Aim To investigate the method of culturing vascular smooth muscle cells (VSMC) of coronary arteries derived from experimental diabetic rats and to establish the model of VSMC for the study of chronic cardiovascular diseases of diabetes. Method After the establishment of the model of diabetic rats, the coronary arteries of rats were separated carefully to culture VSMC using the method of explanting culture. Results The primary culture of VSMC was performed successfully by the method mentioned above. The cells could be isolated by different kinds of digestive enzyme in three steps. 7 ~10 days afterward, the cells overlapped in layers and displayed the typical ‘hill and valley’ pattern and positive immunochemical staining for smooth muscle actin. Conclusion VSMC from experimental diabetes proliferates faster than normal VSMC and its cultivating condition is strictly limited. The morphology of diabetic VSMC is as same as that of normal rats.

Objective To investigate the effect of heparin on the apoptosis and proliferation of human myeloid leukemia cell line K562 induced by vincristine.Methods K562 cells were pretreated by heparin for 1h,then cultured with 0.05 mg/L vincristine in 37℃ 5% CO2 for 24 h.Apoptosis of KS62 cells was evaluated by Hoechst 33342 staining,flow cytometer and DNA agarose gel electrophoresis after culture for 24 hours.The effect of heparin on KS62 cell proliferation and toxicitv was determined by Trypan blue staining and MTT assay.Results In the apeptosis induced group,the apeptosis rate was 40.10% dected by Hoechst 33342 fluorescence staining.The hepafin in different concentrations was found to be able to inhibit the apoptosis of K562 cells triggered by vincristine and the apoptosis rate was 32.47%,29.7%,25.5%,19.53% in the heparin groups of 25,50,100,200 U/ml,respectively.The apeptosis rate was significantly lower in the apeptosis induced group than in the heparin groups of 25,50,100,200 U/ml(P＜0.05).The typical DNA ladder could be found in the apoptosis-induced group,and the DNA ladder gradually disappeared along with the increase of heparin(5～200 U/ml).The sub-G1 peak of K562 cells could be found in the induced group by FACS and the apoptosis rate was 21.61%.In the heparin groups of 25,50,100,200 U/ml,the sub-G1 Peak of K562 cells gradually dropped and the apoptosis rate was 13.64%,11.75%,8.59%,6.03%(P＜0.05),respectively.After K562 cells were incubated with different hepafin concentrations(5～200 U/ml)for 24 hours,there was no difference compared with the normal control group in both the total live cell numbers and the cell proliferation rate measured by trypan blue staining and MTT assay(P＞0.05).Conclusion The results suggested that heparin had no influence on KS62 cell toxicity and proliferation,but may inhibit the apoptosis of KS62 cells induced by vincristine.

OBJECTIVE: To evaluate the relationship between katG gene mutation and isoniazid (INH) resistance and to develop a rapid screening method of point mutation in the katG gene associated with MTB resistance. METHODS: Twenty-four clinical isolates of MTB with 8 INH resigtance isolates and 16 INH-sensitive isolates were analyzed by PCR-RFLP, with the H(37)Rv reference strain as the control. RESULTS: G-->C point mutations were detected in 7 of 8 isoniazid-resistant strains and no gene mutation was shown in 16 isoniazid-sensitive isolates. The sensitivity and specificity were 87.5 % and 100 % respectively. No katG gene sequence deletion was observed in any specimen. CONCLUSION: Our results suggest katG gene mutation is one of the most important mechanisms of INH-resistant TB. PCR-RFLP may be useful in detection of katG gene mutation.

Objective To establish the HeLa cell line that can stably express EYFP fluorescent protein as the model for anion channel blocker (halide ion) screening ,which lays the foundation for high throughput screening of anion channel blocker (halide ion) .Methods Through gene recombination technology ,a new lentivirus vector which can express mutant protein YFP (EYFP‐H148Q/I152L) and puromycin resistance ,was built .The mixture of lentivirus vector and packaging plasmid was transfected into 293T cells to produce lentivirus particles . After infection of HeLa cells by the lentivirus particles ,puromycin was used to screen the cells as YFP‐positive HeLa cell line .Then cell amplification was carried out after purification and efficiency of EYFP‐H148Q/I152L was further detected by Real‐time quantitative PCR (RT‐PCR) and Western blot .We then verified the activity of EYFP‐HeLa transfected cell line as a screening model of anion channel blocker .Results Gene sequencing verified that EYFP‐H148Q/I152L was successfully inserted into lentivirus vectors .RT‐PCR and Western blot results showed that the target gene was overexpressed in HeLa cells . The specific yellow fluorescence of EYFP of HeLa cells could be observed under fluorescence microscope with the efficiency of nearly 100% . I- (low permeability ) solution stimulated the opening of anion (halogen) channels ,and the yellow fluorescence was quenched by I - flow into cells . Conclusion The EYFP‐HeLa cell line can stably express EYFP yellow fluorescent protein and is sensitive to the internal flow of I - .Therefore ,it can be used as an ideal screening model of anion channel blocker (halide ion) .

Objective To study the bacterial types and their drug resistance in intra-abdominal infections after pancreatic surgery,and to evaluate the appropriate treatment measures.Methods 113 patients who underwent pancreatic surgery from Jan 2012 to Dec 2012 in our hospital were included into this study.The drainage liquid from the surgical sites were collected for bacterial culture and drug susceptibility tests.Results The incidence of intra-abdominal infections was 39.8％ (45/113).There were 54 pathogenic strains of bacteria isolated,including 49 strains of gram-negative bacteria (90.7％),4 strains of gram-positive bacteria (7.4％),and 1 strain of fungus (1.9％).The top three pathogens were Pseudomonas aeruginosa (50.0％),Acinetobacter baumannii (14.8％) and Singular deformation bacteria (1 1.1％).Most gram-negative bacteria were sensitive to Polymyxin B and Aminoglycoside antibiotics (＞ 70％),but they were resistant to Imipenem and Cephalosporin which were commonly administered.Pancreatic fistula was closely related to intra-abdominal infections.Concluusions A gram-negative bacteria,Pseudomonas aeruginosa,was the predominant organism in intra-abdominal infections after pancreatic surgery in our hospital.The situation of drug-resistance was still severe.More effective measures should be taken to prevent growth of resistant strains such as using antibiotics according to drug sensitivity and avoiding empirical single use of broad-spectrum antibiotics.Pancreatic fistula commonly led to intra-abdominal infections.

Objective To study the impact of lymph node metastasis on prognosis of patients with pancreatic cancer and to evaluate predictors of postoperative survival of these patients.Methods The clinical data on patients with pancreatic cancer who underwent pancreatic cancer radical surgery in our hospital from January 2002 to December 2013 were reviewed and analyzed.Data on lymph node metastasis,number of lymph node dissection,number of positive lymph nodes and positive lymph ratio were analyzed.Results Of 101 patients,the 6-month,1-year and 2-year survival rates were 84.2％,56.6％ and 28.5％,respectively.The median survival was 13.8 months.Univariate and multivariate analyses showed lymph node metastasis,a positive lymph node ratio,number of lymph node dissection and positive lymph nodes were independent influential factors of prognosis.Results of subgroup analysis showed the number of lymph node dissection was a prognostic factor for pNO patients,while a positive lymph ratio had no impact on survival of pN1 patients.In the subgroup of patients with pancreatic head cancer,lymph node metastasis was associated with prognosis but not in the subgroup of patients with pancreatic body and tail carcinoma.Conclusions For patients with pancreatic head cancer,lymph node metastasis was closely correlated with prognosis.In addition,factors including lymph node metastasis,number of lymph node dissection,a positive lymph node ratio and number of positive lymph nodes were independent influential factors of prognosis for patients with pancreatic head cancer.However,for pN1 patients,a positive lymph node ratio has no influence on prognosis.

Objective To analyze the risk factors of pancreatic fistula after pancreaticoduodenectomy,in order to provide evidence to reduce post-operative complication in clinical practice.Methods The clinical data of 352 patients with malignancy who received pancreaticoduodenectomy at the Shanghai Renji Hospital from September 2009 to September 2012 were retrospectively analyzed.The patients were divided into pancreatic fistula group and non-pancreatic fistula group.Peri-operative risk factors of pancreatic fistula after pancreaticoduodenectomy were analyzed by univariate and multivariate logistic regression analysis.Results Forty-nine cases of pancreatic fistula occurred,and the incidence rate of pancreatic fistula was 13.9％ (49/352).Univariate and multivariate logistic regression analysis showed sex,age,history of diabetes,operation time,intra-operative blood loss,vessel reconstruction,pancreatic tube placement,anastomosis time,type of digestive tract reconstruction were not risk factors of pancreatic fistula; however,brittle pancreatic tissue,diameter of pancreatic duct ＜3 mm,pre-operative total bilirubin level ＞ 171 μmol/l,duration of preoperative jaundice ＞ 8 weeks,pre-operative albumin level ＜30 g/L were the independent risk factors of pancreatic fistula (P ＜ 0.05).Conclusions Brittle pancreatic tissue,small pancreatic duct,high level of serum bilirubin,long duration of preoperative jaundice,low level of serum albumin are the independent risk factors of pancreatic fistula after pancreaticoduodenectomy.

Objective Discussion leukoreduction of red blood cells suspended in two different storage bag placement and he-molysis rate impact on the supernatant free hemoglobin (FHb),to ensure the clinical transfusion is safe and effective.Meth-ods Selected 20 donors to sample 400 ml whole blood per person to make leukodepleted red blood cells,which were evenly divided into 10 bags.The 10 bags were randomly divided into two groups,one to the upright position,one group of horizon-tal.The two groups were stored under the same conditions.Respectively,in the 7,14,21,28,35 day,randomly removed one storage bag from each group,FHb and red blood cell hemolysis rate were measured and analyzed statistically.Results FHb and hemolysis rate results stored in the first 21 days of testing,uprightgroup were (217.310±48.477)mg/L and (0.250± 0.056)%,respectively horizontal group (173.972±39.027)mg/L and (0.189±0.045)%,the results set upright than hori-zontal group,the results were statistically(t=3.114,P =0.003<0.05 and t=3.798,P =0.001<0.05),the difference was statistically significant.Conclusion In the blood storage period,storage bags can be placed horizontally to reduce the de-struction of red blood cells,blood storage is more favorable.

OBJECTIVETo determine the inhibitory effect of 5-aza-2'-deoxycytidine (5-Aza-CdR) on the growth of human colon carcinoma cells and xenografts in nude mice, to observe its effect on CDH13 gene expression and methylation in the xenografts, and to explore the possible mechanisms.

METHODSHuman colon carcinoma cell line HCT116 cells were treated with 5-Aza-CdR, and the cell morphology was observe by phase contrast microscopy. The cell growth was assessed by MTT assay. A tumor-bearing mouse model was generated by subcutaneous inoculation of human colon carcinoma HCT116 cells into nude mice. The tumor growth in the nude mice was observed, the CDH13 gene expression and its methylation status in the tumors were detected using methylation specific PCR (MSP), RT-PCR, Western blotting and immunohistochemistry.

RESULTSAfter treatment with 5-Aza-CdR, the inhibition rate of the growth of cultured HCT116 cells was increased as the concentration was increasing. The growth of the xenografts in nude mice was significantly inhibited, and the methylated CDH13 gene was reactivated. After 4 weeks of 5-Aza-CdR treatment, no significant difference was found between the body weights of nude mice in the 5-Aza-CdR group [(18.06 ± 1.29) g] and control group [(17.07 ± 0.84) g], (P > 0.10), and the average volume of xenografts of the 5-Aza-CdR group was (907.00 ± 87.29) mm(3), significantly smaller than the (1370.93 ± 130.20) mm(3) in the control group (P < 0.005). No expression of CDH13 gene was found in the control group. The expression of CDH13 gene in the 5-Aza-CdR group was increased along with the increasing concentration of 5-Aza-CdR.

CONCLUSIONS5-Aza-CdR inhibits the growth of human colon cancer cells in culture and in nude mice, and induces the cancer cells to re-express CDH13 in nude mice. Its mechanism may be that demethylation of the methylated CDH13 promoter induced by 5-Aza-CdR restores CDH13 expression and thus inhibits the tumor growth in nude mice.

Animals ; Antimetabolites, Antineoplastic ; pharmacology ; Azacitidine ; analogs & derivatives ; pharmacology ; Cadherins ; genetics ; metabolism ; Cell Proliferation ; drug effects ; DNA Methylation ; HCT116 Cells ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; RNA, Messenger ; metabolism ; Tumor Burden ; drug effects

Objective:To analyze the mean levels of skeletal muscle mass and strength in elderly patients with type 2 diabetes mellitus(T2DM), and to investigate the effects of chronic inflammatory factors and oxidative stress on them.Methods:A cross-sectional study was conducted on 120 patients with T2DM aged over 60 years and 126 elderly patients without diabetes(the control group). Skeletal muscle mass, strength and serum levels of chronic inflammatory factors interleukin-6(IL-6), tumor necrosis factor-α(TNF-α)and 8-hydroxy-2′-deoxyguanosine(8-OHdG)were determined, and their effects on skeletal muscle mass and strength in elderly patients with T2DM were analyzed.Results:Compared with the control group, grip strength decreased in elderly patients with T2DM(25.03±7.85)kg vs.(29.52±7.73)kg( P<0.01), and skeletal muscle mass decreased(21.36±5.46)kg vs.(22.01±5.22)kg with no significant difference( P>0.05). Serum levels of 8-OHdG were higher in elderly patients with T2DM than in the control group(3.08±0.26)ng/L vs.(2.59±0.16)ng/L( P<0.01). Correlation and regression analysis results showed that 8-OHdG was an influencing factor for muscle strength in elderly patients with T2DM( R2=0.457)and that height and weight could be influencing factors for skeletal muscle mass in elderly patients with T2DM( R2=0.822). Conclusions:Skeletal muscle mass and strength decline in elderly T2DM patients, probably as a result of increased levels of oxidative stress.These findings may serve as evidence for sarcopenia intervention in elderly T2DM patients.