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.
ObjectiveTo investigate the expression and significance of cysteine-rich protein 61 (Cyr61) in patients with chronic obstructive pulmonary disease (COPD).MethodsBetween September 2017 and September 2018, 27 patients with benign tumor needing to surgical therapy, were divided into COPD group (15 patients) and non-COPD group (12 patients), according to lung function. Lung tissues were selected at the distance at least 5 cm from the tumor. The levels of Cyr61, interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in serum were determined by enzyme-linked immunosorbent assay. Meanwhile, the expressions of Cyr61 in lung tissues were measured by immunohistochemistry technology between two groups. Furthermore, correlations among Cyr61, IL-8, MCP-1, smoking index, forced expiratory volume in the 1st second as percentage of predicted values (FEV1%pred), scores of COPD Assessment Test (CAT) were analyzed.ResultsSerum Cyr61, IL-8, MCP-1 levels were significantly higher in patients with COPD than in the non-COPD group (P<0.05), (2409.80±893.87)pg/mL, (76.27±10.53)pg/mL, (173.67±42.64)pg/mL vs. (1065.42±158.83)pg/mL, (57.33±8.29)pg/mL, (138.42±27.62)pg/mL, respectively. By immunohistochemistry technology, the expression levels of Cyr61 in lung epithelial cells and in lung macrophage cells of COPD patients were higher than in the non-COPD group (P<0.01). Positive correlations were found between serum IL-8, serum MCP-1, CAT scores, smoking index and serum Cyr61 (r=0.674, 0.566, 0.602, and 0.755, P=0.006, 0.028, 0.018, and 0.003, respectively) in COPD group. Furthermore, in COPD group, there were also positive correlations between serum IL-8, serum MCP-1, CAT scores, smoking index and intrapulmonary Cyr61 (r=0.542, 0.635, 0.809, and 0.580, P=0.037, 0.011, 0.001, and 0.038 respectively). Inverse correlation was found between serum Cyr61 and FEV1%pred (r=–0.772, P<0.01), and the same as between intrapulmonary Cyr61 and FEV1%pred (r=–0.683, P<0.01).ConclusionsCyr61 highly expresses in serum and in lung tissues of patients with COPD, and its expression is correlated with lung function of patients. The results indicate that Cyr61 may interact with IL-8 and MCP-1 in the pathogenesis of chronic obstructive pulmonary disease.
ObjectiveTo observe the effect of Crocin on structure and the expression of tumor necrosis factor-alpha; (TNF-alpha;) and interleukin-1beta; (IL-1beta;) in rat retina after injury by ischemia-reperfusion. Methods A total of 80 Sprague-Dawley male rats at the age of 8 -10 weeks were divided into control group, model group, low-dose Crocin group and high-dose Crocin group, with 20 rats in each group. The rats of control group were not treated. The rats in model, low-dose Crocin and high-dose Crocin group were induced with normal saline by anterior chamber perfusion creating a retinal ischemia-reperfusion (RIR) model. The rats of the low-dose Crocin and highdose Crocin group received intraperitoneal injection with different doses of Crocin solution (5 mg/kg, or 50 mg/kg) 30 minutes prior to ischemic injury and one time per day after successful RIR. Optical microscopy was used to observe the retinal structure. Enzymelinked immunosorbent assay (ELISA) was used to measure the expression of TNF-alpha; and IL-1beta; 6, 12, 24 and 48 hours after RIR. ResultsThe retinal structure of control group was normal. Pathological changes were found in the RIR model and low-dose Crocin group, such as retinal edema, disorganized structure and loosely packed cells. The degree of pathological changes in lowdose Crocin group was less than the RIR model group. The retinal structure of high-dose Crocin group was similar to the control group. The expression of TNF-alpha; was the highest at 24 hours after modeling, while the expression of IL-1beta; was the highest at 12 and 48 hours after RIR modeling. Six, 12, 24 and 48 hours after RIR modeling, compared with the control group, the TNF-alpha; expression of model (t=5.42, 7.94, 9.32, 9.18;P＜0.05 ), low-dose Crocin (t=3.94, 4.12, 4.98, 3.84;P＜0.05) and high-dose Crocin group (t=2.13, 2.34, 2.96, 2.78;P＞0.05) were increased. Compared with the RIR model group, the TNF-alpha; expression of low-dose Crocin (t=3.95, 4.56, 4.01, 5.12) and high-dose Crocin group (t=5.23, 7.65, 7.74, 7.63) was decreased. Compared with the control group, the IL-1beta; expression of model (t=7.23, 7.87, 7.15, 15.60), low-dose Crocin (t=5.65, 5.10, 5.54, 6.87;P＜0.05) and high-dose Crocin group (t=4.38, 5.21, 4.56, 4.75) was increased (P＜0.05). Compared with the model group, the IL-1beta; expression of low.dose Crocin group was decreased significantly 48 hours after RIR modeling (t=7.56,P＜0.05); but it decreased significantly at each time point in high-dose Crocin group (t=6.94, 5.36, 6.05, 10.50;P＜0.05). Conclusion Crocin can improve the retinal pathologic changes, while down-regulating TNF-alpha; and IL-1beta; expression in RIR rats.
ObjectiveTo examine and compare the value of procalcitonin (PCT), C-reactive protein (CRP) and interleukin (IL)-6 in diagnosing fetal sepsis in premature neonates. MethodsPreterm neonates with premature rupture of membrane between January 2010 and September 2012 were screened, and the serum levels of PCT, CRP and IL-6 were detected in the first day of life. All preterm neonates were divided into two groups according to the development of sepsis (45 cases with sepsis and 39 cases without sepsis). ResultsThe levels of PCT, CRP and IL-6 in premature neonates with sepsis were all significantly higher than those without sepsis. The cut-off value of PCT in diagnosis of sepsis was 2.14 μg/L, with a sensitivity and specificity of 76% and 85% respectively; the cut-off value of CRP in diagnosis of sepsis was 7.90 mg/L, with a sensitivity and specificity of 67% and 61% respectively. For IL-6, the cut-off value in diagnosis of sepsis was 13.80 ng/L, and its sensitivity and specificity were high to 90% and 94%, respectively. ConclusionIL-6 is the most reliable biochemical marker for the detection of early-onset sepsis in preterm neonates with premature rupture of membrane.
Objective To investigate the impacts of cytokines （interleukin-4，IL-4；tumor necrosis factor-α，TNF-α） and medications of bronchial asthma （dexamethasone，aminophylline，salbutamol） on the activity of histamine N-methyltransferase（HMT） in tracheal epithelial cells.Methods BEAS-2B bronchial epithelial cells were cultured and treated with different concentration of TNF-α, IL-4, dexamethasone, salbutamol and aminophylline respectively. The activity of HMT in BEAS-2B cells was determined by high performance liquid chromatography.Results The activity of HMT in tracheal epithelial cells was (50±7) pmol?min-1?mg pro-1.TNF-α and IL-4 lowered the activity of HMT significantly at the concentration equal to or higher than 1 ng/mL and 5 ng/mL respectively，and reached the maximum inhibitory effect at the level of 10 ng/mL.Dexamethasone and aminophylline could ameliorate distinctly the inhibitory effect of TNF-α on the activity of HMT, while salbutamol had no significant inhibitory effect.Conclusions TNF-α and IL-4 exert the lowering effect on the activity of HMT，which would be one important cause of airway hyperreactivity.Glucocorticoids and theophyllines are administered to treat asthma partly due to its relieving mechanism of TNF-α negative effects on HMT.
Objective To study the regulative effect of angelica sinensis on cellular immune function in perioperative patients with obstructive jaundice. Methods Fourteen patients with obstructive jaundice were injected with angelica before and after operation for 14 days. The activity of IL-2 and the expression of IL-2R in lymphocytes in peripheral blood were measured, respectively. Results The activity of IL-2 and the expression of IL-2R decreased significantly in patients with obstructive jaundice (P＜0.01). The activity of IL-2 and the expression of IL-2R in peripheral blood lymphocyte increased significantly before and after operations (after treatment using angelica) (P＜0.01), though there was a little decrease after operation but they were still higher than that befor using angelica.Conclusion It maybe useful to use angelica to improve the cellular immune function in patients with obstructive jaundice.
Objective To observe the effect of celastrol on the secretion of interleukin (IL)-17 in peripheral blood mononuclear cells in patients with sympathetic ophthalmia (SO), and its possible mechanisms. Methods Venous blood samples were extracted from 10 cases of sympathetic ophthalmia patients and 10 health objectives. The peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation and then were divided into 4 groups. Group A (control group): PBMCs of health objectives; Group B: PBMCs of SO patients; Group C: PBMCs of SO patients with 0.5 μmol/L celastrol in the medium; Group D: PBMCs of SO patients with 1 μmol/L celastrol in the medium. After culturing the cells for 3 days, the supernatant of 4 groups was collected, and the levels of IL-23 and IL-17 were detected by enzyme-linked immuno sorbent assay (ELISA). Then, the 50 ng/ml rIL-23 was added into the medium of group A which was the group A1; the 50ng/ml rIL-23 and 1 μmol/L Cela were added into to the medium of group A which was the group A2. For the medium of group B, the 50 ng/ml rIL-23 was added into the medium which was the group B1; the 50 ng/ml rIL-23 and 1 μmol/L celastrol were added into to the medium of group B which was the group B2. After culturing for 3 days, the supernatant of cells of these 4 groups was collected, and the levels of IL-17 were detected by ELISA. Results In group A, the levels of IL-23 and IL-17 were (228.43±17.27) pg/ml and (220.55±31.15) pg/ml respectively. In group B, the levels of IL-23 and IL-17 were (513.85±36.46) pg/ml and (866.77±72.92) pg/ml respectively. In group C, the levels of IL-23 and IL-17 were (381.07±20.93) pg/ml and (517.43±54.87) pg/ml respectively. In group D, the levels of IL-23 and IL-17 were (237.14±17.97) pg/ml and (242.89±34.09) pg/ml respectively. Between group A and D, there was no statistically significant difference in IL-23 or IL-17 level (P＞0.05); but when comparing other groups, the differences were statistically significant (P＜0.05). The levels of IL-17 in group A1 and group A2 were (428.43±24.53) pg/ml and (229.15±23.28) pg/ml and the difference was statistically significant (P＜0.05). The levels of IL-17 in group B1 and group B2 were (1373.39±89.51) pg/ml and (571.01±94.88) pg/ml and the difference was statistically significant (P＜0.05). Conclusion Celastrol can inhibit the secretion of IL-17 by PBMCs in SO patients via inhibiting the secretion of IL-23.
ObjectiveTo investigate the effects and clinical significance of edaravone on serum tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1) in elderly patients with obstructive sleep apnea hypopnea syndrome (OSAHS).MethodsA total of 90 elderly patients with moderate to severe OSAHS confirmed by polysomnography were recruited from North China University of Science and Technology Affiliated Hospital in February 2016 to October 2017. According to random number table method the OSAHS patients were randomly divided into group A (n=30), group B (n=30) and group C (n=30). Group A received continuous positive airway pressure treatment for six months, group B received edaravone therapy and continuous positive airway pressure treatment for six months, and group C only received edaravone therapy for six months. The changes of serum TNF-α, IL-6 and ICAM-1 were detected by enzyme-linked immunosorbent assay before and after treatment.ResultsThe differences of serum TNF-α, IL-6 and ICAM-1 before treatment in the three groups were not statistically significant (P>0.05). Compared with before treatment, the levels of serum TNF-α, IL-6 and ICAM-1 decreased in the three groups (P<0.05). After six months of treatment, the levels of serum TNF-α, IL-6 and ICAM-1 decreased in group A and group B compared with group C (P<0.05), and decreased significantly in group B compared with group A (P<0.05).ConclusionEdaravone can inhibit the expressions of serum TNF-α, IL-6 and ICAM-1 in elderly patients with moderate to severe OSAHS, and thereby reduce vascular endothelial dysfunction and injury.
Objective To observe the levels of malonaldehyde (MDA) , interleukin-8 (IL-8) and tumor necrosis factor-α(TNF-α) in lung tissues of subjects with or without chronic obstructive pulmonary disease (COPD) , and investigate their roles in the the pathogenesis of COPD. Methods The content of MDA, IL-8 and TNF-αin lung tissues of smokers with COPD (n=9) , ex-smokers with COPD (n=8) , non-smokers with COPD (n=7) , healthy smokers (n=9) , healthy ex-smokers (n=8) and healthy nonsmokers (n=6) was measured with enzyme-linked immunosorbent assay ( ELISA) and corrected by creatinine. Results The levels of MDA, IL-8 and TNF-α in lung tissues of the COPD patients were significantly higher than those in the healthy subjects (Plt;0.05) , which were also significantly higher in the smokers when compared with the non-smokers (Plt;0.05) , whether suffering from COPD or not. In the COPD patients, not the levels of IL-8 but MDA and TNF-αin lung tissues of the smokers were significantly higher than those in the ex-smokers (Plt;0.05) ; whereas in the healthy cases, no statistical significance was revealed between the smokers and the ex-smokers on the levels of MDA and IL-8 in lung tissues except TNF-α( Pgt;0.05) . Conclusion The abnormal increase of MDA, IL-8 and TNF-αin lung tissues caused by chronic smoking may play an important role in the the pathogenesis of COPD.
Objective To investigate the effects of ginkgo biloba extract （GBE） on expressions of IL-1β, IL-6,and TNF-α in the pancreas and brain tissues of rats with severe acute pancreatitis （SAP）, and further to explore the pathogenesis of SAP and the efficacy of GBE on brain injury. Methods Fifty-four Winstar rats were randomly divided into normal control group, model group, and treatment group, with 18 rats for each group. For rats in the normal control group, only conversion of pancreas was performed by abdomen opening , followed by wound closure immediately. For rats in the model group and treatment group, 5% sodium taurocholate hydrate were injected under pancreatic capsule to establish SAP model, and then GBE and normal saline were infected into intra-abdomen repeatedly every 8 hours, respectively. At 6 h, 12 h, and 24 h after the model establishment, experimental samples were extracted and serum amylase was detected. Pathogenic scoring for pancreas tissues was performed under light microscopy, and immunohistochemistry method was employed to detect the expression levels of IL-1β, IL-6, and TNF-α in pancreas and brain tissues. Results For the treatment group, both serum amylase and pancreas scoring were significantly lower than those of the model group （P＜0.01）. At 24 h after model establishment, the expressions of IL-1β, IL-6, and TNF-α of pancreas tissues in model group were significantly higher than those at 6 h and 12 h （P＜0.05 or P＜0.01）, but no significant differences wereobserved in treatment group （P＞0.05）. The expressions of IL-1β, IL-6, and TNF-α of brain tissues in model group were significantly higher than those at 6 h and 12 h （P＜0.05 or P＜0.01）, but in treatment group decreased （P＜0.05 or P＜0.01）. The expressions of IL-1β, IL-6, and TNF-α in the treatment group were significantly lower than those of the model group at same time （P＜0.01）. Conclusions During SAP, the expressions of IL-1β, IL-6 and TNF-α in pancreas and brain tissues increased obviously. GBE showed suppressing and scavenging effects on IL-1β, IL-6 and TNF-α in pancreas and brain tissues.