CN102899287B - Method for inducing differentiation from mesenchymal stem cells to cartilage cells and application of mesenchymal stem cells in osteoarthritis - Google Patents

Abstract

The invention relates to a method for inducing differentiation from mesenchymal stem cells to cartilage cells and application of the mesenchymal stem cells in osteoarthritis, and provides a method for preparing umbilical cord mesenchymal stem cells differentiated to the cartilage cells, mesenchymal stem cells differentiated to the cartilage cells and application of the prepared drug prepared by the mesenchymal stem cells in treatment of the osteoarthritis. The method comprises the following steps of: inducing and culturing the mesenchymal stem cell in a 15ml taper-bottom centrifuge tube by using a globule method; and slightly shaking the centrifuge tube for one time every 24 hours to separate cell sphere from the bottom of the tube, wherein the differentiation inducing culture medium is prepared by adding 100mu g/ml of sodium pyruvate, 10ng/ml TGFbeta3, 100nM of dexamethasone, 25mu g/ml of vitamin C, 40mu g/ml of proline and 1*ITS and 1 of premix into a high-glucose dulbecco modified eagle medium (DMEM). A non-bracket cartilage built by the method can be used for repairing damaged articular cartilages.

Description

A kind of inducing mesenchymal stem cell is to the method for chondrocyte's differentiation and the application in osteoarthritis thereof

Technical field

The present invention relates to the stem cells technology field, specifically, relate to the preparation method of the UC-MSCs of chondrocyte induction and the application in osteoarthritis thereof.

Background technology

Joint cartilage is a kind of heavy burden reticular tissue, and often because of tumour, motion, degeneration or geriatric disease cause damage; Yet joint cartilage self repair ability is limited, has caused very big difficulty for the clinical cure cartilage defect.The method that had occurred multiple treatment cartilage defect in recent years comprises from body chondrocyte cell transplantation, microfracture and inlays plasty, but these methods have its limitation separately.In recent years, tissue engineering bone/cartilage becomes the new focus of repair of cartilage research, and (Mesenchymal stem cells MSCs) is its most promising current seed cell to mescenchymal stem cell.MSCs derives to grow early stage mesoderm and an ectodermic class multipotential stem cell, finds in marrow at first.At present the main source of MSCs is adult's marrow, but adult's bone marrow MSCs cell quantity and proliferation and differentiation potential descends with the increase at age, and viral infection rate is higher, and the collection of donor MSCs must the row bone marrow puncture, and the source is restricted.Discover that MSCs extensively exists at the multiple tissue of human fetal and birth back, comprises periosteum, fat, amniotic fluid, corium, tooth, skeletal muscle, tire lung, tire liver and bleeding of the umbilicus etc.Under the subsidy of the natural fund of previous country " umbilical cord mesenchymal stem cells biological nature, hematopoiesis support research and the application in transplanting " (30570905), the collagenase of our application enhancements and the direct digestion method of enzyme that pancreatin combines, by the magnetic bead screening, from umbilical cord, obtained a large amount of MSCs.And set up a kind of method that can separate amplification umbilical cord MSCs, and operation is simple (ZL200910242375.8).According to present method, 90% mature umbilical cord can be isolated MSCs, obtains 1 * 10⁹The MSCs cell fully satisfies clinical and needs experimental study.

Prior art is not still with the trial of umbilical cord source mescenchymal stem cell to the chondrocyte induction differentiation, we are with the seed cell of umbilical cord source mescenchymal stem cell as us, it is induced to differentiate into no support cartilage, and its ability of repairing impaired joint cartilage studied, have the important clinical directive significance.

Summary of the invention

The present invention provides the method for a kind of inducing mesenchymal stem cell to chondrocyte's differentiation in first aspect, wherein, in the 15ml conical centrifuge tube, use bead method inducing culture mescenchymal stem cell, and rocked centrifuge tube gently once every about 24 hours, the cell ball was separated with the pipe end.

In a specific embodiment, the inductive differentiation medium that uses adds 100 μ g/ml Sodium.alpha.-ketopropionates as the sugared DMEM of height, 10 ng/ml TGF β 3,100 nM dexamethasone, 25 μ g/ml vitamins Cs, 40 μ g/ml proline(Pro), 1 * ITS+1 premix; Inducing the differentiation culture time is 21 days.

Preferably, described mescenchymal stem cell is umbilical cord source mescenchymal stem cell.

Aspect second of the present invention, provide the mescenchymal stem cell of prepared according to the methods of the invention to chondrocyte's differentiation.

Preferably, described mescenchymal stem cell is umbilical cord source mescenchymal stem cell.

Aspect the 3rd of the present invention, provide the application of mescenchymal stem cell in the medicine of preparation treatment osteoarthritis to chondrocyte's differentiation.

Preferably, described mescenchymal stem cell is umbilical cord source mescenchymal stem cell.

The present invention has following advantage:

1. adopt bead method inducing culture mescenchymal stem cell, can make no support cartilage, rock centrifuge tube gently once every about 24 hours, the cell ball was separated with the pipe end, guaranteed that the cell ball fully contacts with inductive differentiation medium, the mescenchymal stem cell differentiation state homogeneous in the cell ball.

2. use TGF β 3 to replace TGF β 1 commonly used in the inductive differentiation medium, reached and better induced effect.

3. umbilical cord source mescenchymal stem cell is easy with respect to the bone marrow mescenchymal stem cell separation preparation of routine, and the source is abundant.

Description of drawings

Fig. 1 is to the Toluidine blue staining result of the UC-MSCs bead of chondrocyte induction differentiation after 21 days, and red-purple proves the chondrocyte.

HE coloration result after the UC-MSCs cell suspension treatment of Fig. 2 rat bone arthritis model warp-wise chondrocyte induction.

A normal control group; The B model group; C joint cavity inner cell suspension treatment group 1; D joint cavity inner cell suspension treatment group 2; E tail vein injection cell suspension treatment group 3; F tail vein injection cell suspension treatment group 4.

Embodiment

Embodiment 1 umbilical cord source mescenchymal stem cell is induced to the chondrocyte

1. MSC growth medium individual layer amplification umbilical cord source mescenchymal stem cell:

The MSC growth medium (adds 13.5ml DF12 in per 75 square centimeters of culturing bottles, 10% human serum, 20 ng/ml EGF, 5ng/ml FGF2) cultivates P3 for umbilical cord source mescenchymal stem cell (UC-MSCs), the cell growth reaches 70-80% and merges, after using D-Hank ' s washing lotion to wash twice, adding 1.5ml pancreas enzyme-EDTA (0.25% pancreatin-0.02% EDTA) digests, microscopically is observed, and when cell presents the ball state, stops digestion with the 1.5ml human serum, cell is sucked in 50 milliliters of centrifuge tubes, fully behind the mixing, get 200 microlitres counting and determine cell quantity, all the other cell suspensions are centrifugal, 1000rpm, 10min.Outwell the supernatant after centrifugal, go down to posterity with the ratio of 1:3, be cultured to P4 generation, cell growth reaches 70-80% merges, use D-Hank ' s washing lotion to wash twice after, adding 1.5ml pancreas enzyme-EDTA digests, microscopically is observed, when cell presents the ball state, stop digestion with the 1.5ml human serum, cell is sucked in 50 milliliters of centrifuge tubes, fully behind the mixing, centrifugal, 1000rpm, 10min.Outwell the supernatant after centrifugal, add 30ml D-hank ' s washing lotion, recentrifuge behind the mixing is outwelled supernatant, adds 30ml D-hank ' s washing lotion again, behind the mixing, takes out 200 microlitres counting, and all the other cell suspensions are centrifugal, 1000rpm, 10min.Remove supernatant after centrifugal.

2. MSC growth medium re-suspended cell is adjusted cell concn to 1 * 10⁶/ ml cell.

3. Pellet(bead method) culturing cell: 1ml is contained 1 * 10⁶The cell suspension of individual cell adds in the 15ml conical centrifuge tube, and centrifugal (240g, 5 minutes) put into 37 ℃ with centrifuge tube, 5% CO₂In the cell culture incubator, after about 24 hours, rock centrifuge tube gently, the cell ball was separated with the pipe end, (high sugared DMEM adds 100 μ g/ml Sodium.alpha.-ketopropionates to change the adding inductive differentiation medium afterwards, 10 ng/ml TGF β, 3,100 nM dexamethasone, 25 μ g/ml vitamins Cs, 40 μ g/ml proline(Pro), 1 * ITS+1 premix (10 μ g/ml Insulin, 5.5 μ g/ml transferrin, 5ng/ml selenium, 5.35 μ g/ml linoleic acid), rocked centrifuge tube gently once every 24 hours, the cell ball is separated with tube wall, cartilage division culture medium of replacing in per two days.After 21 days, take out bead, Toluidine blue staining, as shown in Figure 1, red-purple proves the chondrocyte.

Embodiment 2 is to the application of UC-MSCs in osteoarthritis of chondrocyte induction

With obtain among the embodiment 1 to the cell ball of chondrocyte induction in centrifuge tube after the II Collagen Type VI enzymic digestion half an hour with 3ml 2% (w/v), 1000rpm, after 5min was centrifugal, being inoculated in floorage after resuspended with inductive differentiation medium similarly to Example 1 was 25 cm²Culturing bottle in amplification cultivation, cover with the back with the digestion of 0.5ml pancreas enzyme-EDTA, make the UC-MSCs cell suspension to chondrocyte induction.

Select healthy male Wistar rat, body weight 200 ± 50 g, 60.Be divided into the ⑴ intraarticular injection at random to the UC-MSCs cell suspension treatment group 1 of chondrocyte induction; ⑵ intraarticular injection is to the UC-MSCs cell suspension treatment group 2 of chondrocyte induction; ⑶ tail vein injection is to the UC-MSCs cell suspension treatment group 3 of chondrocyte induction; ⑷ tail vein injection is to the UC-MSCs cell suspension treatment group 4 of chondrocyte induction; ⑸ control group (intraarticular injection cell suspension solvent); ⑹ model group; ⑺ normal group, wherein preceding four groups every group 10, three groups every group 6 of backs.Modeling method is the Chloral Hydrate by 0.3 ml/100 g abdominal injection, 10 %, and after the anesthesia, rat bilateral knee joint injection 4 % papoid poloxamer phosphate buffer solutions place 2h in 50 ℃ of self-control baking boxs with lower limb, namely finish modeling after the end.The 1st week after modeling is finished was 0 week, respectively at 0,2,4 weeks injection cell suspension.Intraarticular injection group 1 and tail vein injection group 3 every injection 100 μ l 4 * 10⁷Individual/ml, intraarticular injection group 2 and tail vein injection group 4 every injection 100 μ l 2 * 10⁷Individual/ml, control group is respectively at 0,2,4 weeks injection, 100 μ l cell suspension solvents.All treated animals were put to death in the 6th week, drew materials.

After treating for 6 weeks, anaesthetize by the Chloral Hydrate of 0.3 ml/100 g body weight abdominal injection, 10 %.Abdominal aortic blood, centrifugal 10 minutes of 3000 r/min, separation of serum, 4 ℃ of preservations, standby.

Pathologic sampling: get complete right knee joint, remove surrounding skin, muscle, 10 % formalin fixed, the routine paraffin wax embedding, HE dyeing, the histopathology of observing the joint under the light microscopic changes, as shown in Figure 2.A normal control group, diagram normal cartilage structure, cartilage surface is more smooth, and continuity is good, and top layer, the layer of dividing a word with a hyphen at the end of a line, radiating layer, 4 layers of clear in structure of calcification layer can be distinguished, chondrocyte's marshalling, the matrix even dyeing, damp line is complete; The B model group, articular cartilage surface is coarse, chondrocyte and the arrangement disorder of visible dehydration pyknosis necrosis around the visible more crack, top layer, crack, each layer structure is difficult for differentiating; C joint cavity inner cell suspension treatment group 1, articular cartilage surface is smooth than model group, and reduce in the crack, top layer, but the chondrocyte fills the air and increases, and damp line owes complete; D joint cavity inner cell suspension treatment group 2, coloration of substrates shoals, and dyeing is owed evenly, and recovery situation is weaker than joint cavity inner cell suspension treatment group 1; E tail vein injection cell suspension treatment group 3, the joint cartilage structure develops to normal cartilage, but matrix dyeing still owes even; F tail vein injection cell suspension treatment group 4, the articular chondrocytes necrosis, recovery situation is inferior to tail vein injection cell suspension treatment group 3.

Through above-mentioned to the joint cartilage PATHOMORPHOLOGICAL OBSERVATION OF PULLORUM, the joint cartilage for the treatment of group than model group be improved significantly, and the cartilage injury most pronounced effects of high dosage tail vein injection cell suspension treatment osteoarthritis, to sum up, the effect that has the impaired joint cartilage of reparation to a certain degree to the UC-MSCs cell of chondrocyte induction.

Claims (4)

1. an inducing mesenchymal stem cell is to the method for chondrocyte's differentiation, and its step comprises: 1ml is contained 1 * 10⁶The cell suspension of individual mescenchymal stem cell adds in the 15ml conical centrifuge tube, and centrifugal 5 minutes of 240g puts into 37 ℃ with centrifuge tube, 5%CO₂In the cell culture incubator, after about 24 hours, rock centrifuge tube gently, the cell ball was separated with the pipe end, change the adding inductive differentiation medium afterwards; Rocked centrifuge tube gently once every 24 hours, the cell ball is separated, inductive differentiation medium of replacing in per two days with the pipe end; After 21 days, take out bead.

2. the described method of claim 1, wherein, the inductive differentiation medium that uses adds 100 μ g/ml Sodium.alpha.-ketopropionates as the sugared DMEM of height, 10ng/ml TGF β 3,100nM dexamethasone, 25 μ g/ml vitamins Cs, 40 μ g/ml proline(Pro), 1 * ITS+1premix.

3. claim 1 or 2 described methods, wherein, described mescenchymal stem cell is umbilical cord source mescenchymal stem cell.

4. the described method of claim 3, its step comprises:

1) at each 75cm²Add 13.5ml growth of mesenchymal stem cells culture medium culturing P3 in the culturing bottle for umbilical cord source mescenchymal stem cell, the cell growth reaches 70-80% merges, use D-Hank ' s washing lotion to wash twice after, adding the 1.5ml pancreas enzyme-EDTA digests, microscopically is observed, and when cell presents the ball state, stops digestion with the 1.5ml human serum, cell is sucked in 50 milliliters of centrifuge tubes, fully behind the mixing, get 200 microlitres counting and determine cell quantity, all the other cell suspensions are centrifugal, 1000rpm, 10min; Outwell the supernatant after centrifugal, go down to posterity with the ratio of 1:3, be cultured to P4 generation, cell growth reaches 70-80% merges, use D-Hank ' s washing lotion to wash twice after, adding 1.5ml pancreas enzyme-EDTA digests, microscopically is observed, when cell presents the ball state, stop digestion with the 1.5ml human serum, cell is sucked in 50 milliliters of centrifuge tubes, fully behind the mixing, centrifugal, 1000rpm, 10min; Outwell the supernatant after centrifugal, add 30ml D-hank ' s washing lotion, recentrifuge behind the mixing is outwelled supernatant, adds 30ml D-hank ' s washing lotion again, behind the mixing, takes out 200 microlitres counting, and all the other cell suspensions are centrifugal, 1000rpm, 10min; Remove supernatant after centrifugal;

Wherein, described growth of mesenchymal stem cells substratum is that DMEM/F12 adds 10% human serum, 20ng/ml EGF, 5ng/ml FGF2;

2) with growth of mesenchymal stem cells substratum re-suspended cell, adjust cell concn to 1 * 10⁶Cell/ml;

3) bead method inducing culture cell: 1ml is contained 1 * 10⁶The cell suspension of individual cell adds in the 15ml conical centrifuge tube, and centrifugal 5 minutes of 240g puts into 37 ℃ with centrifuge tube, 5%CO₂In the cell culture incubator, after about 24 hours, rock centrifuge tube gently, the cell ball was separated with the pipe end, change the adding inductive differentiation medium afterwards; Rocked centrifuge tube gently once every 24 hours, the cell ball is separated, inductive differentiation medium of replacing in per two days with the pipe end; After 21 days, take out bead;

Wherein, described inductive differentiation medium is that high sugared DMEM adds 100 μ g/ml Sodium.alpha.-ketopropionates, 10ng/ml TGF β 3,100nM dexamethasone, 25 μ g/ml vitamins Cs, 40 μ g/ml proline(Pro), 1 * ITS+1premix.

Images