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Objective:To evaluate the role of epidermal growth factor (EGF) in repair of lung tissues in mice with acute respiratory distress syndrome (ARDS).Methods:Fifty SPF male C57BL/6 mice, aged 6-8 weeks, weighing 21-23 g, were divided into 5 groups ( n=10 each) using a random number table method: control group (group C), EGF group, LPS+ PBS group, LPS+ EGF group and AG1478+ LPS+ EGF group.PBS 0.1 ml was intraperitoneally injected in group C. EGF 10 μg (0.1 ml) was intraperitoneally injected in group EGF.The equal volume of PBS and EGF 10 μg was intraperitoneally injected at 12 h after tracheal infusion of LPS in group LPS+ PBS and group LPS+ EGF, respectively.EGF receptor (EGFR) antagonist AG1478 1 mg was intraperitoneally injected, 30 min later LPS was tracheally instilled, and 12 h later EGF 10 μg was intraperitoneally injected in group AG1478+ LPS+ EGF.ARDS model was developed by endotracheal instillation of LPS 3 mg/kg.The mice were sacrificed on the 1st and 5th days after development of the model, and lung tissues were obtained for microscopic examination of the pathological changes which were scored after HE staining.Bronchoalveolar lavage was performed on 5th day after development of the model and before sacrifice, and bronchoalveolar lavage fluid (BALF) was collected to detect total protein concentration (by BCA method) and IL-6 and TNF-α concentrations (by enzyme-linked immunosorbent assay). Lung tissues were obtained for determination of the wet/dry lung weight ratio (W/D ratio), expression of lung surfactant associated protein C (SP-C) and proliferating nuclear antigen (PCNA) (by immunofluorescence method), and expression of EGFR, phosphorylated EGFR (p-EGFR), protein kinase B (Akt), and phosphorylated Akt (p-Akt) (by Western blot). Results:Compared with group C, the pathological score, W/D ratio, concentrations of total protein, IL-6 and TNF-α in BALF and neutrophil count were significantly increased, the number of cells co-expressing SP-C and PCNA was increased, and p-EGFR/EGFR and p-Akt/Akt ratios were increased in group LPS+ PBS ( P<0.01), and no significant change was found in the indexes mentioned above in group EGF ( P>0.05). Compared with group LPS+ PBS, the pathological score, W/D ratio, concentrations of total protein, IL-6 and TNF-α in BALF and neutrophil count were significantly decreased, the number of cells co-expressing SP-C and PCNA was increased, and p-EGFR/EGFR and p-Akt/Akt ratios were increased in group LPS+ EGF ( P<0.01). Compared with group LPS+ EGF, the pathological score, W/D ratio, concentrations of total protein, IL-6 and TNF-α in BALF and neutrophil count were significantly increased, the number of cells co-expressing SP-C and PCNA was decreased, and p-EGFR/EGFR and p-Akt/Akt ratios were decreased in group AG1478+ LPS+ EGF ( P<0.01). Conclusions:EGF can promote the repair of lung tissues in mice with ARDS, and the mechanism may be related to activation of EGFR signaling pathway and promotion of proliferation of alveolar epithelial cell type Ⅱ.
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Objective:To investigate the repair effect of amphiregulin (Areg) on injured lung tissue in mice with acute respiratory distress syndrome (ARDS) and its underlying mechanism.Methods:The ARDS mouse model was made by tracheal infusion of lipopolysaccharide (LPS), and bronchoalveolar lavage fluid (BALF) was extracted for 7 consecutive days. Adult male C57BL/6 mice were randomly (random number) divided into 5 groups ( n=4 per group): (1) Control group; (2) Areg group: mice were treated intraperitoneally (i.p.) with recombinant Areg; (3) LPS+PBS group; (4) LPS+Areg group; and (5) LPS+Anti-Areg group; mice were instilled with LPS, then were injected i.p. with PBS, Areg or Areg neutralization antibody (Anti-Areg) 30 min later. Lung tissue and BALF were extracted at day 1, 3, 5 and 7 after ARDS. HE staining was used to evaluate the pathological changes of lung tissues. The total protein content in BALF was detected by BCA method, and the concentrations of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1β and immunoglobulin M (IgM) were determined by ELISA method. The phosphorylated levels of epidermal growth factor receptor (EGFR) and expressions of proliferating cell nuclear antigen (PCNA) and surface proteins-C (SP-C) were tested by Western blot. The immunofluorescence was used to detect the co-expression of PCNA and SP-C in lung tissues. One-way analysis of variance was used to compare the mean values of normally distributed measurement data between groups. Comparisons between groups were performed using the least significant difference t-test. Results:Compared with that at before modeling [(51.05±2.47) pg/mL], Areg concentrations were increased significantly at day 1 [(71.97±6.51) pg/mL; P<0.01] and day 3 [(147.58±7.56) pg/mL, P<0.01] in the BALF after ARDS. At day 1 after ARDS, there were significant interstitial edema, neutrophil infiltration and alveolar collapse in the LPS+PBS group and LPS+Areg group. Compared with the LPS+PBS group at day 3, 5 and 7, the pathological changes of lung tissues were notably improved in the LPS+Areg group, while were more serious in the LPS+Anti-Areg group. Compared with the control group, the LPS+PBS group had higher levels of neutrophil number, total protein, IgM, TNF-α, IL-1β, and IL-6. However, Areg treatment significantly reduced the levels of these indicators. Moreover, the expressions of PCNA (1.34±0.10), SP-C (1.48±0.10) and p-EGFR (0.92±0.032) in the LPS+Areg group were significantly up-regulated compared with those in the LPS+PBS group (0.88±0.03, 1.06±0.15, and 0.68±0.03, all P<0.01). And compared with the LPS+PBS group, PCNA and SP-C double positive cells were significantly increased in the LPS+Areg group, but decreased in the LPS+Anti-Areg group. Conclusions:Areg enhances the proliferation of alveolar typeⅡ epithelial cells by activating EGFR pathway, therefore promotes the repair of lung tissues during ARDS development.
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Vibrio splendidus is an opportunistic pathogen in aquaculture. It can infect a variety of aquaculture animals and has caused huge losses to the aquaculture industry. In this study, a novel and efficient method for detecting V. splendidus was developed by combining the exonuclease Ⅲ amplification strategy with a nucleic acid test strip developed based on gold nanoparticles-labeled DNA probe. The results could be directly visualized by naked eyes, and this system overcame the difficulty in preparation of the monoclonal antibody used in conventional immunostrip. Upon optimization of experimental conditions, the detection limit of the strip was 5 ng/mL for the synthetic oligonucleotide DNA fragment and 10 ng/mL for the actual genomic DNA sample of V. splendidus. This test strip was more sensitive compared with the PCR method and was specific for the detection of V. splendidus. The rapid preparation of nucleic acid strip and the efficient detection of V. splendidus open a new way for the prevention and control of aquatic diseases.
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Animals , DNA Probes , Gold , Metal Nanoparticles , Vibrio/geneticsABSTRACT
Objectives To observe the effect of amphiregulin (Areg) via lateral ventricle injection on focal cerebral ischemia/reperfusion (I/R) injury in rats and to investigate its possible mechanism. Methods A total of 96 3-month old health specified pathogen free SD rats were randomly divided into 6 groups (n=16 in each group):sham operation group (sham group),only exposure of common carotid artery and bifurcation;I/R group,making I/R model;solvent control group,lateral ventricle injection of standard protein solution(5 μl);Areg group,lateral ventricle injection of Areg(2 μg/5 μl);AG1478 group [AG1478,a blocker of Areg receptor epidermal growth factor receptor(EGFR),lateral ventricle injection of AG1478 (2.5 μg/5 μl);Areg combined AG1478 (AAG) group,lateral ventricle giving AG1478 (2.5 μg/5 μl),and then giving Areg (2 μg/5 μl) after 30 mm.The model of focal cerebral I/R injury was induced after 30 min administration of the above last 4 groups.After 24 h of reperfusion,the volume of cerebral infarction, the neurobehavioral score and the number of apoptotic cells in the brain tissue were compared among the groups. After 6 h of reperfusion,the phosphorylation levels of EGFR and protein kinase B(Akt)in ischemic brain tissue were detected. Results Compared with the sham group,the cerebral infarction volume and the number of apoptotic cells in brain tissue were increased significantly,while the neurobehavioral score was decreased(all P<0.05).Compared with the I/R group,the volume of cerebral infarction,the number of apoptotic cells in the brain tissue were decreased significantly,and the neurobehavioral score was increased in the Areg group,the levels of EGFR and Akt phosphorylation were significantly higher (all P <0. 05). Compared with the I/R group,the volume of cerebral infarction and the number of apoptotic cells of the AG1478 group were increased,the levels of EGFR and Akt phosphorylation were decreased(all P<0.05);Compared with the Areg group,the volume of cerebral infarction and the number of apoptotic cells of the AAG group and AG1478 group were increased significantly,and the levels of EGFR and Akt phosphorylation were decreased significantly(all P<0.05). Conclusions Areg reduces the infarct volume in ischemic brain tissue,improves nerve function,and inhibits apoptosis by activating EGFR-Akt signaling pathway. Therefore,it has some protective effect for cerebral I/R injury.
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Objective To investigate the role of spinal cord chemokine CXC ligand13(CXCL13) in the formation of rat bone cancer pain(BCP).Methods Twenty healthy female SD rats weighing 160-200 g were divided into four groups(n=5):sham operation group(S),BCP group(BP),small interference RNA(siRNA) negative control(NC-siRNA) group (NC) and CXCL13-siRNA group(CS).Normal saline was given by tibial medullary cavity injection in the S group.The tibial BCP model was established by tibial medullary cavity injection of equivalent Walker-256 breast cancer cells in the group BP,NC and CS.NC-siRNA lentivirus and CXCL13-siRNA lentivirus were injected intrathecally in the group NC and CS respectively.The mechanical pain threshold was measured on 1 d before model construction and on postoperative 7,9,14,21 d.The rats were killed after pain threshold measurement.The spinal cord and tibial tissue were taken.The co-expression of spinal CXCL13,microglia specific marker Iba-1 and neuron specific neucleoprotein NeuN was determined by using the immunofluorescence double standard staining,and expressions of CXCL13 and ionized calcium binding adaptor molecule-1 (Iba-1) protein and mRNA in spinal cord were detected by Western blot and RT-PCR;the HE staining microscopy was adopted to observe the tibial bone structure destroy situation.Results Compared with group S,the mechanical pain threshold in theBP group and NC group was decreased on 7-21 d after inoculation,CXCL13 expression in neuron was significantly increased and microglia was obviously activated,the expression of CXCL13 and Iba-1 protein and mRNA was significantly elevated (P＜0.05);compared with the NC group,the mechanical pain threshold on 9-21 d after model construction in the CS group was significantly increased,CXCL13 expression in neurons was significantly decreased,microglia activation was decreased and expression of CXCL13 and Iba-1 protein and mRNA was significantly decreased(P＜0.05);HE staining showed that the model groups appeared the tumor growth in bone marrow cavity,moreover which was eroded outwards and destroyed bone cortex,but no abnormality was found in the S group.Conclusion Spinal cord CXCL13 is involved in the BCP formation in rats by activating microglia.
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Objective To study the clinical efficacy of Baofukang suppository and recombinant human inter -feron α-2b in the treatment of HPV infection with chronic cervicitis ,and to observe the prognosis ,thus to provide reference for its clinical treatment .Methods 120 patients with chronic cervicitis with HPV infection were selected . All patients were divided into observation group and control group by random number table method ,60 cases in each group.The observation group was treated with Baofukang combined with recombinant human interferon α-2b,and the control group was treated with recombinant human interferon α-2b.The patients were evaluated before treatment ,at the end of treatment and 3 months after treatment.The cure rate,total effective rate,recurrence rate,incidence rate of adverse reactions were observed and compared .Results In the observation group ,26 cases were cured ,the cure rate was 43.33％.In the control group ,15 cases were cured ,the cure rate was 25％.The cure rate of the observation group was higher than that of the control group ,the difference was statistically significant (χ2 =4.482,P<0.05).The total effective rate of the observation group was 98.33％,which was higher than 80.00％ of the control group,the differ-ence was statistically significant (χ2 =10.438,P <0.05).The negative rate of HPV in the control group was 38.33％,which was lower than 56.67％ in the observation group,the difference was statistically significant (χ2 = 4.043,P<0.05).The incidence rate of adverse reactions in the observation group was 11.67％,which in the control group was 10.00％,the difference was not statistically significant (χ2 =0.086,P >0.05).After treatment for 3 months,the recurrence rate was 0.00％in the observation group and 1.67％in the control group,the difference was not statistically significant (χ2 =1.008,P >0.05).Conclusion The combination of Baofukang suppository and recombinant human interferon α-2b showed good curative effect and prognosis in the treatment of chronic cervicitis with HPV infection ,and it is worthy to be popularized in clinical practice .
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Objective To know the vaccination status of BCG in Gaoxin district of Chongqing and explore the associated impact factors on seroconversion rate .Methods PPD was used to test the seroconversion status after PDG vaccination in infants under 3 months .Results There was no significant differences of seroconversion rate between different sex ,birth weight and vaccination age (P>0 .05) .But the rate was significantly higher in infants with normal birth weight than that in fetal macrosomia which was signif-icantly higher than that in low-birth weight .The rate was significantly decreased in infants of 1-2 months ,2-3 months and 0-1 months in order .The rate of infant with vaccination scar was significantly higher than that without scar (χ2 =80 .057 ,P<0 .01) . Conclusion There was no significant differences of seroconversion rate between different sex ,birth weight and vaccination age ,and the scar can indirectly show the seroconversion condition of vaccination .
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The protein production system using a baculovirus Antheraea pernyi nucleopolyhedrovirus (AnpeNPV) as a gene expression vector and its host insect as a natural bioreactor was successful established and its excellent performance in the protein production has been demonstrated. In this paper, the system is used to produce recombinant human epidermal growth factor (rhEGF), which have been widely used in medical and cosmetic treatment. A recombinant AnpehEGF virus has been constructed by replacing the viral polyhedrin gene with the rhEGF gene, and then injected it to Samia cynthia ricini pupae. Amplification and expression of rhEGF gene in the pupae was clearly detected by PCR, Western blot and ELISA analyses. These analyses have also revealed that rhEGF in the pupae was significantly increased at 6 days post-infection, and reached maximum level at the 12th day. The concentrations of rhEGF were 19.77, 24.90, 618.59 and 1 952.46 ng/g pupae at 3, 6, 9 and 12 days post-infection, respectively. However, the rhEGF concentration reduced at later stage (days 15). The rhEGF in the pupae could be purified using ammonium sulfate precipitation and Ni-NTA agrose affinity chromatography. Results demonstrate that Samia cynthia ricini pupae can be used as a bioreactor to produce rhEGF and, if successfully improved, will be a novel method of rhEGF production with lower cost and more efficient.
Subject(s)
Animals , Humans , Amino Acid Sequence , Bombyx , Genetics , Metabolism , Epidermal Growth Factor , Genetics , Genetic Vectors , Genetics , Molecular Sequence Data , Nucleopolyhedroviruses , Genetics , Recombinant Proteins , GeneticsABSTRACT
Sixty-eight patients with pneumoconiosis combined with chronic obstructive pulmonary disease underwent large volume lavage in one lung under double cavity tracheal intubation and intravenousinhalant anesthesia. The vital signs of patients were recorded before, 10, 30min after and at the end of lavage. Results showed that the vital signs were stable during the lavage; and after the lavage all patients had relief significantly from the symptoms of dyspnea, polypnea and cough. Our results indicate that general anesthesia with bilateral lung ventilation are a safe and effective method in large volume whole lung lavage for treatment of pneumoconiosis patients combined with chronic obstructive pulmonary disease.
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Objective To investigate the effect of stellate ganglion block(SGB) on the plasma concentration of noradrenaline(NE) in rabbits suffering from acute pain and the possible mechanism.Methods Fourteen healthy rabbits of both sexes weighing 2.5 2.8 kg were anesthetized with 20% urethane 1 g?kg -1 . Spontanous breathing was maintained. Right stellate ganglion was exposed aseptically. An epidural catheter was fixed with one end placed close to stellate ganglion and the other end outside the neck through a hole on the skin for administration of drugs. One week later 3% formalin 0.2ml was injected subcutaneously into plantar region of the right paw. Pain response was observed . 60 min after formalin injection 0.25% bupivacaine 0.5ml was injected through catheter (bupivacaine group n=7) while in control group (n=7) normal saline 0.5ml was injected. The effect of SGB was confirmed by ptosis and miosis. Blood samples were taken from edge vein of the ear 10 min before (T 0) and 10(T 1), 30(T 2), 50min(T 3) after subcutaneous injection of formalin and 10min(T 4), 30min(T 5), 50min(T 6) after bupivacaine or normal saline injection for determination of plasma NE concentration by radioimmunoassay.Results Plasma NE concentration increased significantly after subcutaneous formalin injection and peaked at T 1,then decreased slightly at T 2 and T 3. In group B, plasma NE concentration decreased significantly after bupivacaine injection, while in control group there was no significantly change in plasma NE concentration after normal saline injection.Conclusions SGB reduces the increased plasma NE concentration in rabbits suffering from acute pain. This may be the possible mechanism of analgesia provided by SGB.
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Objective To investigate the effect of stellate ganglion block (SGB) on substance P of spinal cord in rabbits following formalin stimulation and the possible mechanism. Methods Rabbits of both sexes weighting 2.5-3.0 kg were anesthetized with 20% urethane 1 g?kg-1 . Spontanous breathing was maintained. Right stellate ganglion was exposed aseptically. An epidural catheter was fixed with one end placed closed to stellate ganglion and the other end outside the neck through a hole on the skin for administration of drugs. One week later, nineteen healthy rabbits were randomly divided into three groups: sham operation group (group A, n = 5), SGB group (group B, n=7) and control group (group C, n = 7) . In group B and group C, 3 % formalin 0.2 ml was injected subcutaneously into plantar region of the right paw. Pain response was observed. 1h after formalin injected, 0.25% bupivacaine 0.5 ml was injected through the catheter in group B while normal saline 0.5 ml was injected in group C. The effect of SGB was confirmed by ptosis and miosis. 1h after using of bupivacaine or normal saline, rabbits were deeply anesthetized and chest was opened, 1 000 ml of normal saline was infused via left ventricle then followed by 2000-2500 ml of 4% paraformaldehyde in 0.1mol/L phosphate buffer pH7.4 infusion for fixation of tissue, 60 min later spinal cord of cervical 6-8(C6-8) and thoracic 6-8(T6-8 ) were removed. In group A the spinal cord were extracted similar to the other groups. The content of SP in spinal cord use immumohistochemistry technique of strept avidin biotin enzyme complex (SABC) to display. Results Immunoreaction of SP were distributed mainly in lamina Ⅰ and Ⅱ of dorsal horn of spinal cord. Compared with group A, the optical density(OD) of SP in cervical slices were decreased significantly in group B and group C(P 0.05 ). Conclusions The content of SP in spinal cord decreased significantly after subcutaneous injection of formalin in rabbits. SGB can reverse the phenomena partly and increase the content of SP of cervical spinal cord in rabbits following formalin stimulation. This may be the possible mechanism of analgesia provided by SGB.
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Objective To investigate the effect of ketamine on spatial learning and memory in a rat model of acute pain produced by an incision in plantar area. Methods Seventy-two 3-month old male SD rats weighing 200-250g were randomly divided into 3 groups: (1) control group (C) received no plantar incision (n = 24); (2) acute pain group (M) received an incision in the sole of the hindpaw according to the method described by Brennan (n = 24) and (3) ketamine group (K) received plantar incision and intraperitoneal ketamine 10 mg?kg-1 every day for 7 days starting from the day when plantar incision was made ( n = 24). In group C and M normal saline (NS) was given i.p. instead of ketamine. The three groups were further divided into 2 subgroups according to the time when Morris water maze (MWM) testing was started-1 week (C1, M1,K1) or 3 weeks (C3, M3, K3) after plantar incision. MWM test was performed 4 times a day for 6 consecutive days. The latent periods and swimming distances were recorded automatically by MWM monitoring system. Six days after the last MWM testing the animals were anesthetized and killed. The hippocampus was removed for microscopic examination. Results (1) MWM test: From the 1st to the 6th day of testing, the latent period and swimming distance in group K1 were significantly longer than those in group C1 and M1 (P