Effect of vascular endothelial growth factor on bone marrow-derived mesenchymal stem cell proliferation and the signaling mechanism / 南方医科大学学报

<p><b>OBJECTIVE</b>To observe the effect of vascular endothelial growth factor (VEGF) on bone marrow-derived mesenchymal stem cell (MSC) proliferation and explore the signaling mechanism involved.</p><p><b>METHODS</b>MSC culture was performed following the classical whole bone marrow adhering method. The characteristics of MSC were identified by induction of multi-lineage differentiation and flow cytometry for surface marker analysis (CD34, CD45, CD29, and CD90). Following the addition of 50 nmol/L wortmannin, 50 µmol/L PD98059, 30 µmol/L SB203580, 10 µmol/L H89, 20 µmol/L Y27632, 1 µmol/L rapamycin, 10 µmol/L straurosporine, 6 nmol/L Go6976, or 50 µmol/L Pseudo Z inhibitors in the cell culture, the MSC were treated with 20 ng/ml VEGF and the changes of the cell proliferation rate was measured with MTT assay.</p><p><b>RESULTS</b>Cultured MSC were capable of multi-linage differentiation and did not express VEGF-R, CD29 or CD90. Treatment with 20 ng/ml VEGF obviously promoted MSC proliferation, and this effect was inhibited partially by p38 mitogen-activated protein kinase (MAPK) inhibitor rapamycin, PD98059, SB203580, Go6976, and straurosporine.</p><p><b>CONCLUSIONS</b>VEGF promotes MSC proliferation in close relation to the AKT-PKC pathway, in which PKC signal pathway may play the central role.</p>

In vivo anti-obesity effect of the agonist for receptor VPAC1 / 生理学报

It was hypothesized that the VPAC1 agonist may exert anti-obesity functions because VPAC1 is involved in the anorexigenic effects and the anti-inflammatory function of pituitary adenylate cyclase-activating polypeptide (PACAP)/vasoactive intestinal polypeptide (VIP). Furthermore, our in vitro test showed that the expression of VPAC1 increased significantly after the 3T3-L1 adipocytes were differentiated, and that incubation of adipocytes with VPAC1 agonist (10-1 000 nmol/L per 1x10(6) cells) resulted in stimulation of lipolysis. To test the effect of VPAC1 agonist [Lys15, Arg16, Leu27]-VIP (1-7) GRF (8-27) on diet-induced obesity (DIO), we further designed the following two in vivo experiments: (1) Mice were fed on high-fat diet (HFD) and intraperitoneally (i.p.) treated with VPAC1 agonist simultaneously for 28 d; (2) Mice were given HFD for 35 d, and subsequently fed on the same HFD and i.p. treated with VPAC1 agonist for the next 28 d. The physiological indices, including body weight, weight of white adipose tissue, plasma glucose and blood lipid, were collected. The results showed that treatment with VPAC1 agonist inhibited ingestion significantly and prevented the elevations in body weight and the weights of the white adipose tissues (epididymal and dorsal) induced by HFD. The increases in plasma glucose, cholesterol, triglycerides and LDL induced by HFD were also down-regulated in mice treated with VPAC1 agonist. VPAC1 agonist treatment also improved the glucose tolerance. Therefore, VPAC1 agonist treatment inhibits the development of the obesity induced by HFD and helps to improve the morbidities associated with DIO.

Preparation and Identification of Recombinant Vasoactive Intestinal Peptide / 中国生物工程杂志

In order to prepare the recombinant vasoactive intestinal peptide (VIP) using intein mediated rapid purification system,the cDNA encoding the recombinant VIP was designed and synthesized according to the preference of E.coli,and then was cloned into the expression vector PTWIN. The recombinant plasmid PTWIN-VIP was transformed into expression host E.coli strain ER2566.The fusion protein consisting of the recombinant VIP,intein and chitin binding domain was expressed and purified by chitin affinity chromatography. The target peptide was released from the fusion protein by changing the temperature and the pH of the cleavage buffer. The molecular weight of the recombinant VIP was determined by the mass spectrometry and the results was conformity with the theoretical value. The preliminary bioactivity assay indicated that the recombinant VIP decreased the serum resistin levels significantly in LPS-induced acute inflammation. The preparation and the characterization of anti-inflammatory effects of the recombinant VIP layed the foundation for its further application.

Cloning and Expression of the New Gene Recombinant RMBAY Against Type-2 Diabetes and Its Production Optimization / 中国生物工程杂志

Constructing prokaryotic expression vector pKY-RMBAY by gene recombination and research its optimizing productive conditions.By PCR technology synthesizing the gene of the RMBAY with preference codon of E.coli and the RMBAY gene was inserted into high efficiency expression vector pKYB-MCS.Expressed fusion proteins in E.coli ER2566 were purified with Chitin-Beads column.Fusion proteins binding on Chitin-Beads was cut on N-terminus of intein due to the induction of ?-mercaptoethanol and the target peptide RMBAY was released.The RMBAY was identified by mass spectrum.Experiment results showed RMBAY can be high efficiently expressed in E.coli ER2566,with optimizing productive conditions the yield of the RMBAY may be 6.7mg/L fermentation product and its purity is greater than 98%.The molecular weight of RMBAY is 3.887 kDa by mass spectrum and that accords with its theory value.