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.

Object To determine the structures of three saponins isolated from leaves of Aralia elata (Miq ) Seem Methods HPLC and electrospray ionization and tandem mass spectrometry were used for purification and structure identification Results Three saponins were identified as congmunoside Ⅴ, congmunoside Ⅶ, and congmunoside Ⅹ Conclusion These saponins are obtained from the leaves of A elate for the first time

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.

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 analyze the differences in paraoperative morbidity and lymph node dissection between thoracoscopic esophagectomy and open procedure.Methods From October 2012 to April 2014,207 patients with esophageal cancer underwent surgery.125 patients underwent video-assisted esophagectomy,and 82 underwent open procedure.In the minimally invasive group,there were 109 thoracoscopic cases and 16 thoracolaparoscopic cases.Results There were significant differences between the thoracoscope group and the open group in atelectasis(0.8％ vs.7.3％,P ＜ 0.05),pleural effusion (0 vs.4.9％,P ＜ 0.05),acute respiratory distress (0 vs.6.1％,P ＜ 0.05),ligation of thoracic duct (3.2％ vs.15.9 ％,P ＜ 0.05),recurrent laryngeal nerve paralysis (19.2％ vs.32.9％,P ＜ 0.05),c hylothorax (0 vs.4.9％,P ＜ 0.05),number of lymphonode along the right recurrent laryngeal nerve lymphatic chains[1.91 ± 0.73 vs.1.12 ± 0.81,P ＜ 0.05)] and achievement ratio(97.6％ vs.89.0％,P ＜0.05) and number of lymphonode along the left recurrent laryngeal nerve lymphatic chains (0.93 ± 0.71 vs.1.76 ± 0.84,P ＜ 0.05) and achievement ratio(52％ vs.76.8％,P ＜ 0.05).No significant differences were observed in pneumonia,anastomotic leak,thoracic abscess,esophago-tracheal fistula,re-laparotomy,re-thoracotomy,wound infection,arrhythmia,cardia failure,renal failure,hepatic inadequacy,cerbral infarction,and mortality(P ＞ 0.05).There were also no significant differences in number of lymphonode and achievement ratio of periesophagel lymph nodes,subcarinal lymph nodes and hilar lymph nodes (all P ＞ 0.05).Conclusion The thoracoscopic esophagectomy has some obvious advantage associated with less pulmonary complications,lower injury of thoracic duct and recurrent laryngeal nerve,more lymphonode and higher achievement ratio along the right recurrent laryngeal nerve lymphatic chains.But it has still a larger space for improvement of lymphadenectomy along the left recurrent laryngeal nerve lymphatic chains.

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

AIM: To observe the inhibitory effect of antisense eukaryotic expression vectors for c-myc on rat airway smooth muscle cells. METHODS: Antisense and sense eukaryotic expression vectors for c-myc pcDNA3-myc-antisense and pcDNA3-myc-sense were constructed. Lipofectin was used to introduce antisense and sense eukaryotic expression vectors for c-myc into rat. The inhibitory effect was assayed by MTT cell proliferation assay. Cell cycles were detected by flow cytometry technology. The expression of c-Myc was detected by immunohistochemistry. RESULTS: The results showed that antisense eukaryotic expression vector for c-myc inhibited rat airway smooth muscle cells proliferation. Rat airway smooth muscle cells were prohibited in S phase and the expression of c-Myc was decreased after antisense eukaryotic expression vectors for c-myc were transfected into cells. CONCLUSION: Antisense eukaryotic expression vectors for c-myc inhibit rat airway smooth muscle cell proliferation. [

Objective To investigate the changes of peripheral blood tacrolimus concentration and lymphocyte subsets in the uterus transplant recipient,and provide the evidence for monitoring the immune status after uterus transplantation.Methods The peripheral blood tacrolimus concentrations of the uterus transplant recipient during 1 year after transplantation were measured with the microparticle enzyme immunoassay (MEIA).Meanwhile,the whole blood cell counts and lymphocyte subsets were determined by the blood analyzer and flow cytometer,respectively.Results The blood tacrolimus concentrations of the uterus transplant recipient in the first month and second month after transplantation were (13.51 ± 3.92) ng/mL and (15.58 ± 1.19) ng/mL,respectively.The lymphocyte absolute counts were normal before transplantation.At the fifth day after transplantation,the counts of CD3 + T lymphocytes,CD4 + T lymphocytes,CD8 + T lymphocytes and NK cells and the ratio of CD4/CD8 were significantly decreased.One week after transplantation,the counts of CD4 + T lymphocytes were recovered to the normal range and maintained,but its recovery was slower than that of CD8 + T lymphocytes.The ratio of CD4/CD8 ranged from 0.4 to 0.8 during 10 days after transplantation,and increased and maintained between 0.8 and 1.1 after that.The counts of NK cells increased gradually from the 10th day after transplantation,but still did not recover to the level before transplantation even at the 20th day after transplantation.However,the counts and percentages of B lymphocytes did not decrease but increased at the fifth day after transplantation,and recovered to normal gradually from the 10th day after transplantation.There was no significant correlation between the CD3 + T lymphocyte count and blood tacrolimus concentration.Conclusion The dynamic changes of blood lymphocyte subsets and tacrolimus concentration exist in the uterus transplant recipient,which need to be further verified by a large amount of clinical data.

Objective To purify native and recombinant heparin-binding hemagglutinin(HBHA)protein,and investigate the activity of HBHA polyclonal antibody against aggregation of Bacillus CalmetteGuerin(BCG)induced by HBHA.Methods After growing BCG to the stationary phase in the 7H9 liquid medium,the native HBHA protein(nHBHA)was obtained by CL-6B column chromatography.At the same time,the HBHA gene fragment was cloned and expressed by transforming Escherichia coli BL-21.Then the polyclonal antibody against rHBHA was prepared by immunizing rabbit.Different comcentration of the HBHA protein was added to the BCG liquid medium,and the aggregation of the BCG was observed.Then,add the HBHA protein that incubated with anti-HBHA antibodies to the BCG culture medium and observe the aggregation of BCG.Results The purity of native HBHA was 99% and the concentration was 1.016 mg/ml.The expressed product contained 36% of total somtic protein.After purified,the purity of the recombinant HBHA protein was 97.1% and the concentration was 10.98 mg/ml.Both the rHBHA and nHBHA could induce the aggregation of BCG.When then concentration of nHBHA is 0.2μg/ml,BCG could be induced to aggregate,while the rHBHA concentration is 2μg/ml could induce the aggregation.Both aggregations could be suppressed by the polyclonal antibody against rHBHA.Conclusions The native and recombinant HBHA are successfully obtained.It is proved that the rHBHA could induce the aggregation of BCG similar as nHBHA,and polyclonal antibody against rHBHA could also suppress the activity of nHBHA.It suggested that rHBHA could be further used in clinical diagnosis and vaccination.

Objective To detect the isoniazid (INH) and rifampin (RIF) resistance of Mycohaeterium tuberculosis isolates in the single tube with multiplex-polymerase chain reaction-single strand conformation polymorphism(muhi-PCR-SSCP) system. Methods According to the sequences of inhA, katG and rpoB genes of the Mycohacterium tuberculosis, three pairs of oligonucleotide primes were designed to examine the INH and RIF resistance with the multi-PCR-SSCP. The validity of the newly developed method was evaluated with 116 clinical isolates of Mycohacterium tuberculosis( 70 isolates that were INH-resistant and 66 isolates that were RIF-resistant). Results The validity of the method was assessed with multiplex PCR-SSCP with the bacteria culture with susceptibility test as golden standard. The three genes, katG, inhA and rpoB, in the 116 clinical isolates and H37Rv strain were amplified successfully in single PCR reactions,except 4 isolates with katG deletion mutants. Compared with strain H37Rv, forty-six isolates had katG gene mutations, thirteen had inhA mutations and fifty-eight had rpoB mutations. Thirty-eight isolates had simultaneous katG and rpoB mutations and 4 isolates had both inhA and rpoB mutations. Four isolates had inhA and katG mutations and 2 isolates had mutations in all three genes simultaneously. The sensitivity of the newly developed multiplex-PCR-SSCP assay was 80% and 82% for INH and RIF, respectively. The specificity of the assay was 100% and 92% for INH and RIF, respectively. Conclusion Muhiplex-PCRSSCP provides a rapid, specific and cost-effective method of detecting multidrug-resistant TB. It laid a solid foundation for the further study of drug resistant gene.