OBJECTIVETo explore the effect and mechanism of hirudin on atherosclerotic plaques in apolipoprotein E knockout (ApoE(-/-)) mice.

METHODSTotally 24 ApoE(-/-) mice, 7-8 weeks old were fed with high fat diets. They were randomly divided into the recombinant hirudin treatment group (drug group) and the model group according to body weight and different dens, 12 in each group. Twelve C57BL/6J mice, 7-8 weeks old fed with high fat diet were recruited as the normal control group. Recombinant hirudin (0.25 mg/kg) was intraperitoneally injected to mice in the drug group from the 10th week old once every other day for five successive weeks. Equal volume of normal saline was injected to mice in the model group. Mice in the normal control group received no treatment. All mice were sacrificed after fed with high fat diet until they were 20 weeks old. Serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), high-sensitive C-reactive protein (hs-CRP), E-selectin, interleukin-6 (IL-6), and stromal metalloproteinase-2 (MMP-2) were detected. The plaque/lumen area and extracellular lipid composition/ plaque area were analyzed by HE staining and morphometry. Changes of signaling molecules in store-operated calcium channels, including stromal interacting molecule 1 (STIM1), Orail protein, and transient receptor potential channel 1 (TRPC1) were determined by Western blot. Results Lipid plaque formed in the aorta vessel wall of 20-week old mice in the model group. Compared with the normal control group, serum levels of TC, TG and LDL increased (P<0.01), hs-CRP, E-selction, IL-6, and MMP-2 obviously increased (P<0.01, P<0.05) in the model group; expression levels of STIM1, TRPC1, and Orail significantly increased (P<0.01). Compared with the model group, the plaque/lumen area and the extracellular lipid composition/plaque area significantly decreased in the drug group (P<0.05, P<0.01); serum levels of TC and LDL, hs-CRP, E-selction, IL-6, and MMP-2 obviously decreased (P<0.05, P<0.01); expression levels of STIM1, TRPC1, and Orail were significantly down-regulated (P<0.05, P<0.01).

CONCLUSIONHirudin could significantly improve lipids and endothelial functions of ApoE(-/-) mice, down-regulate expression levels of STIM1, Orai1, and TRPC1, and thus delaying the occurrence and development of atherosclerosis.

Animals ; Aorta ; Apolipoproteins E ; metabolism ; Atherosclerosis ; C-Reactive Protein ; Cholesterol ; Diet, High-Fat ; Drugs, Chinese Herbal ; E-Selectin ; Hirudins ; metabolism ; Interleukin-6 ; Lipids ; Lipoproteins, HDL ; Lipoproteins, LDL ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Plaque, Atherosclerotic ; metabolism ; Recombinant Proteins ; metabolism ; Triglycerides

Animals ; Carbon Tetrachloride ; toxicity ; Dinoprostone ; blood ; Ginsenosides ; pharmacology ; therapeutic use ; Liver ; ultrastructure ; Liver Cirrhosis, Experimental ; drug therapy ; enzymology ; immunology ; Male ; Panax ; Phospholipases A ; biosynthesis ; genetics ; Phospholipases A2 ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Tumor Necrosis Factor-alpha ; biosynthesis

PBL(Problem-Based Learning, PBL) is a problem-oriented and effective supplementary teaching method. PBL is giving a great help to improve self-learning, communication and cooperation, thinking and problems solving abilities for the students. In the process of PBL teaching, attention should be paid to two important items. One item is the role transfer for the teacher. Teacher is only a guider in PBL teaching, teacher should avoid excessive interfere of the process for keeping the passion and enthusiasm of the students. Meanwhile, students should always be realized that they are the main part in PBL teaching, they should not depend on their teacher too much. Another important item is how to find and solve the frequently encountered problems, in order to avoid students wandering from the subject, and lead them toward the main goal to get effective teaching and learning.

Objective To study the changes of plasma cystatin C level (PcyC),and evaluate the effects of the joint use of atorvastatin and probucol on PcyC and severity of coronary lesion in patients with borderline lesion of coronary artery.Methods One hundred and thirty consecutive patients with borderline coronary lesion assessed by quantitative coronary angiography were enrolled into borderline coronary lesion group (BCL),and another 136 subjects without coronary lesion were enrolled as controls (CTR).And in the meantime,the subjects in BCL group were randomized (closed envelope method) into routine treatment subgroup ( RTT,n =60),and combined treatment subgroup in which patients were treated with atorvastatin 20 mg plus probucol 1.0 g daily in addition to routine medication ( CBT,n =70) for 6 months.There were no statistical differences in basic clinical features between two subgroups.PcyC,high-sensitive C-reactive protein (hs-CRP),total cholesterol (TC),low-density lipoprotein cholesterol (LDL-C),high-density lipoprotein cholesterol ( HDL-C ) and triglycerides (TG) were determined.Of them,104 patients in BCL group rechecked by coronary angiography.Comparison of biomarkers carried out between two groups by using a number of independent-sample t-test and analysis of variance.For enumeration data,chi-square test was used to compare mean values of biomarkers between groups. P ＜ 0.05 was considered statistically significant.Results PcyC levels were significantly higher in BCL group than those in CTR group ( P ＜ 0.05 ).Compared with RTT subgroup,levels of PcyC,TC,LDL-C,TG and hs-CRP were more significantly decreased in CBT subgroup (P ＜ 0.05,P ＜ 0.01 ).Moreover,there was a trend of slight decrease in the mean percent of stenosis (MPS) of coronary artery with borderline lesion in RTT subgroup treated for 6 months,whereas more marked decrease in the MPS of coronary artery with borderline coronary lesion in CBT subgroup treated for 6 months ( P ＞ 0.05 ; P ＜ 0.05 ).Conclusions Cystatin C plays an important role in the pathogenesis of coronary artery,and PcyC is associated with severity of coronary lesion,the combination of atorvastatin and probucol decreases the PcyC level,and it may be the treatment of choice for borderline lesion of coronary artery.

P300/CBP-associated factor(PCAF),an important member of histone acetyltransferase family(HATs) within eukaryotic cells,is capable of inducing the acetylation of histone,promoting the transcription of specific genes and involving in many biological effects.In the present study,full-length cDNA of PCAF was inserted into plasmid pGEX-5x-1,then the soluble protein GST-PCAF was expressed in E.coli BL21(DE3) after the optimization of inducing conditions.The recombinant protein was further purified with affinity chromatography and tested the activity by in vitro acetylation assays.High efficient PCAF protein produced by this method could serve for the study on the role of PCAF in gene regulation and the interaction between PCAF and other proteins.

Objective To evaluate the relationship between plasma cystatin C concentration (PcyC) and coronary artery diseases (CAD). Method A total of 126 subjects with CAD evidenced by coronary angiography admitted from April 2007 to March 2009 were divided into three groups: stable angina pectoris (SAPs, n = 34),unstable angina pectoris (UAPs, n = 56) and acute myocardial infarction (AMIs, n = 36), according to the diag-nostic criteria of CAD set by WHO. Another 34 subjects without CAD were taken as controls. There were no statis-tical differences in demographics among four groups. Serum lipids profile, uric acid (UA), PcyC and high-sensi-tive C-reactive protein (hs-CRP) were determined. And in the meantime, all patients were followed up for six months and adverse cardiovascular events were recorded. Comparisons were made between groups with a number of independent-sample t -tests. Data were processed with analysis of variance to test the differences in means among four groups, and the means were compared with chi-square test. Statistical significance was established at a P val-ue of less than 0.05. Results Cystatin C levels were significantly higher in UAPs than that in SAPs and in controls (P < 0.05), but were much lower than that in AMIs (P < 0.05). And much higher concentration of hs-CRP was found in UAPs (P < 0.05) and in AMIs (P < 0.01). Cystatin C was positively and significantly corre-lated with age, hs-CRP, WBC, creatinine and UA (r > 0, P < 0.05), whereas a significantly negative correla-tion with high-density lipoprotein cholesterol was found (r = - 0.227, P < 0.05). These coefficients were obvi-ously high for creatinine (r = + 0. 612), and WBC (r = + 0.459). During the period of six-month follow-up, 26 patients with adverse cardiovascular events were found, and had significantly higher cystatin C levels than 22 con-trols at admission (P < 0.01). Conclusions Cystatin C plays a pivotal role in the course of CAD, and the PcyC is a strong predictor for the risk of cardiovascular events.

OBJECTIVETo investigate the effects of matrine on the anti-tumor efficiency of H22 murine hepatocarcinoma cell-based vaccine modified by TIM2 gene in vivo.

METHODThe combinant eukaryotic expression vector pIRES2-EGFP-TIM2 was constructed and transfected into H22 cells by lipofectamin. The monoclone of the positive H22-TIM2 cells and negative control H22-EGFP cells were selected by G418 pressure and limited dilution method in turn. The H22 whole-cell-based vaccine were inoculated to establish the tumor-bearing mouse model, and its oncogenicity and immunogenicity were observed in vivo. Then the matrine was administered to the tumor-bearing mice inoculated by H22-TIM2 cells, H22-EGFP cells and H22 cells, and the inhibitory effect of matrine on tumor was studied.

RESULTThe co-expression of EGFP protein and TIM2 mRNA were detected in H22-TIM2 cells. The rate of tumor formation in mice injected of H22-TIM2 cells was 41%, lower than that of H22 cells and H22-EGFP cells injection (92%) in mice. The growth of tumor were significantly inhibited vaccinated with H22-TIM2 cells in mice. The inhibitory rate of tumor (IR) was 69.2% in mice of H22-TIM2 group, higher than that of mice treated with matrine and H22 cells injection, the later was 67.5%. Matrine could dramatically strengthen the anti-tumor efficiency of H22 cells modified by TIM2 gene, with the highest tumor inhibitory rate (IR) (90.6%) in all the experimental mice. The spleen index, populations of CD4-positive lymphocytes and the ratio of CD4-positive to CD8-positive lymphocytes of spleen in mice vaccinated of H22-TIM2 cells were obviously higher than those in the other groups.

CONCLUSIONThe oncogenicity of H22 cells is markedly impaired after modified by TIM2 gene. Matrine can strengthen the inhibitory effect of H22-TIM2 cells on tumor in mice. These data give us important clues to further study the biological role of TIM2 gene in tumor immunity and explore the molecular mechanism of matrine in suppressing tumor.

Alkaloids ; administration & dosage ; pharmacology ; Animals ; Antineoplastic Agents, Phytogenic ; administration & dosage ; pharmacology ; Carcinoma, Hepatocellular ; drug therapy ; genetics ; immunology ; metabolism ; Cell Line, Tumor ; Female ; Gene Expression ; drug effects ; Humans ; Male ; Membrane Proteins ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Neoplasms, Experimental ; Quinolizines ; administration & dosage ; pharmacology ; Spleen ; drug effects ; immunology

BACKGROUNDThe plasma cystatin C concentration (PcyC) has been demonstrated to have prognostic value in acute coronary syndrome, but the study of PcyC in patients with borderline coronary lesions is limited. Moreover, the effects of atorvastatin and probucol on PcyC and the severity of coronary lesions are unknown. This study was to evaluate the effects of the combination of atorvastatin and probucol on PcyC and severity of coronary lesion in patients with borderline coronary lesions.

METHODSOne hundred and thirty consecutive patients with borderline coronary lesions (40% to 60% isolated single stenosis assessed by quantitative coronary angiography) were enrolled into the borderline coronary lesion (BCL) group, and one hundred and thirty-six subjects without coronary lesions comprised the controls (CTR). The subjects in the BCL group were randomized into routine treatment (RTT, n = 60), and combined treatment with atorvastatin 20 mg plus probucol 1.0 g daily added to routine medication (CBT, n = 70), both groups were treated for 6 months continuously. The levels of PcyC, high-sensitive C-reactive protein (hs-CRP), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG) were determined. One hundred and four subjects in the BCL group were rechecked by coronary angiography.

RESULTSPcyC levels were significantly higher in the BCL group than in the CTR group; (2003.26 ± 825.73) ng/ml vs. (1897.83 ± 664.46) ng/ml (P < 0.01). Compared with patients in the RTT group, the levels of PcyC, TC, LDL-C, TG and hs-CRP were significantly lower in the CBT group (P < 0.05). Moreover, there was a trend towards a slight decrease in the RTT patients, (54.38 ± 10.67)% vs. (50.29 ± 9.89)% (P > 0.05), and a significant decrease in the CBT patients, (53.65 ± 9.48%) vs. (40.38 ± 12.93)% (P < 0.05), in the mean percent stenosis of borderline coronary lesions before and after six months of treatment.

CONCLUSIONSCystatin C played an important role in the development of coronary artery disease, and was associated with the severity of coronary lesions. The combination of atorvastatin and probucol decreased PcyC levels, and could be the treatment of choice.

Aged ; Anticholesteremic Agents ; therapeutic use ; Atorvastatin Calcium ; Coronary Disease ; blood ; drug therapy ; pathology ; Cystatin C ; blood ; Female ; Heptanoic Acids ; therapeutic use ; Humans ; Male ; Middle Aged ; Probucol ; therapeutic use ; Prospective Studies ; Pyrroles ; therapeutic use

OBJECTIVETo investigate the effects of matrine on the anti-tumor efficiency of TIM2 gene-modified murine hepatocarcinoma H22 cells.

METHODSA combined eukaryotic expression vector pIRES2-EGFP-TIM2 was constructed and transfected into H22 cells by lipofectamin. The monoclone of positive H22-TIM2 cells and negative control H22-EGFP cells transfected with pIRES2-EGFP vector were selected by G418 pressure and limited dilution method in turn and were inoculated to establish the tumor-bearing mouse model. Next, matrine was administered to the tumor-bearing mice and the inhibitory effect of matrine was determined.

RESULTSThe co-expression of EGFP protein and TIM2 gene was detected in H22 cells selected after TIM2 gene transfecion. After subcutaneous injection of H22-TIM2 cells, the rate of tumor formation (41%) was lower than that of H22 cells and H22-EGFP cells injection (92%) in mice. The tumor growth was significantly inhibited in mice vaccinated with H22-TIM2 cells. After the experiment was completed, the volume of tumors in mice of H22-TIM2 group was 31.34 +/- 9.21 mm3, smaller than those in H22-EGFP group (98.25 +/- 25.23)mm3 and H22 cells group (114.08 +/- 36.45)mm3 (P < 0.01). Matrine dramatically enhanced the anti-tumor efficiency of TIM2 gene-modified H22 cells, with the highest tumor inhibitory rate (IR) 90.6% among the H22-TIM2 group, matrine treatment group and H22-EGFP cells combined with matrine treatment group (69.2%, 67.5% and 70.8%, respectively) in the experimental mice.

CONCLUSIONThe tumorigenesity of H22 cells has been markedly impaired after modification by TIM2 gene. Matrine can enhance its inhibitory effect on tumors of H22-TIM2 cells in vivo. These data indicate importance to further study on the biological role of TIM2 gene in tumor immunity and explore the molecular mechanism of matrine in suppressing of tumor growth.

Alkaloids ; pharmacology ; Animals ; Antineoplastic Agents, Phytogenic ; pharmacology ; Cell Line, Tumor ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; metabolism ; Liver Neoplasms, Experimental ; metabolism ; pathology ; Membrane Proteins ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Quinolizines ; pharmacology ; Recombinant Proteins ; genetics ; metabolism ; Transfection ; Tumor Burden ; drug effects

BACKGROUND: The clinical experience of the treatment of the sternoclavicular joint dislocation and peripheral fracture is relatively lacking, but its incidence is increasing yearly. At present, there are few studies on the anatomy and biomechanics of the sternoclavicular joint in and outside China, and no systematic anatomical measurements of the sternoclavicular joint are reported. OBJECTIVE: To provide a biological reference for the clinical diagnosis and treatment of sternoclavicular joint dislocation or peripheral fractures by studying the anatomy and biomechanics of the sternoclavicular joint. METHODS: (1) A total of 16 specimens (32 sides) of adult antiseptic and moist cadaveric specimens were selected. The complete manubrium, bilateral clavicle and surrounding tissues of sternoclavicular joint were anatomically separated, and repair to bone-ligament-bone specimen models. (2) The areas of manubrium articular surface and the medial clavicular articular surface of all specimens were measured by the ink pattern combined with grid counting method. (3) The morphological features of the anterior and posterior sternoclavicular ligaments of the specimens in this group were observed, and the length, width and thickness were measured and analyzed statistically. (4) The left and right sternoclavicular joints of each specimen were randomly paired into A and B groups. Group A received simply cutting of anterior sternoclavicular ligament. Group B received simply cutting of posterior sternoclavicular ligament. Before and after cuting off the ligament, the anterior and posterior load experiments were performed on the anatomical sites with the same force arm length and perpendicular to the distal clavicle. The angles of joints and load-angle regression line slopes were compared between the two groups in the anterior and posterior directions load. RESULTS AND CONCLUSION: (1) The area of articular surface of manubrium (239.00±28.78 mm2) was smaller than the area of medial articular surface of the clavicle (482.56±44.89 mm2), and the difference was statistically significant (t=-40.105, P < 0.001). (2) The length, width and thickness of the anterior sternoclavicular ligament were (17.56±1.94 mm), (15.54±1.42 mm) and (1.93±0.32 mm), and the length, width and thickness of the posterior sternoclavicular ligament were (17.21±1.86 mm), (15.97±1.17 mm), and (2.07±0.29 mm) respectively;there was no significantly statistical difference in the length, width and thickness between them (P > 0.05). (3) Before cutting the ligaments, when the loads were 2, 4, 6, 8, and 10 N, the angle backwards of joint caused by loads in the forward direction was less than the angle of forwards of joints caused by loads in the backward direction, but only when the loads were 6, 8, and 10 N, the difference between them was statistically significant (P < 0.05). The slope of the regression line of load-angle for the loads in the forward direction was less than the slope of the regression line of load-angle for the loads in the backward direction, with statistical difference (F=31.413, P < 0.001). After the ligaments were cut, when the loads were 2, 4, 6, 8 and 10 N in the forward direction in group A and group B, the backward angulation of joint in group A was less than that in group B (P < 0.05). The slope of the load-angled regression line in group A was less than that in group B (F=52.224, P <0.001). When the loads in the backward direction in group A and group B were 2, 4, 6, 8 and 10 N, the forward angulation of joint in group A was greater than that in group B (P < 0.05), and the slope of the load-angled regression line in group A was greater than that in group B (F=12.503, P=0.008). (4) These results suggest that contact area between the articular surface of the medial clavicle and the articular surface of the manubrium is narrow, which determines the instability of the joint itself. The sternoclavicular ligament is extremely important for maintaining the joint stability. The forward angulation of joint restriction effect of sternoclavicular ligament was weaker than that of the backward angulation, also because of the joint in the anatomical position of the natural forward angulation, so the sternoclavicular joint was prone to anterior dislocation. It is necessary to pay attention to the repair and reconstruction of sternoclavicular ligament when sternoclavicular joint dislocation or peripheral fractures are treated by operations.