To construct a subtractive cDNA library of genes transactivated by NS5A protein of hepatitis C virus with suppression subtractive hybridization technique, the mRNA was isolated from HepG2 cells transfected with pcDNA3 1(-) NS5A and pcDNA3 1(-) empty vector, respectively, then the cDNA was synthesized. After restriction enzyme RsaI digestion, small sized cDNAs were obtained. Then the tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. After the tester cDNA was hybridized with the driver cDNA twice and underwent two times of nested PCR, it was then subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain JM109. The cDNA was sequenced and analyzed in GenBank with Blast search after PCR. The amplified library contained 121 positive clones. Colony PCR showed that 115 clones contained 200- 1 000 bp inserts. Sequence analysis was performed in 90 clones, and the full length sequences were obtained with bioinformatics method. Altogether 46 kinds of coding sequences were acquired, which consisted of 31 kinds of known and 15 kinds of unknown ones. The obtained sequences might be target genes transactivated by NS5A protein of HCV, among which some genes coding proteins involved in cell cycle regulation, cell apoptosis, signal transduction pathway and tumour development. The results indicated that the subtractive library of genes transactivated by NS5A protein of HCV was constructed successfully, which brought some new clues for studying the biological functions and pathogenesis of the viral proteins

Objective To compare the effect on preserving rat amputated extremities during the cold preservation between the UW solution and HTK solution.Methods Thirty healthy adult Wistar rats were randomly divided into 3 groups (10 rats in each group) for producing the models of amputated limbs of rats.The three groups were separately irrigated the amputated limbs with UW solution,HTK solution and saline from femoral artery.After irrigation,the extremities were preserved in the refrigerator at 4 ℃.Samples of skeleton muscle tissue were taken for pathological and biochemical examination every 6 hours since the amputation.Results After perfusion,HE:changes of organizational structures of skeletal muscle were lighter in experimental groups than in control group (saline-perfusion group) at the same time.While samples in UW solution-perfusion group has lighter changes of organizational structure than those in HTK solution-perfusion group.TEM:at 12 h,all the three groups have shown the mitochondria edema.At 24 h,the myofibril in saline-perfusion group become fragments.Biochemical examination:at 24 h,indexes in salineperfusion group,HTK solution group and UW solution group were:ALP (U/L) 3.62 ± 1.32,3.37 ± 0.84 and 2.68 ± 1.59,respectively;ALT(U/L) 542.25 ± 129.36,521.82 ± 97.32 and 462.53 ± 74.18,respectively;AST (U/L) 2200.12 ± 687.61,2002.20 ± 632.27 and 1742.87 ± 396.09,respectively;CK(U/L) 313190.83 ± 42041.32,283173.50 ± 31488.87 and 271319.67 ± 41147.52,respectively;LDH (U/L) 32409.50 ± 4253.20,30382.50 ± 2337.18 and 30047.83 ± 4628.78,respectively;GLU(mmol/L) 0.242 ± 0.041、0.240 ± 0.044 and 0.252 ± 0.049,respectively;LAC (mmol/L) 1.790 ± 0.160,1.792 ± 0.196 and 1.993 ± 0.366,respectively;SOD(U/mL) 80.82 ± 9.46,91.62 ± 14.97 and 73.71 ± 10.60,respcctively.There were statistical difference among the UW solution-perfusion group and centrol group in ALP and GLU (P ＜ 0.05).UW solution-perfusion group has better results than HTK solution-perfusion group in SOD (P ＜ 0.05).Conclusion Amputated limbs irrigated with organ preservation solution as a way to protect skeletal muscle had great effects on lightening tissue damage and keeping tissue active.Between the two usual organ preservation solution,UW solution had a stronger protective effects on the amputated limbs than HTK solution.

Purpose:To study the effect of the herbal decoction Qingyi Xiaoji Formula(QYXJ) on the proliferation of human pancreatic cancer cell line SW1990 in vivo and to explore the mechanism of its functions by means of cDNA microarray.Methods:Tumor-burdened nude mice were randomized into control group,5-FU group,and QYXJ groups at different dosages.After treatment,inhibiting rates of tumor growth were calculated.Tumor mRNA of the control group and the QYXJ group at moderate dose was extracted.The fluorescent cDNA probes were prepared,labeled with two different dyes Cy3 and Cy5,and then hybridized with cDNA microarray and scanned for fluorescent intensity.The genes with different expression were identified through the analysis of gene expression profile.Results:Inhibition rates of tumor growth in the QYXJ groups were 21.31%,38.16% and 29.09%,in the dose of 18 g/kg,36 g/kg,and 72 g/kg respectively.7 genes with reduced expression were identified,the functions of which were oncogene,protein translation and synthesis,DNA synthesis and repair,cell signal transduction,etc.Conclusions:QYXJ decoction may inhibit the proliferation of pancreatic cancer in vivo,possibly by blocking the action of an oncogene and its downstream signaling,or by regulation of protein synthesis in cancer cells.

Objective To understand the genotoxicity of organic extracts (OEs) of fishpond water containing sewage.Methods Male Kunming mice were randomly divided into different groups according to the body weight,6 in each group.The OEs were administrated to the mice through peritoneal injection at the doses of 25.00 L/kg,12.50 L/kg and 6.25 L/kg respectively for 3 consecutive days.The negative and positive control groups were treated with DMSO and cyclophosphamide (CP) respectively.The DNA damage was tested by using un-fluorescent staining comet assay.Results The tailed-cells rates and tail length in each OEs treated group were significantly higher than those in the negative control group (P

Objective:To observe the ultrastructure of biological living skin equivalent in vitro.Methods:Keratinocytes and fibroblasts were sourced from the back skin of legally aborted human foetus.Fibroblasts were seeded into bovine type I collagen gel and cultured for 3 days.Keratinocytes were seeded on the surface of collagen gel and cultured for another 2 days,then the equivalent skin was exposed to air-liquid interface to form a protective cornified layer.Histological structure and ultrastructure of the equivalent skin were observed by light microscope and transmission electron microscope.Results:The morphogy of equivalent skin of full thickness consisted of epithlium and dermis.Epithelium was made up of stratum basale,stratum spinosum,stratum granulosum and stratum corneum.lntercellular bridge existed within the cells of different layers.Some ultrastructures in epithelium such as desmosome,Lamelle,tonofibrils and lipid droplet were found in the equivalent skin.Conclusion:Tissue-engineered skin may have the features of natural skin.

Objective To construct a subtractive cDNA library of genes differentially expressed in human lymphoma cell line Jurkat cells treated with phosphonoformate (PFA), and to clone genes associated with its immune regulation. Methods Using suppression subtractive hybridization (SSH) technique, the mRNA was isolated from Jurkat cells treated with phosphonoformate or 0 9 percent sodium chloride, respectively, and then cDNA was synthesized. After restriction enzyme RsaI digestion, small sized cDNAs were obtained. Then tester cDNA was subdivided into two portions and each was ligated with different cDNA adaptor. The tester cDNA, which was hybridized with driver cDNA twice and amplified by nested polymerase chain reaction (PCR) twice, was subcloned into T/A plasmid vectors to set up the subtractive cDNA library. Amplification of the library was carried out with E. coli strain JM109. The clones, which were selected randomly, were amplified by PCR, and then they were sequenced and analyzed in GenBank with Blast search. Results The subtractive cDNA library of genes differentially expressed in Jurkat cells treated with PFA was constructed successfully. The amplified library contained 46 positive clones, which contained 200- 1 000 bp of inserts. Fourteen clones were analyzed by sequencing and bioinformatics, which were identified as eleven known genes and three genes with unknown function. Conclusions The subtractive cDNA library of genes differentially expressed in Jurkat cells treated with PFA using SSH technique was constructed successfully, which brought some new clues for the study of the immune regulation mechanism of PFA

Objective:To study the clinical features of severe acute pancreatitis(SAP) in aged patients. Methods: The clinical data of 78 aged patients(55-84 years old) with SAP were analyzed and compared with those of 107 young SAP patients(22-54 years old) hospitalized in our hospital over the past 10 years. Results: Most aged patients were associated either with gall stones or idiopathy and chronic diseases, especially hypertension. The incidences of all kinds of clinical symtoms in aged SAP patients were lower than those of the yound SAP patients (P

Objective To explore the value of neopterin (Np) in early diagnosis and risk stratification of sepsis.Methods A total of 82 patients admitted to the emergency department from April 2013 to February 2014 were enrolled in the study.They were divided into two groups:sepsis-free group (n =8) and sepsis group (n =74).Patients' APACHE Ⅱ scores were calculated within 24 hours after admission,and then their plasma levels of Np and procalcitonin (PCT) were detected and analysis was carried out to find the correlation between plasma levels of Np,PCT and acute physiology and chronic health evaluation Ⅱ score (APACHE Ⅱ score) in two groups.And 74 patients in sepsis group were further divided into three subgroups according to their APACHE Ⅱ score:Ⅰ subgroup (score ＜ 15,n =27),Ⅱ subgroup (score 15 to 24,n =32),and Ⅲ subgroup (score ＞ 25 or more,n =15).The differences in plasma Np and PCT levels were compared among three subgroups and correlation analysis of Np levels with PCT levels and APACHE Ⅱ scores was carried out.Results The plasma levels of Np,PCT and APACHE Ⅱ scores in sepsis group were higher than those in sepsis-free group (P ＜ 0.05).In sepsis group,the plasma levels of Np and PCT were gradually increased with the increasing severity of sepsis (P ＜ 0.05);In sepsis group,the plasma levels of Np,PCT and APACHE Ⅱ scores were positively correlated (P ＜0.01).The area under receiver operating characteristic curve (ROC) of Np was O.79 and the area under ROC of PCT was 0.75,both of which had high diagnostic accuracy.Conclusions Neopterin has greatly useful value in early diagnosis and risk stratification of sepsis.

Objective To assess the outcomes of involved field irradiation (IFI) and elective nodal irradiation (ENI) in patients older than 70 years with esophageal squamous-cell carcinoma(SCC) receiving radical IMRT,and to determine whether IFI is feasible in these patients.Methods Totally 79 patients older than 70 years diagnosed with SCC of esophagus without distant metastases were collected.48 patients were received IFI,and the other 31 patients were treated with ENI.With a median follow-up time of 24 months,disease-free survival,overall survival,patterns of failure,irradiated lung dose and radiation pneumonitis were observed and compared between IFI and ENI groups.Results In IFI and ENI arm,the disease-free survival rates of 1,2,3 years were 60.4％,34.9％,29.7％ and 64.5％,54.0％,35.0％,respectively(P ＞0.05).The 1-,2-,and 3-year survival rates were 72.9％,43.4％,31.5％ for the IFI arm,and 73.0％,53.0％,38.3％ for the ENI arm(P ＞ 0.05).The ENI arm had a tendency to expand survival,but the two arms had no significant difference (P ＞ 0.05).The patterns of failure also had no difference between the two arms.Distant failure,local failure,uninvolved nodal failure in IFI arm were 22.9％,27.0％,4.2％,while in ENI arm were 25.8％,0,19.4％,all of them had no significant difference (P ＞ 0.05).However,the lung V5,V20,mean lung dose in ENI arm were higher than that in IFI and all of them had a significantly difference (t =4.66,29.90,15.63,P ＜ 0.05).The radiation pneumonitis rates were higher in ENI than in IFI arm.The rates of degree 1-2 and degree 3 were 22％,19％,and 13％ and 4％ in the two arms,respectively,with a significantly difference(x2 =4.55,4.77,P ＜ 0.05).Conclusions It is feasible that IFI for definitive IMRT in the elderly patients older than 70 years with SCC,because it got similar disease-free survival and overall survival but with less lung doses along with decreased radio-pulmonary lesion when compared with ENI.

Objective To determine the effect of individualized oral hygiene instruction (OHI) on volatile sulfur compounds (VSCs) level in mouth air of patients with chronic periodontitis.Methods The individualized oral hygiene instruction was provided for 31 patients with chronic periedontitis (Organoleptic score was over two points).The VSCs and plaque index (PLI) were evaluated pre-OHI and post-OHI.Results The score of PLI decreased from 3.09±0.44 to 2.63±0.46 significandy (P<0.05).The organoleptic score (OS) decreased from 2.68±0.70 to 2.35±0.75 significandy (P<0.05).While there was no significant difference on the VSCs level pre-OHI and post-OHI (P<0.05).Conclusions Individualized oral hygiene instruction is helpful to remove plaque and improve oral hygiene.But the effect of OHI on VSCs level in mouth air is limited.