Effect of phosphatase PHLPP1 gene transfer on the proliferation of human umbilical vein endothelial cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the constituent expression of PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1) in human umbilical vein endothelial cells (HUVECs) and the effect of PHLPP1 gene transfer on the proliferation of the cells in vitro.</p><p><b>METHODS</b>Cultured HUVECs were transfected with pcDNA3-GFP or pcDNA3HA-PHLPP1 via lipofectamine 2000. The cell proliferation ability was determined by cell counting and MTT colorimetric assay, and Western blotting was used to detect the protein expression of PHLPP1 in the cells.</p><p><b>RESULTS</b>No PHLPP1 protein was detected in the non-transfected cells or pcDNA3-GFP-transfected cells. pcDNA3HA-PHLPP1 gene transfection significantly increased PHLPP1 expression in the HUVECs (P<0.01), but the cell proliferation status remained unchanged (P>0.05). The absorbance of the cells measured by MTT assay was 0.134-/+0.0152, 0133-/+0.014 and 0.137-/+0.016, with cell counts of (8.293-/+0.962)x10(5), (7.937-/+0.101)x10(5) and (8.127-/+0.112)x10(5), respectively, showing no significant differences between the 3 groups (P>0.05).</p><p><b>CONCLUSIONS</b>Phosphatase PHLPP1 may not be the most important signal protein in the regulation of HUVEC proliferation.</p>

Expression of human apolipoprotein A- I in baculovirus-insect cell system / 生物工程学报

Apolipoprotein A- I is the major apolipoprotein in high-density lipoprotein known to have a wide range of physiological functions, the best-studied one of which is in regulating cholesterol metabolism and preventing arteriosclerosis. Human blood has been the only source of this protein. To facilitate further research and application, it is essential to produce it through genetic engineering. In the current research, the baculovirus-insect cell system was used to overexpress human apolipoprotein A- I . Two recombinant baculoviruses were constructed. The first one expressed a pro form of apoA- I lacking native signal peptide. The recombinant protein was found to remain mainly inside cells in the early phase of infection, while being largely excreted to the medium late in infection. The second one used a heterologous signal peptide, snake phospholipase A2 inhibitor alpha subunit signal peptide, to lead the secretion of mature apoA- I. In contrast to the first virus, recombinant apoA- I was found in the culture medium at the early phase of virus infection. The mature apoA- I was purified from culture medium using Phenyl Sepharose hydrophobic interaction chromatography (HIC) and eluted with water and Propylene. This work shows that snake phospholipase A2 inhibitor a subunit signal peptide can be used to secret human apoA- I in insect cells, but the efficiency of its secretion is limited when the expression level is high.