| Objective:With preparation of anti-IL-17 antibody,we will observe its effect on ovariectomized rats and Bisphosphonate-related Osteonecrosis of the Jaw in rats.Our results will provide experimental evidence for improving the success rate of dental implants in osteoporosis patients.Methods:1.Construction of prokaryotic expression system was used to express rat IL-17.After purification and identification by SDS-PAGE,anti-IL-17 monoclonal antibodies were generated through immunization and general hybridoma technology.2.The effects of anti-IL-17 antibody on the regulation of osteoclast differentiation factors of rat osteoclast were measured in vitro.The mRNA of RANKL?OPG?TNF-??M-CSF?IL-6?IL-1 a was measured by Realtime-PCR to test the effect of anti-IL-17 antibody on osteoclast differentiation.3.The expression of cathepsinK and MMP9 was measured by Realtime-PCR?ELIS A?Western-Blot to test the effect of IL-17 and anti-IL-17 antibody on osteoclast cells.4.12-week old Female SD rats were randomly divided into OVX group,sham group?OVX+IL-17mAb group?OVX+ALN group?OVX+IL-17mAb+ALN group.Just fat tissue was removed in sham group,other groups were ovariectomized.After drug intervention,the data from testing weight?serum biochemical indexes?bone mineral density?bone biomechanics,was statistically analyzed by SPSS 13.0.5.Rats model of Bisphosphonate-related Osteonecrosis of the Jaw was established by using Zometa combined with tooth extraction.Rats were randomly divided into the Zometa group?Zometa+IL-17mAb group?control group.After model was established and drug intervention,the content of IL-17 in the serum of rats was identified by indirect ELISA,and the percentage of rats with exposed jaw bone was statistically analyzed.6.The VH?VL genes were amplified by RT-PCR from PBMC of 42 people ' s blood.The ScFv genes were constucted by overlop PCR,then the ScFv genes were sequentially cloned into pCANTAB5E vector to construct ScFv phage antibody library.After few rounds of screening,antigen-specfic ScFvs were identified byELISA?westen-blot.Results:1.The IL-17 gene was cloned into vector pET-32a,then the plasmid pET-32a-IL-17 was transfected into BL21 for expression under the induction of IPTG.The expression products were purified by NI-NTA and identified by SDS-PAGE.The purifty of purified IL-17 was about 90%.2.IL-17 was immunized to BALB/c mouse,and a neutralizing antibody(named 6H2)was obtained by using cell fusion technique.The antibody subtype was IgGl.The binding affinity of the antibody(KD=1.98E-10)was analysed by ProteOn XPR36 interaction array system.3.Compared to the control group,the mRNA levels of RANKL?TNF-??M-CSF?IL-6?IL-1? of osteoblasts were increased significantly with the stimulation of IL-17,but the mRNA level of OPG was decreased significantly.The effect of IL-17 was inhibited by anti-IL-17antibody.4.Compared to the control group,the expression levels of cathepsinK and MMP9 of osteoclasts were increased significantly with the stimulation of IL-17.The effect of IL-17 was inhibited by anti-IL-17 antibody.5.The levels of ALP were appreciably changed by anti-IL-17 antibody(p<0.05).The levels of bone mineral density and bone biomechanics of anti-IL-17 antibody group were better than OVX group and near to Sham group.The combination treatment of IL-17mAb and ALN have certain synergies.6.The results suggested that the inflammation of soft tissue has been reduced and the number of BRONJ rats was decreased significantly in IL-17mAb group.Data from anti-IL-17 antibody group was better than Zometa group.The percentage of rats with exposed jaw bone and IL-17 level were reduced significantly than those from Zometa group(p<0.05).7.A human ScFv phage antibody library was constructed and a neutralizing antibody(clone65)against IL-17 was successfully obtained,which provided an experimental foundation for further study.Conclusions:1.The monoclonal antibody(6H2)against IL-17 was successfully generated.2.We observed that IL-17 adjusted the expression of RANKL?OPG?M-CSF?IL-1??IL-6?TNF-? to regulate the differentiation of osteoclast.3.We observed that IL-17 stimulated the expression of cathepsinK?MMP9 in osteoclast cells to enhance the ability of osteoclasts degradation of bone matrix.4.Anti-IL-17 antibody can prevent osteoporosis.The combination treatment of anti-IL-17 antibody and ALN has certain synergies.5.Anti-IL-17 antibody can reduce the incidence of BRONJ.6.A neutralizing antibody(clone65)against IL-17 was successfully obtained,which could inhibit the binding of IL-17R to immobilized IL-17. |
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