Objective: To confirm the effect of alcohol-induced apoptosis of AC16 cardiomyocytes and the relationship with the alcohol concentration and the action time.To investigate the expression levels of miR-186-5p and X-linked inhibitor of apoptosis protein(XIAP) along with the variational apoptosis level in alcohol -treated AC16 cardiomyocytes with different concentrations. To explore miR-186-5p regulated apoptosis of alcohol-induced cardiomyocytes using XIAP as the target gene.Methods:Flow cytometry analyzed the expression levels of cardiomyocyte apoptosis. Western blot and real-time PCR detected respectly the protein and RNA expression levels of miR-186-5p and XIAP in alcohol-treated AC16 cardiomyocytes. Luciferase reporter gene experiment verified that XIAP is a direct target gene of miR-186-5p.Results:Alcohol induced cardiomyocyte apoptosis, and the levels of apoptosis were dependent on alcohol concentration and duration of time. Alcohol intake can increase the expression levels of miR-186-5p and decrease the expression levels of XIAP in AC16 cardiomyocytes. miR-186-5p was involved in alcohol-induced the process of AC16 cardiomyocyte apoptosis.XIAP can inhibite alcohol-induced AC16 cardiomyocyte apoptosis.miR-186-5p targets XIAP as a target gene to regulate alcohol-induced cardiomyocyte apoptosis.Conclusion:The levels of apoptosis in AC16 cardiomyocytes increase after alcohol treatment and increase further with the increase of alcohol concentration and the prolongation of the action time. The expression levels of miR-186-5p increase, and the expression levels of XIAP decrease in alcohol-treated AC16 cardiomyocytes. miR-186-5p regulates apoptosis of alcohol-induced cardiomyocytes using XIAP as the target gene.
Objective: To investigate the effect of small interfering RNA (siRNA) -mediated bone morphogenetic protein7 (BMP7) gene silencing on osteogenic differentiation of porcine aortic valve interstitial cells induced by osteogenic induction medium,and to provide a theoretical basis for the intervention and treatment of calcific aortic valve disease.Methods:The non-CAVD valvular tissues (non-CAVD group) were taken from patients with surgical treatment of aortic dissection,the CAVD valvular tissues (CAVD group) were taken from patients undergoing aortic valve replacement because of calcified aortic valve stenosis.The expression levels of BMP7 and Runx2 in the non-CAVD group and CAVD group were tested by immunohistochemistry and Western blot. Healthy domestic pigs were sacrificed and the aortic valve leaflets were aseptically removed immediately.The aortic valve interstitial cells(VICs) were isolated by continuous collagenase digestion,its morphological characteristics were observed and the phenotypes were identified by immunofluorescence staining. VICs were transfected with BMP7-siRNA by liposome method.The expression of BMP7 at mRNA and protein levels in the VICs transfected with BMP7-siRNA was detected by qPCR and Western blot,respectively.The conditioned medium induced osteogenic differentiation of VICs for the establishment of calcification model of aortic valve interstitial cells in vitro,then Alkaline phosphatase (ALP)staining and Alizarin red S staining was used to evaluate the cell early and late osteogenic differentiation abilities.The mRNA and protein levels of Runx2,OCN and OPN were determined by qPCR and Western blot,respectively.The protein levels of p-drosophila mothers against de-capentaplegic 1/5/8 (p-Smad1/5/8) was also determined by Western blot.Results:The expression of BMP7 and Runx2 in CAVD group was significantly higher than that in non-CAVD group.The primary porcine aortic valve interstitial cells were successfully isolated and the staining of α-smooth muscle actin (α-SMA) and Vimentin were positive,the staining of von Willebrand factor (vWF) was negative.The expression of BMP7 at mRNA and protein levels in the VICs transfected with BMP7-siRNA was significantly decreased,and the cell early and late osteogenic differentiation abilities were significantly decreased.The mRNA and protein levels of Runx2,OCN and OPN were significantly reduced.Meanwhile,the protein levels of p-Smad1/5/8 were down-regulated.Conclusion:BMP7 gene silencing obviously inhibits the osteogenic differentiation of aortic valve interstitial cells induced by osteogenic induction medium.The BMP7/Smads signaling pathways may play an important role in these processes.
To improve the conversion efficiency of glucoside to small molecule compounds, the gene encoded β-glucosidase from Lactobacillus paracasei TK1501 was inserted into pET28a(+), and further transformed into E. coli BL21(DE3) for heterologous expression. The recombinant enzyme, which purified by nickel affinity chromatography, is conferred with the high specific activity of 675.56U/mg, and the molecular weight of 86.63kDa.The biochemical characterization of this recombinant enzyme shows that it exhibit the highest bio-activity in 30℃ and pH of 6.5, and the β-glucosidase activity was barely inhibited by Mg 2+ and Ca 2+, but largely by Cu 2+ with even no catalytic activity. It was also found that this enzyme possess a broad substrates specificity toward genistin, daidzin, daidzein, geniposide, salicin, heptosporin, polydatin and arbutin. Finally, The kinetics characteristics shows that Km and Vmax of this enzyme are 1.44mmol/L and 58.32mmol/(L·s), respectively, and the catalytic coefficient (kcat) is 3 982/s using β-pNPG as the substrate. The all results above show that the β-Glucosidases from Lactobacillus paracasei TK1501 play important roles in the process of hydrolysis of soybean isoflavone and synthesis of glycosides.
Objective: Construct hepatic-specific knockout mice model of OTUB1, the important deubiquitinase of ovarian tumor domain(OTU) protease superfamily, preliminarily analysis the phenotype of hepatic-specific OTUB1 knockout mice model and explore the physiological function of OTUB1 in liver metabolism.Methods:A mouse model of conventionally disrupting OTUB1 gene in liver using Cre/Loxp system was generate. The obtained OTUB1 fl/fl transgenic mice were crossed with Alb-Cre mice and PCR was used to identify the genotype of its offspring. Furthermore, liver-specific OTUB1 knockout mice were obtained by self-crossing the offspring and PCR was used to identify the genotype. At the same time, OTUB1 protein expression level was detected in tissues and organs of adult mice, include liver and other major organs, from hepatic-specific OTUB1-knockout (HCKO) mice and the control group (control, NC) littermate mice, and Western blot were used to detected and evaluated OTUB1 protein levels. The data indicated whether the hepatic-specific OTUB1 knockout mouse model was successfully constructed. Once comfirmed OTUB1 was truly mutant expression in the liver of HCKO mice, histopathological examination was performed and analyzed the morphology of liver, stomach and spleen, which was analyzed whether there was any spontaneous pathological change existed. In addition, the main biochemical indicators of the liver were detected and analyzed by serum to reflect liver lipid metabolism function in HCKO mice. Moreover, the level of blood glucose metabolism control was recorded and compared between HCKO mice and NC littermate mice through the Glucose Tolerance Test (GTT). Results:The genomic sequencing and Western blot analysis showed that OTUB1 was significantly deleted only in the liver of HCKO mice, but the protein expression level of OTUB1 in other tissues was unchanged at all, where the Alb-Cre transgene is not expressed, such as the kidney, spleen, fat and muscle, which proved that the hepatic-specific OTUB1 knockout mouse model was successfully constructed. Genotyping the offspring of OTUB1 hepatic-conditional knockout mice showed its were born normally. Also, these HCKO mice stayed healthy, without spontaneous histopathological abnormalities in embryonic development. Moreover, the total cholesterol levels in biochemical indicators were significantly lower in HCKO mice less than in NC mice, indicating that the OTUB1 affects liver lipid metabolism level to a certain extent. In glucose tolerance test, the blood glucose level of HCKO mice decreased rapidly after reaching its highest level, suggesting that the homeostasis of liver blood glucose depended on the regulation of OTUB1.Conclusions:The hepatic-specific OTUB1 knockout mouse model was successfully established by Cre/Loxp technology strategy, which are essential for research deubiquitinase OTUB1 in physiological condition,as well as provide an important animal model for studying the physiological functions and regulatory mechanisms of OTUB1 in the liver.
Objective: To construct a red fluorescent protein-green fluorescent protein-murine LC3 fusion multi-lentiviral expression vector (PCDH-Duo-mRFP-eGFPph-LC3rat, PCDH-Duo),which can be used to stably monitor the changes of autophagy flux and overexpression genes. Changes in autophagic flow were observed in the mouse peritoneal macrophage Raw264.7 stable strain.Methods:The mRFP-eGFPph-LC3rat fusion gene was synthesized by PAS and cloned into the lentiviral expression vector PCDH-CMV-MCS-EF1a-copGFP. After the recombinant plasmid was correctly analyzed by PCR, enzyme digestion and sequencing, the lentivirus was packaged. Raw264.7 cells were transfected, and stable cells were obtained by FACS. The reliability of the eGFP protein expression system was confirmed by CQ autophagy inhibition model and Western blot.Results:The recombinant plasmid of PCDH-Duo lentivirus was successfully constructed, which was coated with lentivirus and obtained stable cell line of Raw264.7. The expression of double fluorescent protein was stable. After induction by 3mmol/L CQ for 6h, it was stable and accurate. The phasing changes.Conclusion:The dual-fluorescence multi-function autophagic flux monitoring system based on lentivirus system was successfully constructed, which provides a convenient and powerful tool for studying the relationship between autophagy and coding genes and non-coding genes.
B. circulans 251 β-CGTase was applied to trehalose preparation, and the trehalose yield was increased from 50.4% to 71.9%. In order to further improve the conversion rate of substrates, B. circulans 251 β-CGTase mutants with improved affinity for maltose as a disproportionation acceptor were screened thorugh error-prone PCR and high-throughput screening. Mutant M234I with higher affinity for maltose was selected with a low concentration of 4,6-ethylidene-p-nitrophenyl-indole-D-maltoheptaose (EPS) chromogenic method. The wild-type β-CGTase and the mutant enzyme M234I were purified, and the enzymatic properties were characterized. The specific activity of the mutant M234I was 345.25U/mg (disproportionation activity), while that of the wild type was 357.63U/mg. The maltose Km vaule of the mutant M234I was 0.258 2mmol/L, which was only 54.4% of the wild type (0.474 9mmol/L), which indicates that its affinity for maltose was significantly improved. The optimum temperature and the optimum pH of the mutant did not change much compared with those of the wild type. The mutant M234I was used in the multi-enzyme complex system to produce trehalose with maltodextrin (DE 16) as substrate, the result showed that the trehalose yield was up to 74.9%, which was 3% higher than that of the wild-type β-CGTase.
CD133(Prominin-1) is one of members which are the five-time transmembrane glycoprotein Prominin family. CD133 was originally used as a specific marker for the screening of human hematopoietic stem and progenitor cells, which was subsequently used to isolate and identify specific cell subsets of various cancer stem cells. Many studies have shown that CD133 is a prognostic marker for tumor therapy, interacts with substances such as vascular endothelial growth factor, participates in signal transduction in cell pathways, and plays an important role in maintaining retinal morphology and function. Depending on whether or not it binds to a glycosylation epitope of CD133, antibodies related to CD133 can be classified into glycosylated antibodies, non-glycosylated antibodies, and other antibodies that are not indicated to bind to a glycosylated epitope. The recent years’ research of CD133 was focused,the Prominin family, and the functions, related antibodies and related research methods of CD133 were reviewed.
Although new vaccines such as DNA vaccines, recombinant vaccines, and subunit vaccines obtained through modern biotechnology have been improved in safety compared to conventional vaccines, they also have certain defects. One of the biggest defects is that the immunogenicity is often low, and the efficacy of the immune body cannot be achieved. It is necessary to add an adjuvant or a carrier to improve the immune efficacy. Therefore, it is very important to develop an effective, safe and effective adjuvant to enhance the specific immune response. With the continuous development of adjuvants, aluminum adjuvant, oil emulsion adjuvant, microbial adjuvant, propolis adjuvant, levamisole adjuvant, liposome adjuvant, traditional Chinese medicine adjuvant and peptide adjuvant have been used as immunoadjuvants and its mechanism of action has also been studied more and more thoroughly. Through animal immunization experiments, it was found that peptide immunoadjuvant not only enhances specific immune response, but also has the effect of immune enhancer, and is easy to obtain, easy to transport and preserve, and has high safety, which may be a major direction of future adjuvant research.
