CN106119375A

CN106119375A - The medicine of a kind of regulating and controlling effect to articular cartilage degeneration and verification method

Info

Publication number: CN106119375A
Application number: CN201610525458.8A
Authority: CN
Inventors: 斯海波; 曾羿; 沈彬; 裴福兴; 杨静; 周宗科; 康鹏德
Original assignee: 沈彬
Current assignee: West China Hospital of Sichuan University
Priority date: 2016-07-05
Filing date: 2016-07-05
Publication date: 2016-11-16
Anticipated expiration: 2036-07-05
Also published as: CN106119375B

Abstract

The invention discloses a kind of medicine to articular cartilage degeneration regulating and controlling effect and verification method, this is miRNA 140 to the medicine of articular cartilage degeneration regulating and controlling effect, is verified the regulating and controlling effect of rat knee joints cartilage degeneration by the regulating and controlling effect in osteoarthritis chondrocytes, the regulating and controlling effect in osteoarthritic joint liquid, joint cavity injection miRNA 140.The present invention separates and cultivates people's normally and in various degree OA chondrocyte in vitro, its expression increases the weight of to gradually decrease with OA degree, severe OA chondrocyte is expressed minimum, in OA joint fluid, miRNA 140 expresses compared with normal group and significantly reduces, joint cavity injection miRNA 140agomir can significantly slow down Collagen II PD in articular cartilage tissue, the most substantially suppression MMP13 and ADAMTS 5 protein expression, plays obvious retarding action to cartilage of rats regression.

Description

The medicine of a kind of regulating and controlling effect to articular cartilage degeneration and verification method

Technical field

The invention belongs to medicine technology field, particularly relate to the medicine of a kind of regulating and controlling effect to articular cartilage degeneration and test Card method.

Background technology

Osteoarthritis (OA) is a kind of common disease with Progressive symmetric erythrokeratodermia articular cartilage degeneration as principal character, clinical manifestation master Arthralgia, deformity and the moving obstacle etc. that Progressive symmetric erythrokeratodermia to be increases the weight of.OA sickness rate and teratogenesis, disability rate height, but its morbidity machine System is still not clear at present, also lacks effective treatment means.Recent study find microRNA (miRNA) can by with target base Because of (mRNA) combine cause the latter to degrade or Translational repression, regulate and control the mrna expression of mammal about 1/3, cell proliferation, The aspects such as differentiation, aging and apoptosis play an important role.MiRNA-140 has cartilage specificity, steady in people's normal articular cartilage Fixed expression, normal steady state and function to articular cartilage play important regulating and controlling effect.At present about miRNA-140 in OA process In expression rule remain dispute, but generally believe that articular cartilage is played important protective effect in OA occurs evolution by it. Therefore, miRNA-140 can be as a point of penetration for the treatment of OA, but presently relevant research mostly is chondrocytes in vitro cell experiment or base Because knocking out/transgenic animal experiment, concrete grammar and the approach directive function of further investigation miRNA-140 treatment OA are had by this Limit.

Summary of the invention

It is an object of the invention to provide medicine and the verification method of a kind of regulating and controlling effect to articular cartilage degeneration, it is intended to Solve the concrete grammar of current miRNA-140 treatment OA and the problem that approach directive function is limited.

The present invention is achieved in that a kind of medicine to articular cartilage degeneration regulating and controlling effect, and this is to articular cartilage degeneration The medicine of regulating and controlling effect be miRNA-140.

Another object of the present invention is to provide a kind of and verify that miRNA-140 regulation and control in osteoarthritis chondrocytes are made By method it is:

First verify that miRNA-140 is normal people and expression rule in osteoarthritis (OA) chondrocyte；

Secondly the optimum condition of miRNA-140 transfected with human chondrocyte is screened；

Finally verify miRNA-140 up-regulated or lower the regulating and controlling effect to people's OA chondrocyte and mechanism.

Further, verify that miRNA-140 expression rule in osteoarthritis chondrocytes and regulating and controlling effect method are concrete For:

(1) according to Kellgren and Lawrence (KL) OA iconography grade scale, collector normal (KL 0 grade) and Gently (KL 1-2 level), in (KL 3 grades), severe (KL 4 grades) OA fresh cartilage specimen, in-vitro separation, cultured cartilage cell are gone forward side by side Row is identified；

(2) use fluorescence quantitative PCR method detection normal and miRNA-140 expression in OA chondrocyte, analyze its table Reach Changing Pattern；

(3) miRNA-140mimic using variable concentrations (0,25,50,100 and 200nM) transfects chondrocyte, is turning After dye different time points (24,48 and 72h) by PCR and Western blotting method detection cell Collagen II and MMP13 gene and protein expression level, the screening miRNA-140mimic transfection optium concentration of chondrocyte and effect detection are The good time；

(4) miRNA-140mimic or miRNA-140inhibitor using optium concentration transfects normal and OA cartilage is thin Born of the same parents, detect cell Collagen II, MMP13, ADAMTS-5, Sox9 and Runx2 gene and protein expression water at Best Times Flat, compare after miRNA-140 raises or lowers and respectively organize said gene and protein expression difference in chondrocyte, analyze miRNA- The regulating and controlling effect of 140 pairs of OA chondrocytes and molecular mechanism.

Another object of the present invention is to provide a kind of and verify miRNA-140 regulating and controlling effect in osteoarthritic joint liquid Method is:

Detection people is normal and the expression rule of miRNA-140 in OA joint fluid, and verifies miRNA-140 expression and OA The dependency of the order of severity.

Further, checking miRNA-140 in the regulating and controlling effect method in osteoarthritic joint liquid particularly as follows:

According to KL imaging diagnosis standard, collect normal and light, in, each 10 examples of severe OA patient's Knee Joint Fluid specimen；

Use the expression of miRNA-140 in quantitative real-time PCR each group of joint fluid of detection, use single factor test ANOVA variance analysis is than compared with normal and miRNA-140 differential expression in OA patient articular liquid in various degree；

By miRNA-140 relative expression quantity and KL classification in Spearman rank correlation rank test checking joint fluid it Between dependency.

Another object of the present invention is to provide a kind of checking joint cavity injection miRNA-140 that rat knee joints cartilage is moved back The regulating and controlling effect method become into: by building rat knee joints OA model identifying.

Further, the checking joint cavity injection miRNA-140 regulating and controlling effect concrete grammar to rat knee joints cartilage degeneration For:

With 3 monthly age fully-developed SPF level SD rats as object, take rat 12, wherein 3 be only used as normal control, its Yu and 9 set up rat knee joints OA model only with the method for excision medial meniscus and medial collateral ligament, after surgery the 4th, 8 And respectively put to death 3 in 12 weeks, marked to OA model by gross examination of skeletal muscle, tissue section strain (HE+ toluidine blue) and Mankin ' s Identify；

Take rat 42, wherein 6 be only used as normal control, be left intact；Remaining 36 be randomly divided into experimental group and Matched group, often organizes each 18, when modeling postoperative 1 week by joint puncture to experimental group rat knee joints intracavitary administration 5nmol/100 μ l miRNA-140agomir, the miRNA-140control of matched group injection same dose, observe two groups of rats With or without complication after joint cavity injection；

Within postoperative 4th, 8 and 12 weeks, 6 rats of each execution are often organized, by gross examination of skeletal muscle, histochemical stain (HE+ in modeling Toluidine blue) and Mankin ' s methods of marking compare two groups of rat knee joints cartilage degeneration degree；

By immunohistochemical staining (Collagen II, MMP13 and ADAMTS-5) and protein expression average optical Value (MOD) compares correlative protein expression situation in two groups of rat knee joints cartilaginous tissues, verifies joint cavity injection miRNA-140 pair The regulating and controlling effect of rat knee joints cartilage degeneration and molecular mechanism.

Present invention success separates and cultivates people's normally and in various degree OA chondrocyte in vitro, and miRNA-140 is normally Stable expression in chondrocyte, its expression increases the weight of to gradually decrease with OA degree, expresses minimum, group in severe OA chondrocyte Between difference statistically significant (F=56.32, p < 0.05).

Increasing with miRNA-140mimic concentration and transfection time extends, Collagen II gene and protein expression are gradually Increase, and MMP13 gene and protein expression gradually decrease.When concentration is 50nM transfection 72h, Collagen II and MMP13 base Cause and protein expression level the most statistically significant compared with matched group difference (p < 0.05).

50nM miRNA-140mimic transfection chondrocyte after 72h detection, compared with matched group, Collagen II and Sox9 gene expression increases, differential expression statistically significant (p < 0.05) in normal and light, moderate OA chondrocyte；And MMP13, ADAMTS-5 and Runx2 gene expression reduce, wherein MMP13 in normal and slight OA chondrocyte, Runx2 in Differential expression statistically significant (p < 0.05) in degree OA chondrocyte.In severe OA chondrocyte, said gene is expressed relatively Matched group no difference of science of statistics (p > 0.05).After miRNA-140inhibitor transfection, said gene expression variation tendency is contrary.

72h detection after 50nM miRNA-140mimic transfection, compared with matched group, in normal and OA chondrocyte at different levels Collagen II protein expression level the most substantially increases, difference the most statistically significant (p < 0.05)；And MMP13 and ADAMTS- 5 protein expression levels reduce, wherein MMP13 differential expression in OA chondrocytes at different levels the most statistically significant (p < 0.05), ADAMTS-5 is differential expression statistically significant (p < 0.05) in normal and light, moderate OA chondrocyte.miRNA- After 140inhibitor transfection, above-mentioned protein expression variation tendency is contrary.

MiRNA-140 is stable in people's normal articular cartilage cell to express, and its expression increases the weight of to gradually decrease with OA degree.

The optium concentration of miRNA-140mimic transfected with human articular chondrocytes is 50nM, detection transfection optimal time Between for transfection after 72h.

Raise miRNA-140 expression in human articular chondrocytes and can promote that Collagen II and Sox9 expresses, suppress simultaneously MMP13, ADAMTS-5 and Runx2 express, thus play the effect stoping and repairing chondrocyte OA sample to change, and this acts on Gently, moderate OA chondrocyte becomes apparent from.

All can detect that miRNA-140 expresses in normal and OA joint fluid, in OA joint fluid, miRNA-140 expresses calibration Often group significantly reduces, group difference statistically significant (F=15.28, p < 0.05).

MiRNA-140 expression in joint fluid increases the weight of to gradually decrease with OA degree, and is notable negative with KL classification Close (r=-0.92, p < 0.05).

People normally and all can detect that miRNA-140 expresses in OA joint fluid, and its expression increases the weight of gradually to subtract with OA degree Few, and be notable negative correlation with the OA order of severity.

Being successfully established rat knee joints OA model, average modeling operating time is 10.60 ± 1.80min/, operative incision Healing in 1 week the most after surgery, average Mankin ' s scoring in postoperative 4th, 8 and 12 weeks is respectively 6.50 ± 1.52,10.17 ± 0.75 And 12.83 ± 0.75.

After joint cavity injection miRNA-140agomir or miRNA-140control, without rat, the infection of joint or dead occurs The complication such as die.

Experimental group cartilage of rats regression journey is found at postoperative 4th, 8 and 12 weeks of modeling, gross examination of skeletal muscle and histochemical staining Spend and be substantially lighter than matched group, experimental group rat each time point articular cartilage tissue Mankin ' s scoring (3.50 ± 0.55,6.33 ± 0.82 and 8.67 ± 1.21) being below matched group (6.83 ± 1.17,10.50 ± 1.05 and 13.17 ± 0.75), difference all has system Difference (p < 0.05) learned by meter.

With modeling time lengthening, Collagen II expresses and gradually decreases, and MMP13 and ADAMTS-5 expresses and gradually increase. Postoperative 4th, 8 and 12 weeks of modeling, in experimental group cartilage of rats tissue, Collagen II positive expression intensity was higher than comparison Group, and MMP13 and ADAMTS-5 positive expression intensity is less than matched group.Experimental group each time point Collagen II SABC MOD value is respectively 11.03 ± 1.54,7.88 ± 1.66 and 4.83 ± 1.26 (× 10^-3), matched group is respectively 7.60 ± 1.21, 4.45 ± 1.35 and 1.47 ± 0.31 (× 10^-3), two group differences the most statistically significant (p < 0.05).The each time point of experimental group MMP13 SABC MOD value is respectively 2.93 ± 0.51,5.05 ± 0.98 and 7.92 ± 1.52 (× 10^-3), matched group is respectively 5.63 ± 1.07,8.43 ± 1.09 and 11.70 ± 1.56 (× 10^-3), group difference the most statistically significant (p < 0.05).Experiment Organize each time point ADAMTS-5 SABC MOD value and be respectively 2.60 ± 0.62,3.70 ± 1.05 and 8.85 ± 1.73 (× 10^-3), matched group is respectively 4.13 ± 0.75,9.72 ± 2.04 and 18.17 ± 5.20 (× 10^-3), group difference all has statistics Meaning (p < 0.05).

Being successfully established rat OA model, joint cavity injection miRNA-140agomir is safe, feasible.

Joint cavity injection miRNA-140agomir can substantially slow down cartilage of rats regression process.

Joint cavity injection miRNA-140agomir can significantly slow down Collagen II PD in articular cartilage tissue, The most substantially suppression MMP13 and ADAMTS-5 protein expression, plays obvious retarding action to cartilage of rats regression.

Accompanying drawing explanation

Fig. 1 is the checking miRNA-140 regulating and controlling effect side in osteoarthritis chondrocytes that the embodiment of the present invention provides Method flow chart.

Detailed description of the invention

In order to make the purpose of the present invention, technical scheme and advantage clearer, below in conjunction with embodiment, to the present invention It is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not used to Limit the present invention.

Below in conjunction with the accompanying drawings and the application principle of the present invention is further described by specific embodiment.

A kind of medicine to articular cartilage degeneration regulating and controlling effect, the medicine of the regulating and controlling effect of articular cartilage degeneration is by this miRNA-140。

As shown in Figure 1: the present invention provides a kind of and verifies miRNA-140 regulating and controlling effect side in osteoarthritis chondrocytes Method is:

S101: first verify that miRNA-140 is normal people and expression rule in osteoarthritis (OA) chondrocyte；

S102: secondly screen the optimum condition of miRNA-140 transfected with human chondrocyte；

S103: finally verify miRNA-140 up-regulated or lower the regulating and controlling effect to people's OA chondrocyte and mechanism.

Further, checking miRNA-140 regulating and controlling effect method in osteoarthritis chondrocytes particularly as follows:

The present invention provides a kind of miRNA-140 of checking in the regulating and controlling effect method in osteoarthritic joint liquid to be:

The present invention provides a kind of and verifies the joint cavity injection miRNA-140 regulating and controlling effect side to rat knee joints cartilage degeneration Method is: by building rat knee joints OA model and identifying.

Below in conjunction with test and specific embodiment, the application principle of the present invention is further described.

Osteoarthritis (Osteoarthritis, OA) is a kind of with Progressive symmetric erythrokeratodermia articular cartilage damage, subchondral bone change, pass Joint periphery hyperosteogeny is formed and synovitis sexually revises the degenerative disease being characterized, be apt to occur in many joint big, movable of bearing a heavy burden (as Knee joint, hip, spinal column, ankle and hands etc.), common with old and middle-aged patients.With OA course advancement can engender arthralgia, deformity and The symptoms such as moving obstacle, disability rate may be up to 53%.China's large population base, and stepped into aging society, OA number of patients Will be more and more, but the current concrete pathogenesis about OA is still not clear.Chondrocyte (Chondrocyte) is articular cartilage In tissue, unique cell component, can synthesize the various kinds of cell epimatrix including collagen protein, proteoglycan and noncollagen protein (Extracellular matrix, ECM) composition.In normal articular cartilage, at chondrocyte anabolism and catabolism In a kind of dynamic equilibrium, this is to maintaining articular cartilage normal steady state and function to play an important role, and is regulated by many factors.When This poised state is broken or during accompanying regulation system dysfunction, and chondrocyte metabolic is unbalance, will promote chondrocyte eventually End differentiation and ECM degraded, as do not repaired or stop this process in time, OA sample changes to cause articular cartilage to occur the most at last.

MicroRNA (miRNA) is the non-coding tiny RNA of a class high conservative, is about 22 nucleotide, can be in endochylema Combine with target gene (mRNA) 3 ' end untranslated region (Untranslated regions, UTRs) with complementary type wholly or in part Cause said target mrna degraded or translation to be suppressed, regulate and control the mRNA translation of mammal about 1/3, at cell proliferation, break up, move The aspects such as shifting, aging and apoptosis play an important role.Recent study finds, miRNA-140 has cartilage specificity, people just Often stable expression in articular cartilage, stable state and function to articular cartilage play specific regulating and controlling effect.At present about miRNA- The 140 expression Changing Patterns in OA cartilage remain dispute, and portion's research finds that in OA cartilaginous tissue or cell, miRNA-140 expresses Compared with normal cartilaginous tissue or Leukopenia, but also studies have reported that miRNA-140 expresses compared with normal cartilage in OA cartilage and increases.

Therefore, in order to explore miRNA-140 expression rule in OA chondrocyte and regulating and controlling effect, this part further Testing the expression that will first detect that miRNA-140 is normal people and in OA chondrocyte, next screens miRNA- The optimum condition of 140mimic transfected with human chondrocyte, finally explores miRNA-140 up-regulated or lowers and OA normal to people The regulating and controlling effect of chondrocyte and molecular mechanism.

2 materials and method

2.1 experiment material

2.1.1 people's fresh cartilage specimen

According to Kellgren and Lawrence (KL) imaging diagnosis standard, OA is divided into 0-4 level (table 1).From Sichuan University's West China Hospital orthopaedics and emergency department choose and plan to implement above knee amputation art or total knee arthroplasty (Total knee Arthroplasty, TKA) patient, collect normal (KL 0 grade), slight OA (KL 1-2 level), moderate OA (KL 3 during operation Level) and severe OA (KL 4 grades) condyle of femur cartilage samples each 5-10 example.All specimen collections all obtain patient and/or its family members' Agree to and sign Informed Consent Form.

Table 1 osteoarthritis Kellgren-Lawrence (KL) X-ray film grade scale

2.1.2 main agents

Other reagent are domestic or Import Analysis pure level high-quality reagent.

2.1.3 the preparation of main solution

1) 0.2% collagenase solution: 20mg collagenase powder+10ml DMEM (high sugar) culture medium containing 5%FBS, prepares Afterwards with aseptic disposable filter (aperture 0.2 μm) filtration sterilization, now join before using.

2) 20%FBS culture medium: 20ml FBS+80ml DMEM (high sugar) culture medium, mixing, joins 100ml, subpackage every time Rear 4 DEG C of preservations, are finished and join.

3) 1% Toluidine blue staining liquid: after toluidine blue powder 0.2g+2ml 70% ethanol fully dissolves, then add 18ml 1%NaCl solution (1gNaCl+100ml distilled water, matching while using), mixing, filter paper filtering.

2.1.4 key instrument, equipment

Other instrument and equipments are domestic or import introducing equipment in recent years, Huaxi Hospital Attached to Sichuan Univ Technology Park carry Supply.

2.1.5 consumptive material is tested

The experiment consumptive material such as the Tissue Culture Flask/ware of all size, centrifuge tube, liquid-transfering gun rifle head, cell sieve, filter screen is state Produce or import high-quality product.

2.2 experimental technique

2.2.1 chondrocyte In vitro culture and qualification

2.2.1.1 cartilaginous tissue specimen collection

During in June, 2014 in August, 2015, collect fresh cartilage tissue specimen in Huaxi Hospital Attached to Sichuan Univ, whole Process strict aseptic technique, concrete grammar is as follows:

1), after TKA art exposing condyle of femur, cut in flakes with scalpel and take condyle of femur cartilage.To above knee amputation art patient, limb After body is cut down, knee joint district sterilization paving aseptic towel, cut after exposing condyle of femur and take cartilaginous tissue.Disinfectant solution is avoided during drawing materials Or other zest liquid contacts district's cartilage of drawing materials.

2) the cartilage sheet taken off is placed in clean aseptic kidney basin, with normal saline (Normal saline, NS) repeatedly Flush three times, be transferred to fill in the 50ml sterile centrifugation tube of DMEM (high sugar) culture medium, be transported to the most as early as possible Laboratory carries out cell separation.

2.2.1.2 Primary chondrocyte separates and cultivates

Using trypsin+collagenase digestion separation Primary chondrocyte, concrete grammar is as follows:

1) take appropriate cartilaginous tissue specimen to put in 50ml sterile centrifugation tube, add a small amount of culture medium to flooding lid specimen.By nothing Cartilage sheet is shredded into < 1mm by bacterium long handle tissue shear³Fritter, clean 2 times with aseptic NS, centrifugal after remove NS, blot.

2) add 0.25% trypsin solution of more than 3 times of volumes, shake up, persistent oscillation digestion in 37 DEG C of water-baths 15-30min, centrifugal (1200r/min, 3min), sucking supernatant, aseptic NS cleans 2 times.

3) add 2% more than 3 times of volumes collagenase solutions, shake up, in 37 DEG C of water-baths vibration digestion 4 hours with On, substantially disappear until piece of tissue, till Digestive system becomes cloudy.

4) it is sieved through filter with 200 mesh aseptic rustless steel cell, collects filtrate, centrifugal (1500r/min, 10min), suck supernatant Liquid, with aseptic NS suspendible sedimentation cell, repeats centrifugal 1 time.

With 20%FBS culture medium suspendible sedimentation cell, by cell density (0.5-2) × 10⁵Individual/ml kind enters culture bottle or training Support in ware and cultivate, be placed in 37 DEG C, 5%CO₂Incubator continues cultivate, within every 2-3 days, change liquid 1 time, when cell covers with culture bottle/ware During diapire, trypsin solution digests, passes on.

2.2.1.3 chondrocyte passes on

1) until chondrocyte proliferation to when covering at the bottom of culture bottle more than 80%, sop up old culture medium, add PBS liquid and clean 2-3 Secondary.

2) according to culture bottle/ware size, add appropriate 0.25% trypsin solution, jog, make Digestive system flow through all carefully Cellular surface, examines under a microscope after digesting 1-3 minute in incubator, when most cells kytoplasm retraction, gap increase and become When circle is floating, adds equal-volume 20%FBS culture medium and terminate digestion；Draw culture medium in bottle/ware with suction pipe, blow the most successively Beat bottle/ware parietal cell, collect cell suspension and move in 15ml centrifuge tube, centrifugal (1500r/min, 3-5min).

3) abandon supernatant, add the culture medium re-suspended cell containing 20%FBS, inoculation, put and incubator continues cultivate, microscope The upgrowth situation of lower observed and recorded chondrocyte, changes liquid 1 time in every 2-3 days.

2.2.1.4 chondrocyte is frozen and recovers

" to freeze slowly and to melt soon " when cell cryopreservation and recovery, concrete grammar is as follows:

1) frozen first 1 day routine changes liquid, and (Dimethylsulfoxide, DMSO analyze to take dimethyl sulfoxide before peptic cell Pure), FBS and culture medium be with the proportions cell-preservation liquid of 1:2:7.

2) sucking old culture fluid, PBS 2-3 time, by preceding method peptic cell.

3) taking appropriate cell-preservation liquid re-suspended cell precipitation, piping and druming makes cell be uniformly distributed gently, and cell counting count board counts, Adjust and preserve final concentration of (5-10) × 10 of cell in liquid⁶/ ml, is distributed into cell suspension in the aseptic cryopreservation tube of 1.5ml, rotation The tightening seal mouth of pipe, indicates relevant information, puts into cell cryopreservation box and be placed in-80 DEG C of refrigerators preservation, is shifted by cryopreservation tube after 1-3 days To-150 DEG C of Refrigerator stores.

4) during recovery, cell cryopreservation tube is taken out from-150 DEG C of refrigerators, put into immediately in the case of guaranteeing ferrule In 37 DEG C of water-baths, jog makes its content melt as early as possible.

5) the rapid culture medium sucked in 50ml centrifuge tube by cell suspension and drip more than 10 times of volumes, gently after mixing Low-speed centrifugal (1000r/min, 3min), removes supernatant, cleans with culture fluid 2 times again.

6) with 20%FBS culture medium suspendible sedimentation cell, inoculating after counting, put and continue in incubator to cultivate, next day is changed Culture fluid, later routine changes liquid, observation.

2.2.1.5 chondrocyte is identified

Chondrocyte identifies methods such as using light Microscopic observation, Toluidine blue staining and II Collagen Type VI immunofluorescence dyeing, Specific as follows:

(1) the chondrocyte form of basis of microscopic observation In vitro culture, density, adherent and growing state etc..

(2) Toluidine blue staining

1) sterilizing blood cover plate is put in six orifice plates, inoculate proper density chondrocyte quiescent culture, basis of microscopic observation Chondrocyte fusion rate on blood cover plate, tests cell climbing sheet when cell confluency reaches about 80%.

2) going culture medium, PBS washes 3 times, adds 4% paraformaldehyde phosphate buffer to flooding blood cover plate completely, fixes for 4 DEG C 45-60min, sucks paraformaldehyde, and distilled water washes 3 times.

3) add 1% Toluidine blue staining liquid and be completely soaked cell climbing sheet, dye 1.5-2 hour under room temperature, remove unnecessary dye Liquid, distilled water washes 3-5 time, each 3-5min.

4) successively through each 1min of 70% → 80% → 90% → dehydrated alcohol, dehydration.

5) basis of microscopic observation staining conditions, repeats first two steps if desired, dries, neutral gum mounting after dyeing is satisfied, Basis of microscopic observation, adopt figure.

(3) II Collagen Type VI immunofluorescence dyeings

1) make cell climbing sheet by preceding method and fix.

2) add 0.5%TritonX-100 incubated at room temperature 10-20min, PBS to wash 3 times, each 3-5min.

3) 1%BSA is now joined, wet box, close 30min, abandon confining liquid for 37 DEG C, add rabbit source property polyclone Collagen II and resist Body (1:100), every creep plate 40 μ l, put wet box, 4 DEG C are overnight.

4) PBS washes 3 times, each 5min, adds Alexa FluorR 488F (ab ') 2fragment of goat anti- Rabbit IgG (H+L) two anti-(1:200), every creep plate 40 μ l, put wet box, 37 DEG C of lucifuges 1h.

5) PBS washes 3 times, each 5min, adds 1 μ g/ml DAPI dye liquor, incubated at room temperature 5min.

6) PBS washes 3 times, each 5min, neutral gum mounting, fluorescence microscopy Microscopic observation, adopts figure.

2.2.2 miRNA-140 detection in normal and OA chondrocyte

2.2.2.1 the total miRNA of cell extracts

Operating according to microRNA Purification Kit description, concrete grammar is as follows:

1) six orifice plate cultured cartilage cells reach more than 80% to fusion rate, go culture medium, PBS to wash 3 times, blot, and every hole adds 300 μ l Buffer RL, gently shake, pat orifice plate, crack 5min, lysate is transferred to the 1.5ml without RNAase pollutes and is centrifuged In pipe, often pipe adds 150 μ l 96-100% ethanol, and vortex instrument mixes 10s.

2) Collection Tube and Large RNA Removal Column is assembled, then above-mentioned mixed liquor is turned Move to Column, centrifugal (14000g, 1min), it is ensured that after mixed liquor is all by Column, filtrate is transferred to nothing In the 1.5ml centrifuge tube that RNAase pollutes, add 350 μ l 96-100% ethanol, vortex instrument mixes 10s.

3) Collection Tube and microRNA Enrichment Column is assembled, then by above-mentioned mixed liquor Take half and be transferred to Column, centrifugal (> 3500g, 1min), it is ensured that after mixed liquor is all by Column, abandon filtrate, again Assemble Column and Collection Tube.The step for of repetition, complete tiny RNA and collect.

4) 400 μ l Wash Solution A are added microRNA Enrichment Column, centrifugal (14000g, 1min), it is ensured that after Wash Solution A is all by Column, abandons filtrate, re-assembly Column and Collection Tube, is repeated 2 times.

5) microRNA Enrichment Column is cleaned, it is ensured that after Wash Solution A is all by Column, Abandon filtrate, re-assembly Column and Collection Tube.Recentrifuge 2min, makes gel column be completely dried, abandons Collection Tube, inserts microRNA Enrichment Column in supporting 1.7ml Elution Tube.

6) 50 μ l Elution Solution A are added to Column, centrifugal (200g, 2min), it is ensured that Elution Solution A, all by Column, collects eluent.

7) repetition first two steps are to guarantee at utmost to collect microRNA if desired, and the microRNA sample that will collect is protected There are-70 DEG C of refrigerators, the used time takes out.

2.2.2.2 the total miRNA concentration of cell measures and integrity detection

1) 2 μ l total miRNA sample, microplate reader detectable concentration are taken.

2) the OD260/OD280 ratio of total miRNA is in the range of 1.8-2.2ng/ μ l, detects 3 times, averages.

3) 3% agarose gel is prepared: 0.3g agarose powder adds 30ml TAE ((Tris-acetic acid, 0.04M Tris-second Acid, 0.001M EDTA) solution, after masking foil sealing, microwave oven height fire is to boiling, repeatable operation until all dissolving clarification, then Add 2 μ l Golden well stock solutions, mixing.

4) glue is poured in applicable mould, plug the comb in applicable aperture, cool down 30min, extract comb, gel is moved to In electrophoresis tank, pour 0.5 × TBE electrophoretic buffer into, allow liquid level exceed glue surface about 1-2mm.

5) draw 6 × Laoding buffer of 2 μ l, the 2 total microRNA of μ l, supply 12 μ l with the water without RNAase, Mixing, micro-centrifugal 5s, point sample, every hole applied sample amount 10 μ l.

6) negative pole end (black), voltage 150v, electrophoresis 20min are put in hole.

7), after electrophoresis terminates, under handheld ultraviolet light, observe electrophoretic band, start visible 28S, 18S, 5S bar from nose end Band, 28S band brightness is about 2 times of 18S brightness.

2.2.2.4 reverse transcription RT

The total miRNA that will extract, utilizes random primer and reverse transcriptase reverse transcription to become cDNA.With Fermentas company Revert Aid^TMFrist Strand cDNA Synthesis Kit, expands in PCR instrument, and condition is as follows:

1) on ice chest, following solution is premixed:

2) brief centrifugation, 70 DEG C of pretreatment 5min, then cooled on ice.

3) sequentially add:

4) brief centrifugation, uses PCR instrument to carry out reverse transcription after setting condition, and condition is as follows:

5) after reverse transcription completes, cDNA is placed in-20 DEG C of Refrigerator stores, and pending fluorescence quantitative PCR detection is used.

2.2.2.5 fluorescence quantitative PCR detection

1) qPCR reaction system preparation:

2) qPCR reaction condition is as follows:

3) reaction system is carried out on quantitative real time PCR Instrument, after the increasing reaction of 39 circulation expansions terminates, draws amplification dynamic Force diagram, determines amplification cycles number (Ct value).

4) method utilizing relative quantification carries out data statistics: Δ Ct1=experimental group genes of interest Average Ct values-internal reference base Because of Average Ct values, Δ Ct2=matched group genes of interest Average Ct values-matched group reference gene Average Ct values, Δ Δ Ct=Δ Ct1-Δ Ct2, the relative expression quantity of genes of interest is then 2^-ΔΔCt。

2.2.2.6PCR detection product gel electrophoresis

Concrete grammar is the same.

2.2.3miRNA-140mimic/inhibitor transfected with human chondrocyte conditional FP tree

The microRNA products instruction operation provided according to Rui Bo bio tech ltd, transfection liquid is all turning Now join before dye, strict aseptic technique.

2.2.3.1 transfection reagent is prepared

According to riboFECT^TMThe explanation operation of CP Transfection Kit test kit, concrete grammar is as follows:

1) brief centrifugation before using, PBS water dilution Buffer (10 ×), preparation Buffer (1 ×).

2) taking out Reagent in vortex oscillator after abundant vibration, room temperature is placed, and uses after being allowed to return to room temperature.

2.2.3.2 preparation variable concentrations miRNA-140mimic transfects liquid

All operations follows strictly RNA operation rules, and concrete grammar is as follows:

1) 5nmol miRNA-140 lyophilized powder is taken out, use front brief centrifugation, be configured to 250 μ with the distilled water of sterilizing L (20nM) stores liquid, and subpackage is stored in-80 DEG C of refrigerators, uses front taking-up, it is to avoid multigelation.

2) with 1 × Buffer (v2) dilution miRNA-140mimic (v3), mix gently, add Reagent (v4), blow gently Play mixing, do not vibrate, incubated at room temperature 0-15min.

3) mixed liquor is added in cell culture fluid (v1), mix gently.

4) in variable concentrations transfection liquid process for preparation, various reagent dosages are shown in Table 2 (6 orifice plates).

Table 2 variable concentrations miRNA-140mimic transfection liquid preparation program (6 orifice plate)

2.2.3.3miRNA-140mimic/inhibitor chondrocyte is transfected

1) inoculation right quantity slight OA chondrocyte is in 6 orifice plates, and every hole adds the culture medium without antibiotic, makes transfection Time cell density can reach 30-50%.

2) going old culture medium, experimental group adds the miRNA-140mimic of 2ml variable concentrations and transfects liquid, and matched group is the most more Change culture medium, without any transfection reagent, shake up gently, in incubator continue cultivate 24-72h.

2.2.3.4miRNA-140mimic/inhibitor transfection chondrocyte effect detection

2.2.3.4.1PCR detection Collagen II and MMP13 gene expression dose

1) with 6 orifice plate cultured cartilage cells, culture medium, PBS is gone to wash 2 times.

2) every hole adds 1ml Trizol, repeatedly blows and beats cell lysis, is proceeded to by cell pyrolysis liquid without RNAase pollution In 1.5mlEP pipe, left at room temperature 5min.

3) often pipe adds 200 μ l chloroform, sealing, and acutely vibrate 20s, and room temperature stands 5min, be centrifuged (4 DEG C, 12000g, 15min).

4) liquid in pipe is divided into three layers from top to bottom, and upper strata is total serum IgE liquid, careful absorption upper strata aqueous phase liquid, turns Enter in the 1.5ml EP pipe that new enzyme without RNAase pollutes.

5) adding equal-volume isopropanol, mixing, room temperature stands 10-15min, centrifugal (4 DEG C, 12000g, 10min).

6) removing supernatant, add 75% ethanol 1ml of pre-cooling, mixing is placed on 10min on ice chest, centrifugal (4 DEG C, 12000g, 5min), remove supernatant, under room temperature, be dried 5-10min.

7) 15-30 μ l DEPC water is added, mixing ,-80 DEG C of preservations, total rna concentration to be measured and subsequent detection.

8) total rna concentration mensuration, RNA integrity detection, reverse transcription, quantitative fluorescent PCR and PCR primer gel electrophoresis method Ditto.

2.2.3.4.2Western blotting detects Collagen II and MMP13 protein expression level

(1) total protein extraction

1) 6 orifice plates cultivate human chondrocytes, go culture fluid, PBS to wash 2 times, exhaust PBS, and (RIPA buffers to add 1ml lysate Liquid: PMSF=100:1,100mmol), cell scrapes collection cell and lysate in new 1.5mlEP pipe, and ice chest stands 1.5-2h, fully crack.

2) centrifugal (4 DEG C, 12000rpm, 10-15min), the careful supernatant drawn after being centrifuged, subpackage is transferred to clean EP Guan Zhong, adds 4 × Loading Buffer boiling water boiling 10min, remaining-80 DEG C of preservations by a part of supernatant.

(2) BCA method detection protein concentration

1) quantification of protein is carried out according to BCA protein quantification test kit operating instruction.

2) microplate reader wavelength is set as 562nm, and 96 orifice plates are put in microplate reader detection, makes standard curve, then root Sample protein concentration is drawn according to the light absorption value of testing sample.

(3) SDS-PAGE electrophoresis

1) 12% separation gel needed for preparing electrophoresis by condition set forth below adds each group, mixing, encapsulating.

12% separation gel formula

2) after 30-50min, remove photoresist upper strata dehydrated alcohol being sufficiently absorbed through with absorbent paper, concentrates by the preparation of following condition Glue, fills concentration glue, is plugged comb immediately between glass plate, until after concentrating gelling admittedly, being extracted by comb gently, rushing with distilled water Wash glue hole.

Standard concentrates glue formula

3) take same amount of protein groups tissue samples sample-loading buffer (Loading buffer) polishing, to same volume, to fill Divide mixing.

4) after adding enough electrophoretic buffers in electrophoresis tank, being joined by protein sample in each loading hole, loading is complete Bi Hou, covers electrophoresis tank lid, switches on power, and arranges deposition condition, and adjustment voltage, to 80V, starts electrophoresis, until protein band When running to separation gel, voltage is transferred to 110V.

5) electrophoresis is terminated when electrophoresis to bromophenol blue goes to electrophoresis plate bottom.

(4) transferring film

1) pvdf membrane soaks 5min with methanol in advance, is taken out by glass plate from electrophoresis tank, by glass while flowing water flushing Glass plate is pried open, and scrapes off concentration glue gently, is dipped in transferring film liquid by the glue stripped out.

2) membrane-transferring device placement order is followed successively by foam-rubber cushion, filter paper, glue, the pvdf membrane of activation, filter paper, foam-rubber cushion, and glue needs Aliging with filter paper, roll the bubble removed photoresist and between filter paper and glue and film simultaneously with glass rod, close membrane-transferring device, by correct side To putting it into transfer groove, add appropriate ice bank, constant current 400mA to the edge of transfer groove, carry out transferring film operation.

3) determining the transferring film time according to the size of target protein, transferring film takes out rapidly film after completing, in TBST buffer Washing 3min, confining liquid closes 1h.

(5) immunoreation

1) pvdf membrane after trace closes 1h or 4 DEG C overnight by 5% defatted milk powder solution incubated at room.

2) anti-it is diluted to debita spissitudo, after incubated at room temperature pvdf membrane 1-2h, with TBST at room temperature decolorization swinging table by one On wash 3 times, then wash once with TBS, 10min.

3) anti-it is diluted to two anti-working solutions by proper proportion with confining liquid by the two of HRP labelling, film is put into wherein, then Shaken at room temperature hatches 1h.With washing on TBST at room temperature decolorization swinging table 3 times, each 10min, then wash once with TBS, 10min.

(6) development and fixing reaction

After adding chromogenic substrate, by X-ray developing method, the signal on transfer film is transferred on X-ray.

(7) semi-quantitative analysis

Scan above-mentioned film, then with Image J software analysis target protein band and the gray value of internal reference, carry out semidefinite Component analysis.

2.2.4miRNA-140 regulating and controlling effect and the mechanism to OA chondrocyte

2.2.4.1 cultivate normal and OA chondrocyte method be the same.

2.2.4.2miRNA-140mimic/inhibitor/control transfection is normal and OA chondrocyte

(1) experiment packet often group chondrocyte is divided into: blank group, miRNA-140mimic transfection group, miRNA- 140inhibitor transfection group and miRNA-140control transfection group.

(2) transfection

Transfection concentrations is the optimal transfection concentrations that early stage filters out, and miRNA-140inhibitor transfection concentrations is mimic 2 times of concentration, control transfection concentrations is with mimic concentration, and blank group normally changes culture medium, without any transfection Reagent, concrete transfection method is the same.

2.2.4.3 transfection detection

(1) PCR detects Collagen II, MMP13, ADAMTS-5, Sox9 and Runx2 gene expression

1) concrete grammar is the same.

2) relevant primer design

(2) Western blotting detects Collagen II, MMP13 and ADAMTS-5 protein expression

Concrete grammar is the same.

2.3 statistical analysis

All data all use SPSS 22.0 software to carry out statistical analysis, and measurement data uses mean ± standard deviation to represent, Compare employing Mann-Whitney U inspection between two groups of groups, between many groups group, compare employing single factor test ANOVA variance analysis.p<0.05 It is set to and has significant difference.

3 results

3.1 human articular chondrocytes are cultivated and identify

3.1.1 cartilaginous tissue Specimen origin patient basic condition

(KL 0 grade), moderate (KL 3 grades) and severe is collected normally from Huaxi Hospital Attached to Sichuan Univ orthopaedics and emergency department (KL 4 grades) OA patient femur's condyle cartilage samples totally 36 example, patient's basic document is shown in Table 3.

Table 3 cartilaginous tissue Specimen origin patient's basic document

3.1.2 chondrocyte form and dyeing are identified

(1) morphological observation

Observe under inverted microscope, it is seen that adherent people's normal chondrocyte ovalize, polygon, add with OA degree Weight, cellular morphology changes, spindle shape shape cytosis, is similar to fibroblast-like cells.

(2) Toluidine blue staining

Taking chondrocyte creep plate row Toluidine blue staining to be placed on and just put basis of microscopic observation, chondrocyte core dye is for dark blue Color, Cytoplasm dye is bluish violet, and stained positive shows, meets chondrocyte Toluidine blue staining feature.

(3) Collagen II immunofluorescence dyeing

Take chondrocyte creep plate row Collagen II immunofluorescence dyeing to be placed on and just put fluorescence microscopy Microscopic observation, soft In osteocyte kytoplasm, visible a large amount of green fluorescences, meet chondrocyte Collagen II immunofluorescence dyeing feature.

3.2miRNA-140 the expression Changing Pattern in people's OA chondrocyte

MiRNA-140 relative expression quantity in chondrocyte is shown in Table 4.MiRNA-140 is in normal articular cartilage cell Stable expression, expresses equal compared with normal group in OA chondrocyte and significantly reduces, group difference statistically significant (F=53.62, p <0.001).Increasing the weight of with OA degree, miRNA-140 expresses and is gradually lowered, and in severe OA chondrocyte, miRNA-140 expresses minimum.

Table 4 people is normal and miRNA-140 relative expression quantity in OA chondrocyte

3.3miRNA-140 the optimal transfection concentrations of transfected with human articular chondrocytes and detection time

3.3.1miRNA-140mimic the optium concentration of chondrocyte is transfected

Increasing with miRNA-140mimic concentration, Collagen II gene and protein expression are gradually increased, and MMP13 base Cause and protein expression gradually decrease.Compared with blank group, when miRNA-140mimic concentration is 50nM, Collagen II, MMP13 gene and protein expression difference the most statistically significant (p < 0.05).When miRNA-140mimic concentration is more than 50nM Time, Collagen II gene and protein expression increase further, and MMP13 gene expression declines further, but with 50nM group phase Ratio, the many not statistically significants of difference.Therefore, this research select 50nM as miRNA-140mimic transfected with human chondrocyte Optium concentration.

3.3.2miRNA-140mimic the Best Times of chondrocyte effect detection is transfected

In blank group, Collagen II and MMP13 gene and protein expression the most relatively 24h after cultured chondrocytes 72h Group increased.Compared with 24h group, after miRNA-140mimic transfection 72h, Collagen II gene and protein expression are the most notable Increase, and MMP13 gene and protein expression substantially reduce, difference the most statistically significant (p < 0.05).Therefore, this research selects After transfection, 72h is as the Best Times of miRNA-140mimic transfected with human chondrocyte effect detection.

3.4miRNA-140 expresses up/down and adjusts OA chondrocyte related gene and the regulating and controlling effect of protein expression

According to previous step the selection result, use 50nM miRNA-140mimic or 100nM miRNA-140inhibitor Transfection is normal and after OA chondrocyte after 72h, Collagen II, MMP13, ADAMTS-5, Sox9 and Runx2 in detection cell Gene and protein expression.

3.4.1miRNA-140 express up/down to adjust Collagen II, MMP13, ADAMTS-5, Sox9 and Runx2 gene Express regulating and controlling effect in each group of chondrocyte, each gene expression of miRNA-140control group nothing compared with blank group Notable difference (p > 0.05).

Compared with miRNA-140control group, miRNA-140mimic group Collagen II and Sox9 gene expression increases Adding, both are differential expression the most statistically significant (p < 0.05) in normal and light, moderate OA chondrocyte；And MMP13, ADAMTS-5 and Runx2 gene expression reduce, wherein MMP13 in normal and slight OA chondrocyte, Runx2 soft in moderate OA Differential expression statistically significant (p < 0.05) in osteocyte.In severe OA chondrocyte, miRNA-140mimic group these 5 Gene expression relatively miRNA-140control group no significant difference (p > 0.05).

Compared with miRNA-140control group, miRNA-140inhibitor group Collagen II and Sox9 gene table Reach minimizing, wherein Collagen II differential expression in normal, slight and severe OA chondrocyte statistically significant (p < 0.05) (, Sox9 is differential expression statistically significant (p < 0.05) in normal and severe OA chondrocyte；And MMP13, ADAMTS-5 and Runx2 gene expression increase, wherein MMP13 and ADAMTS-5 express in OA chondrocytes normal and at different levels poor Different the most statistically significant (p < 0.05), and Runx2 differential expression in normal, slight and moderate OA chondrocyte has statistics Meaning (p < 0.05)

3.4.2miRNA-140 express up/down to adjust Collagen II, the impact of MMP13 and ADAMTS-5 protein expression

Compared with blank group, miRNA-140mimic group Collagen II protein expression substantially increases, normal people And differential expression the most statistically significant (p < 0.05) in OA chondrocyte at different levels；And MMP13 and ADAMTS-5 protein expression is bright Aobvious minimizing, wherein MMP13 differential expression in OA chondrocytes at different levels statistically significant (p < 0.05), ADAMTS-5 is just Often, differential expression statistically significant (p < 0.05) in slight and moderate OA chondrocyte.

Compared with blank group, miRNA-140inhibitor group Collagen II protein expression reduces, normal and Differential expression the most statistically significant (p < 0.05) in chondrocytes at different levels；And MMP13 and ADAMTS-5 protein expression is more blank Matched group increases, differential expression the most statistically significant (p < 0.05) in chondrocytes normal and at different levels.

4 discuss

Osteoarthritis (OA) is a kind of common degenerative disease, and its pathogenesis is still not clear, articular cartilage degeneration or After chondrocyte terminal differentiation, regeneration capacity is the most weak, the most still without effective treatment means.Thin at normal articular cartilage In born of the same parents, anabolism and catabolism are in a kind of dynamic balance state, and are regulated by many factors.In recent years find MicroRNA-140 has cartilage specificity, plays an important role, simultaneously in the normal steady state maintaining articular cartilage and function Participate in regulation and control OA to develop.In knee cartilage tissue, find that OA group miRNA-140 expression compared with normal group substantially subtracts Few, it has further been found that miRNA-140 knock out mice articular cartilage when 1 monthly age just shows OA sample and changes, and process LAN The transgenic mice of miRNA-140 not only changes without OA sample, and the even antigen to induction OA also shows certain opposing work With.On the contrary, Swingler and Wheeler et al. is by than compared with normal (fracture of femoral neck) and OA (row replacement of total hip) stock In bone articular cartilage, miRNA-140 expresses change, found that miRNA-140 to express compared with normal cartilage in OA cartilage obvious Increasing, this just makes miRNA-140 expression Changing Pattern in articular cartilage play arguement.First have detected that people is normal and OA is soft In osteocyte, miRNA-140 expresses situation of change, and chondrocyte used is all from knee cartilage tissue, found that miRNA- 140 expressions increase the weight of to gradually decrease with OA degree, and result of study is consistent.Occur in hip, knee joint source cartilaginous tissue or cell The completely contradict reason of this contradictory outcome of miRNA-140 expression rule is unclear, may be different relevant from cartilage samples source, Document there is no at present and compare the report of miRNA-140 expression variation tendency in the different articular cartilage such as hip, knee joint.Because hip, knee joint Joint bear a heavy burden big, OA incidence rate is of a relatively high, afterwards in can attempt between comparative analysis different parts articular cartilage The expression change difference of miRNA-140, perhaps can carry for miRNA-140 effect in OA pathogenesis and successive treatment research For new clue.

Articular cartilage degeneration is one of principal character of OA, many with II Collagen Type VI (Collagen II) and albumen the most again The ECM such as sugar (Aggrecans) are degraded to main.MMP-13 and ADAMTS-5 is presently considered to be in cartilage matrix digestive enzyme most important Hydrolytic enzyme, L et al. is it has also been found that miRNA-140 is a negative regulatory factor of MMP13, and using, proinflammatory factor IL-1 is beta induced soft During osteocyte OA sample changes, miRNA-140 and MMP13 expresses all to be increased, and miRNA-140 can suppress MMP13 to express, he Use further luciferase reporter gene analysis to find miRNA-140 directly can hold UTR site be combined with MMP13 3 '.? Determine in normal and OA chondrocyte after the expression rule of miRNA-140, further by miRNA-140mimic or MiRNA-140inhibitor transfection is raised or is lowered chondrocyte miRNA-140 and expresses, found that raise miRNA-140 Expression can reduce MMP13 gene and protein expression in cell, and wherein protein expression difference becomes apparent from, and this is with miRNA-140's Mechanism of action is consistent, and expresses at post-transcriptional level modulin after being i.e. combined with said target mrna.Thus it is further characterized by miRNA-140 Can be expressed by suppression extracellular matrix degrading enzyme MMP13 and stop and repair the change of chondrocyte OA sample.

In addition to MMP13, it was found that miRNA-140 raises also can suppress ADAMTS-5 gene and protein expression, although gene Expression reduces relatively matched group no difference of science of statistics, but protein expression reduces and relatively compares in normal and light, moderate OA chondrocyte Group has significant difference, and this also further illustrates miRNA-140 and mainly expresses at post-transcriptional level suppression ADAMTS-5.M etc. People finds in testing in vitro that proinflammatory factor IL-1 β intervenes can reduce chondrocyte miRNA-140 expression, increase ADAMTS-5 table Reach；After improving miRNA-140 expression by gene transfection, this inducing action of IL-1 β substantially weakens.It is further discovered that miRNA-140^-/-In mice, ADAMTS-5 expresses increases, and reduces in miRNA-140 transgenic mice, external luciferase Reporter gene assays is it has also been found that ADAMTS-53 ' end UTR includes miRNA-140 binding site.Therefore, suppression ADAMTS-5 table Reaching also is miRNA-140 one of the mechanism of action that stops articular cartilage degeneration.

Sox9 is considered as one of central transcription factor of regulation and control Chondrogenesis, participates in regulation and control mescenchymal stem cell (Mesenchymal stem cells, MSCs) to cartilage thin bone cell differentiation process, the most also controllable Collagen II, Ⅸ, the cartilage specificity albumen such as Ⅺ expression, maintain chondrocyte normal proliferative speed and stop chondrocyte hypertrophic differentiation. Sox9 expresses compared with normal cartilage in OA cartilaginous tissue and reduces, and after reticent Sox9 gene, MMP13 expresses will be increased, and Sox9 can lead to Crossing the expression combining starting region distinguished sequence promotion miRNA-140, this also studies at Sox9 gene knockout and transgenic mice In be proved.Therefore, generally believe that Sox9 is the upstream regulatory factor of miRNA-140 at present.It has however been found that rise chondrocyte Middle miRNA-140 expresses and also can promote that Sox9 expresses and increase, and relatively matched group is poor in normal and light, moderate OA chondrocyte Different statistically significant；On the contrary, lowering chondrocyte miRNA-140 expression also can suppress Sox9 to express, in normal and severe OA In chondrocyte statistically significant compared with matched group difference.Whether this illustrates that miRNA-140 can also feed back tune as downstream elements Joint Sox9 expresses？K etc. find that suppression miRNA-140 may result in Sox9 protein expression level and reduces, and Sox9mRNA expresses also Without significant change, prompting miRNA-140 can raise Sox9 at post-transcriptional level and express.Also it is found miRNA-140 gene expression Rise can suppress RALA (Small GTPases in TGF-β path) to express, and RALA suppression can cause Sox9 protein expression obvious Increase, but Sox9mRNA expresses and is not significantly affected.As it was previously stated, the study find that Sox9 expresses under miRNA-140 Reducing after tune, especially in normal and severe OA chondrocyte, this seemingly has with K et al. result of study and conflicts.Due to the present invention In do not detect the change of Sox9 protein expression, subsequent experimental will improve coherent detection further, further investigation miRNA-140 and The Sox9 effect of regulation mutually in chondrocyte and mechanism.Additionally, also there is Sox9 can suppress Runx2 transcriptional activity, and the latter can Induction of chondrocytes hypertrophic differentiation, this will be discussed in detail below^]。

Runx2 is to regulate and control one of osteoplastic central transcription factor, in high expressed in hypertrophic chondrocyte, and normally Articular chondrocytes is expressed hardly.Confirmed Runx2 can be combined with corresponding gene promoter promotion MMP13, Collagen Ⅹ and alkali phosphatase (ALP) are expressed, thus promote chondrocyte terminal differentiation and endochondral ossification process^[21]。 Study carefully its mechanism of action, Runx2 mainly by be combined with Smad formation Runx2-Smad complex work: Smad3 Yu Runx2 knot After conjunction, Runx2 transcriptional activity is suppressed^[28]；And after Smad1 with Runx2 is combined, Runx2 transcriptional activity is activated^[29]。Smad1、 Smad3 is the key protein in TGF-β/BMP signal path Smads approach^[5], this also illustrates the Runx2 regulation and control to chondrocyte Effect is at least partly realized by regulation and control TGF-β/BMP signal path.Tuddenham etc. are in research long bone and flat bone shape Find during one-tenth that miRNA-140 can pass through inhibition of histone deacetylase 4 (histone deacetylase 4, HDAC4) Promote that Runx2 expresses.But, Miyaki et al. is at miRNA-140^-/-Growth in Rats plate or articular cartilage do not find HDAC4 gene or protein expression change^[15].In our current research, after miRNA-140 raises, the expression of Runx2 reduces, in moderate OA In chondrocyte statistically significant compared with matched group difference, miRNA-140 in osteogenetic process of report can with Tuddenham for this Promote that Runx2 expresses conclusion contrary.This also points out the miRNA-140 may be right in osteogenetic process and during cartilage degeneration Runx2 plays different regulating and controlling effects, miRNA-140 Yu Runx2 mutually regulation relation during articular cartilage degeneration is still Need to be furtherd investigate further.

In sum, this part finds: in human articular chondrocytes, miRNA-140 expresses and increases the weight of gradually to subtract with OA degree Few, and raise miRNA-140 in OA chondrocyte by gene transfection and can promote that Collagen II and Sox9 expresses, press down simultaneously MMP13, ADAMTS-5 and Runx2 processed express, thus play the effect stoping and repairing chondrocyte OA sample to change, and this effect Light, moderate OA chondrocyte becomes apparent from.This suggests that, miRNA-140 perhaps can be as preventing and one for the treatment of OA Important point of penetration, but the research that miRNA-140 treats OA at present mostly is chondrocytes in vitro cell experiment and gene knockout/transgenic Zoopery, this is limited to the practice guiding action exploring miRNA-140 treatment OA.Find at rat knee joints intracavitary administration MiRNA-210 can express promote by improving VEGF (VEGF) and FGF2 (FGF2) Enter ACL and meniscus injury reparation.Therefore, articular cavity application miRNA-140 perhaps can provide new research for OA gene therapy Method and theoretical foundation, but there is no pertinent literature report at present, in subsequent experimental, miRNA-140 articular cavity will be applied soft The effect of bone regression and mechanism are further.

5 conclusions

(1) miRNA-140 is stable in people's normal articular cartilage cell expresses, and increases the weight of its expression with OA degree and gradually subtracts Few.

(2) optium concentration of miRNA-140mimic transfected with human articular chondrocytes is 50nM, and detection transfection is The good time is 72h after transfection.

(3) raise miRNA-140 expression in human articular chondrocytes and can promote that Collagen II and Sox9 expresses, simultaneously Suppression MMP13, ADAMTS-5 and Runx2 express, thus play the effect stoping and repairing chondrocyte OA sample to change, and are somebody's turn to do Act in light, moderate OA chondrocyte and become apparent from.

Below in conjunction with miRNA-140 expression rule in osteoarthritic joint liquid, the present invention is further described.

1, material and method

1.1 experiment material

1.1.1 human knee joint liquid

According to KL diagnostic imaging standard by OA classification (same to Part I), from Huaxi Hospital Attached to Sichuan Univ orthopaedics in-patient department and Normal, slight, moderate and each 10 examples of severe OA patient's Knee Joint Fluid specimen, row articular cavity medicine are collected in outpatient service joint puncture room Injection patient only collects the joint fluid before injection for the first time.All specimen collections all obtain the agreement of patient and/or its family members also Signature Informed Consent Form.

2.1.2 main agents

Other reagent are domestic or Import Analysis pure level high-quality reagent.

1.1.3 key instrument

1.1.4 major experimental consumptive material

Centrifuge tube, rifle first-class experiment consumptive material that all size pollutes without RNase are domestic or import high-quality product.

1.2 experimental technique

1.2.1 Knee Joint Fluid collection, subpackage and preservation

1) joint fluid be transferred quickly to after extracting out 15ml aseptic, without in the centrifuge tube of RNAase, be centrifuged (3000rpm, 5min)。

2) taking supernatant subpackage to 1ml without in the 1.5ml centrifuge tube of RNAase, be placed in liquid nitrogen container and preserve, the used time takes out.

1.2.2 the total miRNA of joint fluid extracts

Operating according to microRNA Purification Kit test kit description, concrete grammar is as follows:

1) 100 μ l joint fluids are transferred in the 1.5ml centrifuge tube without RNAase pollution, add 250 μ l Buffer RL, whirlpool Mix on rotation agitator, 15s.

2) add 150 μ l 96-100% ethanol, vortex instrument mixes 10s.

3) Collection Tube and Large RNA Removal Column are assembled, then by cell pyrolysis liquid with Alcohol mixeding liquid is transferred to Column, centrifugal (14000g, 1min), it is ensured that after mixed liquor is all by Column, retains flowthrough。

4) flowthrough is transferred in the 1.5ml centrifuge tube without RNAase pollution, adds 350 μ l 96-100% second Alcohol, vortex instrument mixes 10s.

5) Collection Tube and microRNA Enrichment Column is assembled, then upper step is obtained Mixed liquor takes half and is transferred to Column, centrifugal (＞ 3500g, 1min), it is ensured that after mixed liquor is all by Column, abandon Flowthrough, re-assemblies Column and Collection Tube.

6) repeat previous step, complete tiny RNA and collect.

7) 400 μ l Wash Solution A are added microRNA Enrichment Column, centrifugal (14000g, 1min), it is ensured that after Wash Solution A is all by Column, abandon flowthrough, re-assembly Column and Collection Tube。

8) repeat previous step 2 times, clean microRNA Enrichment Column, it is ensured that Wash Solution A After all by Column, abandon flowthrough, re-assembly Column and Collection Tube.

9) recentrifuge 2min, makes gel column be completely dried, and abandons Collection Tube, by microRNA Enrichment Column inserts supporting 1.7ml Elution Tube.

10) 50 μ l Elution Solution A are added to Column, centrifugal (200g, 2min), it is ensured that Elution Solution A, all by Column, collects eluent.

11) first two steps are repeated if desired to guarantee at utmost to collect microRNA.

12) the microRNA Sample preservation collected was taken out in-70 DEG C of refrigerators, used time.

1.2.3 joint fluid total microRNA concentration and integrity mensuration'

Concrete grammar is the same.

1.2.4 reverse transcription RT

Concrete grammar is the same.

1.2.5 fluorescence quantitative PCR detection

Concrete grammar is the same.

1.2.6PCR detection product gel electrophoresis

Concrete grammar is the same.

1.3 statistical analysis

Use SPSS 22.0 software to carry out statistical analysis, use single factor test ANOVA variance analysis to miRNA-between many groups Compare between 140 relative expression quantity groups, use Spearman rank correlation rank test to analyze miRNA-140 relative to table simultaneously Dependency between the amount of reaching and OAKL classification.P < 0.05 thinks there is significant difference.

2 results

2.1 joint fluid Specimen origin patient's basic conditions

(KL 0 grade), light is collected normally altogether from Huaxi Hospital Attached to Sichuan Univ orthopaedics in-patient department and outpatient service joint puncture room Degree (KL1-2 level), moderate (KL 3 grades) and severe (KL 4 grades) each 10 examples of OA patient's Knee Joint Fluid specimen, patient's basic document is shown in Table 5, each group typical case's X sheet.

Table 5 joint fluid Specimen origin patient's basic document

In 2.2 human knee joint liquid, miRNA-140 expresses Changing Pattern

All can detect that miRNA-140 expresses in normal and OA joint fluid, in OA joint fluid, miRNA-140 expresses the most relatively Normal group significantly reduces, group difference statistically significant (F=15.275, p < 0.001).Increase the weight of with OA degree, miRNA-140 Expression gradually decreases, and in severe OA joint fluid, miRNA-140 expresses minimum (table 6).

The relative expression quantity of miRNA-140 in table 6 human knee joint liquid

In 2.3 human knee joint liquid, miRNA-140 expresses the correlation analysis with the OA order of severity

Use Spearman rank correlation rank test to analyze to find, miRNA-140 relative expression quantity in human knee joint liquid It is notable negative correlation (r=-0.924p < 0.001) with KL classification.

3 discuss

OA is apt to occur in many joints big, movable of bearing a heavy burden, such as knee joint, hip, spinal column (cervical vertebra and lumbar vertebra), ankle and hands etc., Chang Weidan Send out or the morbidity of several joint^[6].Gently, moderate OA mainly based on expectant treatment, therapeutic modality includes physical therapy, oral anti-inflammatory analgesic Medicine (NSAIDs class medicine) and cartilage nutrient drug (glucosamine, chondroitin sulfate etc.), joint cavity injection hyaluronic acid sodium or Steroid hormone etc..Severe OA patient articular's pain and deformity are substantially, big to Quality of Life, effect of conservative treatment is not good enough and Easily recurrence, for relief of symptoms and quality of making the life better, joint replacement has become the first-selected therapeutic scheme of patient greatly^[7]。 Although joint replacement surgery can finally be alleviated affected joints pain, improve function of joint, but operation there is also a lot of complication, than As patient body and spirit are hit bigger, postoperative there is certain time convalescent period, have certain infection, thrombosis hematoma to be formed And prosthetic loosening equivalent risk, it is contemplated that the person of lasting a long time there is also artificial joint and the problem such as overhauls, and the most also can patient simultaneously Cause certain burden.Therefore, find and can effectively delay, stop the new method of even repairing articular cartilage regression be all the time The emphasis of OA correlational study and difficult point.As described in forward part is tested, existing discovery miRNA-140 can be as a weight for the treatment of OA Want point of penetration, but because OA is often single-shot or the morbidity of several joint, the risk of system application miRNA-140 treatment OA and effect are all Uncertain, and currently also temporarily without the relevant report of system application miRNA, so, topical application miRNA-140 perhaps has more Feasibility.

MiRNA-140 is stable in human articular cartilage to express, and its expression increases the weight of to gradually decrease with OA degree, and we just guess Survey and whether joint fluid there is also miRNA-140 expression？Present invention demonstrates miRNA-140 to express in human knee joint liquid, And its expression increases the weight of to be gradually lowered with OA degree, becoming notable negative correlation with the OA order of severity, this is thin in articular cartilage with it Expression Changing Pattern in born of the same parents is consistent.For in joint fluid miRNA-140 carry out source problem, there is no at present pertinent literature report. Chondrocyte is unique cell component in cartilaginous tissue, and ripe miRNA-140 synthesizes wherein.In OA cartilaginous tissue, Chondrocyte quantity reduces, and miRNA-140 synthetic quantity also just reduces.Thus it is speculated that miRNA-140 may source in Knee Joint Fluid Chondrocyte in articular cartilage tissue, but concrete source and generation mechanism thereof need further.Additionally, joint fluid is by closing The secretion of Jie Nei synovial tissue produces, and can enter cartilage matrix by osmosis and provide nutritional support for chondrocyte, since mesh Before have proven to miRNA-140 and can express in joint fluid, then miRNA-140 the most also can penetrate into cartilage base with joint fluid Matter acts on chondrocyte？Consulting literatures finds, the most temporarily without relevant report.

Cartilage degeneration is mainly degraded to principal character with chondrocyte terminal differentiation, ECM again, and MMP-13 and ADAMTS-5 It it is most important hydrolysis in extracellular matrix degrading enzyme.Front portion experiment has been proven that miRNA-140 up-regulated can promote to close In joint chondrocyte, Collagen II expresses, suppresses MMP13 and ADAMTS-5 to express, and plays bright to the change of chondrocyte OA sample Aobvious prevention and repair.In conjunction with this part experimental result, it is presumed that carried by articular injection of exogenous miRNA-140 In high joint fluid, miRNA-140 expression the most also can play similar effect.Consulting literatures finds that there is no miRNA-140 at present closes The research report of joint chamber topical application.S et al. is at rat knee joints intracavitary administration miRNA-210 and it is to anterior cruciate ligament The reconstruction effect that (Anterior cruciate ligament, ACL) damages, finds that articular cavity application miRNA-210 can Significantly improve ligament VEGF (VEGF) and the expression of FGF2 (FGF2), promote blood Pipe generates, and the final ACL of quickening repairs^[4].Kawanishi et al. is further discovered that rat knee joints chamber injection miRNA-210 subsequently Meniscus white area injury repairing can be promoted by raising Collagen II, VEGF and FGF2 expression in meniscal cells^[5]。

Therefore, propose to assume based on early stage result and existing document support: can be effective by joint cavity injection miRNA-140 Delay, stop even repairing articular cartilage regression.In order to verify that this, it is assumed that will verify at next step, first builds rat knee joint OA model, then injects rat miRNA-140agomir by joint puncture, finally selects appropriate time point in OA process Observe rat knee joints cartilage degeneration degree and explore its mechanism of action.

4 conclusions

People normally and all can detect that miRNA-140 expresses in OA joint fluid, and its expression increases the weight of gradually to subtract with OA degree Few, it is notable negative correlation with the OA order of severity.

Below in conjunction with joint cavity injection miRNA-140 to the regulating and controlling effect of rat knee joints cartilage degeneration and mechanism to this Bright further illustrate.

1 material and method

1.1 experiment material

1.1.1 laboratory animal

3 monthly age fully-developed SPF level male SD rats totally 54, purchased from Da Shuo animal Science and Technology Ltd., body weight 300 ±20g.Rat is raised in Sichuan University's Experimental Animal Center after buying, and ambient temperature maintains 16 DEG C-25 DEG C, and relative humidity is tieed up Holding at 60%-80%, conventional feeds and hello water, 4/cage sub-cage rearing, before experiment, at least adaptability is fed one week.All animals Operation is all performed by Sichuan University's management of laboratory animal regulations.

2.1.2 main agents

Other reagent are domestic or Import Analysis pure level high-quality reagent.

1.1.3 the preparation of main solution

0.5% Toluidine blue staining liquid: after toluidine blue powder 0.1g+2ml 70% ethanol fully dissolves, then add 18ml 1%NaCl solution (1gNaCl+100ml distilled water, matching while using), mixing, filter paper filtering.

2.1.4 key instrument

1.2 experimental technique

1.2.1 rat knee joint OA model is set up

Taking rat of the same age 12, wherein 3 be only used as normal control, 9 carry out the modeling of knee joint OA.Modeling operation process exists Complete between Sichuan University's Experimental Animal Center SPF level SD rat operation, use and cut off medial collateral ligament+excision medial meniscus Method^[4], whole operation process strict aseptic technique, concrete modeling method is as follows:

1) preoperative title rat body weight, 10% chloral hydrate consumption needed for anaesthetizing by 3-4ml/kg Rapid Dose Calculation, through abdominal cavity Injecting anesthetic rat.

2) after anesthesia is satisfied, centered by knee joint, shave except the most about 4cm and left and right about 2cm size area with shaving machine Hair.

3) by preserved skin rat supine position is on operating board, in addition to operated hind limb, remaining limbs is fixed.

4) picking 5% betagen solution sterilization knee surgery preserved skin district 2 times with cotton balls, paving makes aseptic disposable hole by oneself Towel.

5) cutting downwards skin with No. 15 scalpels along patella and patellar ligament medial border stringer, otch is about 0.5-1cm, cuts Open subcutaneous each layer and Musclar layer, until exposing joint capsule.

6) with eye scissors, joint capsule is cut off inside patella and patellar ligament, before passivity separates shin and intercondylar fossa fat pad, With aseptic cotton balls pressing haemostatic when operation process has hemorrhage.

7) stretch knee joint, patella is pushed to outside post-buckling knee joint, fully exposes medial meniscus anterior angle and inner side Ligamena collateralia, dips in aseptic NS with cotton balls in operation process and keeps joint to moisten.

8) cut off medial collateral ligament, outward turning tibia with stylet, fully expose medial meniscus anterior angle and body, use ophthalmology Cut and carefully extract medial meniscus, during being somebody's turn to do, notice that sharp knife and shears are sure not to injure femoral condyles cartilage.In inspection is cut Side meniscus is the most complete, especially meniscus relief angle.

9), after determining and completely cutting medial meniscus, betagen solution and NS rinse successively, stretch knee joint, reset kneecap Bone, sews up joint capsule, 5-0 wire discontinuous sewing skin incision continuously with 5-0 absorbable thread, and betagen solution sterilization is cut again Mouthful.

10) postoperative lumbar injection penicillin (40,000 u/ml) 1ml prevents to infect, and divides cage by number, put back to after rat is clear-headed Rat room routine is raised, and makes it the most movable and feed water inlet.

Within postoperative 1st, 2,3 days, check rats wound, if any sepage or redness, give otch with betagen solution and disappear Poison.

1.2.2 rat knee joint OA model is identified

Modeling Post operation the 4th week, 8 weeks and 12 weeks, the method anaesthetized by excess puts to death rat, each time point execution 3 Only,

Take 3 normal rats as normal control.Surgical exposure knee joint, is identified by gross examination of skeletal muscle and histological observation Rat knee joint OA modeling is the most successful.

1.2.2.1 gross examination of skeletal muscle

Fully exposing knee joint femoral condyle, perusal also records condyle of femur heavy burden district under the same conditions with digital camera Articular cartilage degeneration situation.The main contents of gross examination of skeletal muscle include: the smoothness of articular cartilage face and color and luster, whether cartilage surface There are crack, defect, ulcer and degree thereof, if having subchondral bone exposed.

1.2.2.2 tissue slice is observed

(1) specimen sampling and process

1), after gross examination of skeletal muscle terminates, it is rapidly separated modeling knee joint, carefully cuts periarticular soft tissues, it is ensured that condyle of femur Completely, it is immediately placed in the 50ml centrifuge tube filling enough 10% paraformaldehydes, floods knee joint specimen completely, fixing 24- 48h。

2) it is transferred to knee joint specimen to fill in the 50ml centrifuge tube of enough 20%EDTA liquid, decalcification 4 weeks, changes weekly Decalcifying Fluid.

3) take out knee joint specimen, repair the irregular osseous tissue of condyle of femur near-end with clean sharp knife blade, along between condyle Nest center line sagittal plain cuts condyle of femur open, abandons condylus lateralis femoris, retains medial condyle, continues decalcification 2 weeks.

4) after decalcification terminates with tap water flowing water rinse 12-24h, step by step acetone dehydration, dimethylbenzene transparent.

5) embed after waxdip.

6) cut into slices along sagittal plane, cut into slices every 300 μm, thickness 5 μm, it is stored in 4 DEG C of refrigerators standby.

(2) HE dyeing

1) paraffin section is placed in 60 DEG C of 20min rewarmings, then dewaxes through dimethylbenzene (I) and dimethylbenzene (II) each 10min.

2) successively through dimethylbenzene (I), (II) each 5min, 100% ethanol 2min, 95% → 80% → 75% ethanol is each 1min, distillation washing 1min.

3) brazilwood extract dyeing liquid soaking and dyeing 15min, tap water rinses 30s.

4) 1% acidic alcohol differentiation 30s, tap water rinses 30s.

5) promoting blue immersion profit and return blue 10-30s, tap water soaks 15min or 50 DEG C of warm water soaking 5min.

6) 5% eosin stains liquid dyeing 2min.

7) successively through 95% (I) → 95% (II) → 100% (I) → 100% (II) each 1min of ethanol, dehydration.

8) successively through dimethylbenzene carbolic acid → dimethylbenzene (I) → dimethylbenzene (II) each 1min, transparent.

9) neutral gum mounting.

(3) Toluidine blue staining

1) paraffin section is placed in 60 DEG C of 20min rewarmings, then through dimethylbenzene (I) → dimethylbenzene (II) each 15min dewaxing.

2) successively through 100% (I) → 100% (II) → 95% (I) → 95% (II) → 80% → 75% each 1min of ethanol, Distillation washing 1min.

3) after section being put into 0.5% aqueous Toluidine Blue solution 10min.

4) tap water flowing water rinses 2min.

5) acetone differentiation, stops when understanding to bone chondrocyte structure.

6) successively through 70% → 80% → 90% → 95% (I) → 95% (II) → 100% (I) → 100% (II) ethanol Each 1min, dehydration.

7) successively through dimethylbenzene (I) → dimethylbenzene (II) each 10min, transparent.

8) neutral gum mounting.

(4) tissue slice is observed

Often the tissue slice of HE and Toluidine blue staining that group specimen randomly selects same position carries out histological observation, main Observation index is wanted to include Cartilage tissue constructs, chondrocyte quantity, coloration of substrates and tide line integrity, and according to improvement Mankin ' s method carries out histological score (table 7).

Table 7 articular cartilage tissue Mankin ' s grade form detailed rules and regulations

1.2.3 rat knee joints intracavitary administration miRNA-140agomir

Modeling postoperative 1 week, row joint puncture injection in the case of modeling operative incision heals substantially, this process is four Completing between river university Experimental Animal Center SPF level SD rat operation, whole process strict aseptic technique, concrete grammar is as follows:

1.2.3.1 laboratory animal packet

This part needs rat 42 altogether, and wherein 6 rats are left intact as Normal group, and 36 rat OA build Mould Post operation is randomly divided into miRNA-140agomir group and miRNA-140control group, often group 18.

1.2.3.2miRNA-140agomir injection preparation

5nmol miRNA-140agomir lyophilized powder is taken out, uses front brief centrifugation, be configured to the distilled water of sterilizing 100 μ l injection, now join before experiment every time.MiRNA-140control injection compound method is the same.

2.2.3.3 intraarticular injection miRNA-140agomir

1) rat body weight, 10% chloral hydrate consumption needed for anaesthetizing by 3-4ml/kg Rapid Dose Calculation, warp are weighed before operation Intraperitoneal injection of anesthesia rat.

2) after anesthesia is satisfied, by rat supine position on operating board, in addition to operated hind limb, remaining limbs is carried out solid Fixed.

3) taking knee space midpoint inside patellar ligament is entry point, picks 5% betagen solution sterilization knee joint with cotton balls Operating space, joint.

4) aseptically with microsyringe puncture enter articular cavity, inject 100 μ l miRNA-140agomir or MiRNA-140control injection.

5), after gently pressing with aseptic cotton balls and pulling out pin, continue compressing 2-5min, prevent injection from leaking outside.

Divide cage to put back to rat room routine after rat is clear-headed by number to raise, make it the most movable and feed water inlet.

1.2.4 the action effect of articular cartilage degeneration is detected by intraarticular injection miRNA-140

By preceding method, miRNA-140agomir group and miRNA-140control group modeling postoperative 4th week, 8 weeks and Within 12 weeks, respectively putting to death 6 rats, 6 normal rats are as comparison.Surgical exposure knee joint, is cut by gross examination of skeletal muscle, pathological tissue Sheet dyeing and Cell immunohistochemical staining method detection joint cavity injection miRNA-140agomir are to rat knee joints cartilage degeneration Action effect and molecular mechanism.

1.2.4.1 gross examination of skeletal muscle

Fully exposing knee joint femoral condyle, perusal also records condyle of femur heavy burden district under the same conditions with digital camera Articular cartilage degeneration situation.The main contents of gross examination of skeletal muscle include: the smoothness of articular cartilage face and color and luster, whether cartilage surface There are crack, defect or ulcer and degree thereof, if having subchondral bone to expose.

1.2.4.2 tissue slice is observed

(1) HE dyeing

Concrete grammar is the same.

(2) Toluidine blue staining

Concrete grammar is the same.

(3) Mankin ' s histological score

Concrete grammar is the same.

1.2.4.3 immunohistochemistry views

Take tissue slice and carry out immunohistochemical staining, observe in cartilaginous tissue Collagen II, MMP13 and ADAMTS-5 protein expression situation, and corresponding protein expression is carried out sxemiquantitative divide by calculating positive region average optical density value Analysis.

(1) immunohistochemical method

1) paraffin section is placed in 60 DEG C of oven cooking cycle 20-30min rewarmings.

2) dimethylbenzene (I) → dimethylbenzene (II) each 20min is sequentially passed through, then sequentially pass through 100% → 95% → 80% → 70% ethanol respectively soaks 5min, and tap water flowing water rinses 5min, distilled water flushing 1 time.

3) PBS 3 times, each 5min.

4) every section drips appropriate 3%H₂O₂, incubated at room temperature 10min, deactivating endogenous peroxydase.

5) PBS 3 times, each 5min.

6) drip the one of suitable dilution ratio and resist 30 μ l, fully cover tissue, put in wet box 4 DEG C of refrigerator overnight.

7) rewarming in 37 DEG C of baking boxs, 20min, PBS 3 times, each 5min.

8) PBS is removed, every section dropping 30 anti-working solutions of μ l bis-, incubated at room temperature 30min.

9) PBS 3 times, each 5min.

10) removing PBS, every section dropping 30 freshly prepared DAB of μ l, dye under room temperature 5-10min, sees under microscope Examining dye levels, terminate dyeing in good time, tap water rinses 30min.

11) brazilwood extract dyeing liquid redyes 5min, and 0.5% acidic alcohol differentiation 10s, tap water rinses 15min and returns indigo plant.

12) sequentially pass through 95% ethanol (I) → 95% ethanol (II) each 5min, toning dehydration, then sequentially pass through 100% second Alcohol (I) → 100% ethanol (II) → dimethylbenzene (I) → dimethylbenzene (II) each 5min, tap water rinses 5min, transparent.

13) neutral gum mounting.

(2) immunohistochemistry results analysis

Each group immunohistochemical staining section all shoots and preserves picture under identical conditions, uses Image-Pro Plus (IPP) 6.0 software arranges lower detection every pictures cartilage layers positive region gross area (Area (sum)) identical and always amass Light splitting density value (Integrated optical density, IOD (sum)), calculates average optical density value (Mean optical Density, MOD)=IOD (sum)/Area (sum), protein expression is carried out semi-quantitative analysis.3 positives of each specimen selection Region carries out averaging after detection calculates, and last Statistics Application software carries out statistical analysis to group difference situation.

1.3 statistical analysis

Using Excel to collect data, SPSS 22.0 software carries out statistical analysis to data.Employing is compared between two groups of groups Mann-Whitney U checks, and p < 0.05 is considered as statistically significant.

2 results

2.1 rat knee joint OA models are identified

2.1.1 ordinary circumstance after rat knee joint OA modeling

Rat knee joint OA models average operative time 10.60 ± 1.80min, clear-headed in all rats 2h the most after surgery, operation Otch is healing in 1 week the most after surgery, without the infection of joint and rat cadavers.

2.1.2 gross findings

Respectively put to death rat 3 during modeling Post operation the 4th, 8 and 12 weeks, separately take 3 normal rats and do normal control.Expose stock Perusal articular cartilage degeneration degree use digital camera Taking Pictures recording after bone condyle, each time point typical case's articular cartilage figure Picture.

Normal rat: condyle of femur cartilage surface is smooth, bright color.

Postoperative 4 weeks: condylus medialis femoris cartilage surface is coarse, and color and luster is thin out, have no the most damaged and ulcer, without substantially Hyperosteogeny is formed.

Postoperative 8 weeks: the articular cartilage noticeable wear of condylus medialis femoris heavy burden district, there is ulcer, cartilage in partial rat articular surface Defect, subchondral bone matter expose, and the most visible hyperosteogeny is formed.

12 weeks after operation: condylus medialis femoris articular cartilage heavy wear, subchondral bone matter exposes, the most visible substantially hyperosteogeny shape Become.

2.1.3 histochemical evidence

Take condylus medialis femoris row HE and Toluidine blue staining after pathological section, and use Mankin ' s histological score pair Cartilage degeneration is evaluated, and result is as follows.

(1) HE dyeing

Each time point typical case's HE colored graph picture.

Normal rat knee cartilage: after HE dyes, staining cartilaginous matrix is homogeneous, pinkiness, chondrocyte is distributed Regular, in blueness.Articular cartilage tissue structure is clearly demarcated, can be divided into top layer, layer of dividing a word with a hyphen at the end of a line, radiating layer and calcification layer.Top layer cartilage is thin Born of the same parents' small volume, arranges in flat；Divide a word with a hyphen at the end of a line layer and radiating layer chondrocyte becomes larger and becomes round, arrange sparse；Calcification layer is soft Osteocyte volume is maximum, and being close to tide line is class columnar arrangement.

Postoperative 4 weeks: cartilage surface fibrous tissue covers substantially, top layer and middle layer cells shrank, and the thin-skinned bone of partial joint is thin Born of the same parents come off, and cartilage layers is thinning, and chondrocyte increases in diffusivity on the whole, and with a large amount of gathering cell masses, tide line obscures, portion Disjunction is split.

Postoperative 8 weeks: articular cartilage damage to calcification layer and subchondral bone intersection, chondrocyte quantity reduced, and visible Downright bad disintegrate cell, tide line structure is disorderly, fracture.

12 weeks after operation: articular cartilage damage increases the weight of further, cartilage destruction exposes to subchondral bone layer, subchondral bone, closes Nodal section destroys district and sees that fiber covers, and few chondrocyte remains, tide heading line off.

(2) Toluidine blue staining

Each time point Representative cresyl amine indigo plant colored graph picture.

Normal rat knee cartilage: after Toluidine blue staining, staining cartilaginous matrix is homogeneous, in navy blue color, cartilage Cell distribution rule.Articular cartilage tissue structure is clearly demarcated.

Postoperative 4 weeks: cartilage surface fibrous tissue covers substantially, top layer and middle layer cells shrank, partial exfoliation, and cartilage layers becomes Thin, chondrocyte increasing number, with gathering cell mass, tide line obscures, portion fractures, and cartilage matrix colours light to moderate fall Low.

Postoperative 8 weeks: articular cartilage increased wear, cartilage surface was damaged, and part is up to radiating layer, and chondrocyte quantity subtracts Few, tide line structure is disorderly, fracture, and cartilage matrix coloring moderate reduces to severe.

12 weeks after operation: articular cartilage damage increases the weight of further, few chondrocyte remains, and cartilage destruction to calcification layer is even Subchondral bone intersection, large area subchondral bone exposes, tide heading line off, and residual matrix coloring severe reduction does not even colour.

(3) Mankin ' s histological score

Model postoperative different time points rat knee joints cartilage Mankin ' s histological score and be shown in Table 8.Normal rat joint Cartilage Mankin's scoring is 0, model postoperative 4,8 and 12 weeks respectively 6.50 ± 1.52,10.17 ± 0.75 and 12.83 ± 0.75。

After table 8 modeling, two groups of rat knee joints cartilage Mankin ' s histological score of different time points compare

The impact on rat knee joint cartilage degeneration of the 2.2miRNA-140 joint cavity injection

When rat knee joint OA models postoperative 1 week, operative incision substantially heals (ditto), now after anesthetized rat, passes through joint Chamber puncture injection 50nmol/100 μ l miRNA-140agomir or miRNA-140control, observes rat ordinary circumstance.Build Respectively putting to death rat 6 for two groups during mould Post operation the 4th, 8 and 12 weeks, 6 normal rats are as comparison.By gross examination of skeletal muscle, tissue Section statining and immunohistochemical staining are observed and compare miRNA-140agomir and miRNA-140control articular cavity note Penetrate rear cartilage of rats regression situation.

2.2.1 rat ordinary circumstance after joint cavity injection miRNA-140

All rats are all clear-headed in the postoperative 2h of joint puncture.Until experiment terminates, joint cavity injection miRNA- All there is not the complication such as the infection of joint or death in 140agomir or miRNA-140control rat.Therefore, joint cavity injection MiRNA-140 is safe, feasible.

2.2.2 gross findings

Two groups of typical case's articular cartilage images of each time point.Normal rat condyle of femur cartilage surface is smooth, bright color.

Postoperative 4 weeks condylus medialis femoris cartilage surfaces of miRNA-140control group become coarse, and color and luster is dimmed；Postoperative 8 weeks Time the articular cartilage noticeable wear of condylus medialis femoris heavy burden district, part occurs that ulcer, cartilage defect and hyperosteogeny are formed；During 12 weeks after operation Condylus medialis femoris articular cartilage serious wear, subchondral bone matter large area exposes, and the most visible substantially hyperosteogeny is formed.

During postoperative 4 weeks of miRNA-140agomir group, condylus medialis femoris cartilage surface is the most smooth, and color and luster is partially dark, articular surface without Breakage and ulcer, formed without obvious hyperosteogeny；Within postoperative 8 weeks, condylus medialis femoris heavy burden district articular cartilage surface slightly shows coarse, even See that aphtha and hyperosteogeny are formed；The abrasion of condylus medialis femoris articular cartilage, rough surface, partially visible ulcer and bone during 12 weeks after operation Superfluous formation.Therefore, from generally observing, joint cavity injection miRNA-140agomir can substantially delay cartilage of rats regression Process.

2.2.3 Histological results

(1) HE dyeing

Two groups of each time point typical case's HE colored graph pictures.Normal rat knee cartilage is after HE dyes, and cartilage matrix contaminates Color is homogeneous, chondrocyte distribution rule, and articular cartilage tissue structure is clearly demarcated.

Postoperative 4 weeks cartilage surface fibres visible tissues of miRNA-140control group cover, and cartilage layers is thinning, part cell Shrink, articular surface chondrocyte comes off, and chondrocyte increases on the whole, and with gathering cell mass, tide line obscures, and part is disconnected Split；Visible joint cartilage destruction when postoperative 8 weeks, part is as deep as calcification layer or subchondral bone intersection, cartilage layers inner cell quantity Significantly reducing and visible non-viable non-apoptotic cell, tide line structure is disorderly, fracture；During 12 weeks after operation, articular cartilage damage increases the weight of further, soft Osteoplaque weares and teares totally, and few chondrocyte remains, and subchondral bone exposes, and destruction district, partial joint face fibres visible covers, tide line Disappear.

Postoperative 4 weeks cartilage structures of miRNA-140agomir group are normal, and chondrocyte without significant change or slightly increases Many, have no obvious cell gathering phenomenon, tide line is complete；When postoperative 8 weeks, cartilage layers is the most thinning, rough surface, chondrocyte quantity Normally or slightly reducing, partially visible gathering cell mass, part tide line is disorderly；During 12 weeks after operation, articular cartilage damage increases the weight of, portion Point top layer comes off, and chondrocyte quantity significantly reduces, and tide line structure is disorderly, fracture.

(2) Toluidine blue staining

Two groups at each time point Representative cresyl amine indigo plant colored graph picture.Normal rat knee cartilage is through Toluidine blue staining After, staining cartilaginous matrix is homogeneous, chondrocyte distribution rule, and articular cartilage tissue structure is clearly demarcated.

Postoperative 4 weeks visible chondrocyte increasing number of miRNA-140control group are with gathering cell mass, cartilage matrix Colour light to moderate reduction；When postoperative 8 weeks, layer of articular cartilage is the most thinning, and cartilage destruction is deepened further, chondrocyte Quantity significantly reduces, and cartilage matrix coloring severe reduces, and part severe reduces；Cartilage layers almost all abrasion during 12 weeks after operation, Few chondrocyte residual, large area subchondral bone exposes, and residual matrix coloring severe reduction does not even colour.

During miRNA-140agomir group 4 weeks, chondrocyte quantity is without significantly reducing, slight fall after cartilage matrix coloring is normal Low；When postoperative 8 weeks, layer of articular cartilage is thinning, and chondrocyte quantity reduces, and cartilage matrix coloring mild to moderate reduces；12 weeks after operation Time cartilage wear increase the weight of, cartilage layers is thinning, rough surface, cell quantity reduce, in coloration of substrates to severe reduce.

(3) Mankin ' s histological score

Two groups of Mankin ' s histological score in different time points are shown in Table 9, each time point miRNA-140agomir group Mankin ' s scoring is substantially below matched group, difference statistically significant (p < 0.05).Visible, histological observation is demonstrate,proved further Real joint cavity injection miRNA-140agomir can substantially delay cartilage of rats regression process.

9 liang of table group rat different time points articular cartilage Mankin ' s histological score compares

2.2.4 immunohistochemistry results

Respectively put to death rat 6 during OA modeling Post operation the 4th, 8 and 12 weeks, take condylus medialis femoris row after tissue slice immune Histochemical method detects, Collagen II, MMP13 and ADAMTS-5 protein expression situation in light Microscopic observation cartilaginous tissue, sun Property is expressed as brown yellow granule, realizes protein expression by IPP6.0 computed in software positive region average optical density value (MOD) value Semi-quantitative analysis, and compare miRNA-140agomir group and miRNA-140control group Collagen II, MMP13 and ADAMTS-5 protein expression.

(1) regulating and controlling effect that Collagen in cartilaginous tissue II is expressed by joint cavity injection miRNA-140agomir

Collagen II protein expression situation in light Microscopic observation cartilaginous tissue is visible big in normal rat cartilaginous tissue Amount brown color positive expression, is predominantly located at top layer, divide a word with a hyphen at the end of a line layer and radiating layer.

4 weeks after surgery positive expressions of miRNA-140control group substantially weaken, and divide a word with a hyphen at the end of a line layer and radiating layer weakens and becomes apparent from； When postoperative 8 weeks, positive expression weakens further, only at the cartilage top layer destroyed and a small amount of positive expression seen from layer of dividing a word with a hyphen at the end of a line；After surgery When 12 weeks, cartilage layers disappears totally, only remains the positive expression that minimal amount of chondrocyte companion is faint.

MiRNA-140agomir group 4 weeks after surgery still seen from stronger positive expression；When postoperative 8 weeks, positive expression weakens, Mainly radiating layer is expressed and is weakened；During 12 weeks after operation, positive expression weakens further, but the most visible on irregular cartilage top layer To certain positive expression.

MOD quantitative analysis results is pointed out, and miRNA-140agomir group each time point postoperative MOD value is all remarkably higher than MiRNA-140control group, difference statistically significant (table 10).Thus illustrate, increase the weight of with articular cartilage degeneration degree, soft In osseous tissue, Collagen II expresses and gradually decreases；And by joint cavity injection miRNA-during articular cartilage degeneration 140agomir can significantly slow down Collagen II PD in cartilaginous tissue.

Collagen II expression in two groups of rabbit cartilage tissues of table 10 different time points (MOD, × 10^-3)

(2) regulating and controlling effect that MMP13 in cartilaginous tissue is expressed by joint cavity injection miRNA-140agomir

MMP13 protein expression situation in light Microscopic observation cartilaginous tissue, visible minimal amount palm fibre in normal rat cartilaginous tissue Yellow positive expression, is dispersed in and is distributed in divide a word with a hyphen at the end of a line layer and radiating layer.

4 weeks after surgery positive expressions of miRNA-140control group are remarkably reinforced, and deep layer enhancing becomes apparent from；When postoperative 8 weeks Positive expression further enhances, and also shows strong positive and express in the articular surface of destruction；When 12 weeks after surgery, cartilage layers abrasion is tight Weight, in the cartilaginous tissue of remaining, visible strong positive is expressed.

4 weeks after surgery visible significantly positive expressions of miRNA-140agomir group, but low compared with matched group；When postoperative 8 weeks Positive expression has strengthened, and the enhancing of cartilage deep layer becomes apparent from；During 12 weeks after operation, cartilage shallow-layer positive expression strengthens.

MOD quantitative analysis results is pointed out, and miRNA-140agomir group each time point postoperative MOD value is substantially less than MiRNA-140control group, difference statistically significant (table 11).Thus illustrate, MMP13 in normal articular cartilage in low Horizontal expression, increases the weight of with articular cartilage degeneration degree, and in cartilaginous tissue, MMP13 expresses and is gradually increased；And at articular cartilage degeneration During can significantly inhibit MMP13 protein expression in cartilaginous tissue by joint cavity injection miRNA-140agomir.

MMP13 expression in two groups of rabbit cartilage tissues of table 11 different time points (MOD, × 10^-3)

(3) regulating and controlling effect that ADAMTS-5 in cartilaginous tissue is expressed by joint cavity injection miRNA-140agomir

In normal rat cartilaginous tissue, visible a small amount of brown yellow granule is expressed, and is dispersed in and is distributed in cartilaginous tissue holostrome.

4 weeks after surgery positive expressions of miRNA-140control group are remarkably reinforced, and top layer is obvious；Positive table when postoperative 8 weeks Reaching and further enhance, shallow-layer is expressed and is better than deep layer；When 12 weeks after surgery, cartilage layers is the most thinning, in the cartilaginous tissue of remaining Visible strong positive is expressed.

During miRNA-140agomir group 4-8 week after surgery, positive expression has strengthened, and is concentrated mainly on deep layer；Postoperative 12 During week, cartilage holostrome is expressed and is remarkably reinforced.

MOD quantitative analysis results is pointed out, and miRNA-140agomir group each time point postoperative MOD value is substantially less than MiRNA-140control group, difference statistically significant (p < 0.05) (table 12).Thus illustrating, ADAMTS-5 is in normal cartilage In low expression level in tissue, increasing the weight of with articular cartilage degeneration degree, in cartilaginous tissue, ADAMTS-5 expresses and is gradually increased；And ADAMTS-5 egg in cartilaginous tissue can be significantly inhibited by joint cavity injection miRNA-140agomir during articular cartilage degeneration White expression.

Two groups of rabbit cartilage tissue ADAMTS-5 expressions of table 12 different time points (MOD, × 10^-3)

Articular cartilage degeneration is one of principal character of OA, many with II Collagen Type VI (Collagen II) and albumen the most again The ECM such as sugar (Aggrecans) are degraded to main.MMP-13 and ADAMTS-5 is most important hydrolytic enzyme in extracellular matrix degrading enzyme, L Crinis Carbonisatus Existing miRNA-140 is a negative regulatory factor of MMP13, changes using proinflammatory factor IL-1 beta induced chondrocyte OA sample Cheng Zhong, miRNA-140 can suppress MMP13 to express；They use luciferase reporter gene analysis to find miRNA-140 further Can directly be combined with MMP13 3 ' end UTR site.M et al. finds in testing in vitro that proinflammatory factor IL-1 β intervention can reduce soft Osteocyte miRNA-140 expresses, increases ADAMTS-5 expression；After improving miRNA-140 expression by gene transfection, IL-1 β This inducing action substantially weaken.It is further discovered that at miRNA-140^-/-In mice, ADAMTS-5 expresses increases, and Reducing in miRNA-140 transgenic mice, external luciferase reporter gene analysis is it has also been found that ADAMTS-53 ' end UTR includes MiRNA-140 binding site.Therefore, ADAMTS-5 expression inhibiting is also the effect machine that miRNA-140 stops articular cartilage degeneration One of system.In the present invention, by joint puncture injection of exogenous miRNA-140, found that induce at rat OA model During, in articular cartilage, Collagen II loses relatively matched group and substantially slows down, and MMP13 and ADAMTS-5 expresses substantially quilt Suppression, this is consistent with cell experiment in Part I and the past transgenic/Gene Knock-Out Animal Model experimental result.Accordingly it is also possible to Conclude that in this experiment, joint cavity injection miRNA-140agomir delays the effect of articular cartilage degeneration to be at least partly logical Cross MMP13 and ADAMTS-5 in suppression articular cartilage to express and realize.

The present invention has been successfully established rat OA model, and joint cavity injection miRNA-140agomir is safe, feasible.

The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all essences in the present invention Any amendment, equivalent and the improvement etc. made within god and principle, should be included within the scope of the present invention.

Claims

1. the medicine to articular cartilage degeneration regulating and controlling effect, it is characterised in that this regulating and controlling effect to articular cartilage degeneration And the medicine of mechanism is miRNA-140.

2. the medicine miRNA-140 to articular cartilage degeneration regulating and controlling effect verified described in claim 1 is in osteoarthritis Regulating and controlling effect method in chondrocyte, it is characterised in that checking regulating and controlling effect method in osteoarthritis chondrocytes is:

First verify that miRNA-140 is normal people and expression rule in osteoarthritis chondrocytes；

3. verify the regulating and controlling effect method in osteoarthritis chondrocytes as claimed in claim 2, it is characterised in that checking MiRNA-140 regulating and controlling effect method in osteoarthritis chondrocytes particularly as follows:

(1) according to Kellgren and Lawrence OA iconography grade scale, collector is normal and light, in, severe OA new Fresh cartilage samples, in-vitro separation, cultured cartilage cell are also identified；

(2) use fluorescence quantitative PCR method detection normal and miRNA-140 expression in OA chondrocyte, analyze it and express change Law；

(3) miRNA-140mimic using variable concentrations transfects chondrocyte, after transfection different time points by PCR and Western blotting method detection cell Collagen II and MMP13 gene and protein expression level, screen miRNA- The 140mimic transfection optium concentration of chondrocyte and effect detection Best Times, described variable concentrations be 0,25,50,100 and 200nM；

(4) miRNA-140mimic or miRNA-140inhibitor using optium concentration transfects normal and OA chondrocyte, Cell Collagen II, MMP13, ADAMTS-5, Sox9 and Runx2 gene and protein expression level is detected at Best Times, than Relatively miRNA-140 is each after raising or lowering organizes said gene and protein expression difference in chondrocyte, analyzes miRNA-140 to OA The regulating and controlling effect of chondrocyte and molecular mechanism.

4. the medicine miRNA-140 to articular cartilage degeneration regulating and controlling effect verified described in claim 1 is in osteoarthritis Regulating and controlling effect method in joint fluid, it is characterised in that miRNA-140 is at the regulating and controlling effect in osteoarthritic joint liquid in checking Method is:

Detection people is normal and the expression rule of miRNA-140 in OA joint fluid, and verifies that miRNA-140 expression is serious with OA The dependency of degree.

5. checking miRNA-140 as claimed in claim 4 is in the regulating and controlling effect method in osteoarthritic joint liquid, its feature Be, checking miRNA-140 in the regulating and controlling effect method in osteoarthritic joint liquid particularly as follows:

By Spearman rank correlation rank test checking joint fluid between miRNA-140 relative expression quantity and KL classification Dependency.

6. the medicine joint cavity injection miRNA-140 to articular cartilage degeneration regulating and controlling effect verified described in claim 1 Regulating and controlling effect method to rat knee joints cartilage degeneration, it is characterised in that joint cavity injection miRNA-140 is to rat in this checking The regulating and controlling effect method of knee cartilage regression is: by building rat knee joints OA model and identifying.

7. the checking joint cavity injection miRNA-140 regulating and controlling effect to rat knee joints cartilage degeneration as claimed in claim 6 Method, it is characterised in that the regulating and controlling effect method of rat knee joints cartilage degeneration is had by this checking joint cavity injection miRNA-140 Body is:

With 3 monthly age fully-developed SPF level SD rats as object, take rat 12, wherein 3 be only used as normal control, remaining 9 The method using excision medial meniscus and medial collateral ligament sets up rat knee joints OA model, the 4th, 8 and 12 after surgery Week each execution 3, is marked by gross examination of skeletal muscle, tissue section strain and Mankin ' s and identifies OA model；

Take rat 42, wherein 6 be only used as normal control, be left intact；Remaining 36 are randomly divided into experimental group and comparison Group, often organizes each 18, when modeling postoperative 1 week by joint puncture to experimental group rat knee joints intracavitary administration 5nmol/ 100 μ l miRNA-140agomir, the miRNA-140control of matched group injection same dose, observe two groups of rat joint cavity With or without complication after injection；

Within postoperative 4th, 8 and 12 weeks, 6 rats of each execution are often organized, by gross examination of skeletal muscle, histochemical stain and Mankin ' in modeling S methods of marking compares two groups of rat knee joints cartilage degeneration degree；

Phase in two groups of rat knee joints cartilaginous tissues is compared by immunohistochemical staining and protein expression average optical density value Closing protein expression situation, checking joint cavity injection miRNA-140 is to the regulating and controlling effect of rat knee joints cartilage degeneration and molecule machine System.