率明显高；感染和间质性肺炎是移植后死亡的主要原因。     

肾移植术后微嵌合状态的研究

目的探讨肾移植术后微嵌合体的状态及其与移植肾长期存活的的关系。方法采集以男性为供者的70例女性肾移植患者术后外周血白细胞，用套式PCR扩增单拷贝SRY基因来鉴定患者血中是否存在微嵌合体。结果70例肾移植术后0．5～10年的女性患者微嵌合体阳性率为58．6％（41／70），2年以内（68％）及5年以上（72％）与2～5年（44．4％）的阳性率比较有明显差异（P〈0．05）。结论肾移植术后不同时间段嵌合细胞的阳性率有明显差别；长期的微嵌合状态与移植肾长期存活有一定的关系。     

造血干细胞移植术后患者巨细胞病毒感染发生率及临床转归

采用免疫组化法对100例异体造血干细胞移植（Allo-HSCT）和14例自体造血干细胞移植（Auto-HSCT)术后患者、17例非移植患者以及27例正常供者CMVpp6抗原血症检出率进行分析。100例Allo-HSCT术后患者随访观察18（4－36）个月。结果：AlloHSCT者91％，在不同病期出现CMVpp65阳性（CMV－I），其中56％出现CMV疾病；急性移植物抗宿主病（GVHD）发生率为54％，时间为25（11－27天）；慢性GVHD52.9%，CMV相关性死亡率为21％，在各种死因中占主要地位。CMV－I在异体移植后好发，而在AutoHSCT术后患者、非移植患者以及正常人群极为少见，提示，CMV感染与急慢性GVHD呈显著相关性，是影响预后的主要因素。     

冻存胎儿胸腺移植治疗恶性肿瘤的初步观察

本文介绍46例恶性肿瘤患者实施冻存胎儿胸腺移植前后的E玫瑰花形成细胞（E－RFC）水平的变化。移植后较移植前E－RFC水平升高（p＜0．05）。根据移植前E－RFC水平，我们将46例患者分为3组：第1组，E－RFC水平在40％以下者（7例）2组，E－RFC水平在41％－60％之间者（24例）；第3组，E－RFC水平在61％以上者（15例）。移植后各组E－RFC水平较移植前平均升高值分别为：22．2％（p＜0．01）、7％（p＜0．01）、－5．1％（0．2＞p＞0．1）。从结果看出，通过冻存胎儿胸腺移植可以提高恶性肿瘤患者的E－RFC水平，是E－RFC水平在60％以下者。E－RFC水平在61％以上者实施冻存胎儿胸腺移植无提高E－RFC水平作用。     

良恶性胸水细胞端粒酶活性的研究

目的：探讨端粒酶活性检测在良恶性胸水中的诊断价值。方法：采用TRAP－PCR－ELISA定量及TRAP－PCR银染定性法，对32例恶性胸水和29例良性胸水样本进行端粒酶活性分析，并将结果与胸水细胞学诊断结果进行比较。结果：端粒酶定性和定量结果均显示癌性胸水样本中端粒酶活性水平显高于良性胸水样本（P＜0．05）。端粒酶鉴别良恶性胸水的敏感性为71．8％（23／32），特异性为89．7％（3／29）；细菌学检查的敏感性为68．8％（22／32），特异性为100％（0／29）；两无显性差异（P＜0．05）。在22例细胞学阳性胸水样本有19例端粒酶阳性，2例细胞学可疑胸水标本1例端粒酶阳性，10例细胞学阴性胸水样本有3例端粒酶阳性。端粒酶检测和细胞学检查的符合率为82．6％，若2联合应用，胸水诊断的敏感性可提至84．4％。结论：端粒酶活性检测，可能在良恶性胸水的鉴别诊断方面，有重要的辅助诊断价值。     

创面异体移植培养表皮细胞后存活的判定

应用４种方法判定烧伤创面异体移植的培养表皮细胞存活情况：１．聚合酶链反应（ＰＣＲ）查Ｙ染色体特异性ＤＮＡ片段；２．ＰＣＲ扩增ＰＭＣＴ１１８长度多态性基因位点；３．ＰＣＲ／ＳＳＯＨＬＡ－ＤＱａ分型；４．ＤＮＡ指纹。共检测１６例病人的２７个活检标本，均证实有供者的ＤＮＡ，最长时间为移植后９２天。初步实验结果表明：培养表皮异体移植后可存活较长时间。     

非亲缘非清髓造血干细胞移植成功治疗急性淋巴细胞白血病首例报告

为探讨非亲缘非清髓异基因造血干细胞移植（URD－NAPBSCT）在治疗急性白血病中（AL）的作用，1例HLA完全相合的急性淋巴细胞白血病病人接受了URD－NAPBSCT治疗。结果病人顺利度过造血抑制期并获得完全性植入，移植后发生了皮肤Ⅱ度GVHD和间质性肺炎，经治疗后痊愈。结果初步表明，URD－NAPBSCT较简便安全，可为急性白血病提供新的有效治疗手段。     

非清髓异基因造血干细胞移植治疗急性白血病的临床研究

采用非清髓预处理（环胞霉素A、环磷酰胺、阿糖胞苷及CD3单克隆抗体或抗淋巴细胞球蛋白）的异基因外周造血干细胞移植治疗急性白血病5例。5例病人均顺利度过造血抑制期并移植成功。2例例经嵌合性植入转为完全植入,另3例为嵌合性植入。5例中2例发生Ⅱ～Ⅲ度GVHD,经治疗后治愈。5例中4例仍无病存活。结果表明,非清髓异基因造血干细胞移植简便安全,并发症少,疗效较好,为急性白血病的治疗提供了新手段。     

非清髓异基因造血干细胞移植后急性白血病复发5例报告

目的 研究分析非清髓异基因外周造血干细胞移植(NAST)后急性白血病复发的相关因素。方法 29例急性白血病患者NAST后5例复发，男2例，女3例，中位数年龄36岁(18～59岁)。非清髓预处理方案：环磷酰胺、阿糖胞苷及CD3单克隆抗体，3例患者在此基础上加用氟达拉滨。结果 5例均顺利渡过造血抑制期。2例早期形成供者造血细胞完全嵌合体(FDC)并在FDC状态下复发。3例移植早期形成供受者混合造血细胞嵌合体(MC)，2例转为FDC后复发，1例在稳定MC状态下复发。5例中4例复发前无急性或慢性移植物抗宿主疾病(GVHD)，另1例NAST后发生Ⅱ度aGVHD并在皮肤慢性GVHD未治愈状态下复发。5例中1例行2次NAST治疗，在第2次完全缓解，但于2个月后再次复发，放弃治疗。另4例未再继续治疗，2例分别于复发后1和2个月死亡，2例仍带病存活。结论 NAST简便安全，并发症少，白血病复发率无明显升高，为治愈白血病提供了新手段。     

DNA typing in cases of blood chimerism

A chimera is an organism whose cells derive from two or more distinct zygote lineages. and therefore two different blood cell populations circulate in one individual. To point out the potential pitfalls in forensic analysis, a set of triplets (a girl and two boys) who revealed blood chimerism was investigated with four STR systems using PCR. The results indicated that a DNA profile based on DNA extracted from blood can lead to a false determination because the band pattern of each triplet contained a mixture of the original genotype and the genotype of the siblings. Additional investigations on biological materials other than blood must be made in order to find out the real genetic characteristics of each child. Received: 17 July 1998 / Received in revised form: 27 November 1998     

STR typing of skin swabs from individuals after an allogeneic hematopoietic stem cell transplantation

One of the pre-requisites for forensic DNA analysis is the fact that all nucleated cells of a person carry the same genetic information. However, this is not the case for individuals who have received an allogeneic hematopoietic stem cell or bone marrow transplantation, as all new cells formed by the bone marrow no longer show the genetic information of the recipient but that of the donor, while all other cells still carry the original information before transplantation. Thus, STR typing of a blood sample after successful transplantation yields a DNA profile that differs from the recipient’s original profile and corresponds to the donor genotype instead. Evidence from a routine case suggests that transplanted individuals may show donor alleles in skin swabs, as well. In order to examine this issue more closely, various skin swabs from 28 patients who have received an allogeneic hematopoietic stem cell transplantation were examined in this study. Swabs from the right and left palm, the back of the hand, one of the two upper arms, and the neck were collected from each person. Ninety-one of the 140 resulting swabs delivered useful results. All of those samples showed mixtures of recipient and donor DNA with different mixture ratios and the proportions of donor and recipient alleles revealed inter- and intra-individual differences. Those results were discussed with respect to graft versus host disease.

     

HLA基因序列电泳多态性分析及其在骨髓移植中的应用

利用聚合酶链反应和聚丙烯酰胺凝胶电泳技术建立了一套人白细胞抗原配型技术，包括ＨＬＡ－ＤＲ单链构象多态性，异二聚体分析和ＨＬＡ－ＤＲ，ＤＰ－ＤＮＡ链构象多态性分析技术。对６３例骨髓移植供，受体进行了分析，其中经血清学确定为ＨＬＡ相合的１５对ＰＣＲ－ＳＣＰ分析完全相合；５对因病人血细胞数目和功能异常而供，受体血清不方法无法分型，ＰＣＲ－ＳＣＰ分析确定３对相合，２对不相合，病人临床缓解后重复血清学ＨＬＡ     

异基因外周血造血干细胞移植治疗极重度骨髓型和肠型急性放射病的临床报告

目的 探讨异基因外周血造血干细胞移植在肠型和极重度骨髓型放射病救治中的作用和地位.方法 山东"10·21" 60Co 辐射事故中2例病人受到意外照射,病例A受照射剂量20～25Gy,诊断为"肠型放射病",病例B受照射剂量9～15Gy,诊断为"极重度骨髓型放射病".经联合环磷酰胺、抗淋巴细胞球蛋白和氟达拉滨预处理,2例分别行HLA半相合及全相合外周血造血干细胞移植.采用环孢霉素A和骁悉方案(病例A加用CD25单抗和供者间充质干细胞)预防移植物抗宿主病(GVHD).结果 2例均移植成功,供体完全存活,移植后9～11天白细胞开始恢复,2周后白细胞恢复正常、骨髓造血重建成功.2例均未发生移植排斥和GVHD.病例A照射后33天死于败血症和多器官功能衰竭.病例B照射后75天死于心衰为主的多器官功能衰竭.结论 HLA相合及半相合外周血造血干细胞移植救治极重度骨髓型和肠型急性放射病是完全可能和可行的,联合免疫抑制剂预处理对促进供体稳定植入是必要的,环孢霉素A、骁悉和CD25单抗及供者间充质干细胞对预防GVHD有重要作用.     

Identification of tetragametic human chimerism by routine DNA profiling

Chimerism in humans is defined as the presence of two genetically different cell lines within the same organism. It is usually an acquired condition that is restricted to certain tissues and can be explained by therapeutic interventions such as blood transfusion or the transplantation of allogenic hematopoietic cells. Implications of such patients for forensic DNA testing have been described in the literature. In some rare cases, true inherited chimerism is observed. This so called tetragametic chimerism occurs via the fertilization of the two ova by two spermatozoa, followed by the fusion of early embryos and the development of an organism with intermingled cell lines. Such examples have been found in mice and other mammalian species including humans. We describe a phenotypically normal woman in whom tetragametic chimerism (46,XX/46,XX) was unexpectedly identified by STR typing during routine DNA profiling. Cytogenetic analysis proved to be a valuable tool for both independent confirmation and direct visualization of the two coexisting cell lines.

     

Massively parallel sequencing of 17 commonly used forensic autosomal STRs and amelogenin with small amplicons

The next-generation sequencing (NGS) method has been utilized to analyze short tandem repeat (STR) markers, which are routinely used for human identification purposes in the forensic field. Some researchers have demonstrated the successful application of the NGS system to STR typing, suggesting that NGS technology may be an alternative or additional method to overcome limitations of capillary electrophoresis (CE)-based STR profiling. However, there has been no available multiplex PCR system that is optimized for NGS analysis of forensic STR markers. Thus, we constructed a multiplex PCR system for the NGS analysis of 18 markers (13CODIS STRs, D2S1338, D19S433, Penta D, Penta E and amelogenin) by designing amplicons in the size range of 77–210 base pairs. Then, PCR products were generated from two single-sources, mixed samples and artificially degraded DNA samples using a multiplex PCR system, and were prepared for sequencing on the MiSeq system through construction of a subsequent barcoded library. By performing NGS and analyzing the data, we confirmed that the resultant STR genotypes were consistent with those of CE-based typing. Moreover, sequence variations were detected in targeted STR regions. Through the use of small-sized amplicons, the developed multiplex PCR system enables researchers to obtain successful STR profiles even from artificially degraded DNA as well as STR loci which are analyzed with large-sized amplicons in the CE-based commercial kits. In addition, successful profiles can be obtained from mixtures up to a 1:19 ratio. Consequently, the developed multiplex PCR system, which produces small size amplicons, can be successfully applied to STR NGS analysis of forensic casework samples such as mixtures and degraded DNA samples.     

外周造血干细胞移植治疗51例恶性血液病的临床研究

目的探讨外周血造血干细胞移植(PBSCT)技术治疗恶性血液病的疗效及安全性。方法 51例恶性血液病患者中,34例行亲缘全相合异基因PBSCT(allo-PBSCT);5例行亲缘半相合allo-PBSCT,12例行自体PBSCT。结果全部患者均成功植入并快速重建造血。亲缘全相合allo-PBSCT中Ⅰ～Ⅱ度急性GVHD的发生率为26.4%,未见Ⅲ～Ⅳ度急性GVHD;慢性GVHD的发生率为54.1%。亲缘半相合allo-PBSCT中Ⅰ～Ⅲ度急性GVHD的发生率为32.1%,未见Ⅳ度急性GVHD;慢性GVHD的发生率为74.5%。本组死亡11例,总病死率为21.6%。结论 auto-PBSCT、亲缘HLA配型完全相合与半相合allo-PBSCT均具有造血重建快、aGVHD发生率较低、疗效较好、安全可行及住院时间短的优点,尤其亲缘半相合allo-PBSCT为难治性白血病患者开辟了新的治疗途径。