Purpose　The aim is to develop a method for the analysis of micro-scale amino acids in biological samples.Methods　After having been extracted and dried,the residues were dissolved in a running buffer or a distilled water and acetonitrile.The samples were loaded into column for 5,25 or 50s at a hydrodynamic injection and were separated by capillary electrophoresis.The effect of different dissolvent on the sample stacking was observed.Results　A 100-fold improvement in the amount of material that can be injected into a capillary column without loss of resolution was shown.Conclusion　The marked effect of sample stacking was achieved by the using distilled water and acetonitrile(1∶1,v/v) as dissolvent.

Objective To establish a rapid assay for the determination of methotrexate (MTX) in serum.Method The assay was based on the ultraviolet absorbance of methotrexate at 306 nm. The separation of the drug was done by high performance capillary electrophoresis (HPCE). The bare fused-silica capillary was 60 cm in total length, 50.5 cm in efficient length and 75?m in diameter. The voltage of 25 kV was applied. The running buffer was 75 mmol/L phosphate, pH7.4. The performance of methodology was evaluated.Result The complete separation of MTX was achieved within 10 min. The linearity of the assay was from 1.1 ?mol/L to 1100.0 ?mol/L. The minimal detection limit was 0.55 ?mol/L.The recovery of MTX was from 88.2% to 98.2%. Within-run precision was 4.2% and between-run precision was ~5.4%. Conclusion The result indicated that the method was an effective method for clinical and scientific research with advantages of rapidity, simplicity and accuracy.

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 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 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 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 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 study the role of indocyanine green(ICG)fluorescence imaging in laparoscopic partial splenectomy (LPS).Methods:The data of 4 patients who underwent ICG fluorescence imaging technology for LPS at Beijing Luhe Hospital Affiliated to Capital Medical University from May 2017 to May 2020 were retrospectively analyzed. There were 3 females and 1 male, aged 46, 41, 27 and 12 years respectively. The extents of spleen preservation were compared between ICG fluorescence imaging with ordinary white light during operation. The residual splenic remnants were tested with fluorescence imaging after splenectomy, which showed fluorescence fading indicating good vascular perfusion.Results:ICG fluorescence imaging was performed on 4 patients. The operation time ranged from 180.0 to 250.0 min, and the intraoperative blood loss ranged from 40.0 to 200.0 ml. The postoperative hospital stay ranged from 4 to 14 days. There were no serious complications. Postoperative histopathology showed: splenic cyst ( n=1), splenic hemangioma ( n=2), and splenic laceration ( n=1). Conclusions:ICG fluorescence imaging technology had a significant role to play in partial splenectomy. This study showed this technique to improve safety of laparoscopic partial splenectomy.

OBJECTIVETo screen the genes related to the occurrence and development of varicosis of the great saphenous vein in the patients with primary deep vein valve insufficiency.

METHODSUsing mRNA fluorescent differential display reverse transcriptive polymerase chain reaction (FDD-RTPCR), different genes expressed in the varicose great saphenous veins in patients with primary deep vein valve insufficiency and corresponding normal human tissues were compared. Differentially expressed cDNA fragments confirmed by Northern blot were compared and then cloned into the pGEM-Teasy vector. Positive clones were selected and sequenced. All the sequences were put into GenBank and analyzed by BLASTN software to search for their genetic origins.

RESULTSAltogether 37 different cDNA fragments were obtained and 30 of which were confirmed by Northern blot. Analysis of the sequences by BLASTN software showed that C(610) fragment (NO. 18 cDNA clone) shared 96% homology with the mRNA sequence of the human Mckusick-Kaufman syndrome gene (MKKS gene).

CONCLUSIONC(610) fragment is highly homologous with the mRNA sequence of the human MKKS gene and is closely related to the development of varicosis of the great saphenous vein in patients with primary deep vein valve insufficiency.

Base Sequence ; Blotting, Northern ; Cloning, Molecular ; Group II Chaperonins ; Humans ; Lower Extremity ; blood supply ; Molecular Chaperones ; genetics ; Molecular Sequence Data ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Saphenous Vein ; Sequence Homology, Nucleic Acid ; Varicose Veins ; etiology ; genetics ; Venous Insufficiency ; complications ; genetics

Objective To compare the molluscicidal effects between“Luo-wei”(TDS),a plant molluscicide in 4 percent, and metaldehyde and niclosamide(MNSC)in the field. Methods A natural ecological environment with Oncomelania hupensis was selected as the test area,the test concentrations of TDS and MNSC were 2.5 g/m3 and 2 ml/m3 respectively by the immersion method;the test doses of TDS and MNSC were 3 g/m2 and 2 ml/m2 respectively by the spray method;the doses of WPN in a control group were 2 g/m3 and 2 g/m2 respectively by the two methods above-mentioned. The molluscicidal effects between TDS and MNSC were compared by using the synchronous design method and parallel comparative method. Results In the TDS group,the death rate of snails was 90.70%by immersion for 24 hours,reached to 81.40%after spraying for 7 days,and there were no significant differences among the observation time points in molluscicidal effects(P>0.05). One day after the spraying,the death rate of snails was less in the TDS group compared with that in the MSCN group(P<0.01),but the death rates of snails were similar in both groups 3 days after the spraying(P>0.05). In the MSCN group,the death rate of snails was 99.17%by immersion for 24 hours,reached to 66.07% by spraying for 1 day. In the WPS group,the death rate of snails was 97.15% by immersion for 24 hours,reached to 71.43%after spraying for 1 day,and there were no significant differences(both P>0.05). Conclusion TDS has a good molluscicidal activity and stable efficacy,and the molluscicidal effect of TDS is similar to that of MSCN in the filed, but the molluscicidal sensitivity of TDS is lower than that of MSCN.