ObjectiveTo investigate the expressions of IL-10,tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in serum and lung tissue of COPD rats in order to elucidate the potential mechanism of airway inflammation. MethodsForty-five healthy adult male SD rats were randomly divided into a COPD model group (n=30) and a normal control group (n=15). The COPD rat model was established by intratracheal instillation of lipopolysaccharide (LPS) and exposure to cigarette smoke for 28 days. The concentrations of IL-10,TNF-α and IFN-γ in serum and lung tissue were measured by ELISA. ResultsTNF-α level of serum and lung tissue in the COPD model group increased significantly compared with the control group(P<0.05),while the levels of IFN-γ and IL-10 decreased significantly[serum:(44.68±8.67) ng/L vs. (75.96±10.59) ng/L;lung tissue:(64.55±9.03) ng/L vs. (94.06±8.71) ng/L,P<0.01]. The level of IL-10 in serum and lung tissue was negatively correlated with TNF-α (serum:r=-0.67,lung tissue:r=-0.80,P<0.01). The level of IL-10 in serum and lung tissue was positively correlated with IFN-γ (serum:r=0.64,lung tissue:r=0.72,P<0.01). The level of IL-10 in serum and lung tissue was negatively correlated with the percentage of neutrophils(serum:r=-0.70,lung tissue:r=-0.67,P<0.01). ConclusionIn COPD rats,down regulation of IL-10 plays an important role in regulation of airway inflammation.
Objective To construct recombinant adenovirus vector co-expressing human interleukin (hIL)-10 and green fluorescent protein (GFP) for study of the expression of genes of interest in vascular smooth muscle cells （VSMCs）. Methods hIL-10 cDNA was amplified from pUCm-T/hIL-10 cDNA using polymerase chain reaction (PCR), and cloned into shuttle plasmid pShuttle-IRES-hrGFP-1. Kanamycin resistance screeninged for recombinant plasmids, which were linealized with PmeⅠand transformed into BJ5183-AD-1 containing pAdEasy-1 by electroporation after determining the insert’s sequence correct by NotⅠ and XholⅠdigestion， sequencing and basic local alignment search tool (BLAST). Prepared recombinant adenovirus plasmids were transformed into XL10-Gold cells. Amplified plasmids were transfected to AD-293 cells for packaging after being linearized with PacⅠ. PCR was used to determine target gene; The titer of the recombinant adenovirus was measured. VSMCs were transfected by recombinant adenovirus and viewed under fluorescence microscope. hIL-10 concentration in transfected VSMCs supernant was measured by enzyme linked immune sorbent assay (ELISA). Results Recombinant shuttle plasmids contained interest gene. Recombinant adenovirus had 30 kb and 3 kb fragments after digestion with PacⅠ. PCR indicated that the recombinant adenovirus contained interest gene. The titer of recombinant adenovirus was 3×10１0 efu/ml. Transfected VSMCs had GFP expression and hIL-10 concentration in supernatant was 25 ng/106 cells. Conclusion The recombinant adenovirus co-expressing hIL-10 and GFP is successfully constructed and could effectively express in VSMCs, this lays the foundation for the gene therapy of vascular intimal hyperplasia.
Objective To construct a regulatable plasmid containing single chain fusion gene of murine interleukin-12 (mIL-12) which was regulated with mifepristone (RU486) and explore its expression in vitro. Methods The p40 and p35 subunit sequence of mIL-20 were respectively obtained from the plasmid GCp35Ep40PN by polymerase chain reaction (PCR) and they were cloned into pCA14 plasmid after introducing a linker by overlap PCR. The single chain mIL-12 gene was comfirmed by sequencing and subcloned into pRS-17 vector which contains RU486 regulator cassette. The positive clone named pRS-RUmIL-12 was identified by restriction endonuclease digestion and PCR. Lipofectamine 2000 was used to transfect the pRS-RUmIL-12 to HEK293 cells followed by manufacturer’s recommendations. The protein concentration of mIL-12 induced with RU486 in supernatant of the transfected HEK293 cells was measured by ELISA. Results The sequence of single chain mIL-12 what we obtained was the same as the expected result. The results of restriction endonuclease digestion and PCR showed that the RU486-inducible regulatory vector (pRS-RUmIL-12) was successfully constructed. No significant mIL-12 protein concentration in supernatant of HEK293 cells activation was measured without the inducer RU486, whereas higher concentration of the mIL-12 protein was observed in the presence of RU486. The relationship of concentration of the mIL-12 protein and RU486 was positive correlated under definite range. Conclusion A regulatable eukaryotic expression plasmid of mIL-12 single chain fusion gene was constructed, which could be used in the further research of gene regulation and gene therapy.
【Abstract】ObjectiveTo explore the effect of glutamine on immune function of rat with obstructive jaundice and its possible mechanism. MethodsFifty male Wistar rats were randomly divided into three groups: Control group (n=10)， obstructive jaundice group (n=20) and glutamine treatment group (n=20). The serum concentration of TNF-α, IL-10 was detected by using radioimmune method. Liver function was measured through automated biochemistry analyzer. The animal model of obstructive jaundice was established by ligating the rat’s common bile duct. Bacteria cultures were performed with the rat’s tissues of lung, spleen, liver and kidney respectively. ResultsCompared with control group, obstructive jaundice group showed statistically lower serum level of TNF-α, and statistically higher serum level of IL-10, TBIL, ALT and AST during the first and the second week after ligation of common bile duct. During the first and second week after administration of glutamine, the serum TNF-α of glutamine treatment group was statistically higher than that in control group and obstructive jaundice group. Meanwhile, glutamine treatment group showed statistically lower serum level of IL-10, TBIL, ALT and AST than obstructive jaundice group. There were statistically less bacteria translocations in glutamine treatment group than those in obstructive jaundice group. Conclusion Glutamine can increase the immune function by changing serum concentration of TNF-α, IL-10 and decrease the bacteria translocation.
【Abstract】Objective To study the influence of early hemofiltration on plasma concentrations of proinflammatory cytokines TNF-α and IL-1β and their transcription levels in severe acute pancreatitis (SAP) pigs. Methods The model of SAP was induced by retrograde injection of artificial bile into pancreatic duct in pigs. Animals were divided randomly into two groups: SAP hemofiltration treatment group (HF group, n=8) and SAP no hemofiltration treatment group (NHF group, n=8). TNF-α and IL-1β plasma concentrations were measured by ELISA. Their transcription levels in the tissues of pancreas, liver and lung were assayed by semi-quantitative reverse transcription polymerase chain reaction. Results After hemofiltration treatment, the plasma concentrations of TNF-α and IL-1β increased gradually but were lower than those of NHF group at the same time spot 〔at 6 h after hemofiltration treatment, (618±276) pg/ml vs (1 375±334) pg/ml and (445±141) pg/ml vs (965±265) pg/ml, P＜0.01〕. At 6 h after hemofiltration treatment, the transcription levels of TNF-α and IL-1β in tissues of pancreas, liver and lung were lower than in NHF group (57.8±8.9 vs 85.7±17.4, 48.0±8.1 vs 78.1±10.2, 46.2±9.6 vs 82.4±10.5; 55.9±9.0 vs 82.2±15.7, 40.6±9.2 vs 60.0±10.6, 35.7±9.8 vs 58.1±9.3, P＜0.01). Conclusion Early hemofiltration can reduce TNF-α and IL-1β plasma concentrations and transcription levels in SAP pigs.
ObjectiveTo find out an effective method for amplification and purification of dendritic cells(DC) from peripheral blood of patients with pancreatic carcinoma. MethodsPeripheral blood mononuclear cells were purified from peripheral blood of health volunteers(control group,10 cases) and patients with pancreatic carcinoma (experimental group,12 cases) with incubation of granulocyte/macrophage colonystimulating factor(GMCSF) and interleukin4(IL4).The quality of DC were detected by immumofluorescence method and the expression levels of HLADR and B72 on DC were detected by flow cytometer after and before DC incubation with GMCSF and the IL4. ResultsThe expression level of HLADR and B72 of DC in experimental group were significantly less than those in control group(P＜0.01).DC in experimental group was significantly proliferated in the presence of GMCSF and IL4(P＜0.01).On day 7,the expression level of HLADR and B72 of DC in experimental group were significantly increased(P＜0.01) and there was no difference versus control group(Pgt;0.05).ConclusionIt is suggested that combination of GMCSF and IL4 can selectively and effectively enhance proliferation and immune function of DC from peripheral blood of patient with pancreatic carcinoma.
Objective To study the change in serum levels of soluble CD14, tumor necrosis factor-α, E-selectin, interleukin-10 and mean arterial pressure, as well as their relationship to infection during the pathophysiologic process in endotoxemia of rabbits. Methods Sixteen rabbits were randomly divided into two groups: group A, as a control group; group B, endotoxemia group. The model of rabbit with endotoxemia were used. Endotoxin at a dose of 1.5 mg/(kg·h) or 3 mg/(kg·h) was continuously infused through external jugular vein within 2 hours, 1 hour respectively. The change of levels of serum soluble CD14, tumor necrosis factor-α, interleukin-10 and E-selectin were observed at 0 (time before infusion of endotoxin), 30, 60, 120, 180, 240, 300 minutes, while mean arterial pressure was measured by polygraphy system. Results In the group B,there was an increase of content of soluble CD14,tumor necrosis factor-α,interleukin-10 and E-selectin following 30, 120 minutes respectively,and mean arterial pressure was lower than that of group A at same time points. Conclusion The results suggest that soluble CD14,tumor necrosis factor-α,interleukin-10 and E-selectin may play an important role during the change of infection and that these changes may be closely related with severe infection.
Objective To investigate the effects of cimetidine on the red cell immune function and interleukin-2(IL-2) in rats with obstructive jaundice. Methods Sixty SD rats were divided into bile duct ligation(BDL) group, cimetidine therapy (BDLC) group and sham operation(SO) group respectively. The red cell immue function and serum IL-2 level were determined with the red cell yeast-rosttes test and radioimmunoassay respectively. Results The red blood cell C3b receptor rosette rate(RBC-C3bRR), the red blood cell immune complex rosette rate(RICR), the red blood cell C3b receptor rosette-forming excited rate(RFER) and serum IL-2 level were significantly lower in BDL group as compared with SO group, the red blood cell C3b receptor rosette-forming inhibitory rate(RFIR) in BDL group was higher than that of SO group. After 7 days’ cimetidine therapy RBCC3bRR, RICR, RFER and IL-2 became higher than those of BDL group, but RFIR was lower than that of BDL group. Conclusion Supplemental cimetidine can significantly enhance the impaired red cell immune function and IL-2 production in rats with obstructive jaundice.
Objective To evaluate the effect of inducing differenation of all trans retinoic acid (ATRA) on immunologic function of patients with gastric cancer. Methods T-lymphocyte subsets(T-Ls) and interleukin-2 receptor(sIL-2R) of 56 patients with gastric cancer after treatment of ATRA were studied. Results In radical gastric cancer resection group, the serum CD3,CD4 and CD4/CD8 rate were higher and sIL-2R were lower than those in the control group, after treatment of ATRA, CD3,CD4 and CD4/CD8 rate and sIL2R were as high as those in the control group. In the non-operative or palliative gastric resection group, CD3,CD4 and CD4/CD8 rates were increased markedly and the serum sIL-2R was decreased significantly than those in the control group. Conclusion ATRA inducing differenation can improve the immunity of the patients with gastric cancer.
To evaluate the effect of intraperitoneal hyperthermo-chemotherapy (IPHC) on immunologic function of patients with gastrointestinal malignant tumor (GMT). The authors determined the serum T-lymphocyte subsets (T-LS) and interleukin-2 receptor (sIL-2R) of 32 patients with GMT after IPHC, and compared the two indexes with 20 healthy control group. Results: Before IPHC the serum CD＋3, CD＋4, CD＋8 and CD＋4/CD＋8 were higher and sIL-2R were lower than control group, after IPHC, CD＋3, CD＋4, CD＋8 and CD＋4/CD＋8 increased obviously (Ｐ＜0.01) and the serum sIL-2R decreased significantly (Ｐ＜0.01). Conclusion: The IPHC can improve the patients immunologic function.