Objective:To investigate the practicality of normal rat serum to be used as a negative control of medicine-loaded serum in testing protein expression profile. Methods:PBMC were collected from patients with internal heat due to Yin deficiency type SLE.PBMC of experimental group were cultured with 20% rat serum,while PBMC of control group were cultured with 20% RPMI 1640 medium.SELDI-TOF-MS was adopted to detect protein expression profile of PBMC after incubation. Results:PBMC of experimental group showed differential expression of thirty-two proteins compared with control group,11 of which showed statistically significant difference.The potential protein biomarkers of 7027,5270,2908,4128,2311Da were established for difference detection between two groups.By bioinformatics method the protein with peak mass/charge ratios of 7027Da was identified as HLADRB1 protein coded by HLADRB1 gene. Conclusion:Protein expression profiling studies have shown that normal rat serum itself had regulation function on protein expression profile by PBMC of SLE patients.It is necessary to take it into consideration when we analyze the effect of medicine-loaded serum.

Objective To study the polymorphisms of human leukocyte antigen (HLA)-A, B and DRB1 genes and haplotypes at high-resolution level in Northern Chinese Han population. Methods 631 randomly selected Northern Chinese Han individuals were genotyped by polymerase chain reaction-sequence based typing (PCR-SBT) method for their HLA-A, B and DRB1 genes, and then the frequencies of alleles and haplotypes were estimated by maximum-likehood estimation method. Results Thirty HLA-A alleles were observed and five common alleles with frequencies higher than 0.05 which included A * 1101, A * 2402, A* 0207, A * 0201 and A * 3303 accounted for 69.26% of the total for HLA-A locus. Sixty-four alleles were seen at HLA-B locus, and B* 4001,B* 4601,B* 5801 and B* 1502 were the most frequent with frequencies higher than 0.05 which comprised 38.11% of the total. Among the forty-one HLA-DRB1 alleles, seven alleles with frequencies higher than 0.05 included DRB1 * 0901, DRB1 * 1501, DRB1 * 1202,DRB1 * 0701, DRB1 * 0803, DRB1 * 1101 and DRB1 * 0405, which accounted for 59.98% of the total. 499 A-B, 659 B-DRB1 and 2 426 A-B-DRB1 haplotype were,observed,respectively. 38 A-B, 39 B-DRB1 and 31 A-B-DRB1 haplotypes showed frequencies higher than 0.005 which comprised 59.82%,53.69% and 32.38% of the total. The most frequent haplotype was A * 0207-B * 4601, B * 4601-DRB1 * 0901 and A * 0207-B* 4601-DRB1 * 0901 for each type. Conclusion The presented allelic frequencies and haplotype diversities at high-resolution level in Northern Chinese Han population could be served as reference standard for the anthropology, legal medicine, transplantation matching and disease association studies.

This study was aimed to develop and establish an efficient method for high through-put automatically extracting genomic DNA from EDTA-anticoagulated whole blood samples, and to utilize this method in routine rSSO HLA genotyping by luminex flow array assay, the genomic DNA was extracted automatically from 400 microl blood samples by using TECAN DNA workstation and 96-well plate with 2 ml volume per well. The yield and purity of each DNA sample was tested by UV-spectrophotometer, the integrity of these DNA samples were run electrophoresis on the agarose gel. Each DNA sample was subjected to PCR amplification and hybridization using One lambda rSSO HLA-A, -B and -DRB1 commercial kit, the fluorescent intensity for positive bead and negative bead hybridized with HLA-A, -B and -DRB1 PCR products were calculated and analyzed. The results showed that the mean yield and purity (A260/A280) of genomic DNA extracted from 400 microl whole blood samples were 3.217+/-0.715 microg and 1.710+/-0.103 respectively. The molecular weight was more than 15 kb in size and the fluorescent intensity for positive bead hybridized with HLA-A, -B and -DRB1 PCR products of each sample was >600 RFU, however, the fluorescent intensity for negative bead for each sample was <50 RFU. It is concluded that the highly qualified genomic DNA can be extracted automatically from blood samples of marrow-donors by using TECAN DNA workstation, and the extracted DNA samples are suitable for high through-put HLA genotyping by luminex flow array assay and other downstream transplant immunological and molecular biological experiments.

目的探讨高甘油三脂(TG)对机采血小板保存的影响。方法使用全自动血细胞分离机(MCS+)采集浓缩血小板(PC)24份,用高浓度TG贫血小板同种异体血浆(PPP)制备血小板悬浮于高TG血浆的模型,调整每份PC的TG终浓度分别为正常人群中值(2.3 mmol/L)的1倍、2倍和3倍,用低浓度PPP调整PC的TG使其终浓度为正常人群中值的0.7倍作为对照。样本于22℃保存,并分别于d 0、1、2、3、4、5测定胶原诱导的血小板聚集率和血小板膜CD62P和CD63的阳性表达率。结果随着保存期的延长,各组的血小板聚集率呈下趋势;相同保存时间内,TG含量越高血小板聚集率降低越明显;在保存的d 1—5,高TG组与对照组相比差异有统计学意义。与此同时,CD62P和CD63的阳性表达率呈上升趋势;在保存的d 0—5,高TG组的CD62P和CD63的阳性表达率>对照组。结论悬浮于高甘油三脂血浆的血小板体外保存研究表明,高含量TG不利血小板的保存质量,可增加血小板的活化和降低血小板聚集功能。

OBJECTIVE:To explore the distributive characteristics for leukemia and to provide scientific reference for its prevention and intervention.METHODS:Microsoft SQL 2005 databases was used to make a mathematical analysis of 3708 patients with leukemia in Chinese Marrow Donor Program (CMDP) from 2000 to 2006. The distributive characteristics were calculated by sex, age and area of patients with leukemia and then compared by constituent ratio and relative ratio statistics method.RESULTS:A total of 3708 cases of leukemia were registered for waiting donor during the period 2000-2006 in CMDP, the age of patients were from 7 months to 69 years, the median age of diagnosis was 24.5 years, standard deviation was 6.7-years-old; males suffered more than females, and the ratio was 1.95: 1 (2451/1257). There were 1202 patients with acute lymphoblastic leukemia (ALL), 1066 with acute myeloid leukemia (AML), 1435 with chronic myeloid leukemia (CML), 5 with chronic lymphoblastic leukemia (CLL), CML was the most common patients. The distributive of 3708 patients with leukemia peak was from 15 to 30 years age group, 542 patients were at the age of 15 years, 559 patients were at the age group above 20 years, 514 patients were at the age above 25, 522 patients were at the age over 30-years-old. ALL patients were accounted for 49.36% (613/1242), AML patients accounted for 27.78% (245/1242), CML patients accounted for 22.78% (283/1242), CLL patients accounted for 0.08% (1/1242) in the age group of under 20 years (childhood group). All subjects were mainly in childhood patients with leukemia; The distributive of patients with leukemia in 30 areas were different, leukemia patients were not registered in one area, 494 patients were at the highest peak, 101 patients were in the median.CONCLUSION:The majority of leukemia patients for waiting stem cell transplantation were registered among children and the adolescents groups, males were suffered more than the females. For children, the major type of leukemia was ALL, being necessary to pay more attention to the education of health, and the precaution of leukemia. The distributive of patients with leukemia for waiting stem cell transplantation was different in 30 areas, and the peak region of leukemia should be in Jiangsu, Guangdong, Shangdong, and Zhejiang provinces.

Aim: To identify human leukocyte antigen (HLA)-DRB1*1454 allele and HLA-DRB1 exon 3 sequence information in the Chinese population, which is significant for organ transplantation, cell transplantation, and human genetics. Methods: Polymerase chain reaction sequence-based typing (PCR-SBT) was used to identify HLA-DRB1 alleles from 58 donor-recipient individuals who would undergo haemopoietic stem cell transplantation. Medium to high resolution polymerase chain reaction-reverse sequence specific oligonucleotide probe (PCR-RSSOP) was used to identify HLA-DRB1 alleles from 1 268 healthy donors from Guangdong province. The some ambiguous results of HLA-DRB1*14-associated alleles were confirmed by high resolution polymerase chain reaction-sequence-specific primer typing (PCR-SSP). Results: HLA-DRB1*1403, 1406, 1410, 1412, 1418, 1425 and 1454 alleles were detected in 1 268 healthy donors. HLA-DRB1*1454 was confirmed in 8 ambiguous results of HLA-DRB1*1401/1434/1454 alleles, and HLA-DRB1*1454 was one of common alleles of HLA-DRB1*14 allele group in Guangdong population. HLA-DRB1*14 exon 3 sequence information was confirmed to be polymorphic in Chinese population. Conclusion: HLA-DRB1*1454 and exon 3 of DRB1 are confirmed to be polymorphic in Chinese population, further elucidating that HLA-DRB1 exon 3 sequence information is important for Han population and some minority groups.

Objective To evaluate the heterozygous ambiguity resolution primers (HARPs) method in resolving ambiguous genotyping results of human leukocyte antigen (HLA) genes in Chinese Hart population, and choose some appropriate HARPs primers. Methods HLA-A, HLA-B and HLA-DRB1 genes of 416 southern Chinese Han individuals were genotyped by sequence-based-typing(SBT) method and then the ambiguous genotyping samples were sequenced again by HARPs primers provided by American Atria company. Results The percentage of ambiguous genotyping samples resolved by HARPs for HLA-A, HLA-B and HLA-DRBI locus was 86.3% (132/153), 73.9% (130/176) and 38.1% (85/223) respectively. Among them, 48.5% (64/132)HLA-A, 80.0% (104/130)HLA-B and all HLA-DRB1(85/85)samples only need one primer, 47.7 % (63/132)HLA-A and 20.0% (26/130)HLA-B samples need two primers. Three to six different HARPs primers can resolve more than 90% ambiguities. Conclusion HARPs is a convenient method and could be a routine method to resolve ambiguities for HLA-A, HLA-B and HLA-DRB1 genes genotyped by SBT in Chinese Han population.

ObjectiveTo analyze the polymorphism of HLA-A,B,and DRB1 alleles and their haplotypes in Chinese Man population.Methods Frequencies of HLA-A,B and DRB1 alleles and haplotypes were estimated by maximum-likelihood estimation method based on the genotypes of 2183 Chinese Man bone marrow donors.ResultsA total of 18 HLA-A alleles,44 HLA-B alleles and 15 HLA-DRB1 alleles were detected in Man population,and the most frequent alleles were A*02,A*11,A*24,A*30,A*33,B*13,B*35,B*46,B*51,B*40(B60),B*40(B61),B*15(B62),DRB1*04,DRB1*07,DRB1*08,DRB1*09,DRB1*11,DRB1*12,DRB1*13,DRB1*14 and DRB1*15.A*30-B*13,A*02-DRB1*15,B*13-DRB1*07 and A*30-B*13-DRB1*07 were the most frequent haplotypes in Man population for A-B,A-DRB1,B-DRB1 and A-B-DRB1 haplotype,respectively.The number of haplotypes with frequency ≥ 0.01 for A-B,A-DRB1 and B-DRB1 haplotype was 31,24 and 27,respectively,and ≥ 0.005 for A-B-DRB1 haplotype was 32.There were 14 in A-B,3 in A-DRB1,14 in B-DRB1 and 38 in A-B-DRB1 haploypes that showed strong linkage disequilibrium with ALD≥0.40.ConclusionsThe distribution of HLA-A,B and DRB1 alleles and haplotypes in Man population was similar to that in Northern Chinese Han population.

AIM: To identify human leukocyte antigen (HLA)-DRB11454 allele and HLA-DRB1 exon 3 sequence information in the Chinese population, which is significant for organ transplantation, cell transplantation, and human genetics.METHODS: Polymerase chain reaction sequence-based typing (PCR-SBT) was used to identify HLA-DRB1 alleles from 58 donor-recipient individuals who would undergo haemopoietic stem cell transplantation. Medium to high resolution polymerase chain reaction-reverse sequence specific oligonucleotide probe (PCR-RSSOP) was used to identify HLA-DRB1 alleles from 1 268 healthy donors from Guangdong province. The some ambiguous results of HLA-DRB114-associated alleles were confirmed by high resolution polymerase chain reaction-sequence-specific primer typing (PCR-SSP).RESULTS: HLA-DRB11403, 1406, 1410, 1412, 1418, 1425 and 1454 alleles were detected in 1 268 healthy donors.HLA-DRB11454 was confirmed in 8 ambiguous results of HLA-DRB11401/1434/1454 alleles, and HLA-DRB11454 was one of common alleles of HLA-DRB114 allele group in Guangdong population. HLA-DRB114 exon 3 sequence information was confirmed to be polymorphic in Chinese population.CONCLUSION: HLA-DRB11454 and exon 3 of DRB1 are confirmed to be polymorphic in Chinese population, further elucidating that HLA-DRB1 axon 3 sequence information is important for Han population and some minority groups.

急性非淋巴细胞白血病(AML)是我国白血病分类构成中最常见的一类,其疾病的发生、发展机制目前尚不十分清晰,可能与遗传因素、外界环境因素和自身免疫因素等有关.人类白细胞抗原(HLA)与肿瘤的免疫监视作用密切相关,HLA与白血病相关性研究国内外文献均有报道[1-3],但结论不一。

This study was aimed to discover the novel HLA recombination haplotypes and investigate the distribution of haplotypes in Chinese Han population. Based on the HLA-A, B, DRB1 typing results of 179 family members, 791 haplotypes were assigned by the mode of inheritance. The results showed that a total of 4 novel recombinant haplotypes in HLA-DRB1 locus region were observed in 4 families, which ratio of paternal to maternal chromosomes was 3:1. The recombination ratio between HLA-DRB1 and HLA-A or B loci was 0.92% (4/433). There were a total of 362 kinds of HLA-A, -B, -DRB1 haplotypes to be confirmed in Chinese Han partial population. A33-B58-DR17, A2-B46-DR9, A30-B13-DR7, A11-B13-DR15, A11-B75-DR12 and A2-B46-DR14 were the most common haplotypes that was consistent with the distribution of HLA alleles in unrelated donors. There were A1-B63-DR12, A29-B46-DR15, A1-B61-DR10, A34-B35-DR9, A29-B54-DR4, A23-B13-DR16 and A34-B62-DR15 haplotypes and so on, which were rare haplotypes not yet reported in Chinese. It is concluded that the HLA-A-B-DRB1 haplotypes would be confirmed by analysis of their family pedigree. The results obtained in this study are basic data for study of Chinese anthropology, organ transplantation and disease correlation analysis.

Objective To explore the distributive eharacterisfics for leukemia and to provide scientific reference for its prevention and intervention.Methods Microsoft SQL 2005 databases was used to make a mathematical analysis of 3708 patients with leukemia in Chinese Marrow Donor Program (CMDP) from 2000 to 2006.The distributive characteristics were calculated by sex,age and area of patients with leukemia and then compared by constituent ratio and relative ratio statistics method.Results A total of 3708 cases of leukemia were registered for waiting donor during the period 2000-2006 in CMDP,the age of patients were from 7 months to 69 years,tlle median age of diagnosis was 24.5 years,standard deviation was 6.7 years old:males suffered more than females,and the ratio was 1.95:1(2451/1257).There were 1202 patients with acute lymphoblastic leukemia (ALL),1066 with acute myeloid leukemia (AML),1435 with chronic myeloid leukemia (CML),5 with chronic lymphoblastic leukemia(CLL).CML was the most common patients.The distributive of 3708 patients with leukemia peak was from 15 to 30 years age group,542 patients were at the age of 15 years,559 patients were at the age group above 20 years,514 patients were at the age above 25,522 patients were at the age over 30 years old.ALL patients were accounted for 49.36%(613/1242),AML pailents accounted for 27.78%(245/1242),CML patients accounted for 22.78%(283/1242),CLL patients accounted for 0.08%(1/1242)in the age group of under20 years (childhood group).All subjects were mainly in childhood patients with leukemia:The distributive of patients with leukemia in 30 areas were different,leukemia patients were not registered in one area,494 patients were at the highest peak,101 patients were in the median.Conclusion The majority of leukemia patients for waiting stem cell transplantation were registered among children and the adolescents groups,males were suffered more than the females.For children,the major type of leukemia was ALL,being necessary to pay mofe aaention to the education of health,and the precaution of leukemia-The distributive of patients with leukemia for waiting stem cell transplantation was different in 30 areas,and the peak region of leukemia should be in Jiangsu,Guangdong,Shangdong,and Zhejiang provinces.

OBJECTIVE:To analyze the difference between the frequencies of HLA-A-B, B-DRB1 and A-B-DRB1 haplotype, as well as their linkage disequilibrium pattern in patients with acute lymphoblastic leukemia(ALL) and healthy controls from Northern Chinese Han.METHODS:The frequencies of HLA-A-B, B-DRB1, A-B-DR haplotypes and linkage disequilibrium were estimated by Expectation Maximization method based on the genotypes of 643 patients with ALL and 2 0359 unrelated healthy donors, and the statistical significance between the two groups were estimated by chi-square test. Linkage disequilibrium was analyzed with population genetic methods.RESULTS:The most common HLA-A-B, B-DRB1, and A-B-DR haplotypes were A30-B13, A2-B46, A33-B58, B13-DR7, B46-DR9, B52-DR15, B58-DR17, A30-B13-DR7, A33-B58-DR17 and A1-B37-DR10 in both groups. The frequencies of A30-B13, A2-B46, A33-B44, B13-DR7, A30-B13-DR7 and A2-B46-DR9 haplotypes and linkage disequilibrium value were significantly decreased (P<0.05) in the patient group than that in the control group. On the other hand, the frequencies of A2-B52, A31-B61, A24- B8, B60-DR9, B27-DR4, B52-DR14, B44-DR17, B27-DR12 and A11-B27-DR12 haplotypes and linkage disequilibrium value were significantly increased (P<0.05) in the patient group than that in the control group.CONCLUSION:There are some common and positive linkage disequilibrium haplotypes in both the ALL patients and the healthy donors in Northern Chinese Han. Interestingly, some haplotypes and their linkage disequilibrium patterns had significantly different distributions between the two groups. The study provided basic data for the relationship of ALL and HLA haplotype and for finding the HLA-A, B, DR matching donors.

This study was aimed to establish the quantitative analysis of hIL-2 in culture supernatant by multifunctional Luminex 100. The lymphocytes were separated from ACD-anticoagulated peripheral blood by density gradient method. The lymphocytes were stimulated with PHA for 48 hours, and frozen at -20 degrees C The relative fluorescence units of standard preparations and samples were detected by multifunctional Luminex 100, and the sample concentrations were calculated by standard curve. The results indicated that the regression equation of standard preparation is Lg (RFU) = 1.547 + 0.867 LgC. ANOVA F = 301.7427, p < 0.05 (nu = 6). The analysis of variance showed F = 301.7427, p < 0.05 (nu = 6). The test of regression coefficient showed t = 17.3707 (nu = 6), p < 0.05. It is concluded that method for induction and measurement of human IL-2 in vitro is established. The standard curve established by this way is statistically significant. There is linear relationship between the concentration of hIL-2 and fluorescence intensity.

目的研究流式磁珠反向SSO法HLA-A、B、DRB1分型结果判读中出现的常见问题,并提出相应的解决策略。方法对3 219名随机抽取的深圳市无关造血干细胞供者及组织配型患者的DNA采用流式磁珠反向SSO法进行HLA-A、B、DRB1低分辨基因分型,模棱两可结果用PCR-序列特异性引物(Sequence Specific Primer,SSP)法及PCR-SBT测序方法等方法复核确认。结果3 219份样本中发现有95份(95/3 219)28种类型的模棱两可结果,其中HLA-A位点有3(3/28)种,HLA-B位点有25(25/28)种,91.6%的模棱两可结果其样本的荧光值在阳性控制线上,本研究观察到的模棱两可结果通过基因频率分析、PCR-SSP法及PCR-SBT基因测序等方法得以定型。结论建立流式磁珠反向SSO法HLA-A、B、DRB1基因分型模棱两可结果解决策略,对提高HLA数据分析的速度和分型的准确性有重要的意义。

Objective To analyse the human leukocyte antigen(HLA)-A, B, DRB1 allele and haplotype frequencies in Jiangxi Chinese Han unrelated bone marrow donors.Methods Frequencies of HLA-A,B,DRB1 allele and haplotype were calculated by maximum likelihood estimator method,which based on the genotype data of 2 210 Jiangxi Han unrelated bone marrow donors.Results A total of 72 HLA alleles were observed in Jiangxi Han bone marrow donors and the most frequent were A*02, A*11, A*24, A*33, B60, B*46, B*13, B*58, B*51, B75,B62,DRB1*09,DRB1*12, DRB1*15, DRB1*04, DRB1*08, DRB1*11, DRB1*14 and DRB1*13. Among 358 kinds of A-B haplotype, there were 26 kinds included A2-B46, A11-B60 and A33-B58 with frequency higher than 0.01,and 68 kinds with statistically significant and positive linkage disequilibrium. Moreover, among 420 kinds of B-DR haplotype, there were 22 haplotypes included B46-DR9, B13-DR15 and B75-DR12 with frequency higher than 0.01,and 82 haplotypes with statistically significant and positive linkage disequilibrium. Only 386 kinds of A-B-DR haplotype were informative with frequency higher than or equal to 4.52 per 10 000 in 2 518 haplotypes, and a total of 39 kinds of A-B-DR haplotype included A2-B46-DR9, A2-B46-DR8, A11-B13-DR15, A33-B58-DR13,A33-B58-DR17 and A30-B13-DR7 were the most frequent with frequency higher than 0.005.Conclusion Distribution of HLA-A,B,DRB1 alleles and haplotypes in Jiangxi Chinese Han is relatively closed to Southern Chinese Han population and also showed its own characterization. The frequencies of HLA allele and haplotype from this study may be served as the reference standard for Jiangxi Chinese Han.

OBJECTIVE:To analyze human leukocyte antigen (HLA) polymorphism and search for new alleles in Chinese Han population bone marrow registry donors.METHODS:DNA-based HLA genotyping methods were used including PCR-SSP, BST and molecular cloning.RESULTS:A total of 6965 unrelated donors, 4707 from South China origin and 2258 from north, were typed for HLA-A, B, and DRB1 loci. Seventy-two specificities of HLA alleles were identified. The HLA-A25, A34, A74, B41, B42, B53, B73 and B81 that were rarely reported in previously Chinese population studies were identified in this study. Estimation of gene frequency indicated that the blank gene frequency was less than 0.2% for HLA-A, 0.25% for HLA-B and 0.70% for HLA-DRB1 loci. Three novel alleles were identified and officially assigned by the World Health Organization (WHO) Nomenclature Committee as A*0253N, A*1114 and B*5610.CONCLUSION:Large-scale DNA-based HLA genotyping used in bone marrow registry donors is highly accurate and reliable for estimating gene frequency and searching for new alleles. The discrepancy of HLA gene distribution between South and North China Han population showed the necessity of setting the more regions in South and North China to screen the bone marrow registry donors for bone marrow transplant.