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The detection rate of gallbladder adenomyomatosis has gradually increased, but the accuracy of preoperative diagnosis is low. Most doctors tend to expand the operation indications because they are worried about the carcinogenesis. But there are still great controversies on the key issues such as whether it is cancerous, operation indications and how to follow up for non-surgical patients. This article will review these key issues.
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Objective To summarize the clinical characteristics of hepatic lymphoepithelioma-like carcinoma,and to explore the diagnosis and treatment strategies of hepatic lymphoepithelioma-like carcinoma.Methods A retrospectively analysis on 13 patients with liver lymphoepithelioma-like carcinoma in Union Hospital,Tongji Medical College,Huazhong University of Science and Technology,between March 2005 and May 2019 were carried out.Results 8 cases were male,5 were female,median age was 45years (27 to 68 y).There were 8 cases of intrahepatic cholangiocytic lymphoepithelioma-like carcinoma,4cases of hepatocytic lymphoepithelioma-like carcinoma,and 1 case of mixed hepatocytic and cholangiocytic lymphoepithelioma-like carcinoma.All patients received partial hepatectomy and postoperative comprehensive treatment.The patients were followed from 6 months to 7 years.Only one patient died,and the other patients were all in a tumor-free state.Conclusion Primary hepatic lymphoepithelioma-like carcinoma is a rare liver cancer.It is confirmed mainly by pathological examination and immunohistochemistry.With surgery as the main treatment,prognosis is usually fair.
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Objective To summarize the clinical characteristics of adult patients with residual yolk duct,and to explore the diagnosis and treatment strategy of residual vitelline duct in adults.Methods A retrospective analysis on 11 adult cases with residual vitelline duct in our hospital between June 2012 and May 2017 was carried out.Results 8 cases were males,3 cases were females,and median age was 50 years (18-57 y).2 cases were vitelline cyst,9 cases were Meckel diverticulum.2 cases were with ectopic tissue,3 cases with ulcer bleeding,1 case with secondary intra-abdominal hernia and intestinal obstruction,2 cases with secondary infection.The pathological diagnosis of Meckel diverticulum was consistent with preoperative diagnosis.There were no major postoperative complications.The patients were followed up from 6 months to 2 years.Conclusion Most of the residual vitelline duct in adults are Meckel diverticulum and vitelline duct cyst.Resection of residual vitelline duct is the main treatment method.
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Objective To study the results using T-tube and retroperitoneal space drainage to manage perforating injury of the distal common bile duct(PIDC) during common bile duct(CBD) exploration.Methods We retrospectively analyzed the clinical data of 12 patients who were diagnosed to suffer from PIDC during CBD exploration from 2010 to 2012.Result All these 12 patients who received T-tube and retroperitoneal space drainage,gastrointestinal decompression,nutritional support and antibiotics recovered uneventfully.Conclusion Given that the CBD was unobstructed,T-tube and retroperitoneal space drainage was an good treatment for patients with PIDC.
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Objective To analyse risk factors for bile leakage after liver resection.Methods Between January 2011 and December 2012,469 patients underwent elective hepatectomy.We prospectively collected and retrospectively analyzed demographic data,pathological variables,and perioperative variables.Univariate analysis screened the related factors of bile leakage after liver resection.Multivariate analysis identified the independent risk factors of postoperative bile leakage.Results 469 patients were included in the analysis.The prevalence of bile leakage was 22.6％ (n =106).Univariate analysis identified the following risk factors as male gender,portal hypertension,steatosis,cirrhosis,ChildPugh grade,ascites,operative time,intraoperative transfusion,intraoperative blood loss,portal triad clamping,microwave solidification,lymphadenectomy,number of tumor,tumor margin,tumor capsular,diameter of tumor,portal vein invasion or portal branch thrombosis,number of abdominal drains.Multivariate analysis identified 4 independent risk factors for postoperative bile leakage:Cirrhosis [OR =13.2 (2.3,76.9),P =0.004],steatosis [OR =73.1 (17.7,301.5),P ＜ 0.001],infusion volume of the surgery day [OR=1.0 (1.0,1.0),P=0.019] and diameter of tumor [OR=1.2 (1.1,1.3),P=0.003].Conclusions Cirrhosis,steatosis,transfusion volume of the surgery day,and tumor size were risk factors for bile leakage after major liver resection.
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Objective To summarize the diagnosis and treatment of one case of combined heart liver transplantation. Methods On November 24, 2011, one case of combined heart-liver transplantation was performed on a patient with Ebstein's anomaly and tricuspid valve replacement after 5 years,complicated with congestive cirrhosis,liver failure dccompensation,preoperative heart failure Ⅲ degree and B grade of liver function Child-Pugh score. The operation was done with the graded cardiopulmonary bypass assisted mode:first creating the vena cava-aortic bypass to complete heart transplantation, second creating the femoral vein-ascending aorta bypass to complete liver transplantation,and third stopping and neutralizing.The aortic cross-clamping time was 54 min and the an hepatic phase was 38 min.The total time of three times of cardiopulmonary bypass was 199 min and the total time-consuming of operation was 517 min. The patient was given basiliximab +methylprednisolone for immune induction therapy, and tacrolimus + mycophenolate mofetil +prednisone solution for anti-rejection. After operation, liver protecting treatment, anti-infection therapy and nutrition support therapy were given.Results The recipient died of multiple organ failure after 78 days.The mechanical ventilation treatment duration for this recipient was 78 days and ECMO adjuvant therapy for postoperative hypoxemia time lasted 63 days.Conclusion The combined heart liver transplantation is an effective measures for treatment of heart and liver failure.
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Objective To assess the safety and efficacy of an olive oil-based lipid emulsion for parenteral nutrition in patients after hepatectomy.Methods Thirty-one postoperative patients with elective hepateetomy were randomized to receive isonitrogenous,isocalorie parenteral nutrition over 6 days after liver lobectomy(48-72hours)with either olive oil-based lipid emulsion(study group,n=15)or standard soybean oil emulsion(control group,n=16).The liver function and plasma protoins were assessed using peripheral venous blood collected before surgery,one day after surgery,and 7 days after surgery.The safety profiles of emulsion supports and postoperative rehabilitation were also assessed.Results The preoperative serum levels of total bilirubin,direct bilirubin,alanine amiotransferase,aspartate aminotransferase,alkaline phosphatase,total protein,albumin,and prealbumin were comparable between the two groups(all P>0.05).Although the Postoperative safety profile and liver function were not significantly different between two groups(all P>0.05),plasma total proteins,albumin,and prealbumin returned to the normal levels significantly faster in the study group than in control group[(57.57±9.84)g/L vs.(47.76±6.53)g/L,P=0.000;(31.29±3.11)g/L vs.(26.34±4.87)g/L,P=0.000;(0.188±0.059)g/L vs.(0.103±0.037)g/L,P=0.000]on the 7th Postoperative day,and the Postoperative hospital stay was also significantly shorter in the study group[(13.1±1.2)d vs.(15.2±1.1)d,P=0.041].The incidence of postoperative complications in study group and control group was 26.7%and 31.3%.respectively.Conclusions Treatment with the new olive oil-based lipid emulsion is weU tolerated in hepatectomy patients.It can speed up plasma proteins recovery and may shorten postoperative hospital stay,although it does not remarkably decrease the incidence of postoperative complications.
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AIM: To investigate the effect of mfn2 on mitochondrial function in steatosis hepatocytes. METHODS: Plasmid pEGFP-mfn2 was transfected into hepatocyte strain L02 by Lipofectamine 2000 in vitro, then the steatosis model of hepatocytes was establish by oleic acid induction. RT-PCR was used to evaluate mRNA expression and Western blotting was use to detect the protein expression. ATP level was determined by firefly luciferase bioluminescent. ROS production was measured by fluorescence probe DCFH-DA. Chondrosome transmembrane potential of L02 was observed by labeling of JC-1 and FCM. RESULTS: The stable expression of ectogenesis mitofusin2 in L02 cells was confirmed by RT-PCR and Western blotting. In the model of oleic acids induced lipid formation, Mfn2 obviously inhibited the descent of chondrosome transmembrane potential and ATP level, and increased ROS production in L02 cells. CONCLUSION: Up-regulated expression of mfn2 attenuates mitochondria dysfunction caused by oleic acids induced lipid formation.
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Objective To summarize the clinical experience of hepatectomy with hepatic artery resection and reconstruction using gastroduodenal artery during radical resection of hilar cholangiocarcinoma.Methods From Dec.2004 to Dec.2008,nine cases of hilar cholangiocarcinoma with hepatic artery invasion were subjected to radical resection comhined with tumor invaded hepatic artery resection and reconstruction using gastroduodenal artery.The clinical data of these patients were reviewed.Results Nine cases underwent hilar cholangiocarcinoma radical resection with hepatic artery resection,immediate hepatic artery reconstruction using gastroduodenal artery end to end anastomosis while hepatic artery resection exceeding 1 cm.One patient underwent partial resection of the portal vein and repair using autogenous segment of great saphenous vein.Roux-en-Y hepaticojejunostomy was performed in 9 patients with intrahepatic bile duct stents in 8 patients.All patients suffered from postoperative transient SIRS and recovered within 2-3 days after operation.One patient experienced massive bleeding from the upper alimentary tract 3 day after operation and the bleeding was controlled afterwards.The blood flow in the reconstructed hepatic arteries monitored by Doppler was normal two weeks after operation.There was no inhospital mortality.9 patients were followed up for 1-4 years,the median survival time is 23 months (6 months to 32 months).Conclusion Hepatic artery can be reconstructed using gastroduodenal artery during a radical resection of hilar cholangiocarcinoma,and hepatic artery reconstruction decreases the postoperative complications.
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Objective To investingate the effect of survivin shRNA on chemotherapy resistance in GBC-SD cells. Methods They were divided into three groups of GBC-SD, GBC-SD/EGFP and GBC-SD/survivin. MTT assay was used to detect cell viability in the 3groups. mRNA and protein of survivin were tested by RT-PCR and Western blot. Then the cells were treated with proper construction DDP. The cell survival rate and IC_(50) were determined with MTT, cell apoptosis detected by FACS and the alteration of nucleus observed by TUNEL. Meanwhile, caspase-3 activity was determined using the colorimetric method. Results Cell viability was reduced remarkably in GBC-SD/survivin and survivin expression was decreased obviously. After being treated with DDP, cell survival rate and IC_(50) were decreased obviously in GBC-SD/survivin, apoptotic rate elevated remarkably compared with other groups. There were brownly nucleuses in three groups. Caspase-3 activity increased first and then decreased, but it exceeded in GBC-SD/survivin than that in other two groups. Conclusion The survivin shRNA can down-regulate the expression of survivin in GBC-SD cells remarkably and improve the sensibility to chemotherapy.
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Objective: The impact of parenteral fish oil lipid emulsion on liver function and nutritional status of malignant tumors of the liver and gallbladder patients. Methods: From December 2007 to A-pril 2008, 32 post-operative hepatobiliary cancer patients were randomly divided into control and study groups. Two groups were treated with isocaloric, isonitrogenic parenteral nutrition and the study group was added fish oil lipid emulsion. Comparison of plasma protein, glucose, jaundice index, transaminase, ALP and the rate of infection complications was made betweent the two groups. Results: The blood glucose, transaminase and ALP levels were not significantly different between the two groups. But the plasma proteins and bilirubin levels were improved significantly (P < 0.05) with reduced infection complication in the study group. Conclusion : Fish oil lipid emulsion is conducive to the recovery of post-operative liver and gallbladder cancer patients in live function and nutritional status.
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Objective To investigate the role of toll-like receptor 4 (TLR4) of endothelium or bone marrow derived cells in the acute lung injury (ALI) induced by lipopolyscccharide (LPS) in mice with reciprocal bone marrow transplantation. Method Chimeric mice were produced by reciprocal bone marrow transplantation between TLR4mut/mut and TLR4+/+ mice and divided into 4 groups: WT/WT (recipient/donor),WT/Mutant, Mutant/WT and Mutant/Mutant group. Six to eight weeks following transplantation, LPS was injected inot mice's tail vein in order to produce ALI model,and mice were sacrificed five hours later on.Samples of lung tissues were taken for the following analysis of wet/dry weight (W/D), lung permeabifity index (LPI), myeloperoxidase (MPO),levels of cytokines (TNF-α, IL-1β) and adhesion molecules (ICAM-1). Results Lung injury in the Mutant/Mutant mice was the mildest in the 4 groups. And lung injury in WT/Mutant mice was more serious than that in Mutant/WT mice. levels of MPO and ICAM-1 in WT/Mutant mice were much higher than those of Mutant/WT. In addition,the expression of ICAM-1 in WT/Mutant mice is comarable to that in WT/WT mice. Mutant/WT mice expressed higher levels of TNF-α and IL-1β than WT/Mutant mice. Conclusions Endothelial cell derived TLR4 plays ker-nel role in ALI induced by LPS via lung PMN recruitment,although bone marrow cells derived TLR4 are more im-portant for the release of cytokines.
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BACKGROUND: Effects of embryonic stem cell-derived hepatocyte-like cell transplantation on oncogenicity of differentiated hepatocyte-like cells and biochemical metabolism of liver should be further studied.OBJECTIVE: To evaluate the therapeutic efficacy of embryonic stem cell-derived hepatocyte-like cell transplantation on the acute liver failure.DESIGN: Randomized controlled study.SETTING: the First Affiliated Hospital of Sun Yat-sen University.MATERIALS: This study was performed at the Central Laboratory, the First Affiliated Hospital of Sun Yat-sen University from January 2005 to February 2006. D3-ES cells extracted from the mice which underwent transfection of green fluorescent protein were graciously presented by professor Huang, Ophthalmology Center of Sun Yat-sen University. Forty 6-week-old D3-129 mice of clean grade and irrespective of gender were provided by Experimental Animal Center of Sun Yat-sen University [certification: SCXK (yue) 2004-0011]. The experimentzl animals were disposed according to ethical criteria.Transforming growth factor, basic fibroblast growth factor, and hepatocyte growth factor were provided by Gibco BRL Company, USA.METHODS: Transforming growth factor, basic fibroblast growth factor, and hepatocyte growth factor were combined to differentiate D3-ES cells into hepatic cells. Cell suspension was poured into liver capsule of 20 mice with 2.0×10(6)cells per mouse. Another 20 mice that determined as the controls were injected with saline. Twenty-four hours later, intraperitoneal injection of 5 μL/20 g carbon tetrachloride was used to induce acute liver failure and to observe quality of life and mean survival time. Twenty-four hours after acute liver failure, vena cava posterior blood was drawn to detect total bilirubin,glutamate-pyruvate transaminase, albumin, blood glucose, pro-time prothrombin time, and other hepatic functional parameters. By scarification, hepatic samples were obtained to evaluate oncogenesis condition, and then HE staining and immunohistochemistry were adopted to detect growth of transplanted cells and albumin expression.MAIN OUTCOME MEASURES: Quality of life, average survival time, hepatic functional parameters, growth of transplanted cells, and oncogenesis condition.RESULTS: Quality of life and average survival time: After the onset of acute liver failure, mice in the control group had incoordination and other symptoms of central nervous system. In addition, 14 mice in the control group and 8 in the transplantation group had abdominal dropsy. Average survival time in the control group was significantly shorter than that in the transplantation group (23, 62 hours, P<0.05). Hepatic functional parameters: Levels of total bilirubin and glutamate-pyruvate transaminase in the control and transplantation groups were higher than those before modeling; levels of albumin and blood glucose were lower than those before modeling; pro-time prothrombin time was significantly longer than that before modeling(P<0.01). Furthermore, levels of total bilirubin and glutamate-pyruvate transaminase in the transplantation group were lower than those in the control group; blood glucose in the transplantation group was higher than that in the control group, and pro-time prothrombin time in the transplantation group was significantly shorter than that in the control group (P<0.05). Growth of transplanted cells and oncogenesis condition: Pathological section demonstrated that structure of liver tissue was not changed remarkably, and tumor was not formed. Moreover, transplanted cells and hepatocyte-like cell were well arranged and combined to express albumin.CONCLUSION: Embryonic stem cell-derived hepatocyte-like cell transplantation can improve quality of life, prolong survival time of model mice with acute liver failure; additionally, transplanted cells may well support biochemical metabolism of liver tissue.
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The evolution of hepatocellular carcinoma (HCC) is a compound process which involves many kinds of genes and transductional pathways. The expression of the peptidyl-proplyl- isomerase PIN1 gene, the mutation in exon 3 of β-catenin and its correspondent abnormal expression and their roles in the hepatocellular carcinogeneisis were investigated. Among 29 pair cases of HCC and non-carcinoma tissues, the expression of PIN1 gene was detected by immunochemical staining. Mutations in exon 3 of β-catenin gene and differential expression of β-catenin gene were investigated by the methods of PCR-SSCP, direct sequencing and immunohistochemical technique as well. The results indicated: (1) 44.8% (13/29) cases of HCC presented higher level of PIN1 gene expression than non-cancerous tissues (x2 =32.63, P＜0.05), especially in cytoplasm and nucleus, while there was lower level of PIN1 expression in non-cancerous tissues; (2) 58.6% (17/29) HCC tissues showed β-catenin protein accumulation in cytoplasm and nucleus. 46.2% (6/13) HCC tissues indicated β-catenin protein accumulation with higher level of PIN1 expression, while 53.8% (7/13) HCC tissues indicated β-catenin protein accumulation with lower level or trace of PIN1 expression (x2 =0.00, P＞0.05); (3) 24.1% (7/29) of primary tumor lesions carried gene mutations in exon 3 of β-catenin, and accompanied by β-catenin protein accumulation. There was no mutation in non-cancerous tissues. All the mutation presented in tissues with low level of PIN1 expression. There was no mutation of β-catenin gene in tissues with high PIN1 expression level (x2=58.12, P＜0.05). So it was postulated that the increase of PIN1 gene expression could promote hepatocellular carcinogenesis via a way different from β- catenin gene mutation.
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In order to investigate the origin of neointimal smooth muscle cells in transplant arteriosclerosis in rat aortic allograft, sex-mismatched bone marrow transplantation was performed from male Wistar rats to female Wistar rats. Four weeks after transplantation, the aortic transplant model was established by means of micro-surgery in rats. The recipients were divided into 4 groups: female Wistar-female Wistar aortic isografts, female SD-female Wistar aortic allografts, male SD-male Wistar aortic allografts, female SD-chimera Wistar aortic allografts. Eight weeks after transplantation, aortic grafts were removed at autopsy and processed for histological evaluation and immunohistochemistry. The results indicated that excessive accumulation of α-SMA-positive smooth muscle cells resulted in significant neointima formation and vascular lumen stricture in rat aortic allografts.Neointima assay revealed that the neointimal area and NIA/MA ratio of transplanted artery were significantly increased in all of aortic allograft groups as compared with those in aortic isograft group (P＜0.01). Neointimal smooth muscle cells were harvested from cryostat sections of aortic allograft by microdissection method. The Sry gene-specific PCR was performed, and the result showed that a distinct DNA band of 225 bp emerged in the male-male aortic allograft group and chimera aortic allograft group respectively, but not in the female-female aortic allograft group. It was suggested that recipient bone-marrow cells, as the origin of neointimal smooth muscle cells, contributed to the pathological neointimal hyperplasia of aortic allograft and transplant arteriosclerosis.
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In order to investigate the origin of neointimal smooth muscle cells in transplant arteriosclerosis in rat aortic allograft, sex-mismatched bone marrow transplantation was performed from male Wistar rats to female Wistar rats. Four weeks after transplantation, the aortic transplant model was established by means of micro-surgery in rats. The recipients were divided into 4 groups: female Wistar-female Wistar aortic isografts, female SD female Wistar aortic allografts, male SD-male Wistar aortic allografts, female SD-chimera Wistar aortic allografts. Eight weeks after transplantation, aortic grafts were removed at autopsy and processed for histological evaluation and immunohistochemistry. The results indicated that excessive accumulation of alpha-SMA-positive smooth muscle cells resulted in significant neointima formation and vascular lumen stricture in rat aortic allografts. Neointima assay revealed that the neointimal area and NIA/MA ratio of transplanted artery were significantly increased in all of aortic allograft groups as compared with those in aortic isograft group (P<0.01). Neointimal smooth muscle cells were harvested from cryostat sections of aortic allograft by microdissection method. The Sry gene-specific PCR was performed, and the result showed that a distinct DNA band of 225 bp emerged in the male-male aortic allograft group and chimera aortic allograft group respectively, but not in the female-female aortic allograft group. It was suggested that recipient bone-marrow cells, as the origin of neointimal smooth muscle cells, contributed to the pathological neointimal hyperplasia of aortic allograft and transplant arteriosclerosis.
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The evolution of hepatocellular carcinoma (HCC) is a compound process which involves many kinds of genes and transductional pathways. The expression of the peptidyl-proplyl-isomerase PIN1 gene, the mutation in exon 3 of beta-catenin and its correspondent abnormal expression and their roles in the hepatocellular carcinogeneisis were investigated. Among 29 pair cases of HCC and non-carcinoma tissues, the expression of PIN1 gene was detected by immunochemical staining. Mutations in exon 3 of beta-catenin gene and differential expression of beta-catenin gene were investigated by the methods of PCR-SSCP, direct sequencing and immunohistochemical technique as well. The results indicated: (1) 44.8% (13/29) cases of HCC presented higher level of PIN1 gene expression than non-cancerous tissues (chi2=32.63, P0.05); (3) 24.1% (7/29) of primary tumor lesions carried gene mutations in exon 3 of beta-catenin, and accompanied by beta-catenin protein accumulation. There was no mutation in non-cancerous tissues. All the mutation presented in tissues with low level of PIN1 expression. There was no mutation of beta-catenin gene in tissues with high PIN1 expression level (chi2=58.12, P<0.05). So it was postulated that the increase of PIN1 gene expression could promote hepatocellular carcinogenesis via a way different from beta-catenin gene mutation.
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In order to construct an expression vector carrying small hairpin (sh) RNA (shRNA) for toll-like receptor 4 mRNA and a reporter gene of enhanced green fluorescence protein (EGFP) and study the inhibition of cytokine release by RAW264.7 cell induced by lipopolysaccharide (LPS)stimulation through transfection and expression of shRNA targeting TLR4 gene via the RNAi mechanism, the reporter gene plasmid pEGFP-C1 (4.7 kb) and psiRNA-hHlneo (2979 bp) were used. The H1 promotor and double Bbs Ⅰ restrict endoenzyme site were cloned from plasmid psiRNA-hHlneo and reconstructed them into plasmid pEGFP-C1 in the Mlu Ⅰ restrict endoenzymic site, forming plasmid pEGFP-H1/siRNA, which contained Bbs site and reporter EGFP gene. Then an oligonuclear hairpin sequence targeting TLR4 gene was designed by internet tool and inserted into the plasmid pEGFP-H 1/siRNA forming plasmid pEGFP-H 1/TLR4-siRNA. After transfection of pEGFP-H1/TLR4-siRNA into RAW264.7 cells, tumor necrosis factor-alpha (TNF-α) release by the cells after stimulation by LPS was detected. The results showed that the constructed pEGFP-H1/TLR4-siRNA carrying hairpin RNA for TLR4 gene and reporter EGFP gene were proven to be right by restriction endonuclease analysis. The expression of EGFP gene was (50.37±8.23) %and after transfection of the plasmid pEGFP-H1/ TLR4-siRNA the level of TNF-α released by RAW264.7 cell was down regulated. It was concluded that shRNA targeting TLR4 gene could inhibit the TNF-α release by RAW264.7 cells evoked by LPS.
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In order to construct an expression vector carrying small hairpin (sh) RNA (shRNA) for toll-like receptor 4 mRNA and a reporter gene of enhanced green fluorescence protein (EGFP) and study the inhibition of cytokine release by RAW264.7 cell induced by lipopolysaccharide (LPS) stimulation through transfection and expression of shRNA targeting TLR4 gene via the RNAi mechanism, the reporter gene plasmid pEGFP-C1 (4.7 kb) and psiRNA-hHlneo (2979 bp) were used. The H1 promotor and double Bbs I restrict endoenzyme site were cloned from plasmid psiRNA-hH1neo and reconstructed them into plasmid pEGFP-C1 in the Mlu I restrict endoenzymic site, forming plasmid pEGFP-H1/siRNA, which contained Bbs site and reporter EGFP gene. Then an oligonuclear hairpin sequence targeting TLR4 gene was designed by internet tool and inserted into the plasmid pEGFP-H1/siRNA forming plasmid pEGFP-H1/TLR4-siRNA. After transfection of pEGFP-H1/TLR4-siRNA into RAW264.7 cells, tumor necrosis factor-alpha (TNF-alpha) release by the cells after stimulation by LPS was detected. The results showed that the constructed pEGFP-H1/TLR4-siRNA carrying hairpin RNA for TLR4 gene and reporter EGFP gene were proven to be right by restriction endonuclease analysis. The expression of EGFP gene was (50.37+/-8.23) % and after transfection of the plasmid pEGFP-H1/ TLR4-siRNA the level of TNF-alpha released by RAW264.7 cell was down regulated. It was concluded that shRNA targeting TLR4 gene could inhibit the TNF-alpha release by RAW264.7 cells evoked by LPS.
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The eukaryotic expression of human arresten gene and its effect on the proliferation of in vitro cultured vascular smooth cells (VSMCs) in vitro were investigated. COS-7 cells were transfected with recombinant eukaryotic expression plasmid pSecTag2-AT or control plasmid pSecTag2 mediated by liposome. Forty-eight h after transfection, reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of arresten mRNA in the cells, while Western blot assay was applied to detect the expression of arresten protein in concentrated supernatant. Primary VSMCs from thoracic aorta of male Sprague-Dawley rats were cultured using the tissue explant method, and identified by immunohistochemical staining with a smooth muscle-specific anti-alpha-actin monoclonal antibody before serial subcultivation. VSMCs were then co-cultured with the concentrated supernatant and their proliferation was detected using Cell Counting Kit-8 (CCK-8) in vitro. The results showed that RT-PCR revealed that the genome of arresten-transfected cells contained a 449 bp specific fragment of arresten gene, suggesting the successful transfection. Successful protein expression in supernatants was confirmed by Western blot. CCK-8 assay showed that the proliferation of VSMCs were inhibited significantly by arresten protein as compared with control cells (F=40.154, P<0.01). It was concluded that arresten protein expressed in eukaryotic cells can inhibit proliferation of VSMCs effectively in vitro, which would provide possibility to the animal experiments.