Construction of a targeting adenoviral vector carrying AFP promoter for expressing EGFP gene in AFP-producing hepatocarcinoma cell

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,

Synthesis of endotoxin receptor CD14 protein in Kupffer cells and its role in alcohol-induced liver disease

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.

Intestinal damage mediated by Kupffer cells in rats with endotoxemia

AIM: To determine the in vivo effects of phagocytic blockadeof Kupffer cell (KC) on the release of proinflammatorycytokines in small intestinal lesion and on the integrity ofintestinal tract by using gadolinium chloride (GdCI3) duringearly endotoxemia.METHODS: Wistar rats were divided into three groups: GroupA, rats were injected with endotoxin (F. coli O111:B4, a doseof 12 mg.kg-1) only; Group B, rats were pretreatedintravenously with 25 mg of GdCl3 per kg 24 h are givenendotoxin; and Group C, sham operation on ly.All animalswere sacrificed 4 h after endotoxin injection.Mn portion ofthe rats of three groups, bile duct was cannulated, which thebile was collected externally. Morphological changes of ileumwere observed under light microscopy and electronicmicroscopy. The KC were isolated from rats by collagenaseperfusion and in KC, expression of TNF-α and IL-6 mRNAwere determined by RT-PCR analysis. Plasma and bile TNF-αand IL-6 Levels were determined by enzyme-linkedimmunosorbent assay (ELISA).RESULTS: In group A, there were neutrophil infiltrationand superficial epithelial necrosis of the ileal villi, sloughingof mucosal epithelium, and disappearance of some villi. Ingroup B, the ileal mucosal damage was much reduced. whichin group C, no significant morphological changes were seen.GdCl3 pretreatment decreased significantly the expressionof TNF-α and IL-6 mRNA in group B (4.32±0.47 and 4.05±0.43) when compared to group A (9.46±1.21 and 9.04±1.09) (P＜0.05). There was no significant expression of TNF-α and IL-6 mRNA in group C (1.03±0.14 and 10.4±0.13).In rats of group A, the levels of TNF-α and IL-6 in bile andplasma were 207±29 ng. L-1, 1032±107 ng. L-1, 213±33ng. L-1, and 1185±127 ng. L-1, respectively. In group B, theywere 113±18 ng. L-1, 521±76 ng. L-1, 147±22 ng. L-1, and572±54 ng. L-1, respectively. In group C, they were 67±10ng. L-1, 72±13 ng. L-1, 109±18 ng. L-1, and 118±22 ng. L-1respectively. There were significant difference between thethree group (P＜O.05).CONCLUSION: KC release cytokines TNF-α and IL-6causing damage to the integrity of intestinal epithelium andplay a crucial role in the initiation and progression of intestinalmucosal damage during early endotoxemia.

Lipopolysaccharide induced synthesis of CD14 protein and its gene expression in hepatocytes during endotoxemia

AIM:To observe synthesis of CD14 protein and expressionof CD14 mRNA in hepatic tissue and hepatocytes of ratsduring endotoxemia.METHODS:The endotoxemia model of Wistar rat wasestablished by injection of a dose of lipopolysaccharide(LPS)(5mg·kg^(-1),Escherichia coil O111:B4)via the tailvein,then the rats were sacrificed after 3,6,12 and 24 h inbtaches.Hepatocytes were isolated from normal and LPS-injected rats by in situ collagenase perfusion technique andwere collected to measure the expression of CD14 mRNAand synthesis of CD14 protein by reverse transcription-polymerase chain reaction(RT-PCR)or Western blotanalysis.The binding of fluorescein isothiecyanate(FITC)-CD14 polyclonal antibody to isolated hepatocytes was alsoassessed by flow cytometric analysis(FCM).RESULTS:In the rats with endotoxemia,the expressions ofCD14 mRNA in hepatic tissue and isolated hepatocytes werestronger at 3,6,and 12 h than that in control rats(3.48±0.15,5.89±0.62,4.33±0.18,vs 1.35±0.14 in hepatictissue,P<0.01;4.12±0.17,6.24±0.64,4.35±0.18,vs1.87±0.15 in hepatecytss,P<0.01).The synthesis of CD14protein in hepatic tissue and isolated hepatoeytes increasesalso obviously in 6 and 12 h when compared to that incontrol rats(13.27±1.27,17.32±1.35,11.42±1.20,vs 7.34±0.72 in hepatic tissue,P<0.01;14.68±_+1.30,17.95±1.34,11.65±1.19,vs 7.91±0.70 in hepatocytoes,P<0.01).FCM showed that mean fluorescence intensity(MFI)andnumbers of FITC-CD14 positive cells in the rats withendotoxemia increased obviously at 3,6,12 and 24h whencompared with normal control group(43.4%,70.2%,91.4%,32.6% vs4.5%,P<0.01).CONCLUSION:LPS can markedly promote the synthesis ofCD]4 protein and up-regulate the expression of CD14 mRNAin isolated hepatocytes and hepatic tissue.Liver might be amain source for soluble CD14 production duringendotoxemia.

Protective effect of nitric oxide induced by ischemic preconditioning on reperfusion injury of rat liver graft

AIM: Ischemic preconditioning (IP) is a brief ischemic episode,which confers a state of protection against the subsequent long-term ischemia-reperfusion injuries. However, little is known regarding the use of IP before the sustained cold storage and liver transplantation. The present study was designed to evaluate the protective effect of IP on the long-term preservation of liver graft and the prolonged anhepatic-phase injury.METHODS: Male Sprague-Dawley rats were used as donors and recipients of orthotopic liver transplantation. All livers underwent 10 min of ischemia followed by 10 min of reperfusion before harvest. Rat liver transplantation was performed with the portal vein clamped for 25 min. Tolerance of transplanted liver to the reperfusion injury and liver damage were investigated. The changes in adenosine concentration in hepatic tissue and those of nitric oxide (NO) and tumor necrosis factor (TNF) in serum were also assessed.RESULTS: Recipients with IP significantly improved their one-week survival rate and liver function, they had increased levels of circulating NO and hepatic adenosine, and a reduced level of serum TNF, as compared to controls. Histological changes indicating hepatic injuries appeared improved in the IP group compared with those in control group. The protective effect of IP was also obtained by administration of adenosine,while blockage of the NO pathway using Nco-nitro-L-arginine methyl ester abolished the protective effect of IP.CONCLUSION: IP appears to have a protective effect on the long-term preservation of liver graft and the prolonged anhepatic-phase injuries. NO may be involved in this process.

Prolongation of liver allograft survival by dendritic cells modified with NF_(-k)B decoy oligodeoxynucleotides

AIM: To induce the tolerance of rat liver allograft by dendritic cells (DCs) modified with NF-κB decoy oligodeoxynucleotides (ODNs).METHODS: Bone marrow (BM)-derived DCs from SD rats were propagated in the presence of GM-CSF or GM-CSF+IL-4 to obtain immature DCs or mature DCs. GM-CSF+IL-4-propagated DCs were treated with double-strand NF-κB decoy ODNs containing two NF-κB binding sites or scrambled ODNs to ascertain whether NF-κB decoy ODNs might prevent DC maturation. GM-CSF-propagated DCs, GM-CSF+NF-κB decoy ODNs or scrambled ODNs-propagated DCs were treated with LPS for 18 h to determine whether NF-κB decoy ODNs could prevent LPS-induced IL-12 production in DCs. NF-κB binding activities, costimulatory molecule (CD40, CD80, CD86) surface expression, IL-12 protein expression and allostimulatory capacity of DCs were measured with electrophoretic mobility shift assay (ENSA),flow cytometry, Western blotting, and mixed lymphocyte reaction (MLR), respectively. GM-CSF-propagated DCs, GM-CSF+IL-4 -propagated DCs, and GM-CSF+NF-κB decoy ODNs or scrambled ODNs-propagated DCs were injected intravenously into recipient LEW rats 7 d prior to liver transplantation and immediately after liver transplantation.Histological grading of liver graft rejection was determined 7 d after liver transplantation. Expression of IL-2, IL-4 and IFN-γ, mRNA in liver graft and in recipient spleen was analyzed by semiquantitative RT-PCR. Apoptosis of liver allograft-infiltrating cells was measured with TUNEL staining.RESULTS: GM-CSF-propagated DCs, GM-CSF+NF-κB decoy ODNs-propagated DCs and GM-CSF+ scrambled ODNs-propagated DCs exhibited features of immature DCs, with similar low level of costimulatory molecule(CD40, CD80,CD86) surface expression, absence of NF-κB activation,and few allocostimulatory activities. GM-CSF+IL-4-propagated DCs displayed features of mature DCs, with high levels of costimulatory molecule (CD40, CD80, CD86) surface expression, marked NF-κB activation, and significant allocostimulatory activity. NF-κB decoy ODNs completely abrogated IL-4-induced DC maturation and allocostimulatory activity as well as LPS-induced NF-κB activation and IL-12 protein expression in DCs. GM-CSF+NF-κB decoy ODNs-propagated DCs promoted apoptosis of liver allograft-infiltrating cells within portal areas, and significantly decreased the expression of IL-2 and IFN-γ mRNA but markedly elevated IL-4 mRNA expression both in liverallograft and in recipient spleen, and consequently suppressed liver allograft rejection, and promoted liverallograft survival.CONCLUSION: NF-κB decoy ODNs-rnodified DCs canprolong liver allograft survival by promoting apoptosis of graft-infiltrating cells within portal areas as well as down-regulating IL-2 and IFN-γ mRNA and up-regulating IL-4 rnRNA expression both in liver graft and in recipient spleen.

Role of NF-kB in multiple organ dysfunction during acute obstructive cholangitis

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.

Augmented regeneration of partial liver allograft induced by nuclear factor-kB decoy oligodeoxynucleotides-modified dendritic cells

AIM:To investigate the effect of NF-κB decoy oligodeoxynuleotides (ODNs) - modified dendritic cells (DCs) on regeneration of partial liver allograft.METHODS:Bone marrow (BM)- derived DCs from SD rats were propagated in the presence of GM-CSF or GM-CSF+IL-4 to obtain immature DCs or mature DCs, respectively. GMCSF-propagated DCs were treated with double-strand NF-κB decoy ODNs containing two NF-κB binding sites or scrambled ODNs. Allogeneic (SD rat to LEW rat) 50% partial liver transplantation was performed. Normal saline (group A),GM-CSF-propagated DCs (group B), GM-CSF+IL-4-propagated DCs (group C), and GM-CSF+NF-κB decoy ODNs(group D) or scrambled ODNs -propagated DCs (group E) were injected intravenously into recipient LEW rats 7 days prior to liver transplantation and immediately after transplantation.DNA synthesis (BrdU labeling) and apoptosis of hepatocytes were detected with immunostaining and TUNEL staining postoperative 24h, 48h, 72h and 84h,respectively. Liver graft-resident NK cell activity, hepatic IFN-γ mRNA expression and recipient serum IFN-γ level at the time of the maximal liver allograft regeneration were measured with ^51Cr release assay, semiquantitative RT-PCR and ELISA, respectively.RESULTS: Regeneration of liver allograft was markedly promoted by NF-κB decoy ODNs-modified immature DCs but was significantly suppressed by mature DCs, the DNA synthesis of hepatocytes peaked at postoperative 72h in group A, group B and group E rats, whereas the DNA synthesis of hepatocytes peaked at postoperative 84h in group C rats and 48h in group D rats, respectively. The maximal BrdU labeling index of hepatocytes in group D rats was significantly higher than that in the other groups rats.NF-κB decoy ODNs-modified immature DCs markedly suppressed but mature DCs markedly promoted apoptosis of hepatocytes, liver-resident NK cell activity, hepatic IFN-γ mRNA expression and recipient serum IFN-γ production.At the time of the maximal regeneration of liver allograft,the minimal apoptosis of hepatocytes, the minimal activity of liver-resident NK cells, the minimal hepatic IFN-γ mRNA expression and serum IFN-y production were detected in group D rats. The apoptotic index of hepatocytes, the activityof liver- resident NK cells, the hepatic TFN-γ mRNA expression level and the serum IFN-γ level in group D rats were significantly lower than that in the other groups rats at the time of the maximal regeneration of liver allograft.CONCLUSION:The data suggest that the augmented regeneration of partial liver allograft induced by NF-κB decoy ODNs-modified Des may be attributable to the reduced apoptotic hepatocytes, the suppressed activity of liverresident NK cells and the reduced IFN-γ production.