mRNA and Protein Expression of AXIN,β-Catenin, MMP7 and MMP9 and Their Relationship in Lymphoma Cells / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the mRNA and protein expression of axin inhibitor (AXIN), β-chain protein (β-catenin), matrix metalloproteinase-7 (MMP-7) and matrix metalloproteinase-9 (MMP-9), and their relationship in lymphoma cells.</p><p><b>METHODS</b>The expressions of MMP-7, MMP-9, β-catenin and AXIN in lymphoma cell lines were detected by Western blot and RT-PCR. Moreover, the lymphoma cells with relatively low expression of AXIN were grouped and were transiently transfected by using pcDNA5-His-β-catenin and pCMV5-HA-AXIN; the protein and mRNA expression of MMP-7, MMP-9 and β-catenin in lymphoma cells was detected by Western blot and RT-PCR, respectively; the cell infiltration and migration ability in group with stable ligh expression of AXIN, group of interfering stable high expression of AXIN and blank control group were analyzed by transwell experiment.</p><p><b>RESULTS</b>The AXIN negatively correlated with MMP-7, MMP-9 and β-catenin expression in lymphoma cell lines. After the up-regulation of AXIN, the mRNA expression of MMP7, MMP-9 and β-catenin in Raji cells all not significantly changed, while the pratein expression of MMP-7, MMP-9 and β-catenin all significantly decreased (P<0.05); after the up-regulation of β-catenin, the mRNA and protein expression of MMP-7, MMP-9 was also up-regulated significantly (P<0.05). After interfering the AXIN, the mRNA expression of MMP-7, MMP-9 and β-catenin in group with stable high expression of AXIN all not changed significantly, while protein expression of MMP-7, MMP-9 and β-catenin was down-regulated significantly (P<0.05); after interfering the β-catenin, the protein and mRNA expression of MMP-7 and MMP-9 in group with stable high expression of AXIN all were down-regulated significantly(P<0.05).</p><p><b>CONCLUSION</b>The up-regulation of AXIN expression in lymphoma cells can lead to decrease of β-catenin expression and the resuts in significant decrease of MMP-7 and MMP-9 expression, there by plays a role to block the infiltration and migration of lymphoma cells.</p>

¹H-NMR based metabonomic approach to evaluate anti-coagulant effect of Danggui Sini decoction / 中国中药杂志

To study the anti-coagulant effect and influence of danggui Sini decoction (DSD) on rat's plasma endogenous metabolites by animal experiment and ¹H-NMR based metabolomics method. After intragastric administration of Danggui Sini Decoction for 7 days, Plasma thrombin time (TT) was measured. Rat plasma metabolic fingerprint in two groups was analyzed using ¹H-NMR, based on which the principal component analysis( PCA) and orthogonal partial least-squares discriminant analysis(OPLS-DA) models for metabonomic analysis. Potential biomarkers were screened by using variable importance in the projection (VIP) and T test. DSD could prolong TT of the rat significantly (P < 0.05). Compared with control group, six kinds of endogenous metabolites in DSD group change significantly (P < 0.05), among which isobutyrate, carnitine and phenylalanine content had an upward trend (P < 0.01) and lysine, Histidine and cholesterol content had a downward trend (P < 0.05). It is likely that carnitine, phenylalanine, Histidine and cholesterol are the potential metabolic markers in the anti-coagulant process and DSD affects the platelet aggregation and the expression of tissue factor and fiber protease by regulating the energy, amino acid and lipid metabolism.

Correlations of 24 biochemical markers in seminal plasma with routine semen parameters / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the correlations of 24 biochemical markers in the seminal plasma with routine semen parameters.</p><p><b>METHODS</b>According to the WHO5 standards, we analyzed the routine semen parameters of 66 subfertile men, including the semen volume, sperm concentration, total sperm count, sperm motility, and the percentage of progressively motile sperm (PR). Based on the calibration and quality control measures and using an automatic biochemistry analyzer or electrolyte analyzer, we detected 24 biochemical markers in the seminal plasma of the patients, including total protein (TP), albumin (Alb), globulin (Glb), uric acid (UA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP), γ-glutamyltransferase (GGT), lactate dehydrogenase (LDH), creatine kinase (CK), alpha hydroxybutyrate dehydrogenase (αHBDH), adenosine deaminase (ADA), glucose (Glu), triglyeride (TG), total cholesterol (TC), urea nitrogen (UN), creatinine (Cr), high-sensitive C-reactive protein (hsCRP), K+, Na+, Cl- , Ca, Mg, and phosphorus (P). Then we analyzed the correlations of the 24 biochemical markers with routine semen parameters.</p><p><b>RESULTS</b>The levels of the TP, Alb, and Glb proteins in the seminal plasma were positively correlated with sperm concentration, so was that of Alb with the total sperm count, and the AST and LDH activities with sperm concentration and total sperm count. The AKP activity in the seminal plasma was correlated negatively with the semen volume, but positively with sperm motility. The αHBDH activity exhibited a positive correlation with both sperm concentration and total sperm count, with a coefficient of correlation (r) above 0.7. The UN level was correlated negatively with the semen volume, so was the Cr level with the semen volume, sperm concentration, and total sperm count, and the Glu level with sperm concentration and total sperm count. The TG level was correlated positively with the semen volume, but negatively with sperm motility. The levels of seminal plasma ALT, GGT, ADA, UA, TC, CK, and hsCRP showed no correlation with the above-mentioned semen parameters. None of the seminal plasma K+, Na+, Ca, Mg, and P levels was found correlated with semen parameters except the Cl- level, which was negatively correlated with the semen volume.</p><p><b>CONCLUSION</b>Many biochemical markers in the seminal plasma are closely related to routine semen parameters, indicating that these biochemical components may play roles in spermatogenesis, sperm maturation, and physiological metabolism.</p>

Establishment and evaluation of an automatic method for seminal plasma gamma-L-glutamyl transpeptidase detection / 中华男科学杂志

<p><b>OBJECTIVE</b>To establish an automatic method for seminal plasma gamma-L-glutamyl transpeptidase (GGT) detection and evaluate its accuracy, repeatability and linear range.</p><p><b>METHODS</b>We detected the GGT activity in the seminal plasma by rate assay, and established the detection parameters on an automatic biochemical analyzer. Then, we evaluated the reagent blank absorbance, accuracy, repeatability and linear range of the automatic method, and compared the results obtained from the method and the seminal plasma GGT detection kit (Xindi Biological Pharmaceutical Engineering Co., Ltd, Nanjing, China) commonly used in clinical laboratories.</p><p><b>RESULTS</b>The average absorbance of reagent blank was 0.0476, and the average change rate of blank absorbance (deltaA/min) was 0.000168. The coefficients of variation (CV) for 3 seminal plasma samples with high, middle and low GGT activity detected for 10 times, respectively, were 0.26%, 4.83% and 1.60%. The accuracy of the automatic method was evaluated by a comparison test, and the relative deviation for each concentration point of 40 seminal plasma samples ranged from 13.38% to 11.05%, which met the requirement of < 15%. There was a good linear relationship (r > 0.99) when the seminal plasma GGT activity was between 299 and 1 833 U/L. A significant positive correlation was found between the seminal plasma GGT detection kit (a colorimetric method) as the control and the automatic method as the test reagent in the results of 115 seminal plasma samples (r = 0.981, P < 0.01), with a Kappa value of 0.776 (P < 0.05) and a coincidence rate of 90.43%.</p><p><b>CONCLUSION</b>The established automatic method to detect seminal plasma GGT activity has a low reagent blank, good repeatability and accuracy, and fine concordance with the colorimetric method commonly used in clinical laboratories. It is simple, rapid and suitable for screening large numbers of samples, avoids the necessity of diluting the seminal plasma sample, and saves a lot of manpower and reagents.</p>

Advances in researches on polymorphonuclear neutrophil elastase in semen / 中华男科学杂志

Reproductive tract infection is one of the important factors of male reproduction. Polymorphonuclear neutrophil elastase (PMNE) in semen, as a marker of male reproductive tract inflammation, especially recessive infection, potentially affects male fertility. The concentration of PMNE in semen is correlated significantly not only with semen white blood cell count and seminal plasma ROS level, but also with the levels of other inflammation related cytokines, such as IL-6, IL-8, and TNF-alpha. Furthermore, PMNE has a negative impact on sperm quality by decreasing sperm motility, increasing the percentage of morphologically abnormal sperm and interfering with DNA integrity. PMNE inhibitors in semen can form a compound with PMNE, and the imbalanced proportions of the two may promote the development of chronic inflammation, and consequently lead to male infertility. At present, PMNE in semen is detected mainly by enzyme immunoassay, but this method still needs to be standardized, and the diagnostic standards to be unified.

Preparation and identification of recombinant human neutrophil elastase / 中华男科学杂志

<p><b>OBJECTIVE</b>To prepare a purified recombinant human neutrophil elastase (HNE) using genetic engineering technology, and pave the way for the preparation of the antibody to HNE and establishment of semen HNE detection methods.</p><p><b>METHODS</b>HNE mRNA was obtained from human peripheral blood granulocytes with specific HNE primers, and the cDNA of HNE was cloned into the plasmid pGEX-2T to derive a recombinant plasmid pGEX-2T/HNE. After PCR identification, double-enzyme digestion and gene sequencing, the recombinant plasmid was transferred into competent Escherichia coli DH5alpha and further induced to express the recombinant fusion protein GST/HNE by isopropyl beta-D-thiosulfate galactosidase (IPTG). The recombinant fusion protein was cleaved by thrombin and further purified with glutathione agarose beads to obtain purified recombinant HNE.</p><p><b>RESULTS</b>The recombinant plasmid pGEX-2T/HNE was successfully prepared and transferred into E. coli DH5o; the expression of the recombinant fusion protein GST/ HNE was successfully induced by IPTG at 18 degrees C overnight; and the purified recombinant protein HNE was successfully obtained by thrombin cleavage and purification of glutathione agarose beads.</p><p><b>CONCLUSION</b>The acquirement of purified recombinant HNE has prepared the ground for the preparation of the antibody to HNE and establishment of semen HNE detection methods.</p>

Determination of uric acid in seminal plasma and correlation between seminal uric acid and semen parameters / 中华男科学杂志

<p><b>OBJECTIVE</b>To establish the method of seminal uric acid (UA) determination and investigate the correlation between the seminal UA level and semen parameters.</p><p><b>METHODS</b>The method of seminal UA determination was established by modifying the method of serum UA detection, and its intraassay coefficient of variation (CV) and the difference of the results between different technicians were also observed to evaluate the acceptability of the method. In the meanwhile, the correlations of the seminal UA level with the patients' age, abstinence time, semen volume, pH, sperm concentration, motility, the percentage of grade a and b sperm, and the percentage of morphologically normal sperm.</p><p><b>RESULTS</b>The intraassay CV was 9. 16% for the method of seminal UA detection, and there was no significant difference in the UA level detected by 2 technicians (P = 0.541). The seminal UA level was positively correlated with the percentage of morphologically normal sperm (r = 0.350, P = 0.025) , with a tendency of positive correlation with sperm motility (r = 0.147, P = 0.085) and the percentage of grade a and b sperm (r = 0.156, P = 0.068), but not with other parameters such as semen volume, pH, sperm concentration, abstinence time and the patients' age.</p><p><b>CONCLUSION</b>An acceptable method of seminal UA determination could be established by modifying the method of serum UA detection. Sperm morphology, motility and the percentage of grade a and b sperm might be related to the level of seminal UA.</p>

Cloning and sequence analysis of Chlamydia trachomatis heat shock protein 10 / 中华男科学杂志

<p><b>OBJECTIVE</b>To obtain Chlamydia trachomatis heat shock protein (cHSP) 10 gene from clinical secretion samples.</p><p><b>METHODS</b>cHSP10 gene was amplified from 20 cases of clinical secretion samples with positive gold-labelling by specific primers of cHSP10 and identified by sequence analysis.</p><p><b>RESULTS</b>cHSP10 full-length gene was amplified from 1 of 20 cases of clinical secretion samples with positive gold-labelling. cHSP10 gene encoding 102 amino acids contains 306 bp, which nuclotide at position 194 changes from T to A, leading to the change of corresponding amino acid.</p><p><b>CONCLUSIONS</b>cHSP10 gene may be cloned from clinical secretion samples with positive gold-labelling, which make it possible to further construct expression plasmid of recombinant cHSP10.</p>

Advances in the studies of Chlamydia trachomatis infection in males / 中华男科学杂志

Chlamydia trachomatis(Ct) infection in reproductive tract is one of the most common sexually transmitted diseases at present. However, relatively fewer studies are made on Ct infection in men. The paper reviews the epidemiology of Ct infection in males, Ct infection and male diseases, Ct infection and male infertility, and the detection of Ct infection in men. It aims at providing a theoretical basis and a practical guide for the prevention and control of Ct infection in men.