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

World Journal of Gastroenterology 2002年第1期8卷 124-127页

作者： Sheng-WeiLi jian-pinggong 等 DepartmentofGeneralSurgery TheSecondCollegeofClinicalMedicine&TheSecondAffiliatedHospitalofChongqingUniversityofMedicalSciencesChongqing400010China DepartmentofGeneralSurgery TheSecondCollegeofClini

AIM:To observe synthesis of CD14 protein and expressionof CD14 mRNA in hepatic tissue and hepatocytes of ratsduring ***: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 *** 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 *** 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 *** might be amain source for soluble CD14 production duringendotoxemia.

关键词：内毒素血症脂多糖 CD14蛋白诱导合成基因表达肝细胞动物实验

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Intestinal damage mediated by Kupffer cells in rats with endotoxemia

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World Journal of Gastroenterology 2002年第5期8卷 923-927页

作者： jian-pinggong Chuan-XinWu DepartmentofGeneralSurgery TheSecondCollegeofClinicalMedicin&theSecondAffiliatedHospitalofChongqingUniversityofMedicalScience400010 DepartmentofGeneralSurgery TheSecondCollegeofClinicalMedicin&theSeco

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 ***: Wistar rats were divided into three groups: GroupA, rats were injected with endotoxin (F. coli O111:B4, a doseof 12 ***-1) only; Group B, rats were pretreatedintravenously with 25 mg of GdCl3 per kg 24 h are givenendotoxin; and Group C, sham operation on *** animalswere sacrificed 4 h after endotoxin *** 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 ***3 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).

关键词：炎症细胞内毒素血症动物实验肠道狭窄 ELISA法

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

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World Journal of Gastroenterology 2004年第1期10卷 73-76页

作者： jian-pinggong BingTu WeiWang YongPeng Shou-BaiLi Lu-NanYan DepartmentofGeneralSurgery HuaxiHospitalSichuanUniversityChengdu610041SichuanProvinceChina DepartmentofGeneralSurgery theSecondAffiliatedHospitalofChongqingMedicalUniversityChongqing400010China

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 ***: 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 ***: 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 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.

关键词：一氧化氮缺血预处理再灌注损伤肝移植动物模型

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

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World Journal of Gastroenterology 2004年第4期10卷 573-578页

作者： Ming-QingXu Yu-PingSuo jian-pinggong Ming-ManZhang Lue-NanYan DepartmentofGeneralSurgery WestChinaHospitalSichuanUniversityChengdu610041SichuanProvinceChina WestChinaSecondUniversityHospital SichuanUniversityChengdu610041SichuanProvinceChina

AIM:To investigate the effect of NF-κB decoy oligodeoxynuleotides (ODNs) - modified dendritic cells (DCs) on regeneration of partial liver ***: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 *** 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, ***: 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 ***-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 *** the time of the maximal regeneration of liver allograft,

关键词：同种异体肝移植术核因子-κB 树突状细胞 NK细胞细胞免疫功能排斥反应

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

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World Journal of Gastroenterology 2004年第16期10卷 2361-2368页

作者： Ming-QingXu Yu-PingSuo jian-pinggong Ming-ManZhang lu-NanYan DepartmentofGeneralSurgery WestChinaHospitalSichuanUniversityChengdu610041SichuanProvinceChina WestChinaSecondUniversityHospital SichuanUniversityChengdu610041SichuanProvinceChina

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 *** 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 ***: 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 allo

关键词：肝脏同种异体移植物树枝状细胞 NF-κB 脱氧核苷酸手术治疗

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Expression of toll-like receptor 4 and MD-2 gene and protein in Kupffer cells after ischemia-reperfusion in rat liver graft

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World Journal of Gastroenterology 2004年第19期10卷 2890-2893页

作者： YongPeng jian-pinggong Chang-AnLiu Xu-HongLi LingGan Shou-BaiLi DepartmentofHepatobiliarySurgery theSecondAffiliatedHospitalofChongqingMedicalUniversityChongqing400010China

AIM: To investigate the expression of toll-like receptor 4(TLR4) and MD-2 gene and protein in Kupffer cells (KCs)and their role in ischemia-reperfusion (IR) injury of ratliver ***: KCs were isolated at 0 (control group), 2, 12,24 h (IR group) following IR in rat liver graft, mRNA expressionof TLR4 and MD-2 was detected by RT-PCR analysis, proteinexpression of TLR4/MD-2 was detected by flow cytometric(FCM) analysis, and tumor necrosis factor-(~ (TNF-(~) levelin supernatant was measured by ELISA. Then isolated KCswere incubated with anti-TLR4 polyclonal antibody (anti-TLR4 group), and TNF-(~ level was measured ***: The mRNA and protein expression of TLR4/MD-2 and the level of TNF-(~ in IR group increased significantlyat 2 h following IR, and reached the maximum at 12 h, andslightly decreased at 24 h, but were still significantly higherthan those in the control group (P

关键词：基因表达受体4 MD-2基因蛋白质枯否氏细胞局部缺血多次灌注液老鼠肝移植抗体

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

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World Journal of Gastroenterology 2004年第2期10卷 186-189页

作者： Yu-JunShi jian-pinggong Chang-AnLiu Xu-HongLi YingMei CanMi Yan-YingHuo DepartmentofGeneralSurgery theSecondCollegeofClinicalMedicineandtheSecondAffiliatedHospitalofChongqingUniversityofMedicalSciencesChongqing400010China DepartmentofPathoiogy BasicMedicalCollegeChongqingUniversityofMedicalSciencesChongqing400016China InstituteforRadiology MilitaryAcademyofMedicalSciencesBeijing100850China

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 ***: 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 *** of a normal liver cell line (LO2),a hepatocarcinoma cell line (HepG2) and a cervical cancer cell line (HeLa) were transfected with the *** blot and fluorescence microscopy were used to detect the expression of the EGFP gene at mRNA or protein level in three different cell ***:The 0.3kb AFP promoter was synthesized through PCR from the human *** AFP promoter and EGFP gene were directly inserted into the plasmid pshuttle as confirmed by restriction digestion and DNA *** blot showed that EGFP gene was markedly transcribed in HepG2 cells,but only slightly in LO2 and HeLa *** addition,strong green fluorescence was observed in HepG2 cells under a fluorescence microscopy,but fluorescence was very weak in LO2 and HeLa ***: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,

关键词：肝细胞癌聚合酶链反应 AFP EGFP基因基因序列重组肾病毒受体

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

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World Journal of Gastroenterology 2003年第1期9卷 179-183页

作者： BinTu jian-pinggong Hu-YiFeng Chuan-XinWu Yu-JunShi Xu-HongLi YongPeng Chang-AnLiu Sheng-WeiLi DepartmentofGeneralSurgery theSecondCollegeofClinicalMedicine&theSecondAffiliatedHospitalofChongqingUniversity'ofMedicalScience74LinjiangRoadChongqing400010China DepartmentofGeneralSurgery theSecondCollegeofClinicalMedicine&theSecondAffiliatedHospitalofChongqingUniversity'ofMedicalScience74LinjiangRoadChongqing400010China

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 *** changes of vital organs were observed under light and electron ***-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 *** 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 *** of NF-κB activation in various tissues increased in AOC group when compared to other two *** 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^(-

关键词：核因子-κB 急性阻塞性胆管炎多器官功能失调病理机制动物模型

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

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World Journal of Gastroenterology 2003年第3期9卷 622-626页

作者： Li-LiDai jian-pinggong Guo-QingZuo Chuan-XinWu Yu-JunShi Xu-HongLi YongPeng WuDeng Sheng-WeiLi chang-AnLiu DepartmentofDigestiveDisease theSecondCollegeofClinicalMedicine&theSecondAffiliatedHospitalofChongqingUniversityofMedicalScience74LinjiangRoadChongqing400010China DepartmentofGeneralSurgery theSecondCollegeofClinicalMedicine&theSecondAffiliatedHospitalofChongqingUniversityofMedicalScience74LinjiangRoadChongqing400010China

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 ***: 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 ***: Ethanol administration led to a significantsynthesis of endotoxin receptor CD14 protein and its gene expression in KCs, which maybe result in the pathologica

关键词：内毒素受体 CD14蛋白 Kupffer细胞酒精性肝病肝功能发病机制

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