Objective To increase the sensitivity of quantitative PCR using DNA polymerase in combination with aptamer 6-10.Methods The quantitative PCR was performed using DNA polymerase mixed with aptamer 6-10 or specific antibody.The low detection limit of each modified method was compared with that of the conventional method.Results The quantitative PCR using aptamer 6-10(200 nmol/L) or specific antibody increased the low detection limit of human death receptor 5(DR5) plasmid from 105 copies/?l to 103 copies/?l.Melting curves showed that each method had minimal nonspecific amplification when the concentration of DR5 plasmid was 105 copies/?l;however,when the concentration of DR5 plasmid was 103 copies/?l,the conventional method had nonspecific amplification,whereas the method using aptamer 6-10 had minimal nonspecific amplification.Conclusions Aptamer 6-10 of DNA polymerase can increase the sensitivity of quantitative PCR.

Objective: To study the new device and technology in prepartion of superfined Chinese herbal medicine powders. Methods: Improving the Trost jet mill was to divide continually the feed and the coarse grains classified from a circular disc into two two phase flow by means of a especial four way pipe in mixing. and to impact with high speed oppositely each other, the superfined powders are prepared. Results: The superfined powders of ginseng, boat fruited sterculia seed and balsam pear are successfully prepared to guarantee 10～15?m in diameter. Conclusions: It is very effective to pulverize Chinese herbal medicine with this device in mass production.

AIM: To investigate the amount and patterns of expressing CD69 , IL-4 and IFN-? on TCRV?24 +NKT cells, and compare with that of CD3 +T cells from human peripheral blood in response to in vitro stimulation. METHODS: The whole blood was stained with three-color immunofluorescence directly or after cultured with PDB+ionomycin(Ion) for 6 h, then the mononuclear cells were separated by lysing red blood cells. The expression rates of CD69, IL-4 and IFN-? on TCRV?24 +NKT cells and CD3 +T cells were estimated by flow cytometer. RESULTS: As a proportion of mature T cells, the ratio of TCRV?24 + NKT cells to CD3 +T cells was about(1.34?0.42)%. The expression rates of CD69 on TCRV?24 + NKT cells and CD3 +T cells in response to PDB + Ion for 6 h were (96.71?1.33)% and (98.60?0.47)%, respectively, while the ratio were (11.47?2.86)% and (1.07?0.45)% in the unstimulated group, and there were significant difference between them. The expression rates of IL-4 and IFN-? on TCRV?24 +NKT cells stimulated with PDB+Ion for 6 h were (48.62?2.44)% and (46.65?8.91)%, respectively ,which were significantly higher than that of unstimulated group [(31.57?3.31)%, (13.45?6.29)%] and that of stimulated CD3 +T cells, though the expression rates on stimulated CD3 +T cells were significantly higher than that of unstimulated CD3 +T cells. CONCLUSIONS: There is small amount of NKT cells in adult human peripheral blood. The expression rates of IFN-? and IL-4 on these lymphocytes are higher than CD3 +T cells, suggesting that NKT cells are important immunomodulatory cells in special microvironments.

AIM: To investigate the effect of berberine (Ber) on the activation and proliferation of T lymphocytes and its mechanism of action. METHODS: Whole peripheral blood from normal subjects was stimulated with phytohemagglutinin (PHA) or phorbol ester (PDB) plus ionomycin (Ion) and the expression levels of CD69 and CD25 were evaluated with flow cytometry after the staining with appropriate fluorescent monoclonal antibody. The distribution of cell cycles was analyzed by propidium iodide staining and dead cells by 7-aminoactinomycin live staining. RESULTS: 100 ?mol/L and 50 ?mol/L of Ber had significant inhibition of the expression of CD69 on T cells stimulated with PDB plus Ion or PHA, while effect of 25 ?mol/L Ber was not significant. And as time of action extended, the extent of inhibition decreased. For the expression of CD25, Ber at the concentrations as above all exerted significant inhibitory effect in a dose-dependent manner. Moreover, Ber could block lymphocytes cell cycle progression from G_0/G_1 phase to S and G_2/M phase without phase specificity. Besides, live staining analysis revealed that Ber did not have significant cytotoxicity on lymphocytes. CONCLUSIONS: Ber significantly inhibits the expression of early and mid activation antigens of T cells and also blocks the progression of lymphocytes cell cycles. These results suggest that Ber exerts immunosuppression effect through inhibiting the activation and proliferation of T cells.

Objective To observe the effect of compound rehmannia on SOD and ATP enzyme in the brain of SAM-P/8 dementia mice and the change of apoptosis neuron cells.Methods SAM-P/8 mice were divided randomly into three groups:experimental group,treatment group and model group.Mice in experimental group were lavaged by compound rehmannia(2ml/day,individual).Mice in treatment group were lavaged by donepezil hydrochloride(400mg/kg,individual),and mice in normal group and model contrast group were lavaged by equal sodium chloride.After 3 weeks,we observed mice′s behavior in every group and investigated the changes of SOD,Na+-K+-ATP enzyme by immunohistochemistry and investigated the changes of apoptosis cells peak by FCM.Results Compound rehmannia could improve learning and memory ability of the SAM-P/8 mice,strengthen SOD,Na+-K+-ATP activity and reduce apoptosis neurons in brain.Conclusion Compound rehmannia can significantly improve the Alzheimer's mice learning and memory ability,strengthen the energy of brain cells,and increase resistance to free radical antioxidant capacity,reduce the neuron apoptosis and has a good relatively protection and care to SAM-P/8 mice brain neurons.

AIM: To construct the mammalian cell expression vector for enhanced green fluorescent protein (EGFP) and HLA-A*0201 fusion protein and analyze its expression and subcellular localization in the transfected K562 cells. METHODS: The HLA-A*0201 cDNA was cloned by RT-PCR and the gene was inserted into pEGFP-N1 to construct a vector for the fusion protein. The expression of the fusion protein in K562 cells transfected with the vector was evaluated by flow cytometry and its subcellular localization was investigated by confocal microscopy. RESULTS: The full-length encoding region of HLA-A*0201 cDNA was cloned from two HLA-A2 positive donors and the expression vector for the HLA-A*0201-EGFP fusion protein was constructed by PCR using a primer pair to introduce a Kozak sequence before ATG and the stop codon was deleted. Five hours after K562 cells was transfected with the vector, the expression percentages of HLA-A*0201 and EGFP were 25.12?2.26 and 27.37?3.59, respectively and no significant increase was observed after 24 h. The fusion protein was predominantly located on the membrane with low level distribution within the cells. In contrast, no HLA-A*0201 but only EGFP was detected in the empty vector transfected K562 cells and the EGFP was dispersed within the cells. CONCLUSIONS: The expression vector for HLA-A*0201-EGFP fusion protein was constructed and the fusion protein expressed in K562 cells was primarily distributed on the membrane. The results suggest that the transfected K562 cells are potential antigen-presenting cells.

Objective To study the correlation between N-terminal pro-brain natriuretic peptide (NT-proBNP) and cardiac diastolic and systolic function. Methods 60 New Zealand male rabbits were randomly assigned to two groups. The abdominal aorta 1 cm just above right renal artery were constricted in operated group. The clinical manifestation of heart failure was observed, and cardiac structure and function were investigated with echocardiography. Concentration of serum NT-proBNP was determined regularly. Results The concentration of NT-proBNP was correlated with Sm（r=－0.635,P=0.000）, Em（r=－0.693,P=0.000）, E/Em（r=0.688,P=0.000）, E/Vp（r=0.707, P=0.000）, and EF（r=－0.395,P=0.001）. Conclusion Concentration of serum NT-proBNP could prognose the diastolic and long axis systolic function of left ventricle.

Objective To establish an animal model of heart failure with preserved ejection fraction(EF)resulting from hypertension.Methods35 male New-Zealand rabbits were randomly assigned to 2 groups:constriction was performed at the abdominal aorta 1 cm just above right renal artery in operated group(n=25),and the sham-operated animals were taken as control group(n=10).The clinical manifestations of heart failure were followed up carefully.Systolic and diastolic function,and systemic hemodynamics were investigated with echocardiography and left ventricle catheter.ResultsIn operated group,marked ventricular hypertrophy was observed 2 weeks after operation.4 weeks after operation,the clinical manifestations of heart failure presented,and IVRT and LVEDP increased,and-dp/dt max decreased.Tissue doppler image indicated that e/a ratio of annulus of mitral valve decreased(P<0.05).However,EF and +dp/dt max were preserved.ConclusionThe operated animals presented the clinical manifestation of heart failure,and diastolic function of left ventricle injured,however,ejection fraction was preserved,which corresponded with the character of heart failure with preserved ejection fraction resulting from hypertension.

Objective To investigate the status of cellular immunity from Th cell polarization in pa-tients with different courses of condyloma acuminatum(CA).Methods The isolated PBMC were polarized by PHA and self-plasma for72hours,then followed by two-color immunofluorescent staining with anti-CD4-PE and anti-CCR5-FITC,or with anti-CD4-FITC and anti-CCR3-PE.Finally the stained cells were analyzed by flow-cytometry.Results The percentages of Th1/Th2cells of the short-course group and long-course group were(25.82?2.22)%/(14.80?1.14)%and(12.20?1.37)%/(13.74?0.99)%,respectively;the differ-ences between normal control and two CA groups were significant(P

Objective:To analyze the incidence, biochemical and molecular characteristics, and gene mutation spectrum of neonatal methylmalonic acidemia (MMA) in Shaanxi province.Methods:This study involved 146 152 newborns undergoing neonatal screening for methylmalonic acidemia by tandem mass spectrometry in Northwest Women's and Children's Hospital from January 2014 and December 2019. Clinical manifestations and follow-up data of newborns diagnosed with MMA and their acylcarnitine profiles and gene mutations were analyzed. According to whether they had elevated homocysteine or not, these patients were divided into two groups, the complicated group and the isolated MMA group. The control neonates were those excluded from having methylmalonic acid by re-examination. Kruskal-Wallis and Mann-Whitney U test was conducted for statistical analysis. Results:(1) Twenty-one cases of MMA were confirmed with an incidence of 1/6 960, including 11 cases (52.4%) of isolated MMA (isolated MMA group) and 10 (47.6%) complicated by elevated homocysteine (complicated group). Eight patients in the isolated group had symptoms within one month after birth, mainly feeding difficulties, vomiting, drowsiness, poor response and infection, and five died. Patients in the complicated group were all diagnosed before developing typical clinical symptoms, and no developmental abnormalities were reported during follow-up. (2) Blood propionyl carnitine and its ratios to acetylcarnitine and free carnitine in the isolated MMA and complicated groups were higher than those in the control group [ M (min-max), 9.26 (3.70-37.78) μmol/L and 7.27 μmol/L (3.58-13.62 μmol/L) vs 4.51 μmol/L (1.48-8.69 μmol/L), H=23.239; 1.12 (0.32-2.43) and 0.74 (0.36-1.90) vs 0.25 (0.09-0.45), H=47.061; 0.94 (0.12-1.92) and 0.56 (0.18-1.03) vs 0.17 (0.06-0.38), H=36.868; all P<0.001]. The blood methionine level in the complicated group was significantly lower than that in the isolated MMA group [7.64 μmol/L (3.40-19.25 μmol/L) vs 24.22 μmol/L (10.73-56.55 μmol/L), U=3.000, P<0.001]. (3) All 21 patients carried complex heterozygous mutations or homozygous mutations in pathogenic genes, including 15 distinct MMUT mutations and 13 distinct MMACHC mutations. In the isolated MMA group, the most common mutation was c.323G>A (p.Arg108His) in the MMUT gene with a positive rate of 13.6%, and an unreported mutation, c.1676+11A>G, with unidentified clinical significance, was also found. The most common mutations in the complicated group were c.609G>A (p.Trp203Ter) and c.567dupT (p.Ile190fs) in the MMACHC gene, and the positive rates were both 20.0%. Moreover, two unreported variants, c.430-2A>C and c.648_650delAGA (p.216_217delSEinsS), were detected and suspected to be pathogenic. Conclusions:MMA is not uncommon in Shaanxi province. Children with isolated MMA tend to be more severe clinically. The identification of hotspot mutations, including c.609G>A (p.Trp203Ter) and c.567dupT (p.Ile190fs) in MMACHC gene and c.323G>A (p.Arg108His) in MMUT gene, provides a foundation for further genetic screening, counseling, and prenatal diagnosis, and is conducive to reduce the mortality and disability rate of neonatal MMA.