Objective To clone human survivin gene and express survivin-an inhibitor of apoptosis proteins in Pichia pastoris eukaryotic expression system. Methods Full length survivin gene was amplified by PCR using survivin specific primers. The verified survivin gene by sequencing was subcloned into pPic9k. The recombinant plasmid was linearized by restriction enzyme cutting. The linear survivin gene was introduced into GS115/ His-cells by electroporation. The transformants were transferred onto YPD plates that contained different concentrations of G418 for screening the positive clones. The integrated survivin gene in positive clones was confirmed by PCR. Selected transformants were cultured in BMMY medium with 1 % methanol for inducing the expression of survivin protein. The expressed survivin protein was analyzed by ELISA. Results The cloned human survivin sequence was the same as that in the GeneBank. The recombinant pPic9k-survivin was constructed in Picha pastoris eukaryotic expression vector. After the linear digestion, the recombinant vector was introduced into GS115/ His-cells, the 6 positive clones against G418(4 mg/mL) were obtained and confirmed by PCR; the highest survivin protein was expressed when expressed for two days in 1 % methanol BMMY medium. Conclusions Improved survivin protein yield may be reached by modifying the experimental conditions. This will help to further study the biological functions of survivin and its roles in tumor developments.

Osteoarthritis (OA) is the most common form of arthritis, and is the major cause of pain and chronic disability worldwide, causing enormous social and economic burden.Once OA was considered as a 'wear-and-tear' condition and obesity is considered to be one of the most powerful predisposing factors of OA in the weight-bearing joints.However, studies have also linked obesity to OA in non-weightbearing areas, suggesting systemic effects exerted by metabolic factors other than simple local biomechanics perhaps play a role in the high prevalence of osteoarthritis in obese population.Recent studies have shown that systemic metabolic abnormalities, including obesity, diabetes, hypertension, lipid metabolism disorders, atherosclerosis and cardiovascular disease, play an important role in OA pathological process.Metabolic diseases promote the incidence and development of OA through a variety of ways, inducing causing low-grade systemic inflammation, increasing release of adipokines, anabolic cytokines and inflammatory mediators, leading to glucose and lipid metabolism disorders of chondrocytes, upregulating of cartilage extracellular matrix degrading enzymes, raising oxidative stress injury, increasing apoptosis of articular chondrocytes and reducing the cartilage and subchondral bone nutrition supply.These metabolic changes ultimately accelerate the damage of cartilage and promote the incidence and development of OA.Further research on OA and metabolic diseases, has the potential to provide new ideas and methods for the prevention and treatment of OA.

Objective To study the effects of glucosamine sulfate on nitric oxide(NO)production induced by interleukin(IL)-1β in human osteoarthritis chondrocytes(HOC),and explore the possible mechanism.Methods Chondrocytes were harvested from 10 osteoarthritis patients undergoing total knee replacement(TKR)operation.Human recombinant IL-1β(5 μg/L)and glucosamine sulfate GS in different concentrations(0.2 mmol/L,2.0 mmol/L,20.0 mmol/L)were administrated into cell culture medium for 24 h.The content of NO was detected by enzyme-linked immunosorbent assay(ELISA).The mRNA and protein expression of inductive nitric oxide synthetase(iNOS)were measured by RT-PCR and Western blot,respectively.Results Stimulation of HOC with IL-1β enhanced production of NO and expressions of iNOS mRNA and protein(t=-14.81,-45.38,all P＜0.01).Pretreatment with 2.0 and 20.0 mmol/L GS showed a dose-dependent inhibition of IL-1β induced NO production(F=12.43,P＜0.05)and the expression levels ofiNOSmRNA(F=142.28,P＜0.05)and protein(F=78.08,P＜0.01).20.0 mmol/L GS alone did not influence NO production(t =-0.17,P＞ 0.05).Conclusions GS may inhibit the synthesis of NO induced by IL-1β in HOC through down-regulate mRNA and protein expressions of iNOS.

Objective To evaluate the result of open rotator cuff repair and to identify potential preoperative or intraoperative factors that could affect the outcome.Methods 43 cases(44 shoulders)with rotator cuff tear who undergone open repair surgery between May 1987 and October 2002 were retrospectively studied.There were 31 shoulders ruptured in supraspinatus, 9 shoulders in supraspinatus and infraspinatus,3 shoulders in supraspinatus and subscapulafis and 1 in supraspinatus, infraspinatus and subscapularis.And there were 13 shoulders torn with small size, 18 shoulders with middle size, 10 shoulders with large size and 3 shoulders with massive size.Ruptured rotator cuff was repaired with tendon to bone suture in 25 shoulders,with tendon to tendon suture in 7 shoulders, and combined suture in 11 shoulders.One shoulder was repaired by deltoid transfer.After an average 88.2-month-long follow-up(from 52 to 250 months),we evaluate the postoperative shoulder function with University of California at Los Angeles(UCLA)score.The patient's subject satisfaction was assayed by visual analogue scale (VAS).The risk factors as age, sex,dominant side, trauma history,preoperative duration and tear size were analyzed.Results The average UCLA score was 11.5±2.8 preoperatively and 29.7±5.3 at follow-up, with 36 shoulders reaching excellent or good and 2 shoulders ranking poor.According to VAS,the subject satisfaction rate was as high as 89%,being positively related to UCLA score significantly(r=0.72,P＜0.01).Preoperative duration(r=-0.332,P=0.028)and tear size(r=-0.404,P=-0.007)was negatively relative with surgical results.Age,sex,dominant side,trauma history had no effect on prognosis.Conclusion Open rotator cuff repair could acquire satisfying outcome.Preoperative duration and tear size was negatively relative with surgical results.

Objective To explore the effects of sertraline combined with attribution retraining in post-stroke depression and recovery of neurological function.Methods A total of seventy-eight patients with post-stroke depression were randomly divided into research group and control group.The research group was treated by sertraline combined with attribution retraining and the control group was treated by sertraline only for 8 weeks.Depressive symptoms were assessed using the Hamilton Depression Rating Scale (HAMD) and recovery of neurological functions using the National Institute of Health Stroke Scale (NIHSS) at baseline and at the end of the 24,44,64 and 84 week.Results Repeated measure ANOVA for the total scores of HAMD showed that the main effect of time,the main effect of group and the interactive effect of time and group were significant (P＜0.05).Total scores of HAMD in the research group were significantly lower than those in the control group at the end of the 2nd,44,64 and 84 week [(18.25±4.27) vs.(20.81±4.63),(15.94±3.47)vs.(18.12±4.51),(12.85±3.12) vs.(16.54±3.70),(10.42±3.66) vs.(13.09±3.59),P＜0.05].HAMD Total scores in two groups were showed significantly decreased on each time point after treatment (P＜0.01).Effective rates (using reduction rate of HAMD total scores as evaluation) were more significant in the research group than those in the control group at the end of the 8th week (77.5％ vs.52.6％,P＜0.05).The repeated measure ANOVA showed that the main effect of time in NIHSS total scores was significant (P＜0.01).The main effect of group and the interactive effect of time and group in NIHSS total scores were not significant (P＞0.05).Total scores of NIHSS in two groups significantly decreased on each time point after treatment (P＜0.01).Conclusions Sertraline combined with attribution retraining can significantly relieve post-stroke depression and attribution training does not have obvious effects on the recovery of neurological function.

[Objective]To investigate and compare the effects of standard gamma nail (SGN) and proximal femoral nail anti-rotation (PFNA) in the treatment of osteoporotic intertrochanteric fractures in the elderly.[Method]From May 2002 to September 2005,55 patients with intertrochanteric fractures were treated with SGN.Sixty-eight patients were treated with PFNA from November 2005 to June 2008.The effects of the two implants about perioperative complications,bone healing and the function of the operated hip joint were compared.[Result]No statistically significant difference was found between the two groups in blood loss,blood transfusion,infection,deep venous thrombosis and hospital stay.One patient developed femoral head avascular necrosis postoperatively in Gamma nail group.The incidence of postoperative hip pain in Gamma nail group was higher than that in the PFNA group,but there was no significant difference in the Harris score.[Conclusion]Gamma nail and PFNA nail are reliable methods to treat geriatric intertrochanteric fractures,with advantages of minimal iatrogenic injury,stable fixation and few complications.

Objective To evaluate the efficacy and the effects of sevoflurane induced hypotension on balance of cerebral oxygen supply and consumption and on cerebral energy metabolism. Methods Forty patients undergoing elective procedures were randomly divided into three groups. Group Ⅰ :sevoflurane induced hypotension ; group Ⅱ: sevoflurane maintained normal arterial tension ; group Ⅲ: nitroprusside induced hypotension . In groupⅠ and Ⅲ MAP was decreased to 50%-60% of baseline ,was kept for 40 min either by sevoflurane inhalation or by nitroprusside infusion. MAP ,HR,ECG were monitored continuously, and radial arterial blood samples and jugular blood samples were taken synchronously for measuring blood gas and blood lactate ,SOD and MAD levels. Results In group Ⅰ during induced hypotension Da jvO 2 showed reduction ,but in group Ⅲ Da jvO 2 increased significantly. There were no significant changes in blood lactate level and in the cerebral arteriovenous differences of MAD and SOD in three groups.Conclusions The sevoflurane induced hypotension has no adverse effect on cerebral oxygen balance and the cerebral perfusion . The cerebral energy metabolism is well maintained . Sevoflurane can safely applied to controlled hypotension during cerebral neurosystem.

A rapid, reliable and sensitive pressurized capillary electrochromatography-Laser induced fluorescence ( pCEC/LIF ) method with trifluoroacetic acid ( TFA ) pre-column derivation for simultaneous determination of four aflatoxin ( AFB1 , AFB2 , AFG1 , AFG2 ) was developed. This method included separation on a capillary column packed with 1. 8μm C18 particles using 0. 05% FA aqueous solution/methanol (55:45, V/V) as mobile phase at a pump flow rate of 0. 05 mL/min when the split ratio was 1:300. Under the optimum conditions including running voltage of 15 kV, excitation wavelength of 375 nm and emission wavelength of 450 nm, the baseline separation of four aflatoxins was achieved within 10 minutes. The limits of detection (LODs) were 0. 02, 0. 016, 0. 008 and 0. 01 μg/L for AFG1, AFB1, AFG2, AFB2(S/N=3), respectively. The linear detection ranges of AFG1 , AFB1 , AFG2 , AFB2 were 0. 1-10, 0. 1-10, 0. 1-3 and 0. 1-3 μg/L with correlation coefficients (R2) of 0. 9999, 1. 0000, 0. 9995 and 0. 9997, respectively. The established method was applied to analyze the peanut butter, and the recoveries of standard addition experiment were between 90 . 0% and 112 . 0% for all analytes ( RSDs=0 . 5%-1 . 9%) .

Objective To clone survivin gene, prepare its monoclonal antibody(McAb) and check its expression in liver carcinoma cells.Methods Survivin gene cDNA was amplified by RT-PCR from human breast carcinoma cell line MCF-7 and constructed into prokaryocytic expression vector.Fusion-protein of human Survivin was expressed and used for immunizing BALB/C mice.The spleen cells from immunized mice were fused with SP2/0 cells and selectively cultured with HAT medium.ELISA and Western blot were used to screen and identify the McAbs.Immunocytochemical staining was applied for survivin expression in liver carcinoma cells.Results The full survivin gene was cloned. The hybridoma cell that secret specific monoclonal antibody against human surviving was identified.The immunoglobulin subclasses of the McAbs were IgG1.Western blot showed that the McAbs against survivin can specifically react with MS-Survivin fusion protein. The positive reaction was found in hepatocarcinoma cell line HepG2 and hepatocarcinoma tissue by immunocytochemical staining.Conclusion The McAbs against the human Survivin were successfully prepared by a MS2-Survivin fusion protein expressed by E.coli. and the McAb had positive reaction with HepG2 and hepatocarcinoma tissue. It may be a useful tool to study the functions of survivin and check clinical cancer samples.