BACKGROUND: Leptin is a kind of polypeptide hormone secreted by fatty tissue, previous studies have shown that leptin plays a certain role in the formation of atherosclerosis.OBJECTIVE: To investigate the effects of leptin on the expression of tumor necrosis factor-alpha (TNF-α) in RAW264.7 cells, and investigate the possible mechanism from the angle of the change of nuclear factor-κB (NF-κB) activity.DESIGN: A controlled observational experiment.SETTING: Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: The experiments were carried out in the Department of Biochemistry and Molecular Biology, Tongji Medical College, and the Department of General Surgery, Union Hospital affiliated to Huazhong University of Science and Technology between April 2005 and February 2006.The cultured RAW264.7 cells were divided into leptin treated groups treated with different concentration (12.5, 25, 50, 100 μg/L), I kappa B kinase inhibitor group and blank control group. Each group had 3 sub-wells,and the experiments were repeated for 3 times.METHODS: Mouse macrophage RAW264.7 cells were incubated into a 6-well plate at the density of 109 cells L-1, cultured in RPMI-1640 culture medium containing bovine serum of 0.1 in volume fraction. When the cells grew to 80%, serum-free culture medium Opti-MEM was changed to culture for another 24 hours, and then the cells were divided into leptin groups treated with different concentration (12.5, 25, 50, 100 μg/L), I kappa B kinase inhibitor group and blank control group. After the cells were incubated with leptin for 4 hours, the expression of TNF-α mRNA expression in RAW264.7 cells was detected with reverse transcription-polymerase chain reaction (RT-PCR). After the RAW264.7 cells were incubated with leptin for 1, 3, 6 and 9 hours, the expression of TNF-c protein expression was detected with double antibody sandwich enzyme-linked immunosorbent assay (ELISA). After the RAW264.7 cells were incubated with leptin for different times, the activity of NF-κB was detected analyzed with electrophoretic mobility shift assay. Another, the RAW264.7 cells were treated with or without 50 μmol/L leptin and/or 100 μmol/L PS1145 (I kappa B kinase specific inhibitor)divided into four groups: blank control group, I kappa B kinase specific inhibitor PS1145 (10 μmol/L) treated group, leptin (50 μmol/L) treated group, leptin (50 μmol/L) + PS1145 (10 μmol/L) group. Aftere incubated for 6 hours, the activity of NF-κB and expression TNF-α mRNA were detected respectively.MAIN OUTCOME MEASURES: ① Effect of leptin of different concentration on the expression of TNF-α mRNA and protein in RAW264.7cells; ② Effect of leptin of different concentration on the activity of NF-κB in RAW264.7 cells; ③ Influence of inhibition I kappa B kinase activity inhibition on expression of TNF-a induced by leptin in RAW264.7cells.RESULTS: ① After the RAW264.7 cells were treated with leptin of different concentration, the TNF-α mRNA level was elevated in a dose-dependent manner, and it reached the peak value emerged in the 50 μg/L leptin treated group. ② The expression of TNF-α protein increased in dose-dependent and time-dependent manners, and it reached the peak val ue at 6 hours in the 50 μg/L leptin treated group. ③ The activity of NF-κB was also positively correlated with the leptin concentration, and it was the highest value at 6 hours treated with 50 μg/L leptin (P ＜ 0.05). ④ I kappa B kinase activity inhibition only partially suppressed the leptin induced elevation of TNF-α expression induced by leptin.CONCLUSION: Leptin can increase the expression and secretion of TNF-α in RAW264.7 cells directly in both dose-dependent and time-dependent manners, and the mechanism may be correlated with the activated NF-κB induced by leptin. It may be one of the mechanisms of atherosclerosis induced by leptin.

After the monoclonal antibody drug come out,it has played an important role in the clinical treatment,especially in tumor therapy.With the further understanding of monoclonal antibody,it has been applicated in the food, transgenic animal and plants,plant virology aspect. However,rare research of natural medicine have been reported in China.The monoclonal antibody-based enzyme-linked immunosorbent assay method, which is sensitivity,accuracy,fastness and simpler,can be used in quantitative analysis of bioactive compound in the natural products. It also can be used in isolation and purify effective component of the natural medicine, the pharmacokintic study,the quanlity control, screening candidacate drug and expanding the resources of medicinal plant It is important to exploit and utilizate the natural medicine,and promote the progess of modern chinese medicine.

Adipose tissue is a readily available source of adult stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. Peptide hydrogel is a novel biomaterial which provides three-dimensional microenvironments for a variety of cells for tissue grafting. In this study, adipose-derived stem cells (ADSCs) were isolated from rats, seeded into the peptide hydrogel polymer scaffolds and cultured in Neurobasal (NB) media supplemented with B27, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Ten days after the culture, some cells were expanded into clonal populations in which the expression of both Nestin and Brdu was detected but only Brdu expression was detected in the cells that were not expanded into clonal populations. Our results suggested that ADSCs in peptide hydrogel polymer scaffolds can be induced to differentiate into cells capable of expressing the neuron-associated markers, self-renewal and self-propagation.

Objective To study the expression of tumor type M2 pyruvate kinase(tumor M2-PK) in nonsmall cell lung cancer (NSCLC) and their correlation with treatment response and prognosis.Methods The concentration of tumor M2-PK in plasma was detected by ELISA in 106 healthy controls and 83 NSCLC patients.The patients were followed for 24 months.Results The patients after surgical operation showed marked reduction in plasma tumor M2-PK level(13.5 U/ml vs 25.4 U/ml,P

Objective To establish the methodology of flow cytometry for detecting human cells in human/goat chimerisra.Methods Human hemopoietic stem/progenitor cells (CD+34 cells) or MIG-tranadueed-GFP CD+34 cells were transplanted into the peritoneal cavity of fetal goats in utero to obtain human/goat chimera modeL The peripheral blood cells from the chimeras were labeled with multiple mouse anti-human antibodies and the monoelonal antibodies that were specific for human but had not or only minimal cross-reaction with goat were screened as the primary antibodies for routine analysis in flow cytometry.Human cord blood was proportionally (25% ,50% ,75%,100%) added into the blood of the untransplanted goats and the cells were labeled with CD+34 monoclonal antibody.The region and size of the "gate" were chosen based on to the distribution of CD+34 cells or human cord blood.One human/goat chimera marked with GFP (MIG goat) was sacrificed and the substantial liver cells from its perfused liver were analyzed for the GFP+cells percentage and DNA contents by flow cytometry.Results CD7,CD15,CD38,CD45CD20CD34CD14and GPA monoclonal antibodies were chosen as the primary antibodies in rou tine detection by flow cytometry.The size and area of the "gate" were also defined.29.1% (29100/100 000 ) of the substantial liver cells from the MIG goat expressed GFP.DNA content analysis showed that the GFP+ cells obtained from the liver of MIG goat mainly manifested two peaks that were correspond to those of human.Conclusions Flow cytometry is rapid,simple and effective for the investigation of differentiation,homing and biological characteristics of stem cells in vivo.The selections of suitable surface antibodies and the "gate" are very important for detecting human cells accurately in the human/goat chimerism.

Objective To observe the glucose utilization in adipose tissue and skeletal muscle during catch-up growth, and to explore the mechanism of catch-up growth of adipose tissue. Methods Male Wistar rats were randomly divide into normal control group(NC)and catch-up group(RN). Rats in RN group received 50%of food consumed by NC group for 4 weeks, then were re-fed spontaneously as the rats in NC group. In the end of the fifth week(NC1 group and RN1 group)and the sixth week(NC2 group and RN2 group), the experiment was performed. [3]-2-deoxy-glucose was used for detecting the glucose uptake rate. RT-PCR and Western-blot were used for detecting the levels of mRNA and membrane protein of glucose transporter-4(Glut4). Results The glucose uptake rates in adipose tissue and skeletal muscle of RN1 group increased by 189.6%(P<0.01)and reduced by 36.5%(P<0.05)respectively, as compared with NC1 group. After 2 weeks of catch-up growth, the glucose uptake rates in adipose tissue of RN2 group increased by 157.3%(P<0.01)and decreased by 41.5%(P<0.05) in skeletal muscle as compared with NC2 group. However. no significant difference in Glut4 mRNA levels in muscle or in adipose tissue between NC and RN groups were found. The membrane protein of GIut4 after insulin-stimulating in RN1 group and RN2 group reduced by about 46.5%(P<0.01)and 32.1%(P<0.05)in muscle and increased by 116.5%(P<0.01)and 89.9%(P<0.01)in adipose tissue respectively. Conclusion There exists the redistribution of glucose from skeletal muscle to adipose tissue during the early stage of catch-up growth, which results in the catch-up growth of adipose tissue. This change is induced by the tissue-specific alteration of insulin-stimulated Glut4 protein translocation.

Objective To study the correlation between the post-transplant renal allograft function and the variation of serum cystatin C (CyC) concentration in renal allograft recipients. Methods One hundred and ninety-three renal allograft recipients accepted the same combination immunosuppressive regimen of tacrolimus, mycophenolate and prednisone were enrolled into the study. Patient's serum and urine samples were collected on day 5 post-transplant to detect serum cystatin C, serum and urine creatinine (SCr). Correlation analysis was used to analyze correlation between CyC concentration and SCr concentration or the calculated creatinine clearance rate (CkCCr) by using the Cockcroft-Gault equation and urine creatinine clearance rate (CCr). Specificity and sensitivity of using the CyC concentration to evaluate glomerular filtration rate (GFR) were calculated as well.Results The mean concentrations of serum CyC and SCr on day 5 post-transplant were (1.91±1.2)mg/L and (174.0±129.1)μmol/L, respectively. While the CCr and CkCCr were (67.9±27.3)ml/min and (68.1±27.8)ml/min, respectively. Forty-two patients had a CyC concentration below 1.25 mg/L, 102 patients'CyC concentrations were between 1.25 and 2.0 mg/L and 49 patients'CyC concentrations were above 2. 0 mg/L. As for SCr, 62 patients had a concentration below 125 μmol/L, 83 patients'concentrations were between 125 and 200 μmol/L and 48 patients'concentrations were above 200 μmol/L. For CkCCr, there were 52 cases with a concentration above 80 ml/min, 96 cases with a concentration between 80 and 60 ml/min and 45 cases with a concentration below 60 ml/min. Serum CyC concentration had a negative correlation with CkCCr (r=-0. 907, P＜0. 001) and had a significantly positive correlation with SCr concentration (r=0. 886, P＜0. 001). SCr had a significantly negative relationship with CkCCr (r=-0. 889 ,P＜0. 001). Serum CyC had higher correlation with CkCCr than the correlation between SCr and CkCCr. The ROC curves showed that areas under curve of CyC, SCr, CCr and CkCCr were 0. 877, 0. 771, 0. 832 and 0. 909, respectively. Specificity and sensitivity of CyC, SCr,CCr and CkCCr were 69.3%, 96.1%, 77.1%, 71.3%and 91.6%, 52.2%, 67.5%, 84.6%, respectively.Conclusions Serum CyC concentration elevates earlier than SCr concentration when there is slight renal function impairment. Serum CyC concentration might become a more sensitive marker to evaluate the post-transplant renal allograft function in renal transplant recipients.

Objective To evaluate and analyze the clinical effect of comprehensive rehabilitation therapy after arthroscopic rotator cuff repair using suture-bridge technique for full-thickness rotator cuff tears.Methods Forty-one patients (20 males,21 females; mean age 52.2 years) with full-thickness rotator cuff tears were treated with arthroscopic rotator cuff repair using suture-bridge technique between June 2010 and January 2012 in our hospital.After arthroscopic rotator cuff repair,the patients were randomly assigned to a treatment group (21 patients) or a control group (20 patients).The treatment group received systematic rehabilitation therapy including rehabilitation education,physical modalities treatment and rehabilitative training additionally,while the control group only accepted the routine rehabilitation therapy including stretching and muscle strength training.The outcome was evaluated at 6 months after surgery,by employing visual analogae scale (VAS),the range of motion (ROM) testing of shoulder joint flexion and rotation,the rating scale of University of California at Los Angeles (UCLA),and the shoulder index of American shoulder and elbow surgeons (ASES).Results The mean follow-up period was 15.6 months (8-24 months).Prior to intervention,there was no significant difference in any parameter between the two groups (P ＞ 0.05).Six months later,all scores of assessments changed:in treatment group VAS (1.7 ± 1.5),ROM [flexion (168.3±31.3)°,rotation (47.2±11.2)°],UCLA(30.7 ±4.13) and ASES (85.1 ±15.67); in control group VAS(3.8±2.2),ROM[flexion (121.2 ±53.6)°,rotation (32.9 ±14.9)°],UCLA(18.3 ±4.94) and ASES (36.4 ± 17.70).Significant changes occurred in both groups in all the parameters after treatment when compare to baseline (P ＜ 0.05).Conclusions Comprehensive rehabilitation therapy is an effective approach for improving motor ability of the shoulder in patients after arthroscopic rotator cuff repair with suture-bridge technique for their full-thickness rotator cuff tears.

Objective: To explore correlation among clinic blood pressure (CBP), ambulatory blood pressure (ABP) and cardiovascular diseases in diabetic populations.Methods: A total of 336 patients complicated with type 2 diabetes mellitus, who received 24h ambulatory blood pressure monitoring, were selected.According to complicated with coronary heart disease or stroke or not, they were divided into cardiovascular disease group (CVD group, n=122) and no cardiovascular disease group (NCVD group, n=214).Blood lipids, blood pressure, CBP and ABP etc.were compared between two groups;according to median of 24h mean SBP (122mmHg), they were divided into <122mmHg group (n=168) and ≥122mmHg group (n=168), incidence of cardiovascular diseases was compared between these two groups.Results: (1) Compared with NCVD group, there were significant rise in age, percentages of smoking and hypertension, and plasma hsCRP level in CVD group (P<0.05 or <0.01);for ambulatory blood pressure,there were significant rise in levels of 24h mean SBP(mSBP) [(119.8±8.7)mmHg vs.(124.4±9.6) mmHg], daytime SBP (dSBP)[(121.4±9.3) mmHg vs.(128.0±10.3) mmHg] and nighttime SBP(nSBP) [(114.4±4.2) mmHg vs.(120.8±4.7) mmHg] in CVD group, P<0.01 all;there was no significant difference in CBP between two groups;(2) compared with <122mmHg group, there were significant rise in percentages of stroke (20.2% vs.25.0%) and total cardiovascular diseases (32.7% vs.39.9%) in ≥122mmHg group, P<0.01 both;(3) Logistic regression analysis indicated that diabetic patients no matter complicated with hypertension or not, 24h mean SBP was always an independent risk factors of diabetic patients complicated cardiovascular diseases (OR=1.83, 1.36, P<0.05 all).Conclusion: ABP is superior to CBP in predicting cardiovascular risk in patients with diabetes, and 24h mean SBP may be a good ABP index to predict cardiovascular risk.

[Objective] The present study was to evaluate the association of serum total cholesterol level and prognosis in patients with acute left heart failure and associated mechanisms.[Methods] Sixty-eight patients due to acute episode of left heart failure prospectively enrolled,and baseline data and biochemical parameters were collected.After discharge,patients were follow-up for 1 month and they were divided into two groups (with and without cardiovascular events).Differences between groups were evaluated and the association of serum total cholesterol level and cardiovascular events were analyzed by logistic regression analysis.[Results] The mean age was 57.3 ± 12.6 years old and 52 cases were male patients accounting for 76.5 ％.Among these patients,46 had a diagnosis of coronary heart disease (67.6 ％),10 rheumatic heart disease (14.7 ％),12 dilated cardiomyopathy (17.7％),38hypertension (55.9％) and 24 diabetes mellitus (35.3％).After 1 month's follow up,39 patients (57.4％) had experienced cardiovascular events,36 cases were re-hospitalized,and 3 died from heart failure.Compared to those with cardiovascular events,event free individuals were younger and were less likely smokers (P ＜ 0.05).In addition,event free group had lower serum levels of N-terminal pro-BNP and C-reactive protein (P ＜ 0.05) while serum levels of total cholesterol and albumin were significantly higher (P ＜ 0.05).There was no significant difference in medication between these two groups.After adjusted for age,gender,smoking,systolic blood pressure,serum albumin level,diabetes,hypertension and medications,increased total cholesterol level was independently associated with better prognosis with odds ratio of 0.91 (95 ％ confidence interval 0.80-0.96).Further adjusted for C-reactive protein,the association was attenuated to non-significance,with odds ratio of 0.97 (95 ％ confidence interval 0.87-1.09).[Conclusion] Adequate serum total cholesterol level was beneficial for improving short-term cardiovascular outcomes in patients with left heart failure and the potential mechanisms might be related to cholesterol effects on improving nutritional status and anti-inflammation.