AIM:To construct a recombinant adenoviral vector carrying AFP promoter and EGFP gene for specific expression of EGFP gene in AFP producing hepatocellular carcinoma (HCC) HepG2 cells.METHODS: Based on the Adeno-X^TM ex...AIM:To construct a recombinant adenoviral vector carrying AFP promoter and EGFP gene for specific expression of EGFP gene in AFP producing hepatocellular carcinoma (HCC) HepG2 cells.METHODS: Based on the Adeno-X^TM expression system, the human immediate early cytomegalovirus promoter (PCMV IE)was removed from the plasmid, pshuttle,and replaced by a 0.3 kb (α-fetoprotein (AFP) promoter that was synthesized by polymerase chain reaction (PCR).The enhanced green fluorescent protein (EGFP) gene was inserted into the multiclone site (MCS),and then the recombinant adenovirus vector carrying the 0.3kb AFP promoter and EGFP gene was constructed.Cells of a normal liver cell line (LO2),a hepatocarcinoma cell line (HepG2) and a cervical cancer cell line (HeLa) were transfected with the adenovirus.Northern blot and fluorescence microscopy were used to detect the expression of the EGFP gene at mRNA or protein level in three different cell lines.RESULTS:The 0.3kb AFP promoter was synthesized through PCR from the human genome.The AFP promoter and EGFP gene were directly inserted into the plasmid pshuttle as confirmed by restriction digestion and DNA sequencing.Northern blot showed that EGFP gene was markedly transcribed in HepG2 cells,but only slightly in LO2 and HeLa cells.In addition,strong green fluorescence was observed in HepG2 cells under a fluorescence microscopy,but fluorescence was very weak in LO2 and HeLa cells.CONCLUSION:Under control of the 0.3kb human AFP promoter, the recombinant adenovirus vector carrying EGFP gene can be specially expressed in AFP-producing HepG2 cells,Therefore,this adenovirus system can be used as a novel,potent and specific tool for gene-targeting therapy for the AFP positive primary hepatocellular carcinoma,展开更多
AIM: To observe the synthesis of endotoxin receptor CD14 protein and its mRNA expression in Kupffer cells (KCs), and evaluate the role of CD14 in the pathogenesis of liver injury in rats with alcohol-induced liver dis...AIM: To observe the synthesis of endotoxin receptor CD14 protein and its mRNA expression in Kupffer cells (KCs), and evaluate the role of CD14 in the pathogenesis of liver injury in rats with alcohol-induced liver disease (ALD).METHODS: Twenty-eight Wistar rats were divided into two groups: ethanol-fed group and control group. Ethanol-fed group dextrose instead of ethanol. Two groups were sacrificed at 4 wk and 8 wk, respectively. KCs were isolated and the synthesis of CD14 protein and its mRNA expression in KCs were determined by flow cytometric analysis (FCM)or the reverse transcription polymerase chain reaction (RTPCR) analysis. The levels of plasma endotoxin and alanine transaminase (ALT) were measured by Limulus Amebocyte Lysate assay and standard enzymatic procedures respectively, and the levels of plasma tumor necosis factor (TNF)-α and interleukin (IL)-6 were both determined by ELISA. The liver pathology change was observed under light and electric microscopy.RESULTS: In ethanol-fed group, the percentages of FITCCD14 positive cells were 76.23 % and 89.42 % at 4 wk and 8 wk, respectively. Compared with control group (4.45 %and 5.38 %), the difference was significant (P＜0.05). The expressions of CD14 mRNA were 7.56±1.02 and 8.74±1.37 at 4 wk and 8 wk, respectively, which were significantly higher compared with the control group (1.77±0.21 and 1.98±0.23)(P＜0.05). Plasma endotoxin levels at 4 wk and 8 wk increased dramatically in ethanol-fed rats (112±15 IU/L and 147±22 IU/L) than those in the control animals (31±12 IU/L and 33±9 IU/L) (P＜0.05). In ethanol-fed rats, the levels of wk, respectively which were significantly higher than those fed rats, there were marked pathological changes including steatosis, cell infiltration and necrosis. No marked pathological changes were seen in control group.CONCLUSION: Ethanol administration led to a significantsynthesis of endotoxin receptor CD14 protein and its gene expression in KCs, which maybe result in the pathological changes of liver tissue and hepatic functional damages.展开更多
AIM:To elucidate the role of NF-kB activation in the development of multiple organ dysfunction(MOD)during acute obstructive cholangitis(AOC)in rats. METHODS:Forty-two Wistar rats were divided into three groups:the AOC...AIM:To elucidate the role of NF-kB activation in the development of multiple organ dysfunction(MOD)during acute obstructive cholangitis(AOC)in rats. METHODS:Forty-two Wistar rats were divided into three groups:the AOC group,the group of bile duct ligation(BDL group),and the sham operation group(SO group).All the animals in the three groups were killed in the 6th and 48th hour after operation.Morphological changes of vital organs were observed under light and electron microscopy.NF-κB activation was determined with Electrophoretic Mobility Shift Assay(EMSA).Arterial blood gas analyses and the serum levels of lactate dehydrogenase(LDH),alanine aminotransferase(ALT),blood urea nitrogen(BUN)and creatinine were performed.The concentrations of TNF-α and IL-6 in plasma were also measured. RESULTS:The significant changes of histology and ultrastructure of vital organs were observed in AOC group. By contrast,in BDL group,all the features of organs damage were greatly reduced.Expression of NF-κB activation in various tissues increased in AOC group when compared to other two groups.At 6 h,the arterial pH in three groups was 7.52±0.01,7.46±0.02,and 7.45±0.02,and the blood pCO_2 was 33.9±0.95 mmHg,38.1±0.89 mmHg,38.9±0.94 mmHg,there was difference in three groups(P<0.05).At 48 h,the blood pH values in three groups was 7.33±0.07, 7.67±0.04,and 7.46±0.03,and blood HCO_3^- was 20.1±1.29 mmol·L^(-1),26.7±1.45 mmol·L^(-1)and 27.4±0.35 mmol·L^(-1),there was also difference in three groups(P<0.05).In AOC group, Levels of LDH,ALT,BUN and creatinine were 16359.9±2278.8 nkat·L^(-1),5796.2±941.9 nkat·L^(-1),55.7±15.3 mg/dl,and 0.72± 0.06 mg/dl,which were higher than in SO group(3739.1± 570.1 nkat·L^(-1),288.4±71.7 nkat·L^(-1),12.5±2.14 mg/dl,and 0.47±0.03 mg/dl)(P<0.05).Levels of plasma TNF-α and IL-6 in AOC at 48 h were 429±56.62 ng·L^(-1)and 562±57 ng·L^(-1), which increased greatly when compared to BDL group (139±16 ng·L^(-1),227±43 ng·L^(-1))and SO group(74±10 ng·L^(-1), 113±19 ng·L^(-1))(P<0.05). CONCLUSION:The pathological damages and the NF-κB activation of many vital organs exised during AOC.These findings have an important implication for the role of NF-κB activation in MOD during AOC.展开更多
基金Supported by the Key Program of Medical Science Foundation of Chongqing Public Health Bureau,[2001] 01-1-018
文摘AIM:To construct a recombinant adenoviral vector carrying AFP promoter and EGFP gene for specific expression of EGFP gene in AFP producing hepatocellular carcinoma (HCC) HepG2 cells.METHODS: Based on the Adeno-X^TM expression system, the human immediate early cytomegalovirus promoter (PCMV IE)was removed from the plasmid, pshuttle,and replaced by a 0.3 kb (α-fetoprotein (AFP) promoter that was synthesized by polymerase chain reaction (PCR).The enhanced green fluorescent protein (EGFP) gene was inserted into the multiclone site (MCS),and then the recombinant adenovirus vector carrying the 0.3kb AFP promoter and EGFP gene was constructed.Cells of a normal liver cell line (LO2),a hepatocarcinoma cell line (HepG2) and a cervical cancer cell line (HeLa) were transfected with the adenovirus.Northern blot and fluorescence microscopy were used to detect the expression of the EGFP gene at mRNA or protein level in three different cell lines.RESULTS:The 0.3kb AFP promoter was synthesized through PCR from the human genome.The AFP promoter and EGFP gene were directly inserted into the plasmid pshuttle as confirmed by restriction digestion and DNA sequencing.Northern blot showed that EGFP gene was markedly transcribed in HepG2 cells,but only slightly in LO2 and HeLa cells.In addition,strong green fluorescence was observed in HepG2 cells under a fluorescence microscopy,but fluorescence was very weak in LO2 and HeLa cells.CONCLUSION:Under control of the 0.3kb human AFP promoter, the recombinant adenovirus vector carrying EGFP gene can be specially expressed in AFP-producing HepG2 cells,Therefore,this adenovirus system can be used as a novel,potent and specific tool for gene-targeting therapy for the AFP positive primary hepatocellular carcinoma,
基金the National Natural Science Foundation of China,No.39970719,30170919
文摘AIM: To observe the synthesis of endotoxin receptor CD14 protein and its mRNA expression in Kupffer cells (KCs), and evaluate the role of CD14 in the pathogenesis of liver injury in rats with alcohol-induced liver disease (ALD).METHODS: Twenty-eight Wistar rats were divided into two groups: ethanol-fed group and control group. Ethanol-fed group dextrose instead of ethanol. Two groups were sacrificed at 4 wk and 8 wk, respectively. KCs were isolated and the synthesis of CD14 protein and its mRNA expression in KCs were determined by flow cytometric analysis (FCM)or the reverse transcription polymerase chain reaction (RTPCR) analysis. The levels of plasma endotoxin and alanine transaminase (ALT) were measured by Limulus Amebocyte Lysate assay and standard enzymatic procedures respectively, and the levels of plasma tumor necosis factor (TNF)-α and interleukin (IL)-6 were both determined by ELISA. The liver pathology change was observed under light and electric microscopy.RESULTS: In ethanol-fed group, the percentages of FITCCD14 positive cells were 76.23 % and 89.42 % at 4 wk and 8 wk, respectively. Compared with control group (4.45 %and 5.38 %), the difference was significant (P＜0.05). The expressions of CD14 mRNA were 7.56±1.02 and 8.74±1.37 at 4 wk and 8 wk, respectively, which were significantly higher compared with the control group (1.77±0.21 and 1.98±0.23)(P＜0.05). Plasma endotoxin levels at 4 wk and 8 wk increased dramatically in ethanol-fed rats (112±15 IU/L and 147±22 IU/L) than those in the control animals (31±12 IU/L and 33±9 IU/L) (P＜0.05). In ethanol-fed rats, the levels of wk, respectively which were significantly higher than those fed rats, there were marked pathological changes including steatosis, cell infiltration and necrosis. No marked pathological changes were seen in control group.CONCLUSION: Ethanol administration led to a significantsynthesis of endotoxin receptor CD14 protein and its gene expression in KCs, which maybe result in the pathological changes of liver tissue and hepatic functional damages.
基金the National Natural Science Foundation of China, No.39970719,30170919
文摘AIM:To elucidate the role of NF-kB activation in the development of multiple organ dysfunction(MOD)during acute obstructive cholangitis(AOC)in rats. METHODS:Forty-two Wistar rats were divided into three groups:the AOC group,the group of bile duct ligation(BDL group),and the sham operation group(SO group).All the animals in the three groups were killed in the 6th and 48th hour after operation.Morphological changes of vital organs were observed under light and electron microscopy.NF-κB activation was determined with Electrophoretic Mobility Shift Assay(EMSA).Arterial blood gas analyses and the serum levels of lactate dehydrogenase(LDH),alanine aminotransferase(ALT),blood urea nitrogen(BUN)and creatinine were performed.The concentrations of TNF-α and IL-6 in plasma were also measured. RESULTS:The significant changes of histology and ultrastructure of vital organs were observed in AOC group. By contrast,in BDL group,all the features of organs damage were greatly reduced.Expression of NF-κB activation in various tissues increased in AOC group when compared to other two groups.At 6 h,the arterial pH in three groups was 7.52±0.01,7.46±0.02,and 7.45±0.02,and the blood pCO_2 was 33.9±0.95 mmHg,38.1±0.89 mmHg,38.9±0.94 mmHg,there was difference in three groups(P<0.05).At 48 h,the blood pH values in three groups was 7.33±0.07, 7.67±0.04,and 7.46±0.03,and blood HCO_3^- was 20.1±1.29 mmol·L^(-1),26.7±1.45 mmol·L^(-1)and 27.4±0.35 mmol·L^(-1),there was also difference in three groups(P<0.05).In AOC group, Levels of LDH,ALT,BUN and creatinine were 16359.9±2278.8 nkat·L^(-1),5796.2±941.9 nkat·L^(-1),55.7±15.3 mg/dl,and 0.72± 0.06 mg/dl,which were higher than in SO group(3739.1± 570.1 nkat·L^(-1),288.4±71.7 nkat·L^(-1),12.5±2.14 mg/dl,and 0.47±0.03 mg/dl)(P<0.05).Levels of plasma TNF-α and IL-6 in AOC at 48 h were 429±56.62 ng·L^(-1)and 562±57 ng·L^(-1), which increased greatly when compared to BDL group (139±16 ng·L^(-1),227±43 ng·L^(-1))and SO group(74±10 ng·L^(-1), 113±19 ng·L^(-1))(P<0.05). CONCLUSION:The pathological damages and the NF-κB activation of many vital organs exised during AOC.These findings have an important implication for the role of NF-κB activation in MOD during AOC.
