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 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 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 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.

Background The aspheric intraocular lenses(IOLs)can reduce ocular spherical aberration to some degree.However,the clinical effect depends more on the IOL proper alignment.It becomes more important to study the IOL position in eye,Objective This study was to analyze the position alteration of IOL after phacoemulsification combined with implantation of one-piece soft IOLs.Methods In this prospective control study,80 eyes of 40 patients with age-related cataract were enrolled.The phacoemulsification with IOL implantation was performed in all the eyes.Decentration and tilt of IOL in the nasal superior,superior temporal,inferior temporal and nasal inferior quadrants(the intersection point of the system optical axis and the IOL maximum cross plane were regarded as the ordinate origin)were measured by rotating Scheimpflug camera(Pentacam Oculus)in 3 months postoperatively under the mydriasis condition.Written informed consent was obtained from each subject prior to this trial.Results In the right eye group,the IOL decentered toward temporal in 26 eyes(65％)and infratemporal in 16 eyes(40％).IOLs tilted temporally in the horizontal plane in 37 eyes(92.5％)and tilted inferiorly in the vertical plane in 34 eyes(85.0％).In the left eye group,IOLs decentered temporally 33 eyes(82.5％)and 20 IOLs (50％)infratemporally,IOLs tilted temporally in the horizontal plane in 37 eye(92.5％)and 36 IOLs(90％)tilted inferiorly in the vertical plane.There was no statistical difference for the intercomparsion of horizontal/vertical decentration in various quadrant in the right eye(F =0.221,0.792,P＞0.05).The obvious elevated horizontal decentration was found in the supertemporal and infratemporal quadrants compared with supernasal quadrant in the left eyes but there was no significant difference in the vertical decentration among 3 quadrants(F=0.576,P＞0.05).Decentrations were positively correlated with the tilt in both horizontal and vertical plane(right eye horizontal plane:r=0.374,P=0.002;right eye vertical plane:r=0.402,P=0.001 ;left eye horizontal plane:r=0.377,P=0.002；left eye vertical plane:r=0.347,P=0.002).Conclusions The one-piece soft IOLs(Adapt AO)decenter toward temporal mostly in 3 months after surgery,especially infratemporally in the eye.And the optical axis of the IOL tilt toward infratemperol mostly in both right and left eyes.The decentration and tilt are consisted in the corresponding direction between the right and left eyes.The position of the IOLs showed mirror symmetry between right and left eyes.The IOLs decentration show the positively correlation to tilt whatever in horizontal and vertical plane.

380 mmHg) while the PaCO2 increased slightly at T5 and decreased gradually. The PCWP decreased slightly at T2, T3 , T4 and increased quickly at T5 , T6, levelling with T0 at T9. PVRI was on gradual increase beginning ahepatic stage and falling to the level of T0 at T7. The changes of PAP were similar with PCWP, the value at T6 being higher than that at T3, T4 significantly. Conclusion DO2 -directed hemodynamic management protects pulmonary function from damage during LTx.

Talk teaching is an effective form of teaching research activity.It focuses on the teachers,teaching ideas and their abilities of curriculum development,curriculum designing and teaching practice.It is of great significance in optimizing pathophysiologic teaching,improving the teaching effect and teachers'quality.It is worth discussing and promoting.Talk teaching should be the basic skill necessary to the teachers of basic medicine.

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 construct a fusion gene (h1a-spaO) encoding H1a-SpaO protein of Sal-monella paratyphi A ( S.paratyphi A) and to express it in prokaryotic expression system , then to further ana-lyze the immunoprotective effects of the expressed protein rH 1a-SpaO.Methods The h1a-spaO fusion gene formed from separate h1a and spaO genes was amplified by PCR using flexible peptide sequence-containing linking primers and then sequenced after T-A cloning.A prokaryotic expression system for expressing h1a-spaO fusion gene was constructed by using the genetic engineering technique .The expressed protein rH1a-SpaO was examined by SDS-PAGE.The antigenicity and immunoreactivity of rH1a-SpaO protein were deter-mined by Western blot assay .The ability of rH1a-SpaO antiserum agglutinating S.paratyphi A strains was detected by micro-Widal′s test.The immunoprotective effects of rH 1a-SpaO against the lethal dose challenge of S.paratyphi A strains were analyzed in a mouse model and that were compared with those by using equal dose of individual recombinant protein H1a and SpaO (rH1a and rSpaO) as the immunogens, respectively. Results The h1a-spaO fusion gene was 100%identical with the individual h1a or spaO gene in nucleotide and amino acid sequences .The constructed prokaryotic expression system could express the recombinant pro-tein rH1a-SpaO with an advantage of high efficiency .rH1a-SpaO protein was able to react with rH 1a or rSpaO antiserum.Moreover, rH1a-SpaO antiserum also could efficiently recognize rH 1a and rSpaO as well as agglutinate Salmonella paratyphi A strains by binding with H-antigen.The immunoprotective rate (93.3%) in mice pre-immunized with 100 μg of rH1a-SpaO protein was significantly higher than that in those pre-immunized with equal dose of rH1a (60.0%) protein or rSpaO protein(53.3%) (P<0.05).Conclusion The recombinant fusion protein rH 1a-SpaO showed more stronger immunoprotective function than the individ-ual rH1a or rSpaO protein , which could be used as an effective antigen for the development of bi -valent para-typhoid A vaccine or typhoid/paratyphoid capsular polysaccharide-protein combined vaccine .