Objective To increase the killing effect of aminolevulinic acid (ALA)-based photodynamic therapy (PDT) on a human skin squamous cell carcinoma cell line A431 by poly lactic-co-glycolic acid nanoparticles (PLGA NPs).Methods ALA-loaded PLGA NPs (ALA PLGA NPs) were prepared using a modified double emulsion solvent evaporation method,and characterized in terms of size,encapsulation efficiency,loading capacity and morphology.Transmission electron microscopy was carried out to observe the morphology of A431 cells after uptake of ALA PLGA NPs.To optimize incubation time,multi-mode microplate reader was used to describe the fluorescence kinetics of protoporphyrin Ⅸ generated by A431 cells during 24 hours of incubation with 0.1 mmol/L ALA,1 mmol/L ALA,2.7 g/L ALA PLGA NPs containing about 0.1 mmol/L ALA,and PLGA NPs without ALA separately.Some A431 cells were divided into 10 groups:control group receiving neither treatment nor irradiation,0.1 and 1 mmol/L ALA dark/PDT group incubated 0.1 and 1 mmol/L ALA respectively,ALA PLGA NP dark/PDT group incubated with ALA PLGA NPs of 2.7 g/L,PLGA NP dark/PDT group incubated with PLGA NPs of 2.7 g/L,simple irradiation group irradiated with a He-Ne laser (wavelength:635 nm,power density:8.6 mW/cm2,energy density:8 J/cm2) only.The dark groups were kept in darkness strictly by wrapping in aluminum foil,and PDT groups were irradiated using a He-Ne laser.After another 24 hours of culture following irradiation,methyl thiazolyl tetrazolium (MTF) assay was conducted to estimate the survival rate of cells.To study the effect of ALA PLGA NPs on cell apoptosis,some A431 cells were divided into three groups:control group receiving neither treatment nor irradiation,ALA-PDT group and ALA PLGA NP PDT group receiving PDT after incubation with ALA and ALA PLGA NPs respectively.Flow cytometry was performed to detect the apoptosis of A431 cells at 12 and 24 hours,separately,after the photodynamic therapy.Data were statistically analyzed using SPSS 13.0 software package by means of a t test.Results The prepared ALA PLGA NPs were spherical with a mean particle size of (65.6 ± 26) nm,encapsulation efficiency of (65.8 ± 7.2) ％,and drug loading capacity of (0.62 ± 0.27)％.ALA PLGA NPs could be uptaken by A431 cells and gathered in the cytoplasm.The PpIX fluorescence kinetic study showed that the fluorescence intensity increased with time within 24 hours in A431 cells incubated with ALA or ALA PLGA NPs.After 6 and 24 hours of incubation,the A431 cells incubated with 2.7 g/L ALA PLGA NPs showed a significant increase in the protoporphyrin Ⅸ fluorescence intensity compared with those incubated with 0.1 mmol/L ALA (both P ＜ 0.01).Further more,the survival rate of A431 cells was statistically lower in the ALA PLGA NP PDT group than in the 0.1 mmol/L ALA PDT group at 6 and 24 hours (t =35.685,5.262,respectively,both P ＜ 0.01).Elevated apoptosis rate was observed in the ALA PLGA NP PDT group compared with the ALA PDT group at 12 ((13.10 ± 0.50)％ vs.(4.90 ± 0.13)％,t =9.074,P＜ 0.01) and 24 ((30.17 ± 1.02)％ vs.(11.6 ± 0.59)％,t =9.095,P ＜ 0.01) hours.Conclusions ALA PLGA NPs can promote the formation of protoporphyrin Ⅸ,strengthen the killing effect of ALA-PDT on A431 cells in vitro,and enhance the apoptosis induced by ALA-PDT in tumor cells.

Objective To study the signal transduction pathway genes in acute respiratory distress syndrome(ARDS) of rats by gene chip. Methods Total RNAs were isolated from lungs of normal and ARDS rats respectively. The RNAs were purified by oligotex. Both mRNAs from two kinds of tissues were reversely transcribed to cDNA with incorporation of fluorescent-labelled dUTP to prepare the hybridization probes. The mixed probes were hybridized to cDNA microarray. Picture signals of fluorescence in gene array were scanned and compared by CapitlBio Molecule Annotation System V4.0. Quantitative fluorescence RT-PCR was used to validate the results of genechip. Results The results showed that there were 2 genes up-regulated and 9 genes down-regulated in lung tissue of ARDS rats,and these genes were involved in 11 signal transduction pathways. Conclusion Many genes of signal transduction pathways expressed in ARDS transcription profiles of rats.

Objective:To evaluate the immunogenicity and protective effect of a multicomponent recombinant protein vaccine EPRHP014 constructed independently and provide a scientific basis for developing new tuberculosis (TB) vaccine and effective prevention and control of TB.Methods:Three full-length Mycobacterium ( M.) tuberculosis protein antigens (EsxH, Rv2628, and HspX) and two epitope-predicted and optimized epitope-dominant protein antigens (nPPE18 and nPstS1) were selected, from which five protein antigens were used to construct a protein antigen composition EPRHP014, including a fusion expression multi-component protein antigen (EPRHP014f) and a multi-component mixed protein antigen (EPRHP014m) formed with the five single protein using clone, purification, and purification respectively. Multicomponent protein vaccines EPRHP014f and EPRHP014m were prepared with aluminum adjuvant, and the BCG vaccine was used as a control. ELISA detected the titer of serum-specific antibodies, the secretion of various cytokines was detected by ELISpot and Luminex, and immune protection was observed by the M.tuberculosis growth inhibition test in vitro. The results were statistically analyzed by t-test or rank sum test, and P<0.05 was considered a statistically significant difference. Results:Mice Immunized with EPRHP014m and EPRHP014f could produce highly effective IgG antibodies and their subtypes IgG1 and IgG2a, and the antibody titers were similar to those of mice immunized with BCG, with no statistical significance ( P>0.05). The number of spot-forming cells (SFC) secreting IFN-γ and IL-4 induced by EPRHP014f group was significantly higher than those by EPRHP014m group and BCG group ( P<0.05), but there was no significant difference in the number of SFC for IFN-γ and IL-4 induced between EPRHP014m group and BCG group ( P>0.05). The secretion levels of GM-CSF and IL-12p70 induced by the EPRHP014m group were higher than those of the BCG group ( P<0.05), but there was no significant difference in the levels of IL-6 and IL-10 induced between EPRHP014m group and BCG group ( P>0.05). There was no significant difference in the secretions of IL-6, IL-10, IL-12, and GM-CSF between the EPRHP014f and BCG groups ( P>0.05). EPRHP014m group, EPRHP014f group, and BCG group had obvious antibacterial effects in vitro, and the difference was insignificant ( P>0.05). Conclusion:Both EPRHP014f and EPRHP014m can induce strong humoral and cellular immune responses in mice after immunization, and have a strong ability to inhibit the growth of M. tuberculosis in vitro, indicating that the antigen composition EPRHP014 has good potential in the development and application of TB vaccine.

Objective:To preliminarily evaluate the immunogenicity and efficacy of two novel tuberculosis vaccine candidates (a fusion multicomponent protein EPDPA015f and a mixed multicomponent protein EPDPA015m) and to provide a new antigen combination for the development of tuberculosis vaccines.Methods:Recombinant plasmids for the expression of EPDPA015f and EPDPA015m proteins were constructed. Six-week-old BALB/c mice were immunized with EPDPA015f or EPDPA015m in combination with aluminium adjuvant (50 μg/mouse) for three times with an interval of 10 d. The mice were sacrificed 10 d after the last immunization to collect blood and spleen samples. Serum antibody titers and cytokine levels were measured by ELISA, Luminex technique and enzyme-linked immunospot assay (ELISPOT). Mycobacterial growth inhibition assay (MGIA) was used to detect the ability of mouse splenocytes to inhibit the growth of Mtb in vitro. One-way analysis of variance and t-test were used for statistical analysis. Results:Both EPDPA015f and EPDPA015m could induce the production of various cytokines and IgG antibodies at a high level. The levels of cytokines related to Th1 (IL-2, TNF-α, IFN-γ), Th2 (IL-4, IL-6, IL-10) and Th17 (IL-17) as well as other proinflammatory cytokines (GM-CSF, IL-12) were higher in the EPDPA015f group than in the adjuvant group ( P<0.05). The titer of IgG antibody induced by EPDPA015f was as high as 1∶4×10 6. The results of MGIA showed that the numbers of Mtb (lgCFU) in the PBS, adjuvant, EPDPA015f and EPDPA015m groups were 3.46±0.11, 3.51±0.06, 2.98±0.09 and 3.19±0.08, respectively. The number of colonies in the EPDPA015f group was the least as compared with that in the other three groups ( P<0.001, P<0.001, P<0.01). Conclusions:The vaccine candidate EPDPA015f could elicit more comprehensive and high-level cellular and humoral immune responses, and exhibited superior in vitro inhibitory activity against the growth of Mtb. EPDPA015f had the potential to be used as a preventive vaccine or a booster vaccine