1.Establishment of genotyping method for human platelet antigens of HPA-15 system by PCR-SSP.
Yue-Kang CHEN ; Da-Cheng LI ; Da-Ming WANG ; Qian LI ; Zhi-Hui DENG
Journal of Experimental Hematology 2008;16(1):185-188
This study was aimed to establish the reliable genotyping method of human platelet antigens of HPA-15 system by PCR-SSP and to use this assay in the further HPA genotyping of volunteer platelet donors. 3 sequence-specific primers recommended by the 11th Platelet Genotyping and Serology Workshop on behalf of International Society of Blood Transfusion (ISBT) were synthesized. The concentration of each primer pair, the concentration of Mg(2+) and the PCR conditions were adjusted to optimize the conditions so that HPA-15 system could be specific amplified. The accuracy and reliability of the developed assay was evaluated and confirmed by typing the coded DNA samples provided by the 11th Platelet Genotyping and Serology Workshop. As a parallel control, a total of 50 volunteer platelet donors in Shenzhen were genotyped by both our assay and the G&T commercial kit at HPA-15 system. 10 coded samples distributed by the 11th Platelet Genotyping and Serology Workshop were genotyped by established PCR-SSP method. The results showed that a concordance rate of 100% was observed between the results obtained by established PCR-SSP method and the results provided by ISBT report. The HPA gene frequencies observed in 50 randomly-selected platelet donors in Shenzhen were 0.5100 and 0.4900 for HPA-15a and HPA-15b respectively. In conclusion, PCR-SSP assay established in our study provides a simple, rapid and accurate method for HPA-15 system genotyping, which assay is suitable for routine clinical HPA genotyping and shows a broad prospect in its further applications.
Antigens, CD
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genetics
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immunology
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Antigens, Human Platelet
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genetics
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GPI-Linked Proteins
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Genotype
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Humans
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Isoantigens
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genetics
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immunology
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Neoplasm Proteins
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genetics
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immunology
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Polymerase Chain Reaction
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methods
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Polymorphism, Single-Stranded Conformational
2.Development of a high-throughput sequence-based typing assay for human leukocyte antigen loci.
Qiong YU ; Da-ming WANG ; Zhi-hui DENG
Chinese Journal of Medical Genetics 2012;29(3):323-327
OBJECTIVETo develop a reliable assay for simultaneous sequence-based typing (SBT) of HLA-DPA1 and HLA-DPB1, and to apply it for the study of allelic polymorphisms in southern Chinese Han population.
METHODSBased on full-length HLA-DPA1 and HLA-DPB1 allelic sequences, locus-specific PCR primers were designed and applied to amplify the target sequence encompassing the entire exon 2 of HLA-DPA1 and HLA-DPB1. PCR products were purified with magnetic beads, and run through an ABI 3730 DNA sequencer. Genotypes were assigned with an Assign 3.5 SBT software.
RESULTSThe target sequences of HLA-DPA1 and HLA-DPB1 were both amplified with the PCR procedure. Little background and noise was observed in the derived sequences. Among 176 non-related healthy individuals, 4 HLA-DPA1 alleles with the frequencies of DPA1*02:02 (0.589) > DPA1*01:03 (0.284) > DPA1*02:01 (0.096) > DPA1*04:01 (0.031) were identified. In addition, 14 HLA-DPB1 alleles, including 4 common alleles (with a frequency of more than 5%, namely DPB1*05:01, DPB1*02:01, DPB1*04:01 and DPB1*02:02), 7 alleles with a frequency ranging from 1%-5% and 3 alleles with a frequency of less than 1% were identified. The results of HLA-DPB1 genotyping were all in accordance with the typing results derived from an Atria AlleleSEQR HLA-DPB1 kit.
CONCLUSIONA reliable technique has been established for simultaneous genotyping of HLA-DPA1 and HLA-DPB1, which may have a broad application in population and disease association studies.
Alleles ; Base Sequence ; DNA Fingerprinting ; methods ; Gene Frequency ; Genotype ; HLA-DP alpha-Chains ; genetics ; HLA-DP beta-Chains ; genetics ; High-Throughput Nucleotide Sequencing ; methods ; Humans ; Molecular Sequence Data ; Polymorphism, Genetic
3.Effects of TNF-?on PPAR-?2 mRNA expression and adiponectin secretion in 3T3-L1 adipocytes
Da-Tong DENG ; You-Min WANG ; Ling LIU ; Guo-Ping HU ; Ming-Gong YANG ; Qi-Mei SHE ; Chang-Jiang WANG
Chinese Journal of Endocrinology and Metabolism 1985;0(02):-
Undifferentiated and differentiated 3T3-L1 adipocytes were treated with 100 ng/ml tumor necrosis factor-?(TNF-?),and peroxisome proliferator-activated receptor-?2 (PPAR-?2) mRNA expression and adiponectin secretion in cultured cells were measured.The results showed that TNF-?suppressed PPAR-?2 mRNA expression and adiponeetin secretion in 3T3-L1 adipocytes (P
4.High through-put genomic DNA isolation technique and its application in HLA genotyping for samples from bone marrow donor program.
Da-Ming WANG ; Si TANG ; Zhen LI ; Xi CHENG ; Su-Qing GAO ; Zhi-Hui DENG
Journal of Experimental Hematology 2009;17(5):1265-1268
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.
Biological Specimen Banks
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Bone Marrow
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DNA
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isolation & purification
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DNA Primers
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Genotype
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HLA Antigens
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genetics
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High-Throughput Screening Assays
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methods
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Humans
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Living Donors
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Nucleic Acid Hybridization
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methods
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Oligonucleotide Array Sequence Analysis
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methods
5.Long-term visual outcome of dense bilateral congenital cataract.
He-hua YE ; Da-ming DENG ; Yi-yong QIAN ; Zhi LIN ; Wei-rong CHEN
Chinese Medical Journal 2007;120(17):1494-1497
BACKGROUNDDense congenital cataracts often cause severe visual impairment. The results of long-term follow-up of dense bilateral congenital cataract in China have not been well documented. The purpose of this study was to evaluate the long-term visual function in children who underwent cataract extraction for dense bilateral congenital cataract in southern part of China.
METHODSMedical records of children who underwent surgery of dense bilateral congenital cataract between January 1992 and December 2000 at Zhongshan Ophthalmic Center of Sun Yat-sen University were retroactively reviewed. In 38 children available for current follow-up, best corrected visual acuity (BCVA) and stereoscopic vision, as well as nystagmus, strabismus, and other complications, were evaluated. The mean follow-up period was 107.6 months (range 60 to 167 months).
RESULTSThe mean age of cataract extraction and secondary intraocular lens implantation were 5.6 months (range 3 to 12 months) and 4.2 years (range 2.4 to 15 years), respectively. The mean BCVA was 0.25 in the better eye and 0.16 in the fellow eye. Stereoscopic vision was absent in all patients, and 3 children had simultaneous perception. Nystagmus was detected in all cases and strabismus in 35 cases. A high correlation was found between timing of cataract extraction and final BCVA of the better eye (r = -0.55, P = 0.00). A statistically significant difference was found in BCVA between post- and pre-treatment of amblyopia (t = 5.65, P = 0.00).
CONCLUSIONSLong-term visual function in children with dense bilateral congenital cataract was poor when cataract surgery was performed at age of 3 months or later. Earlier cataract surgery with adequate optical rehabilitation contributed to better visual outcome.
Adolescent ; Cataract ; congenital ; Cataract Extraction ; Child ; Child, Preschool ; Follow-Up Studies ; Humans ; Infant ; Retrospective Studies ; Visual Acuity
6.Effect of selenium-enriched garlic on chronic gastritis of the glandular stomach of Mongolian gerbils induced by H. pylori.
Lian-Kun GU ; Ping ZHOU ; Jing ZHOU ; Ru-Ming WANG ; Wen-Jie YANG ; Da-Jun DENG
Chinese Journal of Preventive Medicine 2007;41 Suppl():104-107
OBJECTIVETo detect the therapeutic effect of selenium-enriched garlic (SeG) on chronic gastritis.
METHODSChronic gastritis was induced of the glandular stomach of male Mongolian Gerbils via gastric instillation of H. pylori TN2 strain once every 4 days for 5 consecutive times followed by random classification into six groups. Fresh SeG suspension was administrated daily at dosages of 4.70, 1.5, 0.47, 0.15 g.kg(-1).d(-1) for four weeks. The gerbils in the positive control group were treated with omeprazole, clarithromycin, and amoxicillin for one week. The gerbils were killed for pathological examination four weeks after SeG-treatment.
RESULTSChronic gastritis (CAG), low-grade dysplasia or gastric intraepithelial neoplasia (DYS/GIN) were observed among 77% and 38.5% of the 13 H. pylori-treated animals in the negative control group, respectively; whereas 40% and 26.7% in the positive control group (n = 15), respectively. The incidences of CAG and DYS/GIN in the SeG groups (n = 21 - 27) were reduced dose-dependently, 16.7% - 38.7% and 11.1% - 14.3% for CAG and DYS/GIN, respectively.
CONCLUSIONSeG administration inhibits the development and progression of CAG induced by H. pylori remarkably.
Animals ; Chronic Disease ; Disease Models, Animal ; Garlic ; Gastritis ; drug therapy ; microbiology ; Gerbillinae ; Helicobacter Infections ; drug therapy ; Helicobacter pylori ; Male ; Phytotherapy ; Selenium ; therapeutic use
7.Correlation of killer immunoglobulin-like receptor gene diversity with nasopharyngeal carcinoma in Chinese southern Han population.
Liang LU ; Shi-Zheng JIN ; Da-Ming WANG ; Su-Qing GAO ; Zi-Hui DENG
Journal of Experimental Hematology 2011;19(3):798-800
The objective of this study was to elucidate the correlation of killer immunoglobulin-like receptor (KIR) gene diversity with nasopharyngeal carcinoma (NPC) in the Chinese southern Han population. KIR genotyping of peripheral blood samples from 67 patients with NPC and 77 randomly-selected healthy controls was performed by PCR-SSP, the relative risk (RR) value was calculated by means of Wolf method. The results showed that the KIR2DL3 gene frequency in NPC patient group was significantly lower than that in healthy controls (χ²>3.84, p < 0.05, RR = 0.08), whereas the KIR2DS5 and KIR2DL5B gene frequencies in patient group were significantly higher than those in healthy controls (χ²>3.84, p < 0.05, RR > 1), the other KIR gene frequencies were no statistically different between two groups. It is concluded that the KIR2DL3, KIR2DS5 and KIR2DL5B genes may be correlated with pathogenesis of NPC in the Chinese southern Han population.
Adult
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Aged
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Asian Continental Ancestry Group
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genetics
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Case-Control Studies
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Female
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Gene Frequency
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Genotype
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Humans
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Male
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Middle Aged
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Nasopharyngeal Neoplasms
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genetics
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Receptors, KIR
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genetics
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Receptors, KIR2DL3
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genetics
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Receptors, KIR2DL5
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genetics
8.Investigation and analysis of a common allele HLA-C*08:22 frequency in the Chinese southern Han population.
Zi-Qian BAO ; Da-Ming WANG ; Zhi-Hui DENG ; Yuan-Ping XU
Journal of Experimental Hematology 2011;19(6):1493-1495
This study was purposed to investigated and analyze the allelic frequency of a common allele HLA-C*08:22 in the southern Chinese Han population. A total of 32 samples with the C*08:01:01/08:22 ambiguous results previously identified in 163 unrelated southern Chinese Han population by routine sequencing based typing (SBT) at exons 2 - 4 of HLA-C gene were subjected to HLA-C SBT at exons 5 and 6 using our in-house method. Forty C*08:01:01-positive unrelated donor/recipient pair identified before the C*08:22 allele were officially nomenclatured and released by the World Health Organization (WHO) Nomenclature Committee for Factors of HLA System, were re-sequenced at exons 2 - 6 of HLA-C gene by our in-house SBT method. The allele assignment was accomplished with the Assign 3.5 SBT software. The results showed that three samples were identified as C*08:22-positive in the 32 samples with C*08:01:01/08:22 ambiguous results, the allele frequency of C*08:22 was 0.92% in the southern Chinese Han population. Retrospective analysis indicated that 2 donor/recipient pairs previously identified as C*08:01:01-positive were actually C*08:22-positive in the 40 tested donor/recipient pairs. It is concluded that the novel C*08:22 allele is the common allele in southern Chinese Han population, it can't be considered as rare allele and is ruled out for the samples with C*08:01:01/08:22 ambiguous results.
Alleles
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Asian Continental Ancestry Group
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genetics
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China
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Gene Frequency
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Genetics, Population
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HLA-C Antigens
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genetics
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Humans
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Sequence Analysis, DNA
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Unrelated Donors
9.An experimental study of the effect of burying testis in thigh pocket on spermatogenesis.
Da-li WANG ; Yu-ming WANG ; Zai-rong WEI ; Hong ZHENG ; Fei DENG ; Zhen-yu GAO
Chinese Journal of Plastic Surgery 2007;23(5):419-421
OBJECTIVETo explore the effect of burying testis in thigh pocket on spermatogenesis.
METHODSGuizhou miniature male pigs at child-bearing period were randomly divided to receive operation of scrotum incision and dissection with the testis burying in thigh pocket (experimental group) or without (control group). 3 months later, testis biopsy was performed on 2 pigs from each group for pathological examination. Then every male pig from both experimental (n = 6) and control group (n = 6) got a mating partner and lived together for 3 months. The fertility of the male pigs was observed. 6 months after operation, testis biopsy was performed again on all the animals from both the groups.
RESULTSBoth at 3 months and 6 months after operation, the pathological examination showed the spermatogenic cells of all stage in contorted seminiferous tubules markedly decreased with no mature sperm in experimental group, while normal spermatogenic cells with mature sperm in control group. After the male pigs lived with mating partners for 3 months, no female pigs staying with the experimental group became pregnant, but the male pigs in control group had a normal fertility.
CONCLUSIONSBurying testis in thigh pocket impedes spermatogenesis in the miniature male pig. So burying testis in thigh pocket is not recommended for patients with scrotum skin defect who wish to remain fertile.
Animals ; Female ; Fertility ; Male ; Pregnancy ; Pregnancy Rate ; Scrotum ; Skin ; injuries ; Spermatogenesis ; Swine ; Swine, Miniature ; Testis ; physiology ; Thigh ; surgery
10.Study on the origin of Rana temporaria for quality Oviduetus Ranae.
Yi-ping LI ; Jin-gang ZHANG ; Miao LI ; Chun-mei LIU ; Da-cheng JIANG ; Ming-lu DENG
China Journal of Chinese Materia Medica 2003;28(1):15-17
OBJECTIVETo determine the origin of Rana temporaria for quality Oviduetus Ranae in the light of historical documents and modern researches on the classification of Rana temporaria chensinensis.
METHODWorks of Chinese meteria medica of all ages, related historical documents and reports from home and abroad on researches of R. temporaria chensinensis were consulted, sorted out, analyzed and summarized.
RESULTThe original Shange recorded in the works of Chinese meteria medica is R. temporaria chensinensis, which is the independent species, not one of species of European forest frogs. R. temporaria chensinensis is divided into 4 subspecies: R. temporaria chensinensis, Lanzhou, Kangding, and Changbaishan. The origin of R. temporaria is Changbaishan subspecies of R. temporaria chensinensis.
CONCLUSIONChangbaishan subspecies of R. temporaria chensinensis is determined as the origin for quality Oviduetus Ranae.
Animals ; Female ; History, 18th Century ; History, 19th Century ; History, 20th Century ; History, Ancient ; Materia Medica ; history ; isolation & purification ; Oviducts ; chemistry ; Rana temporaria ; anatomy & histology ; Ranidae ; anatomy & histology ; classification ; Species Specificity ; Terminology as Topic