Capillary electrophoresis has high sensitivity and high separation efficiency, and mass spectrometry can provide structural information of samples. The combination of capillary electrophoresis and mass spectrometry can form powerful capabilities on qualitative and quantitative analysis. The presence of capillary electrophoresis-mass spectrometry makes it possible to accurately analyze the structure and molecular weight of bioactive components at the nanoscale sample level, or even at the single-cell level, which is an indispensable technology platform for the analysis of biological samples. With the development of this technique, the applications of capillary electrophoresis-mass spectrometry began to emerge in the field of clinical testing.

Objective To investigate the relationship between the onset of cerebral infarction in anterior and posterior circulation and the "three high" risk factors in Guangzhou communities. Methods From Jan. 2014 to Jan.2016,367 patients from Guangzhou communities were diagnosed with new cerebral infarction by head diffu-sion weighted imaging(DWI).The data were divided into two groups of anterior and posterior circulation,and uni-variate and multivariate methods were used to analyze the relationship between the "three high" and other risk factors and the onset of cerebral infarction in anterior and posterior circulation.The data of anterior circulation cere-bral infarction were further divided into two subgroups of lacunar and non-lacunar infarction,and the same statisti-cal methods were employed to analyze differences of risk factors between the two subgroups. Results The frequen-cies of hypertension(P = 0.040)and large atherosclerotic infarction(P = 0.012)were significantly higher,and the serum high-density lipoprotein(HDL)level(P = 0.045)was significantly lower in posterior circulation than those in anterior circulation,respectively;and the onset of posterior circulation cerebral infarction was more associ-ated with the incidence of hypertension(OR = 1.767,P = 0.035)and the decrease of HDL(OR = 0.380,P =0.021). In anterior circulation,the levels of systolic blood pressure(SBP)(P = 0.011)and diastolic blood pres-sure(DBP)(P=0.000),as well as the frequency of large atherosclerotic infarction(P=0.000)in non-lacunar infarction subgroup were significantly higher than those in lacunar infarction subgroup respectively,and the onset of non-lacunar infarction was more closely related to increased SBP levels(OR=1.045,P=0.001). Conclusions Among the"three high"risk factors,the onset of posterior circulation cerebral infarction is more closely related to the incidence of hypertension and the decrease of HDL.In anterior circulation,and the onset of non-lacunar infarc-tion is more closely related to the increased levels of DBP and SBP,especially to the elevated levels of SBP.

Objective To analyze the gene mutation and clinical characteristics of BRCA 1/2 by high resolution melting curve ( HRM ) in breast cancer patients and high risk groups , and to discuss the application value for BRCA 1/2 mutation detection by using HRM curve analysis in people at high risk of breast cancer.Method The clinical control analysis was used ,Peripheral blood samples of 52breast cancer patients,their first-degree relatives (52 cases consisting of high risk group ) and 40 healthy people without family history of breast cancer cases were collected from Anhui Province Tumor Hospital or Bozhou People ′s Hospital during March 2015 to June 2016.To establish an effective method for BRCA 1/2 mutation detection by using HRM curve, and the mutation positive results were validated by gene sequencing .To analyze the correlation between the of BRCA 1/2 gene mutation and the risk factors , a logistic method was used .Results 9 cases of BRCA1/2 gene mutations were found in 52cases of breast cancer patients and the mutation rate was 17.3%.5 cases of BRCA1/2 gene mutations were found in 52 cases of breast cancer high risk groups and the mutation rate was 9.6%.In 40 healthy people who without family history of breast cancer cases ,we found only 1 gene mutation case.All the mutation positive results detected by HRM curve analysis were matched with gene sequencing results .BRCA1/2 gene mutations and the risk factors such as primiparous age and chronic mammary gland disease have a certain correlation .In the 9 cases of BRCA1/2 gene mutations , we found 5 cases of BRCA1/2 gene mutations in their first-degree relatives, with the consistency of 44.4%(4/9).Conclusion Thebreast cancer patients′s first-degree relatives have a high mutation rates on BRCA1/2 gene and they can be the key screening objects .HRM curve analysis can be used in health screening and risk assessment at the breast cancerhigh risk groups .

Objective To establish a novel method for detecting circulating tumor cells (CTCs) in phripheral blood of lung cancer patients with high sensitivity and specificity.Methods Experimental study.42 cases of initial treatment patient who underwent resection and diagnosed to be non-small cell lung cancer by biopsy were studied,including 7 patients at stage Ⅰ,9 patients at stage Ⅱ,16 patients at stage Ⅲ and 10 patients at stage Ⅳ.As a control group,20 cases of healthy volunteers were selected.A series of experiments was conducted to determine the efficiency of tumor cells isolation,in which varied concentration (50,100,200,500,1000 cells) of A549 cells spiked into 2 ml peripheral blood drawn from healthy donors.The blood was removed of unwanted erythrocytes by lysis buffer,and made the rest of nucleated cells incubate with anti-EpCAM magnetic beads,then separated and enriched by a specific detector.All epithelia cells were retained on a slide because of a magnetic force and identified by H&E staining protocol.On the basis of cell recovery rate we calculated the sensitivity of tumor cells isolation.20 blood samples taken from healthy individuals were also detected to validate the specificity of this method.Samples of 42 patients with lung cancer were assayed for CTCs detection by above method.The correction of CTCs quantity with the patients' clinical features,for example,ages,gender,clinical stage,tumor size was analyzed in lung cancer patients by chi-square statistics.The correction of recovery cells with the spiked cells were assayed by linear correlation.Results The recovery rate was ranging from 68％ to 82％ by spiking varying numbers of A549 lung cancer cells into 2ml blood samples of healthy volunteers.Regression analysis of number of recovered vs.spiked A549 cells yielded a regression equation of Y =0.6419X + 8.8875.The number of CTCs detected has signification correlate with the cells spiked (R2 =0.9916,P ＜ 0.05),Eighteen of the 42 patients (43％) were found have CTCs in peripheral blood.The detection rate of lung cancer cells was 0 at stage Ⅰ,the detection rate of lung cancer cells was 11.1％ at stage Ⅱ,the detection rate of lung cancer cells was 62.5％ at stage Ⅲ and the detection rate of lung cancer cells was 70％ at stage Ⅳ.The positive rate of CTCs has no signification correlate with ages and gender of patients and tumor size (P ＞ 0.05),has signification with the clinical stage (P ＜ 0.05).None of the peripheral blood samples of the 20 healthy subjects analyzed was found to have CTCs.Conclusions This novel immunomagnetic separation technology is a sensitive and specific method,which provides a new tool allowing for feasible and specific detection of CTCs in lung cancer patients.The level of CTCs increases with the clinical stage and tumor size increased,which has important value to discover the early stage micrometastasis and redefine the clinical stage.But further multicenter and large sample clinical research are needed to confirm its clinical value.

Objective To investigate the correlation between the expression level of BLM and its clinical significance in leukemia.Methods 125 bone marrow specimens of inpatients and outpatients with leukemia were collected in Anhui provincial hospital from January 2011 to December 2011.125 leukemia patients were diagnosed and classified into acute leukemia (AL,n =66) and chronic myelogenous leukemia (CML,n =59) by Morphologic and Immunologic criteria,5 non-tumor individuals were included as control group.The BLM mRNA expressions were by reverse transcription-polymerase chain reaction(RT-PCR).The specimens were devided into groups according to the age,gender,leukemia type,peripheral blood leukocyte counts,hepatomegalia and(or) splenomegaly,fusion gene,chromosome karyotype,whether first visit and transplantation.The expression of BLM gene in each group and the correlation with above factors were retrospectively analysed.The statistical methods such as chi-square test,single factor variance analysis,t test and Pearson correlation test were mainly used.Results BLM mRNA was detected in leukemia.In bone marrow cells,the BLM gene expression was positive in 71 patients and negative in 54 patients.But none of 5 non-tumor bone marrow cells expressed BLM gene.The difference of BLM expression between patients and controls was statistically significant in two groups,i.e.peripheral blood leukocyte counts and fusion gene (x2 =14.730,22.399 ; P ＜ 0.05),but there is no statistical significant differences in other groups.The expressions of BLM mRNA in leukemia patients who had been treated with chemotherapy were lower than those newly diagnosed (0.1788 ± 0.1091 vs 0.3276 ± 0.2016 ; P ＜ 0.05).Moreover,BLM mRNA level in post-bone marrow transplant patients was lower than those not treated (0.1271 ± 0.1009 vs 0.2902 ±0.2034 ; P ＜ 0.05).Pearson correlation analysis showed that higher BLM mRNA expression positively correlated with fusion gene (r =0.357,P ＜ 0.01) and chromosome abnormality (r =0.279,P ＜ 0.05).Conclusion The BLM mRNA expression level of measurement can be used as judgment for leukemia patients disease severity and the index of prognosis,testing the level may provide a basis for clinical and curative effect judgment.

Objective To establish a real-time fluorescence quantitative PCR method for detection of the different expression level of FPGS in methotrexate enantiomer-resistant A549 cell lines,and observe FPGS mRNA expression in patients with leukemia.Methods A real-time fluorescence quantitative PCR method for detection FPGS mRNA was established using SYBR Green Ⅰ as fluorescence and β-actin as reference.The method was evaluated by Ct,correlation coefficient,slope,repeatability curve,melting curve and amplification efficiency curve.The expression levels of FPGS gene in methotrexate enantiomer-resistant A549 cell lines and methotrexate resistant leukemia cells in bone marrow were detected by the method.Results The standard curves had a high linear relationship between cycle threshold and template concentration.The correlation coefficients of FPGS and β-actin were 0.996 8 and 0.998 7,and the slopes were -3.595 and -3.740,respectively.The inter-coefficient of variation was from 1.27% to 2.95%.The intra-coefficient of variation was 3.82%.The method was characterized with specific melting curve and similar amplification efficiency(slope was 0.021 7).The relative contents of FPGS mRNA were(3.51 ±0.66),(0.16 ±0.01) and(1.00 ±0.31) in L-(+)-MTX/A549 cells(L),D-(-)-MTX/A549 cells(D)and A549 parent cells,and there was statistically difference among the three groups(F = 64.45 ,P＜ 0.01)Statistical difference was observed between L and D(q =9.29,P＜0.01).After treated with MTX,the expression level of FPGS mRNA was(0.35 ± 0.04) in methotrexate resistant leukemia patients,compared with(1.00 ± 0.44) before treatment.Statistical difference was observed(t = 8.83 ,P＜ 0.01).Conclusions The real-time fluorescence quantitative PCR is suitable for the quantification of FPGS.The expression levels of FPGS in methotrexate resistant leukemia cells in bone marrow and drug resistant cells are different.Two enantiomer forms of methotrexate may play different roles in drug resistance mechanisms.

Objective To develop an assay with polyclonal antibodies against a fragment derived from human albumin for determination of a peptide in urine, and to provide an early diagnostic tool for GVHD. Methods A small peptide composed of 16 amino acids (LVRYTKKVPQVSTPTL) was synthesized artificially. The immunogen was then diluted into 100 μg/kg body weight of rabbit. Subcutaneous injection in the immune animals was performed on both sides of spine and groin with 2.5 ml antigen suspension for three times, in order to prepare the polyclonal antibodies. The peptide antigen was then labeled with fluorescein isothiocyanate (FITC), and detected by LIF-CE-IA with the pre-prepared antibodies. Results The titer of serum. The migration time of the labeled antigen was 5.93 min, while the migration time of antigen-antibody complex was 6.47 min. The linear range of the method was 16 to 512 ng/ml, and the minimum detection limit was 10 ng/ml. Conclusions The polyclonal antibodies against the peptide antigen was isolated successfully, which possessed high titer and specificity. These results indicated that the assay was simple and rapid and could be applied for the early diagnosis of patient with GVHD.

Objective To establish a rapid and convenient method for determination of genomic DNA methylation in cells.Methods Five standard substances (dC, mdC, dA, dT and dG) were separated by high-performance capillary electrophoresis.Bare fused-silica capillary was used and eletrophoresis buffer was 48 mmol/L NaHCO3 with 60 mmol/L SDS, pH 9.6.The temperature of separation was controlled at 25 ℃ and a voltage of 20 kV was applied.The separation of the mixture was performed at a wavelength of 256 nm with UV-Vis detection and injection time was 5 seconds at 0.7 psi.Under optimal condition,genomic DNA methylation in methotrexate drug-resistant A549 cells was detected.Results The optimal condition was made by adjusting SDS concentration(40, 60, 80 mmol/L), pH value of running buffer(9.4,9.6, 9.8), voltage(15, 17, 19, 20, 22 kV), injection time(5, 10, 15, 20, 30 s) and capillary temperature(15, 20, 25, 30 ℃).The method for determination of genomic DNA methylation in cells was established.Five substances were completely separated by high-performance capillary electrophoresis in 10 mins.Intra-day coefficient of variation was less than 0.2% and inter-day coefficient of variation was less than 2%.The minimal detection limit was 2 μmol/L.Percentage of mdC in A549 parent cells was (4.80 ±0.52) %.Percentage of mdC in 15, 30, 40 μmol/L methotrexate drug-resistant A549 cells were (4.20±0.44) %, ( 3.70 ± 0.36 ) %, (3.10±0.35 ) %, respectively.Conclusions Genomic DNA methylation can be quantificated by high-performance capillary electrophoresis efficiently, rapidly, conveniently and sensitively.Genomic DNA methylation in methotrexate drug resistance cells decreases significantly.

Objective:To investigate the effect of traditional Chinese medicine (TCM) icariin (ICA) on metastatic phenotype of methotrexate (MTX)-resistant lung cancer A549 cells and elucidate the action mechanism and therapeutic value of ICA. Methods:The half inhibition concentration(IC_(50))value of ICA in inhibiting the growth of A549/MTX cells was measured by MTT assay. The colony formation rates and the morphology of cell cluster of A549/MTX and ICA-treated A549/MTX cells were determined by double-layer soft agar colony formation assay. The migration abilities of A549/MTX and ICA-treated A549/MTX cells were evaluated by cell scratch assay. The invasion ability of cells was tested by using Transwell chamber assay. Results:MTT assay showed that the IC_(50) value of non-toxic ICA plus MTX was reduced compared with that induced by equal dose of MTX (35.50±1.85 μmol/L vs 52.17±2.25 μmol/L). The colony formation rate of ICA-treated A549/MTX cells was 0.94±0.09, less than that of A549/MTX cells (1.56±1.07, P<0.05). Cell scratching assay demonstrated that the migration capability of A549/MTX cells was stronger than that of ICA-treated A549/MTX cells at 72 h (P<0.05). Transwell experiment revealed that more A549/MTX cells passed through artificial basement membrane than ICA-treated A549/MTX cells (P<0.05), indicating that the invasion capability of ICA-treated A549/MTX cells was weaker than that of A549/MTX cells.Conclusion:TCM ICA can reverse the metastatic phenotype of MTX-resistant A549 cells.

Objective To explore the feasibility of fluorescence quantitative PCR(FQ-PCR)in prenatal diagnosis of the fetal RhD genotyping using free DNA from RhD-negative pregnant women.Methods The fetal RhD gene was amplified from 78 RhD-negative pregnant women with single fetus maternal plasma (gestation from 11 to 40 weeks).Rhe existence of fetal DNA was confirmed by amplification ofnine different polymorphic short tandem repeat loci(STR)and sex-determining region Y chromosome(SRY)gene.Exon5,7,10 and intron 4 were amplified by real-time polymerase chain reaction with TaqMan probe.The results of fetal RhD genotyping were evaluated retrospectively by the serologic analysis of infant cord blood.Results Among the 78 specimens,the SRY positive signals were detected from samples of 41 and were all identified male fetal through 8ex observation after newborn infants delivered from the women enrolled.The mean concentration of SRY gene reached(214.7±120.9)eopies/ml.RhD genotyping results of 70 cases were in complete concordance with the resets through serological detection of fetal cord blood after delivery.In addition,5 cases were false-positive.3 cages were considered inconclusive.The coincidence rate was 90%(70/78).From 5 false-positive cases,4 cases were identified as RhDel phenotype by detecting RHDl227A allele gene.The final accuracy rate of FQ-PCR was 95%(74/78)in the fetal RhD genotyping.Conclusion FQ-PCR analysis for noninvasive prenatal of fetal RhD genotyping could be useful in prevention and diagnosis of hemolytic disease of newborn.