Effects of serum starvation on cell cycle synchronization in primary human umbilical vein endothelial cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the optimal starvation conditions of human umbilical vein endothelial cells (HUVECs) and establish a highly efficient and stable method for separating HUVECs.</p><p><b>METHODS</b>HUVECs harvested from human umbilical cords by digestion with 0.1% collagenase II for 15 min were cultured in endothelial culture medium (ECM) containing 5% fetal bovine serum (FBS), 1% endothelial cell growth factor (ECGS) and 1% penicillin/streptomycin solution(P/S) at 37 degrees celsius; in 5% CO2. The cells were observed for cell morphology under an inverted microscope and identified with immunofluorescence assay. The purity of HUVECs was detected using flow cytometry (FCM). The cell cycles of HUVECs cultured in the presence of 0, 0.1%, 0.5%, and 1% FBS for 0, 6, 12, 18, and 24 h were analyzed with flow cytometry.</p><p><b>RESULTS</b>s The purity of HUVECs harvested by digestion with 0.1% collagenase II reached 99.67%. The primary HUVECs showed a cobblestone or volute appearance in vitro. Immunocytochemistry showed that HUVECs highly expressed VIII-related antigen. Cell culture in the presence of different concentrations of FBS for 6 h resulted in 70% G0/G1 phase cells, which increased to 80%-90% at 12 h of cell culture, and further to around 95% at 18 and 24 h.</p><p><b>CONCLUSION</b>Digestion with 0.1% collagenase II can obtain high-purity primary HUVECs. Culturing HUVECs in serum-free medium for 12 h can result in a high purity (over 80%) of G0/G1 phase cells.</p>

Silence of VEGFR2 expression mediated by PEI/siRNA complexes / 药学学报

The aim of this paper is to report the study on gene silencing efficiency of siRNA targeted against mouse VEGFR2 (siVEGFR2) in vitro mediated by polyethyleneimine (PEI) and its anti-tumor effect in vivo. CY3-labeled siRNA was compounded into PEI and transfected into MS1 cells. Confocal microscopy was used to image the subcellular distribution of siRNA in MS1 cells. Semi-quantitative RT-PCR and Western blotting were used to evaluate VEGFR2 gene silencing induced by siVEGFR2/PEI complexes. A tumor-bearing nude mice model was established to compare the anti-tumor effect after delivered by local and systemic routes. siVEGFR2/PEI complex-transfected cells exhibited much fluorescence in cytoplasm with no evidence of nuclear accumulation. The expression levels of VEGFR2 mRNA and protein in PEI-transfected cells were significantly down-regulated compared with that in blank group, the silencing efficiency were 28.2% and 23.6% respectively. The tumor sizes in mice intratumorally injected with siVEGFR2/PEI complexes (189.429 +/- 17.562 mm3) were reduced definitely compared to that in mice injected with siVEGFR2/PEI complexes via vein route (315.507 +/- 20.491 mm3), or to saline groups (365.844 +/- 20.713 mm3). The study demonstrated that PEI could effectively transfect siRNA into cells and silence the VEGFR2 gene expression. Intratumoral delivery is more suitable for non-targeted modified PEI/siRNA complexes to inhibit the tumor growth in vivo. The present data lay a solid foundation to further study on the gene silencing mechanism for PEI-medicated RNAi and its anti-tumor efficiency in vivo.

Effects of sustained-release alpha-lipoic acid tablet on blood lipid, blood sugar and insulin in hyperlipidemic New Zealand rabbits / 南方医科大学学报

<p><b>OBJECTIVE</b>To evaluate the effect of sustained-release alpha-lipoic acid tablets (SRLA) on blood lipid, glucose and insulin levels in hyperlipidemic New Zealand rabbits.</p><p><b>METHODS</b>Twenty-four New Zealand rabbits were randomized into normal diet group, high-fat diet group, and high-fat diet + SRLA (300 mg/tablet) group with corresponding feed. At the beginning and 4 weeks after the feeding, the serum levels of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), blood glucose, and serum insulin were measured, and insulin sensitivity index (ISI) was calculated.</p><p><b>RESULTS</b>Four weeks after feeding with high-fat diet, the insulin levels was elevated and the ISI lowered in the New Zealand rabbits, indicating successful establishment of the animal model of hyperlipidemia. Compared with the high-fat diet group, the serum levels of TG, TC, LDL-C and insulin were significantly reduced (P<0.05), and the ISI was significantly increased (P<0.05) in high fat diet + SRLA group. But no statistically significant difference was found in the blood glucose among the 3 groups.</p><p><b>CONCLUSION</b>SRLA can significantly correct blood lipid and insulin disorders in hyperlipidemic New Zealand rabbits and prevent the occurrence of insulin resistance and hyperlipidemia.</p>

Plasma membrane-related Ca(2+)-ATPase-1 gene silencing promotes insulin secretion in islet beta cells NIT / 南方医科大学学报

<p><b>OBJECTIVE</b>To assess the effect of RNA interference-mediated gene silencing of plasma membrane-related Ca(2+)-ATPase-1 (PMR1) gene on the insulin secretion in islet beta cells NIT-1 in vitro.</p><p><b>METHODS</b>A small interfering RNA duplex (siPMR1) corresponding to the nucleotides 337-357 of mouse PMR1 cDNA was introduced into NIT-1 cells via liposomes. The gene silencing effect was assessed by RT-PCR, and the total insulin level in the transfected cells was measured by radioimmunoassay.</p><p><b>RESULTS</b>Transfection with siPMR1 resulted in obviously reduced PMR1 expression and increased insulin secretion in NIT-1 cells.</p><p><b>CONCLUSION</b>The synthesized siPMR1 can significantly silence the expression of PMR1 and promote the secretion of insulin in the islet cells in vitro, which shed light on further studies of RNAi-based therapy of diabetes.</p>

Construction of small interfering RNA targeting mouse vascular endothelial growth factor receptor-2: its serum stability and gene silencing efficiency in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a small interfering RNA (siRNA) targeting mouse vascular endothelial growth factor receptor-2 (VEGFR2) and study its serum stability and gene silencing efficiency in vitro.</p><p><b>METHODS</b>The synthesized siRNA targeting VEGFR2 (siVEGFR2) diluted in RNase-free water was mixed at a 1:1 ratio with fresh serum and incubated at 37 degrees celsius;. Gel electrophoresis was performed to determine the integrity of siVEGFR2 incubated for different time lengths. Oligofectamine 2000 was used to mediate siVEGFR2 transfection of MS1 cells, and semi-quantitative RT-PCR was used to evaluate VEGFR2 gene silencing effect induced by the siRNA.</p><p><b>RESULTS</b>The naked siRNA incubated in serum underwent gradual degradation with prolonged incubation time and became virtually undetectable after 24 h. Transfection of MS1 cells with siVEGFR2 significantly down-regulated the expression of VEGFR2 mRNA in comparison with the blank group and control siRNA transfection group (P<0.001), while no significant difference was found in VEGFR2 mRNA levels between the latter two groups (P=0.157).</p><p><b>CONCLUSION</b>The naked siRNA is unstable in serum and not suitable for direct administration in vivo. The designed siRNA can effectively silence the VEGFR2 gene expression in MSI cells in vitro.</p>

Interleukin-1β inhibits collagen synthesis and promotes its decomposition in cultured cardiac fibroblasts / 生理学报

The present study aimed to investigate the effects of interleukin-1β (IL-1β) at different doses on collagen synthesis and decomposition in cultured cardiac fibroblasts from neonatal Sprague-Dawley rat. Cardiac fibroblasts were treated with IL-1β (0.01, 0.1, 1, 10, 100 ng/mL) for 24 h. Cell DNA synthesis was measured by (3)H-thymidine ((3)H-TdR) incorporation and collagen synthesis was measured by (3)H-proline ((3)H-Pro) incorporation. Matrix metalloproteinase (MMP) activity was measured by gelatinase zymography. MMP-2, MMP-9 protein expressions were measured by Western blot. mRNA expressions of MMP-2 and MMP-9 were detected by reverse transcription-polymerase chain reaction (RT-PCR). Compared with that in the control group, the incorporation of (3)H-TdR and (3)H-Pro decreased in high-dose IL-1β groups (≥0.1 ng/mL) but not in low-dose IL-1β group (0.01 ng/mL). IL-1β significantly increased MMP-2 and MMP-9 activities. IL-1β (0.01-100 ng/mL) also dose-dependently increased the protein and mRNA expressions of MMP-2 and MMP-9 (P<0.05, P<0.01), respectively. These results suggest that IL-1β decreases collagen synthesis and MMP activities through transcriptional and posttranslational processes via degrading collagen in a dose-dependent way. Elevation of IL-1β is possibly involved in the process of ventricular remodeling after myocardial infarction, and the concentration of IL-1β is possibly a major factor which affects the extent of ventricular remodeling.

High-performance liquid chromatography for determining plasma alpha-lipoic acid in New Zealand rabbits / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a method for determining the content of alpha-lipoic acid in New Zealand rabbit plasma.</p><p><b>METHODS</b>Alpha-lipoic acid in the plasma samples was purified by solid-phase extractor and analyzed on an HYPERSIL C18 column with isocratic mobile phase consisting of potassium dihydrogen phosphate-acetonitrile (50:50, v/v) at a flow rate of 1.0 ml/min and detection wavelength of 230 nm.</p><p><b>RESULTS</b>The standard curve was linear in the range of 5-100 microg/L (r=1) and the average recovery was 77.4%-82.1%. The relative standard deviations of intra-day and inter-day assay were within 1.5%-8.9%.</p><p><b>CONCLUSION</b>The method is sensitive, accurate and simple for determining plasma alpha-lipoic acid levels in New Zealand rabbits.</p>

Synthesis and characterization of chitosan-DNA nanoparticles as gene carriers / 南方医科大学学报

<p><b>OBJECTIVE</b>To prepare chitosan nanoparticles (NPs) as gene carriers and study its pharmaceutical characteristics and gene transfection efficiency in vitro.</p><p><b>METHODS</b>The plasmid expressing green fluorescent protein (pGFP) was used as the reporter gene, and the chitosan-pGFP NPs were prepared using a complex coacervation process. The binding of pDNA was evaluated by agarose gel electrophoresis analysis, and the encapsulation rate was determined with colorimetry. The size distribution and polydispersity of the NPs were measured by nanoparticle size analyzer, and their morphologies observed by atomic force microscope. The transfection studies were performed with LoVo cells in vitro.</p><p><b>RESULTS</b>The results of gel electrophoresis demonstrated full binding of chitosan with the pDNA by electrostatic interaction. The encapsulation rates for these NPs all exceeded 90%. The morphology of the chitosan NPs was mostly spherical and well distributed, with a mean diameter of about 209 nm and polydispersity of 0.32. The in vitro transfection of chitosan-pGFP NP was efficient in LoVo cells and the expression of green fluorescent proteins was observed under fluorescent microscope.</p><p><b>CONCLUSIONS</b>Chitosan NP prepared by complex coacervation can bind to the pDNA efficiently with high encapsulation rate and diameter distribution of 100 to 500 nm. These NPs allow efficient delivery of the reporter genes into cells in vitro for their expression. The chitosan-pDNA NPs may serve as an effective non-viral carrier for delivery of nucleotides into cells.</p>