Objective To evaluate the value of detection of interleukin 28B (IL28B) rs12979860 by allele-specific PCR (AS-PCR) for the prediction of antiviral treatment hepatitis C patients.Methods One hundred seventy-four blood samples were random collected from hospitalized patients with hepatitis C,who came from department of infectious diseases,Renmin Hospital of Wuhan University from May 2011 to May 2012.Two pairs of specific primers were designed for rs12979860 gene polymorphisms,and one mutated base was introduced to the second or third site of the end of 3' with the reverse primer.rs12979860 gene polymorphism of 30 cases with hepatitis C was detected by AS-PCR,and gene sequencing was further used to verify the consistency of the two methods in parallel.Then,the frequency distribution of different rs12979860 genotypes with 174 cases were analyzed by the AS-PCR method in the population.Results The genotype CC,CT or TT of rs12979860 with 30 cases could be well identified by both AS-PCR and gene sequencing,and the coincidence rate was 100％ (x2 =60.0,P ＜ 0.01).Compared to gene sequencing,both of the sensitivity and specificity of AS-PCR were 100％.Compared to the control (CC genotype),TT genotype detection sensitivity by AS-PCR was 10-5,while sequencing sensitivity was 2 × 10-1.rs12979860 polymorphism in the TT,CC and CT genotype distribution in the Chinese population frequencies were 3.45％ (6/174),13.2％ (23/174) and 83.3％ (145/174),respectively.Conclusion AS-PCR can quickly,accurate,reliable,economic and efficiently detect IL28B rs12979860 gene polymorphism of hepatitis C in patients,which could predict the effect of antiviral therapy on patients with hepatitis C.

Objective To evaluate and analyze the clinical application value of detection of Warfarin-related gene polymorphisms,cytochrome P450 2C9 (CYP2C9) and Vitamin K epoxide reductase complex subunit 1 (VKORC1) polymorphisms.Methods From July of 2011 to July of 2012,the blood samples were randomly collected from 140 lung cancer patients from Department of Oncology in Renmin Hospital of Wuhan University.These lung cancer patients were diagnosed through imaging examination and pathological examination.CYP2C9 and VKORC1 polymorphisms were detected in 70 patients (studied group) but not detected in the other 70 patients (control group) before they used warfarin.According to known gene sequences of CYP2C9 and VKORC1,specific primers were designed to genotype the CYP2C9 *2 and CYP2C9 * 3 alleles as well as the VKORC1-1639G ＞ A polymorphism through PCR amplification and DNA sequencing.Meanwhile,the distribution of these alleles in the studied group was analyzed.The clinical significance of detection of these polymorphisms was evaluated by comparing the proportion of patients within the therapeutic INR (International Normalized Ratio) range between control and genotype-guided dosing groups using Chi square test after 2 and 4 weeks of Warfarin therapy.Results Based on the results of agarose gel electrophoresis of PCR products and DNA sequencing,the primers for CYP2C9 and VKORC1 polymorphisms were indeed specific to these SNPs (CYP2C9 * 1,CYP2C9 * 2 and CYP2C9 * 3 ;VKORC1-1639GG,VKORC1-1639AG and VKORC1-1639AA) and both of the specificity and sensitivity of these primers are 100％,thus contributiug for genotyping these alleles.The distribution of CYP2C9 * 1/* 1 was 100％,CYP2C9 * 1/* 2,CYP2C * 1/* 3,CYP2C9 * 2/* 2,CYP2C9 * 3/* 3 and CYP2C9 * 2/* 3 were 0％.The distribution of VKORC1-1639AG,VKORC1-1639AA and VKORC1-1639 GG were 10％,90％ and 0％ respectively.2 weeks after the treatment of Warfarin,85.7％ patients in the genotype-guided dosing group reached the stable therapeutic INR range,which was significantly higher than that in the control group (48.6％,x2 =21.9,P ＜ 0.01); 4 weeks later,all patients (100％) were inside the stable therapeutic INR range whereas only 65 patients (92.9％) in the control group reached the therapeutic INR range.No haemorrhage or thromboembolic events occurred in both groups.Conclusions CYP2C9 and VKORC1 polymorphisms can be accurately detected by PCR reaction with the designed primers and the subsequent DNA sequencing in patients with lung cancer.This method is validated to be reliable.The genotyping of the Warfarin-related genes detective method can effectively guide Warfarin-dosing.

Objective:To research the clinical effects of ulinastatin in the treatment of sepsis induced acute renal injury and its possible mechanisms.Methods:114 cases of patients with sepsis induced acute kidney injury from 2014.02 ～ 2016.08 were selected and randomly divided into the control group (n=57) and experimental group (n=57) according to the draw method,the control group was given conventional treatment,while the experimental group was treated by ulinastatin based on the control group,the urine urinary injury molecule-1 (KIM-1),atrialnatriuretic peptide (ANP),cyscatin-c (CYS-C),interleukin l,6 (IL-1,IL-6),c-reactive protein (CRP),tumor necrosis factor-α(TNF-α),nitric oxide (NO),endothelin 1 (ET-1),immunoglobulin A,G,M (IgA,IgG,IgM) levels,APACHE-Ⅱ score were compared between two groups before and after the treatment.Results:After treatmented,the urine of KIM-1,ANP,serum of CYS-C,IL-l,IL-6,CRP,TNF-α,ET-1 levels and APACHE-Ⅱ score of experimental group were significantly lower than those of the control group (P＜0.05).The serum NO,IgA,IgG,IgM levels of experimental group were significantly higher than those of the control group (P＜0.05).Conclusion:Ulinastatin could significantly relieve sepsis induced acute renal injury,which might be related to the inhibition of inflammatory response,improvement of the renal blood flow and immune function.

Objective To study the structures of the plasmids of Klebsiella pneumoniae KF3 at the genome metagenome level througth with whole plasmid DNA sequencing, to analyze the functional genes carried by plasmid and to identify the correlation of resistance and pathogenicity between the plasmids and the host strains. Methods The alkaline lysis method was used to extract plasmids. We constructed the small insert pUC18 library and the large insert Forsmid library, sequenced and used the Phred / Phrap / Consed package to assemble these sequences and gained a complete sequence. The open reading frame(ORFs) were predicted by the Glimmer software and annotated, analyzed the functions of these genes. Results We successfully constructed the pUC18 library and the Fosmid libraries for the plasmid DNA and obtained three circular double-stranded DNA plasmids: pKF3-70 (69 477 bp), pKF3-90 (91 327 bp) and pKF3-147 ( 147 416 bp). There were drug resistant genes, conjugative transfer genes and mobile DNA elements identified on three plasmids. Conclusion The three plasmids of KF3 could be transferred among different strains. It would lead to the dissemination of the resistant genes.

Objective: To explore the clinic-pathological significance of microRNA-145 expression in human cervical cancer and its ef-fects on Wnt/β-catenin signaling pathway. Methods: Real-time PCR was used to detect the expression of microRNA-145 in 62 cervical cancer samples. The correlation between microRNA-145 expression and the clinic-pathological parameters and its prognostic signifi-cance was analyzed. MicroRNA-145-expressing plasmid and non-sense plasmid were transfected into human cervical cancer HeLa cells, assigned into overexpressed microRNA-145 group and control group. Immunofluorescence staining was performed to detect the expression location of β-catenin. Top Flash luciferase reporter assay was performed to investigate the effects of microRNA-145 on the transcriptional activity of TCF/LEF and direct interactions with Cateninδ-1. Western blot was used to detect the effects of microRNA-145 on the expression of Cateninδ-1, C-MYC, and CyclinD1. Results: The patients with low microRNA-145 expression showed poorer prognosis [(41.28 ± 2.00) months vs . (46.06 ± 0.95) months, P<0.05]. β-catenin immunofluorescence was distributed within the cyto-plasm in the microRNA-145-overexpressed HeLa cells, but mainly within the nucleus and cytoplasm in the control cells. The luciferase reporter system indicated that the transcriptional activity of TCF/LEF was inhibited in the microRNA-145-overexpressed HeLa cells, and validated Cateninδ-1 was a target of miR-145. The expression of Cateninδ-1, C-MYC, and CyclinD1 was decreased in the microRNA-145-overexpressed HeLa cells. Conclusions: microRNA-145 may inhibit cervical cancer progression via Cateninδ-1 and inhibit the Wnt/β-catenin signaling pathway.

Objective:To establish a clinical laboratory diagnostic pathway for hepatitis C covering diagnosis, differential diagnosis, drug toxicity monitoring, and therapeutic and prognostic evaluation, and to explore a new teaching model for laboratory diagnostics based on the clinical laboratory diagnostic pathway.Methods:According to the clinical diagnosis and treatment guidelines for hepatitis C, laboratory testing strategies for different stages of diagnosis and treatment of the disease were formulated to establish a clinical laboratory diagnostic pathway for hepatitis C. The pathway was applied in the teaching for undergraduate medical students of the seven-year program of grade 2019 of The First Clinical College of Wuhan University, with those of grade 2018 as the control to receive traditional teaching. The teaching effect was compared through questionnaires and quizzes in class. The data were analyzed through the t test with the use of SPSS 19.0. Results:A clinical laboratory diagnostic pathway for hepatitis C recognized by clinicians was established, covering the entire process of clinical diagnosis, differential diagnosis, monitoring of drug side effects, and therapeutic and prognostic evaluation. The students of grade 2019 receiving the pathway-based teaching model had significant improvements in teaching quality evaluation indicators ( P<0.05), with the most marked improvement in "having mastered the key and difficult points of this lesson", with a score of (60.90±2.15) points for grade 2018 and (84.80±3.44) points for grade 2019. The total score for teaching evaluation was significantly higher in students of grade 2019 than in those of grade 2018 [(94.02±4.29) vs. (79.21±3.68)] points, P<0.05). Grade 2019 also had a significantly higher classroom quiz score than grade 2018 (94.60±5.63) vs. (78.10±4.92), P<0.01]. Conclusions:We established and applied a clinical laboratory diagnostic pathway of hepatitis C in the teaching model of laboratory diagnostics, which organically integrates laboratory diagnostics and clinical medicine, and significantly improves the quality of teaching.