Enhanced external counterpulsation(EECP) was developed in our laboratory and applied in clinical setting since 1976. Before the year of 1990, the mechanism of this treatment was supposed to promoting collateral circulation by increasing diastolic pressure during counterpulsation. According to a series of investigations, we discovered and confirmed that the flow shear stress were increased obviously during EECP, regulating a series of reaction of shear stress responsive elements, inducing vascular endothelial cell (VEC) repair mechanism, improving VEC function, all of which contribute to the inhibition of development of atherosclerosis. A new generation of EECP device was designed based on the advances above and aimed at promoting the aterial flow shear stress more effectively in turn to protect vascular endothelium.

Objective To separate the CD133 + subpopulation in human hepatocellular carcinoma (HCC) and investigate the tumorigenicity.Methods The human liver cancer tissues were subcutaneously transplanted into nude mice to generate xenograft tumors which were then isolated to prepare single cell suspension.The expression of CD133 + subpopulation was further detected using flow cytometry.The CD133 + subpopulations were separated and depurated with magnetic-activated cell sorting system.Immunofluorescence was performed to identify the histological phenotype of CD133 + subpopulation.The in vitro and in vivo clone formation assay and in vivo xenograft formation assay were performed, respectively.Results Flow cytometry analysis revealed that a percentage of (4.1 ± 0.6) ％ CD133 + cells were detected in xenografts.Immunofluorescence studies showed that (86.8 ± 7.5) ％ of the isolated cells were CD133 +.Compared with CD133-population, CD133 + cells showed a higher capability to generate clone sphere in vitro and a higher tumorigenicity in nude mice (P ＜ 0.05).Conclusion The CD133 + subpopulation in human hepatocellular carcinoma had a potent tumorigenicity and was enriched in cancer stem cells.

AIM: To examine the alteration of local renin-angiotensin system of dogs with myocardial ischemia and external counterpulsation treatment and its mechanism. METHODS: Acute myocardial ischemia was induced by occluding the LAD of ischemic and ECP groups. The tissue renin activity and angiotensin(AngⅡ) level in ischemic myocardium and aorta were measured. The expression of angiotensinogen and renin mRNA were detected by RT-PCR. RESULTS: Renin activity, AngⅡ level in ischemic myocardium and AngⅡ level in aorta of ECP group were lower than those of MI group. Except for renin in ischemic myocardium, angiotensinogen mRNA and renin in ischemic myocardium and angiotensinogen mRNA in aorta of ECP group were reduced to normal level. CONCLUSION: The inhibitory effect of ECP on cardiovascular renin activity and angiotensinogen gene expression could be one of the mechanisms by which ECP protects ischemic myocardium.

AIM: To examine the effect of external counterpulsation(ECP) on renin-angiotensin system(RAS) and hemodynamics in dogs with myocardial ischemia and their relationship. METHODS: Acute myocardial ischemia was induced by occluding the left anterior descending of the dogs. The local renin activity, angiotensin Ⅱ(AngⅡ) level, angiotension-converting enzyme(ACE) activity were tested by biochemical methods and hemodynamics was recorded by a 8-channel physiological recorder. RESULTS: Ischemia could actiivate renin,ACE and AngⅡ in cardiovasculature and ECP reduced them except for renin in ischemic myocardium. Ischemia also could activate RAS in lung and kidney, which play an important role on circulating RAS, and ECP reduced them. Furthermore, ECP could improve hemodynamics and there existed a close relationship between local AngⅡlevel and hemodynamics. CONCLUSION: ECP can reduce local RAS and improve hemodynamics in dogs with myocardial ischemia, which might be one of mechanisms underlying the protective effect of ECP on ischemic myocardium.

Objective:To investigate the correlations between CO-029 expression and cholangiocarcinoma invasion and metastasis, and the further explore the potential mechanism involved.Methods:The constructed lentiviral vector of vshRNA-CO-029 (LV/GFP/CO-029) was used to transfect and screen the stable transfected cholangiocarcinoma cell line HCCC-9810-vshRNA-CO-029 as the silence group, HCCC-9810 cells transfected with the mock plasmid were used as the mock group, and the untransfected cells were used as the control group. Cell scratch assay, Transwell assay and in vivo implantation assay were used to detect the migration, invasion and metastasis of the three groups of cells. Immunoprecipitation and tumor necrosis factor (TNF)-α stimulation assay were used to detect the effect of CO-029 on the expression of EMT-related genes.Results:The scratch healing rate of the silence group was (27.11±4.58)%, which was lower than that in mock group (92.84±6.24)%, the number of cells passing through Matrigel in silence group was (57.15±6.10), which was significantly lower than that in mock group (108.20±9.21) and control group (112.00±10.45), the differences were statistically significant ( all P<0.01). The volume of liver tumors in the silence group of orthotopic xenograft mouse model was (2.17±0.54) cm 3, while the volume of liver tumors in the transplanted simulation group was (0.74±0.15) cm 3, the differences were statistically significant ( P<0.05). The incidence of lung metastasis and the number of lung metastases in the simulated group was 100%(6/6) and (214.17±35.64), respectively, while that in the silence group was 16.7% (1/6) and (41.56±14.15), respectively, the differences were statistically significant (all P<0.05). Co-immunoprecipitation showed that CO-029 can form a complex with TNF-αR1. TNF-α induced the down-regulation of E-cadherin and up-regulation of vimentin and N-cadherin in the mock group, but no significant changes were observed in the silence group. Conclusion:CO-029 expression is positively correlated with tumor invasion and metastasis of cholangiocarcinoma, and could couple with TNF-α to induce EMT, which is a novel well-established potential prognostic and therapeutic target for cholangiocarcinoma metastasis and prognosis intervention.

Objective To study the effect of Tspan 8 on metastasis and invasion of human hepatocellular carcinomas(HCC).Methods RT-PCR and western blot were used to detect the expressions of Tspan 8 in HCC cell lines,HCC and matched nontumorous tissues.The expression of Tspan 8 was then down-regulated by LV/GFP/Tspan 8 in HCC cells.The expressions of Tspan 8 mRNA and protein were determined by RT-PCR and Western blot assay,respectively.The proliferation was examined by MTT,the expression of AMDM12 was assessed by Western blot,and the invasion ability of HCC cells was evaluated by transwells.Results A high level of Tspan 8 was found in high metastatic potential HCC cells,and the expression of Tspan 8 in HCC tissues was much higher than that in the matched nontumorous tissues. Down-regulation of Tspan 8 had no influence on the proliferation of HCC cells (P＞0.05),while it inhibited the expression of ADAM12 and the invasive ability of HCC cells (P＜0.01,P＜0.01 respectively).Conclusion Tspan 8 played an important role in invasion and metastasis of human hepatocellular carcinomas and down-regulation by LV/GFP/Tspan 8 inhibited the invasiveness of human hepatocellular carcinoma cells.

Objective To investigate the effect of occult HBV infection (OBI) on carcinogenesis of cryptogenic hepatocellular carcinoma.Methods Samples of hepatocellular carcinoma (HCC) and pericarcinomatous tissues obtained after hepatectomy from January 2011 to November 2013 at the Third Affiliated Hospital of Sun Yat-Sen University were collected.They were divided into two groups:the cryptogenic HCC group (the CH group,n =26) and the HBV related HCC group (the HH group,n =40).These samples were compared with the normal liver tissues obtained in 30 patients.HBV DNA was identified by the nested polymerase chain reaction and the immunohistochemical method was taken to examine the hepatitis B virus X protein (HBx) and Yes-associated protein (YAP) expressions.Results OBI was identified in 20 (77.8％) cryptogenic HCC patients and 8 (26.7％) in the control group.There was a significant difference between the two groups (x2 =14.072,P ＜ 0.05).HBV DNA was detected in all the HBV-related HCC patients.The HBx protein expression was mainly located in the cytoplasm of liver cells and liver cancer cells,but YAP was expressed in the nucleus.Both of them showed diffuse brown or tan particles.In the HH group and the CH group,the positive expression rates of HBx protein in the tumorous tissues were 80.0％ and 90.0％,respectively,and 85.0％ and 82.5％ in the nontumorous tissues,but only in 40.0％ in the control group.The positive expression rates of YAP in the tumorous tissues were 65.0％ and 67.5％,respectively,15.0％ and 20.0％ in the nontumorous tissues,respectively,but only in 12.5％ in the control group.The HBx expression in the cancerous tissues and para-cancerous tissues of the HH group and the CH group showed no significant difference (P ＞ 0.05),but the YAP expression in the tumor tissues was significantly higher than that in the nontumorous tissues (P ＜ 0.05).The HBx and YAP expressions in the HH group were comparable to the CH group (P ＞ 0.05).However,their expressions in the cancerous tissue of the HH group and the CH group were significantly higher than in the normal liver tissues (P ＜ 0.05).Conclusion A high prevalence of HBV infection was observed in HBsAg-negative HCC and the high expressions of HBx and YAP might be involved in the process of cryptogenic hepatocellular carcinoma.

AIM: To investigate the effects of external counterpulsation (ECP) on nitric oxide (NO) and nitric oxide synthase (NOS) and the expression of NOS gene in myocardial infarction canines. METHODS: Nineteen healthy dogs were randomly divided into three groups ie. controls, ischemia group, ischemia and ECP group. Serum NO concentrations and myocardium NO levels and NOS specific activity were determined by modified nitrate reductase method. The protein synthesis of sub-type NOS including inducible NOS (iNOS) and endothelial NOS (eNOS) of myocardial tissue were also determined by immunohistochemical method. The constitutive NOS (cNOS) mRNA was measured via in situ hybridization. RESULTS: 120 and 180 minutes after the ligating of LAD, serum NO concentration in ECP groups were higher than those in ischemic groups (P

Objective To evaluate the effects of surgical treatment of acute deep vein thrombosis (DV T) of lower extremity. Methods Thirty-six patients with a cute DVT of lower extremity were treated by thrombectomy with thrombolytic,a nticoagulant and compression on the affected lower extremity during and after op eration. Results No death and serious complications happened i n this series. Thirty-one patients were followed-up for 2 to 20 months with a n average of 9 months. Symptoms disappeared totally in 23 patients , 8 patien ts had slight edema in the lower extremities. Conclusions Com bination of thrombectomy the thrombolytic and anticoagulant agent,and comp ression of the affected lower extremity is a safe and effective method in the t reatment of acute DVT of lower extremity.

Objective To investigate the portal vein embolization (PVE) and portal vein ligation (PVL) in liver regeneration of rats with hepatic fibrosis.Methods Fifty rats with liver fibrosis were prepared,including 10 rats were randomly chosen as pre-operative control group.The other 40 rats were divided into two groups:PVE group (A1,n =20) and PVL group (A2,n =20).We chose to embolize and ligate the right portal vein,respectively.The blood samples were obtained at different end points for measuring ALT and AST levels.Each liver lobes and whole liver were weighed,and non-embolized liver lobe/whole liver weight ratio,non-ligated liver lobe/whole liver weight were caculated at different end points.The samples from liver with/without embolization or ligation were were stained with hematoxylin-eosin,and the changes of microstructure of liver were observed.Immunostained for PCNA and Ki-67 were performed.Results Transient elevation of postoperative ALT and AST levels were noted in each group.Serum ALT and AST reached the peak on the first day in both of PVE and PVL groups [ALT,A1 (66.5 ±6.3) U/L vs(491.5 ± 48.0) U/L,A2 group(62.8 ±5.7) U/L vs(433.7 ±41.0) U/L;AST,A1group (113.4 ± 12.5) U/L vs (685.2 ±65.7) U/L,A2 group (110.4 ± 11.1) U/L vs(623.9 ±75.2) U/L,P＜0.05),and started to decrease on the third day,recovered to the pre-operative level on the fourteenth day (P ＞ 0.05).The weight percentage of non-embolized and non-ligated liver lobes/whole liver after PVE and PVL increased.There was no significant difference between the first day and pre-operative levels (P ＞ 0.05).Nevertheless,there were significant differences observed from the third,seventh,fourteenth days (A1 group,50.2 ± 5.0,57.7 ±5.7,61.8 ±6.6;A2group,49.6 ±3.5,55.7 ±6.9,63.0±5.1,respectively)compared with preoperative groups (A1 group,34.4 ± 4.0;A2 group,34.4 ± 4.0) (P ＜ 0.05).There was no significant difference between group A1 and A2 in each time point (P ＞0.05).The PCNA and Ki-67 were positive in hepatocytes and increased after operation,reached the peak in the third day (P ＜ 0.05),decreased slowly and restored to the normal level in the fourteenth day after operation,meanwhile,there was no significant difference between group A1 and A2 (P ＞ 0.05).Conclusions Fibrosis rats had the ability of regeneration in the contralateral part of the liver after selective PVE and PVL and there was no significant difference on the proliferative degree.Therefore,the safety and reliability of PVE and PVL in inducing liver regeneration in rats with liver fibrosis were confirmed.