CN107353333A

CN107353333A - A kind of bone protectiveness molecule Semaphorine3A and its pharmacy application

Info

Publication number: CN107353333A
Application number: CN201710517341.XA
Authority: CN
Inventors: 张燕; 滕月; 尹战海; 李婧; 李坤
Original assignee: First Affiliated Hospital of Medical College of Xian Jiaotong University
Current assignee: First Affiliated Hospital of Medical College of Xian Jiaotong University
Priority date: 2017-06-29
Filing date: 2017-06-29
Publication date: 2017-11-17

Abstract

The invention discloses a kind of bone protectiveness molecule Semaphorine3A and its pharmacy application, and the molecule protein sequence is as shown in SEQ.ID.NO.1.Semaphorine3A provided by the invention and RA is closely related, and Semaphorine3A processing can promote macrophage M2 polarization, suppress endothelial cell, synovial cell and osteoclast function.Restructuring Semaphorine3A adenovirus intervention can effectively prevent, treat mouse rheumatoid arthritis.

Description

A kind of bone protectiveness molecule Semaphorine3A and its pharmacy application

Technical field

The invention belongs to biomedicine technical field, and in particular to a kind of bone protectiveness molecule Semaphorine3A and its Pharmacy application.

Background technology

Rheumatoid arthritis (RA) is a kind of chronic generalized autoimmune disease based on arthropathy, its disease Reason is characterized as that chronic inflammation, cellular infiltration and the synovial membrane screen of synovium of joint are formed, chondro-osseous progressive destroys, and ultimately results in Arthrocleisis, deformity, dysfunction.Rheumatoid arthritis disability rate is high, has a strong impact on China's mid-aged population physiological health And quality of life, the tremendous economic of family and society burden is caused, it is serious to take China's health resource.

Traditional RA treatment means include internal medicine anti-inflammatory treatment, surgery villusectomy and joint replacement etc..Anti-inflammatory and Immune formulation treatment is only capable of alleviating acute stage RA arthropathy, not good enough for middle and advanced stage RA curative effects.Villusectomy has certain Curative effect, but complication is more, and patient recovery times' length, postoperative synovial membrane easily regenerates.Artificial joint replacement is only adapted to high point The displacement of section, Shou Zhong little, and function of joint is recovered in the postoperative part that is only capable of.

Gene therapy in recent years is that RA treatment opens new route.The main thought of gene therapy is will to be controlled with coding The channel genes of the property treated albumen specifically in tissue, enable the patient to routinely produce this human cytokines, reach treatment Purpose.

Gene therapy research at present on RA is more.For example, by adenovirus vector by insulin-like growth factor-i (IGF-1) horse cartilage cell is imported in vitro, then this cell by modification is imported with to the femoro-patellar articulation of 15mm Cartilage breakdowns In, matrix synthesis and repair of cartilage can be promoted.For another example, mouse CTLA4-FasL is merged by recombinant adeno-associated virus (AAV) Gene injection can effectively suppress the arthritic progress of mouse in AIA models in mouse ankle joint.

Clinical test on RA gene therapies was implemented early in 1996.Univ. of Pittsburgh is by IL-1R α channel genes Into the 2nd~5 metacarpophalangeal joints of RA end late stage patients, its feasibility and security to RA patient's treatment specify that. Targeted Genetics company evaluation contains TNFR：The effect of Fc single-stranded rAAV2 viral therapies RA and security, hair The clinical symptoms of existing treated target joint are preferably improved, and rAAV carriers are safe.

The success of clinical test provides strong support for the exploration of RA gene therapies, at present RA gene therapy research hair Exhibition is very fast, obtains larger breakthrough, but there is also some problems：1. lack preferable RA animal models, current animal mould Type only has single episode or the development in a stage of arthritis.2. the carrier with target gene may send out target device Outside official, target gene, which is difficult to stabilization, expresses, and there is also problem for the regulation and control of target gene.3. lack preferable carrier.Make at present With it is most be retrovirus, adenovirus and non-virus carrier.Retrovirus can stablize express, but in vivo transfection efficiency it is low, And random integration is on recipient cell chromosome；Adenovirus transfection rate is high, but can not express steadily in the long term；Non-viral load The expression time of body gene is short, and the transfection efficiency in active somatic cell is relatively low.Currently without a carrier can it is safe and stable, have The expression of effect ground.It is believed that with the constantly improve of carrier, gene regulation and transfection efficiency, gene therapy will turn into treatment RA effective means.

The content of the invention

It is an object of the invention to provide a kind of bone protectiveness molecule Semaphorine3A and its pharmacy application, the molecule It is negatively correlated with RA course of disease indexs with the low expression in RA patients serums, synovial fluid, it is a kind of new RA marks.

The present invention is to be achieved through the following technical solutions：

The invention discloses a kind of bone protective protein molecule Semaphorine3A, its amino acid sequence such as SEQ ID Shown in NO.1.

The invention also discloses above-mentioned bone protective protein molecule Semaphorine3A to prepare treatment/prevention rheumatoid Application in the arthritic medicine of property.

Preferably, described medicine is regulation medicine of the macrophage to anti-inflammatory M2 type polarizations.

Preferably, described medicine is suppression endothelial cell migration and the medicine of signal activation.

Preferably, the medicine that described medicine is attacked for suppression synovioblast.

Preferably, described medicine is secreted to suppress synovioblast IL-6 and promotes the medicine of its apoptosis.

Preferably, described medicine is the differentiation of suppression osteoclast, the medicine of function.

Invention additionally discloses for the recombined adhenovirus containing above-mentioned bone protective protein molecule Semaphorine3A.

Invention additionally discloses in order to which above-mentioned recombined adhenovirus is in the medicine for preparing treatment/prevention rheumatoid arthritis Application.

Preferably, described medicine is the medicine for the treatment of/prevention Serum-induced type rheumatoid arthritis.

Compared with prior art, the present invention has technique effect beneficial below：

Involved Semaphorine3A provided by the invention is a kind of cell exocrine albumen, is had in RA serum, cunning Low expression in film liquid, the negatively correlated characteristic with the RA courses of disease, it is a kind of new RA marks.Semaphorine3A is specified in RA Middle effect can provide new biological target selection to effectively improve RA clinical efficacies.

Semaphorine3A provided by the invention and RA development is closely related, Semaphorine3A regulation macrophages pole Changing, be allowed to anti-inflammatory M2 type macrophage differentiations, Semaphorine3A suppresses endothelial cell migration and signal activation, Semaphorine3A suppresses synovioblast invasion and attack, IL-6 secretes and promotes its apoptosis, and Semaphorine3A suppresses broken Bone cell differentiation and function.Recombined adhenovirus containing Semaphorine3A can preventing/treating mice serum induction type rheumatoid Arthritis.

Involved Semaphorine3A provided by the invention provides new for design resisting rheumatoid arthritis medicine Target spot, and on this basis, to design and develop directly against rheumatoid arthritis new drug.

Brief description of the drawings

Fig. 1-1 is RA patients serum's Semaphorine3A expressions；

Fig. 1-2 is RA patient's synovial fluid Semaphorine3A expressions；

Fig. 1-3 is RA patients serum Semaphorine3A expressions and RF correlations；

Fig. 1-4 is RA patients serum Semaphorine3A expressions and DAS28 correlations；

Fig. 1-5 is RA patients serum Semaphorine3A expressions and CRP correlations；

Fig. 2-1 is the expression that Semaphorine3A suppresses macrophage M1 polarization marker molecules Nos2mRNA；

Fig. 2-2 is the generation that Semaphorine3A suppresses macrophage M1 polarization marker molecules ROS；

Fig. 2-3 is the generation that Semaphorine3A suppresses macrophage M1 polarization marker molecule IL-1 β；

Fig. 2-4 is the secretion that Semaphorine3A suppresses macrophage M1 polarization marker molecule TNF-αs；

Fig. 2-5 is the secretion that Semaphorine3A suppresses macrophage M1 polarization marker molecules IL-6；

Fig. 3-1 is the expression that Semaphorine3A promotes macrophage M2 polarization marker molecules Arg1mRNA；

Fig. 3-2 is the expression that Semaphorine3A promotes macrophage M2 polarization marker molecules Ym1mRNA；

Fig. 3-3 is the expression that Semaphorine3A promotes macrophage M2 polarization marker molecules Fizz1mRNA；

Fig. 3-4 is the expression that Semaphorine3A promotes macrophage M2 polarization marker molecules CD206mRNA；

Fig. 3-5 is the expression that Semaphorine3A promotes macrophage M2 polarization marker molecules CD163mRNA；

Fig. 3-6 is that Semaphorine3A promotes macrophage M2 polarization marker molecule Arginase activity；

Fig. 4 is that Semaphorine3A suppresses macrophage STAT3 phosphorylations；

Fig. 5-1 is Semaphorine3A inhibition of endothelial cell proliferation；

Fig. 5-2 is Semaphorine3A inhibition of endothelial cell proliferation quantitative analyses；

Fig. 5-3 is that Semaphorine3A suppresses endothelial cell migration；

Fig. 5-4 is that Semaphorine3A suppresses endothelial cell migration quantitative analysis；

Fig. 5-5 is that Semaphorine3A suppresses endothelial cell ERK, AKT phosphorylation；

Fig. 6-1 is that Semaphorine3A suppresses synovioblast invasion and attack；

Fig. 6-2 is that Semaphorine3A suppresses synovioblast invasion and attack quantitative analysis；

Fig. 6-3 is that Semaphorine3A suppresses synovioblast secretion IL-6；

Fig. 6-4 is that Semaphorine3A promotes synovioblast apoptosis；

Fig. 7-1 is that Semaphorine3A suppresses osteoclast marker molecule NFATc1mRNA expression；

Fig. 7-2 is that Semaphorine3A suppresses osteoclast marker molecule integrin β 3mRNA expression；

Fig. 7-3 is that Semaphorine3A suppresses osteoclast marker molecule Src mRNA expression；

Fig. 7-4 is that Semaphorine3A suppresses osteoclast marker molecule CathepsinK mRNA expression；

Fig. 7-5 is that Semaphorine3A suppresses osteoclast marker molecule protein expression；

Fig. 8-1 is that Semaphorine3A suppresses osteoclast formation；

Fig. 8-2 is that Semaphorine3A suppresses osteoclast formation quantitative analysis；

Fig. 9-1 is that Semaphorine3A suppresses osteoclast function；

Fig. 9-2 is that Semaphorine3A suppresses osteoclastic bone depression alveole area；

Fig. 9-3 is that Semaphorine3A suppresses osteoclastic bone depression alveole depth；

Figure 10-1 is that Semaphorine3A suppresses STA mouse Arthritis development indexes；

Figure 10-2 is that Semaphorine3A suppresses STA mouse ankle thickness；

Figure 10-3 is that Semaphorine3A suppresses STA mouse sole thickness；

Figure 11-1 is that Semaphorine3A suppresses STA mouse ankle-joint osteoclasia degree；

Figure 11-2 is that Semaphorine3A suppresses STA mouse ankle-joint bone densities；

Figure 11-3 is that Semaphorine3A suppresses STA mouse change of serum C TX levels；

Figure 12-1 is that Semaphorine3A suppresses STA mouse ankle-joint section TRAP positive cell numbers；

Figure 12-2 is that Semaphorine3A suppresses the section TRAP positive cell number quantitative analyses of STA mouse ankle-joint；

Figure 12-3 is that Semaphorine3A suppresses the section osteoclast number quantitative analysis of STA mouse ankle-joint；

Figure 12-4 is that Semaphorine3A suppresses the section osteoclast surface area quantitative analysis of STA mouse ankle-joint.

Embodiment

With reference to specific embodiment, the present invention is described in further detail, it is described be explanation of the invention and It is not to limit.

1st, expression of the clear and definite Semaphorine3A in RA patients serums, synovial fluid, and the correlation with the RA courses of disease

Experimental method：

A. 100 μ l samples product are added per hole or sample is (general during detection to use 1:50-1:400 dilution), per sample extremely Less plus diplopore, 37 DEG C are put to be incubated 2h (should be capped to avoid evaporating, on plate or plate is lain in into bottom has the metal wet box of wet gauze In), discard liquid in hole；

B. the antibody of 100 μ l biotin labelings is added, puts 37 DEG C of incubation 1h；

C. liquid in hole is discarded, is crossed with cleaning solution and is washed one time and (cleaning solution is filled into plate hole, 1-2min is placed, intermittently shakes Dynamic, soak time can not arbitrarily shorten), liquid in hole (being patted dry after liquid can be got rid of on cleaning towel or blotting paper) is blotted, Repeat washing 3 times；

D. 100 μ l HRP enzyme labelled antibodies, 37 DEG C of incubation 1h are added；

E. washing 3 times is repeated；

F. adding 90 μ l tmb substrates liquid, (matching while using, working concentration are 10mg/5ml absolute ethyl alcohols, put 37 DEG C of lucifuges and put Put 30min；

G. the μ l terminating reactions of terminate liquid 50 are added per hole, now blueness is vertical turns yellow, in determination experiment result in 5min.Inspection Survey wavelength is 450nm.Advanced line blank hole system returns to zero during detection.

H. result judges：In the result that in white background, directly detects by an unaided eye.Color is deeper in reacting hole, positive degree Stronger, negative reaction is colourless or extremely shallow.

Fig. 1-1 is RA patients serum's Semaphorine3A expression results, as can be seen that patient's RA blood from Fig. 1-1 Clear Semaphorine3A expressions compare low compared with OA.Fig. 1-2 is RA patient's synovial fluid Semaphorine3A expression knots Fruit, as can be seen that patient's RA synovium of joint liquid Semaphorine3A expressions are low compared with OA controls from Fig. 1-2.Meanwhile point The horizontal correlations with RA progress indexs RF, DAS28, CRP etc. of RA serum Semaphorine3A are analysed, find RA patients serums Semaphorine3A levels and the negatively correlated (figure of RF negatively correlated (Fig. 1-3) and DAS28 negatively correlated (Fig. 1-4) and CRP 1-5), explanation：RA extents are more serious, and Semaphorine3A levels are lower, and Semaphorine3A can comment as one The marker molecule of valency RA extents.

2nd, Semaphorine3A promotes macrophage to be converted to M2 types

A. the separation and culture of bone marrow macrophage (BMM)

The C57BL/6 mouse of six week old are taken, the neck that breaks is put to death.Mouse is dissected under superclean bench, takes its femur.By femur Two is cut, and 5ml DMEM nutrient solutions are drawn with 1ml syringes, inserts in the ossis being exposed, carries out soft rush Wash, until ossis becomes translucent.The liquid rinsed is filtered with the filter screen of 20 mesh, removes big cell mass. Transfer liquid lightly blows and beats cell dispersion into 10ml centrifuge tubes, with suction pipe.With 5ml M-CSF containing 100ng/ml, 10%FBS Cell is resuspended with the α MEM nutrient solutions of 1% mycillin, is inoculated in blake bottle, is cultivated in 37 DEG C 5% DEG C of incubator.

B. the extraction of cell total rna

Tissue Culture Dish is placed on ice, and the TRIzol of 500 μ l precoolings is added per ware, is blown and beaten, is transferred to new repeatedly with rifle L.5ml in centrifuge tube, it is stored at room temperature 5min.12000rpm centrifuges 15min at 4 DEG C, takes supernatant, is transferred to new l.5ml centrifugation Guan Zhong.100 μ l chloroforms are added, acutely mixes, is stored at room temperature 5min.12000rpm centrifuges 15min at 4 DEG C, and liquid is in 3 after centrifugation Layer, the liquid of upper strata colourless aqueous phase layer is carefully passed in new l.5ml centrifuge tube.Add 0.5ml isopropanols, fully mix ,- 10min is stood at 20 DEG C.12000rpm centrifuges 10min, supernatant discarding at 4 DEG C.Precipitation is washed with 75% ethanol, is dried in the air at room temperature It is dry, it is dissolved in 20 μ l DEPC water.Take appropriate RNA to be diluted in 100 μ l DEPC water, determine OD values under 260nm and 280nm.

Calculate RNA concentration formula be：Total rna concentration (μ g/ml)=OD260 × 40 × 100, OD260/OD280 are 1.8 ~2.0 think that purity is preferable, think there is albumen or phenol pollution during less than 1.8.

C.RNA reverse transcriptions are into cDNA

1 μ g cell total rnas are taken, reverse transcription is carried out with Promega reverse transcription reagent box.The reverse transcription reaction system such as institute of table 1 Show：

Table 1

D. by 20 μ l reverse transcription end-product moisturizings to 300 μ l, it is template to take 5 μ l, is carried out in 25 μ l reaction systems Realtime PCR are expanded, and amplification system is as shown in table 2：

Table 2

Amplification condition is：95 DEG C of pre-degeneration 10s, followed by two-step method PCR：95 DEG C of denaturation 5s, 60 DEG C of annealing/extensions 34s, carry out 40 circulations.

E. the extraction of total protein of cell and quantitative

Culture dish is placed in the culture medium on ice, discarded in ware, the PBS of 1ml precoolings is carefully added into along ware wall, is gently floated Wash, discard the liquid of residual, so again rinse cell one time.The cell pyrolysis liquid of 150 μ l precoolings is added, will with cell scraper Cell scrapes off, and is transferred in 1.5ml centrifuge tubes.30min is incubated on ice, is during which shaken 6 times, is allowed cell fully to crack.At 4 DEG C 12000rpm centrifuges 20min, takes supernatant, this albumen carried.

4 μ l protein stostes are taken, 20 μ l are diluted to lysate.By the A liquid in BCA kits and B liquid with 50:1 ratio Example is mixed into working solution, adds in 96 orifice plates, adds 200 μ l per hole.20 μ l protein standard substances or the protein sample diluted are added, It is sufficiently mixed, 37 DEG C of incubation 30min.Under wavelength 562nm, the absorbance of determination sample and standard items.According to protein standard substance Concentration and absorbance make standard curve, calculate the concentration of protein sample.

F.Western blot

Prepare 10% separation gel and 3% concentration glue.The μ g of protein sample 20 are taken, add 5 appropriate × sample-loading buffer, Boil 10min after mixing, loading, electrophoresis about 15min under 120V.Glue is taken out from electrophoresis tank, soaked in transfering buffering liquid 10min.NC films are taken out, 2min is dyed in Ponceaux dye liquor, observe transfer effect.With the TBS containing 0.5%Tween-20 (TBST) dyestuff on film is washed away, is enclosed in the polybag containing 5% skimmed milk power TBST, room temperature slowly shakes 1h；E. phase is pressed Dilution proportion primary antibody is answered in TBST, film is enclosed in the polybag containing appropriate primary antibody, 4 DEG C overnight.TBST washes film 10min × 3 It is secondary, with TBST with 1:The 20000 corresponding infrared ray secondary antibody of dilution proportion, film is enclosed in the polybag containing appropriate secondary antibody, room It is warm slowly to shake 1h.TBST washes film 15min × 3 time, and scanning analysis are carried out on Odessy looms.

Experimental result is as shown in Fig. 2-1 to Fig. 2-5, it can be seen that Smaphorine3A pre-processes macrophage, The macrophage M1 polarization marker molecule Nos2mRNA expression (Fig. 2-1) induced by LPS/IFN- γ can effectively be reduced, ROS is produced (Fig. 2-2) and IL-1 β (Fig. 2-3), TNF-α (Fig. 2-4), IL-6 (Fig. 2-5) secretions, suppress macrophage M1 polarization.

As can be seen that Smaphorine3A pretreatment macrophages, can effectively strengthen by IL-4 in from Fig. 3-1 to Fig. 3-5 Induction macrophage M2 polarization marker molecule Arg1 (Fig. 3-1), Ym1 (Fig. 3-2), Fizz1 (Fig. 3-3), CD206 (Fig. 3-4), CD163 (Fig. 3-5) mRNA expression, and Arginase are active (Fig. 3-6), promote macrophage M2 polarization.

Fig. 4 is the result figure that Semaphorine3A suppresses macrophage STAT3 phosphorylations, from fig. 4, it can be seen that Smaphorine3A pre-processes macrophage, can effectively reduce by the LPS/IFN- γ STAT3 induced phosphorylation.

3rd, Smaphorine3A suppresses endothelial cell, synovial cell's migration and activation

Endothelial cell proliferation activity after A.Edu dyeing detection Smaphorine3A processing

HUVEC cells are seeded to 24 orifice plates, and give Smaphorine3A (10nM) processing.Cultivated in every hole in cell Add Edu dyeing liquors to be dyed, stop dyeing after 4h.Nutrient solution is abandoned, adds the paraformaldehyde fixers of 300 μ l 4%, room temperature Stand 30min.The glycine that the 2mg/ml of Fresh is added per hole is incubated 10min.PBS is washed 3 times, each 5min.Abandon Clearly, 300 μ l 0.5%TritonX-100 are added per hole and carries out permeabilized processing.PBS is washed 3 times, each 5min.According to the form below 3 is suitable Sequence prepares the 1 × Apollo reaction solutions for being not less than 50 μ l per hole.

Table 3

PBS is washed 3 times, each 5min.Dyeing 10min is carried out with DAPI.PBS is washed 3 times, each 5min.Fragmentation is moved To slide, mounting.In the micro- Microscopic observation result of confocal fluorescent.Positive cell represents new proliferative cell and excited in green Mark is that DPAI staining cells represent total cell number under fluorescence, includes proliferative cell and nondividing proliferative cell in purple Dyeing is blueness under color exciting light.As a result as shown in Fig. 5-1,5-2, Smaphorine3A pretreated group EdU positive cell percentages Compare control group to have reduced, inhibition of endothelial cell proliferation effect is obvious.

Endothelial cell migration activity after B.Wound healing experiment detection Smaphorine3A processing

HUVEC cells are seeded to 24 orifice plates, and give Smaphorine3A (10nM) processing, until cell growth is to complete Converge entirely.Cell surface is streaked with 200 μ l pipette tips, PBS surface cast-off cells twice, change fresh serum-free media continuation Cultivate 48h.Micro- Microscopic observation is migrated to the cell of damage field.And with LAS EZ software analysis cell migration distances.As a result As shown in Fig. 5-3,5-4, Smaphorine3A pretreated groups cell migration distance is reduced, and Smaphorine3A effectively suppresses endothelium Cell migration.From Fig. 5-5 as can be seen that Smaphorine3A suppresses endothelial cell ERK, AKT phosphorylation of VEGF inductions.

C. the FLS cell-invasive activities after vitro invasion experiment detection Smaphorine3A processing

4 DEG C of the Matrigel liquefaction overnight in -80 DEG C will be frozen.Serum free medium and matrigel press 1：5 dilutions, often Hole adds 50 μ l (about 50 μ g/ are per room) and mixed, the equal 4 DEG C of ice baths operation of the above.Transwell is placed in 24 well culture plates, is added Each 100 μ l Matrigel dilutions of upper chamber.It is put into 37 DEG C of incubators and is incubated 2h, white layer occurs to Matrigel solidifications, with nothing Blood serum medium is washed 1 time.Vitellophag, PBS are washed 3 times, count, cell suspension is made into the serum free medium containing BSA, per hole 100 μ l cell suspensions are added, adding 500 μ l in lower chambers contains 20%FBS conditioned mediums.After 12h being cultivated in 37 DEG C of incubators Hypoxic processing, continue to cultivate 24h.Take out cell to be washed 2 times with PBS, interior ventricular cell is carefully wiped with cotton swab.5% glutaraldehyde Fixed 5min, 0.1% violet staining 30min.The cell for moving to small outside is counted under inverted microscope, each sample is random 10 visuals field are counted to average.

Experimental result is as shown in Fig. 6-1 to 6-4, the Smaphorine3A pretreated groups cell it can be seen from Fig. 6-1,6-2 Invasive ability reduces, and Smaphorine3A effectively suppresses synovioblast invasion and attack.It can be seen from Fig. 6-3 Smaphorine3A effectively suppresses synovioblast secretion inflammatory factor IL-6.It can be seen from Fig. 6-4 Smaphorine3A can promote synovioblast apoptosis.

4th, Semaphorine3A suppresses osteoclast differentiation and function

A.TRAP dyeing monitoring amount of osteoclast

5% glutaraldehyde room temperature fixes cell 30min, and PBS is washed 3 times, utilizes the TRAP staining kits of Sigma companies to carry out Specification is shown in dyeing, in detail operation.As shown in Fig. 8-1, Fig. 8-2, Smaphorine3A can effectively suppress osteoclast formation.From Fig. 7-1 to-7-5 can be seen that Smaphorine3A suppress osteoclast differentiation marker molecule NFATc1mRNA expression (Fig. 7- 1), integrin β 3mRNA express (Fig. 7-2), Src mRNA expression (Fig. 7-3), CathepsinK mRNA expression (Fig. 7- 4), Smaphorine3A suppresses osteoclast differentiation (Fig. 7-5).

B. the alveole that is recessed forms experimental monitoring osteoclast function

Cell is inoculated on osteocomma, carries out osteoclastic induction, with 0.5N NaOH processing osteocomma 1min after 6 days, is brushed away with denticle Except the cell on osteocomma.Add appropriate peroxidase-conjugated wheat germ agglutinin (20mg/ml) after PBS is fully rinsed, on osteocomma Lucifuge dyes 1h, is taken a picture after chromogenic reagent 10min.Experimental result Smaphorine3A it can be seen from Fig. 9-1 to Fig. 9-3 Suppress osteoclast and form bone depression alveole in bone surface, suppress the area (Fig. 9-2) and depth (Fig. 9-3) of depression alveole, say Bright Smaphorine3A suppresses osteoclastic bone resorption function.

5th, the effect of Semaphorine3A recombined adhenovirus treatment mouse rheumatoid arthritis

It is prepared by A.STA models

Etherization mouse, every μ l K/BxN serum of mouse peritoneal injection 200 or control serum.Noted respectively at D2, D5 Penetrate Semaphorine3A recombined adhenovirus or GFP comparison virus (10⁹PFU/ is only), injection site is proximal tibia ossis.In From next day, rear solid end and ankle thickness are measured daily with slide measure.Blood is collected in D9 anesthetized mices and is used for Serological testing, is broken Neck takes hind leg to be fixed for the analysis of μ CT scan after putting to death mouse.

The RA clinics for injecting Semaphorine3A recombined adhenovirus group mouse it can be seen from Figure 10-1 to Figure 10-3 are commented Divide and be significantly lower than control group (Figure 10-1), its ankle-joint thickness (Figure 10-2), sole thickness (Figure 10-3) are considerably less than control group. Illustrate that Semaphorine3A can effectively delay the RA of STA model mouses to be in progress.

B.μCT

Use the Scanco μ CT40 scanner scanning mouse hind leg samples of Switzerland's Scanco Medical company As.Scanning Threshold is set to 250.3D reconstructions are carried out to scanning result using VG studio Max2.2 softwares, voxel size are set to 18mm。

As shown in Figure 11-1, the ankle-joint osteoclasia degree for injecting Semaphorine3A recombined adhenovirus group mouse is obvious Mitigate.As shown in Figure 11-2, the ankle-joint bone density of injection Semaphorine3A recombined adhenovirus group mouse substantially increases.Such as Shown in Figure 11-3, the horizontal obvious reductions of change of serum C TX of Semaphorine3A recombined adhenovirus group mouse are injected.Result above is said Bright, Semaphorine3A recombined adhenovirus can effectively reduce RA osteoclasia degree, play bone protective effect.

C. histological examination bone slice

10% neutral formalin fixed sample, sample is placed in 14%EDTA decalcifications processing 10d, carried out after FFPE HE and TRAP dyeing.Utilize BioQuant OsteoII software analysis amount of osteoclast, osteoclast surface area/bone surface product The parameters such as tectology.

As shown in Figure 12-1, Figure 12-2, the ankle-joint section TRAP of injection Semaphorine3A recombined adhenovirus group mouse Positive cell number substantially reduces.As shown in Figure 12-3, Figure 12-4, its ankle-joint section osteoclast number substantially reduces.Say It is bright：Semaphorine3A recombined adhenovirus protects Bones and joints by suppressing osteoclast differentiation and function.

Nucleotides sequence list

<110>No.1 Hospital Attached to Medical College, Xi'an Communication Univ

<120>A kind of bone protectiveness molecule Semaphorine3A and its pharmacy application

<160> 1

<210> 1

<211> 771

<212> PRT

<400> 1

Met Gly Trp Leu Thr Arg Ile Val Cys Leu Phe Trp Gly Val Leu Leu

1 5 10 15

Thr Ala Arg Ala Asn Tyr Gln Asn Gly Lys Asn Asn Val Pro Arg Leu

20 25 30

Lys Leu Ser Tyr Lys Glu Met Leu Glu Ser Asn Asn Val Ile Thr Phe

35 40 45

Asn Gly Leu Ala Asn Ser Ser Ser Tyr His Thr Phe Leu Leu Asp Glu

50 55 60

Glu Arg Ser Arg Leu Tyr Val Gly Ala Lys Asp His Ile Phe Ser Phe

65 70 75 80

Asp Leu Val Asn Ile Lys Asp Phe Gln Lys Ile Val Trp Pro Val Ser

85 90 95

Tyr Thr Arg Arg Asp Glu Cys Lys Trp Ala Gly Lys Asp Ile Leu Lys

100 105 110

Glu Cys Ala Asn Phe Ile Lys Val Leu Lys Ala Tyr Asn Gln Thr His

115 120 125

Leu Tyr Ala Cys Gly Thr Gly Ala Phe His Pro Ile Cys Thr Tyr Ile

130 135 140

Glu Ile Gly His His Pro Glu Asp Asn Ile Phe Lys Leu Glu Asn Ser

145 150 155 160

His Phe Glu Asn Gly Arg Gly Lys Ser Pro Tyr Asp Pro Lys Leu Leu

165 170 175

Thr Ala Ser Leu Leu Ile Asp Gly Glu Leu Tyr Ser Gly Thr Ala Ala

180 185 190

Asp Phe Met Gly Arg Asp Phe Ala Ile Phe Arg Thr Leu Gly His His

195 200 205

His Pro Ile Arg Thr Glu Gln His Asp Ser Arg Trp Leu Asn Asp Pro

210 215 220

Lys Phe Ile Ser Ala His Leu Ile Ser Glu Ser Asp Asn Pro Glu Asp

225 230 235 240

Asp Lys Val Tyr Phe Phe Phe Arg Glu Asn Ala Ile Asp Gly Glu His

245 250 255

Ser Gly Lys Ala Thr His Ala Arg Ile Gly Gln Ile Cys Lys Asn Asp

260 265 270

Phe Gly Gly His Arg Ser Leu Val Asn Lys Trp Thr Thr Phe Leu Lys

275 280 285

Ala Arg Leu Ile Cys Ser Val Pro Gly Pro Asn Gly Ile Asp Thr His

290 295 300

Phe Asp Glu Leu Gln Asp Val Phe Leu Met Asn Phe Lys Asp Pro Lys

305 310 315 320

Asn Pro Val Val Tyr Gly Val Phe Thr Thr Ser Ser Asn Ile Phe Lys

325 330 335

Gly Ser Ala Val Cys Met Tyr Ser Met Ser Asp Val Arg Arg Val Phe

340 345 350

Leu Gly Pro Tyr Ala His Arg Asp Gly Pro Asn Tyr Gln Trp Val Pro

355 360 365

Tyr Gln Gly Arg Val Pro Tyr Pro Arg Pro Gly Thr Cys Pro Ser Lys

370 375 380

Thr Phe Gly Gly Phe Asp Ser Thr Lys Asp Leu Pro Asp Asp Val Ile

385 390 395 400

Thr Phe Ala Arg Ser His Pro Ala Met Tyr Asn Pro Val Phe Pro Met

405 410 415

Asn Asn Arg Pro Ile Val Ile Lys Thr Asp Val Asn Tyr Gln Phe Thr

420 425 430

Gln Ile Val Val Asp Arg Val Asp Ala Glu Asp Gly Gln Tyr Asp Val

435 440 445

Met Phe Ile Gly Thr Asp Val Gly Thr Val Leu Lys Val Val Ser Ile

450 455 460

Pro Lys Glu Thr Trp Tyr Asp Leu Glu Glu Val Leu Leu Glu Glu Met

465 470 475 480

Thr Val Phe Arg Glu Pro Thr Ala Ile Ser Ala Met Glu Leu Ser Thr

485 490 495

Lys Gln Gln Gln Leu Tyr Ile Gly Ser Thr Ala Gly Val Ala Gln Leu

500 505 510

Pro Leu His Arg Cys Asp Ile Tyr Gly Lys Ala Cys Ala Glu Cys Cys

515 520 525

Leu Ala Arg Asp Pro Tyr Cys Ala Trp Asp Gly Ser Ala Cys Ser Arg

530 535 540

Tyr Phe Pro Thr Ala Lys Arg Arg Thr Arg Arg Gln Asp Ile Arg Asn

545 550 555 560

Gly Asp Pro Leu Thr His Cys Ser Asp Leu His His Asp Asn His His

565 570 575

Gly His Ser Pro Glu Glu Arg Ile Ile Tyr Gly Val Glu Asn Ser Ser

580 585 590

Thr Phe Leu Glu Cys Ser Pro Lys Ser Gln Arg Ala Leu Val Tyr Trp

595 600 605

Gln Phe Gln Arg Arg Asn Glu Glu Arg Lys Glu Glu Ile Arg Val Asp

610 615 620

Asp His Ile Ile Arg Thr Asp Gln Gly Leu Leu Leu Arg Ser Leu Gln

625 630 635 640

Gln Lys Asp Ser Gly Asn Tyr Leu Cys His Ala Val Glu His Gly Phe

645 650 655

Ile Gln Thr Leu Leu Lys Val Thr Leu Glu Val Ile Asp Thr Glu His

660 665 670

Leu Glu Glu Leu Leu His Lys Asp Asp Asp Gly Asp Gly Ser Lys Thr

675 680 685

Lys Glu Met Ser Asn Ser Met Thr Pro Ser Gln Lys Val Trp Tyr Arg

690 695 700

Asp Phe Met Gln Leu Ile Asn His Pro Asn Leu Asn Thr Met Asp Glu

705 710 715 720

Phe Cys Glu Gln Val Trp Lys Arg Asp Arg Lys Gln Arg Arg Gln Arg

725 730 735

Pro Gly His Thr Pro Gly Asn Ser Asn Lys Trp Lys His Leu Gln Glu

740 745 750

Asn Lys Lys Gly Arg Asn Arg Arg Thr His Glu Phe Glu Arg Ala Pro

755 760 765

Arg Ser Val

770

Claims

A kind of 1. bone protective protein molecule Semaphorine3A, it is characterised in that its amino acid sequence such as SEQ ID NO.1 It is shown.
2. the bone protective protein molecule Semaphorine3A described in claim 1 is preparing treatment/prevention rheumatoid joint Application in scorching medicine.
3. application as claimed in claim 2, it is characterised in that described medicine is thin to anti-inflammatory M2 types for regulation macrophage The medicine of born of the same parents' polarization.
4. application as claimed in claim 2, it is characterised in that described medicine activates to suppress endothelial cell migration and signal Medicine.
5. application as claimed in claim 2, it is characterised in that the medicine that described medicine is attacked for suppression synovioblast Thing.
6. application as claimed in claim 2, it is characterised in that described medicine is secreted to suppress synovioblast IL-6 And promote the medicine of its apoptosis.
7. application as claimed in claim 2, it is characterised in that described medicine breaks up to suppress osteoclast, the medicine of function Thing.
8. the recombined adhenovirus containing the bone protective protein molecule Semaphorine3A described in claim 1.
9. application of the recombined adhenovirus in the medicine for preparing treatment/prevention rheumatoid arthritis described in claim 8.
10. application as claimed in claim 9, it is characterised in that described medicine is treatment/prevention Serum-induced type rheumatoid The arthritic medicine of property.