Objective To construct prokaryotic expression plasmid of human recombinant soluble TNF-related apoptosis inducing ligand(rsTRAIL),then to investigate the effects of rsTRAIL on apoptosis in non-small cell lung carcinoma(NSCLC).Methods The encoding sequence for rsTRAIL was amplified with RT-PCR and cloned into PQE30 vector to establish the prokaryotic expression system.The competent cells of host strain of M15 were transformed by the recombinant plasmid.The expression of the target protein was induced with IPTG and purified by Ni2+-NTA agarose column.rsTRAIL was added in the media of A549 and H460wt cells,then the viability was examined by MTT assay.Apoptosis of H460wt cells was observed under fluorescence microscopy.The apoptotic rate of tumor cells was examined by FACS.Results The cloned fragment of rsTRAIL was 100% consistent with that reported in GenBank.The expressed protein with molecular weight of 21 000 in SDS-PAGE as expected was obtained and recognized by a commercial McAb.The apoptotic rate of H460wt cells after treated with rsTRAIL for 24 h was 43.2%.Cislatin enhanced the effect of rsTRAIL on A549 cells.Conclusion The rsTRAIL is obtained after Ni affinity chromatograph.rsTRAIL has a strong cytotoxic activity against NSCLC and cisplatin may enhance the antitumor effect of rsTRAIL.

Objective To discuss the effects of capsular tension ring after phacoemulsification combined with IOL implantation on tilt and decentration of IOL in high myopia patients with cataract by ultrasonic biomicroscope (UBM).Methods A total of 36 cases (40 eyes) with high myopia and cataract underwent phacoemulsification combined with IOL implantation were chosen.The average axial length was 26.88 mm.The patients were divided into implant group (19 eyes,the capsular tension ring was implanted) and control group (21 eyes,the routine surgery was performed).The patients were examined by conventional slit lamp,and the best corrected visual acuity (BCVA)was measured at pre-operation and postoperative 6 months.Tilt and decentration were measured horizontally and vertically,and total tilt and decentration were calculated by geometry method.Results The postoperative BCVA in the two groups were all better than the pre-operation,there was no statistical difference in the preoperative and postoperative BCVA between two groups (all P ＞ 0.05).The horizontal,vertical and total decentration the implant group were (0.15 ± 0.07) mm,(0.30 ± 0.40) mm,(0.11 ±0.02)mm,respectively,which in the control group were (0.26 ± 0.19)mm,(0.32 ±0.60) mm,(0.24 ± 0.97) mm,respectively.The horizontal,vertical and total tilt in the implant group were 0.02° ±0.11°,0.70° ±0.25°,0.21° ±0.74°,respectively,which in the control group were 0.11 ° ± 0.31 °,1.09° ± 0.20°,1.24° ± 0.97°,respectively.There were statistical differences in the horizontal,total tilt and decentration between two groups (all P ＜ O.05),but no statistical difference in the vertical tilt and decentration (P ＞ 0.05).Conclusion The capsular tension ring can stable the IOL position after surgery in high myopia and cataract patients.

Objective To compare clinical efficacy of inverse less invasive stabilization system (LISS) and Gamma nail in treating unstable femoral intertrochanteric fractures combined with lateral wall fractures.Methods Fifty-two patients with intertrochanteric fractures associated with lateral wall fractures followed up for at least 12 months after surgical treatment between June 2010 and June 2013 were included in this retrospective cohort study.According to the different internal fixation,the patients were assigned to inverse LISS group [16 males and 8 females;(62.5 ± 12.4)years] and Gamma nail group [17 males and 11 females;(60.4 ± 18.6)years].According to the AO classification,there were 6 patients with A2.2 type,5 A2.3 type,5 A3.1 type,6 A3.2 type and 2 A3.3 type in inverse LISS group,and there were 4 patients with A2.2 type,7 A2.3 type,9 A3.1 type,5 A3.2 type and 3 A3.3 type in Gamma nail group.Opcration time,total blood loss (intraoperaive plus occult blood loss),hospital length of stay,bone healing,time of commencing full weight-beating and complication incidence were compared between the two groups.Harris hip score was recorded postoperatively.Results All patients were followed up for 24-36 months (mean,30.2 months).Operation time in Gamma nail group was shorter than that in inverse LISS group (P ＜ 0.05),while relatively more blood loss was found in Gamma nail group(P ＜0.05).There were no significant differences in hospital length of stay and bone healing timebetween the two groups (P ＞ 0.05).Patients in Gamma nail groups started earlier full weight-bearing than inverse LISS group (P ＜0.05).Harris score was (86.1-± 12.4)points in inverse LISS group one year after operation,not significantly different from (83.3 ± 11.2) points in Gamma nail group (P ＞ 0.05).Complication of one patient with coxa vara and one bone nonunion in inverse LISS,showing no significant from one patient with screw breakage and one femoral head osteonecrosis in Gamma nail group (P ＞ 0.05).Conclusions Both techniques are effective in treatment of intertrochanteric fractures combined with lateral wall fractures and achieve similar results in function recovery.But inverse LISS is eccentric fixation,so too early weight-bearing should not be over-emphasized.

Objective:To observe the change of CD4+CD25+regulatory T cells and Th17 cells in mice with experimental anti-phospholipid antibody syndrome ( EAPS ) .Methods: EAPS model was established by immunizing BALB/c mice with recombinant humanβ2 glycoprotein 1 (rhβ2GP1).The levels of serum anti-β2 glycoprotein 1 (anti-β2GP1),anti-cardiolipin antibody (aCA),IL-17,IL-2,IL-6 and TGF-βwere tested by ELISA.The rate of abortion,activated partial thromboplastin time (APTT) and platelet count were also detected.Flow cytometry was applied to detect the percentages of the CD4+CD25+regulatory T cells and Th17 cells in peripheral blood mononuclear cells.Results:Compared with the control group,the levels of anti-β2 GP1,aCA,IL-17,IL-2 and IL-6 were significantly increased,the rate of abortion was increased,APTT time was prolonged and the levels of TGF-βand platelet count were de-creased in model mice (P<0.05).No significant difference was detected of percentage of Treg cells in PBMC at the eighth weeks in model group (P>0.05),but percentage of Treg cells was lower than that in control group after 12 weeks (P<0.05);the percentage of Th17 cells in model group was higher than that in control group (P<0.05).In addition,the ratio of Treg/Th17 cells was lower in model mice than that in control group.Conclusion: The imbalance of CD4+CD25 Treg/Th17 cells may participate in the pathogenesis of EAPS.

Objective To explore the application of ABCD3 score on stratifying the antithrombotic treatment strategy in patients with capsular warning syndrome (CWS).Methods The clinical features of 15 patients with CWS were analyzed retrospectively,and the risk of stroke were evaluated by ABCD3 score and to guide the treatment of Stratifying antithrombotic therapy.The status of patients hospitalized,discharged and discharged after 90 d were evaluated.Results The frequency of patients with CWS accounted for 2.51％ (15/ 597) of all patients with transient ischemic attack(TIA),and the mean age in patients with CWS was (70.27 ±8.29) years old.The duration of the first onset was (10-30) min,the mean time was (17.33±1.53) min,and ABCD2 score was 5.0-9.0 points,mean score was 7.00±0.26 points,and the total episodes of CWS were 51 times during 24 hours,the mean duration was (18.13 ± 15.36) minutes ((3.0-60.0) min).All 15 cases presented with limb hemiparesis.Of them,9 cases had dysarthria,5 case with ipsilateral facial palsy.All 15 cases CWS patients showed no signs of cortical deficit.The mean NIHSS score at onset was 1.0-6.0 points,mean scores was 3.20±0.31 points.Fourtheen patients were treated with clopidogrel plus aspirin,and 2 cases with administration of the loading dose 300 mg of clopidogrel,1 case was treated with clopidogrel plus aspirin orally followed by intravenous rt-PA thrombolysis.The average hospital periods of all 15 patients was (7.67±0.29) days.The NIHSS score were 0 point at discharge.There was no symptomatic intracranial hemorrhage or death within 90 days follow-up periods.Conclusion CWS is prone to develop a completed stroke.Stratified antithrombotic therapy guiding by ABCD3 score may decrease the risk of ischemic stroke.

Objective To construct an eukaryotic expression vector for shRNA targeting WT1 gene.Methods Firstly, one site targeting human WT1 gene in gene bank was selected according to the guidelines for the selection of highly effective siRNA sequence and then BLAST test was performed. Secondly,a pair of oligonucleotides fragments were synthesized and annealed to double strand. Thirdly, the double strand was cloned into plasmid vector pGenesil-1. Finally, the recombinant plasmid was identified by restriction enzyme digestion and sequencing analysis. Results Enzyme cutting, and sequencing analysis have confirmed that the construction was successful. Conclusion WT1shRNA is constructed which is the fundament to further research of WT1 gene.

Objective To observe the clinical effect of Sangpa Zhike decoction in the treatment of acute exacerbations of chronic bronchitis. Methods 180 patients with acute exacerbation of chronic bronchitis admitted to Autonomous Prefecture Hospital of traditional Chinese medicine(TCM)in Changji,Xinjiang,from August 2012 to August 2013 were enrolled. They were divided into Sangpa Zhike decoction treatment group and western medicine treatment control group by random number table,90 cases in each group. General treatments were given to the two groups. In the Sangpa Zhike decoction treatment group,additionally only oral Sangpa Zhike decoction was given(composition:Morus alba 10 g,loquat leaf 10 g,Houttuynia 10 g,honey aster 15 g,honey coltsfoot flower 6 g,Zhejiang Fritillaria 12 g,perilla 10 g,white mustard seed 6 g,Platycodon grandiflorum 10 g,Rhizoma Cynanchi Stauntonii 12 g,tangerine peel 6 g,Stemona 6 g). The ingredients were mixed in water and boiled to form a decoction,one dose daily,divided into two parts to be taken twice a day. In the western medicine treatment group,the infection was controlled by western medicine and the drugs to eliminate cough and phlegm,etc were used. The therapeutic course in both groups was 7 days. The therapeutic effect for treatment of TCM syndromes and the cough quantized integration score in the two groups were observed after treatment. Results The cure rate of Sangpa Zhike decoction treatment group was significantly higher than that of western medicine treatment control group〔70.00%(63/90)vs. 33.33%(30/90),P<0.01〕. Before treatment,the cough symptom quantization integral scores in the two groups had no statistically significance, but after treatment,the scores in the two groups were significantly lower than those before treatment. And the score in Sangpa Zhike decoction treatment group was decreased more significantly (1.66±1.12 vs. 4.36±2.32, P<0.01). Conclusion Sangpa Zhike Decoction has obvious curative effect in the treatment of acute attack of chronic bronchitis.

Objective To investigate the effect to the expression of VEGF in human leukemic cell line by WT1gene silencing.MethodsK562 cells were transfected with WT1shRNA Plasmid.Transfection efficiency was detected by flow cytometIy,and the succeed transfered cells Were sorted.WT1 and VEGF mRNA variation were measured by fluorescence RT-PCR.VEGF concentrations in the cell growth medium were detected by ELISA.Results 48 hours after transfection,the transfection efficiency was 65％±4.5％.WT1shRNA positive cells were sorted by flow cytometry.Both the mRNA level of WT1 and VEGF and VEGF concentrations in cell growth medium were significantly decreased than the control group.ConclusionWT1 gene silence can down-regulate the expression of VEGF in K562 cells which suggest that WT1 gene may take part in leukemia angiogenesis through up-regulation of VEGF.

Objective To study the safety and efficacy of low dose Peginterferon-alpha-2a (PEG-INF-α-2a) combined with Ribavirin treating chronic hepatitis C in renal transplant recipients.Methods A total of 13 cases of HCV hepatitis were randomly divided into treatment and control groups.Seven cases in treatment group were given PEG-INF-α-2a (90 μg/week) and ribavirin (600mg/day) for 16 to 48 weeks,and the rest 6 cases in control group were subjected to general liver protection and anti-inflammatory treatment. All patients were followed up for more than 2 years.Results There were 5 cases getting early response in treatment group for 16 weeks,including four cases of complete response and no non-effects response patients. In 4 cases voluntarily receiving treatment for 48 weeks,1 case had facial muscle myalgia and increased Cr level at 35th week,humoral graft rejection was confirmed pathologically,and the treatment was terminated; 1 case had recurrence of HCV RNA replication and PEG-INF-α-2a was withdrawn at 38th week.As results,5 patients in the treatment group obtained complete response after two years,including 2 cases whose HCV-IgG had got negative,HCV RNA replication was significantly lower than in the control group,and the average Cr higher than in control group (P＞ 0.05). There were adverse reactions during this treatment protocol: fever,muscle myalgia,agranulocytosis, anemia and humoral graft rejection.Conclusion The efficacy of low lose PEG-INF α-2a combined with ribavirin is definite in the treatment of chronic HCV hepatitis in kidney transplant recipients.The 16-week treatment duration is reasonable.It is remarkable that PEG-INF-α-2a may cause humoral graft rejection and Cr crawling.

BACKGROUND: Classic isolation method of bone marrow mesenchymal stem cells (BMSCs) is Percoll density gradient centrifugation. Blood cell component was removed. However, this method is complicated. Preparation density was needed when isolating dog bone marrow. Moreover, centrifugation was frequent, which had a great damage to cells. OBJECTIVE: To establish methods of the isolation, proliferation, culture and osteoinduction of canine BMSCs, and observe the in vitro proliferation and ability to osteoinduction differentiation. METHODS: 10 mL bone marrow was extracted from dog posterior superior iliac spine, heparin anticoagulation, diluted using Hanks juice, treated with 1.077 g/mL Ficoll solution 3 mL, and centrifuged at 2 000 r/min for 20 minutes. Karyocytes were absorbed to form white cloudlike layering interface, and then centrifuged twice using DMEM supplemented with fetal bovine serum, incubated at 12×10~4/cm~2 at 37 ℃ in a 5% CO_2 incubator. Following subculture, cells were incubated in DMEM containing dexamethasone, β-sodium phosphoglycerol and ascorbic acid 2-phosphate. Immunocytochemical staining and immunofluorescence staining were utilized to detect osteocalcin, osteopontin and type Ⅰ collagen expression in osteoblasts. Alkaline phosphatase staining and alizarin red staining were performed. RESULTS AND CONCLUSION: 1.077 g/mL Ficoll density gradient centrifugation was used to isolate karyocytes that were significant compared with Percoll solution. Obtained BMSCs had high purity, good growth and the mean doubling time was 24 hours. Following in vitro osteogenic incubation of dog BMSCs, osteocalcin, osteopontin and type Ⅰ collagen showed positive expression. Alkaline phosphatase staining demonstrated bluish-green cytoplasm. Alizarin red staining showed red nodes in extracellular matrix, and could differentiate into osteoblasts in vitro.