| Letinous edodes is a species of basidiomycetes of Umbelliferaeand the world's second largest edible fungus.As early as one thousand years ago,Letinous edodes was included in the "Compendium of Materia Medica".Modern medicine found that letinous edodes has lots of functionality,like anti-tumor,anti-oxidation,protect the liver,regulate blood pressure and anti chemical substances.With the deepening of the research on letinous edodes,lentinan as the main active ingredient in letinous edodes has also been found,the efficacy of enhanced immune function are further explored.However,as one kind of large molecular polysaccharide,lentinan has many shortcomings,such as lentinancannot exist in the body for a long time,and it is metabolized rapidly.Moreoverits production requires large doses of medication.Carbon nanotubes are one of the hot spots in the field of nano materials.The properties of high specific surface area,hollow porous structure,good biological membrane permeability and high chemical stability make it highly potential in the field of biological materials.However,theircarbon skeletons make carbon nanotubesvery hydrophobic and insoluble,which limits the biomedical application of pure carbon nanotubes.In this study,the carbon nanotubes were used as a carrier,and the lentinanwas grafted onto the carbon nanotubes by covalent bonds to enhance the water solubility of carbon nanotubes.Then,in vitro,the effect of the lentinan-modified carbon nanotubes on the activity and differentiation of splenic lymphocytes and dendritic cell was studied.In addition,the study also explored the effect of lentinan-modified carbon nanotubesfor cellular immunity and humoral immunity as an adjuvant.This research contains four parts as follows:Experiment 1 Preparation and characterization oflentinan-modified carbon nanotubes The lentinan-modified carbon nanotubes was synthesized by covalentlyattaching lentinan to nanotubes,and precise detailed study of the surface characteristics of the complex.First of all,using high resolution transmission electron microscope to observe and measure the micro morphology of the lentinan-modified carbon nanotubes.Then the connection mode of lentinan and carbon nanotubes was detectedby Fourier transform infrared spectroscopy and ultraviolet spectrophotometer.The graft yield of lentinan-modified carbon nanotubeswas determined by a thermogravimetric analyzer.Finally,the adsorption capacity was determined by BCA kit and particle size analyzer.The resultsindicated the average diameter of the lentinan-modified carbon nanotubes was 12nm,which was higher than that of carboxylic carbon nanotubes by 8nm,and there were many more attachments on the outer graphite sheets and inside the lentinan-modified carbon nanotubes.The red shift of UV spectrum and the comparison of infrared spectra proved that the lentinan is connected with the carbon nanotubes by ester bond.The curve of thermal gravity analysis showed that the grafting ratio of lentinan was 9.1%.The zeta potential of the lentinan-modified carbon nanotubesplus OVA was-26mV,which was higher than that of lentinan-modified carbon nanotubes.BCA kit results showed that each of the 3 weight of lentinan-modified carbon nanotubes can adsorb up to 2 of the weight of OVA.Experiment 2 The effect of lentinan-modified carbon nanotubes on spleen lymphocytes of mice in vitro In order to detect the effect of lentinan-modified carbon nanotibes on cell viability.Three different concentrationsoflentinan-modified carbon nanotubes and the corresponding concentration of carbon nanotubes and lentinan were added to spleen lymphocytes of mice in vitro,and the changes of lymphocyte activity in the spleen and the change of cell phenotype were determined with MTT method and flow cytometer.The results showed that the three drugs had no significant effect on the activity of cells at low concentrations.Same as carboxyl carbon nanotubes,lentinan-modified carbon nanotubes cannot significantly impact the proliferation of lymphocytes at medium concentrationsbutboththe carbon nanotubesat high concentration showed a significant inhibitory effect.The comparison of cell phenotype showed that the carboxyl carbon nanotubes had a certain influence on cellular immune activity in vitro,but the lentinan-modified carbon nanotubes had no such influence.Experiment 3 The effects of lentinan-modified carbon nanotubes on dendritic cells To evaluate the maturation effect of lentinan-modified carbon nanotubes on murine dendritic cells,mouse bone-marrow-derived dendritic cells were chosen as a model,and then differentiated into dendritic cells by rmG-CSF and rmIL-4.After 7 days of culture,the cells were stimulated by lentinan-modified carbon nanotubes for 48h.Then,the expression rates of MHC-II,CD80 and CD86 on cell surfacewere determined,and the secretion of cytokines in supernatant of cell culture medium was tested.The dendritic cell werecultivated for 24 h with the constructs of FITC-labeled OVA,then the phagocytic function of dendritic cell was detectedby laser scanning confocal microscope.The results showed that lentinan-modified carbon nanotubes could not only significantly improve the expression of CD86,CD80 and MHC-II of DCs,but also increase the secretion of cytokines,at the same time,lentinan-modified carbon nanotubes can significantly increase the dendritic cell's ability of antigen intaking.Experiment 4 The immune response of mouse immunized by OVA withlentinan-modified carbon nanotubesas adjuvantOne hundred and ninty-two 4-weeks-old mouse were randomly divided to 8 groups,namely,three concentrations oflentinan-modified carbon nanotubes,carboxylic carbon nanotubes,lentinan,complete freund's adjuvant and PBS as adjuvants.The mice were inoculated subcutaneously 3 times at a 2 week interval.The blood of vaccinated and control mice were collected at 7,14,21,and 28 days after the last immunization.The concentration changes of IgQ IgG1,IgG2a,IL-4,IL-6,TNF-? and IFN-? were determined by ELISAmethod.The proliferation of spleen lymphocytesstimulatingwith PHA and LPSrespectively were measured by MTTmethod.The expression of CD4+ and CD8+ in lymphocytesof spleen was determined by flow cytometryat 7 and 21 days after the last immunization.Sciatic and popliteal lymph nodes of vaccinated andcontrol mice at 24 and 48 h after the first immunization were collected todetermine dendritic cell activation in the lymph nodes.The results showed that lentinan-modified carbon nanotubes could significantly increase the secretion of cytokines and improve the cellular immune level.In summary,lentinan-modified carbon nanotubes are a potential adjuvant with excellentactivity. |
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