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Objective To construct a recombinant poxvirus vector vaccine, rVTTδTK-RBD, and to evaluate its safety and immunogenicity. Methods The receptor-binding domain (RBD) gene was synthesized with reference to the gene sequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and was inserted into the polyclonal site of the self-constructed recombinant plasmid pSTKE, to construct the recombinant poxvirus shuttle vector pSTKE-RBD. This was then transfected into BHK-21 cells pre-infected with the vaccinia virus Tiantan strain (VTT). The recombinant poxvirus rVTTδTK-RBD was successfully obtained after several rounds of fluorescence phage screening. The effect of rVTTδTK-RBD on the body mass of BALB/c mice was detected after immunizing mice by intra-nasal vaccination. The levels of specific and neutralizing antibodies produced by rVTTδTK-RBD on BALB/c mice were analyzed after immunizing mice intramuscularly. The effect of rVTTδTK-RBD on T cell subsets in BALB/c mice was detected by flow cytometry. Results Through homologous recombination, enhanced green fluorescent protein (EGFP) screening marker, and multiple rounds of fluorescent phosphorescence phage screening, a recombinant poxvirus rVTTδTK-RBD, expressing RBD with deletions in the thymidine kinase (TK) gene, was successfully obtained, which was validated by PCR. The in vivo experiments on BALB/c mice showed that rVTTδTK-RBD was highly immunogenic against SARS-CoV-2 and significantly reduced toxicity to the body compared to the parental strain VTT. Conclusion The recombinant poxvirus vaccine rVTTδTK-RBD against SARS-CoV-2 is successfully constructed and obtained, with its safety and immunogenicity confirmed through various experiments.
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Animals , Mice , SARS-CoV-2/genetics , COVID-19 , Vaccines, Synthetic/genetics , Genes, Reporter , Bacteriophages , Mice, Inbred BALB CABSTRACT
Objective@#To establish depression model in female C57BL / 6 mice using chronic unpredictable stress + sleep deprivation + castration and observe the change of behavior and neuroendocrine of this model to provide basis for screening anti-depressant drugs.@*Methods@#To simulate the etiopathological mechanism of perimenopausal depression or depression symptoms, C57BL/6 female adult mice were exposed to chronic unpredictable stress + sleep deprivation + castration to establish perimenopausal depression model. The mice were randomly divided into perimenopausal group, model group, sham operation group, which of behavioral and neuroendocrine differences were observed.@*Results@#(1)Compared with the sham operation group((21.48±0.79)g, (0.02±0.06), (93.02±6.17)%) and the perimenopausal group((22.02±0.15)g, (0.06±0.09)score, (90.30±11.68)%), the body weight ((20.16±1.03)g), coat state score (0.39±0.04) and sucrose preference((58.11±11.97)%) in the model group had significant differences(all P<0.05). (2)Compared with the perimenopausal group and sham operation group, the horizontal movement, the vertical movement score and the percentage of central cells in the open field experiment, the percentage of immobility state and immobility state in forced swimming and tail suspension experiment of model mice were all significantly different (P<0.05). (3)Compared with the perimenopausal group and the sham-operation group, the latent period of eating, the total time of exploration and single exploration in NSFT had significant difference(P<0.05). (4)Compared with the sham operation group((46.16±7.72)pmol/ml, (320.77±23.19)ng/ml) and the perimenopausal group ((9.75±1.77)pmol/ml, (386.42±37.58)ng/ml), serum estradiol ((9.99±0.37)pmol/ml) were elevated and corticosterone ((426.31±105.01)ng/ml) were increased in the model group, which had significant differences (P<0.05).@*Conclusion@#C57BL/6 female mice show depression-like behavior after ovariectomy, 4 weeks of chronic unpredictable stress and sleep deprivation intervention.
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Objective To explore the anti-post-traumatic-stress-disorder(anti-PTSD)effects of YL-IPA08,a selective 18kDa translocator protein(TSPO)ligand.Methods The time-dependent stress sensitization(TDS)method was used to establish a rat mod?el of PTSD.The open field test(OFT)was used to evaluate the locomotor activity in rats.The contextual freezing(CF)measurement, elevated plus maze(EPM)test and novel objective recognition(NOR)test were used to evaluate the PTSD-like behaviors in rats.The concentration of allopregnanolone in rat hippocampus was detected by ELISA. Results Compared with the control group,neither TDS nor drug treatment affected the locomotor activity in rats(P>0.05).However,PTSD rats showed significant PTSD-like behaviors with enhanced CF time in CF mearsurement,decreased open arm time and open arm entries in EPM test,and dropped object recogni?tion index in NOR test(P<0.05 or 0.01).Moreover,the concentration of hippocampus allopregnanolone was decreased in PTSD rats (P<0.05).YL-IPA08(0.05-0.1mg/kg)or positive drug sertraline(15 mg/kg),administered intragastrically after establishment of PTSD model,significantly reversed the above PTSD-like behavioral changes(P<0.05 or 0.01)and increased the concentration of hip?pocampus allopregnanolone in PTSD rats.Conclusion YL-IPA08 could improve the PTSD-like behavior in rats,and the mechanism may be related to the increased allopregnanolone level in hippocampus.
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Objective To explore the role of 18 ku translocator protein (TSPO) in the anti-post-traumatic-stress-disorder(PTSD) effects of YL-IPA08 and the value of TSPO as a potential pharmacological target using gene knock out mice.Methods The PCR method was used to genotype TSPO wild type (WT) mice and knock out (KO) mice.Foot shock was used to establish a well-accepted mouse model of PTSD,the open field test (OFT) was used to evaluate the locomotor activity in mice,and freezing measurement was used to evaluate the PTSD-like fear behavior in mice.Results Compared with TSPO WT mice,KO mice had no expressible TSPO gene,but showed similar locomotor activity to WT mice after PTSD modeling.On day 1,day 5 and day 16 after PTSD modeling (day-1-day 0),both WT and KO mice showed significant PTSD-like behavior with enhanced freezing time.However,8 d treatment (day 0-day 7) of YL-IPA08 (0.3 mg/kg,once daily) or positive drug sertraline (15 mg/kg,once daily) after PTSD modeling significantly reduced freezing time selectively in WT mice,but not in KO mice.Conclusion It has been found for the first time that TSPO WT and KO mice can show the same sensitivity to PTSD modeling (namely the same PTSD-like behavior performance).Interestingly,TSPO can mediate the anti-PTSD effects of YL-IPA08.Therefore,the present study provides direct evidence for the value of TSPO as an potential pharmacological target for PTSD.
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OBJECTIVE@#To study expressions of netrin-1 and its receptor UNC5C in female precocious puberty rat hypothalamus, and explore its effect on precocious puberty process.@*METHODS@#Forty female one-week-old SD rats were randomly divided into four groups: experimental group A (precocious puberty early youth), experimental group B (precocious puberty medium youth), group A (normal pre-puberty), group B (normal early youth) with 10 rats in each group. Precocious puberty experimental rats were induced with Danazol and rats in control group were injected with saline. Uterus and ovaries were removed, specimens were weighed, uterus index and ovarian index were calculated, and amount of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were detected from the blood by ELISA. Real-time PCR was used to detect netrin-1 and its receptor UNC5C, as well as hypothalamic gonadotropin-releasing hormone (GnRH) mRNA expression in hypothalamus tissues; and then, a co-immunoprecipitation study of interactions between netrin-1 and its receptor UNC5C was carried out.@*RESULTS@#Relative target gene expression levels of control group A, control group B, experimental group A, and experimental group B (with β -actin as an internal control for normalization) were as follows: Netrin-1: 3.5±0.9, 5.4±0.7, 4.9±1.0, 5.3±0.3; UNC5C: 0.8± 0.04, 1.7±0.2, 1.82±0.23, 1.58±0.4; GnRH: 1.2±0.3, 2.7±0.3, 2.4±0.7, 3.2±0.4.@*CONCLUSIONS@#LH and FSH concentrations, netrin-1 and its receptor expression are increased in precocious puberty animal models.
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Objective: To study expressions of netrin-1 and its receptor UNC5C in female precocious puberty rat hypothalamus, and explore its effect on precocious puberty process. Methods: Forty female one-week-old SD rats were randomly divided into four groups: experimental group A (precocious puberty early youth), experimental group B (precocious puberty medium youth), group A (normal pre-puberty), group B (normal early youth) with 10 rats in each group. Precocious puberty experimental rats were induced with Danazol and rats in control group were injected with saline. Uterus and ovaries were removed, specimens were weighed, uterus index and ovarian index were calculated, and amount of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were detected from the blood by ELISA. Real-time PCR was used to detect netrin-1 and its receptor UNC5C, as well as hypothalamic gonadotropin-releasing hormone (GnRH) mRNA expression in hypothalamus tissues; and then, a co-immunoprecipitation study of interactions between netrin-1 and its receptor UNC5C was carried out. Results: Relative target gene expression levels of control group A, control group B, experimental group A, and experimental group B (with β -actin as an internal control for normalization) were as follows: Netrin-1: 3.5±0.9, 5.4±0.7, 4.9±1.0, 5.3±0.3; UNC5C: 0.8± 0.04, 1.7±0.2, 1.82±0.23, 1.58±0.4; GnRH: 1.2±0.3, 2.7±0.3, 2.4±0.7, 3.2±0.4. Conclusions: LH and FSH concentrations, netrin-1 and its receptor expression are increased in precocious puberty animal models.
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10.3969/j.issn.1000-8179.2013.12.005
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ObjectiveTo explore the effect of budesonide nebulization in treatment of patients with chronic obstructive pulmonary disease and ventilator-associated pneumonia. MethodsA total of 49 cases with COPD and VAP was randomly divided into two groups:the control group consisted of 24 patients received salbutamol nebulization on the routine treatment.The observe group consisted of 25 patients received budesonide plus salbutamol bulization on the routine treatment.Variables such as TNF-αt and IL-8 in the bronchial alveolar lavage fluid(BALF),and efficacy of pneumonia and clinical healing rate were observed. ResultsThe levels of TNF-α and IL-8 in BALF of the observe group were significantly lower than those in the control group after 72h(all P<0.05).The days of symptoms,signs and bedside chest X-ray improved,off-machine time,extubation time and the days of inpatient were statistically shorter in observe group compared with the control group(all P<0.05).But the clinical cure rate of the observe group was no significant improvement. ConclusionThe nebulization of budesonide could suppress the local pulmonary inflammation and reduce the time of machine ventilation and improve prognosis in COPD patients with VAP.
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Objective To explore the relationship between KISS-1 gene and metastasis of bladder carcinoma,and to study the effect of the stable expression of KISS-1 gene on the invasion of bladder carcinoma cell line T24.Methods Fluorescent quantitative PCR was used to detect the expression of KISS-1 mRNA in primary bladder carcinoma without metastasis and primary bladder carcinoma with metastasis.Recombinant vector pIRES2-AKS-1 was constructed and transfected into T24 cells.Single clone of stably transfected cells was screened,and the changes in the invasive ability of T24 cells was detected after transfection.Results The expression level of KISS-1 mRNA in primary bladder carcinoma with metastasis was significantly lower than that in primary bladder carcinoma without metastasis(P<0.05).The expression of KISS-1 protein in the single clone of stably transfected cells increased significantly,and the invasive ability significantly decreased(P<0.05).Conclusion KISS-1 gene is correlated with the metastasis of bladder carcinoma,and the up-regulated expression of KISS-1 gene can inhibite the invasiveness of T24 cell line.
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<p><b>OBJECTIVE</b>To explore mechanism of S100A8 in the oncogenesis and development of laryngeal cancer.</p><p><b>METHODS</b>Proteins interacting with S100A8 were isolated from laryngeal cancer cell lines Hep-2 by immunoprecipitation assay with anti-S100A8 antibody. The target bands were cut out and identified by maxtrix assisted laser desorption/ionization time of flight (MALDI-TOF). The peptide mass fingerprinting data of the proteins identified were analyzed based on the Mascot database. The NF-kappa B binding sites of the proteins were predicted by P-Match software. The binding ability of one of the proteins to S100A8 was confirmed by co-immunoprecipitation and immunocytochemistry methods.</p><p><b>RESULTS</b>Four proteins interacting with S100A8 were obtained, which were hypothetical protein LOC80154, MHC class I HLA-B, similar to T-box 1 isoform C and sarcolemmal associated protein 1. The four genes were predicted to have NF-kappa B binding sites. MHC class I HLA-B, which is one of targets in NF-kappa B pathway, was first confirmed to have the binding ability to S100A8.</p><p><b>CONCLUSION</b>The novel partners of S100A8 identified in the study might be involved in NF-kappa B pathway. The binding ability of MHC class I HLA-B to S100A8 implies that S100A8 might function as a new member with other proteins including HLA-B in NF-kappa B pathway. These findings provide a new clue to further study on the molecular mechanism of S100A8 in the genesis of laryngeal carcinomas.</p>
Subject(s)
Animals , Humans , Binding Sites , Calgranulin A , Genetics , Metabolism , Carcinoma, Squamous Cell , Genetics , Metabolism , Pathology , Cell Line, Tumor , HLA-B Antigens , Genetics , Metabolism , Laryngeal Neoplasms , Genetics , Metabolism , Pathology , NF-kappa B , Metabolism , Signal TransductionABSTRACT
<p><b>BACKGROUND</b>Laryngeal carcinoma is a common malignant tumor of the upper respiratory tract, and in 95% of cases the tumor is laryngeal squamous cell carcinoma (LSCC). The abnormity of SH3-domain GRB2-like 2 (SH3GL2) gene was found in LSCC. In order to clarify the relationship between SH3GL2 gene and LSCC, we evaluated the expression of the SH3GL2 gene in LSCC.</p><p><b>METHOD</b>Real-time PCR, immunohistochemistry and Western blotting were used to detect the mRNA and protein expression and find the various rules of SH3GL2 gene in LSCC.</p><p><b>RESULTS</b>The result of real-time PCR showed that the expression level of SH3GL2 mRNA in LSCC tissue was apparently down-regulated; immunohistochemical analysis showed that SH3GL2 protein was mainly located in cytoplasm, the rate of positive cells and SH3GL2 protein expression level were fluctuated with the pathological classification of LSCC; the result of Western blotting showed that SH3GL2 protein was down-regulated significantly in LSCC samples, especially in metastatic lymph nodes.</p><p><b>CONCLUSIONS</b>These results suggest that SH3GL2 is a LSCC related gene and its expression level is fluctuated with the pathological classification which indicate that SH3GL2 participates in the development and progression of LSCC. And it may be considered as a novel tumor marker to find both a new anti-oncogene and relative factors of invasion and metastasis of laryngeal carcinoma.</p>
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Humans , Adaptor Proteins, Signal Transducing , Genetics , Blotting, Western , Carcinoma, Squamous Cell , Chemistry , Genetics , Immunohistochemistry , Laryngeal Neoplasms , Chemistry , Genetics , Polymerase Chain Reaction , src Homology DomainsABSTRACT
<p><b>OBJECTIVE</b>With the objective of discovering novel putative chromosomal regions and special genes involved in the carcinogenesis, progression and metastasis of laryngeal squamous cell cancer (LSCC).</p><p><b>METHODS</b>DNA copy profile of LSCC were obtained and analyzed by comparative genomic hybridization (CGH) and a computerized digital image analysis system. cDNA microarray of LSCC was performed and the profile was analyzed by Hierarchical clustering.</p><p><b>RESULTS</b>CGH analysis showed average-12.9 gains and losses of chromosomes in LSCC. Relatively high frequencies of gains were found at 3q15-21 (14/18), 5p12-13 (11/18), 8q22-24 (6/18), 11q12-13 (8/18), 15q21-23 (7/18) and 18p11 (8/18), while those of losses at 1p13-21 (8/18), 3p21-23 (14/18), 5q21-22 (14/18), 9p12-pter (11/18) and 13q21-31 (8/18). Hierarchical clustering analysis showed that the differentially expressed genes were segregated into three groups. Three genes differentially expressed in process I (normal tissue to cancer) and process II (cancer to lymph node metastasis), and the Cy5/Cy3 ratios of twelve genes were either higher than 5.0 or lower than 0.2 in process I or process II. The fifteen special genes were first reported possibly to be the relationships with LSCC. In particular, 4 genes of them, which were cytochrome C oxidase Va, PPBP, EPHX2 and PON1, were first reported to correlate with tumorigenesis. SH3GL2, which was one of the 15 special genes, was located at one of the special chromosome regions, 9p12-pter.</p><p><b>CONCLUSION</b>The important genes and special chromosomal aberrances might provide us a clue for further investigation of carcinogenesis, progression and metastasis in LSCC.</p>