CN105708861A - Novel application of exosome of mesenchymal stem cell source - Google Patents
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Abstract
The invention discloses application of an exosome of a mesenchymal stem cell source in preparation of a drug for treating ankylosing spondylitis. The exosome of the mesenchymal stem cell source has the inhibition effect on both chronic inflammations and pathological osteogenesis and has the advantages of being low in side effect and cost and more durable in effect.
Description
Technical field
The present invention relates to the outer purposes secreting body of source for mesenchymal stem cells, particularly relate to the outer of source for mesenchymal stem cells and secrete body application in the medicine of preparation treatment ankylosing spondylitis.
Background technology
It is a kind of common autoimmune disease that ankylosing spondylitis (is called for short AS), its prevalence is higher, being about 0.2～0.54% in China (can be referring to: RongJ, JieruoG.SpondyloarthritisinChina.Currentopinioninrheumat ology.2013；25 (4): 460-7.).AS is apt to occur in young man, often involves axial bone joint, and Major Clinical and pathological characters are chronic progressive external inflammation (can be referring to: BraunJ, SieperJ.Ankylosingspondylitis.Lancet.2007 with pathologic skeletonization；369 (9570): 1379-90.).But, the pathogenesis of AS is still not clear at present, and its two big relation between pathological characters inflammation and skeletonization is also indefinite, and pathogenesis is unclear causes that current AS treatment means limitation is big, effectiveness is low.Along with the progress of disease, AS patient will lose life and work capacity gradually, bring heavy mental pressure and financial burden to patient, and the development of society is caused tremendous influence and obstruction.Therefore, explore and can alleviate AS inflammation in the patient the Therapeutic Method of pathologic skeletonization can be stoped again particularly important.
The treatment means of current AS includes non-drug therapy, Drug therapy and operative treatment three kinds [15,16].Non-drug therapy mainly includes patient education, reconditioning, naturopathy etc..Drug therapy includes nonsteroidal antiinflammatory drug (such as diclofenac sodium, celecoxib, loxoprofen etc.), alleviates state of an illness antirheumatic (such as sulfasalazine, methotrexate etc.), glucocorticoid (such as corticosteroid etc.), analgesic (such as ibuprofen etc.) and biological preparation (such as tumor necrosis factor antagonists etc.) etc..Operative treatment includes replacement of total hip, spinal osteotomy orthopaedy etc..
In the treatment of AS, non-drug therapy is mainly used in auxiliary treatment, it is impossible to change the final Prognosis of AS patient.Drug treatment, though the pain symptom of nonsteroidal antiinflammatory drug, analgesic energy reduction of patient and level of inflammation, but the pathologic bone formation of patient cannot be improved, also having the side effect such as gastrointestinal reaction, cardiovascular response (can be referring to: Miceli-RichardC, DougadosM.NSAIDsinankylosingspondylitis.Clinicalandexper imentalrheumatology.2002；20 (6Suppl28): S65-6.)；Alleviate state of an illness antirheumatic and be only capable of the periphery arthritic symptom for the treatment of AS patient, cannot improve axial bone inflammation and pathologic skeletonization (can be referring to: vanderHeijdeD, SieperJ, MaksymowychWP, DougadosM, Burgos-VargasR, LandeweR, etal.2010UpdateoftheinternationalASASrecommendationsfort heuseofanti-TNFagentsinpatientswithaxialspondyloarthriti s.Annalsoftherheumaticdiseases.2011；70 (6): 905-8.)；Corticosteroid is simply possible to use in local injection alleviating pain symptom, for systemic inflammatorome level and pathologic ossification also without effect；Biological preparation is treated, particularly tumor necrosis factor antagonists treatment, it it is one of the main Therapeutic Method of current AS, but studies have found that though it can suppress AS inflammation in the patient, but cannot stop and even also can accelerate rachiopathy rationality bone formation, in addition biological preparation medical expense is expensive, some patients is difficult to undertake, also there is infection, tumor, the side effect such as hepatic and renal function is abnormal (can be referring to: vanderHeijdeD, LandeweR, EinsteinS, OryP, VosseD, NiL, etal.Radiographicprogressionofankylosingspondylitisafter uptotwoyearsoftreatmentwithetanercept.Arthritisandrheuma tism.2008；58 (5): 1324-31.).Operative treatment aspect, is mainly used for the treatments such as AS patient spine deformity in late period, hip lesion, it is impossible to reach to cure the target of AS.
Mescenchymal stem cell (mesenchymalstemcells, MSCs) it is derived from a mesoblastic non-hemopoietic system pluripotent stem cell of class, there is now have proven to MSCs except self renewal and multi-lineage potential, also there is very strong antiinflammatory and suppress the ability of panimmunity cell, and can inducing peripheral immunologic tolerance.Research shows that MSCs passes through secretion panimmunity regulatory factor, as IFN-γ, PGE2 etc. (can be referring to: PolchertD, SobinskyJ, DouglasG, KiddM, MoadsiriA, ReinaE, etal.IFN-gammaactivationofmesenchymalstemcellsfortreatme ntandpreventionofgraftversushostdisease.Europeanjournalo fimmunology.2008；38 (6): 1745-55. and SpaggiariGM, AbdelrazikH, BecchettiF, MorettaL.MSCsinhibitmonocyte-derivedDCmaturationandfunct ionbyselectivelyinterferingwiththegenerationofimmatureDC s:centralroleofMSC-derivedprostaglandinE2.Blood.2009；113 (26): 6576-83.), thus suppressing the function of immunocyte (such as T cell, B cell, NK cell, antigen-presenting cell etc.) (can be referring to: CorcioneA, BenvenutoF, FerrettiE, GiuntiD, CappielloV, CazzantiF, etal.HumanmesenchymalstemcellsmodulateB-cellfunctions.Bl ood.2006；107 (1): 367-72. and DiNicolaM, Carlo-StellaC, MagniM, MilanesiM, LongoniPD, MatteucciP, etal.HumanbonemarrowstromalcellssuppressT-lymphocyteprol iferationinducedbycellularornonspecificmitogenicstimuli. Blood.2002；99 (10): 3838-43.).
It is a kind of by the diameter of emiocytosis about 30～100nm for secreting outward body, density range film microcapsule bubble between 1.13～1.19g/ml.Secrete outward the multiple protein similar to source cell of body portability, mRNA, miRNA, the process such as immunomodulating, intercellular communication, cell migration, angiogenesis that participates in (can be referring to: Yanez-MoM, SiljanderPR, AndreuZ, ZavecAB, BorrasFE, BuzasEI, etal.Biologicalpropertiesofextracellularvesiclesandtheir physiologicalfunctions.Journalofextracellularvesicles.20 15；4:27066.).Lai etc. find that the outer body of secreting in MSCs source can reduce myocardial ischemia reperfusion injury, and confirm that the outer miRNA secreting body can promote angiogenesis, the new direction being likely to become treatment cardiovascular disease because secreting body in addition (can be referring to: LaiRC, ArslanF, LeeMM, SzeNS, ChooA, ChenTS, etal.ExosomesecretedbyMSCreducesmyocardialischemia/reper fusioninjury.Stemcellresearch.2010；4 (3): 214-22.).Xin etc. find that the outer body of secreting in MSCs source is by transfer miR-133b to neurocyte, can promote that the growth of neural axon (can be referring to: XinH, LiY, BullerB, KatakowskiM, ZhangY, WangX, etal.Exosome-mediatedtransferofmiR-133bfrommultipotentme senchymalstromalcellstoneuralcellscontributestoneuriteou tgrowth.Stemcells.2012；30 (7): 1556-64.).Filipazzi etc. find that the outer body of secreting in tumor cell source can pass through NK cell-stimulating receptor NKG2D (natural-killergroup2, memberD) cytotoxicity of T cell and NK cell is suppressed, thus the immune system affecting host (can be referring to: FilipazziP, BurdekM, VillaA, RivoltiniL, HuberV.Recentadvancesontheroleoftumorexosomesinimmunosup pressionanddiseaseprogression.Seminarsincancerbiology.20 12；22 (4): 342-9.).
Utilize that MSCs originates outer secrete body treatment disease in, there are two patents, KiangLS etc. adopt the outer pulmonary fibrosis secreting body treatment mice in MSCs source, mice survival rate can be significantly improved, alleviating pulmonary fibrosis pulmonary lesion that bleomycin (bleomycin, BLM) causes (can be referring to: KiangLS, WeiYMS, ShengCT, ChaiLR.USEOFEXOSOMESTOPROMOTEORENHANCEHAIRGROWTHpatentUS 2015024011.2015-01-22.).The outer body of secreting that MSCs is originated by Liu Ming etc. is used for promoting repair in trauma and hair growth, obtaining good result (can be referring to: Liu Ming, body application patentCN104666344A.2015-06-03. in the pharmaceutical preparation of preparation treatment pulmonary fibrosis is secreted) outside Xu Jun, inventors mescenchymal stem cell.
But up to the present, at home and abroad there is no the outer report secreting body treatment AS utilizing MSCs to originate and patent.
Summary of the invention
The weak point that it is an object of the invention to overcome prior art to exist and provide the outer of source for mesenchymal stem cells and secrete body application in the medicine of preparation treatment ankylosing spondylitis.Chronic inflammatory disease and pathologic skeletonization are all had inhibitory action by the outer body of secreting in MSCs source, and have the advantage that side effect is low, cost is low, effect is more lasting.
As the further improvement to technique scheme, described mescenchymal stem cell is mesenchymal stem cells MSCs.
As the further improvement to technique scheme, described mesenchymal stem cells MSCs is prepared by following methods: separation and Extraction mescenchymal stem cell from bone marrow, then obtains through original cuiture and Secondary Culture.
As the further improvement to technique scheme, the extracting method secreting body outside described is: cultivate enough mesenchymal stem cells MSCs, and 48h before secreting body outside extraction stem cell uses PBS cell, changes serum-free medium, continues to cultivate 48h；
Collecting serum-free medium, 300 × g is centrifuged 10min, takes supernatant；2000 × g is centrifuged 10min, takes supernatant；10000 × g is centrifuged 30min, takes supernatant；100000 × g is centrifuged 70min, takes precipitation；The resuspended precipitation of PBS, 100000 × g is centrifuged 70min, collects precipitate and obtain and described secretes body outward.
As the further improvement to technique scheme, the outer concentration secreting body in described medicine is 1.3 × 109～3.5 × 109Grain/milliliter.
As the further improvement to technique scheme, described medicine also comprises pharmaceutically acceptable carrier." pharmaceutically acceptable carrier " include the volume of any and all physical compatibilities, disperse medium, coating material, antibacterium and antifungal, etc. blend absorption delayer etc..The example of pharmaceutically acceptable carrier includes one or more in water, saline, phosphate-buffered saline, dextran, glycerol, ethanol etc. and their combination.In many cases it is preferred to be that isotonic agent such as sugar, polyhydric alcohol or sodium chloride are included in the composition.Pharmaceutically acceptable carrier also can comprise the auxiliary substance of a small amount of shelf life that can improve antibody or antibody moiety or effectiveness, such as wetting agent or emulsifying agent, preservative or buffer.
The medicine of the present invention can be various ways.These forms include such as liquid, semisolid and solid dosage forms, such as liquid solution agent (such as injectable and can infusion solution), dispersant or suspensoid, tablet, pill, powder, liposome and suppository.Preferred form depends on predetermined administering mode and treatment use.Preferably, described pharmaceutical dosage form is injection or lyophilized formulations.Typically preferred medicine is injectable or can infusion solution form.Preferred administering mode is parenteral (such as intravenous, subcutaneous, intraperitoneal, intramuscular) administration.In a preferred embodiment, the medicine of the present invention is given by intravenous infusion or injection.In another preferred embodiment, the medicine of the present invention is given by intramuscular or subcutaneous injection.Also can Supplementary active compounds be incorporated in medicine, in certain embodiments, preparation and/or give altogether altogether together with medicine of the present invention can be used for treating the other healing potion of AS with one or more.Such as, can in conjunction with preparation and/or give altogether altogether together with the medicine of other target by the medicine of the present invention and one or more.
We find in the research of early stage, and the outer body of secreting in MSCs source can substantially suppress propagation and the CTL cytotoxicity of T cell, and induction Th1/Th2 balance tilts to Th2, and cause Treg generates.Body is secreted, it has been found that it has the effect that substantially alleviation AS patient's inflammation and spinal column ossify by AS patient being carried out the outer of intravenous injection MSCs source.Proving first, what utilize that MSCs originates outer secretes body, it is possible to reach the effect for the treatment of AS chronic inflammatory disease in the patient and the big pathologic problems of pathologic skeletonization two simultaneously.This is a kind of brand-new, effective AS Therapeutic Method.
Compared with existing treatment technology, present invention have the advantage that
1) chronic inflammatory disease and pathologic skeletonization are all had inhibitory action by the outer body of secreting in MSCs source.Proving first, what utilize that MSCs originates outer secretes body, it is possible to reach the effect for the treatment of AS chronic inflammatory disease in the patient and the big pathologic problems of pathologic skeletonization two simultaneously.Relative to nonsteroidal antiinflammatory drug, analgesic, alleviation state of an illness antirheumatic, tumor necrosis factor antagonists etc. can only the medicine of amelioration of inflammation, the outer body of secreting in MSCs source has more fully action effect.
2) low side effect.AS medicine previously is only capable of delaying to a certain extent disease progression, it is impossible to finally stops the course of disease, there is also many untoward reaction and side effect simultaneously.The outer body immunogenicity of secreting in MSCs source is low, treats AS patient, does not find the unfavorable conditions such as infection, gastrointestinal reaction, cardiovascular response, tumor generation, hepatic and renal function exception, points out its side effect less.
3) low cost.The outer body of secreting in MSCs source constantly can be secreted by MSCs, relative to biological preparation such as tumor necrosis factor antagonists, less costly.
4) effect is more lasting.The outer body of secreting in MSCs source has the biological characteristics similar to MSCs, simultaneously because its diameter only has about 40～100nm, is not easily retained by organs such as liver, lung, spleens, is easier to circulation in vivo, it is possible to play more lasting therapeutic effect.
Accompanying drawing explanation
Fig. 1 is for secreting outward surface Protein Detection figure；
Fig. 2 is treatment flow chart；
Fig. 3 shows that ankylosing spondylitis companion psoriatic uses the outer facial symptom secreted before body treatment and after treatment, and the face treating up till now patient covers with erythra, and with pain, limitation of activity, after treatment, these symptoms obtain notable alleviation；
Fig. 4 shows that the sacroiliac joint focus of ankylosing spondylitis companion psoriatic is improved；
Fig. 5 shows that the laboratory checking index of ankylosing spondylitis companion psoriatic significantly reduces with disease activity index.
Detailed description of the invention
It is all that the chronic inflammatory disease for AS is treated that existing therapeutic scheme (includes all Drug therapys as above), and attempts by suppressing inflammation to delay pathologic skeletonization.Though these methods can amelioration of inflammation to a certain extent, but pathologic skeletonization cannot be suppressed, the shortcoming such as simultaneously there is also somewhat expensive, patient cannot tolerate, side effect is many.
The present invention is different from existing treatment means, has not yet to see and treats the case of AS with the outer body of secreting in MSCs source, and this Therapeutic Method is possible not only to significantly inhibit inflammation, also can suppress pathologic skeletonization, simultaneously without obvious toxic-side effects.
For better illustrating the object, technical solutions and advantages of the present invention, below in conjunction with specific embodiment, the invention will be further described.
Embodiment
The outer body of secreting in MSCs source is for treating AS chronic inflammatory disease and pathologic skeletonization
(1) volunteer's screening
Choosing 20～30 one full year of life volunteers, project on request checks UP, and after qualified, 30ml bone marrow is extracted in reservation.
(2) separation, identification of M SCs
From the bone marrow obtained, separation obtains MSCs, carries out cellular morphology and phenotypic evaluation, and does microbiologic inhibition tests.
(3) MSCs amplification, secretes outward body and extracts preparation
By qualified MSCs amplification cultivation, according to 4 × 107Cell concentration cultivate enough MSCs, extract outer secrete body before 48h, remove the cell culture fluid containing serum, PBS washes three times, adds serum-free medium, cultivation 48h.
(4) secrete body outside to extract and detection
Collecting serum-free medium, 300 × g is centrifuged 10min, takes supernatant；2000 × g is centrifuged 10min, takes supernatant；10000 × g is centrifuged 30min, takes supernatant；100000 × g is centrifuged 70min, takes precipitation；The resuspended precipitation of PBS, 100000 × g is centrifuged 70min, collects outside precipitation is and secretes body.Take and secrete body sample outside part, carry out Electronic Speculum, surface markers, microbiologic inhibition tests.Secrete outward surface Protein Detection figure as shown in Figure 1.
(5) AS patient enters group
All enter organize case diagnosis all meet ASAS work out mesinae ankylosing spondylitis diagnostic criteria (the MRI positive+at least 1 SpA sign or the HLA-B27 positive+at least two SpA signs), BASDAI >=4, spinal pain VAS scoring >=4, between age requirement 18～40 years old, course of disease time requirement is made a definite diagnosis less than 5 years from first time, chronic low back pain shape at least 3 months, all patient's courses of disease did not accept biological preparation treatment, and tumor markers inspection was all negative.All enter to organize patient and all sign Informed Consent Form, accept that MSCs source is outer secretes body infusion of therapeutic.
(6) vena systemica injection is secreted outside
AS patient is divided into three groups, secretes body treatment group, tumor necrosis factor antagonists (benefit match is general) group and matched group including MSCs source is outer.The outer body treatment group of secreting in MSCs source gave in the 1st～8 week secreting body treatment outside venoclysis, according to 1.3～3.5 × 1010Secreting outward body particle total amount is that a unit (can be referring to: KordelasL, RebmannV, LudwigAK, RadtkeS, RuesingJ, DoeppnerTR, etal.MSC-derivedexosomes:anoveltooltotreattherapy-refrac torygraft-versus-hostdisease.Leukemia.2014；28 (4): 970-3.), intravenous injection is carried out, 2 times weekly, totally 16 times；Tumor necrosis factor antagonists group gave general 25mg/ the subcutaneous injection treatment of benefit match in the 1st～8 week, 2 times weekly, totally 16 times；Matched group gave placebo venoclysis treatment in the 1st～8 week, 2 times weekly, totally 16 times.
(7) therapeutic effect assessment
The indexs such as each group of AS patients serum, detection ESR, CRP, TNF-α, IL-6, IL-17, IL-23 are collected, to assess its current internal level of inflammation respectively at 0,1,2,3,4,5,6,7,8,16,24,32,40,48 weeks.MRI checks, observes local spinal column, sacroiliac joint pathologic skeletonization situation.
(8) interpretation of result
Data in table 1～table 8 show: compare matched group, the outer body treatment group of secreting in MSCs source all can suppress AS level of inflammation in the patient (ESR, CRP, TNF-α, IL-6, IL-17, IL-23 all significantly reduce) with tumor necrosis factor antagonists group, and the outer body treatment group of secreting in MSCs source suppresses inflammation energy force rate tumor necrosis factor antagonists group higher.
Comparing matched group, the outer body treatment group of secreting in MSCs source significantly inhibits AS patient spine pathologic bone formation, and tumor necrosis factor antagonists group does not affect pathologic bone formation progress, and (pathologic ossification is marked: 0 point: normal；1 point: vertebral rim has slight hypertrophy；2 points: the obvious hyperosteogeny of vertebral rim is formed；3 points: side vertebral rim forms bone bridge；4 points: bilateral vertebral rim forms bone bridge, intervertebral fusion).Treatment flow chart is as shown in Figure 2.Fig. 3～Fig. 5 is that the treatment situation map of a wherein ankylosing spondylitis companion psoriatic: Fig. 3 shows that this ankylosing spondylitis companion psoriatic uses the outer facial symptom secreted before body treatment and after treatment, the face treating up till now patient covers with erythra, and with pain, limitation of activity, after treatment, these symptoms obtain notable alleviation；Fig. 4 shows that the sacroiliac joint focus of this ankylosing spondylitis companion psoriatic is improved；Fig. 5 shows that the laboratory checking index of this ankylosing spondylitis companion psoriatic significantly reduces with disease activity index.Table 1～table 8 is as follows.
Table 1: treatment number of cases and effective percentage
| Number of cases | Effective percentage (%) | |
| Add up to | 89 | 68.5 |
| Male AS | 57 | 70.1 |
| Women AS | 32 | 65.6 |
| Early stage AS | 35 | 82.8 |
| Middle and advanced stage AS | 54 | 59.2 |
Note: therapeutically effective finger MSCs source outer secrete body treatment during, Bath ankylosing spondylitis disease activity index assessment (BASDAI) or the ankylosing spondylitis state of an illness mobility assessment (ASDAS) reduce by more than 2.
Table 2: erythrocyte sedimentation rate (ESR) level (mm/h) compares
Table 3:C reactive protein (CRP) level (ng/ml) compares
| MSCs source is outer secretes body treatment group | Tumor necrosis factor antagonists group | Matched group | |
| 0 week | 63.4±2.1 | 60.9±3.8 | 62.0±4.1 |
| 1 week | 61.2±3.9 | 59.4±5.2 | 64.1±1.3 |
| 2 weeks | 57.8±1.7 | 56.6±4.8 | 56.4±4.8 |
| 3 weeks | 45.2±4.7 | 56.1±4.0 | 62.2±2.7 |
| 4 weeks | 43.3±2.9 | 44.3±2.8 | 59.7±2.1 |
| 5 weeks | 32.5±1.8 | 37.5±6.7 | 59.2±7.9 |
| 6 weeks | 20.9±4.9 | 33.8±1.1 | 62.0±3.4 |
| 7 weeks | 17.6±4.5 | 28.4±3.3 | 57.4±0.6 |
| 8 weeks | 7.6±4.5 | 16.4±3.8 | 56.3±4.6 |
| 16 weeks | 9.2±2.2 | 20.2±1.9 | 58.6±2.8 |
| 24 weeks | 17.7±1.6 | 28.4±2.6 | 45.5±5.5 |
| 32 weeks | 27.7±5.3 | 45.3±5.2 | 62.3±1.5 |
| 40 weeks | 39.0±4.7 | 55.4±1.4 | 57.8±4.1 |
| 48 weeks | 44.4±2.5 | 65.7±2.9 | 67.5±2.6 |
Table 4:TNF-alpha levels (pg/ml) compares
| MSCs source is outer secretes body treatment group | Tumor necrosis factor antagonists group | Matched group | |
| 0 week | 475.3±15.6 | 477.5±23.4 | 439.3±22.5 |
| 1 week | 453.1±25.2 | 481.6±15.5 | 401.6±32.0 |
| 2 weeks | 377.8±18.7 | 356.6±24.8 | 456.4±48.8 |
| 3 weeks | 245.2±45.7 | 296.1±47.0 | 382.2±24.7 |
| 4 weeks | 213.3±26.9 | 284.3±28.8 | 349.7±23.1 |
| 5 weeks | 196.8±11.9 | 247.5±12.2 | 380.3±22.3 |
| 6 weeks | 133.6±6.3 | 210.9±14.8 | 402.0±14.4 |
| 7 weeks | 109.3±13.8 | 185.3±13.9 | 369.2±15.8 |
| 8 weeks | 89.3±11.4 | 178.5±22.5 | 392.3±20.1 |
| 16 weeks | 258.2±8.9 | 333.7±32.0 | 382.1±35.9 |
| 24 weeks | 299.4±18.3 | 404.8±21.6 | 389.3±19.4 |
| 32 weeks | 259.7±43.6 | 463.8±34.1 | 467.3±52.0 |
| 40 weeks | 312.5±17.5 | 427.9±22.1 | 484.3±26.7 |
| 48 weeks | 418.4±25.9 | 524.7±28.0 | 419.8±29.6 |
Table 5:IL-6 level (pg/ml) compares
Table 6:IL-17 level (pg/ml) compares
| MSCs source is outer secretes body treatment group | Tumor necrosis factor antagonists group | Matched group | |
| 0 week | 416.4±13.7 | 432.5±19.5 | 418.3±12.7 |
| 1 week | 374.2±24.9 | 399.7±37.8 | 455.4±35.4 |
| 2 weeks | 313.4±22.4 | 384.5±18.5 | 364.7±13.1 |
| 3 weeks | 293.5±13.9 | 346.5±22.3 | 484.2±12.4 |
| 4 weeks | 237.6±6.7 | 311.7±15.9 | 402.0±14.4 |
| 5 weeks | 207.2±16.5 | 264.7±29.0 | 395.3±32.7 |
| 6 weeks | 183.9±29.6 | 199.2±36.3 | 378.4±9.7 |
| 7 weeks | 75.7±26.2 | 148.6±32.8 | 373.7±13.6 |
| 8 weeks | 54.3±11.4 | 128.1±8.1 | 452.5±24.6 |
| 16 weeks | 194.6±14.8 | 223.5±12.1 | 389.2±34.4 |
| 24 weeks | 239.7±23.6 | 289.7±19.5 | 345.6±29.3 |
| 32 weeks | 229.3±9.8 | 333.6±39.7 | 395.5±22.1 |
| 40 weeks | 334.9±26.8 | 368.1±12.4 | 372.6±17.4 |
| 48 weeks | 388.1±14.5 | 445.1±14.3 | 415.7±24.6 |
Table 7:IL-23 level (pg/ml) compares
Table 8: the scoring of rachiopathy rationality bone formation is compared
| MSCs source is outer secretes body treatment group | Tumor necrosis factor antagonists group | Matched group | |
| 0 week | 4.2±1.9 | 4.7±2.8 | 4.4±1.4 |
| 1 week | 4.6±1.5 | 4.2±1.9 | 4.3±0.6 |
| 2 weeks | 4.7±2.0 | 4.7±2.3 | 5.3±1.5 |
| 3 weeks | 5.0±0.4 | 5.1±2.9 | 4.6±1.4 |
| 4 weeks | 5.4±1.5 | 5.2±0.7 | 4.6±0.2 |
| 5 weeks | 4.3±1.1 | 4.2±1.9 | 5.5±1.7 |
| 6 weeks | 4.0±0.7 | 4.6±1.2 | 4.5±1.7 |
| 7 weeks | 4.1±1.6 | 5.3±1.5 | 5.4±0.6 |
| 8 weeks | 3.8±1.7 | 4.4±1.6 | 4.5±1.6 |
| 16 weeks | 4.0±0.2 | 4.5±0.7 | 4.4±1.5 |
| 24 weeks | 3.5±0.8 | 3.9±1.3 | 4.9±1.4 |
| 32 weeks | 2.8±1.3 | 4.8±1.1 | 4.7±1.7 |
| 40 weeks | 2.2±0.3 | 4.8±2.1 | 5.8±2.8 |
| 48 weeks | 2.9±0.2 | 5.3±3.6 | 5.3±1.6 |
Finally be should be noted that; above example is only in order to illustrate technical scheme but not limiting the scope of the invention; although the present invention being explained in detail with reference to preferred embodiment; it will be understood by those within the art that; technical scheme can be modified or equivalent replacement, without deviating from the spirit and scope of technical solution of the present invention.
Claims (8)
1. the outer of source for mesenchymal stem cells secretes body application in the medicine of preparation treatment ankylosing spondylitis.
2. application according to claim 1, it is characterised in that: described mescenchymal stem cell is mesenchymal stem cells MSCs.
3. application according to claim 2, it is characterised in that: described mesenchymal stem cells MSCs is prepared by following methods: separation and Extraction mescenchymal stem cell from bone marrow, then obtains through original cuiture and Secondary Culture.
4. application according to claim 3, it is characterised in that: the extracting method secreting body outside described is: cultivate enough mesenchymal stem cells MSCs, 48h before secreting body outside extraction stem cell, uses PBS cell, changes serum-free medium, continues to cultivate 48h；
Collecting serum-free medium, 300 × g is centrifuged 10min, takes supernatant；2000 × g is centrifuged 10min, takes supernatant；10000 × g is centrifuged 30min, takes supernatant；100000 × g is centrifuged 70min, takes precipitation；The resuspended precipitation of PBS, 100000 × g is centrifuged 70min, collects precipitate and obtain and described secretes body outward.
5. application according to claim 1, it is characterised in that: the outer concentration secreting body in described medicine is 1.3 × 109～3.5 × 109Grain/milliliter.
6. application according to claim 1, it is characterised in that: described medicine also comprises pharmaceutically acceptable carrier.
7. application according to claim 1, it is characterised in that: the dosage form of described medicine is injection or lyophilized formulations.
8. application according to claim 1, it is characterised in that: described medicine is applied to human body by the mode of venoclysis.
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