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OBJECTIVE To design the two isomers of ferrocene （Fc）-coupled cationic peptides （hereinafter referred to as “peptides”） [Fc-K（C8）FFHK and C8-K（Fc） FFHK] and the control peptide [C8-K（C8）FFHK]， and to explore the effects of Fc position isomerization on the self-assembly behavior and antibacterial effect of peptides. METHODS All isomerized peptides were prepared by standard solid-phase synthesis and purified by reversed-phase high-performance liquid chromatography. The stability of the peptide was analyzed by using UV spectrophotometry to detect UV absorption spectra， and Zeta potential analyzer to determine Zeta potential. The secondary structure was characterized by circular dichroism spectrum （CD）， Fourier transform infrared spectrometer （FTIR）， thioflavin T （ThT） fluorescence spectrum and transmission electron microscopy （TEM）. The differences in antibacterial activity and biocompatibility of the 2 kinds of isomerized peptides were evaluated by in vitro reactive oxygen species （ROS） generation test， growth curve determination test， plate method， cytotoxicity assay and hemolysis test. RESULTS Three peptides with purity higher than 95% were synthesized. The stability test results showed that the UV absorption spectra of Fc-K（C8）FFHK and C8-K（Fc）FFHK remained almost unchanged when placed at room temperature for 24 and 96 hours， and their Zeta potential were decreased by 0.3 mV and 0.5 mV， respectively. Secondary structure characterization results showed that Fc-K（C8）FFHK and C8-K（Fc）FFHK were self-assembled to form twisted nanoribbons and short nanofibers， respectively； C8-K（C8）FFHK was assembled into cylindrical nanofibers. The optical spectrum results showed that there were certain differences in the content of structures such as β-sheet and α-helix. The in vitro ROS generation test results showed that ROS generation efficiency of Fc-K（C8）FFHK at pH 6.0 was higher than C8-K（Fc）FFHK. The results of in vitro antibacterial activity showed that for methicillin-resistant Staphylococcus aureus， both the isomeric peptides had similar minimum inhibitory concentration （MIC） values of 50 μg/mL which were far lower than the control peptide （400 μg/mL）. To Escherichia coli， Fc-K（C8）FFHK had better antibacterial activity than C8-K（Fc）FFHK. Finally， cytotoxicity assay and hemolysis test results showed that both isomeric peptides had good biocompatibility. CONCLUSIONS By wangjingwen8021@163.com coupling Fc， the antibacterial activity of cationic self-assembled peptides can be improved. Regulating the position of Fc in the peptide sequence could regulate the self-assembly behavior and antibacterial effect of the self-assembled peptides.
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OBJECTIVE@#Cassiae Semen (CS, Juemingzi in Chinese) has been used for thousands of years in ancient Chinese history for relieving constipation, improving liver function as well as preventing myopia. Here we aimed to elucidate the anti-steatosis effect and underlying mechanism of CS against non-alcoholic fatty liver disease (NAFLD).@*METHODS@#High-performance liquid chromatography (HPLC) was used to identify the major components of CS water extract. Mice were fed with a high-fat and sugar-water (HFSW) diet to induce hepatic steatosis and then treated with CS. The anti-NAFLD effect was determined by measuring serum biomarkers and histopathology staining. Additionally, the effects of CS on cell viability and lipid metabolism in oleic acid and palmitic acid (OAPA)-treated HepG2 cells were measured. The expression of essential genes and proteins involved in lipid metabolism and autophagy signalings were measured to uncover the underlying mechanism.@*RESULTS@#Five compounds, including aurantio-obtusin, rubrofusarin gentiobioside, cassiaside C, emodin and rhein were simultaneously identified in CS extract. CS not only improved the diet-induced hepatic steatosis in vivo, as indicated by decreased number and size of lipid droplets, hepatic and serum triglycerides (TG) levels, but also markedly attenuated the OAPA-induced lipid accumulation in hepatocytes. These lipid-lowering effects induced by CS were largely dependent on the inhibition of fatty acid synthase (FASN) and the activation of autophagy-related signaling, including AMP-activated protein kinase (AMPK), light chain 3-II (LC3-II)/ LC3-1 and autophagy-related gene5 (ATG5).@*CONCLUSION@#Our study suggested that CS effectively protected liver steatosis via decreasing FASN-related fatty acid synthesis and activating AMPK-mediated autophagy, which might become a promising therapeutic strategy for relieving NAFLD.
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The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with high pathogenicity and infectiousness has become a sudden and lethal pandemic worldwide. Currently, there is no accepted specific drug for COVID-19 treatment. Therefore, it is extremely urgent to clarify the pathogenic mechanism and develop effective therapies for patients with COVID-19. According to several reliable reports from China, traditional Chinese medicine (TCM), especially for three Chinese patent medicines and three Chinese medicine formulas, has been demonstrated to effectively alleviate the symptoms of COVID-19 either used alone or in combination with Western medicines. In this review, we systematically summarized and analyzed the pathogenesis of COVID-19, the detailed clinical practice, active ingredients investigation, network pharmacology prediction and underlying mechanism verification of three Chinese patent medicines and three Chinese medicine formulas in the COVID-19 combat. Additionally, we summarized some promising and high-frequency drugs of these prescriptions and discussed their regulatory mechanism, which provides guidance for the development of new drugs against COVID-19. Collectively, by addressing critical challenges, for example, unclear targets and complicated active ingredients of these medicines and formulas, we believe that TCM will represent promising and efficient strategies for curing COVID-19 and related pandemics.
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Objective:To study the characteristics of vaginal microbiota in pregnant women with premature rupture of membranes (PROM) and to establish prediction models for PROM.Methods:This study involved 35 women with preterm premature rupture of membranes (PPROM), 180 with term premature rupture of membranes (TPROM) and 255 term birth cases without premature rupture of membranes (TBWPROM, control group). The V3-V4 hypervariable region sequences in the vaginal samples collected at 16-28 weeks of gestation were detected by 16S rRNA gene next-generation sequencing. The differences in Alpha and Beta diversity, and the attributes and metabolic function prediction of each recognized species among the three groups were analyzed. Subsequently, a random forest model was used to establish the prediction models for PROM using vaginal microbiota species and environmental risk factors.Results:Compared with the control group, the Alpha diversity of the PPROM group was higher (Observed features, P=0.022; Faith_pd index, P=0.024) and Beta diversity was also significantly different (Unweighted UniFrac, P=0.010; Jaccard index, P=0.008). In PPROM cases, Megasphaera genomosp. typeⅠ was significantly increased ( P=0.017) and Lactobacillus mulieris was significantly decreased ( P=0.003). In the patients with TPROM, Megasphaera was significantly increased ( P=0.009) and Lactobacillus mulieris was significantly decreased ( P=0.002). In terms of functional pathways, sulfur oxidation ( P=0.021), methanogenesis from acetate ( P=0.036), L-histidine biosynthesis ( P=0.009), adenosylcobalamin biosynthesis ( P=0.041) and fucose degradation ( P=0.001) were significantly increased in patients with PPROM; L-histidine biosynthesis ( P<0.001) and fucose degradation ( P=0.030) were significantly increased in patients with TPROM. The prediction models were established using the random forest model with vaginal microbiota species and environmental risk factors and the prediction model for PPROM performed well [AUC: 0.739 (95%CI: 0.609-0.869), sensitivity: 0.928, specificity: 0.659, positive predictive value: 0.750, negative predictive value: 0.906], which had a certain reference value. Conclusions:Vaginal microbiota might be related to the development and progression of PROM. Studying the differences in vaginal microbiota might provide a new idea for the prevention and treatment of PROM. Functional prediction provided a direction for further research on the mechanism of PROM. The established prediction model could prevent the occurrence of PPROM and promote maternal and infant health.
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OBJECTIVES@#The high morbidity and mortality of colorectal cancer (CRC) have posed great threats to human health. Circular RNA (circRNA) and microRNA (miRNA), acting as competing endogenous RNAs (ceRNAs), have been found to play vital roles in carcinogenesis. This paper aims to construct a circRNA/miRNA/mRNA regulatory network so as to explore the molecular mechanism of CRC.@*METHODS@#The sequencing data of circRNA from CRC were obtained from Gene Expression Omnibus (GEO). The differential circRNA was screened and its structure was identified by Cancer-specific CircRNA Database (CSCD); the sequencing data of miRNA and messenger RNA (mRNAs) were downloaded from The Cancer Genome Atlas (TCGA) database and the differentially expressed genes were screened; the corresponding miRNA of differential circRNAs were predicted by CircInteractome database; DIANA, Miranda, PicTar, and TargetScan databases were used to predict the target genes of different miRNAs; the target genes from Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were enriched by R language; String database combined with Cytoscape 3.7.2 software was used to construct protein-protein interaction (PPI) network and hub genes were screened; the expressions of mRNAs in the Top10 hub genes were verified in CRC. The network diagrams of circRNAs/miRNAs/mRNAs and circRNAs/miRNAs/Top10 hub mRNAs were constructed by Cytoscape3.7.2. Real-time PCR was used to examine the expression levels of hsa_circRNA_0065173, hsa-mir-450b, hsa-mir-582, adenylate cyclase 5 (ADCY5), muscarinic acetylcholine receptor M2 (CHRM2), cannabinoid receptor 1 (CNR1), and lysophosphatidic acid receptor 1 (LPAR1) in the CRC tissues and the adjacent normal tissues.@*RESULTS@#A total of 14 differential circRNAs were identified, and 8 were found in CSCD; 34 miRNAs targeted by circRNAs were obtained. The PPI network was constructed, and the Top10 hub genes were identified, which were CHRM2, melanin concentrating hormone receptor 2 (MCHR2), G-protein gamma 3 subunit (GNG3), neuropeptide Y receptor Y1 (NPY1R), CNR1, LPAR1, ADCY5, adenylate cyclase 2 (ADCY2), gamma 7 (GNG7) and chemokine 12 (CXCL12), respectively. The expressions of Top 10 hub genes were also verified, and the results showed that the Top 10 hub genes were down-regulated in CRC; the constructed network diagram showed that hsa_circRNA_0065173 may regulate ADCY5, CHRM2, and Hsa-mir-450b by modulating hsa-mir-450b and hsa-mir-582. CNR1 and LPAR1 genes might serve as potentially relevant targets for the treatment of CRC. Real-time PCR results showed that the expression levels of hsa_circRNA_0065173, ADCY5, CHRM2, CNR1 and LPAR1 in the CRC tissues were significantly reduced compared with the adjacent normal tissues (all P<0.05); the expression levels of hsa-mir-450b and hsa-miR-582 were significantly increased (both P<0.05).@*CONCLUSIONS@#In this study, a potential circRNAs/miRNAs/mRNAs network is successfully constructed, which provides a new insight for CRC development mechanism through ceRNA mediated by circRNAs.
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Humans , Colorectal Neoplasms/genetics , Computational Biology/methods , Gene Regulatory Networks , MicroRNAs/genetics , RNA, Circular/genetics , RNA, Messenger/geneticsABSTRACT
Objective:To investigate the effects of different chimerism strategies and different immune ways on the two antigen-dominant regions of Xinjiang hemorrhagic fever virus (XHFV) glycoprotein.Methods:The 5' end was added or not added with interleukin-2 (IL-2) signal peptide and the general-purpose auxiliary T cell epitopes as different design strategies. GcⅠ and GcⅡ and the epitopes previously identified on GcⅠ (Gc 233-248, Gc 241-256 and Gc 281-296) were fused and constructed into the eukaryotic expression vector pVAX1 and the prokaryotic expression vector pET-28a. The recombinant prokaryotic plasmid transformed into E.coli BL21 was induced and purified, and the recombinant eukaryotes were extracted by indirect immunofluorescent assay. BALB/c mice were immunized by protein immunity, gene immunity, and DNA prime-protein boost immunity. The IgG antibody level was measured by ELISA. The immune effect was evaluated by the proliferation of T-lymphocytes and the content of cytokines in the spleen. Results:The results of double enzyme digestion and sequencing showed that eight recombinant plasmids were successfully constructed, and the recombinant eukaryotes were successfully expressed in vitro by fluorescence microscopy. After three times of immunization, the IgG level and the proliferation of T-lymphocytes in the spleen of mice in the experimental group were significantly higher than those in the control group ( P<0.01). The mass concentration test results of Th2 cytokines IL-4 and Th1 cytokines interferon-gamma (IFN-γ) revealed that the response of the DNA prime-protein boost immunity was biased to Th1. Conclusions:The multi-epitope chimeric vaccine of XHFV glycoprotein was successfully constructed, and the target antigen could be expressed effectively in vivo. The immune groups stimulated stronger humoral and cellular immune responses compared with the control group. Among them, the immune effect of pVAX1-ST(GcⅠe+GcⅡ) combined with recombinant protein r(GcⅠe+GcⅡ) was the best, and it is expected to be a new candidate vaccine for XHFV.
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Objective:To study the characteristics and influencing factors of vaginal microbiota in normal pregnant women.Methods:This study was based on a cohort of pregnant women established in Anqing Municipal Hospital Affiliated to Anhui Medical University from February 2018 to February 2020. Vaginal samples of normal pregnant women who met the inclusion and exclusion criteria were ordered by the gestational weeks at sampling. Five samples were randomly selected from each gestational week group and if the samples were less than five, all samples were included. Sequencing of the V3-V4 region of the 16S rRNA gene was performed. Dominant species were analyzed by MicrobiomeAnalyst. Alpha diversity was measured with Chao1, Observed Features, Shannon diversity, Simpson diversity, Faith_pd and Pielou′s Evenness. The dominant status of Lactobacillus was also described and compared. Multiple linear regression and logistic regression were used to analyze the factors influencing vaginal microbiota. Analysis of variance and Kruskal Wallis test were used for statistical analysis of continuous variables, and Chi-square test and Fisher′s exact test were used for categorical data. The differences were considered statistically significant when the P value was less than 0.05. Results:This study enrolled 91 pregnant women (91 vaginal samples) with an average age of (27.37±3.60) years. There were 18, 56 and 17 vaginal samples collected at the median gestational age of 11.93 weeks (the first trimester), 19.43 weeks (the second trimester) and 38.29 weeks (the third trimester), respectively. The relative abundance of Firmicutes and Lactobacillus was 91.30% and 87.67%, respectively. Lactobacillus iners and Lactobacillus crispatus had a relative abundance of 43.95% and 36.33%, respectively. Moreover, Lactobacillus iners-dominated vaginal microbiota was detected in all trimesters. The number of samples with high relative abundance of Lactobacillus iners gradually decreased with gestational age. Lactobacillus crispatus-dominated vaginal microbiota was found in the second and third trimesters and the number of samples with high relative abundance gradually increased during pregnancy. The Alpha diversity of vaginal microbiota had a decreasing trend during the gestation. There were significant differences in Pielou′s Evenness diversity index of vaginal microbiota between different smoking groups ( P<0.05) and in Shannon diversity index between different drinking groups ( P<0.05). There were significant differences in Chao1, Observed Features and Faith_pd diversity index of vaginal microbiota between pregnant women with different education ( P<0.05) and in Shannon and Simpson diversity index between different income groups ( P<0.05). Conclusions:Vaginal microbiota was dominated by Lactobacillus in normal pregnant women. The dominance of Lactobacillus iners gradually decreased, while that of Lactobacillus crispatus increased during gestation. In normal pregnant women, the Alpha diversity of vaginal microbiota was correlated with smoking, drinking, education and family annual income. Smoking cessation and drinking before pregnancy were related to lower Alpha diversity of vaginal microbiota in pregnant women, while lower education and higher family income were associated with higher Alpha diversity.
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Objective:To investigate the prognostic value of systemic inflammatory score (SIS) in prognostic assessment of patients with unresectable metastatic colorectal cancer (mCRC).Methods:The clinical data of 130 patients with unresectable mCRC in Affiliated Haikou Hospital of Xiangya Medical College of Central South University from January 2014 to December 2016 were retrospectively analyzed. The relationship between SIS and clinicopathological characteristics of unresectable mCRC patients was also analyzed. The survival analysis was made by using Kaplan-Meier. The risk factors affecting the prognosis of unresectable mCRC patients were analyzed by using Cox regression model to make univariate and multivariate analysis.Results:According to SIS results, patients were divided into 0-score group (40 cases), 1-score group (58 cases), and 2-score group (32 cases). There were no significant differences in different SIS constitution patients stratified by age, gender, primary tumor location, functional status score, tissue type, RAS gene status, number of metastatic organs, peritoneal spread and molecular targeted therapy (all P > 0.05). Kaplan-Meier survival analysis showed that the 5-year overall survival rates of SIS 0-score, 1-score and 2-score group were 37.5%, 19.0%, 6.3%, respectively; and the difference was statistically significant ( χ2 = 3.152, P<0.01). Cox regression survival analysis showed that female, primary tumor location in right side and SIS (scores of 1-2) were independent risk factors for overall survival in patients with unresectable mCRC (all P<0.05). Conclusion:SIS may be an important indicator for prognostic assessment of patients with unresectable mCRC, and patients with high SIS have poor prognosis.
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Phagosomes undergo the fusion of early endosomes, late endosomes and lysosomes, and then degrade antigens and kill pathogenic microorganisms, which is called phagosome maturation. The functional differences of phagosomes after maturation in different immune cells lead to distinct characteristics on their processing of phagocytic antigens. It has been found that the antigen degradation ability of immune cells is opposite to their antigen presentation capacity through comparing the functional differences of phagosomes from neutrophils, macrophages and dendritic cells after antigens uptake. Among them, dendritic cells have the strongest antigen presentation capacity, followed by macrophages and neutrophils. The in-depth study of immune cell phagosome maturation and antigen presentation will provide new strategies for vaccine development and immunotherapy.
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Varicose veins of the lower extremities are a common disease in vascular surgery and are the result of multiple factors. The clinical manifestations are mainly thickening, tortuosity and dilation of the superficial veins of the lower extremities, which may be accompanied by discomforts such as lower extremity pain and swelling of the lower legs. In severe cases, skin pigmentation of lower extremities, venous ulcers, etc. The current common treatment options include high saphenous vein ligation and stripping, endogenous laser treatment, radiofrequency ablation and foam sclerotherapy, etc. This article reviews some parameter settings and equipment selection that may affect the therapeutic effect and complications of endogenous laser treatment. Hope to help clinicians better choose the device of intracavity laser closure and find the best possible treatment plan for patients.
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【Objective】 To study the application effect of gel adsorbent tank in the production of human prothrombin complex concentrate(PCC). 【Methods】 Six batches of PCC were produced from 1000 L cryoprecipitated plasma, using the same gel twice for adsorption within the tank.The number of gel repeated application was examined by retrospective confirmation, and the adsorption rate, specific activity and residue of finished virus inactivation reagent were determined before and after adsorption. 【Results】 All 6 batches of PPC, produced by the same gel, satisfied quality criteria. Both PPC solution and the gel presented good color. The average activities of coagulation Factors Ⅱ, Ⅶ, Ⅸ and Ⅹ of six batches of PCC were 118.2%, 157.0%, 140.5% and 176.8%, respectively. The The adsorption capacity of coagulation factor Ⅱ, Ⅸ and Ⅹ were both 100% in the first and second adsorption, while coagulation factor Ⅶ were 75% and 81%, respectively. The average specific activity of coagulation factor Ⅸ was 0.7 IU/mg. The average residues of polysorbate 80 and tributyl phosphate products were 0 μg/mL and 33 μg/mL, respectively. The same batch of gel can be repeatedly used up to 6 times during the PCC process. 【Conclusion】 The gel adsorption tank presents good application value in the production of PCC, which can realize process amplification and automatic control.
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Objective:To explore the therapeutic efficacy and safety of conventional radiotherapy and large-segment radiotherapy in patients with early breast cancer undergoing breast-conserving surgery.Methods:A total of 84 early breast cancer patients undergoing breast-conserving surgery treated in Haikou People's Hospital from March 2013 to March 2015 were collected, and they were divided into the conventional group (40 cases) and the large segmentation group (44 cases) according to the radiotherapy method. The patients in the conventional group received conventional radiotherapy at a dose of 2 Gy/time, 25 times in total, with a total dose of 50 Gy, 42 d of total treatment; the patients in the large-scale group received large-scale radiotherapy at a dose of 2.66 Gy/time, 16 times in total, with a total dose of 42.56 Gy, 22 d of total treatment. The effect of radiotherapy, cosmetic effect and adverse reactions of patients in two groups was evaluated. The survival, local recurrence and distant metastasis were also analyzed.Results:There was no statistically significant difference in the total effective rate between the large segmentation group and the conventional group [90.9% (40/44) vs. 95.0% (39/40), χ2 = 1.626, P = 0.205], and there was no statistically significant difference in the excellent rate of beauty effect [90.9% (40/44) vs. 92.5% (37/40), χ2 = 0.069, P = 0.792]. There were no significant differences in the incidence of esophageal mucosal reaction, radiation-induced pneumonia, myelosuppression, advanced skin reaction and acute skin reaction between the conventional group and the large-scale group [2.5% (1/40) vs. 4.6% (2/44), χ2 = 0.255, P = 0.614; 2.5% (1/40) vs. 2.3% (1/44), χ2 = 0.005, P = 0.946; 52.5% (21/40) vs. 52.3% (23/44), χ2 = 0.001, P = 0.983; 2.5% (1/40) vs. 2.3% (1/44), χ2 = 0.005, P = 0.946; 85.0% (34/40) vs. 79.6% (35/44), χ2 = 0.425, P = 0.514). There were no significant differences in 3-year overall survival rate, local recurrence rate and distant metastasis rate between the large-scale segmentation group and the conventional group [90.0% vs. 90.9%, χ2 = 0.020, P = 0.904; 5.0% (12/40) vs. 2.3% (1/44), χ2 = 0.453, P = 0.501; 7.5% (3/40) vs. 6.3% (3/44), χ2 = 0.015, P > 0.05]. Conclusions:Compared with conventional segmented radiotherapy, large-segment radiotherapy regimen can achieve similar efficacy and safety when used as an adjuvant treatment for early breast cancer after breast-conserving surgery. It has the advantages of short treatment cycles and fewer radiotherapy treatments.
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Objective:To evaluate the clinical prognosis of patients with pseudomyxoma peritonei (PMP) after cytoreductive surgery (CRS) combined with hyperthermic intraperitoneal chemotherapy (HIPEC).Methods:The clinical data of 42 patients with PMP after CRS combined with HIPEC in the Affiliated Haikou Hospital of Xiangya Medical College of Central South University from January 2012 to December 2018 was retrospectively analyzed. All patients underwent open surgery CRS combined with HIPEC, the operation condition and prognosis of patients were analyzed.Results:In 42 patients with PMP, the disenminated peritoneal adenomucinosis (DPAM) accounted for 61.9% (26/42), the peritoneal mucinous carcinomatosis (PMCA) accounted for 28.6% (12/42), and the borderline accounted for 9.5% (4/42). The incidence rate of major operative complications (grade Ⅲ-Ⅳ) after CRS combined with HIPEC was 21.4% (9/42). The logistic regression analysis showed that the previous surgery score ( OR = 35.765, 95% CI 2.746-43.986, P = 0.001) and completeness of CRS score ( OR = 23.865, 95% CI 1.345-347.876, P = 0.028) were independent factors influencing major postoperative complications in PMP patients. The overall survival time of 42 patients with PMP was (64.8±4.1) months, and the disease-free survival time was (54.0±4.9) months; the 3-year and 5-year overall survival rates were 80.8% and 65.9%, and the 3-year and 5-year disease-free survival rates were 59.5% and 54.6%, respectively. The difference in overall survival time of patients with different pathological subtypes was statistically significant ( P = 0.022). Conclusion:CRS combined with HIPEC is safe and effective for treatment of patients with PMP, and most of the patients have a good prognosis.
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Objective:To express truncated glycoprotein (Gn, Gn1, Gn2, Gn3, Gc1 and Gc2) of Guertu virus (GTV) in Escherichia coli ( E. coli) cells, and prepare polyclonal antibodies against recombinant proteins Gn-His, Gc1-His and Gc2-His after purification. Methods:Gene fragments encoding Gn, Gn1, Gn2, Gn3, Gc1 and Gc2 of GTV DXM strain were amplified by RT-PCR, and cloned into the prokaryotic expression vector pET-32a (+ ) to construct recombinant expression plasmids. The transformed E. coli BL21(DE3) strains carrying expression plasmids were induced by IPTG to express target proteins, which were identified by SDS-PAGE. Recombinant proteins Gn-His, Gc1-His and Gc2-His were purified by nickel affinity chromatography and detected by Western blot using GTV-positive sheep serum for analysis of their antigenicity. New Zealand white rabbits were immunized with the purified recombinant proteins. The titers and specificity of serum antibodies were analyzed by ELISA. Meanwhile, eukaryotic expression vectors pcDNA3.1-Gn, pcDNA3.1-Gc1/Gc2 were constructed and transfected into mammalian Vero cells to evaluate the binding activity of rabbit polyclonal antibodies by indirect immunofluorescence method. The specific reactivity of serum antibodies to recombinant proteins was detected by Western blot. Results:Restriction enzyme analysis and DNA sequencing confirmed that the recombinant expression vectors of pET-32a-Gn, pET-32a-Gn1/Gn2/Gn3, pET-32a-Gc1/Gc2, pcDNA3.1-Gn and pcDNA3.1-Gc1/Gc2 were constructed successfully. The relative molecular mass ( Mr) of the expressed recombinant proteins Gn-His, Gn1/Gn2/Gn3-His, Gc1/Gc2-His were approximately 63.4×10 3, 37.1×10 3, 31.9×10 3, 30.8×10 3, 40×10 3 and 54.4×10 3, respectively. The recombinant proteins could be recognized by GTV-positive sheep serum. The titers of polyclonal antibodies against GTV Gn, Gc1 and Gc2 were 1∶409 600, 1∶204 800 and 1∶6 400, respectively. Indirect immunofluorescence assay and Western blot showed that the prepared rabbit polyclonal antibodies could specifically react with the proteins expressed in eukaryotic cells and the recombinant proteins. Conclusions:The recombinant GTV glycoproteins Gn-His and Gc1/Gc2-His were efficiently expressed and purified and characterized with good immunity. The prepared polyclonal antibodies had high titers and good specificity. This study provided reference for further studying the biological function and detection methods of GTV glycoproteins and research on vaccines.
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Objective To express and purify two domains GcⅠand GcⅡof Xinjiang hemorrhagic fever virus (XHFV) glycoprotein, and to study its immunogenicity and the effects on immune response in mice. Methods The prokaryotic expression plasmids of pET28a-GcⅠand pET32a-GcⅡwere constructed and transformed into E. coli BL21, respectively. The expression and purification conditions of rGcⅠand rGcⅡproteins were optimized. The antigenicity of the fusion protein was detected by Western Blot and enzyme-linked immunosorbent assay (ELISA). BALB/c mice were immunized by protein immunization and DNA priming-protein boosting. The mice were randomly divided into 5 groups, including pVAX1-GcⅠ+rGcⅠgroup, pVAX1-GcⅡ+rGcⅡgroup, rGcⅠgroup, rGcⅡgroup and saline group (control group) with 7 mice in each group. The serum antibody titer of mice was detected by indirect ELISA, and the immune effect was evaluated by spleen T lymphocyte proliferation assay and cytokine content determination. Results The fusion proteins rGcⅠand rGcⅡwere purified and obtained, which could react with positive serum of sheep and had good antigenicity. After three immunizations, the IgG levels in the serum of each experimental group were significantly higher than those in the control group (all P<0.001). The serum antibody titers of the experimental groups were reached above 1:12800. Among them, the concentration of Th2 type cytokine interleukin-4 (IL-4) in the spleen cell culture supernatant of rGcⅡ[(79.97±7.47) ng/L] and pVAX1-GcⅡ+rGcⅡgroup [(61.43±9.27) ng/L] was significantly higher than (24.29±3.81) ng/L of the control group, respectively (all P<0.01). The highest mass concentration [(42.46 ±2.60) ng/L] of Th1 type cytokine interferon-γ(IFN-γ) was observed in the pVAX1-GcⅡ+rGcⅡ group, which was significantly higher than (20.33±1.67) ng/L of the control group, and the difference was statistically significant (P<0.001). That showed a significant antigen-specific splenic T lymphocyte proliferation (P<0.001). Conclusions The purified recombinant proteins rGcⅠand rGcⅡhave good immunogenicity, which can make the immune system T lymphocytes tend to Th2 response, and pVAX1-GcⅡ combined with recombinant protein GcⅡ can induce better antigen-specific immune effect. And pVAX1-GcⅡ combined with recombinant protein GcⅡis expected to be used as vaccine candidates for the prevention and control of XHFV.
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Objective To investigate a anatomy research and clinic application of defect of thumb by reverse dorsoradial thumb flap of different rotation point.Methods The origin,course,distribution and vascular chain of the first metacarpal dorsoradial artery of thumb from 11 adult cadaveric hand specimens perfused by red latex were explored from September,2012 to December,2016.There were 3 different rotation points:the proximal of the metacarpophalangeal joint,proximal basal of the first proximal phalanx and distal of the first proximal phalanx.Each could be used as reverse flow flap to repair the defect of thumb.Results The first metacarpal dorsoradial artery of thumb originated from the radial artery and the initial diameter was (0.68±0.26) mm,diagonally across the extensor pollicis brevis tendon and then along the radialis part and terminated in the proximal of the first proximal phalanx of the vascular chain.There was a constant communicating branch among the proximal metacarpophalangeal joint,proximal basal of the first proximal phalanx and digital arterial dorsal branch.All the proximal of the metacarpophalangeal joint 4.3 mm to 10.2 mm and proximal basal of the first proximal phalanx 4.9 mm to 7.2 mm could be used as the rotation point of the flap.The flap of the first promixal phalanx blood supply was based on the vascular chain of neurocutaneous.There was a constant dorsal branch of the pollical artery,which was 8.6 mm to 10.3 mm far from the interphalangeal joint,could be used as the rotation point of the flap.Twenty-four cases with soft tissue defects of thumb were repaired by reverse dorsoradial flap.The flap size ranged from 2.0 cm×1.5 cm to 3.5 cm×2.8 cm.The follow-up period was 3 months to 12 months,protective sensations were restored,and skin flap two-point discrimination were 9.0 mm to 12.0 mm.The appearance of the thumbs was satisfactory.Conclusion Different rotation point of reverse dorsoradial flap can successfully repair the defect of thumb.The operation has advantages of simple,reliable blood supply,high success rate and is an ideal option for reconstruction the defect of thumb.
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Objective To investigate the effects of Glycyrrhiza uralensis Fisch L. crude polysac-charides (GUCP) as an adjuvant on the immunity of mice and the immune responses induced by human pap-illoma virus ( HPV)-DNA vaccine. Methods ICR mice were injected with different concentrations of GUCP by different ways to detect the influences of GUCP on body weight, organ indexes and the numbers of immune cells in spleen. C57BL/ 6 mice were co-immunized with HPV-DNA vaccine and GUCP to detect the adjuvant efficacy on antigen-specific cellular and humoral immune responses. Results GUCP in all injected groups had no side effect on mouse body weight and liver, heart, lung and kidney indexes, but intraperitone-al injection of GUCP significantly increased spleen and thymus indexes and the numbers of B cells, CD4+ T cells, macrophages and dendritic cells in spleen. Subcutaneous injection of GUCP significantly increased the numbers of B cells and macrophages in spleen and intragastric administration significantly increased the num-bers of CD4+ and CD8+ T cells in spleen. Furthermore, GUCP as an adjuvant enhanced the antigen-specific CD4+ and CD8+ T cell responses and the levels of IgG, IgG1 and IgG2a induced by HPV-DNA vaccine at a certain degree. Conclusion GUCP enhanced the immunity of mice and the antigen-specific cellular and hu-moral immune responses induced by HPV-DNA vaccine. These results suggested that GUCP might be used as an adjuvant for DNA vaccine.
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AIM: To study the effect of interleukin-1β (IL-1β) on the activity of vacuolar proton pump (V-ATPase) and intracellular pH value in the osteoclasts for exploring the mechanism of IL-1β to promote osteoclastic bone absorption.METHODS: The mature osteoclasts were attained by induction of bone marrow monocytes/macrophages.The osteoclasts were cultured with α-MEM and treated with different concentrations (0.1, 0.3 and 0.5 μg/L) of IL-1β.After cultured for 48 h, the mRNA and protein expression of V-ATPase was detected.The effect of IL-1β on intracellular pH value, activity of V-ATPase and the bone absorption abilities of osteoclasts were examined.RESULTS: The expression level and activity of V-ATPase were significantly increased and the intracellular pH value of the osteoclasts decreased after incubated with IL-1β for 48 h.Under the same culture condition, the bone absorption capacity of the osteoclasts was promoted.The enhanced bone absorption capability of the osteoclasts was accompanied by an increase in the concentration of IL-1β.CONCLUSION: The mechanism of IL-1β participating in pathological bone absorption in the bone and joint inflammatory diseases is that IL-1β increases the expression and activity of V-ATPase, boosts the production and/or transportation of hydrogen ion, leading to increased osteoclastic bone absorption activity, and resulting in bone destruction.
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Objective To observe the effect of sodium butyrate on the invasion and migration of human salivary gland ade‐noid cystic carcinoma cell line ACC‐2 in vitro and to investigate the underlying mechanisms .Methods The cultured ACC‐2 cells were treated with different concentrations of sodium butyrate for 24 h ,and detected the viability rate of the cells by methyl thiazolyl tetrazolium(MTT) assay ;the drug′s influence on invasion and migration on ACC‐2 were detected by Transwell experiment ,while the protein and mRNA expression of HMGB1 and TLR4 explored by Western‐blot and RT‐PCR assay ;the relationship between TLR4 expression and HMGB1 was investigated .Results Compared with control group ,0 .625 ,1 .250 ,2 .500 ,5 .000 ,10 .000 mmol/L groups of sodium butyrate inhibited the proliferation of ACC‐2 cells(P0 .05);only 2 .500 ,5 .000 and 10 .000 mmol/L groups inhibited ACC‐2 cells migration and down‐regulated the protein and mRNA of HMGB1 and TLR4(P<0 .05) .Correlation analysis showed a positive correlation between the TLR4 protein and HMGB1 protein(r=0 .810 ,P<0 .05) .Conclusion Sodium butyrate could inhib‐it ACC‐2 cells proliferation and high concentration gropes inhibit ACC‐2 cells migration ,while reducing HMGB1 and TLR4 mRNA and protein expression ,suggesting that NaB might inhibite ACC‐2 cells migration through down‐regulated the mRNA and protein expression .
ABSTRACT
Background and purpose: Researches demonstrated that the butyric acid sodium salt (sodium butyrate, NaB) has effect on the inhibition of tumor cell proliferation, differentiation and apoptosis-promoting, while the mechanism on salivary adenoid cystic carcinoma(SACC) is still uncertain. This study mainly probed into the impact of different concentration of sodium butyrate on the migration and invasion of SACC cell line ACC-M, and its mechanism of action. Methods:MTT assay explored the optimal concentration of sodium butyrate on the cell ACC-M and the observation of cell growth. Transwell assay was used to detect the effects of sodium butyrate on the ACC-M cells on the aspact of invasion and migration ability. Fluorescence real-time quantitative PCR (RT-PCR) and Western blot were used to test respectively the expression of HMGB1, TLR4 mRNA and protein in ACC-M after functioned by 5 group drugs with different concentrations. Results:Compared with the control group, on the one hand, the concentration 0.625, 1.25, 2.5, 5 and 10 mmol/L of sodium butyrate could effectively inhibit cell proliferation and apparently showing concentra-tion-dependence (P<0.05);On the other hand, 5 sets concentration of sodium butyrate could also effectively inhibit invasion and migration ability of ACC-M cells in vitro (P<0.05), as well as reducing the expression of HMGB1, TLR4 mRNA and protein in ACC-M cells (P<0.05). Furthermore related analysis showed that the decline of TLR4 protein expression was positively correlated with inhibition of HMGB1 (r=0.810, P<0.05). Conclusion:Sodium butyrate has an effect on inhibiting ACC-M cell proliferation, signiifcantly reducing ACC-M cell invasion and migration capabilities, and reducing expression of HMGB1, TLR4 mRNA and protein, and both expression amount are positively correlated, Meanwhile the positively correlation suggests that sodium butyrate probably achieve the inhibition ability by lowering the expression of HMGB1, TLR4 mRNA and protein in ACC-M cell.