Adolescent ; Adult ; Aged ; Calcaneus ; injuries ; surgery ; Female ; Fractures, Bone ; surgery ; Humans ; Male ; Middle Aged ; Skin Diseases ; etiology ; prevention & control ; Young Adult

This study was aimed to establish the real-time fluorescent quantitative PCR (RT-qPCR) with erythrocyte Kidd blood group gene for detecting the hematopoietic chimera and to investigate the feasibility of this method. The TaqMan MGB probes and special primers were designed on basis of difference of erythrocyte Kidd blood group alleles, the hematopoietic chimerism was detected by RT-qPCR, the DNA chimerism was simulated by means of dilution of multiple proportions, and the sensitivity analysis was performed. The results showed that the RT-qPCR with erythrocyte Kidd blood group gene could effectively distinguish JK*A and JK*B alleles. There was no significant difference between the theoretic value and the practical measured value by this method (P > 0.05). As 156 donor's cells could be discriminated from 10(4) chimeric cells, this method may effectively detect donor's cells with correlation coefficient 0.998. It is concluded that the established RT-qPCR with erythrocyte Kidd blood group gene shows the feasibility for quantitative detection of hematopoietic chimera, and may be used to quantitatively detect chimera in a certain range.
Chimera ; Erythrocytes ; Humans ; Kidd Blood-Group System ; genetics ; Real-Time Polymerase Chain Reaction

To establish delta block HLA-matching technique, DNA was extracted from whole blood by salting-out method, delta block was amplified by polymerase chain reaction (PCR), and PCR product was detected by GeneScan. The results showed that delta block had polymorphism in 104 samples without sibship of the Han people from Zhejiang province. The range of DNA fragment length was 81-393 bp and could be divided into 4 groups: 81-118 bp, 140-175 bp, 217-301 bp, 340-393 bp. The numbers of DNA fragments were 6-32. It is concluded that the method of delta block matching is reliable and can be applied to select donors for the patients to be transplanted. It is the first time to get delta block data of the Han people in China.
HLA-A Antigens ; genetics ; immunology ; HLA-B Antigens ; genetics ; immunology ; HLA-DQ Antigens ; genetics ; immunology ; HLA-DR Antigens ; genetics ; immunology ; HLA-DRB1 Chains ; Hematopoietic Stem Cell Transplantation ; Histocompatibility Testing ; methods ; Humans

The aim of this study was aimed to investigate the molecular genetic basis for a novel HLA allele, HLA-B*4061, in Chinese population. DNA was extracted from whole blood by salting-out method. The HLA-B exons 1 - 8 of the proband was amplified and the amplified product was cloned using TOPO TA cloning sequencing kit to split the two alleles apart. Both strands of exons 2, 3 and 4 of chosen colonies were sequencing. The PCR-SSP was performed to confirm the mutations detected by sequencing. The sequencing results showed HLA-B alleles of the proband as B*4601 and the novel allele. The sequences of the novel allele have been submitted to GenBank (DQ089628, DQ089629, DQ089630). After HLA blast analysis, the novel allele showed a single nucleotide mismatch with B*400101 in exon 2 at position 272 C-->A, as the results, changing amino acid from Ser to Tyr at codon 67. It is concluded that this allele is a novel one and has been officially named B*4061 by the WHO Nomenclature Committee.
Alleles ; Amino Acid Substitution ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; China ; HLA-B Antigens ; genetics ; immunology ; Histocompatibility Testing ; Humans ; Molecular Sequence Data ; Sequence Analysis, DNA ; Sequence Homology, Nucleic Acid

To analyze killer immunoglobulin (Ig)-like receptor (KIR) gene content and allelic polymorphism in Zhejiang Han population, samples were genotyped by polymerase chain reaction sequence-specific primers (PCR-SSP). The results demonstrated that all 17 KIR genes could be observed in the population. All individuals contained 2DL4, 3DL2 and 3DL3 genes. The frequencies of these genes was 1.00. 2DL1, 2DL3, 2DP1, 3DP1*003, 3DL1, 2DS4*001/002 loci were more common, their frequencies were 0.902, 0.902, 0.902, 0.902, 0.7598, 0.5615 respectively, while the frequencies of 2DL2, 2DL5A, 2DL5B, 2DS1, 2DS2, 2DS3, 2DS4*003, 2DS5, 3DS1 and 3DP1*001/002 were relatively lower. The A KIR haplotype was the most prevalent (74.7%) in Zhejiang Han population and there were 12 different KIR haplotypes in total, the most common was 2 (53.0%). Twenty six different genotypes have been found in the population, AJ (2, 2) and AF (1, 2) showed higher frequencies, followed by AH (2, 5), NN2 (2, 6), AI (1, 5) and AG (1, 1). Fifteen of these genotypes have not been found in Caucasians so far and four new KIR profiles could not be assigned to the haplotypes according to standard assign method. In conclusion, there are distinctive frequencies of KIR gene content, haplotype as well as genotype in Zhejiang Han population.
China ; Gene Frequency ; Genotype ; Haplotypes ; Humans ; Polymerase Chain Reaction ; methods ; Polymorphism, Genetic ; Receptors, KIR ; genetics

The purpose of this study was to establish beta block matching technique. DNA was extracted from whole blood by salting-out method, beta block matching was performed by PCR and GeneScan technique. The results showed that the length of fragments amplificated in 100 samples was different and the range of them was 91-197 bp. Amplification fragments could be divided into four regions: 91-93, 105-113, 125-139 and 177-197 bp respectively. 91 bp DNA fragments could be found in all of samples. The numbers of DNA fragments with different length have been shown high polymorphism and they focused on the range of seven to twenty four. In conclusion, the beta block matching technique is reliable and applicable to the selection of hematopoietic stem cell transplantation donors.
DNA ; genetics ; HLA-B Antigens ; genetics ; HLA-C Antigens ; genetics ; HLA-DR Antigens ; genetics ; HLA-DRB1 Chains ; Hematopoietic Stem Cell Transplantation ; Histocompatibility Testing ; methods ; Humans ; Polymerase Chain Reaction ; methods

In order to construct prokaryotic expression system of MHC classI chain-related gene A (mica) and purify MICA protein, RNAs were extracted from the peripheral blood samples and mica cDNA fragments were obtained by RT-PCR method. The cDNA for mica was ligated with cloning vector by TOPO method. The recombinant cloning vector and prokaryotic expression vector pET-28a were digested by two restriction enzymes and ligated to construct pET-28a-MICA recombinant expression vector, then the pET-28a-MICA vector was transformed and expressed in E. coli BL21 DE3. The recombinant protein was purified by Ni-NTA Spin method. The results showed that the recombinant MICA protein expressed with soluble form in host with pET-28a-MICA vector after IPTG induction. The recombinant target protein was obtained by Ni-NTA spin purification. In conclusion, this study has constructed prokaryotic expression system of mica gene and has purified MICA protein which would help to explore the interaction between MICA and transplantation immunology.
Cloning, Molecular ; Escherichia coli ; metabolism ; Genetic Vectors ; Histocompatibility Antigens Class I ; genetics ; metabolism ; Humans ; Recombinant Proteins ; genetics ; metabolism

This study was purposed to investigate the molecular basis of a para-Bombay phenotype for screening and identification of rare blood group. ABO and H phenotypes of the proband were identified by serological techniques. The exon 6 to exon 7 of ABO gene and full coding region of α-1,2-fucosyltransferase (fut1) gene of the proband were analyzed by polymerase chain reaction and direct sequencing of the amplified fragments. The haplotype of compound heterozygote of fut1 was also identified by cloning sequencing. The results indicated that a rare para-Bombay phenotype was confirmed by serological techniques. Two deletion or insertion variant sites near nucleotide 547 and 880 were detected in fut1 gene. The results of cloning sequence showed that one haplotype of fut1 gene was two bases deletion at 547-552 (AGAGAG→AGAG), and another one was two bases deletion at position 880-882 (TTT→T). Both two variants caused a reading frame shift and a premature stop codon. It is concluded that a rare para-Bombay phenotype is found and confirmed in blood donor population. The molecular basis of this individual is compound heterozygote of two bases deletion on fut1 gene which weaken the activity of α-1, 2-fucosyltransferase.
ABO Blood-Group System ; genetics ; Alleles ; Base Pairing ; Female ; Fucosyltransferases ; genetics ; Genotype ; Heterozygote ; Humans ; Mutation ; Phenotype ; Sequence Deletion

BACKGROUNDA living fetus within the maternal uterus provides an example of allogene tolerance in mammals. Poria cocos Wolf is the main component of many Chinese medicinal combination drugs that have therapeutic effects on recurrent spontaneous abortion and that can maintain pregnancy until delivery. It was hypothesized that this herbal medicine can also prolong allograft survival after organ transplantation. Here, in an in vivo study, we report the anti-rejection effect of the ethanol extract of Poria cocos Wolf (EEPCW) in rats after cardiac allograft implantation.

METHODSTen normal rats were healthy controls. Eighty rats receiving homologous heart transplants were divided into 4 groups of 20 rats each based on type of treatment: olive oil 8 ml.kg(-1).d(-1), EEPCW 25 mg.kg(-1).d(-1), EEPCW 50 mg.kg(-1).d(-1) or cyclosporin A 5 mg.kg(-1).d(-1). Allograft survival was observed in 10 rats from each group. On the seventh day post transplantation, pathological lesions and percentages of CD3+, CD4+, and CD8+ lymphocytes and the CD4+/CD8+ ratio in peripheral blood were assessed in another 10 rats from each group and in 10 normal rats.

RESULTSThe survival time of donor hearts in the two EEPCW groups was significantly prolonged, to (15.9 +/- 2.4) days and (30.0 +/- 0.0) days, respectively, compared with (6.7 +/- 0.8) days in the control group. Pathological lesions in the two EEPCW groups were also less severe, and the percentages of CD3+, CD4+, and CD8+ lymphocytes and CD4+/CD8+ ratio were significantly lower in the EEPCW groups.

CONCLUSIONSAcute rejection of heart transplants and cellular immune reaction can be effectively suppressed using the EEPCW. Taking advantage of novel immunosuppressants derived from Chinese medicinal herbs used to treat abnormal pregnancy provides a hopeful road for future research and treatment in organ transplantation.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Graft Rejection ; prevention & control ; Graft Survival ; drug effects ; Heart Transplantation ; Immunosuppressive Agents ; pharmacology ; Male ; Polyporales ; chemistry ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar

The study was purposed to investigate the molecular genetic basis for HLA novel allele HLA-B*5408N in Chinese population. DNA was extracted from whole blood by commercial DNA extraction kit, the HLA-B exons 2 - 4 of the proband was amplified by allele specific primers PCR and the amplified product was sequenced for exons 2, 3 and 4 bidirectionally. The sequencing results showed HLA-B alleles of the proband as B*1527 and the novel allele. The sequences of the novel allele have been submitted to Genbank (DQ295998, DQ295999, DQ296000). After blast analysis, the novel allele showed a single nucleotide mismatch with HLA-B*5401 in exon 3 at position 553 G-->T, which resulted in an amino acid changing from Glu to premature stop codon at position 161. No the HLA-B54 antigen specificity expression in the proband cells was found using HLA-AB serological Typing Trays. It is concluded that this allele is a novel null allele and has been officially named B*5408N by the WHO Nomenclature Committee.
Alleles ; China ; Exons ; genetics ; HLA-B Antigens ; genetics ; Humans ; Polymerase Chain Reaction ; Sequence Analysis, DNA