ABSTRACT
BACKGROUND: The recombinant adenovirus has certain toxic reactions to cell growth and survival, but the determination of the optimal multiplicity of infection (MOI) is not well documented. OBJECTIVE: To study the effectiveness and toxicity of transfection of bone marrow mesenchymal stem cells (BMSCs) by adenovirus vectors with different MOIs, and to explore the effect on osteogenic capability of the cells. METHODS: BMSCs at passage 5 were infected with enhanced green fluorescent protein (EGFP) via adenovirus vectors with different MOI (0, 50, 100, 150, 200 and 250). Cell morphology and mortality rate were observed and calculated using trypan blue staining method at 24 hours after cell transfection. The transfection efficiency and the relative mRNA expression of EGFP and Runx2 were analyzed respectively by inverted florescent microscopy and real-time quantitative PCR at 48 hours after cell transfection. The activity of BMSCs transfected with different MOIs was evaluated by MTT, and the optimal MOI was determined thereafter. RESULTS AND CONCLUSION: With the increase of MOI, BMSCs showed decreased adherent ability, and even some cells aged and died. The mortality rate of BMSCs transfected for 24 hours at a MOI of 0 to 250 was 5.80%, 6.67%, 7.95%, 7.76%, 10.35% and 11.18%, respectively, indicating that the mortality rate of BMSCs is positively correlated with the MOI. The transfection efficiency changed insignificantly when the MOI was greater than 100. On the contrary, the cellular viability and osteogenic differentiation capability of rabbit BMSCs were receded when the MOI level was up to 200-250. The study discovered that the suitable scope of MOI to transfect BMSCs is 50-150, and the optimal MOI is 100.
ABSTRACT
To reveal the protective and anti-apoptosis effect of compound Ginkgo biloba granules on oxidative stress injury of human umbilical vein endothelial cells (HUVEC). Negative control group, H2O2 model group and 4 drug pretreatment groups (80, 160, 320, 640 mg• L⁻¹) were established. The cell proliferation, morphological changes in each group after oxidative stress injury was detected by MTT assay and through microscope observation respectively. The content of LDH, MDA, SOD and NO and SOD activity in supernatant were detected to judge the protection effect of the drugs on endothelial cells. The protective effect on HUVEC apoptosis was analyzed by Caspase-3 activity test and Annexin V-FITC/PI staining. Western blot was used to observe the expression of apoptosis-related proteins Bcl-2 and Bax. Results showed that 1 200 μmol• L⁻¹ H2O2 can induce oxidative stress injury in endothelial cells and reduce the cell survival rate; cell proliferation inhibition degree is positively correlated with the effect time of H2O2. Besides, 80, 160, 320 640 mg•L⁻¹ compound Ginkgo biloba granules can protect HUVEC from oxidative stress injury, recover the normal proliferation level of cells, improve their state, prohibit cell apoptosis, and can up-regulate and down-regulate the expression level of Bcl-2 and Bax respectively. In conclusion, compound G. biloba granules can protect HUVEC from the oxidative stress injury induced by H2O2, its mechanism may be correlated with inhibition of the mitochondrial apoptotic pathway in HUVEC.
ABSTRACT
The aim of this research is to investigate the protection of PM2.5 infected RAW264.7 cell by traditional Chinese medicine (TCM)--Shenlian(SL) extracts and to establish the damage model. We use cell growth, cell damage and oxidative stress related markers, and inflammatory cytokines as observation index to evaluate the protection of PM2.5 infected RAW264.7 by SL extract. The results showed that 50 mg x L(-1) PM2.5 could cause cell particle deposition, inhibit the growth of cells, and significantly increase the cell supernatant of LDH, NO release quantity and intracellular reactive oxygen species (ROS) level during 4 h and 24 h. In the intervention of SL extract 50, 25, 10 mg x L(-1), the particle deposition of RAW264.7 cells, cell supernatant of LDH, NO, IL(-1) beta release, MCP-1 was significantly decreased, the SOD activity increased significantly. It shows that SL extracts of PM2.5 infected RAW264.7 cell damage has obvious protective effect, the effect may be related to the direct protection of cells, reduce oxidative stress and inflammatory injury.
Subject(s)
Animals , Mice , Cell Line , Drugs, Chinese Herbal , Pharmacology , Macrophages , Oxidative Stress , Particulate Matter , Toxicity , Protective Agents , Pharmacology , Reactive Oxygen Species , MetabolismABSTRACT
Objective To investigate the effect of insulin on D_5 dopamine receptor expression and function in renal proximal tubule (RPT).Methods Immortalized RPT cells and D_5 receptor transfected HEK293 (HEK-D_5) cells were used in the study to investigate the effect of insulin on D_5 receptor expression and function,and those effects were compared in RPT cells from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). The function of D_5 receptor was determined by measurement of the Na~+-K~+-ATPase activity in HEK-D_5 cells. Results Insulin increased D_5 receptor protein expression in a concentration and time-dependent manner in WKY RPT cells,but not in SHR.The basal level of D_5 receptor expression was higher in WKY cells than that in SHR cells. Stimulation with fenoldopam(D_1-like dopamine receptor agonist) inhibited the Na~+-K~+-ATPase activity;pretreat- ment with insulin increased the inhibitory effect of fenoldopam on Na~+-K~+-ATPase activity in HEK-D_5 cells. Conclusion The abnormal regulation of insulin on D_5 receptor expression and function might be involved in the path- ogenesis of essential hypertension.