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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.
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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.
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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.
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Objective To investigate the expression of phosphorylated RB (pRB) in the invasion front of papillary thyroid carcinoma and the clinical pathological implications. Methods Immunohistochemistry was ap-plied in analyzing pRB expression feature and the clinical pathological implications in a total of 82 patients who underwent total or subtotal thyroidectomy and neck lymph node dissection for papillary thyroid carcinoma. Results The displacement of pRB proteins from the nucleus to the cytoplasm was observed in the invasive front of papil-lary thyroid carcinoma , which was an independent predictor of lymph node metastasis in papillary thyroid carci-noma. Conclusion Nuclear-cytoplasmic transport of pRB in the invasive front was an independent predictor of lymph node metastasis in papillary thyroid carcinoma.
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Objective To investigate the role of transmembrane protein C0-029 in epithelial mesenchymal transition (EMT) induced by tumor necrosis factor alpha (TNFα) of intrahepatic cholangiocarcinoma (ICC) cells.Methods RT-PCR and Western blot were conducted to detect the CO-029 expression in ICC cells and tissues.The effect of CO-029 silencing by lenti-virus on EMT induced by TNFα was investigated.Western blot and mass spectrometry after immunoprecipitation were used to confirm whether TNFαR1 can directly or indirectly bind with CO-029 to form complexes in ICC.Results Differential expression of CO-029 was observed in ICC cells and tissues.The expression of CO-029 was significantly reduced by lenti-viral interference in ICC cells,resulting in the failure of TNFα to induce EMT.TNFαR1 in ICC could directly or indirectly form complexes with CO-029.Conclusion CO-029 mediates TNFα/TNFαR1 induced EMT in ICC,which might play an important role in the invasion and metastasis.
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Objective To investigate the expressions of tetraspanin CO-029 in intrahepatic cholangiocarcinoma (ICC) and to find out their clinical significance.Methods RT-PCR and western blot were used to detect the expressions of CO-029 in ICC and their matched para-tumorous tissues from 20 patients with ICC,as well as in the HCCC-9810 cell lines.The expressions of CO-029 were further detected via tissue microarray (TMA) in the pathological specimens of 40 patients with ICC.Correlations between the expressions of CO-029 and the clinicopathologic features and prognosis were analyzed.Results A high level of CO-029 was detected in the 20 patients with ICC and the HCCC-9810 cell lines via western blot and RTPCR.Moreover,the expression levels of CO-029 in the ICC tissues were higher than the matched para-tumorous tissues (P ＜ 0.05).TMA detection revealed the positive expression rate of CO-029 to be 65％ (26/40).The expression level of CO-029 was much higher in the early recurrence group (Time to recurrence,TTR ＜ 1 year) than the non-recurrence group (TTR≥ 1 year).On analysis,the correlations were significant between the expressions of CO-029 and tumor encapsulation,hilar lymph node metastasis,TNM stage and prognosis (P ＜0.05).Conclusions CO-029 was highly expressed in ICC.It had close correlations with recurrence,metastasis and prognosis of patients with ICC.
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BACKGROUND: The feasibility and the mechanism of human umbilical cord blood mesenchymal stem cel transplantation for the treatment of liver cirrhosis need to be discussed in-depth. OBJECTIVE: To observe the effect of human umbilical cord blood mesenchymal stem cel transplantation through portal vein on the liver function and tissue pathological changes of the rats with liver cirrhosis. METHODS: Carbon tetrachloride was used to prepare rat model of liver cirrhosis. After the success of modeling, the rats in the cel transplantation group received portal vein injection of 1 mL 5-bromo-2-deoxyuridine -labeled human umbilical cord blood mesenchymal stem cells (5×106), the model group was injected with the same volume of PBS; the normal rats received 1 mL human umbilical cord blood mesenchymal stem cel transplantation via the portal vein were as the control group. At 4 weeks after transplantation, the rat tail vein blood and liver tissue were obtained for testing. RESULTS AND CONCLUSION: At 4 weeks after cel transplantation, compared with the model group, levels of serum alanine aminotransferase, aspartate aminotransferase and total bilirubin in the cel transplantation group were significantly decreased, while the albumin level was increased significantly (P < 0.01); the liver cel inflammatory necrosis, steatosis and liver fibrosis were improved significantly (P < 0.05 or P < 0.01). Immunohistochemistry and immunofluorescence staining showed that human umbilical cord blood mesenchymal stem cel colonization could be seen in the rat liver tissues of the cel transplantation group and control group, but the number of 5-bromo-2-deoxyuridine-positive cells in cel transplantation group was significantly larger than that in the control group. Reverse transcription-PCR test result showed that the expressions of cytokeratin 18 and albumin mRNA could be observed in the rat liver tissue of the cel transplantation group, but no expression could be seen in the control group. It is visible that human umbilical cord mesenchymal stem cells can improve liver function and pathological damage of liver cirrhosis rats in a certain extent, which may relate with the intrahepatic homing colonization and hepatocyte-like cel differentiation of the transplanted cells in the liver cirrhosis rats.
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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.
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PURPOSE: Tubulointerstitial hypoxia in the kidney is considered a hallmark of injury and a mediator of the progression of tubulointerstitial fibrosis. Hypoxia-inducible factor-1alpha (HIF-1alpha), a master transcription factor in cellular adaptation to hypoxia, regulates a wide variety of genes, some of which are closely associated with tissue fibrosis. The present study set out to characterize urinary HIF-1alpha expressions in patients with lupus nephritis (LN) and to explore whether urinary HIF-1alpha expressions are associated with histologic chronicity changes and renal function. MATERIALS AND METHODS: Urinary HIF-1alpha levels were measured by enzyme-linked immunosorbent assays in 42 patients with LN and in 30 healthy controls. Activity and chronicity indexes as well as tubular HIF-1alpha expressions were analyzed for each specimen. RESULTS: Urinary HIF-1alpha levels were higher in LN patients than in healthy controls (3.977+/-1.696 vs. 2.153+/-0.554 ng/mL, p0.05). CONCLUSION: Urinary HIF-1alpha levels were elevated in LN patients and were associated with histologic chronicity changes and renal function, indicating that HIF-1alpha might contribute to histologic chronicity in LN.
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Adolescent , Adult , Female , Humans , Male , Young Adult , Enzyme-Linked Immunosorbent Assay , Hypoxia-Inducible Factor 1, alpha Subunit/urine , Kidney/metabolism , Lupus Nephritis/urineABSTRACT
Objective To study the expression of nuclear factor-kappaB (NF-κB) and the counts of lymph vessels in laryngeal squarnous cell carcinoma and polyps of vocal cord tissues, and explore their clinicopathologic significance and correlation in the course of laryngeal carcinoma. Methods SP immuno-histochemical method was used to detect the expression of NF-κB and the counts of lymph vessels on the routinely paraffln-embedded sections of the specimens from 50 cases laryngeal squamous cell carcinoma and 10 cases of polyps of vocal cord tissues. Results The positive rate of NF-κB and the counts of lymph ves-sels in laryngeal carcinoma[60. 0% ,( 13.3±3.4)/HP]were significantly higher ( P <0. 05 and P <0. 01respectively) than those in polyps of vocal cord tissues[10.0 % ,(6. 1±3. 8)/HP]. The positive rate of NF-κB and the counts of lymph vessels in well differentiated adenocarcinoma and cases without metastasis were significantly lower( P < 0. 05, P <0. 01 ), compared with poor-differentiated adenoearcinoma and ca-ses with metastasis. The counts of lymph vessels in the NF-κB positive cases were significantly higher than thoseinNF-κBnegativecases[(14.9±4.1)/HPvs (9.8±3.1)/ HP, P <0.01] . Conclusions The expression of NF-κB and the counts of lymph vessels might be important markers to be used to monitor the progression, biological behaviors, metastatic status and prognosis of laryngeal carcinoma. NF-κB might pro-mote lympoangiogenesis in laryngeal squnmous cell carcinoma tissues.
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Objective To investigate the effect of c-myc ASODN on the proliferation and invasion of human bile duct carcinoma cell line QBC939. Methods QBC939 cells was conventionally cultured. C-myc ASODN was designed and transfected into QBC939 cell line. MTT assay and transwell experiment were used to study cell proliferation and invasion of QBC939 cells. Results MTT assay showed that cell survival rate in ASODN group was significantly lower than that in blank group(P < 0.05). Transwell experiment showed that the num-ber of cells penetrated in ASODN group was significantly lower than that in blank group(P<0.01). The cell survival rate and the number of cells penetrated in vechicle group had no difference with blank comparison group(P>0.05). Conclusions C- myc ASODN can inhibit the proliferation and invasion of QBC939 cells.
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Objective To investigate the application of selective hepatopetal blood occlusion techniques in anatomic hepatectomy.Methods We retrospectively reviewed the clinical data of 259 patients with hepatolithiasis or liver tumor undergoing anatomic hepatectomy under selective hepatopetal blood occlusion from January 2006 to December 2009.Results Totally,183 cases with hepatolithiasis and 76 cases with liver tumor underwent anatomic hepatectomy under selective hepatopetal blood occlusion.The average intra-operation blood loss was 210 mL(120-1 600 mL);post-operation incidence of complications and the rate of residual stones was 10.9% and 4.2%,respectively.Thre was no operative death in this series.The intrahepatic recurrence and metastasis rate of liver tumor was 23.6% and the median recurrence was 16.3 months.Conclusions The use of a appropriate selective hepatopetal blood occlusion during anatomic hepatectomy for hepatolithiasis and liver tumors is an effective measure to reduce surgical complications and improve outcome.
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Objective To investigate the protective effect of Calcitonin Gene-related Peptide(CGRP)on ET-1 mediated injury of human hepatocyte.Methods Human liver tissues obtained from patients undergoing partial hepatectomies were randomly divided into five groups:control group,liver perfused with D-Hank's solution group;liver perfused with ET-1 group and three liver perfased with ET plus CGRP(10-6M,10-7M,10-8M)treated groups.Collagenase digestion method was used to isolate human hepatocytes,then hepatocytes were cultured,and the level of MDA and TNF-?,the viability and proliferation of hepatocyte,and the hepatocyte function(ALT,Alb,Urea and LDH)were determined.Results As compared with control group,in ET-1 group,the viability and proliferation of hepatocytes,the level of Alb and Urea declined significantly(P
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Objective To study the operative procedure for stage III and IV hilar cholangiocarcinoma. Methods A crescent shape excision on the edges of multiple hepatic segments followed by a 'skirt edge′ form hepatoenterostomy to drain the multiple hepatic ducts was used to treat unresectable stage III,IV hilar cholangiocarcinoma, . Results (1)the mean survival time was 15.65 months;(2)the patient comfortable index was 81.5%;(4)there was no operative death in the series. Conclusions The 'skirt edge' form hepatoenterostomy is a feasible and effective palliative method for unresectable stage III,IV cholangiocarcinoma.
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Objective To explore the reasons,diagnosis and treatment of residual cholecystitis(RCC) with gallstones. Methods The clinical data of 36 RCC patients with gallstones identified by operation were retrospectively analyzed. Results All the 36 patients were cured by reoperation. Residual cholecystectomy was performed on 8 patients, and residual cholecystecomy plus common bile duct exploration and T tube drainage on 28 patients. Thirty one patients were followed up for 3 months to 12 years,93.55% of the patients had good results. Conclusions The main reason of residual cholecystitis with gallstones was not followed the principle of "identify cut identify" during cholecystectomy .The clinical presentation of RCC is similar to that of cholecystitis with gallstones .The accurate rate of auxiliary examinations is low,so the results of these exammations should be analyzed comprehensivly in the diagnosis. The principle of "identify cut identify" should be followed during the reoperation. The common bile duct and common hepatic duct should be opened first and then the residual gall be resected.
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Objective To study the pathologic changes of the gallbladder in rabbits with calculous cholecystitis and the causes of gallstone recurrence after choleystolithotomy. Methods Twenty- four rabbits were randomly divided into four groups:(1) normal control group (n=5);(2) operative control group(n=5);(3) stone-implanted(S-I) group (n=6),and (4)stone- removed(S-R) group(n=8).The human cholesterol gallstones were implanted into the rabbit gallbladder in stone-implanted and stone- removed rabbits,The pathologic change of the gallbladder was observed by light microscopy 3 months before and after cholecystolithotomy. Results (1)The wet weight of gallbladder in the S-R group [ (1.1?0.06)g] was significant higher than that in S-I group [(0.515?0.1)g] (P0.05);(3)Sever chronic cholecystitis was 33.3% in S-I group 87.5% and in S-R group(P
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Objective To constuct and identify a specific siRNA targeting mucins (MUC1) expression vector.Methods A pair of oligonucleotide completing and coding hairpin MUC1-siRNA synthesized were inserted into pGC silencerTM U6/Neo/RNAi vector.Double enzyme digestion,PCR test and DNA sequencing were used to identify the recombinant plasmid.Then,special MUC1-siRNA was transfected into cholangiocarcinoma cells with Lipofectamine TM 2000.In vitro,MUC1 mRNA and protein expression was tested by RT-PCR and Western Blot respectively.Results Double enzyme digestion,PCR test and DNA sequencing confirmed that the MUC1 specific siRNA expression vector was constructed successfully.After transfection,the expression of MUC1 mRNA and protein in QBC939 (experimental group) decreased significantly (P