ObjectivesTo investigate the clinical value of the melting curve analysis-based PCR assay for the clinical genetic diagnosis and prenatal diagnosis of β-thalassemia.Methods A total of 451 peripheral blood samples,including 372 cases with β-thalassemia phenotypes and 79 cases without β-thalassemia phenotypes,were collected by Liuzhou Municipal Maternity and Child Healthcare Hospital between January 2011 and August 2011.Moreover,another 84 cases,including 16 fetal villi samples (10 - 13 weeks),64 amniotic fluid samples (16 -24 weeks ) and 4 umbilical cord blood samples (above 17 weeks),whose parents were β-thalassemia carriers,were also collected for this assay between June 2011 and September 2011.A double-blind test was done to compare the detection reliability of the melting curve analysis-based assay (24 β-thalassemia mutations can be detected) with PCR-RDB probe assay (17 β-thalassemia mutations can be detected ) and DNA sequencing using these samples.The wildtype,mutant and total concordance rates of the genotyping results were calculated separately among the melting curve analysis based assay,PCR-RDB probe assay and DNA sequencing.Results Among the 451 peripheral blood samples,thirteen mutations and nineteen genotypes were obtained by using melting curve analysis-based assay.447 samples had the same detection results and 4 samples had different detection results by comparing melting curve analysis-based assay with PCR-RDB probe assay,thus,the concordance rate of the sample detection result was 99.1％ (447/451),and the concordance rate of the allele detection result was 99.6％ (898/902).DNA sequencing results of the 4 samples showed that 3 samples had the same genotyping result with melting curve analysis-based assay,and 1 sample had the same genotyping result with PCR-RDB probe assay.A rare β-globin mutation which was not included by melting curve analysis-based assay was not detected.Thus,the genotypes of 450 samples were detected accurately by melting curve analysis-based assay,and the concordance rate of the sample detection between the melting curve assay and DNA sequencing assay was 99.8％ (450/451).Among 84 fetal villi,amniotic fluid,and umbilical cord blood samples,8 mutation types and 18 genotypes were obtained by using melting curve analysis-based assay.All the samples have the same detection results by comparing melting curve analysis-based assay with PCR-RDB probe assay and DNA sequencing,so the concordance rate of the genotyping results was 100％ among the melting curve analysis-based assay,PCR-RDB probe assay and DNA sequencing.Conclusions The melting curve analysis-based PCR assay can detect multiple unknown samples simultaneously,and detect multiple mutations accurately.It is very useful for the genetic diagnosis and prenatal diagnosis of β-thalassemia.

The cholinesterase(ChE)and lactate dehydrogenase(LDH)activities in blood smears of 37 patients were observed before and 20 minutes after electroacupuncture.Hegu and Zusanli were mainly chosen as the acupuncture points.Karnovsky method was used to demonstrate ChE,activity,and tetrazolium-formazan reaction for LDH activity. Before acupuncture,all formed elements of blood showed both ChE and LDH acti- vities.In red blood cells ChE and LDH activities were localized at the cell membrane, while in white blood cells they were found throughout the cytoplasm as colored granu- les.The granulocytes showed greater ChE and lesser LDH activities than the lymph- ocytes.The platelets also showed greater LDH and ChE activities,especially the former. On the whole,platelets and leucocytes,as compared with erythrocytes,showed greater activities for ChE and LDH. Under microscopic observation,according to the amount and color of granules,the degrees of GhE and LDH activities for each kind of blood formed elements before and after acupuncture were recorded as different markes,such as +、++、+++ and so on.The degrees of GhE or LDH activity in various formed elements of blood were compared by means of statistical tests.After acupuncture,both GhE and LDH activities of all blood formed elements were increased(p

70 patients including 60 cases under acupuncture anesthesia and 10 cases under drug anesthesia were observed. Before and 20 minutes after acupuncture the blood samples were taken from the patient ear lobes respectively, and in some patients taken once again 24 hrs after acupuncture. The electroacupuncture point Hegu or Zusanli was mainly adopted. As the method for detection of cell-mediated immunity(CMI) in vitro the improved microtechnique of whole blood for E-rosette (active and nonactive) and lymphocyte transformation tests was used. In performance of active rosetting the total leucocyte count and the differential lymphocyte count were done for calculation of absolute number of active rosette forming cells (RFC). The mean value of increase of active RFC was 12.7?1.43, the decrease was 6.8?1.77 after acupuncture. The increment of the absolute number of active RFC was 175?63.59. However no marked effect on the drug anesthesia group was found. In the lymphocyte transformation assay the increase was 12.7?1.49, the decrease was 7.0?2.19, and the enhancement effect still exhibited 24 hrs after acupuncture. In these tests an increase was mostly found in those with a lower or a usual CMI level; a decrease often found in those with a higher CMI level prior to acupuncture. The increase or decrease level in the results of three kinds of test (active, nonactive RFC and lymphocyte transformation) was similar, the increase range was 12～13%, the decrease range 6～7%. As the former compared with the latter, the promotion was prominent by all means.

OBJECTIVETo explore the absorption, distribution and excretion of 3-Chloro-1,2-propandiol (3-MCPD) in healthy male SD rats after oral administration.

METHODS3-MCPD was administrated with a single oral dosage of 75 mg/kg BW to each rat. Samples of blood, tissues (including liver, kidney, brain and testicle) and excreta were then collected, and analyzed by the GC-MS method to determine 3-MCPD concentrations. The reported value is the mean value of three rats.

RESULTSAt 2 h after the administration, 3-MCPD concentrations in blood, testicle and kidney were (67.46 +/- 7.72), (78.37 +/- 5.15) and (56.21 +/- 3.64) microg/g, respectively. At 24 h, however, the corresponding values changed to (1.07 +/- 0.97) microg/g, (49.43 +/- 28.18) microg/g and (11.41 +/- 2.55) microg/g. During the 24-hour period, 9.74 +/- 3.05% of the given parent compound was excreted in urine, whereas 0.56 +/- 0.22% and 0.28 +/- 0.03% were excreted in feces and bile, respectively, which implies that kidney is a major organ for excretion 3-MCPD.

CONCLUSIONS3-MCPD was quickly absorbed through the alimentary tract and quickly distributed into a number of tissues, and then accumulated in the target organs, especially in the testicle. The excretion of the parent compound was largely through the kidney. It was inferred that 3-MCPD was mainly metabolized in the liver.

Absorption ; Administration, Oral ; Animals ; Brain ; metabolism ; Chemosterilants ; administration & dosage ; analysis ; pharmacokinetics ; Drug Monitoring ; methods ; Gas Chromatography-Mass Spectrometry ; Kidney ; metabolism ; Liver ; metabolism ; Male ; Rats ; Rats, Sprague-Dawley ; Testis ; metabolism ; Tissue Distribution ; alpha-Chlorohydrin ; administration & dosage ; analysis ; pharmacokinetics

Dendritic cell (DC) plays a key role in antitumor immune response. However, there is a deficiency of DC function in the majority of leukemia patients. It is a novel idea that expanding DC in vitro and enhancing their antitumor immune function and DC-based tumor vaccines may be used as an efficient immune therapy for leukemia. In the project, the condition to induce DC from myeloid leukemia cell lines and its anti-leukemia response were investigated. HL-60, K562 and THP-1 cells were cultured with various combinations of cytokines for inducing DC. The morphologic features were analyzed with optical and electron microscopy. The phenotype of DC was detected by FCM with CD1a, CD40, CD80, CD86, HLA-A, B, C and HLA-DR monoclonal antibodies. The ability of DC stimulating lymphocyte proliferation was observed by allo-mixed lymphocyte reaction using (3)H-TdR incorporation. Cytotoxicity assay was measured by (51)Cr-release method. The level of IL-12 and IFN-gamma in supernatant of DC culture was measured by ELISA. It was proved that the DCs derived from K562, HL-60 and THP-1 cells showed a typical morphology of dendritic cell. The induced cells expressed the surface differentiation antigens of DC. A high expression of phenotypes was found in HL-60-DC and THP-1-DC stimulated by GM-CSF + IL-4 + TNF-gamma and K562-DC with GM-CSF + IL-4 + IL-12. The DCs from the 3 leukemia cell lines stimulated allo-MLR and CTL reaction strongly. Different contents of IL-12 were detected in the supernatants of DC culture and IFN-gamma in the coculture of DC and blood mononuclear cells. It is concluded that the myeloid leukemia cells are able to be induced DCs by cytokines in vitro. The different leukemia cells need different cytokines and cultural conditions. DCs derived from leukemia cells express phenotype of antigen-presenting cells. They have the ability of stimulating T lymphocyte proliferation and inducing CTL reaction to clear leukemia cells, and the DCs secrete IL-12 and increase secretion of IFN-gamma by T cells.
Antigens, CD ; immunology ; Antigens, CD1 ; immunology ; B7-2 Antigen ; CD40 Antigens ; immunology ; Coculture Techniques ; Cytokines ; pharmacology ; Cytotoxicity, Immunologic ; drug effects ; Dendritic Cells ; drug effects ; immunology ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; HL-60 Cells ; HLA Antigens ; immunology ; Humans ; Immunophenotyping ; Interferon-gamma ; pharmacology ; Interleukin-12 ; pharmacology ; Interleukin-4 ; pharmacology ; K562 Cells ; Membrane Glycoproteins ; immunology ; Microscopy, Electron ; Time Factors ; Tumor Cells, Cultured ; drug effects ; immunology ; ultrastructure ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo investigate a new method to construct tissue-engineering bone that will be applicable clinically.

METHODSThe cultured 5th generation rabbit bone marrow stroma osteoblasts (MSO) was dissolved in 3% sodium alginate solution (the final concentration of sodium alginate in the solution being 1%, and MSO, 5x10(6)/L), and then inoculated into prepared true bone ceramic (TBC) and gelatinized the bone by dribbling with calcium gluconate. The standard bone defect models were made in 48 adult New Zealand rabbit's both radius. Among the 48 rabbits, 24 were in Groups A and B, in which the left radius was implanted with gelatinized MSO-TBC (Group A) and right radius implanted with autograft-bone (Group B); and the other 24 were in control group whose left radius was implanted with non-gelatinized MSO-TBC (Group C) and right radius implanted with gelatinized TBC (Group D). Outcomes of the implanted bones were assessed by radiology, pathological histology, osteogenetic quantitative analysis, and biomechanics at 2, 4, 8, 12 weeks postoperatively.

RESULTSIn Groups A and B, a satisfactory bone reparation and bony union was noted within 12 weeks. In Groups C and D, bone reparation was not satisfied compared with Group A in terms of ostogenetic quantity and biomechanics.

CONCLUSIONSGelatinized MSO-TBC is an ideal artificial active bone that overcomes TBC shortcomings of fragileness and smooth surface that is not eligible for seed cell's adhesion. It is promising to put into clinical use extensively.

Animals ; Biomass ; Bone Diseases ; diagnostic imaging ; pathology ; therapy ; Bone Marrow Cells ; cytology ; Bone Substitutes ; Ceramics ; Disease Models, Animal ; Female ; Gelatin ; Male ; Osteoblasts ; cytology ; transplantation ; Osteogenesis ; Rabbits ; Radiography ; Radius ; diagnostic imaging ; injuries ; pathology ; surgery ; Stromal Cells ; cytology ; transplantation ; Tissue Engineering ; methods ; Treatment Outcome

OBJECTIVETo screen for carriers of SMN1 gene mutation, which underlies spinal muscular atrophy (SMA), in 4931 pregnant women from Liuzhou region of Guangxi, and to determine the carrier rate.

METHODSCombined denaturing high-performance liquid chromatography (DHPLC) and multiple PCR techniques were used to detect the copy number of SMN1 gene. The carrier frequency was calculated. The spouse of the carrier was also screened, and prenatal diagnosis was provided to the couples who were both positive.

RESULTSAmong the 4931 pregnant women, 61 were found to harbor only one copy of the SMN1 gene, which yielded a carrier rate of 1.2%. Subsequent testing has identified 1 fetus carrying homozygous deletions of the SMN1 gene.

CONCLUSIONThe carrier rate of SMA mutation in Liuzhou region is slightly lower than that of other regions of southern China. DHPLC can effectively screen the carriers of SMA mutation and provide a basis for genetic counseling and prenatal diagnosis.

OBJECTIVETo evaluate the clinical value of multicolor melting curve analysis(MMCA) for detecting genetic mutations in G6PD deficiency.

METHODSA total of 402 peripheral blood samples(256 males and 146 females) were collected from suspected patients or their relatives at the Prenatal Diagnosis Center of Liuzhou Maternal and Child Health Hospital between March 2012 and May 2012. The samples were screened by G6PD/6PGD quantitative ratio testing. The reliability of the assay was evaluated by multiplex probe melting curve assay(which can detect 16 G6PD mutations) and DNA sequencing through a double blind study.

RESULTSOne hundred seventy cases with G6PD/6PGD ratio < 1.0 and 232 cases with G6PD/6PGD ratio ≥ 1.0 were detected by the enzymological method. DNA sequencing has identified 182 wild type samples, 151 hemizygous mutation samples, 5 female homozygous mutation samples, 54 female heterozygous mutation samples and 10 female double heterozygous mutation samples. Multicolor melting curve analysis has detected 185 wild type samples, 148 hemizygous mutation samples, 5 female homozygous mutation samples, 55 female heterozygous mutation samples and 9 female double heterozygous mutation samples. The specificity and sensitivity of G6PD gene mutation detection by multicolor melting curve analysis were 100%(182/182) and 98.6%(217/220), respectively. The positive predictive value and negative predictive value were 99.5%(216/217) and 98.4%(182/185), respectively, and the Youden's index was 0.986. The concordance rate of the sample detection between the melting curve assay and DNA sequencing was 99.0%(398/402). Twenty-one different genotypes were detected by the multicolor melting curve analysis and 24 different genotypes were detected by DNA sequencing. Four samples containing mutations(c.196T>A or c.406C>T) were not detected by multicolor melting curve analysis, which can be attributed to different technical settings of the two methods.

CONCLUSIONMulticolor melting curve analysis for G6PD gene mutation detection is a simple, rapid, sensitive and specific method, which can be used for clinical diagnosis of G6PD deficiency.

Adolescent ; Adult ; Child ; Child, Preschool ; Female ; Glucosephosphate Dehydrogenase ; genetics ; Humans ; Infant ; Infant, Newborn ; Male ; Mutation ; Polymerase Chain Reaction ; methods ; Sequence Analysis, DNA

OBJECTIVETo study the role of dendritic cells (DCs) and macrophages, differentiated from the same individual peripheral blood monocytes, in tumor antigen- presenting.

METHODSDCs and macrophages were differentiated from human peripheral blood monocytes by adding both Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) or GM-CSF only. Then they were loaded with tumor antigen at different concentrations and cocultured with autologous T cells in 96-well flat-bottomed microtiter plates for five days at 37 degrees C, 5% CO(2). (3)H-thymine was added before the culture terminated, and twelve hours later, the cells were gathered to test the cpm value.

RESULTSBoth DCs and macrophages chased with tumor antigen could strongly stimulate the proliferation of autologous T cells, especially DCs. The stimulation effect with 20 microl/ml antigen was the most remarkable and the cmp values were 11,950.3 +/-1621.8, 8,708.5 +/-176.1, 402.5+/-43.1 in DCs group, Macrophages group, and lymphocytes group, respectively.

CONCLUSIONThe antigen presenting role of DCs is stronger than that of macrophages from the same individual.

Antigen Presentation ; immunology ; Antigen-Presenting Cells ; immunology ; physiology ; Antigens, Neoplasm ; immunology ; Carcinoma, Hepatocellular ; immunology ; Dendritic Cells ; immunology ; physiology ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Liver Neoplasms ; immunology ; Macrophages ; immunology ; physiology ; Tumor Cells, Cultured

OBJECTIVETo verify that the trabecular meshwork (TM) in the wall of the eyeball consists of smooth muscle fibers instead of collagen fibers or endothelial cells.

METHODSEighteen fresh eyeballs from 3 rabbits, 3 SD rats and 3 mice were sectioned along the sagittal plane and sliced after paraffin embedding for HE staining, VG staining, Masson staining, α-SMA immunohistochemistry or CD31 immunohistochemistry. These slices were observed under microscope and the structure of the TM was compared with those of scleral collagen fibers, ciliary muscles and endothelial cells.

RESULTSHE staining of the eyeball slices from the 3 animal species resulted in purplish red staining of the TM, which was highly consistent with ciliary muscle fibers. The cell?like structures on the surface of the TM were not clearly outlined, with flat nuclei showing a dark purple staining; these structures did not show obvious boundaries from the TM. Ciliary muscle fibers, which were smooth muscle cells in nature, were aligned in bundles in various directions. The longitudinally sectioned cells were flat and contained purplish cytoplasm and highly flattened nuclei. Scleral collagen fibers were stained dark red with a few fibroblasts sandwiched among them. The long axis of the fibroblasts was in parallel with that of the collagen fibers. The outline of the fibroblast was not clear and the nucleus was flat in dark blue. The vascular endothelial cells presented with different morphologies and contained light purplish cytoplasm and dark nuclei, protruding into the vascular cavity. VG staining of the TM revealed a pale red filamentous structure, and the collagen fibers were stained bright red. Masson staining of the TM showed a reticular structure consisting mainly of dark red fibers intermingled with thin green fibers. Scleral collagen fibers presented with a cord?like green wavy structure. The endothelial cells were green and flat, while the ciliary smooth muscle fibers were purple. In immunohistochemistry for α?SMA, the TM and the ciliary smooth muscle fibers showed a strong positivity in the cytoplasm, while the scleral collagen fibers and vascular endothelial cells showed negative staining; immunohistochemistry for CD31 showed no obvious positive staining in the TM, collagen fibers or ciliary smooth muscle cells from all the animals in spite of slight differences among them.

CONCLUSIONThe TM consists mainly of smooth muscle fibers with a thin layer of peripheral endomysium without endothelial cells.