Objective Analysing the potential relationship of mismatch repair protein MLH3 gene C2531T mutation with oligospermia and azoospermia cases. Methods Using MLH3 C2531T as an example to establish a method of tetra-primer amplification refractory mutation system-PCR (4P-ARMS-PCR); We chose 81 cases of oligospermia, azoospermia patients and 80 normal controls, detecting MLH3 C2531T both using 4P-ARMS-PCR and PCR-RFLP; randomly to select 20% samples to sequence. Results The results suggested higher prevalence of CT + TT genotypes in patients as compared to the controls and association of T allele with disease (P <0. 01); PCR-RFLP results , 4P-ARMS-PCR results and sequence results are fully consistent. Conclusion 4P-ARMS-PCR can be a fast, easy-operating and accurate technology for detection of gene mutation of single nucleotide polymorphism (SNP) ; MLH3 C2531T polymorphism is related to oligospermia and azoospermia.

Objective Analysing the potential relationship of mismatch repair protein MLH3 gene C2531T mutation with oligospermia and azoospermia cases. Methods Using MLH3 C2531T as an example to establish a method of tetra-primer amplification refractory mutation system-PCR (4P-ARMS-PCR);We chose 81 cases of oligospermia,azoospermia patients and 80 normal controls,detecting MLH3 C2531T both using 4P-ARMS-PCR and PCR-RFLP; randomly to select 20% samples to sequence. Results The results suggested higher prevalence of CT+TT genotypes in patients as compared to the controls and association of T allele with disease(P

Objective To establish an inductively coupled plasma‐mass spectrometry (ICP‐MS) method for analysis of elements in peripheral blood of children .Methods A total of 474 healthy children in Hunan area were enrolled in this study ,and six ele‐ments ,including Ca ,Mg ,Fe ,Cu ,Zn and Pb ,in peripheral blood specimens were detected by using ICP‐MS method .Results The levels of elements ,including Ca ,Mg ,Fe ,Cu ,Zn and Pb ,in peripheral blood of healthy children showed skewed distributions ,and no significant differences were found in levels of these elements between male and female children(P>0 .05) .The reference intervals of Ca ,Mg ,Fe ,Cu ,Zn and Pb in peripheral blood of healthy children in this area were 57 .30 -81 .40 mg/L ,30 .40 -44 .80 mg/L , 361 .20-531 .40 mg/L ,848 .10-1 469 .20 μg/L ,2 .68 -6 .54 mg/L and 0 .00 -100 .00 μg/L respectively .Conclusion The ICP‐MS method for simultaneously detecting Ca ,Mg ,Fe ,Cu ,Zn and Pb in peripheral blood of children and reference interval of each ele‐ment are successfully established .

Objective:To improve the immunoblotting of immunoprecipitated proteins and decrease the interference of immunoprecipitation antibody in the interaction of endogenous proteins. Methods:Transient transfect cells with fusion protein expression vector containing the targeted S5b gene and the FLAG tag, the transfected cells or untransfected cells were harvested to study the exogenous or endogenous protein interaction. The total cell lysate was immunoprecipitated by specific antibody. Then the eluted immunocomplex was separated by SDS-PAGE, and the TrueBlot antibody or conventional antibody was used as the secondary antibody for immunoblotting detection of S5b and its partner (Rpt1 and Rpt2). Results:Clear immunoblotting bands for S5b, Rpt1 and Rpt2 were obtained. Conclusion:TrueBlot antibody prefers the immunoblot antibody to immunoprecipitation antibody, and decreases the interruption of immunoprecipitation antibody to display clear protein band.

Laboratory medicine is experiencing rapid development and moving from the edge of medicine stage to the center of medicine.Laboratory medicine has become the component of evidence-based medicine,the pathway to translational medicine and the core of precision medicine.These recent advances have increased the challenges and opportunities for laboratory medicine education.It will be the most urgent task to us how to train innovative personnel, medical laboratory physician, genetic counselor, medical laboratory scientist and personnel needed for personalized medicine.

OBJECTIVE: To explore the effect of fluorouracil (FU) combined with epigenetic drug FK228 (depsipeptide FR901228) on the proliferation, apoptosis and Fas mRNA level in HepG2 hepatoma cell lines. METHODS: There were 4 groups in this experiment: an untreated group, an FK228 treated group, a FU treated group,and a FU combined with FK228 group.Tetrazolium salt colorimetry (MTT) assay was used to assess the cell inhibition rate. Q value of Kingos formula and multi-factor analysis of variance (ANOVA ) were used to judge the combination treatment effect,and flow cytometry was used to detect the apoptosis rate. Fas mRNA level was analyzed by RT-PCR. RESULTS: FK228 or FU would inhibit the growth of HepG2 cells in a concentration-dependent and time-dependent manner. Cell inhibition rates of HepG2 were significantly enhanced in the FU combined with FK228 group, compared with that in the FU treated group alone (P<0.05). Both Q values were more than 1, the 2 drug combinations showed interaction,and FU combined with FK228 had synergistic effect.Compared with the FK228 treated group and the FU treated group, apoptosis rate of HepG2 cells was significantly increased (P<0.05), and the Fas mRNA level was up-regulated in HepG2 cells in FU combined FK228 group (P<0.05). CONCLUSION Combination of FK228 and FU can enhance the proliferation inhibition and apoptosis induction of FU in hepatoma cell lines, up-regulate the Fas mRNA level, and increase the sensitivity of hepatoma cell lines to FU.
Antibiotics, Antineoplastic ; pharmacology ; Antimetabolites, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Depsipeptides ; pharmacology ; Drug Synergism ; Fluorouracil ; pharmacology ; Hep G2 Cells ; Humans ; RNA, Messenger ; genetics ; metabolism ; fas Receptor ; genetics ; metabolism

OBJECTIVE: To develop a rapid detection method for microalbuminuria with size-exclusion high performance liquid chromatography. METHODS: With a new mobile phase, samples were injected onto an Agilent Zorbax GF-250 size exclusion chromatographic column. The method was evaluated and urine albumin of 56 diabetic patients was analyzed. RESULTS: The mobile phase containing 0.1% formic acid and acetonitrile, with 20 μL sample size, 1 mL/min flow rate, 205 nm detection wavelength. The retention time of albumin both in human serum and urine was 1.7 min. The linear range was 5-2000 mg/L. The lower limit of measurement was 2 mg/L. The intra-assay coefficient of variation and the inter-assay coefficient of variation were 3.98% and 4.05% (20 mg/L), 3.55% and 3.60% (200 mg/L), 4.65% and 4.74% (2000 mg/L), respectively. Recovery rates were 95.3%, 98.1%, and 97.2%. Microalbuminuria was detected in 30 samples by high performance liquid chromatography and 15 samples by immunoturbidimetry from 56 patients with diabetic mellitus. CONCLUSION A fast and high sensitivity method, namely size-exclusion high performance liquid chromatography, with mobile phase containing 0.1% formic acid and acetonitrile has been established to analyze microalbuminuria, which can detect more microalbuminuria than other methods and is suitable for clinical routine measurement.
Albuminuria ; diagnosis ; Chromatography, High Pressure Liquid ; Diabetes Mellitus ; urine ; Humans

Objective To investigate the regional distribution of positive results of early screening for women of child-bearing agein the six provinces of southern China(Guizhou,Guangxi,Yunnan,Sichuan, Chongqing and Hunan).Methods The screening of all were performed by red blood cell related parameters、quantitative analysis of hemoglobin、Osmotic fragility test of erythrocyte incubation(one tube method)、dena-tured hemoglobin inclusion body test among 222 645 peoples reproductive age in the six provinces of southern China,and statistical analysis of abnormal screening in different provinces(cities)was carried out.Results A-mong 222 645 cases in the six provinces(cities),abnormal samples were detected in 32074 cases,and the abnor-mal detection rate was 14.39%.The total positive detection rate from high to low according to the order of Guangxi(24.87%),Sichuan(17.53%),Yunnan(14.76%),Hunan(11.53%),Chongqing(10.69%)and Guizhou(8.61%).Among the six provinces(city),the total α and β thalassemia screening positive rate were 16.37%,17.81%,the specific distribution(α/β):Guangxi(7.06% /5.89%),Sichuan(2.74% /3.02%),Hunan (1.87% /2.32%),Yunnan(1.56% /1.57%),Guizhou(1.85% /3.04%)and Chongqing(1.29% /1.97%).The total α and β thalassemia screening suspected positive rate were 50.56%,3.25%,the specific distribution(α/β):Guangxi(11.59% /0.33%),Sichuan(11.27% /0.50%),(9.58% /2.05%)in Yunnan,Chongqing(7.35% /0.08%),(7.12% /0.22%)of Hunan and Guizhou(3.65% /0.07%).Conclusion The six southern provinces (city)positive screening rate distribution is different,the highest in Guangxi,followed by Sichuan,Guizhou is the lowest;suggestions will flow into the southern city and high malaria area included in in screening of thalas-semia.The four-way experiment has a higher positive rate of screening for thalassemia,and screening out the number of abnormal hemoglobin disease,and has great value of reduce the misdiagnosis.In order to further re-duce the misdiagnosis,suggested that serum iron detection into thalassemia screening.Strengthen the screen-ing efforts for thalassemia in high risk families,reducing the birth rate of thalassemia major,improve popula-tion quality,reduce the economic burden of the family and the country.

Objective To investigate the DNA staining efficiencies of 4 kinds of nucleic acid fluorescent dyes,including EB,SYBR Green Ⅰ,Gold View and AO,in single-cell gel electrophoresis (SCGE) assay,and the possibility to use a new nucleic acid fluorescent dye instead of EB.Methods The peripheral blood lymphocytes from healthy individuals were isolated and treated with 0,20,40,60 and 80 μg/mL of H2O2,respectively.Then,the DNA damages of lymphocytes were detected by the neutral SCGE assay.The DNA was stained with EB,SYBR Green Ⅰ,Gold View and AO dyes,respectively,and the staining results were observed and compared under a fluorescence microscope.In addition,the percentages of tail DNA (％ Tail DNA) from different staining methods were analyzed and compared.Results The results of SCGE showed that SYBR Green Ⅰ,Gold View and AO staining could well reflect the DNA damages of lymphocytes,and that the optimal concentrations for SYBR Green Ⅰ,Gold View and AO were 1 ×-5 ×,2 ×-5 × and 2-5 μg/mL,respectively.The regression coefficients for EB,SYBR Green Ⅰ,Gold View and AO were 2.71,2.81,2.73 and 2.75,respectively,which indicated that there was consistent dyeing effect between them.The results of ％ Tail DNA were stable with in 48 hours,and there was no significant difference between the 4 fluoresent dyes (P ＞ O.05).The inter-assay coefficients of variation (CVs) of SYBR Green Ⅰ,Gold View and AO were 6.92％,7.10％ and 8.25％,respectively,which were superior to that of EB (8.35％).The intra-assay CVs of SYBR Green Ⅰ and Gold View were 3.07％ and 2.74％,respectively,which were superior to that of EB (3.59％).Conclusion SYBR Green Ⅰ,Gold View and AO may be used for the nucleic acid fluorescent staining,and especially Gold View is more suitable for instead of EB in SCGE.

Objective To investigate the expressions of phosphorylated H2AX (γH2AX) and p53-binding protein 1 (53BP1) in DNA oxidative damage of human bronchial epithelial (HBE) cells.Methods The HBE cells were treated with 0,25,50,100,200,400 μmol/L of hydrogen peroxide (H2O2) for 1 hour,respectively,and their DNA oxidative damages displaying as double-strand breaks (DSBs) were induced.The viability and apoptosis of HBE cells were measured by the CCK-8 method and flow cytometry,respectively.The expression status of γH2AX and 53BP1 in nucleus of HBE cells was observed by a fluorescence microscope.The expression levels of γH2AX,53BP1 and BRCA1 were determined by western blot.Results Compared with the control (0 μmol/L of H2O2),the via bility of HBE cells treated with 25 μmol/L of H2O2 (1.07 ±0.01) increased,while those with 50,100,200,400 μmol/L of H2O2 (0.97 ± 0.01,0.96 ± 0.01,0.95 ± 0.01,0.94 ± 0.01) decreased significantly (F =50.35,P ＜ 0.01).The apoptosis rates of HBE cells treated with 50,100,200,400 μ mol/L of H2O2 ([7.54 ± 0.57] ％,[7.84 ± 0.68] ％,[8.40 ± 0.50] ％ and [14.03 ± 1.03] ％) were significantly higher than that with 0 μmol/L of H2O2 ([4.65 ± 0.32] ％,F =35.879,P ＜ 0.01).Compared with the control (0 μmol/L of H2O2),the average fluorescence intensity of γH2AX in nucleus of HBE cells treated with 25,50,100,200,400 μmol/L of H2O2 increased significantly (F =223.97,P ＜ 0.01),while those of 53BP1 in nucleus of HBE cells treated with 50,100,200,400 μmol/L of H2O2 decreased significantly (F =117.78,P ＜ 0.01).The results of western blot showed that the expres sion levels of γH2AX increased with the increase of H2O2 concentration,while that of 53BP1 and BRCA1 was on the contrary (F =96.20,21.92 and 11.55,respectively,P ＜0.01).Conclusion In the oxidative damage of HBE cells induced by H2O2,γH2AX may be used as a marker of DNA oxidative damage,while the decreased expression of 53BP1 suggests that other mechanisms to repair the DNA damage sites may exist.