Objective To construct the eukaryotic plasmid expression vector mediated short hairpin RNA(shRNA) interference targeting TLR4 gene,and transfect it into pancreatic adenocarcinoma cell line PANC1,then screen stably transfected clonal cell line.Methods Three shRNA interference expression plasmid vectors targeting the TLR4 gene were constructed,named TLR4-1,TLR4-2,TLR4-3.The shRNA plasmid with highest inhibitory efficiency was selected and transfected into PANC1 cells with liposome.The silencing efficiency and transfection efficiency of TLR4-shRNA was assayed with real-time quantitative PCR and flow cytometry analysis.Monoclonal cell with stable transfection of TLR4-shRNA were selected by geneticin 418 (C418) and limiting dilution analysis.Results Transient transfection efficiency of PANC1 was (46.72 ±5.06) ％.TLR4 mRNA expressions were 0.025 ± 0.004,0.027 ± 0.003,0.019 ± 0.006in cells transfected with TLR4-1,TLR4-2,TLR4-3,respectively,which were significantly lower than that in untransfected group (0.061 ±0.018) and negative control group (0.057 ±0.015,P ＜0.05).The transfection efficiency of TLR4-3 vector in stably transfected clones [(82.79 ±8.16)％] was significantly higher than that of transient transfection (P =0.001 ).The expression of TLR4 mRNA was decreased to 0.010 ± 0.002,which was significantly lower than that of transient transfection ( P =0.001 ).The expression of TLR4 protein was (0.54±0.32) ％,which was significantly lower than that of untransfected cells [( 87.42 ± 5.00 ) ％] and that of negative control [(82.9±5.00)％,P =0.000].Conclusions Stable transfection PANC1 cell lines with TLR4 gene silencing are successfully identified.

Objective To study the inhibition of tumor necrosis factor-alpha(TNF-α)cytokine expression of BV-2 cells induced by hypoxia-reoxygenation injury by siRNA targeting TLR4 gene via the RNAi mechanisms.Method BV-2 mouse microglial cell line was cultured in six-well plates and randomly divided into group N(nor-mal group),group H(hypoxia-reoxygenation),group T(hypoxia-reoxygenation+TLR4-siRNA transfected group),group C(hypoxia-reoxygenation+pEGFP-H1/control-siRNA transfected group)and group B(hypox-ia-reoxygenation+pEGFP-H1 transfected group).Group H,group T,group C and group B were cultured in hy-poxia condition for 3 h followed by reoxygenation for 24 h.The plasma was transfected into BV-2 cells mediated by lipofectamine 2000.The efficiency of transfection were detected by flow cytometry to observe the expression of EGFP.RT-PCR method was used to detect the level of mRNA of TLR4 or NF-кB p65.Westem blot methed was used to test the expression of TLR4 protein.and ELISA was used to test the level of TNF-α in the supernatants.Analysis of variances was used for statistical analysis.Results The expression of EGFP gene waa;(67.58±7.16)% after transfection by flow cytometry analysis.Compared to group N,the TLR4 mRNA,NF-кB p65 mR-NA,TLR4 protein level and the TNF-α quantity in group H,group T,group C and group B increased after the hy-poxia-reoxygenation treatment(P＜0.01).While the expression of the TLR4 mRNA,NF-кB p65 mRNA,TLR4 protein level and the TNF-α quantity in the group T down-regulated compared to group H,group C and group B(P＜0.01).And there were no changes in group C,group B and group H about observation index(P＞0.05).Conclusions The siRNA targeting TLR4 mRNA could inhibit the inflammatory reaction released by BV-2 cells in-duced by hypoxia-reoxygenation stimulation.

The relationship between the hepatic ischemia/reperfusion (I/R) injury and the balance of nitric oxide/endothelins (NO/ET) was studied. The changes of the ratio of NO/ET and the hepatic injury were observed in a rat hepatic I/R model pretreated with several tool drugs. In the acute phase of hepatic I/R injury, the ratio of plasma NO/ET was reduced from 1.58 +/- 0.20 to 0.29 +/- 0.05 (P < 0.01) and the hepatic damage deteriorated. NO donor L-Arg and ET receptor antagonist TAK-044 could alleviate the hepatic I/R injury to some degree, whereas NO synthase inhibitor L-NAME aggravated the damage. It was concluded that the hepatic I/R injury might be related with the disturbance of the NO/ET balance. Regulation of this balance might have an effect on the I/R injury.
Arginine ; Endothelins/*blood ; Liver/*blood supply ; NG-Nitroarginine Methyl Ester ; Nitric Oxide/*blood ; Receptors, Endothelin/antagonists & inhibitors ; Reperfusion Injury/*blood

Whether M3 cholinergic receptor signal transduction pathway is involved in regulation of the activation of NF-kappaB and the expression of chemokine MOB-1, MCP-lgenes in pancreatic acinar cells was investigated. Rat pancreatic acinar cells were isolated, cultured and treated with carbachol, atropine and PDTC in vitro. The MOB-1 and MCP-1 mRNA expression was detected by using RT-PCR. The activation of NF-kappaB was monitored by using electrophoretic mobility shift assay. The results showed that as compared with control group, M3 cholinergic receptor agonist (10(-3) mol/L, 10(-4) mol/L carbachol) could induce a concentration-dependent and time-dependent increase in the expression of MOB-1, MCP-1 mRNA in pancreatic acinar cells. After treatment with 10(-3) mol/L carbachol for 2 h, the expression of MOB-1, MCP-1 mRNA was strongest. The activity of NF-kappaB in pancreatic acinar cells was significantly increased (P<0.01) after treated with M3 cholinergic receptor agonist (10(-3) mol/L carbachol) in vitro for 30 min. Either M3 cholinergic receptor antagonist (10(-5) mol/L atropine) or NF-kappaB inhibitor (10(-2) mol/L PDTC) could obviously inhibit the activation of NF-kappaB and the chemokine MOB-1, MCP-1 mRNA expression induced by carbachol (P<0.05). This inhibitory effect was significantly increased by atropine plus PDTC (P<0.01). The results of these studies indicated that M3 cholinergic receptor signal transduction pathway was likely involved in regulation of the expression of chemokine MOB-1 and MCP-lgenes in pancreatic acinar cells in vitro through the activation of NF-kappaB.

AIM: To study the relationship between the disturbance of nitric oxide/endothelin-1(NO/ET-1) and hepatic ischemia/reperfusion(I/R) injury as well as the regulation of NO/ET-1 system by hepatic ischemic preconditioning(IPC). METHODS: The changes of NO/ET-1 system and their relationship with hepatic I/R injury were compared between I/R group and IPC+I/R group in a rat hepatic I/R model. Two hours after reperfusion, the liver tissues were detected by RT-PCR to see whether there was inducible nitric oxide synthase (iNOS) mRNA expression. RESULTS:In the acute phase of hepatic reperfusion, the ratio of NO/ET-1 was reduced, which was due to a significant reduction of NO - 2/NO - 3 (the metabolic product of NO) and significant elevation of ET-1 in the blood plasma. The content of ALT, AST, LDH and TNF-? in blood plasma, and of MDA in liver tissue were increased but ATP in liver tissue was reduced, the hepatic damage was deteriorated. The protection of the hepatic IPC was concerned with the elevation of the ratio of NO/ET-1 caused by the elevation of NO - 2/NO - 3, and reduction of ET-1 as well. There was no iNOS mRNA detected in the liver tissues.CONCLUSION: Hepatic I/R injury is related to the disturbance of NO/ET-1. The protection of the hepatic IPC in the acute phase might be conducted by its regulation of NO/ET-1 system. The cNOS rather than the iNOS generated the NO in this situation.

Objective To investigate the effect of predeposit autotransfusion in operation of the patients with lumbar disc protrusion.Methods Fifty patients of transfusion with lumbar disc protrusion were assigned into two groups by stratified sampling randomly,30 patients whose blood were predeposited before operation in experimental group,and the other 20 patients whose blood were not predeposited before operation in control group.The blood loss,the blood requirements during operations,the hemotological routine indexes and the complications related to blood transfusion were compared respectively.Results The blood loss of experimental group [ (720 ± 665 ) ml ] perioperative period was lower than that of control group [ ( 1060 ± 558 ) ml ],but there was no significant difference between two groups (P ＞ 0.05 ).All the patients in experimental group went through perioperative period safely without allogenic blood transfusion.Hemoglobin,red blood cell and white blood cell were not significantly different between two groups before and after operation for 3,7 days (P＞ 0.05 ),the platelet count after operation for 7 days was significantly different between two groups (P ＜ 0.05).No complication was observed in experimental group but 1 case with complication was observed in control group.Conclusions Predeposit autotransfusion is an effective to avoid homologous blood transfusion and its complications for the patients with lumbar disc protrusion.Furthermore,the clinical effect is not significantly different between the predeposit autotransfusion patients and the allogenic blood transfusion patients.

Objective To detect the expression of melanoma antigen-1 gene (MAGE-l),melanoma antigen-3 gene (MAGE-3) and explore their clinical significance. Methods The expression of MAGE-1 and MAGE-3 in central tumor tissue, para-tumorous normal mucosa and tissue of resection-border were detected by RT-PCR. Results The expression of MAGE-1 was positive in 10 out of 33 (30.30 %) cases of rectal cancer, 4 out of 33 cases (12.12%) and 4 out of 33 cases (12.12 %) in para-tumorous normal mucosa and tissue of resection-border respectively; the expression of MAGE-3 was positive in 14 out of 33(42.42 %) eases of rectal cancer, 6 out of 33 cases (18.18 % ) and 5 out of 33 cases (15.15 %) in para-tumorous normal mucosa and tissue of resection-border respectively; 7 out of 33 cases (21.21%) was expressed MAGE-1 and MAGE-3 simutaneously; 17 out of 33 cases (51.51%) was expressed at least one of MAGE-1 and MAGE-3;the positive rates of MAGE-1 or MAGE-3 in center tumor tissue were significant higher than those of its para-tumorous and resection-border (P＜0.05). The expression of MAGE-1 or MAGE-3 was not related to age, sex, histological grades, metastasis to lymph nodes, duke stages (P＞0.05). Conclusion As their significance, MAGE-1, MAGE-3 protein may be used as a target molecule of immunotherapy for rectal cancer and indexes for follow-up study of rectal cancer as well.

Objective To investigate the significance of expression of toll-like receptor on medullary system cell and endothelial cell surface in PMN recruiting in severe acute pancreatitis.Method In this study,26 C3H/He-J mice and 26 C3H/He-N mice were derided into 4 groups by bone marrow transplantation:group A, mosaic of medullary system cell TLR4-/- and endothelial cell TLR4+/+,group B, mosaic of medullary system cell TLR4+/+ and endothelial cell TLR4-/- ;group C, homozygote ofTLR4+/+ ;group D, homozygote of TLR4-/-. SAP was induced to all these mice. Amylase, TRIA of granular cell in peripheral blood,TRL4 expression in pancreas, Naphthol AS-D chloroacetate esterase, MPO activity were tested. Results Amylase level increased in all mice, there was no differences of the amylase level among groups, TRIA of granular cell in peripheral blood in group B and C was significant higher than A and D(P<0.05), TLR4 in pancreas were positive in group A and C, negative in group B and D, AS-D count and M PO activity in group A and C were higher than that in group B and D (P < 0. 05 ). Conclusions It was endothelial cell, not peripheral blood granulocyte, that plays a key role in recuiting neutrophils that triggers severe acute pancreatitis.

Objective To determine the correlation between the expression of interleukin 8 (IL 8) and vascularity in human gastric carcinoma speciemens,and to provide a possible new clinical way for anti angiogenesis of tumor . Methods IL 8 mRNA expression was examined with reverse transcription polymerase chain reaction (RT PCR).IL 8 protein secretion level and its specific expression were determined by enzyme linked immunosorbent assay (ELISA).The number of blood vessels in the gastric carcinoma was determined by antibody against factor Ⅷ associated antigen and immunohistochemical analysis. Results Most of the tumors (26/29,89.7%) and metastatic lymph nodes (17/20,85%) expressed IL 8 at higher levels than that in corresponding normal mucosa of resection end of stomach and negative metastatic lymph node.The expression of IL 8 protein and IL 8 mRNA were at the same level.The level of IL 8 mRNA in the gastric carcinoma tissue correlated strongly and positively with vascularization (P= 0.001).Conclusions There is a positive correlation between IL 8 mRNA expression and the vascularity of gastric carcinoma.IL 8 produced by tumor cells may up regulate the microvascularization in the gastric cancer, and enhance the growth,spreading and lymph node metastasis in human gastric carcinoma.So decreasing the IL 8 expression might inhibit the microvascularization,growth and metastasis of gastric cancer.

Objective To investigate the role of expression of tall-like receptor 4 (TLR4) on medullary system cell (neutrophii, platelet) and endothelial cell surface in polymorphonuclear neutrophils (PMN) recruitment in rata with acute necrotizing pancreatitis. Methods C3H/He-J mice and C3H/He-N mice were divided into 4 groups by bone marrow transplantation: mosaic of medullary system cell TLR4-/- and endothelial cell TLR4 +/+ ; mosaic of medullary system cell TLR4 +/+ and endothelial cell TLR4-/-; mosaic of medullary system cell TLR4 +/+ and endothelial cell TLR4 -/-; mosaic of medullary system cell TLR4-/- and endothelial cell TLR4-/-, another control group was also established. ANP was induced in all these groups by intraperitoneal injection of cerulein and caudal vein injection of lipopolysaccharide. Serum amylase, pancreatic tissue naphthol AS-D chloroacetate esterase, MPO activity, TRL4 mRNA expression in peripheral blood granuloeyte was determined by RT-PCR, TRL4 protein expression in pancreatic tissue was measured by immunohistochemisty. Results Compared with that of control group, the levels of serum amylase in the 4 groups all significantly elevated and there was no difference among these 4 groups. Pathological scores of pancreas in the 4 groups were 5.52 ± 1.21, 5.18 ± 1.02, 2.03 ± 0. 82, 1.92 ± 0. 78, respectively; MPO activities were (1.834 ± 0. 170) U/g, (2. 596 ±0. 138) U/g, (0. 367 ±0. 018) U/g, (0. 202 ±0. 018) U/G, respectively; AS-D counts were 66.88 ± 2.17, 75.00 ± 2.43, 21.50 ± 2.38, 20.00 ± 2.19, respectively ; the expressions of TLR4mRNA of granular cell in peripheral blood were 0. 037 ± 1. 047 E-2, 1. 489 ± 8. 084 E-2, 1.470 ± 5. 210E-2, 0. 017 ± 6. 668 E-3, respectively; the 2 groups with endothelial eel1 TLR4 +/+ had strongly positive expression of TLR4 protein in vascular endothelial cell ; while the 2 groups with endothelial cell TLR4-/- had no expression; the pancreatic injuries, MPO activities, AS-D counts and TLR4 protein in the 2 groups with endothelial cell TLR4 +/+ were significantly higher than those in the 2 groups with endothelial cell TLR4-/-. Conclusions It was endothelial cell, not peripheral blood granulocyte, which played a key role in the process of neutrophil recruitment and pathological injure of ANP.