ABSTRACT
The aim of this study was to construct the eukaryotic expression vector of pCMV-Myc-NOVA1 based on NOVA1 gene, and to screen the optimum expression condition after transfecting to PC12 cells, and further to explore the distribution of NOVA1 protein in PC12 cells using cell immunohistochemistry, and to identifyits anti-hypoxia activity. According to the NOVA1 gene sequence of NCBI database, we designed the upstream and downstream primers, and performed polymerase chain reaction (PCR) to amplify the full length cDNA coding sequence using pCR4-TOPO-NOVA1 as a template. The products were digested by restriction endonuclease SalⅠand XhoⅠ, and conjugated to the eukaryotic expression vector ofpCMV-Myc followed by validating by digestion and direct sequencing. Subsequently, the validated pCMV-Myc-NOVA1 was transfected to PC12 cells followed by optimizing of transfection ratio and transfection time, and identified by qPCR, Western blotting and cell immunohistochemistry respectively. After validation by digestion and direct sequencing, the eukaryotic expression vector of pCMV-Myc-NOVA1 was correctly constructed. The optimum transfection ratio of plasmid to Lipo 2000 was 1:2.5, and the optimum transfection time was 72 h. At the optimum transfection condition, the expression level of NOVA1 mRNA and protein significantly increased, and after transfection of pCMV-Myc-NOVA1, NOVA1 protein mainly distributed in cell nucleus and cytoplasm. After 6 h hypoxia, the cell proliferation activity was significantly increased compared to that of the control and pCMV-Myc group. Our findings provided a reference for exploring the mechanism of NOVA1, and also a technical support for potential drug development of NOVA1.
Subject(s)
Animals , Humans , Rats , Blotting, Western , Cell Hypoxia , Genetic Vectors , PC12 Cells , Plasmids , RNA, Messenger , RNA-Binding Proteins , Genetics , Metabolism , TransfectionABSTRACT
OBJECTIVE:To systematically review the efficacy and safety of cinacalcet in the treatment of hemodialysis pa-tients with secondary hyperparathyroidism,and provide evidence-based reference for the clinical treatment. METHODS:Retrieved from Medline,Cochrane Library,EMBase and CBM,randomized controlled trials(RCT)about cinacalcet in the treatment of he-modialysis patients with secondary hyperparathyroidism (SHPT) were collected. Meta-analysis was performed by using Rev Man 5.3.5 software after data extract and quality evaluation by Cochrane systematic Rev Man 5.3.5. RESULTS:Totally 7 RCTs were en-rolled,involving 1 987 patients. Results of Meta-analysis showed cinacalcet can significantly reduce the rate of surgical parathyroid-ectomy[RR=0.23,95%CI(0.06,0.89),P=0.03],incidence of fracture[RR=0.26,95%CI(0.12,0.60),P=0.002] and increase the incidences of hypocalcemia[RR=9.81,95%CI(3.92,4.59),P<0.001],nausea[RR=1.97,95%CI(1.58,2.46),P<0.001] and vomit-ing[RR=1.91,95%CI(1.50,2.42),P<0.001],while it showed no significant effect on the the incidence of all-cause mortality and cardiovascular death. CONCLUSIONS:The clinical efficacy of cinacalcet in the treatment of hemodialysis patients with secondary hyperparathyroidism is good,but there are common adverse reactions such as nausea and vomiting,hypocalcemia.
ABSTRACT
OBJECTIVE:To optimize the formulation of nisoldipine buccal adhesive tablets. METHODS: The formulation was optimized by orthogonal design with the dosage ratio of carbopol to chitosan in drug-containing layer and protection layer, the content of lactose and the tabletting hardness as factors. The bioadhesiveness and in vitro drug release rate of the tablets was measured as well. RESULTS: The optimal formulation was as follows: the ratio of carbopol to chitosan in drug-containing layer and protection layer were 22.5∶22.5 and 33.3∶16.7 mg, respectively; the dosage of lactose was 10 mg, and tabletting hardness was 5 kg. The averge bioadhesiveness of 3 bacthes of optimized preparations was 79.5 g?cm-2, and the average in vitro drug release rate of the tablets in 12 h was more than 95%. CONCLUSIONS: The optimized nisoldipine buccal adhesive tablets enjoy ideal bioadhesiveness and slow-release character.