Two-dimensional electrophoresis(2-DE) is an important technique in proteomics research.The 2-DE system for proteome of Monascus ruber was established by comparing and analyzing the infection caused by different kinds of mediums,lysis buffer and the condition of rehydration.By cultivating the Monascus ruber with YES for 6 days,extracting total protein by TCA-acetone,lysis buffer with 8 mol/L urea,2 mol/L thiourea,4 % CHAPS,1 % DTT and 2 % Bio-lyte,an ideal 2-DE map with higher resolution and better legibility was obtained,which laid a foundation for the further studies on proteome of Monascus ruber.

Objective To verify the reference interval for D-dimer assay and analyze the influence of age and gender on the ref-erence interval.Methods Inclusion criteria for reference individuals were established.60 healthy males and 63 females were enrolled and divided to three groups by age,including 20 to 39 years old group (20 males and 20 females),40 to 59 years old group (20 males and 23 females)and above 60 years old group (20 males and 20 females).Blood samples were drawn in cit-rate sodium anticoagulated tubes and D-dimer concentration was determined by three different coagulation analyzers using o-riginal reagents.According to CLSI guideline C-28-A3,the reference interval for each measurement system from reagent manufacturer was verified and the difference of D-dimer concentration between different age-group and sex-group was ana-lyzed using non-parameters tests.Results All reference intervals were verified for people under age 40,while one reference interval cannot be verified for people from 40 to 59 years old as same as one for people above 60 years old.D-dimer concen-tration increased with age and there was significantly different between 20~39 years old group and 40~59 years old group or above 60 years old group(P<0.05).There was only a significant difference between sex-group for people under age 60(P<0.05).Conclusion D-dimer concentration was associated with age and sex.For people under age 40,the reference inter-val from reagent manufacture can be verified and directly used in laboratory,while for people above age 60,the reference in-terval from reagent manufacture cannot be verified.The cause should be investigated and a new reference interval should be established separately when necessary.

Objective To detect fetal RhCcEe genotype from fetal DNA in maternal plasma for noninvasive prenatal diagnosis.Methods DNA from maternal plasma sample was extracted by use of QIAamp DNA Kit. The existence of fetal DNA was confirmed by amplified fetal SRY gene. The fetal RhCcEe gene was amplified by polymerase chain reaction (PCR) from 30 pregnant maternal plasma. The results of fetal RhCcEe genotype were evaluated retrospectively by the serologic analysis of infant and pregnant woman RhCcEe phenotype.Results Among the 30 samples, 13 were the same phenotypes between mother and infant, 17 were different. When mother phenotypes were RhCC, cc, EE and ee homozygous, the deleted allele gene can be successfully amplified from mother plasma.Conclusion Noninvasive fetal RhCcEe genotyping is reliable. When the mother was homogyzous, genotyping the fetal CcEe alleles was very significant and useful for HDN (hemolysis disease of newborn) diagnosis and therapy.

Objective To investigate the association of B?-fibrinogen gene-455G/A polymorphism and plasma fibrinogen levels with Henoch-Schoenlein purpura(HSP) in children.Methods Sixty-seven children(including 40 boys and 27 girls) with HSP were served as HSP group,age ranging from 5-14 years,with the average age of 9 years.Seventy healthy controls(including 37 boys and 33 girls) were served as healthy controls.Age ranging from 5-13 years,with the average age of 9 years.The B?-fibrinogen gene-455G/A polymorphism was detected in all subjects by polymerase chain reaction-restrictive fragment length polymorphism technique with restrictive enzyme HaeⅢ.Results There were a greater proportion of individuals with the AA,GA,GG genotype in the HSP group comparied with those of the healthy controls(?2=29.5 P0.05].Conclusions The B?-fibrinogen gene-455G/A mutation is correlated with HSP in children,A allele is susceptibility gene of HSP in children.The plasma fibrinogen level is related to HSP in children.

Asian People/genetics* ; Gene Frequency ; Genetics, Population ; Humans ; Microsatellite Repeats/genetics* ; Phylogeny ; Polymorphism, Genetic/genetics*

Objective To clarify the efficacy and safety of cyclosporine A CsA) as salvage therapy in patients with severe active ulcerative colitis (UC) and refractory to steroids.Methods A total of 24 severe active UC patients refractory to steroids and hospitalized from 2006 to 2012,were retrospectively enrolled.Data including demographic features,clinical manifestations,laboratory tests and medications were collected.Results CsA was effective in 15 (62.5％) patients,who did not receive colectomy during 12-week administration.This regimen was tolerable in most patients.Twelve (50.0％) patients reported 16 adverse events,but only one patient withdrew CsA due to intolerance.The rates of adverse events in initial intravenous CsA including 4 mg · kg-1 · d-1,3 mg · kg-1 · d-1 and 2 mg· kg-1 · d-1 were 2/2,9/17 and 1/5 respectively.Responders had higher white blood cell count compared with non-responders (P =0.045).Conclusions CsA could be an effective alternative regimen to colectomy in severe active UC patients who are refractory to steroids.

Adoptive immunotherapy using allogeneic natural killer (NK) cells provides to be useful in recipients after allogeneic hematopoietic stem cell transplantation (Allo-HSCT), but its application has been limited by the inability to obtain sufficient numbers of pure NK cells. This study was aimed to optimize the expansion of high purity NK cells from human peripheral blood. First, the NK cells were isolated from PBMNC by using miniMACS (magnetic cell-selection) and NK Cell Isolation Kit II. Then the isolated cells were cultured in SCEM (Stemline Hematopoietic Stem Cell Expansion Medium, Sigma) supplemented with 10% human AB serum and different combinations of IL-2 and/or IL-12, IL-15 for 15 days. Cultures were fed with fresh media and cytokines every 3 days, and were evaluated for cell expansion, phenotype, and cytotoxicity at the end of the culture period. The results showed that in group IL2 + IL15 and IL2 + IL15 + IL12, cells were expanded 50.46 +/- 4.31 and 52.35 +/- 6.72-fold respectively, much higher than others (P<0.01), but no significant difference between themselves (P>0.05). And the purity of CD3(-)CD56(+) NK cells was over 94% in all groups except the control. The cytotoxicity of expanded NK cells cultured with cytokines was significantly higher than the starting population at different E:T ratio (P<0.01), although the cytotoxicity of IL2 + IL15 + IL12 group was slightly higher than that of IL2 + IL15 group, but no significant difference between themselves (P>0.05). It is concluded that high purity of NK cells can be efficiently expanded in culture with IL2 + IL15.
CD3 Complex ; analysis ; CD56 Antigen ; analysis ; Cell Culture Techniques ; methods ; Cell Differentiation ; drug effects ; Cell Separation ; Cells, Cultured ; Culture Media ; Humans ; Interleukin-15 ; pharmacology ; Interleukin-2 ; pharmacology ; Killer Cells, Natural ; cytology ; immunology ; Leukocytes, Mononuclear ; cytology

OBJECTIVETo explore the expansion method of high purity NK cells from human peripheral blood and explore the changes in biological functions of NK cells after ex vivo expansion.

METHODSNK cells were isolated from peripheral blood mononuclear cells (PBMNCs) by using miniMACS (Magnetic cell-selection) and NK Cell Isolation Kit II, and cultured in SCEM (Stemline Hematopoietic Stem Cell Expansion Medium, Sigma) supplemented with 10% human AB serum and different combinations of interleukin (IL)-2 and/or IL-12, IL-15 for 15 days. Cultures were semi-exchanged with fresh media and cytokines every 3 days. Evaluation for cell expansion, phenotype, perforin and granzyme B mRNA expressions, and IFN-gamma secretion before and after the culture period.

RESULTSCD3(-) CD56(+) cells concentration increased from (11.2 +/- 5.2)% to (94.2 +/- 3.5)%. In group IL-2 + IL-15 and IL-2 + IL-15 + IL-12 group, cells were expanded 50.5 +/- 4.3 and 52.3 +/- 6.7 - fold, respectively, being significantly higher than that in other three groups [(15.4 +/- 1.1 fold in IL-2 group, 19.9 +/- 3.9 fold in IL-2 + IL-12 group, 6.1 +/- 1.0 fold in control group)] (P<0.01), but no significant difference between each other (P>0.05). The purity of CD3(-) CD56(+) NK cells was over 94% in all groups except the control. The perforin and granzyme B mRNA expressions of expanded NK cells in four experimental groups were significantly higher than those of before expansion (P<0.01) and the expressions in IL-2 + IL-15 and in IL-2 + IL-12 + IL-15 group were significant higher than in other three groups (P<0.01) while no significant difference between each other (P>0.05). IFN-gamma levels in the supernatants of four experiment groups were significantly higher than that in control group (P<0.01) and its levels order was IL-2 + IL-15 + IL-12 group > IL-2 + IL-12 group > IL-2 + IL-15 group > IL-2 group (P<0.01).

CONCLUSIONHigh purity NK cells isolated by negative selection using miniMACS can be efficiently expanded with IL-2 + IL-15, and their biological functions were enhanced.

Cell Culture Techniques ; Cell Proliferation ; Cell Separation ; Cells, Cultured ; Granzymes ; metabolism ; Humans ; Interferon-gamma ; metabolism ; Interleukin-12 ; pharmacology ; Interleukin-15 ; pharmacology ; Interleukin-2 ; pharmacology ; Interleukins ; pharmacology ; Killer Cells, Natural ; cytology ; drug effects ; immunology ; metabolism ; Perforin ; metabolism

Objective To study the changes of plasma cystatin C level (PcyC),and evaluate the effects of the joint use of atorvastatin and probucol on PcyC and severity of coronary lesion in patients with borderline lesion of coronary artery.Methods One hundred and thirty consecutive patients with borderline coronary lesion assessed by quantitative coronary angiography were enrolled into borderline coronary lesion group (BCL),and another 136 subjects without coronary lesion were enrolled as controls (CTR).And in the meantime,the subjects in BCL group were randomized (closed envelope method) into routine treatment subgroup ( RTT,n =60),and combined treatment subgroup in which patients were treated with atorvastatin 20 mg plus probucol 1.0 g daily in addition to routine medication ( CBT,n =70) for 6 months.There were no statistical differences in basic clinical features between two subgroups.PcyC,high-sensitive C-reactive protein (hs-CRP),total cholesterol (TC),low-density lipoprotein cholesterol (LDL-C),high-density lipoprotein cholesterol ( HDL-C ) and triglycerides (TG) were determined.Of them,104 patients in BCL group rechecked by coronary angiography.Comparison of biomarkers carried out between two groups by using a number of independent-sample t-test and analysis of variance.For enumeration data,chi-square test was used to compare mean values of biomarkers between groups. P ＜ 0.05 was considered statistically significant.Results PcyC levels were significantly higher in BCL group than those in CTR group ( P ＜ 0.05 ).Compared with RTT subgroup,levels of PcyC,TC,LDL-C,TG and hs-CRP were more significantly decreased in CBT subgroup (P ＜ 0.05,P ＜ 0.01 ).Moreover,there was a trend of slight decrease in the mean percent of stenosis (MPS) of coronary artery with borderline lesion in RTT subgroup treated for 6 months,whereas more marked decrease in the MPS of coronary artery with borderline coronary lesion in CBT subgroup treated for 6 months ( P ＞ 0.05 ; P ＜ 0.05 ).Conclusions Cystatin C plays an important role in the pathogenesis of coronary artery,and PcyC is associated with severity of coronary lesion,the combination of atorvastatin and probucol decreases the PcyC level,and it may be the treatment of choice for borderline lesion of coronary artery.

To investigate the effect of Jiawei Foshou San and its various combined administration on hepatic P450 enzyme activity and hepatocyte morphology in rats. Rats were orally administered with drugs for four weeks and then sacrificed to prepare liver microsomes. The liver microsomes were incubated with the cocktail method; The metabolites were determined with the rapid liquid chromatography with tandem mass spectrometry (LC-MS/MS) to investigate the hepatocyte P450 enzyme activity. In addition, the hepatic pathological changes were observed by using the hematoxylin and eosin (HE) staining. Compared with the control group, the enzyme activity of CYP1A2 and CYP3A4 in the Jiawei Foshou san group showed a significant rise (P < 0.05); the enzyme activity of CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 in the ferulic acid + ligustrazine group and the ligustrazine + tetrahydropalmatine group showed a significant rise (P < 0.05) ; the enzyme activity of CYP1A2, CYP2D6 and CYP2E1 in the ligustrazine group showed a significant rise (P < 0.05); the enzyme activity of CYP3A4 in the ferulic acid group showed a significant reduction (P < 0.05). After the administration with various drugs, the hepatocyte morphologies in the ferulic acid group and the ligustrazine group were normal. The pathological changes were observed in the tetrahydropalmatine group, such as unclear boundary of hepatic lobules, disordered hepatic cell arrangement, blurred edge, anisokaryosis and infiltration of inflammatory cells. The ferulic acid + tetrahydropalmatine group, the ligustrazine + tetrahydropalmatine group and the Jiawei Foshou San group also showed inflammatory infiltration, but with less pathological changes, particularly the Jiawei Foshou San group. The study result shows that Jiawei Foshou San can induce the enzyme activity of CYP1A2 and CYP3A4, and ligustrazine may be the effective substance for inducing CYP1A2. Its combination with ferulic acid and ligustrazine can significantly reduce the liver toxicity of tetrahydropalmatine.
Animals ; Cytochrome P-450 Enzyme System ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Hepatocytes ; drug effects ; enzymology ; Microsomes, Liver ; drug effects ; enzymology ; Rats ; Rats, Sprague-Dawley