| Osteoporosis is a process of bone formation, the rate of osteoclast growth is greater than the growth of osteoblasts. Follicle stimulating hormone(FSH) is a hormone secrete by anterior pituitary basophil granulocytes, It's major role is to promote follicular maturation and it plays an important role in premenopausal and postmenopausal women's bone metabolism regulation. Studies have shown that FSH levels increased of functional decline induced have close relationship with osteoporosis,and the effect of FSH on bone does not rely on changes in estrogen levels, blocking the action of FSH can not only through the inhibition of bone resorption and significantly reduced bone loss, but also to promote bone reconstruction.Among adults and animals, FSHR is expressed mainly in testis support cells and ovarian granulosa cell surface. FSHR is also expressed in the mature person broken bone cells(OC) and broken bone cell body as well as a mononuclear cell surface that becomes potential targets of blocking FSH action. It is reported that FSH and osteoporosis(OP)are closely linked, and has achieved some results in a certain range of blocking the FSH level of elevation in the treatment of osteoporosis. As a new type of single domain antibody, nano- antibody has a low molecular weight, good stability, high solubility, and it is easy to be combined with the receptor to exert further physiological function. The main purpose of this study is to prepare anti-FSHR nano- antibodies in order to investigate the therapeutic effect of FSHR antibody on experimental osteoporosis in rats:1. To obtain anti- FSHR nano antibody by phage display technology from the camel immune antibody libraryWe used FSHR immune llama to build VHH phage display library, after 3 rounds of biopanning selection, 63 positive clones, in which the selection of abundance of the highest two clones VHH-3F9 and VHH-3F19, with lower abundances of a sequenceVHH-3F20 and separate sequence random two VHH-3F8 and VHH-3F10 etc. five of VHH gene sequence, its construction to vector pET22 b and protein expression was induced by IPTG, Ni ion affinity chromatography purified antibody protein, gained more than five anti FSHR nano antibody. Verified by ELISA, only VHH-3F9 and VHH-3F19 can be combined with FSHR, and the VHH-3F9 binding ability is higher. In order to obtain the expression of FSHR in the supernatant and the cytoplasm, the concentration and purity of VHH-3F9 were higher. ELISA and Blot Western detection of the antibody can be combined with the FSHR specific, and VHH-3F9 nano antibody with some of the in vitro blocking ability, the determination of its temperature stability that in 37 C processing 12 h still retains 70% of the activity.2. Preparation of anti-FSHR camel polyclonal antibodyThe prokaryotic expression of purified FSHR234 protein was used as antigen to Xinjiang Bactrian camel were immunized every two weeks strengthen immunity, a total of seven immune. In seventh strengthen immunity 2 weeks, the camels were immunized after blood collection, the indirect ELISA determination of anti FSHR polyclonal antibody in the serum titer reached 128000. We collect a lot of camel immune serum and affinity purified camel not immune serum and immune serum by protein a column. The obtained anti FSHR camel polyclonal antibody IgG. The lowest concentration of FSHR was 0.0625 ?g/ml in camel anti IgG polyclonal antibody ELISA. In detection of anti FSH antibody and cell Caov-3 surface of follicle stimulating hormone receptor(FSHR)combined with and immunocytochemical experiments results show that VHH-3F9 nano antibody and anti FSHR camel polyclonal FSHR were of IgG antibodies and Caov-3 the cell surface binding, and IgG polyclonal antibodies with high capacity, VHH-3F9 is part of the binding capacity.3. Establishment of osteoporosis model in SD rats by bilateral ovarian resectionIn this study, experimental animal SD rats were randomly divided into two groups,respectively, the OVX group and the control group SHAM. The levels of estradiol(E2),follicle stimulating hormone(FSH), Luteinizing Hormone(LH) and bone mineral density(BMD) were detected in ovariectomized rats, and the osteoporosis model was established.The results showed that the level of OVX group was significantly decreased(P<0.01) in group E2 compared with group SHAM after 10 weeks of castration in 2 weeks old rats,and remained stable in 2 to 5 weeks. FSH and LH levels were significantly increased(P<0.05), and in the 2 to 5 weeks to increase the trend slowly, reaching relatively stable level. The bone mineral density(BMD) level of SD rats was measured after 3 weeks of 8weeks, and the results showed that the bone mineral density of OVX group was significantly lower than that of group SHAM(P<0.05), which was verified by the establishment of osteoporosis model.4.Therapeutic efficacy of osteoporosis by FSHR antibodiesUsing tail vein injection in the treatment of bone osteoporosis SD rats, nanobodies VHH-3F9 and camel anti FSHR polyclone antibody IgG as the same dose of 2 mg/kg,leuprorelin(LE) according to 1.6mg / kg and control group was injected with 200?l of PBS. Each group was treated 2 times a week for 6 weeks total. Blood sampling was collected to detect the indexes of serum, and the effect of each group on osteoporosis in SD rats was observed. The experimental results showed that compared with the control group SHAM, the OVX's levels of E2 in the experimental group were significantly lower(p<0.05), but there was no difference between the groups. With the increase of FSH levels in the OVX group, IgG and VHH-3F9 injected with osteoporosis significantly inhibited the FSH, and the IgG effect was stronger than VHH-3F9, but the difference was not significant. OVX+LE group, with the injection of LE time extended, FSH levels weresignificantly reduced(P<0.05), found that the application of LE inhabited FSH. The changes of LH were in accord with the trend of FSH, but the level of LH in group OVX+VHH-3F9 was higher than that in group OVX+IgG. By inhibiting the FSH and achieving the purpose of treatment of osteoporosis.In summary, this study on the basis for the establishment of a SD rat animal model of osteoporosis by from immune phage library screening, high affinity anti FSHR nano antibody and immune camel obtained high titer anti FSHR polyclonal antibody IgG, and is used in the treatment of experimental animals SD rats osteoporosis, to leuprolide acts as an inhibitory control of FSH to determine anti receptor antibodies for the treatment of osteoporosis rats. In order to further prevent osteoporosis screening effective drugs to do a basic research in order to be used for clinical diagnosis and prevention of osteoporosis. |
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