Protective effect of Pollen Typhae extract on diabetic retinopathy in rats / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM:To investigate the protective effects of Pollen Typhae extract on diabetic retinopathy(DR)in rats. <p>METHODS: Fifty SPF rats were randomly divided into five groups: control group with normal feeding, DR group was given the same amount of normal saline by gavage, experimental group A with Pollen Typhae extract 50mg/(kg·d), experimental group B with Pollen Typhae extract 100mg/(kg·d)and experimental group C with Pollen Typhae extract 200mg/(kg·d). Determination of fasting blood glucose in rats of each group. HE staining was used to observe the pathological changes of retina of rats in each group. The expression of IL-6 and TNF-α in the serum of rats were measured by ELISA. The VEGF, VEGFR2 and Ang-1 protein expression in retina tissue were observed by Western blot. The VEGF, VEGFR2 and Ang-1 mRNA expression in retinal tissue were observed by <i>q</i>RT-PCR. <p>RESULTS: Compared with the control group, the fasting blood glucose, the expression of IL-6 and TNF-α in serum and VEGF, VEGFR2 and Ang-1 protein and mRNA expression in retina tissue in DR group and each experimental group were significantly higher(<i>P</i><0.05). Compared with DR group, fasting blood glucose decreased in all experimental groups, and the fasting blood glucose of experimental groups B and C was significantly decreased than that of DR group(<i>P</i><0.05). In addition, the contents of IL-6 and TNF-α in serum, the protein and mRNA expression levels of VEGF, VEGFR2 and Ang-1 in retina tissue in experimental group B and group C were significantly lower than those in group DR(<i>P</i><0.05). The results of HE showed that the structure of retinal photoreceptor cell layer in DR group was obviously destroyed, the cell edema and gap widened, and the retinal histopathology of rat retina in group B and group C were improved in varying degrees. <p>CONCLUSION: Pollen Typhae extract can down-regulate the level of inflammation and the expression of VEGF, VEGFR2 and Ang-1 in DR rats, so as to improve the retinopathy.

Construction of recombinant lentiviral vectors for mouse NMNAT1 gene expression and its interference RNA / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct two recombinant lentiviral vectors carrying mouse NMNAT1 gene and RNAi targeting NMNAT1.</p><p><b>METHODS</b>According to GenBank, the full-length cDNA sequence of mouse NMNAT1, an interfering sequence targeting NMNAT1 and a negative sequence were designed, synthesized and inserted into plasmid pLenti6 lentiviral vector. The viral stock was prepared by cotransfection of plasmids and the packaging plasmid mix to 293T cells. The virus titer was tested by qPCR methods. After infection of Hela cells with these lentiviruses, the expression of NMNAT1 was detected by qPCR and Western blot.</p><p><b>RESULTS</b>All the recombinant plasmids were confirmed by sequencing. The titer of virus was over 2 X10(8) TU/mL. Hela cells infected with lentiviral vector carrying full length NMNAT1 gene successfully expressed high-level NMNAT1. The expression of NMNAT1 reduced to less than 30% after delivery of lentiviral vector carrying RNAi sequence.</p><p><b>CONCLUSION</b>The lentiviral vectors carrying full length NMNAT1 gene and RNAi sequence targeting NMNAT1 have been successfully constructed.</p>

Nicotinamide mononucleotide adenylyltransferase 1 gene NMNAT1 regulates neuronal dendrite and axon morphogenesis in vitro / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Wallerian degeneration is a self-destructive process of axonal degeneration that occurs after an axonal injury or during neurodegenerative disorders such as Parkinson's or Alzheimer's disease. Recent studies have found that the activity of the nicotinamide adenine dinucleotide (NAD) synthase enzyme, nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) can affect the rate of Wallerian degeneration in mice and drosophila. NMNAT1 protects neurons and axons from degeneration. However, the role of NMNAT1 in neurons of central nervous system is still not well understood.</p><p><b>METHODS</b>We set up the culture of primary mouse neurons in vitro and manipulated the expression level of NMNAT1 by RNA interference and gene overexpression methods. Using electroporation transfection we can up-regulate or down-regulate NMNAT1 in cultured mouse dendrites and axons and study the neuronal morphogenesis by immunocytochemistry. In all functional assays, FK-866 (CAS 658084-64-1), a highly specific non-competitive inhibitor of nicotinamide phosphoribosyltransferase was used as a pharmacological and positive control.</p><p><b>RESULTS</b>Our results showed that knocking down NMNAT1 by RNA interference led to a marked decrease in dendrite outgrowth and branching and a significant decrease in axon growth and branching in developing cortical neurons in vitro.</p><p><b>CONCLUSIONS</b>These findings reveal a novel role for NMNAT1 in the morphogenesis of developing cortical neurons, which indicate that the loss of function of NMNAT1 may contribute to different neurodegenerative disorders in central nervous system.</p>

Construction and identification of lentiviral vector for RNA interference targeting STUB1 gene / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct and identification of a lentiviral vector for RNA interference (RNAi) targeting STUB1 gene.</p><p><b>METHODS</b>A pair of complementary small hairpin RNA (shRNA) oligonucleotides targeting STUB1 gene was designed, synthesized and inserted into linearized pMagic 4.0 vector. The recombinant plasmid was identified by double restriction digestion with Age I/EcoR I and DNA sequencing.</p><p><b>RESULT</b>PCR and DNA sequencing showed that the shRNA sequence was successfully inserted into pMagic 4.0 vector. The pMagic 4.0 vector was successfully packaged into lentivirus particles.</p><p><b>CONCLUSION</b>A lentiviral shRNA expression vector and particles targeting STUB1 gene has been successfully constructed for the further study of the STUB1 gene.</p>