Objective　To investigate exogenous ceramide-induced apoptosis in colon carcinoma LoVo cells.Methods　LoVo cells were pretreated for 3 h with or without the presence of phorbol 12-myristate 13-acetate (PMA), a potent protein kinase C (PKC) stimulator, later treated with C2-, C6-ceramide or C2-dihydroceramide, and then passed through gel electrophoresis, Heochest 33342 fluorescence staining and flow cytometry with Annexin V/PI staining. The treated LoVo cells were observed for biochemical and morphologic changes. Results　Treatment with C2- or C6-ceramide in the range of indicated concentrations (10～25 μmol/L) for 12 to 24 hours resulted in apoptosis in LoVo cells, whereas C2-dihydroceramide, which is similar to C2-ceramide in configuration but lacks the trans double bond at C4-C5 of the sphingoid base backbone, did not induce the apoptosis at the same or even higher concentrations, indicating that the ceramide-induced apoptosis was stereospecific. Moreover, the exogenous ceramide-induced apoptosis in LoVo cells was inhibited in part by PMA. Conclusion　Ceramide takes part in the process of apoptotic signal transduction in LoVo cells. PKC may be one of downstream target molecules acted by ceramide.

AIM: To develop a simple, convenience, and inexpensive method on primary culture model of pancreatic islets in rats for the study of anti-diabetic drugs. METHODS: The pancreases of SD rats were separated from the pancreatic duct with cold Hank' s solution and picked. Then the pancreases were cut into pieces and repeatedly digested by collagenase at 37℃ for the short durations of the experiment. The isolated islets were identified by dithizone staining and the viability was evaluated by trypan blue staining. Pancreatic islets were incubated in RPMI 1640 or DMEM for 14 - 16 h, then they were transferred to new culture plates with the same medium mentioned above. Determination of insulin content of su-pernate and cell lysate and the experiment of insulin secretion by stimulation of glucose and implantation of micewith STZ-induced diabetes were used for evaluated the function of islets. RESULTS: The viability of isolated pancreatic islets was more than 95% and the purity of cultured islets was about 85% . The insulin synthesis, secretion and sensitivity of islets stimulate by glucose which were cultured in RPMI 1640 were higher than that in DMEM. The levels of blood glucose recovered to normal in type 1 diabetic mice after islets implantation. CONCLUSION : The islets got in this study have higher purity and viability with the normal biological activity for about 7 days by this method and they can be used as a cell model for the study of diabetes in vitro .

Objective To promote in vitro tissue-plasminogen activator(t-PA)thrombolysis by using microbubbles augmented low frequency ultrasound(US) insonication.Two kinds of microbubbles,albumin-coated vs.lipid-coated were compared.Methods Human blood clot weighted 200～300 mg was made from of 0.8 ml fresh blood.Twenty kHz ultrasound,lipid and albumin-coated microbubbles were applied to separate clots groups with or without t-PA administration.The clots before and after processing were weighed and then the clot dissolution ratios were calculated.Results The simple ultrasound group and the basic t-PA group dissolved((24.72)?(4.83))% and((35.66)?(3.34))% of the clots,respectively.The clot dissolution ratio rised to((42.06)?(4.20))% when coordinated US and t-PA(P(0.05)).Conclusions The in vitro US augmentation of t-PA thrombolysis can be significantly promoted by introduction of microbubbles.But there is no significant thrombolysis difference between albumin and lipid-coated microbubbles.

Aim To explore the inhibitory effects and immunoregulatory activity of the extract from Eupolyphaga sinensis Walker (EFP) on the growth of liver cancer H22 in mice.Methods The chemical composition of extract from Eupolyphaga sinensis Walker(EFP)was separated with water extraction methods.The tumor bearing mouse model was constructed by injecting tumor cells subcutaneously.The EFP was given to the mouse by oral and hypodermic injection to observe its antitumor activity and immunoregulatory activity in vivo.The antibody level of anti-H22 in serum was measured with ELISA methods.The weight,tumor weight,spleen index,liver index were noted,and series of enzymes,such as,MDA,SOD,GPT and GOT activity of mouse serum and liver were measured.Results The extracts from Eupolyphaga sinensis Walker markedly inhibited the proliferation of tumor H22.The EFP constituent purified might be the main antitumor active part of Eupolyphaga sinensis Walker as shown by antitumor test in vitro.After treatment with EFP,the general condition of the test mice was much better than that of control mice,with spleen index,liver index increasing more obviously,antibody level of anti-H22,and MDA,SOD,GPT and GOT activity increasing more,and tumor growing more slowly.Conclusion EFP may be the main antitumor active part of Eupolyphaga sinensis Walker. EFP also has antitumor effect in vivo.EFP can exert antitumor effect in vivo by enhancing antibody level of anti-H22 and the series enzymes to be related to immunity activity.

Aim To investigate the differential expression of apoptosis-associated genes in human gastric cancer MGC803 cells induced by diallyl trisulfide(DATS).Methods Growth inhibition against MGC803 cells was assayed by MTT assay;The apoptosis induced by DATS was assessed by Flow cytometry and fluorescent microscope.The apoptosis-associated gene expression of MGC803 cell treated with DATS was determided by Human Apoptosis Gene Array.Apaf-1 and SODD genes were confirmed by RT-PCR.Results DATS had significant growth inhibitory activity against MGC803 cells,inhibition ratio increased from 11% to 78% at 4,8,12,16 and 24 mg?L-1 for 72 h(P

BACKGROUND: It is the most effective and economical method to reduce the incidence of stroke by conducting the health education concerning the etiological factors or risk factors and therapeutic knowledge of hypertension, diabetes mellitus and hyperlipidemia among the public and improving their knowledge level about the prevention and treatment of stroke.OBJECTIVE: To investigate the cognition of the knowledge concerning the prevention and treatment of stroke among the public in Dongguan area.DESIGN: A cluster sampling and simple randomized sampling survey.SETTINGS: Department of Neurology, the Fifth Affiliated Hospital of Sun Yat-sen University and the Department of Neurology, Affiliated Tungwah Hospital of Sun Yat-sen University.PARTICIPANTS: Between March and June in 2001, totally 2 500 cases were randomly selected by means of cluster sampling combined with simple randomized sampling from the residents and migrant populations no younger than 17 years old in the government organization, enterprises, factories of various forms, Dongguan University of Technology and senior middle schools of urban and rural Dongguan city and the administrative villages of the rural areas of Dongguan city in Guangdong province before March 2003.METHODS: The inventory was modified according to relevant literatures,including demographic data, general knowledge of stroke, and knowledge about the prevention, identification and treatment of stroke, and the pathway to obtain knowledge about the prevention and treatment of stroke.Each item for the knowledge about the prevention and treatment of stroke was marked as 1-4.5 points, and the total score was 60. There was no mark for the pathway to obtain knowledge about the prevention and treatment of stroke. One unit was investigated each time, and 300-600 questionnaires were sent out, 500 questionnaires were randomly handed out to the healthy physical examinees in the Tungwah Hospital.MAIN OUTCOME MEASURES: The correct rate of answers to the questionnaire, the total score of each kind of knowledge about the prevention and treatment of stroke, the total score of each kind of knowledge in the subgroups, and the pathways to obtain knowledge about the prevention and treatment of stroke were mainly observed.RESULTS: Totally 2 320 complete and effective questionnaires were collected back. ① The average correct rate of answers to all the questions was 49.80%, and those of knowledges about identification and treatment were lower, the total score was ＜ 60% in 48.20% of the subjects. ② The level for each knowledge about the prevention and treatment of stroke was lower in males than in females (P ＜ 0.05), but higher in the middle-age and old groups than in the young group (P＜0.05), and it was decreased from various civil servants, free agents, workers, farmers and students in order (P＜0.05), and there was significant difference between students and free agents (P＜0.01); it was also decreased in order from higher educational level to middle and lower ones in order (P＜0.05); it was also higher in the subjects with stroke history than in those without (P ＜ 0.05). ③) After the subjects were subdivided according to gender, age, profession and educational level, the pathways to obtain knowledge about the prevention and treatment of stroke in order were reading newspapers and magazines,watching TV, reading handbooks about science popularization, influence from relatives and friends, promotion and education of medical staff, and the last one was the least in all the groups.CONCLUSION: The level for the knowledge about the prevention and treatment of stroke was lower among the public in Dongguan area during the investigation, especially the knowledges about identification and treatment, so proper measures should be adopted to further develop education on the knowledge about the prevention and treatment of stroke.

BACKGROUND:Nowadays,liver grafts for transplantation are clinically sourced from non-heart-beating donors.Moreover,there is still no uniform determination of safe time limit points for non-heart-beating donor liver in warm ischemia and cold preservation.OBJECTIVE:To evaluate the application safety and curative effects of warm ischemia liver graft affected by varied cold ischemia time (CIT) in liver transplantation.DESIGN,TIME AND SETTING:A randomized controlled observation was performed in the Organ Transplantation Center,First Affiliated Hospital of Sun Yat-sen University between January 2006 and December 2007.PARTICIPANTS:154 cases who underwent non-heart-beating liver transplantation were included in this study.All liver graftsinvolved had a warm ischemia time(WIT) less than 10 minutes.METHODS:All cases were assigned into 3 groups according to CIT:group Ⅰ (CIT＜8 hours,n=58),group Ⅱ (CIT 8-12hours,n=62),and group Ⅲ (CIT 13-16 hours,n=34).The liver grafts were randomly allocated for the patients.Following liver transplantation,the same immunosuppression protocol was employed for each group.MAIN OUTCOME MEASURES:Following surgery,peak level of alanine aminotransferase (ALT),primary graft dysfunction (PGD) after liver transplantation,acute rejection response,biliary complications,vessel complications,perioperative infections,graft and recipient survival rate were compared among 3 groups.RESULTS:Follow-up time was 8-32 months.No PGD was detected in all 154 cases.Group Ⅱ showed postoperative ALT peak levels significantly higher than group Ⅰ (P＜ 0.05).There was no significant difference between groups Ⅰ and Ⅱin terms of acute cellular rejection,perioperative infection,biliary complication,vessel complication,graft survival rate,and recipient survival rate (P＞0.05).Compared with group Ⅰ,the group Ⅲ exhibited significantly increased postoperative ALT peak level,biliary complications,and perioperative infections,and significantly decreased graft and recipient survival rate (P＜0.05).CONCLUSION:Non-heat-beating-liver grafts with less than 10 minutes of warm ischemia can tolerance 12 hours of cold ischemia.More than 12 hours,postoperative liver transplantation complications ascend and,contradictorily,graft and recipient survival rate descend.

Objective To explore the targeting homing capacity of bone-marrow derived mesenchymal stem eells(MSCs) on rabbit myocardial ischemia by intravenous injection of MSCs under the mediation of diagnostic ultrasound and microbubble. Methods Density gradient centrifugation and adherent culture method were used in the isolation and cultivation of MSCs. MSCs were labeled with DAPI. Rabbit myocardial infarction(MI) models were builded by totally ligation of left anterior descending branch of coronary artery. DAPI labeled MSCs were implanted by intravenous injection with or without the mediation of diagnostic ultrasound and microbubbles. Forty-eight hours after cell transplantation, the hearts of MI rabbits were made of frozen section and observed under fluorescent microscope. The DAPI positive cells were counted in the MI and border area of rabbit heart and compared between two groups. Pathological changes of MI area were observed with HE staining under light microscope and transmission electronic microscope. Results The number of DAPI positive cells in MI and border area of rabbit in both groups were counted under fluorescent microscope. There were more DAPI positive cells in the MI area in ultrasound + microbubble + MSCs group (213.2±26.5) than that in the intravenous injection group (146.8±18.78, P<0.01). There were erythrocytes leaking out of the vessels in MI area in HE staining section under light microscope in ultrasound + microbubble + MSCs group while there were nearly none in the intravenous injection MSCs group. The intercellular space of endothelial cells of the vessels wall was increased and serum component leaked out of the vessel wall in ultrasound + microbubble + MSCs under transmission electronic microscope. Conclusions The targeted homing capacity of BM-MSCs in the MI area of rabbit heart can be enhanced under the mediation of diagnostic ultrasound and microbubbles.

Objective To explore the value of diagnostic ultrasound mediated microbubble destruction in improving the myocardial perfusion and left ventricular systolic function when cooperated with the mecsenchymal stem cells(MSCs) transplantation in rabbit myocardial ischemia. Methods One week after myocardial ischemia (MI) modeling,36 rabbits were divided into 3 groups,the control group(group Ⅰ) ,intravenous injection of MSCs group(group Ⅱ) and ultrasound + microbubble + MSCs group (group Ⅲ). Myocardial contrast enhancement (MCE) was performed and quantification analysis of anterior wall was assessed with Photoshop. Left ventrieular systolic function was assessed with M-mode echocardiography and bi-plane Simpson's method. CD34 expression in heart was detected with immunohistochemisty(IHC). Western blotting was applied to detect the level of VEGF in three groups. Results The differences of gray scale analyzed with histogram of Photoshop in anterior wall of ischemia myocardium between the group Ⅰ and group Ⅱ or group Ⅲ were significant,and P value was 0. 032 and 0. 000 , respectively. There were significant differences of FS between group Ⅲ (30. 43±4.09)% and group Ⅱ (26.29±2.93)%, P<0.01, and similar to group Ⅰ (19.28 ± 2.84)%. The difference of EF(%) between group Ⅲ and group Ⅱ was significant [(61.5±5.8 vs 53.6±4. 71), P<0. 05] ,or markedly significant between group Ⅲ and group Ⅰ [(61.5±5.8 vs 42.6± 5.0), P <0.01]. EF(%) assessed with bi-plane Simpson's method was significantly increased from (34.64 ± 4.59) in group Ⅰ to (41.78 ± 4.21) in group Ⅱ and (48.6±3.96) in group Ⅲ. The expression of CD34 assessed with immunohistochemistry was the highest in group Ⅲ. The level of VEGF with western blotting in group Ⅲ was significantly higher than other two groups. Conclusions It is an efficacious transplantation means of MSCs infusion under the ultrasound mediated microbubles destruction in improving the myocardial perfusion and cardiac systolic function.

Objective Modified upper abdominal cluster transplantation (MCT), which was inspired by the classical cluster transplant technique, has been proven more effective and feasible in the treatment of patients with end stage liver diseases associated with insulin-dependent diabetes mellitus (DM) than orthotopic liver transplantation (OLT) alone. In this study, we summarized our experience with MCT in 5 consecutive patients suffering from end stage liver diseases associated with insulin-dependent type 2 DM in our single center.Methods Five patients with hepatitis B-related chronic liver cirrhosis and insulin dependent type 2 DM received MCT in our single center. The biliary and exocrine pancreatic drainage reconstructions were achieved by a Roux-en-Y duodenojejunostomy or a side-to-side duodenojejunostomy. A quadruple immunosuppressive regimen based on tacrolimus including Basiliximab induction, mycophenolate mofetil (MMF) and steroids was used in the early stage post-transplant, and then converted to tacrolimus monotherapy.Results All of the patients experienced an uneventful post-operative recovery. They were rendered independent from insulin therapy shortly after transplantation. The fasting glucose and glycosylated hemoglobin levels were within normal range. In addition, the fasting C-peptide value was increased from much lower than the normal range pre-transplant to within normal range post-transplant and maintained stable since then. However, the third patient suffered from graft verse host disease (GVHD) 20 days post-operatively and died from severe infection on the post-operative 47 days. The other 4 patients had returned to work and a normal lifestyle over 22, 15, 5 and 4 months of follow-up.Conclusion MCT is an effective method in treating patients suffering from end stage liver diseases combined with insulin-dependent type 2 DM. Whether a cluster graft would increase the risk of GVHD needs further investigation.