Feasibility And Functional Analysis Of CD4?CD40 Molecules As Target Molecules For Targeted Treatment Of Spinal Tuberculosis

Objective To investigate the possibility of targeted treatment of spinal tuberculosis,the feasibility and performance of CD4?CD40 as target molecules for spinal tuberculosis were studied.Methods(1)T lymphocyte(CD4)and osteoblasts(CD40)were infected by fluorescent Mycobacterium tuberculosis(H37Ra-GFP)with the Multiplicity of infection(MOI)of 10:1.The ability of infected bacteria and the quantity of TNF-? secreted after infection were measured.(2)The cultured three passages of human osteoblasts were randomly divided into four groups: A,B,C and D.A group was the control group,which was not infected by the fluorescent Mycobacterium tuberculosis.The other three groups that B,C and D were all treatment groups,these groups of cells were infected by Mycobacterium tuberculosis with the different Multiplicity of infection(MOI)10:1?100:1?1000:1.Real-time quantitative PCR and Western blot analysis the expression levels of CD40 molecules in the osteoblast by different MOI of Mycobacterium tuberculosis infection.According to(1),(2)result,a suitable target cell(target molecule)for targeted treatment of spinal tuberculosis was selected.(3)The synthesis of aptamer corresponding to CD40 molecule and the determination of its specific binding ability to osteoblasts.(4)Synthesis and the thermodynamic stability measurement of pRNA-3WJ nanoparticles chimerized with the antisense heterozygous oligonucleotide siLNA sequence(LNA-DNA-LNA Gapmer antisense)of Mce4 a gene silenced in Mycobacterium tuberculosis and aptamer sequence corresponding to CD40 molecule.(5)The specific bindingability of pRNA 3WJ-siLNA(Mce4a)-aptamer(CD40)nanoparticles to osteoblasts was measured by flow cytometry and fluorescence microscopy.Results(1)The ability of osteoblasts to phagocytosis of Mycobacterium tuberculosis was stronger than that of T lymphocytes,and the release of TNF-? in osteoblasts was significantly higher than that of T lymphocytes.(2)The degree of mycobacterium tuberculosis infection in osteoblasts was positively correlated with the MOI.In group A,the expression of CD40 mRNA was found in osteoblasts by real-time quantitative PCR.The expression levels of CD40 mRNA in A,B,C,D groups,there were significant differences(F= 74.005,P<0.05);B,C,D of three groups were compared with the A group,there were significant differences(P<0.05),showed fluorescent Mycobacterium tuberculosis infection groups B,C,D the expression of the CD40 mRNA was higher than the non-infection group A;Compared with B,C and D,there were differences(P<0.05),which showed that the expression of CD40 mRNA from Group B to Group D is up-regulated.And the Western Blot results showed that CD40 molecules levels also correlated well with the CD40 mRNA levels.(3)It was found that the fluorescence value of osteoblasts increased significantly after treatment with aptamer.(4)pRNA 3WJ-siLNA(Mce4a)-aptamer(CD40)nanoparticles were successfully synthesized by stepwise synthesis.The melting point of pRNA 3WJ-siLNA(Mce4a)-aptamer(CD40)nanoparticles was 59 ± 3 ?,while the half-life of pRNA-3WJ nanoparticles in 50% serum was 21.9 hours.(5)The treatment of osteoblast by the pRNA3WJ-siLNA(Mce4a)-aptamer(CD40)nanoparticle group resulted in a significant shift of the peak of Alexa647-A,while the pRNA 3WJ-siLNA(Mce4a)nanoparticle group did not shift the peak of Alexa647-A to the right,and the result of its peak movement was the same as that of the control group.Fluorescence microscopy showed that the experimental group pRNA3WJ-siLNA(Mce4a)-aptamer(CD40)nanoparticles specifically bound to osteoblasts and entered the cells and the control group pRNA 3WJ-siLNA(Mce4a)nanoparticles could notspecifically bind to osteoblasts and enter the cells.Conclusions(1)The results show that osteoblasts and their expressed CD40 molecules are suitable target cells and target molecules for targeted treatment of spinal tuberculosis.(2)In this study,pRNA 3WJ-siLNA(Mce4a)-aptamer(CD40)nanoparticles was successfully synthesized and the thermodynamic properties was stable,which could be used to study the targeted therapy of spinal tuberculosis.(3)The results show that pRNA 3WJ-siLNA(Mce4a)-aptamer(CD40)nanoparticles have good targeting performance.