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@#Objective To investigate the effect of silencing E6-associated protein(E6AP)on the level of p53 protein in human papilloma virus(HPV)negative cervical cancer cells(C33A cells).Methods The siRNA sequence silencing E6AP(siE6AP)and silencing control disordered siRNA sequence(siControl)were transfected into C33A cells with the mediation of LipofectamineTM2000 transfection reagent respectively.The silencing effect of siRNA on E6AP and the expression of p53and cleaved-caspase-3 proteins were detected by Western blot.Results The levels of E6AP protein in C33A cells of siE6AP group were significantly lower(t =-4.597,P<0.05),while the levels of p53 and cleaved-caspase-3 proteins were significantly higher than those of siControl group(t = 4.533 and 7.099 respectively,each P<0.05).Conclusion Silencing of E6AP significantly increased the expression of p53 protein in C33A cells,indicating that silencing of E6AP may restore the activity and function of p53 protein in C33A cells.
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@#Abstract：Objective To improve the replication level of varicella⁃zoster virus（VZV）in human diploid cell line MRC⁃5 and increase the yield of VZV vaccine by reducing the expression of interferon（IFN）related genes via optimizing the cell line MRC⁃5. Methods Interferon receptor 1（IFNAR1）silenced MRC⁃5 cell line（MRC⁃5IFNAR1⁃）was constructed by CRISPR／Cas9 gene editing technology，which was determined for the relative expression of IFNAR1 mRNA，and for those of mRNA of IFN related genes IFNβ and OAS1 after VZV infection by qRT⁃PCR to evaluate the effect of gene silencing. Gene mutation sequences were further identified by sequencing of the silenced sites. The replication of VZV in MRC⁃5 and MRC⁃5IFNAR1⁃ cell lines was compared 168 h after VZV infection by using qRT⁃PCR and plaque formation unit（PFU）assay， to evaluate the effect of MRC⁃5IFNAR1⁃cell line on VZV replication. Results The growth status of MRC⁃5IFNAR1⁃ cell line wasconsistent with that of MRC ⁃ 5 cells，and the relative expression of IFNAR1 mRNA decreased by 73%；The relative expressions of IFNβ and OAS1 mRNA in MRC⁃5IFNAR1⁃ cell line were 61% and 90% lower than those in MRC⁃ 5 cells respectively after VZV infection；In addition，168 h after VZV infection，the level of DNA replication and the titer of VZV increased by 5. 7 folds and 4 folds respectively. Conclusion The successful establishment of MRC⁃5IFNAR1⁃ cell line may be a potential scheme to increase the yield of vaccines based on human diploid cells，and provided a reference for expanding production of VZV vaccine.
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ABSTRACT Purpose To investigate the relationship between atherosclerotic abdominal aortic aneurysm (AAA) and CXC chemokine receptor type 2 (CXCR2). Methods Mouse AAA model was established by embedding angiotensin-II pump (1000 ng/kg/min) in ApoE-/- mice. Mice were received SB225002, a selective CXCR2 antagonist, for treatment. Blood pressure was recorded, and CXCR2+ macrophages were examined by flow cytometry analysis. Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining was performed to detect cell apoptosis of abdominal aortic aneurysms. Macrophages were isolated from ApoE-/- mice and treated with Ang II and/or SB225002. Dihydroethidium staining was carried out to determine reactive oxygen species (ROS) activity. Enzyme-linked immunosorbent assay (ELISA) was performed to determine the production of IL-1β and TNF-α. The corresponding gene expressions were measured using real-time polymerase chain reaction (PCR), western blot, and immunohistochemistry staining. Results We found that Ang II activated the expression of CXCR2 in monocytes during the formation of AAA. Inhibition of CXCR2 significantly reduced the size of AAA, attenuated inflammation and phenotypic changes in blood vessels. Ang II-induced macrophages exhibited elevated ROS activity, and elevated levels of 1β and TNF-α, which were then partly abolished by SB225002. Conclusions CXCR2 plays an important role in AAA, suggesting that inhibiting CXCR2 may be a new treatment for AAA.
Subject(s)
Animals , Mice , Aortic Aneurysm, Abdominal/prevention & control , Aortic Aneurysm, Abdominal/drug therapy , Apolipoproteins E/genetics , Angiotensin II , Receptors, Interleukin-8B , Disease Models, Animal , Macrophages , Mice, Inbred C57BLABSTRACT
Objective To study the effect of arthroscopic internal fixation combined with arthrodesis on patients with advanced ankle arthritis and American Orthopedic Ankle Association Scoring System (AOFAS) and visual analogue scale (VAS). Methods 84 patients with advanced ankle arthritis from January 2012 to January 2015 were randomly divided into experimental group (42 cases) and control group (42 cases) by random number method. The patients in the control group were treated with traditional open ankle arthrodesis, the experimental group under the arthroscopic assisted internal fixation joint fusion. Then compare the time of surgery, intraoperative blood loss, postoperative hospitalization time and complication. The follow-up period was 12 to 36 months. Used the AOFAS score system to evaluate the curative effect. Use VAS to evaluate the degree of ankle pain. Results The operation time and intraoperative blood loss were significantly lower in the experimental group than that in the control group (P < 0.05). The postoperative hospital stay and the time of joint fusion were lower in the experimental group than that in the control group (P < 0.05). The incidence of complication (9.52%) in the experimental group was significantly lower than that in the control group (25.57%) (P < 0.05). The results of follow-up showed that the VAS and AOFAS scores of the experimental group were better than those in the control group (P < 0.05). Conclusion The procedure of arthroscopic endoscopic fusion is short, the bleeding rate is low, the incidence of complications is low, the healing rate is high, and the follow-up effect is accurate. It is suitable for clinical use.
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The drug resistance of microorganisms isolated from laboratory animals never treated with antibiotics is being reported consistently, while the number of laboratory animals used in medicine, pharmacy, veterinary medicine, agriculture, nutrition, and environmental and health science has increased rapidly in Korea. Therefore, this study examined the development of antimicrobial resistance in bacteria isolated from laboratory animals bred in Korea. A total of 443 isolates (7 species) containing 5 Sphingomonas paucimobilis, 206 Escherichia coli, 60 Staphylococcus aureus, 15 Staphylococcus epidermidis, 77 Enterococcus faecalis, 27 Citrobacter freundii, 35 Acinetobacter baumannii were collected from the nose, intestine, bronchus and reproductive organs of ICR mice and SD rats. Of these species, Acinetobacter baumannii and Enterococcus faecalis showed significant antimicrobial resistance according to the minimum inhibition concentration (MIC) in E-test. In case of Acinetobacter baumannii, several isolates showed MIC values 16-128 microg/mL for cefazolin and cefoxitin, and higher resistance (128-512 microg/mL) to nitrofurantoin than that of standard type. Resistance to cefazolin, cefoxitin and nitrofurantoin was detected in 17.14, 20.00, and 8.57% of the Acinetobacter baumannii isolates, respectively. In addition, 44.1% of the Enterococcus faecalis isolates collected from the laboratory animals were resistant to oxacillin concentration of 16-32 microg/mL range, while MIC value of standard type was below oxacillin concentration of 6 microg/mL. These results suggest that in rodent species of laboratory animals, Acinetobacter baumannii are resistance to cefazolin, cefoxitin and nitrofurantoin, whereas those of Enterococcus faecalis were resistance to oxacillin.
Subject(s)
Animals , Mice , Rats , Acinetobacter baumannii , Agriculture , Animals, Laboratory , Anti-Bacterial Agents , Bacteria , Bronchi , Cefazolin , Cefoxitin , Citrobacter freundii , Drug Resistance , Drug Resistance, Microbial , Enterococcus faecalis , Escherichia coli , Intestines , Korea , Mice, Inbred ICR , Nitrofurantoin , Nose , Oxacillin , Pharmacy , Rodentia , Sphingomonas , Staphylococcus aureus , Staphylococcus epidermidis , Veterinary MedicineABSTRACT
Exercise training is highly correlated with the reduced glucose-stimulated insulin secretion (GSIS), although it enhanced insulin sensitivity, glucose uptake and glucose transporter expression to reduce severity of diabetic symptoms. This study investigated the impact of short-term swimming exercise on insulin regulation in the Goto-Kakizaki (GK) rat as a non-obese model of non-insulin-dependent diabetes mellitus. Wistar (W/S) and GK rats were trained 2 hours daily with the swimming exercise for 4 weeks, and then the changes in the metabolism of insulin and glucose were assessed. Body weight was markedly decreased in the exercised GK rats compare to their non-exercised counterpart, while W/S rats did not show any exercise-related changes. Glucose concentration was not changed by exercise, although impaired glucose tolerance was improved in GK rats 120 min after glucose injection. However, insulin concentration was decreased by swimming exercise as in the decrease of GSIS after running exercise. To identify the other cause for exercise-induced insulin down-regulation, the changes in the levels of key factors involved in insulin production (C-peptide) and clearance (insulin-degrading enzyme; IDE) were measured in W/S and GK rats. The C-peptide level was maintained while IDE expression increased markedly. Therefore, these results showed that insulin down-regulation induced by short-term swimming exercise likely attributes to enhanced insulin clearance via IDE over-expression than by altered insulin production.
Subject(s)
Animals , Rats , Body Weight , C-Peptide , Diabetes Mellitus, Type 2 , Down-Regulation , Glucose , Glucose Transport Proteins, Facilitative , Insulin , Insulin Resistance , Insulysin , Running , SwimmingABSTRACT
OBJECTIVE: Ovarian cancer is common a gynecologic malignancy and leading cause of death in women being diagnosed with advanced disease. This study was undertaken to investigate the roles of the proteins related to G1 cell cycle in ovarian carcinogenesis. METHODS: The expression of cyclin Dl, p16, RB and PCNA in DMBA (7, 12-dimethylbenzanthracene)-induced ovarian cancer of rats was analyzed by immuno-histochemistry and Western blot. RESULTS: 1. Twenty-nine tumors were induced in 32 ovaries from 16 rats (90.6%) in the experimental group. The average weight of tumor was 3.35+/-0.73 gm and the average size was 1.84+/-0.17 cm in greatest dimension. The histologic types were adenocarcinomas (n=20), squamous cell carcinomas (n=3), sarcoma (n=4) and combined types (n=3). 2. With respect to the cyclin D1 and PCNA labelling index, ovarian cancers showed significantly higher index than normal ovarian surface epithelium. There were no differences among the cancer types. In Western blot analysis, the expression of cyclin Dl in ovarian cancers was higher than that in normal ovarian surface epithelium. 3. With respect to the p16 and RB labelling index, ovarian cancers showed significantly lower index than normal ovarian surface epithelium. There were no differences among the cancer types. In Western blot analysis, the expression of cyclin Dl in ovarian cancers were lower than that in normal ovarian surface epithelium. 4. Positive correlation was shown among cyclin D1, PCNA. RB was negatively correlated with cyclin D1, PCNA. The p16 had no correlation with cyclin D1, PCNA. CONCLUSION: These results suggest that the deregulation of cyclin Dl, p16, RB and PCNA occur in DMBA induced rat ovarian carcinogenesis and result in tumor progression. Further studies are needed to investigate the role and function of cyclin Dl-p16-RB pathway in human ovarian cancer with this animal model.