Expression levels of TGF-β1 mRNA in renal cortex of control group, diabetic group and taurine group measured by real-time quantitative RT-PCR were (7.0±0.8)×10 -3, (64.4±8.0)×10 -3 and (16.7±2.0)×10 -3, respectively. The corresponding results with end-point RT-PCR method were 0.28±0.12,0.58±0.16 and 0.43±0.10, respectively. Compared with end-point RT-PCR method, real-time quantitative with SYBR Green I was easier, faster, sensitive and specific.

The human tissue kallikrein(KLK)gene family consists of 15 highly conservative serine pro-teases,which is the largest uninterrupted cluster of protease genes in the human genome. Several members of the family are expected to be markers for tumor diagnosis and prognosis. Studies find that the expressions of KLK2-11 and KLK13-15 are abnormal,and the majority of KLKs have potential diagnostic and prognostic values.

Objective To investigate the significance of IL-18R? expression on CD4+ T cells in peripheral blood (PB) of children with Mycoplasma pneumoniae pneumonia (MPP).Methods T cell subtype CD3/CD4/CD8 and expression of IL-18R? on CD4+ T cells in PB from 35 children with MPP and 15 age- and sex-matched control subjects was determined by flow cytometry.Results Compared with that of healthy control, CD3+ and CD4+ positive cells in MPP children were decreased (P0.05).Conclusion There exists cell-mediated immune function disorders and Th1/Th2 imbalance in children with MPP. Th1 immune response is dominant in acute-stage MPP.

Objective To prepare the mAbs against hK6 for establishing a sandwich enzyme-linked immunosorbent assay(ELISA) of hK6,and exploring its clinical value.Methods The hybridoma technique was used to prepare mAbs against hK6.The mAbs were purified and labelled with horseradish peroxidase for the sandwich ELISA method.The sandwich ELISA method was used to detect the serum hK6 concentrations in patients with malignant gastric neoplasm.Then the best antibody pair was selected from coating antibody and enzyme-linked antibody to establish a sandwich ELISA method through the chessboard titrations.Compared with CEA,we explored the feasibility of hK6 as gastric cancer biomarkers.Results A sandwich ELISA method was established for quantifying hK6 in serum.The results showed that the optimal concentration of coating antibody was 5 μg/mL.The optimal concentration of enzyme-linked antibody was 1:2 000.Serum hK6 in the patients with gastric cancer groups[(5.78±1.66)ng/mL] than healthy individuals groups[(3.35±0.67)ng/mL] and those in gastric ulcer groups[(3.59±1.02)ng/mL],the difference was statistically significant(P<0.05).Furthermore,there was no significant difference in values of serum hK6 between patients with gastric ulcer groups and healthy individuals groups(P>0.05).The hK6 positive rate of gastric cancer was 69.70％,and CEA was 45.46％.In the combined detection,the positive rate was 78.79％.Conclusion A sandwich ELISA is established successfully.As a favorable serum biomarker for gastric cancer,the detection of hK6 together with CEA is helpful in the diagnosis of gastric cancer.

Objective To evaluate the ability of Vitek 2 Compact YST identification cards and pyrosequencing analysis for identifying the clinical isolates of yeast-like fungi.Methods Vitek 2 Compact YST identification cams and ITS1 region pyrosequencing analysis were used to identify the clinical isolates of yeast-like fungi at Jinan Military General Hospital in 2011.The strains which could not be identified to species by pyrosequencing analysis were identified again by the method of ITS1 region Sanger sequencing.The strains with inconsistent identified results by YST and pyrosequencing were identified again using API 20C.Results A total of 282 srtains were isolated from various clinical specimens for culturing in 2011.In addition to the three which could only be identified to similar species,other strains could be clearly identified to species by the method of ITS1 reverse pyrosequencing.Three strains which failed identified to species by pyrosequencing couldn't be distinguished fully by the method of Sanger sequencing.There were 7 strains unidentified,14 strains low discrimination(％ id ＜ 80％),and 6 strains misidentification using YST cams.The identification coincidence rate was 90.4％.The identification coincidence rates were various in different species of yeast-like fungi using YST cards.Conclusions The vast majority of clinical isolates of yeast-like fungi could be identified by ITS1 reverse pyrosequencing analysis.Vitek 2 Compact YST caRD basically meets the needs.But attention should be paid to the standard operating and regular internal quality control.For some rare species it is also needed to combine the source of specimen,colonies and characteristics to determine the identification results for the rare stains.

Objective To investigate the distribution and resistant pattern of Enterobacter cloacae to antimicrobial agents for reasonable use of antibiotics .Methods E .cloacae strains were isolated from patients from January 2009 to December 2013 .The strains were identified by VITEK‐2 Compact System of BioMerieux and tested for antimicrobial susceptibility by Kirby‐Bauer disk diffusion method .The results were analyzed .Results A total of 397 nonduplicate E .cloacae strains were isolated during 5 years ,accounting for 5 % of the total gram‐negative isolates . The strains were mainly isolated from sputum (48 .9% ) , followed by secretions (30 .5% )and urine (7 .3% ) .The percentage of E .cloacae strains resistant to all the antibiotics tested was on decline except carbapenems ,cefoxitin and amoxicillin‐clavulanic acid .Carbapenems were relatively more active against E .cloacae strains .The E .cloacae strains showed higher resistance rate toβ‐lactams .Conclusions It is necessary to strengthen the monitoring of clinical isolates to guide rational use of antimicrobial agents .

BACKGROUND:Many domestic and foreign scholars and institutions are studying how to relieve radiation damage and to find the most suitable drug, while ginsenosides as the main pharmacological ingredient of ginseng show significant antioxidant effect. OBJECTIVE:To investigate the ease effect of ginsenosides on human hematopoietic stem cels under different intensities of ionizing radiations. METHODS: The CD34+ hematopoietic stem cels were isolated from the healthy cord blood. Then the cels were divided into normal group and ginsenoside-pretreated group, respectively, exposed under 1, 2, 5 Gy of X-ray irradiations for 24 hours. Cel viability was detected in irradiated hematopoietic stem cels by MTT assay. Apoptosis was estimated using the folowing assays: Annexin-V assay, caspase-3 mRNA and protein levels. The generation of reactive oxygen species was evaluated, in the presence or absence of ginsenoside in liquid cultures of CD34+ human hematopoietic stem cels irradiated with 1-, 2- and 5-Gy X-rays, using a flow cytometry assay. The Nrf-2 mRNA and protein levels were also studied by western blot analysis and RT-PCR, respectively. RESULTS AND CONCLUSION: Ionizing radiation at the therapeutic dose could decrease the viability of CD34+ cels and induce the cel apoptosis, and meanwhile, the activity of intracelular reactive oxygen species also showed a progressive increase that was correlated with the dose of ionizing radiation. However, ginsenoside pretreatment could relieve these above-mentioned effects. Ginsenoside inhibited the increase in caspase-3 activity induced by ionizing radiation, and additionaly, enhanced the mRNA and protein expressions of Nrf-2 in CD34+cels. In conclusion, ginsenoside protects CD34+ hematopoietic stem cels from radiation effects, which is probably correlated with anti-apoptosis and anti-oxidant roles of ginsenosides.

Objective To establish a rapid diagnostic method for the detection of marine vibrios,and then construct a new technology platform for the clinical diagnosis of marine vibrio infection.Methods A pair of PCR primers and a sequencing primer based on the whA gene of V.vulnificus and the toxR genes of V.parahemolyticus and V.alginolyticus were designed respectively,and then the specific DNA fragments were amplified.Next,the single-stranded DNA templates were prepared for pyrosequencing.The obtained base sequence was validated by NCBI alignment.In addition,the 16S rRNA genotyping of V.vulnificus was also performed.Results The PCR primers and sequencing primer of V.vulnificus showed good specificity,and a 167 bp DNA fragment was amplified from 4 strains of V.vulnificus.The pyrosequencing results completely matched with the whA gene sequence of V.vulnificus.Meanwhile,the control strains were negative.A 105 bp DNA fragment and a 134 bp DNA fragment were amplified from 11 strains of V.parahemolyticus and V.alginolyticus,respectively,and the pyrosequencing results were consistent with the expected sequence.In addition,one of 4 strains of V.vulnificus was identified as 16S rRNA-A type,and the other 3 as 16S rRNA-B type.Conclusion The PCR-pyrosequencing method established in this study is a new method for the real-time detection of short nucleotide sequences.It has some advantages such as high throughput,high precision and simple operation,and may be applied to the fast and accurate identification of marine and terrestrial pathogenic bacteria.

Objective To develop a real-time quantitative PCR method for detection of human breast cancer related novel gene-kallikrein gene 6 (klk6) expression and investigate klk6 expression levels in breast cancer tissue.Methods Using Sybr Green I, with GAPDH as reference, a real-time quantitative PCR method was established. klk6 expression levels of 32 normal and breast cancer tissues were detected and analyzed by the method and REST software.Results The amplification efficiencies for GAPDH and klk6 of the real-time quantitative PCR method were 0.90 And 0.95, respectively; inter-coefficient of variation were 1.0%~2.1% and 0.8%~1.2%,respectively; intra-coefficient of variation were 3.2% and 3.9%, respectively. The relative expression levels of klk6 in normal breast and breast cancer tissues were 0.017?0.009 and 0.040?0.017 with GAPDH as reference. Analysis results with REST indicated klk6 expression was up- regulated in breast cancer.Conclusion The real-time quantitative PCR method with Sybr Green I for klk6 mRNA quantification was simple, specific, reproductive and reliable, and could be used to study relationship betweeen klk6 expression and tumor.

OBJECTIVE To discuss the best scheme for fungemia detection by analyzing and comparing the ability and performance characteristics of the BACTEC 9120 automated blood culture system and 4 kinds of blood culture bottles in the detection of simulated fungemia.METHODS Simulated blood culture was produced using 65 fungi isolates from clinical specimens and BACTEC Plus Aerobic/F,Plus Anaerobic/F,Peds Plus/F and Myco/F Lytic blood culture bottles and detected by BACTEC 9120 automated blood culture system.The final inoculum densities were 1-5 CFU/ml blood.The(time to detection TTD) of simulated blood culture with different concentrations of suspension produced using 2 kinds of standard strains and 4 kinds of blood culture bottles was compared.RESULTS From the 260 bottles in this study 216 had growth detected by the BACTEC 9120 blood culture system.The positive rates of BACTEC Plus Aerobic/F and Anaerobic/F,which were 90.77%and 41.54%,respectively,were significantly(P