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Objective To investigate the effects of toll-like receptor 9 (TLR9) agonist CpG ODN2216 on the sensitivity of pancreatic cancer cell line PANC1's to gemcitabine.Methods The immunofluorescence staining method and Western blot method were used to examine the expression of TLR9 protein in PANC1 cells.The changes of sensitivity to gemcitabine after CpG ODN2216 treatment were examined by MTT assay.Results The TLR9 protein was highly expressed in PANC1 cells and the median inhibition concentration of gemcitabine against PANC1 cells was reduced from (1.23 ± 0.14) mg/L to (0.28 ± 0.13) mg/L after CpG ODN2216 treatment,and the difference between the two groups was statistically significant (P ＜0.01).After 0.01,0.10,1.00,10.00 mg/L gemcitabine treatment with CpG 0DN2216,the inhibition rates of PANC1 were (34.4 ±1.3)％,(43.5 ± 2.7)％,(76.3 ± 2.5)％,(95.3 ± 2.2)％ ; and without CpG ODN2216,the inhibition rates of PANC1 were (14.5 ± 0.9) ％,(23.5 ± 1.1) ％,(44.8 ± 1.4) ％,(63.6 ± 1.8) ％,and the difference between the two groups was statistically significant (P ＜ 0.01).Conclusions The sensitivity of PANC1 cells to gemcitabine can be enhanced by CpG ODN2216.
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Objective To investigate the expression and clinical significance of toll-like receptor 9 (TLR9) and hypoxia inducible factor 1 α (HIF-1 α) in pancreatic cancer.Methods The real-time RT-PCR technique,western blot method and immunohistochemical method were used to examine the expressions of TLR9 and HIF-1α in 30 samples of pancreatic cancer,para-cancerous tissues,and 10 samples of normal pancreatic tissues.The relationship between TLR9 and HIF-1 α was determined,and the correlations between their expressions and clinicopathological parameters were measured,and the impact on survival was detected.Results The levels of TLR9 mRNA and HIF-1 α mRNA expression in human pancreatic cancer tissues was 2.32(1.41～3.22) and 2.26 (1.62～ 2.89) folds as many as that in normal tissues.The levels of TLR9 mRNA and HIF-1α mRNA expression in para-cancerous tissues were 1.23 (1.18 ～ 1.28) and 1.36 (1.17 ～1.55) folds as many as that in normal tissues.The expressions in human pancreatic cancer tissues were significantly higher than those in para-cancerous tissues (t =2.642,P =0.023 ; t =4.076,P =0.001).The positive rates of TLR9 and HIF 1α protein were 73.3％ (22/30) and 70.0％ (21/30),and the corresponding values were 33.3％ (10/30) and 36.7％ (11/30) in para-cancerous tissues,while the corresponding values were 20％ (2/10) and 10％ (1/10) in normal tissues,which showed a decreasing trend (x2 =13.99,P =0.001 ;x2 =13.15,P =0.001).The expressions of TLR9 mRNA in human pancreatic cancer tissues was positively associated with HIF 1 α mRNA (r =0.537,P =0.003).The expressions of TLR9 protein was also positively associated with HIF 1α protein (r =0.511,P =0.001).The expressions of TLR9 and HIF 1α were positively correlated with the degree of tumor differentiation,TNM staging and lymph node metastasis,but were negatively correlated with the survival.Conclusions TLR9 and HIF-1α are over-expressed in pancreatic cancer and they are associated with malignant biological behavior and poor prognosis.
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Objective To investigate the expression of HIF-1 α and toll-like receptor 4 (TLR4) in human pancreatic cancer cell line panc-l under hypoxia.To observe the effect of HIF-1α in the regulation of TLR4 expression in pancreatic cancer cells under hypoxic conditions.Methods Cells were placed in airtight chamber or treated with CoCl2 to mimic tumor hypoxic micro environment.mRNA and protein levels of HIF-lα and TLR4 were detected by reverse transcription-polymerase chain reaction(RT-PCR) and flow cytometric analysis.By RNA interference( RNAi ) originated from small interference RNA (siRNA) to use siRNA was transfected into panc-1 cells.Western blot was used to detect gene scilencing effect on HIF-1α.RT-PCR and flow cytometric analysis was used to observe the change of TLR4 gene expression after HIF-1α gene silence.Results Under hypoxia,both mRNA and protein levels of TLR4 were up-regulated.The siRNA targeting HIF-1α gene down-regulated HIF-1α gene in panc-l cells efficiently,and TLR4 gene was down-regulated as well.Conclusions Hypoxia can increase protein level of TLR4 in pancreatic cancer cells.TLR4 signaling pathway together with HIF-1α may promote development of the pancreatic cancer.
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ObjectiveTo construct recombinant adenovirus vector containing human pancreatic and duodenal homeobox factor 1 (PDX1) and detect its expression in human umblical cord mesenchymal stem cells (HUCMSCs). MethodsPDX1 obtained by BgⅢ/XhoI enzyme digestion from pUC57-PDX1 was ligated into the recombinant shuttle vector pShuttle-GFP-CMV to obtain the recombinant shuttle plasmid pShuttle-GFP-CMVPDX1. pShuttle-GFP-CMV- PDX1 was shifted to pAdxsi vector to obtain pAdxsi-GFP-PDX1 virus plasmid. The recombinant plasmid was packaged and amplified in 293 cells. The expression of PDX1 gene and protein in HUCMSCs was detected by fluorescence microscopy, RT-PCB, immunofluorescence, immunohistochemistry, and Western Blot. ResultsPDX1 gene was inserted correctly into shuttle plasmid and the recombinant adenovirus vector was successfully constructed according to the results of sequence and enzyme digestion identification. The adenovirus was effectively transfected into HUCMSCs. RT-PCR verified that PDX1 mRNA was positively expressed in HUCMSCs. Expression of PDX1 protein in the nuclear of HUCMSCs was found by immunofluorescence assay, immunohistochemistry and Western Blot. ConclusionThe adenovirus vector containing PDX1 gene is successfully constructed and effectively expressed in HUCMSCs.
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Objective To investigate risk factors for early postoperative complications after pancreaticoduodenectomy(PD).Methods Clinical data of 221 patients undergoing PD in our hospital between September 2005 and January 2009 were collected and reviewed retrospectively,the risk factors in relation to early postoperative complications were analyzed with SPSS 15.0 software.Results The incidence of early postoperative complications was 17.6％.Univariate analysis showed that age ( ≥65 years),preoperative total serum bilirubin level ( ≥ 171 μmol/L),preoperative serum albumin level ( ≤30 g/L),intraoperative blood transfusion ( ≥ 1000 ml),operation time ( ≥5 h) were the relative risk factors,Logistic regression analysis revealed that age (≥ 65 years),preoperative serum albumin level ( ≤30 g/L),intraoperative blood transfusion ( ≥ 1000 ml),operation time ( ≥5 h) were the independent risk factors for early complications.Conclusions The risk of postoperative complications after PD is still high,so the dicision to perform a PD should be strictly balanced against its risk.For older or poor risk patients,preoperative management should be enhanced.The set up of surgical team may help standardize operation procedures control intraoperative bleeding,therefore decrease the incidence of postoperative complications after PD.
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Objective To detect the expression of Toll-like receptor 9 (TLR9) in pancreatic cancer and to study the effect of CPG ODN2216 on the biological behavior of pancreatic cell carcinoma, and to explore their clinical significance. Methods Immunohistochemical method was used to examine the expression of TLR9 protein in pancreatic cancer tissue and immunofluorescence staining was also performed to detect TLR9 protein expression in pancreatic carcinoma cells. In vitro cell adhesion, wound-healing scrape assay, transwell invasion assay and cell colony formation assay were performed to assess the effect of CPG ODN2216 on the invasive properties of Panc-1 cells. Results TLR9 were highly expressed in the pancreatic cancer tissue and pancreatic carcinoma cells. In vitro experiments as cell spreading assays, cell adhesion, colony formation assay and invasion assays showed the cell adhesion and cell motility properties of CPG ODN 2216 group to be apparently weakened compared with the control group. MTT assay showed cell proliferation ability in the CPG ODN group to be notably decreased, and CPG ODN2216 had inhibitive effects on the growth of panc-1 cells in a dose and time-dependent manner. Conclusions TLR9 gene was correlated with the invasive and metastatic potentials of pancreatic carcinoma. The used of CPG ODN2216 induced the inhibition of migration and invasion of the Panc-1 cell line.
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Objective To detect the expression of Toll-like receptor 9 (TLR9) in pancreatic cancer,and to explore its clinical significance. Methods The real-time PCR, Western blotting and immunohistochemistry were used to examine the expression of TLR9 in 30 samples of pancreatic cancer and the adjacent tissues, and 10 samples of normal pancreatic tissues. The relationships of TLR9 with clinicopathological parameters were analyzed. Results Compared with normal pancreatic tissues, the amplification value of TLR9 mRNA expression was 2.32 fold (1.41 ～ 3.22 ) in human pancreatic cancer, was 1.23 fold (1.18 ～ 1.28) in paracancerous tissues, respectively, and the difference was statistically significant (P ＜ 0.05 ). The percentage of positive cells expressing TLR9 protein in human pancreatic cancer tissues,paracancerous tissues and normal tissues were 73.3% (22/30), 33.3% (10/30) and 20.0% (2/10), and the relative expressions of TLR9 protein were 0.780 ±0. 026,0. 400 ±0.018,0. 173 ±0.043 ,respectively, and the difference was statistically significant( P ＜ 0.05 ). The highly expressed TLR9 was positively correlated with the degree of tumor differentiation, TNM stage and lymph node metastasis. Conclusions TLR9 was highly expressed in pancreatic cancer tissues. TLR9 expression plays a role in the canceration of pancreatic tissues.
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Objective To explore the method how PDX1 gene modified mesenchymal stem cells (MSCs) from human umbilical cord can be differentiated into islet β-like cells in vitro. Methods Recombined adenovirus vectors inserted with PDX1 (Adxsi-CMV-PDX1) was transfected into MSCs and multiple cytokines was combined to induce differentiation. The expressions of PDX1, insulin, ngn3, glut2,NKX6.1 were examined by RT-PCR and immunofluorescence staining. The levels of insulin and C peptide secretion were examined by chemiluminescence immunoassay. Flow cytometry was used to determine the positive rate of insulin cells. Results After Adxsi-CMV-PDX1 transfection and cytokines induction, MSCs were transformed from short spindle shape to long spindle shape and aggregated into islet-like cell clusters.Dithizone staining of these cells showed bright red color. PDX1, ngn3, NKX6.1, insulin, glut 2 mRNA were expressed in cells 17d after induction. Insulin and C peptide were expressed in cytoplasm. The levels of insulin and C peptide in cell culture supernatant were (473.1 ± 51.5)mU/L and (1.61 ± 0.41)ng/ml; the levels of insulin and C peptide secretion were (964.4 ± 68.1) mU/L, (3.72 ± 1.52) ng/mL, respectively, with 25 mmol/L glucose stimulation for one hour. Insulin (+) cells rate (11.6 ± 4.8) %. Conclusions Adxsi-CMV-PDX1 combined with cytokines can induce MSCs from human umbilical cord to differentiate into islet β-like cells.They can secret insulin and C peptide, and have the sensitivity to the stimulation of glucose.
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Objective: To study the mechanism and effect of Chinese medicine Yiqi Tongyang Huatan prescription(益气通阳化痰方,YQTYHT) on hyperlipidemia and atherosclerosis(AS).Methods: The rabbit′s model was established by feeding the food containing high cholesterol,and 32 rabbits were randomly divided into four groups(each n=8): normal group,model group,Huoxue Huayu prescription treatment group(活血化瘀方,HXHY group) and YQTYHT treatment group(YQTYHT group).Before the experiment,at the end of 6 th and 12 th weeks after the experiment,the following items were measured: blood lipids platelet aggregation rate,platelet adhesion rate,malondialdehyde(MDA),endothelin-1(ET-1),nitric oxide(NO),the ratio of the lipid plaque area and the thickness of the endothelial plaque.Results: After feeding the food containing high cholesterol,the blood lipids,platelet aggregation rate,platelet adhesion rate,MDA,ET1,the ratio of lipid plaque area and the thickness of plaque were elevated significantly,while serum NO3-/NO2-decreased significantly in model group.Compared with the model group,the blood lipids were lower in the two treatment groups,and the blood lipid regulating effect in YQTYHT group was significantly better than that in HXHY group.Platelet aggregation rate and platelet adhesion rate of YQTYHT group and HXHY group were lower than those in the model group,but the degree of lowering in YQTYHT group and HXHY group had no significant differences.The serum MDA of YQTYHT group and HXHY group was lower than that in model group,and the lowering effect in YQTYHT group was better than that in HXHY group.YQTYHT group and HXHY group had a significant increase on the level of serum NO3-/NO2-and a decrease on the content of plasma ET-1,furthermore,the effects in the former group were significantly better than those of the latter group.The area of atherosclerotic plaque coverage in aorta and the thickness of plaque of HXHY group were larger than those in YQTYHT group(both P