Objective To isolate and identify exosomes from human serum , explore the feasibility of detecting exosomal miRNA in human serum.Methods Retrospective study.Serum samples from 10 healthy individuals in January 2013 were randomly selected.Besides, from January 2013 to December 2014, serum samples from prostate cancer(PCa) patients (n=20), benign prostatic hyperplasia(BPH) patients ( n=20 ) and healthy controls ( n=20 ) were selected.Exosomes were isolated from these serum samples using ExoQuick , and then identified by using transmission electron microscopy , NanoSight nano particle analyzer and Western Blot for morphology and molecular phenotype.The quality of exosomal RNA was analyzed using Agilent 2100 Bioanalyser.Then quantificational real-time polymerase chain reaction ( qRT-PCR) was carried out to detect miRNAs in different components of human serum ,and nonparametric tests were used for difference analysis.Results Exosomes isolated from human serum showed round or oval vesicles, mainly in diameter 40-100 nm, and with maximum peak distribution of 58 nm.Moreover, they expressed HSP70 and four transmembrane protein CD 63.Agilent 2100 Bioanalyzer results showed that the major RNA component of exosome was about 25nt small RNA.qRT-PCR confirmed that 4 normal miRNAs were expressed in human serum exosome , and the expression of miRNAs in exosome pellets were higher than the whole serum (miR-21, U =16,P =0.007 2; miR-16, U =3,P<0.000 1; miR-20a, U =2,P <0.000 1;let-7a, U=13,P=0.003 2) and exosome-depleted supernatant ( miR-21, U=15,P=0.006 5;miR-16, U=2,P<0.000 1;miR-20a, U=1,P<0.000 1;let-7a, U=10,P=0.002 8).miR-141, the molecular marker of prostate cancer ,were analyzed by qRT-PCR in whole serum samples and serum exosome pellets isolated from the same serum in a cohort of 20 PCa patients , 20 BPH patients and 20 healthy control people.The results showed that , in three groups , exosomal miR-141 expression were all significantly higher than serum circulating miR-141 (Control group, U=66,P=0.000 3; BPH group, U=83,P=0.001 6;PCa group, U=54,P<0.000 1).In addition, the expreession of exosomal miR-141 in PCa patients was significantly higher than BPH patients or healthy controls (3.85 fold, U=74,P=0.000 7 and 4.06 fold, U=70,P=0.000 5).Conclusion Exosome can be efficiently isolated from human serum.Compared with the whole serum , isolation of serum exosome may helpful to improve the detection of circulating miRNA.

Objective To analyze the expression of urine exosomal miR-375 in prostate tumors and investigate its clinical utility.Methods A total of 45 patients with PCa,24 with benign prostate hyperplasia (BPH) and 24 healthy individuals were enrolled into this study.Exosomes were isolated from the urine of PCa,BPH and healthy individuals and the total RNA was extracted from the exosomes.The exosomal miR-375 expression was assessed by quantitative real-time PCR and analyzed with the comparative quantification cycle method (2-△△CT).We performed comprehensive biostatistical analyses to explore the clinical value of miR-375 in prostate cancer.Results The urine exosomal miR-375 expression was significantly downregulated in the patients with PCa compared with BPH and the healthy controls (P ＜ 0.01).No statistically significant difference of the urine exosomal miR-375 expression levels between the patients with BPH and healthy individuals was observed (P ＞ 0.05).The urine exosomal miR-375 expression level was also found to be associated with clinical stage and bone metastasis status of the patients with PCa (P ＜0.05),and with the increase of Clinical stage.The expression level of miR-375 decreased.No significant relationship was detected between miR-375 level and the patient's age,gleason score and serum prostate-specific antigen level (P ＞ 0.05).Receiver operator characteristic analyses demonstrated that the urine exosomal miR-375 expression could better differentiate PCa from BPH patients:AUC 0.715 (95％ CI:0.589-0.842) vs PSA AUC 0.632 (95％ CI:0.492-0.771) (P＜0.01).Conclusion The urine exosomal miR-375 could serve as a non-invasive biomarker for the diagnosis of PCa.

Objective Establishment and development of a novel Single-Nucleotide-Polymorphism TaqMan Real-Time PCR assay for rapid detection of rs12979860 that predicts HCV therapy response.Methods Human genomic DNA were extracted from solid tissues,secretion and plasma before allelic discrimination.With the property of minor groove binding protein (MGB) binding to minor groove of DNA with strong specificity and affinity,primers and MGB probes were particularly designed for differentiation of human genomic frequencies.MGB probe-based real-time PCR was established to increase allelic discrimination using two probes that only differ in one nucleotide of IL28B rs12979860.The specificity was evaluated by fluorescence signal emissions which were selected from two signal channels.And DNA sequencing was used to confirm the genomic polymorphisms.Results TaqMan probe-based SNP real-time PCR increased allelic discrimination using two probes that only differ by one nucleotide of amplicon,which indicated this assay was easily performed regardless of genomic DNA concentration and quality,minimizes sources of error.The sensitivity was as low as 1.5 ng/μl,the amplification efficacy was 97.6％.The genotype frequencies of CC,CT were remarkably different between Caucasian and Mongolian.The dominated genotype of Caucasian was CT,while most Mongolian was carried CC (26/40 vs.40/50,x-2 =18.75,P value ＜ 0.05).However,the genotype between two population showed no relationship with their virological clearance clinically (P value ＞ 0.05).Conclusions This TaqMan MGB assay shows highly specificity,which has potential as a routine diagnostic test for the detection of rs12979860 from various types of samples.This robust assay would be widely used clinically to predict patients' response before anti-HCV treatment in personalized medicine.

Laboratory diagnostics is one of the most fast developing medical sciences.But the teaching quality falls behind due to the traditional education model.Based on the concept of ‘three-stage fusion' of the subject and ‘five abilities' of the training objects,‘three-highlight & stereo' teaching style was established and put through the course,which means to emphasize function,practice,effect in optimizing teaching content and methods,and construct stereo platform including information system,online course,laboratory equipments,research activities and teacher training program.The results showed a significant improvement of the students' knowledge mastering and utilizing capability.And the teaching situation was well re-created.Furthermore,the teaching team was much more powerful than ever.It suggests that ‘three-highlight and stereo' teaching style is a successful explore and practice of teaching reform of laboratory diagnostics on adapting for internationalization of education tendency.

The development of metagenomics revealed a novel role of microorganism in lots of diseases.Emerging researches at home and abroad illustrated that microbiome changes from nasopharynx, oropharynx and/or lung in quality and/or quantity exist in many respiratory diseases including chronic obstructive pulmonary disease, asthma, cystic fibrosis, pneumonia as well as upper respiratory infection, which play an important role in immune system, metabolism and neuroregulation.These research results may provide us new strategy for the diagnosis, therapy, surveillance and prognosis of respiratory diseases.

Objective To shorten the turn around time of positive blood culture results by optimizing the blood culture positive specimen processing flow.Methods In January 26,2015,the microbiology department started the blood culture positive specimen processing flow optimization project,and applied the Lean Six Sigma method in the microbiological process management.The TAT data of 124 positive blood cultures containing Enterobacteriaceae were collected before and after the start of the project in about two months.We analyzed the turnaround time median,mean and standard deviation and reference Z value,process performance index,millions of error opportunities.We decompose the turnaround time into six time periods to find the key points of the process improvement and the influencing factors,and then put forward the reform measures to optimize the blood culture inspection process.MiniTab17.0 statistical software was used to process capability analysis and double sample t test.Results After the implementation of the project,the average turnaround time of the blood culture was shortened from 77.10 h to 64.03 h,improved by 13.06 h(16.94％).Process performance greatly improved in Ppk value increased from 0.49 to 0.88,the benchmark Z value increased from 1.48 to 2.63.After the improvement,except the positive alarm time of blood culture,the mean of the other decomposition time was significantly shorter than before.Conclusions The application of Six Sigma in process management can greatly improve the work efficiency and process performance.This project can save a lot of manpower,material and financial resources,reduce the waiting,shorten turnaround time,that achieve the desired results.

ObjectiveTo screen out the two-component system associated with drug resistance of Mycobacterium tuberculosis by detecting the differential expression of two-component system regulator genes between multidrug resistant Mycobacterium tuberculosis strains and drug sensitive strains. MethodsTotal RNA of MTB was extracted from cultured MTB during the logarithmic phase in the 7H9 brook medium, and then its purity was identified. Reverse transcription was further completed. The expressing levels of TCS response regulators were quantified using SYBR Green I qRT-PCR, which aimed at finding the differential expressions between multidrug resistant strains and sensitive strains. Finally, all of differentially expressed TCS were screened out under the stress of INH, SM and LFA. Results Compared with sensitive strains,multidrug resistant strains of Rv0491, Rv3133c, Rv3143 and Rv3246c were up-regulated 1. 03, 7.11,3.48and 1.37 folds, respectively (t/t' =5. 623, -4. 196, -3. 559 and -3. 016, respectively, P ＜0. 01 ). The expressing level of other regulators had no statistical significance between muhidrug resistant strains and drug sensitive strains. Under the antibiotic pressure, the expression of Rv1027c, Rv3246c and Rv3143 showed significant changes compared with no antibiotic group. ConclusionRv3246c and Rv3143 may be associated with MTB drug resistance and the differentially expressed genes in multi-drug resistant strains may be used as potential drug targets against drug resistant tuberculosis.

Objective To express recombinant HBHAΔC and HBHAΔN protein,and compare the HBHA series protein activity with each other.It will be provide a experimental basis for the research on clinical diagnostic of HBHA.Methods The HBHAΔC and HBHAΔN gene fragments were cloned and expressed by transforming E.coli BL-21.Test the protein heparin binding ability by CL-6B column.And then added protein to the BCG 7H9 culture medium,to observe the induced BCG aggregation.Results nHB-HA,rHBHA and HBHAΔN protein have heparin binding ability.Meanwhile nHBHA,rHBHA and HBHA Δ C protein have in-duced BCG aggregation effect.Conclusion The HBHAΔC and HBHAΔN protein were successfully obtained.It was proved that the HBHA C-terminal could be combined with heparin and the N-terminal involved could induce the aggregation of BCG.This results provide a basis for further study on molecular mechanism of TB infection and clinical application.

Objective To study the characteristics of antimicrobial resistance and molecular epidemiology of Staphylococcus aureus (S .aureus)in the intensive care units(ICUs)of a hospital.Methods Clinical isolates of S .aureus collected from ICUs between January and December 2014 were identified and performed antimicrobial susceptibility testing,then typed by staphylococcal protein A (spa)typing and multilocus sequence typing (MLST) methods.Results Of 160 isolates of S .aureus ,120 (75.00%)were methicillin-resistant S .aureus (MRSA). Resistance rates of MRSA to erythromycin,clindamycin,and levofloxacin were all > 80%;methicillin-sensitive S .aureus (MSSA)were sensitive to cefazolin,resistance rates to erythromycin,clindamycin,and levofloxacin were 62.50%,35.00%,and 10.00% respectively.spa typing and MLST results showed that the main types of 120 isolates of MRSA were ST239-t030,ST239-t037,and ST5-t2460,the major epidemic strains were ST239-t030 (n=105,87.50%),and were isolated from 8 ICUs;MSSA had more types,ST59-t437 were detected only from depart-ment of neurology(n =8)and department of digestive diseases(n =2),ST6-t701 ,ST398-t3625,ST398-t1793,and ST121-t2092 were isolated from departments of neurology(n=7),anesthesiology(n=5),neurosurgery(n=4),and cardiac surgery(n=4)respectively.Conclusion Isolation rate of MRSA in ICUs in this hospital is high,ST239-t030 is the main type,which prevailed in hospital;different types of MSSA have epidemic trends in various departments.

AIM: To observe the antiproliferative effect of c-myc antisense oligonucleotide in rat thymus lymphocytes. METHODS: Rat thymus lymphocytes were separated by Ficoll-Urografin density gradient centrifugation. Lipofectin was used to introduce antisense, sense and mismatched oligonucleotides for c-myc to rat thymus lymphocytes. The antiproliferative effect was assayed by incorporation of -TdR and MTS cell proliferation assay. TR-PCR was used to detect the expression of c-myc mRNA. RESULTS: c-myc antisense oligonucleotide inhibited rat thymus lymphocytes proliferation [(0.14?0.03)A vs (0.32?0.16)A, P