OBJECTIVE:To overcome the deficiency in the current therapies for erectile dysfunction (ED), we designed and synthesized a novel high-efficiency polymer/gene compound drug controlled release system and discussed the feasibility of pH and temperature dually sensitive injectable hydrogel in ED gene therapy.METHODS:We synthesized optimal siRNA gene nanoparticles by characterizing the zeta potential of polylysine (PLL)/siRNA gene compounds, and established a pH and temperature dually sensitive injectable gene compound drug controlled release system via Schiffs reaction between glycol chitosan (GC) and benzaldehyde capped OHC-PEO-PPO-PEO-CHO. Then we demonstrated the sustained release of the system at different temperatures.RESULTS:When the mass ratio of PLL to siRNA was 20:1, the zeta potential of the PLL/siRNA gene compound reached the peak (+23.5 mV) and the siRNA was encapsulated by PLL in the maximal degree. GC and OHC-PEO-PPO-PEO-CHO was crosslinked via benzoicimine reaction when environmental pH was changed from 5.5 to 7.4. The reslease of the siRNA encapsulated in this system kept at a low rate at 37 degrees C, significantly enhanced with the increase of the temperature to 60 degrees C, rising to (122.5 +/- 5.3) microg at 1 000 minutes as compared with (23.8 +/- 6.0) microg at 37 degrees C (P < 0.05).CONCLUSION:The polymer/gene compound drug controlled release system was successfully synthesized, which improved the stability and capacity of gene carriers and achieved siRNA release at different temperatures, promising to be a new approach to the gene therapy of ED.

Objective To investigate the possibility of SLC35F2 inhibition by lentiviral vector-mediated RNA interference (RNAi) and the influence on cell proliferation and apoptosis in lung cancer cell line,and to set up a lung cancer cell line in which SLC35F2 is stably suppressed.Methods The lentiviral vector of siRNA targeted against SLC35F2 (SLC-siRNA) was constructed and transfected into the packaging cells 293T,and the viral supernatant was collected to transfect H1299 cells.After selection by puromycin and culture expansion,the stable cell clones were attained.Quantitative real-time fluorescent polymerase chain reaction (PCR) and Western blotting were used to detect the expression of SLC35F2.The effect of SLC35F2 silencing by RNAi on cell proliferation was quantified by CCK-8 assay.Annexin V-FITC/PI staining was employed to examine the apoptosis.Results Lentiviral vector SLC35F2-shRNA was constructed successfully.As compared with control group,the SLC35F2 expression was decreased to 81.8% in RNA and protein levels.CCK-8 revealed that the inhibition rate of H1299 cells transfected with SLC35F2 was 16.3%,and the apoptosis rate was significantly increased as compared with negative control group ( 14.88% vs 3.16% ,P ＜0.05 ).Conclusion Lentiviral vector-mediated RNA interference targeting against SLC35F2 can effectively inhibit SLC35F2 expression and cell proliferation.The lung cell line in which SLC35F2 gene was stably suppressed was successfully established.

Objective To evaluate the effect of survivin siRNA silencing survivin gene on the apoptosis rate and sensitivity to cisplatin of lung cancer A549 cells.Methotis A549 cells were transfected with survivin siRNA for 48 h.Survivin mRNA expression.apoptosis rate and sensitivity to cisplatin were checked.Reslllts After survivin-siRNA transfection.survivin expression was decreased from 1.17±0.25 to 0.41±0.18 shown as survivin/β-actin mRNA ratio,and apoptosis rate increased from 2.67%to 32.33%,and IC50 to cisplatin decreased from 6.37 t0 2.42 ms/L.Conclusion Survivin gene silencing by specific siRNA in lung cancer A549 cells Can induce apoptosis and sensitize cells to cisplatin.

Objective To explore the effective method of establishing human lung cancer mouse model.Methods Human lung cancer line PAa was administrated subcutaneously in severe combined im- munodeficiency(SCID)mice at a dose of 2×10~6 cells/mouse,which was reconstituted by human lym- phocytes from peripheral blood at a dose of 2.5×10~7 cells/mouse.The latency period and rate of tumor formation was monitored.Results Subcutaneous tumor developed in all mice gave human PBL and PAa cells.The latency period in PAa-PBL-SCID murine model was significantly prolonged,and the tumor size was markedly depressed(P＜0.01),as compared with mice subject to subcutaneous implatantion of PAa cells alone.Two,4 and 6 weeks later,human IgG in mouse blood determined by ELISA was 60.36±5.42,86.65±7.76 and 124.24±5.66 mg/L respectively.On the week 6,immunobistochemical staining revealed presence of remarkable human CD3~+ lymphocytes in murine tumor.Pathological examination showed tumor growth.Conclusion The PAa-PBL-SCID model can be successfully established by combi- nation of intraperitoneal injection of huPBL and subcutaneous injection of lung cancer cells in SCID mice.

Objective To evaluate the effect of ischemia/reperfusion (I/R) on the expression of intercellular adhesion molecule-1 (ICAM-1) in lung and study the relationship between expression of ICAM-1 and polymorphonuclear leukocyte (PMN) infiltration.Methods Single lung in suit warm I/R animal model was used. The rats were subjected to 90 min left lung ischemia followed by 0,1,2,4,8,16 or 24 h of reperfusion separately. At the end of reperfusion, expression of ICAM-1 mRNA and protein was detected by using semi-quantitative RT-PCR and Western blot method. Myeloperoxidase activity in each group was also measured. Results ICAM-1 mRNA expression was increased at 1 h of reperfusion, and decreased to basal level 16 h after reperfusion. Expression of ICAM-1 protein began to increase 4 h after reperfusion, peaked at 8 h of reperfusion and then gradually decreased. Myeloperoxidase activity was positively correlated with ICAM-1 expression during 8 to 24 h of reperfusion (r=0.69, P0.01).Conclusion I/R could upregulated ICAM-1 expression in rat lung, and the PMN infiltration after 8 h of reperfusion was correlated with the expression of ICAM-1 in lung. \;