CN104531615A - Method for collecting mesenchymal stem cells through rat stress fracture model - Google Patents
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Abstract
The invention provides a method for collecting mesenchymal stem cells by building a rat stress fracture model. The method comprises the steps that firstly, the rat stress fracture model fixed by an external fixing support is built, after modeling successes, fracture broken-end tissue is taken out, and the migrating cells are separated and cultured; the mechanical environment for fixing the fractured position is controlled, the mesenchymal stem cells are collected, and fracture healing is promoted. The method is easy to operate, scientific and reasonable, a shearing method is adopted to separate the migrating cells in the fracture broken-end tissues, the method is easy and convenient to implement, the injury to the cells is small, most of the cells in the fracture broken-end tissues can be separated out, operation time is short, the method can be applied to researching of the influence of the fracture healing early stress environment on the collection of the mesenchymal stem cells of the fracture broken end and further researching of indexes like differentiation of the mesenchymal stem cells, the early safe and effective rehabilitation plan of the fracture is guided and determined, and through the proper stress, the joint function is restored to be normal while more mesenchymal stem cells are collected.
Description
Technical field
The present invention relates to a kind of method of raising mescenchymal stem cell, being specifically related to a kind of method of raising mescenchymal stem cell by setting up rat stress fracture model.
Background technology
In injuries of tissues and organs reparation, organizational project and disease treatment field, although the cries such as embryonic stem cell are awfully hot, optimal cell is still autologous tissue's cell.Therefore how to obtain and utilize autologous tissue, autologous stem cells to be the problem that researchist pays close attention to always.
Now, the source of stem cell and utility theory are divided into 3 important directions substantially, and the first, embryonic stem cell (embryonic stem cell, ES); The second, mescenchymal stem cell (mesenchymal stem cells, MSC); 3rd, utilize gene reprogrammed (reprogramming) variant cell culture inductive pluripotent stem cells (induced pluripotent stemcell, iPS).The research of current ES, owing to affecting by ethics dispute, immunological rejection, experimental technique restriction etc., hinders its research and the application in clinical treatment.Mescenchymal stem cell because realizing autologous stem cell transplantation, and has in body the characteristic of moving with proliferation and differentiation and participating in repair process, and is in recent years becoming study hotspot.At present, the mescenchymal stem cell adopted in digestion method diastatic fracture broken ends of fractured bone tissue, this method needs the participation of multiple enzyme, and the operating time is long, and the use of enzyme can reduce vigor, the minimizing cell number of cell more.
In Orthopedic Clinical, apply the diseases such as autologous mescenchymal stem cell (MSC) transplantation treatment necrosis of femoral head and nonunion and achieve certain curative effect.In fractures, along with the innovation of biology internal fixtion theory and the application of new technology, recognize that protection blood is transported, set up stability and fracture reduction three balances the importance setting up union of fracture excellent biomechanical and biological environment.Union of fracture needs suitable biomechanical environment and optimum stress level, large quantity research shows, early stage in union of fracture, fine motion can promote union of fracture: 1. suitable fine motion can promote the release of fracture region capillary vessel formation and PGE2, PGE2 has stronger induced osteogenesis effect, undifferentiated mescenchymal stem cell can be accelerated under aerobic conditions, be constantly divided into scleroblast and chondroblast, promote union of fracture further; 2. osteocyte can experience the stimulation of mechanical stress, and the metabolic activity such as osteocyte, scleroblast increases; 3. prostaglandin(PG) synthesis increases, and promotes bone cell proliferation; 4. the inflammatory phase of fracture repair is extended, inflammatory phase cell and capillary vessel are all more vigorous, discharge many biochemical mediators, the osteogenesis factor etc., the ability making local organization cell be released into bone active material is recovered, thus induction local mescenchymal stem cell propagation, be divided into scleroblast or chondroblast; 5. the biological respinse that fine motion causes take part in and starts the recruiting cells of new bone formation and differentiation, so union of fracture is the most duration of response of mechanics factor promotion union of fracture in early days.But promote that the cytological mechanism of union of fracture is still not fully aware of about fine motion, if illustrate the relation between fine motion and cellular elements activity, likely will promote union of fracture by control mechanics environment, to its further investigation, finally realize fracture patient quantified controlling rehabilitation course, the fine motion that rehabilitation course is caused is in the safety range of favourable union of fracture all the time, and the various fracture complications could effectively avoiding blindly rehabilitation to cause, realize fractures target.
Summary of the invention
The object of the invention is the deficiency existed for prior art, a kind of clinical characters of meeting is provided, simple to operate, the scientific and reasonable method of being raised mescenchymal stem cell by rat stress fracture model, by controlling the mechanical environment of fixing fracture, the mescenchymal stem cell can raising a greater number participates in the process of union of fracture.There are some researches show, at the fracture initial stage, fracture local only has a small amount of scleroblast and chondroblast, if only rely on now osteoblastic quantity, conversion fracture of the femoral shaft healing time needs 200-1000.In fact, various corresponding biologically active substance can be produced by the various cells that trauma fracture activates in union of fracture different steps, greatly accelerate fracture healing process.First be propagation and the differentiation of mescenchymal stem cell, this is prerequisite and the key of union of fracture.Therefore the quantity increasing fracture local mescenchymal stem cell becomes the important foundation promoting union of fracture.The inventive method raises more mescenchymal stem cell by control mechanics environment, and mescenchymal stem cell propagation and differentiation, make scleroblast and chondroblast increasing number, effectively promote union of fracture.The present invention has important clinical meaning to instructing fractures and quantified controlling healing processes of bone fracture.First specify that union of fracture is the critical period of raising mescenchymal stem cell in early days, be also now the critical period of prevention joint accretion, therefore, limb rehabilitating should carry out in early days and need limited amount rehabilitation.The stress of limb rehabilitating activity can make fracture site raise mescenchymal stem cell, can prevent limb muscle atrophy and joint accretion again.Limb rehabilitating activity is the important measures for the treatment of fracture, determines the final effect of fractures.But the fine motion that functional rehabilitation causes must be in the safety range of favourable union of fracture all the time, the inventive method can help the safety range determining limb rehabilitating, thus realizes fracture patient quantified controlling rehabilitation course, realizes fractures target.
Technical solution of the present invention theoretical foundation: union of fracture needs participating in of the mescenchymal stem cell of q.s (MSC), and at the fracture initial stage, fracture local only has a small amount of mescenchymal stem cell, scleroblast and chondroblast, the propagation of mescenchymal stem cell and differentiation, this is prerequisite and the key of union of fracture.Therefore the quantity increasing fracture local mescenchymal stem cell becomes the important foundation promoting union of fracture.Stem cell has and produces reaction to tissue injury, can migrate, proliferation and differentiation participate in repair process.Therefore, union of fracture is the critical period promoting union of fracture in early days, early stage in union of fracture, fracture site is under the acting in conjunction of bone wound and appropriate stress stimulation, the adult stem cell of mescenchymal stem cell (MSC), Surrounding muscles and blood in marrow, will be migrated to fracture by corresponding approach, the stem cell of a greater number can be raised in fracture local like this.The present invention adopts the external fixing rack of suitable fixed stress, establishes the dry fracture site micro-motion model of rat femur, i.e. rat stress fracture model, adopts the cell in shearing method diastatic fracture broken ends of fractured bone tissue, simple and easy to do, very little to cell injury.
Technical solution of the present invention is: first set up the rat stress fracture model that exterior fixing rack is fixing, after modeling success, at different time point, taking-up fracture site tissue, isolate mesenchymal stem cells is also cultivated, for studying the impact that the early stage ambient stress of union of fracture is raised fracture site mescenchymal stem cell, and the further research to Derived from Mesenchymal Stem Cells.
Described exterior fixing rack comprises 4 pieces of draw points and 2 pieces of crossbeams, and 4 pieces of draw points are that comb teeth-shaped is evenly arranged between two pieces of crossbeams, and 2 pieces of crossbeams are fixed together by 2 pieces of Ti alloy miniature screws; Exterior fixing rack axial restraint stress is 4.5N/mm.
Italian Orphfix product selected by described draw point.The femur contact jaw diameter of draw point is 0.8mm, and the other end diameter is 1.2mm; Described crossbeam, material is high-molecular polythene medical material, and its rigidity, elasticity, toughness all meet biomechanics requirement, and has the characteristic of not blocking x light and the unique advantage fixedly securing draw point; Described Ti alloy miniature screw diameter is 2.6mm; Described exterior fixing rack length is respectively 20mm, 5.5mm, 6mm.
A kind of method of being raised mescenchymal stem cell by rat stress fracture model of 1 the present invention, step is:
1) rat stress fracture model is set up
After rat weight, 3% vetanarcol 40mg/kg intraperitoneal injection of anesthesia, operation side femur unhairing preserved skin, sterile drape, operation is carried out under stringent asepsis requirements, from lateral side of femur otch, femur is exposed from musculus lateralis externi gap, first exterior fixing rack is placed, exterior fixing rack crosses over femur total length in femur little tuberosity to condyle of femur, after fixedly completing, cut-out femur is walked crosswise with micro-saw of thickness 1mm between the fixing draw point in 2, inner side, rinse the edge of a knife, with absorbable thread suture muscles tissue, with nonabsorable suturing with thread management skin, Post operation will add the anodyne tramadol hydrochloride of 12mg/500ml in 3 days in the tap water of rat, the clindamycin of operation consent 30min and Post operation 3 days subcutaneous injection 45mg/kg, prevent bacteriological infection, after having performed the operation, rat is freely movable, steel nail every day place routine disinfection,
2) separation of cell and original cuiture
By step 1) method establishment rat stress fracture model, respectively at after modeling when the 6th, 9,12,15,18 day, put to death rat, take out fracture site tissue and be good for side femur, according to method isolated cell below: fracture site tissue phosphate buffered saline(PBS) (PBS) rinsing 2 times of taking-up, is trimmed to pasty state; Repeatedly blow and beat gently with substratum, make it through 200 eye mesh screens, collect migrating cell (migratingcells, MCS) suspension, collects unfiltered material, repeats with substratum resuspended, filter, collect and merge migrating cell suspension, centrifugal rear substratum re-suspended cell; Be separated the medullary cell of rat according to following method, PBS liquid goes out marrow, collects bone marrow cell suspension, adjustment cell concn to 10
6/ ml, add Ficoll parting liquid at 1: 1 by volume, the centrifugal 20min of 1800r/min, draws interface mononuclearcell, wash 2 times, use substratum re-suspended cell, the medullary cell of separation, migrating cell concentration are adjusted to 1 × 10 with PBS
6/ ml, is inoculated in the culturing bottle of poly-lysine bag quilt, moves into 37 DEG C, 5%CO
2incubator in cultivate; After 3 days, full dose more renews nutrient solution, and remove non-attached cell, cell density reaches 80% ~ 90%, namely goes down to posterity.
Described substratum is the DMEM substratum containing 10% foetal calf serum.
2 observe and evaluation method
1) fracture site histocyte counting: respectively at after modeling when the 6th, 9,12,15,18 day, put to death rat, take out fracture site tissue and be good for side femur, with the rinsing of D-Hanks liquid, shear the DMEM substratum that material adds equivalent, Trypan Blue, cell counting.
2) morphological observation: at the morphological feature of inverted microscope (Olympus company) observation of cell lower every day.
3) cell proliferation tracing analysis: by fracture site or derived from bone marrow the 3rd generation cell, with 2 × 10
5cells/ml/ hole is seeded in 24 orifice plates, digests 3 holes, collecting cell every 24h, and with 0.4% trypan blue living cell counting, draw growth curve.And be calculated as follows cell doubling time: TD=t × ［ lg2/ (lgN
t-lgN
0).N
t: collecting cell number; N
0: inoculating cell number; T: incubation time.
4) cell is differentiation-inducing
Osteoinductive differentiation: get fracture site or derived from bone marrow the 3rd generation cell, with 10
5the density in/hole is inoculated in 6-fL plate, treats cytogamy 80%, adds Osteogenic Induction Medium, after induction 21d, and row Alizarin red staining, and staining conditions is observed under inverted microscope.
Become chondrocyte induction differentiation: get sponge or derived from bone marrow the 3rd generation cell, with 10
5the density in/hole is inoculated in 6-fL plate, treats that cell melts 80%, adds into chondrocyte induction substratum, and after induction 21d, row alcian blue dyes, and observes staining conditions under inverted microscope.
Adipogenic induction breaks up: get fracture site or derived from bone marrow the 3rd generation cell, with 10
5the density in/hole is inoculated in 6-fL plate, treats cytogamy 90%, adds adipogenic induction substratum, after induction 21d, and oil red O stain, and staining conditions is observed under inverted microscope.
5) statistical analysis: experimental data all represents with means standard deviation.Use SPSS17.0 software analysis, utilize One-way ANOVA (One-wayANOVA) method to compare, measurement data adopts t inspection, and the comparison of enumeration data adopts χ
2inspection.With P < 0.05 for difference has significant, P < 0.01 difference is very significant.
A kind of method of being raised mescenchymal stem cell by rat stress fracture model provided by the invention, its beneficial effect is:
1) by setting up rat stress fracture model, control the mechanical environment of fixing fracture, raise mescenchymal stem cell, promote union of fracture, present method is simple to operate, scientific and reasonable.
2) relation between stress fine motion and cellular elements activity is demonstrated, can be used for the impact that the early stage ambient stress of research union of fracture is raised fracture site mescenchymal stem cell, and for the further research of the indexs such as Derived from Mesenchymal Stem Cells, the early stage rehabilitation scheme safely and effectively of fracture is determined in guidance, while raising more mescenchymal stem cells by suitable stress, joint function recovery is normal.
3) migrating cell in shearing method diastatic fracture broken ends of fractured bone tissue is adopted, simple and easy to do, very little to cell injury, cell major part in fracture site tissue can be separated, operating time is short, does not need the participation of enzyme, therefore can not reduce the vigor of cell, reduce cell number.
Accompanying drawing explanation
Fig. 1 is rat stress exterior fixing rack structure
Fig. 2 is the rat stress fracture model set up
Fig. 3 is rat stress exterior fixing rack structure iron
Fig. 4 is the morphological feature figure (100 ×) of cell
Fig. 5 is migrating cell source (a under inverted microscope, b, c) vitro differentiation of cell can be tried hard to, Fig. 5 (a) is to fat induction (oil red O stain c, 200 ×), Fig. 5 (b) is to osteogenic induction (Alizarin red staining a, 40 ×), Fig. 5 (c) is to chondrocyte induction (alcian blue dyeing b, 200 ×).
Embodiment
Below in conjunction with drawings and Examples, the inventive method is specifically described.
1 materials and methods
1,1 material
Animal and grouping: test with healthy male SD rat 36, body weight 250 ~ 300g, is provided by Shandong Academy of Medical Sciences.Be divided into control group and experimental group at random, often organize 18.
Experimental group: after external fixing rack is fixing, the complete cross-section generation fine motion of femoral shaft; Control group: after external fixing rack is fixing, femoral shaft is cross-section half only, has bone injury but without fine motion.Two groups of rats are freely movable under identical living environment.
Main raw and equipment:
Rat MSC inductive differentiation medium and nitrite ion are purchased from Guangzhou Cyagengs company;
DMEM substratum, foetal calf serum are purchased from Hangzhou folium ilicis chinensis company;
Dual anti-purchased from Gibco company;
Violet staining liquid, trypan blue and 0.25% trypsinase are purchased from green skies company;
Ficoll parting liquid is purchased from Pharmacia company;
Italian Orphfix product selected by exterior fixing rack draw point, and Titanium Alloy Screw is purchased from Johson & Johnson.
1,2 methods
1,2,1 exterior fixing rack (as shown in Figure 1,3)
Exterior fixing rack comprises the crossbeam that 4 pieces of Italian Orphfix draw points and 2 pieces of high-molecular polythene medical materials are made, 4 pieces of draw points are that comb teeth-shaped is evenly arranged between two pieces of crossbeams, 2 pieces of crossbeams are that 2.6mm Ti alloy miniature screw is fixed together by 2 pieces of diameters, and exterior fixing rack length is respectively 20mm, 5.5mm, 6mm; The femur contact jaw diameter of draw point is 0.8mm, and the other end diameter is 1.2mm.
1,2,2 rat stress fracture model is set up
after rat weight, 3% vetanarcol 40mg/kg intraperitoneal injection of anesthesia, operation side femur unhairing preserved skin.Sterile drape, performs the operation and to carry out under stringent asepsis requirements.From lateral side of femur otch, expose femur from musculus lateralis externi gap, first place exterior fixing rack, exterior fixing rack crosses over femur total length in femur little tuberosity to condyle of femur.After fixedly completing, experimental group is walked crosswise with micro-saw of thickness 1mm and is cut off femur (Fig. 2) between the fixing draw point in 2, inner side, and control group walks crosswise cut-out 1/2 femur with micro-saw of thickness 1mm between the fixing draw point in 2, inner side.Rinse the edge of a knife, with absorbable thread suture muscles tissue, with nonabsorable suturing with thread management skin.Post operation will add the anodyne tramadol hydrochloride of 12mg/500ml in 3 days in the tap water of rat, the clindamycin of operation consent 30min and Post operation 3 days subcutaneous injection 45mg/kg, prevents bacteriological infection.After having performed the operation, rat can be allowed personal movable.Steel nail every day place routine disinfection.
1, the separation and Culture of 2,3 cells
Set up rat stress fracture model, respectively at after modeling when the 6th, 9,12,15,18 day, take out the femur that fracture site tissue and rat are good for side.According to method isolated cell below: the fracture site tissue PBS rinsing 2 times of taking-up, is trimmed to pasty state; Repeatedly blow and beat gently with the DMEM substratum containing 10% foetal calf serum, make it through 200 eye mesh screens, collect migrating cell suspension, collect unfiltered material, repeat with substratum resuspended, filter, collect and merge migrating cell suspension, centrifugal rear substratum re-suspended cell; Go out marrow with PBS liquid, collect bone marrow cell suspension, adjustment cell concn to 10
6/ ml, add Ficoll parting liquid at 1: 1 by volume, the centrifugal 20min of 1800r/min, draws interface mononuclearcell, wash 2 times, use substratum re-suspended cell, the medullary cell of separation, migrating cell concentration are adjusted to 1 × 10 with PBS
6/ ml, is inoculated in the culturing bottle of poly-lysine bag quilt, moves into 37 DEG C, 5%CO
2incubator in cultivate; After 3 days, full dose more renews nutrient solution, and remove non-attached cell, cell density reaches 80% ~ 90%, namely goes down to posterity.
2 observe and evaluation method
2,1 fracture site histocyte counting
respectively at after modeling when the 6th, 9,12,15,18 day, Stochastic choice is put to death by 3 rats, and take out the femur that fracture site tissue and rat are good for side, with the rinsing of D-Hanks liquid, shearing material adds the DMEM substratum of equivalent, Trypan Blue, cell counting.
2,2 morphological observations
At the morphological feature of inverted microscope (Olympus company) observation of cell lower every day.
2,3 cell proliferation tracing analysiss
By fracture site or derived from bone marrow the 3rd generation cell, with 2 × 10
5cells/ml/ hole is seeded in 24 orifice plates, digests 3 holes, collecting cell every 24h, and with 0.4% trypan blue living cell counting, draw growth curve.And be calculated as follows cell doubling time: TD=t × ［ lg2/ (lgN
t-lgN
0).N
t: collecting cell number; N
0: inoculating cell number; T: incubation time.
2,4 cells is differentiation-inducing
Osteoinductive differentiation: get fracture site or derived from bone marrow the 3rd generation cell, with 10
5the density in/hole is inoculated in 6-fL plate, treats cytogamy 80%, adds Osteogenic Induction Medium, after induction 21d, and row Alizarin red staining, and staining conditions is observed under inverted microscope.
Become chondrocyte induction differentiation: get sponge or derived from bone marrow the 3rd generation cell, with 10
5the density in/hole is inoculated in 6-fL plate, treats that cell melts 80%, adds into chondrocyte induction substratum, and after induction 21d, row alcian blue dyes, and observes staining conditions under inverted microscope.
Adipogenic induction breaks up: get fracture site or derived from bone marrow the 3rd generation cell, with 10
5the density in/hole is inoculated in 6-fL plate, treats cytogamy 90%, adds adipogenic induction substratum, after induction 21d, and oil red O stain, and staining conditions is observed under inverted microscope.
2,5 statistical analysis
Experimental data all represents with means standard deviation.Use SPSS17.0 software analysis, utilize One-way ANOVA (One-wayANOVA) method to compare, measurement data adopts t inspection, and the comparison of enumeration data adopts χ
2inspection.With P < 0.05 for difference has significant, P < 0.01 difference is very significant.
3 interpretations of result
3,1 cell quantity
Fracture site tissue is taken out respectively the 6th, 9,12,15,18 day time, cell counts shows, control group and experimental group migrating cell quantity extend all in time and increase, experimental group migrating cell quantity the 18th day time reaches maximum value, and control group migrating cell quantity 15 days time reaches maximum value (as shown in table 1)
Cell quantity × 10 that table 1 rat femur dry fracture site stress and unstressed different time points are caught
6(
x±
s)
Compare with control group, * p < 0.05; Δ p < 0.01
3,2 morphological observation
Start adherent after migrating cell inoculation 24h, part cell presents elongated fusiformis; Full dose is changed liquid and is removed non-attached cell, and now attached cell is single dispersion or cluster growth, and cellular form is even, how in spindle shape, fusiform; After this attached cell breeds rapidly, and cell volume increases, and form becomes spindle shape, Polygons, and cell colony obviously increases, increase.Be cultured to 8 ~ 10 days cells to reach 80% ~ 90% and merge, through going down to posterity, cell shows as more homogeneous spindle shape gradually, and the growth in swirling, and control group is consistent with experimental group, they all with both derived from bone marrow attached cells plesiomorphism (Fig. 4).
3,3 cell proliferation features
Two groups of migrating cells source the 3rd generation cell growth curve be S type substantially, substantially identical with the cell growth curve of derived from bone marrow.After 24h latent period, entered logarithmic phase from the 2nd day, enter stationary phase in the 6th day cell.The doubling time calculating fracture site and bone marrow MSCs in logarithmic phase is respectively 22.6 ± 3.5h(control group), 22.1 ± 4.6(experimental group) and 21.3 ± 5.3h(bone marrow).
3,4 cell differentiation qualifications
After two groups of migrating cell adipogenic induction differentiation qualification induction 48h, little fat is had to ooze in cell existing; After two weeks, fat drips quantity increases and merges mutually, and cell becomes circular or Polygons by fusiformis, and oil red O stain shows a large amount of lipidosiss, and (a), prompting cell is divided into adipocyte to Fig. 5.After Osteoinductive differentiation qualification osteogenic induction, cell volume increases in time and expands, and becomes polygon, cube by spindle shape, and in paving stone shape, many places aggregation growth, induces and define obvious tubercle in latter about 10 days, tubercle size heterogeneity.Inducing culture is after 21 days, and the strip tubercle of visible deposition is orange through Alizarin red staining tubercle, sharpness of border, and prompting has mineralized dentin matrix to deposit (Fig. 5 b).Become chondrocyte induction differentiation identification of cell after differentiation-inducing, cell body becomes circle, and kytoplasm reduces.Be bright green (Fig. 5 c) at induction 21 days alcian blues dyeing visible cell slurry and intercellular substance, point out cell differentiating cartilage-forming cell.
By above-mentioned experiment and result visible, in fracture site tissue, cell quantity reached maximum value 15 days time.Cell in shearing method diastatic fracture broken ends of fractured bone tissue, simple and easy to do, very little to cell injury, the cell major part in fracture site tissue can be separated.The attached cell form that the present invention adopts differential attachment method to be separated is more homogeneous, and purity is higher, although the stem cell purity that differential attachment method is separated is more limited, this method separation and purification stem cell is fast, simple.Experimental result of the present invention confirms that the stem cell be separated has similar cellular form with bone marrow mescenchymal stem cell, multiplication capacity, to adipocyte, scleroblast and chondrogenic differentiation ability.In addition, the present invention has carried out the Phenotypic examination of stem cell in early-stage Study, the low expression hematopoietic stem markers of cell CD34 and CD45, high expression level CD105 and stem cell labeling thing Sca-1, the surface marker similar to mesenchymal stem cells MSCs, is not expressed as myocyte's marker MyoD.In addition, the present invention, to the union of fracture situation of rat stress fracture model, has carried out structure observation, and performance speed of fracture union obviously speeds, and fracture around osteogenic ability is stronger, and area of new bone cell is many and active.
Experimental study of the present invention shows, fracture site is under ambient stress, more migrating cell can be raised, above-mentioned research shows, the surface marker that these migrating cells of raising of fracture site are similar to mesenchymal stem cells MSCs, and can break up to scleroblast and chondroblast direction, consistent with mescenchymal stem cell feature.By setting up rat stress fracture model, to raise the method for mescenchymal stem cell simple in the present invention, is the important method promoting union of fracture.
Claims (4)
1. raising a method for mescenchymal stem cell by setting up rat stress fracture model, it is characterized in that: first set up the rat stress fracture model that exterior fixing rack is fixing, after modeling success, take out fracture site tissue, separated migrates cell is also cultivated;
Described exterior fixing rack comprises 4 pieces of draw points and 2 pieces of crossbeams, and 4 pieces of draw points are that comb teeth-shaped is evenly arranged between two pieces of crossbeams, and 2 pieces of crossbeams are fixed together by 2 pieces of Ti alloy miniature screws; Exterior fixing rack axial restraint stress is 4.5N/mm.
2. method according to claim 1, is characterized in that, step is:
1) rat stress fracture model is set up
After rat weight, 3% vetanarcol 40mg/kg intraperitoneal injection of anesthesia, operation side femur unhairing preserved skin, sterile drape, operation is carried out under stringent asepsis requirements, from lateral side of femur otch, femur is exposed from musculus lateralis externi gap, first exterior fixing rack is placed, exterior fixing rack crosses over femur total length in femur little tuberosity to condyle of femur, after fixedly completing, cut-out femur is walked crosswise with micro-saw of thickness 1mm between the fixing draw point in 2, inner side, rinse the edge of a knife, with absorbable thread suture muscles tissue, with nonabsorable suturing with thread management skin, Post operation will add the anodyne tramadol hydrochloride of 12mg/500ml in 3 days in the tap water of rat, the clindamycin of operation consent 30min and Post operation 3 days subcutaneous injection 45mg/kg, prevent bacteriological infection, after having performed the operation, rat is freely movable, steel nail every day place routine disinfection,
2) separation and Culture of cell
By step 1) method establishment rat stress fracture model, respectively at after modeling when the 6th, 9,12,15,18 day, take out fracture site tissue and be good for side femur, according to method isolated cell below: the fracture site tissue use PBS rinsing of taking-up 2 times, is trimmed to pasty state; Repeatedly blow and beat gently with substratum, make it through 200 eye mesh screens, collect migrating cell (migratingcells, MCS) suspension, collects unfiltered material, repeats with substratum resuspended, filter, collect and merge migrating cell suspension, centrifugal rear substratum re-suspended cell; Be separated the medullary cell of rat according to following method, go out marrow with PBS liquid, collect bone marrow cell suspension, adjustment cell concn to 1 × 10
6/ ml, add Ficoll parting liquid at 1: 1 by volume, the centrifugal 20min of 1800r/min, draws interface mononuclearcell, wash 2 times, use substratum re-suspended cell, the medullary cell of separation, migrating cell concentration are adjusted to 1 × 10 with PBS
6/ ml, is inoculated in the culturing bottle of poly-lysine bag quilt, moves into 37 DEG C, 5%CO
2incubator in cultivate; After 3 days, full dose more renews nutrient solution, and remove non-attached cell, cell density reaches 80% ~ 90%, namely goes down to posterity.
3. method according to claim 1, is characterized in that: described substratum is the DMEM substratum containing 10% foetal calf serum.
4. method according to claim 1, is characterized in that, observation and evaluation method are:
1) fracture site histocyte counting: respectively at after modeling when the 6th, 9,12,15,18 day, put to death rat, take out fracture site tissue, with the rinsing of D-Hanks liquid, shearing material adds the DMEM substratum of equivalent, Trypan Blue, cell counting;
2) morphological observation: under inverted microscope every day observation of cell morphological feature;
3) cell proliferation tracing analysis: by fracture site or derived from bone marrow the 3rd generation cell, with 2 × 10
5cells/ml/ hole is seeded in 24 orifice plates, digests 3 holes, collecting cell every 24h, and with 0.4% trypan blue living cell counting, draw growth curve; And be calculated as follows cell doubling time: TD=t × ［ lg2/ (lgN
t-lgN
0);
N
t: collecting cell number; N
0: inoculating cell number; T: incubation time;
4) cell is differentiation-inducing: Osteoinductive differentiation, the differentiation of one-tenth chondrocyte induction, adipogenic induction differentiation;
5) statistical analysis: experimental data all represents with means standard deviation; Use SPSS17.0 software analysis, utilize One-way ANOVA (One-wayANOVA) method to compare, measurement data adopts t inspection, and the comparison of enumeration data adopts χ
2inspection; With P < 0.05 for difference has significant, P < 0.01 difference is very significant.
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| CN109576334A (en) * | 2018-11-22 | 2019-04-05 | 天津百恩生物科技有限公司 | Detect the kit and method of mescenchymal stem cell immunosuppressive biological effect |
| CN112029710A (en) * | 2020-08-31 | 2020-12-04 | 上海交通大学医学院附属第九人民医院 | Screening method of direct mechanical response cell subset and application thereof |
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