ABSTRACT
Neovascularization is a characteristic manifestation of a variety of retinal diseases. Vascular endothelial growth factor (VEGF) mainly regulates the proliferation and migration of endothelial cells. VEGF receptor 2 (VEGFR2) is the main receptor to mediate this effect. The activation of downstream signals requires the binding of VEGF and VEGFR2, followed by receptor dimerization and autophosphorylation. Blocking this process and inhibiting neovascularization is very attractive treatment ideas. Monoclonal antibodies and fusion protein drugs currently used in ophthalmology can bind free VEGF. In addition, there are also macromolecular antibodies binding VEGFR2 and small molecule tyrosine kinase inhibitors, which is expected to further expand into the field of ophthalmology. Although anti-VEGFR2 therapy is a revolutionary method to inhibit neovascularization, there are no sufficient clinical evidences at present. In-depth understanding of the application status and progress of anti-VEGFR2 in the treatment of retinal neovascular diseases has important clinical significance.
ABSTRACT
Objective:To observe the stoichiometry of vascular endothelial growth factor receptor 2 (VEGFR2) on the retinal vascular endothelial cell membrane by single-molecule fluorescence imaging.Methods:Rhesus monkey retinal vascular endothelial cells (RF/6A) were divided into blank control group (normal culture) and plasmid transfection group [transfected with VEGFR2-green fluorescent protein (GFP) recombinant plasmid]. The expression of GFP in the plasmid transfected group was observed by confocal microscope, and the expression of VEGFR2 in the cells was detected by real-time fluorescent quantitative polymerase chain reaction (qPCR) and Western blot. The fluorescence intensity distribution and bleaching steps of single VEGFR2-GFP molecule on the cell membrane were recorded by single-molecule imaging. The distribution of fluorescence intensity and the number of fluorescence bleaching steps of GFP were recorded.Results:GFP green fluorescence was observed in the transfected cells 12 hours after transfection. qPCR results showed that the expression of VEGFR2 and GFP mRNA in the plasmid transfected group was significantly higher than that in the blank control group ( t=11.240, 12.330; P<0.001, 0.001). Western blot results showed that the expression of VEGFR2 protein in the plasmid transfected group was significantly higher than that in the blank control group ( t=8.346, P<0.01). The results of single-molecule imaging showed that the fluorescence intensity distribution of VEGFR2-GFP on the surface of RF/6A cell membrane without ligand stimulation was bimodal, in which monomer and dimer were 86.0% and 14.0% respectively. By counting the steps of GFP fluorescence bleaching, the proportions of receptor monomer, dimer, trimer, and tetramer were 81.4%, 12.9%, 5.5%, and 0.3% respectively. Conclusion:In the absence of ligands, VEGFR2 coexists in the form of monomers and dimers on the surface of RF/6A cell membrane, and monomers are dominant.
ABSTRACT
Agmatine is an endogenous amine synthesized from the decarboxylation of arginine.It has a rich biological effects and presents in plants, bacteria and mammalian tissues.Agmatine is highly polar and has a low molecular weight.There are no UV and fluorescent absorption groups in agmatine structure, so it is difficult to gasify.In addition, the content of endogenous agmatine is low, and there are many interference components in biological samples, and the general method is difficult to detect.The pre-column derivatization of agmatine with fluorescence reagents not only increase the molecular weight of agmatine, but also make them with fluorescence, which greatly improve the detection sensitivity and enable the endogenous agmatine to be well separated.It is an important method to determine the content of agmatine.In this paper, several kinds of precolumn derivatives are summarized, and the advantages and disadvantages of various derivatives, the best derivation conditions and detection methods were also analyzed comprehensively.The aim is to determine the content of agmatine and to provide methods and ideas for its biological research.
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Objective To develop a HPLC method for determination of pueratin.Methods The separation was carried out on a Waters Symmetry C18 column(4.6 mm×250 mm, 5 μm), the mobile phase was composed of acetonitrile and 1% formic acid(11∶89), the detection wavelength was set at 250 nm, the flow rate was 1.0 ml/min, the column temperature was 30 ℃ and the injection volume was 10 μl.Results The linearity was obtained over 2~40 μg/ml (r=0.999 8) for pueratin.The RSD of precision were less than 2%.The average recovery was between 98% and 103%.Conclusion This HPLC method was simple, accuracy and suitable for the quality control of Jiangzhi Hugan capsule.
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Objective To investigate serum indicators of liver fibrosis in patients with chronic hepatitis in liver fibrosis diagnostic significance.Methods determination of serum HA,LN,PCⅢ,ⅣC was condueted by RIA technology in 100 cases of chonic hepatitis and 40 cases of healthy people,the difference of the findings was in two groups coup ared.Results There was signifieunt difference in terms of serum HA,LN,PCⅢ,ⅣC among normal control,moderafe hepatitis,sever hepatitis and cirrhosis group(P0.05).There was significant difference in ferms of serum HA,LN,PCⅢ,ⅣC among cirrhosis and mild,moderate,serere hepatitis as well as healthy control group(P
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Objective To establish and optimize a method to extract and determine rhynchophylline in a traditional Chinese herb,Ramulus Uncariae cum Uncis. Methods Cold maceration with methanol and supersonic extraction was used to treat the medical material. Chromatography was performed on a Diamonsil C18 column (250 mm?4.6 mm,5 ?m). Gradient elution was employed with a mobile phase consisting of methanol (solution A) and ammonium acetate buffer (0.01 mol/L,pH 6.0 adjusted by ammoniae aqua,solution B) as follows: A∶B from 25∶75 to 50∶50 from 0 to 20 min. The flow rate of the mobile phase was 1.0 ml/min and the column was maintained at 20 ℃. The detector was monitored at 245 nm. Results The calibration curve was linear among the concentration range of 0.25 to 64.0 ?g/ml (y=3.64?104x-2.13?104,r=0.999 5) and the relative recovery ranged from 95.95% to 114.4%. The relative standard deviation (RSD) of the reproducibility test was 6.50% and of the stability test was 4.62%,2.65% and 4.58% from the high density to the low. Conclusion Our method is accurate,reliable and with good reproducibility,and it can be used for determining rhynchophylline in Ramulus Uncariae cum Uncis.