CN106119190A - OncostatinM application in the product that preparation promotes derived mesenchymal stem cells in vitro directed differentiation to be osteocyte - Google Patents

OncostatinM application in the product that preparation promotes derived mesenchymal stem cells in vitro directed differentiation to be osteocyte Download PDF

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CN106119190A
CN106119190A CN201610504749.9A CN201610504749A CN106119190A CN 106119190 A CN106119190 A CN 106119190A CN 201610504749 A CN201610504749 A CN 201610504749A CN 106119190 A CN106119190 A CN 106119190A
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oncostatinm
umbilical cord
cell
mesenchymal stem
stem cells
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孔宁
李云华
孙飞
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Jilin Zhongke Bioengineering Ltd By Share Ltd
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Jilin Zhongke Bioengineering Ltd By Share Ltd
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Abstract

The invention belongs to cellular biological technique and bone tissue engineer technical field, be specifically related to oncostatinM application in derived mesenchymal stem cells in vitro Osteoblast Differentiation.The present invention is experimentally confirmed, oncostatinM has in derived mesenchymal stem cells in vitro Osteoblast Differentiation can raise Osteoblast Differentiation mark ALP, Runx2, OPN and OCN at mRNA, the expression of protein level and calcium ion deposition, promotes that mescenchymal stem cell directed differentiation is the function of osteocyte.The medicine that oncostatinM coordinates mescenchymal stem cell applied research in preparation treatment bone injury medicine to be the diseases such as exploitation preparation treatment osteoporosis, fracture, Cranial defect, bone wound provides material base.

Description

In preparation, oncostatinM promotes that derived mesenchymal stem cells in vitro directed differentiation is osteocyte Application in product
Technical field
The invention belongs to cellular biological technique and bone tissue engineer technical field, be specifically related to oncostatinM and promote in preparation Derived mesenchymal stem cells in vitro directed differentiation is the application in the product of osteocyte.
Background technology
At present, by wound, tumor, infection or fractures improper etc. cause Cranial defect, Fracture Nonunion is in clinical pole For common.Bony site implantation (bone grafting) is treatment Cranial defect, the required means of Fracture Nonunion.
Tradition bone-grafting material is mainly autologous skeleton, allosome skeleton and artificial bone.Autologous skeleton is a kind of more satisfactory Graft, its skeletonization is rapid, without immunological rejection, but its limited source, is often not enough to the Cranial defect of reparation bulk, and adopts Operation wound can be increased during collection and complication may be caused.Homogeneous allogenic bone is big for bone amount, without the autologous complication taking bone, but lives Property composition few, ossification is weak, is only capable of playing filling and support effect after implantation, and fatigue fracture easily occurs, and may cause handing over Fork infects.Artificial bone wide material sources, convenience of drawing materials, do not have homogeneous allogenic bone may cause the danger of cross infection, but can cause Immunity rejection, poor biocompatibility, absorption, alternative Process slowly, then occur that bone does not connect or Cranial defect incidence rate are high.
The fast development of organizational project and regeneration medicine technology in recent years, for solve after tradition bone grafting operation bone formation slowly and The technological difficulties such as new bone formation is bad provide a kind of effectively solution route.Its Research Thinking is mainly from skeletonization support, skeletonization Cytokine three aspect of cell and regulation and control Osteoblast Differentiation is set about, by forming tissue engineering artificial bone, and new bone after solving bone grafting Form problem slowly.Wherein the source of seed cell, the derivant of regulation and control seed cell Osteoblast Differentiation are its key points.Become Osteocyte is classical seed cell source, but there is inconvenience of drawing materials in a large number, has certain damage, multiplication capacity relatively to for district The distinct disadvantage such as weak, it is impossible to meet the needs as Seeding Cells in Bone Tissue Engineering.
Along with scientific and technological development in recent years, stem cell is increasingly becoming the focus of bone tissue engineer research.Originate by genetis method With cell developmental stage, stem cell can be divided into embryonic stem cell and adult stem cell.Former for ethics and safety in utilization Cause, makes the dispute that the research of embryonic stem cell causes in worldwide with application.Adult stem cell refers at fetal development Remaining in the undifferentiated cell in various tissue and internal organs during becoming adult, it still has differentiation potential, simply loses Develop into the ability of complete individuals.At present, the adult stem cell that experiment is most with clinical research is mescenchymal stem cell.
But Derived from Mesenchymal Stem Cells becomes osteoblast to need the induction of chemical induction or somatomedin, warp in prior art The osteogenic induction agent of allusion quotation mainly includes vitamin C, dexamethasone, sodium β-glycerophosphate etc..But inducing mesenchymal stem cell skeletonization The efficiency comparison of differentiation is low.
In view of this, the special proposition present invention.
Summary of the invention
The first object of the present invention is to provide oncostatinM to promote that derived mesenchymal stem cells in vitro directed differentiation is bone in preparation Application in the product of cell.Described oncostatinM has in derived mesenchymal stem cells in vitro Osteoblast Differentiation can raise skeletonization Mark ALP, Runx2, OPN and OCN are in mRNA, the expression of protein level and the effect of calcium ion deposition in differentiation.
The second object of the present invention there are provided oncostatinM and coordinates mescenchymal stem cell at preparation treatment bone injury medicine In application, this application process can be widely used for osteoporosis, fracture, Cranial defect and the preparation of bone wound medicine.
In order to realize the above-mentioned purpose of the present invention, present invention employs following technical scheme:
OncostatinM application in the product that preparation promotes derived mesenchymal stem cells in vitro directed differentiation to be osteocyte.
In the present invention, described product is preferably medicine.
OncostatinM (OSM) is the member of IL-6 cytokine family, is a kind of multi-functional cell regulating factor, can make For various kinds of cell, there is regulator gene activity, cell survival, the potential function breeding and break up.
In the present invention, described oncostatinM mescenchymal stem cell osteoblast differentiation marker protein can be promoted at mRNA and The expression of protein level, makes alkaline phosphatase activities raise and the formation of Mineral nodules, and directional induction is to osteoblast differentiation.
OncostatinM of the present invention is bought by R&D company of the U.S. and is obtained, escherichia coli expression be prepared by recombinant.
Optimal technical scheme of the present invention is: the mescenchymal stem cell obtained by Isolation and culture is inoculated into and adds Basal medium added with oncostatinM and chemical inducer carries out cultivation and obtains osteoblast, described incubation time 6～21 My god.
Described incubation time is preferably 7 days, 14 days or 21 days.
Mescenchymal stem cell is inoculated into by technique scheme and is added with oncostatinM and chemical inducer by the present invention Carry out on basal medium cultivating a period of time, osteoblast differentiation can be promoted by experimental verification mescenchymal stem cell subsequently Marker protein, in mRNA and the expression of protein level, can make alkaline phosphatase activities raise and the formation of Mineral nodules, with Prove that oncostatinM can be with directional induction mescenchymal stem cell to osteoblast differentiation.
Further, the inoculum density of mescenchymal stem cell is 2 × 104cells/cm2～5 × 104cells/cm2。
The inoculum density of described mescenchymal stem cell is preferably 2 × 104cells/cm2。
The source of the mescenchymal stem cell of original acquisition is not enough and content is relatively low.So generally we can be by original The mescenchymal stem cell obtained carries out cultivating amplification, to obtain the requirement of above-mentioned inoculum density as seed cell.
Further, described basal medium includes hyclone and DMEM/F12 culture fluid, wherein, described DMEM/F12 Culture fluid accounts for 80～90% volume ratio, and hyclone accounts for 10～20% volume ratio.
Further, basal medium includes that the hyclone of 10% volume ratio and 90% volume ratio DMEM/F12 are cultivated Liquid.
In the present invention, in described basal medium, DMEM/F12 culture fluid and the mixed volume of hyclone are 100%.
Further, described oncostatinM concentration in basal medium is 0.1～10ng/mL.
OncostatinM concentration in basal medium is typical but non-limiting is 0.1,1 and 10ng/mL.
Further, described chemical inducer includes dexamethasone, sodium β-glycerophosphate and the mixing of ascorbic acid 2 phosphate Being formed, wherein, the content of described dexamethasone is 100nmol/L, and the content of described sodium β-glycerophosphate is 10mmol/L, described The phosphatic content of ascorbic acid 2 is 50mg/L.
Further, described mescenchymal stem cell is umbilical cord mesenchymal stem cells.
Human umbilical cord mesenchymal stem cells has drawn from umbilical cord tissue as childbirth garbage always, draws materials conveniently, it is easy to receive Collection, preservation, freezing, not by ethics and legal restriction.Umbilical cord mesenchymal stem cells rich content, a 30cm's Umbilical cord can get 107Primary cell, and cell is the most original, differentiation capability is strong, and biological property is stable, repeatedly passes on after amplification still Energy keeps vigorous function, can be to test the cell derived sufficient with clinical offer.
So far, umbilical cord mesenchymal stem cells is proved to be autologous and variant cell transplanting, treatment in many-side Preferable cell.Compare with embryonic stem cell, umbilical cord mesenchymal stem cells on biological characteristics seeks peace immunological characteristic with its phase Seemingly, it is the important sources of Seeding Cells in Bone Tissue Engineering.
Meanwhile, as preferable bone seeding cell, umbilical cord mesenchymal stem cells has the following characteristics that (1) umbilical cord mesenchyma is done Cellularity is fairly simple, is the germinal cell without specific function；(2) breed during umbilical cord mesenchymal stem cells In vitro culture Power is strong；(3) umbilical cord mesenchymal stem cells can break up to specific direction under certain conditions, is stably expressed as osteocyte table Type.Having These characteristics to understand umbilical cord mesenchymal stem cells is a kind of preferably bone seeding cell.
The process that above-mentioned umbilical cord mesenchymal stem cells carries out cultivating amplification as seed cell includes: original cuiture, pass on Cultivation, frozen and recovery.
Above-mentioned original cuiture refers to the umbilical cord mesenchymal stem cells that separated at 37 DEG C, the CO of 5%2, the air of 95% Change liquid after cultivating 48h under gas phase, first rinse gently by PBS solution 3 times, to remove most non-attached cell, change basis Culture fluid, 37 DEG C, the CO of 5%2, 95% air conditions under continue cultivate.
Heretofore described basic culture solution refers to the DMEM/F12 culture fluid containing 10% hyclone.
Above-mentioned Secondary Culture refers to pass on when umbilical cord mesenchymal stem cells grows to account for culture dish bottom surface 80%, first Sucking old culture fluid, PBS rinses 2 times, bounces back to most cells kytoplasm with the digestion of cell separation liquid, will be from cultivation When ware diapire comes off, add basic culture solution and terminate digestion, blow and beat gently with suction pipe and make single cell suspension, with the ratio of 1:3 Cell is seeded in another culture dish continuation cultivate.
Above-mentioned frozen refer to when the umbilical cord mesenchymal stem cells after Secondary Culture grows to account for culture dish bottom surface 80%, Digesting and collect cell suspension, 1000rpm is centrifuged 5min, abandons supernatant, with 5 × 106The ratio of cells/ml adds precooling Liquid storage, fully mixes in the cryopreservation tube of rearmounted pre-cooling.By 4 DEG C of 30min ,-20 DEG C of 30min ,-80 DEG C of programs overnight are frozen Deposit.Finally it is placed in liquid nitrogen container preservation.Separated in time recovery cell, observation of cell growing state is answered after Long-term Cryopreservation.
Above-mentioned recovery refers to take out cryopreservation tube from liquid nitrogen container, puts in 37 DEG C of water-baths rapidly and quickly rocks, until frozen Liquid is completely dissolved, and will complete rewarming in 1～2min.Then sucking-off cell suspension inoculation is in culture dish, adds basis and cultivates Liquid, in 37 DEG C, 5%CO2, 95% air gas phase under cultivate, within second day, change liquid.
Further, described umbilical cord mesenchymal stem cells prepares as follows: (1) by umbilical cord soaking disinfection, with It is rinsed with buffer afterwards, the umbilical cord separation umbilical vein after rinsing and umbilical artery, then umbilical cord is cut into umbilical cord tissue block；
(2) in the umbilical cord tissue block sheared, cell separation liquid is added, at 37 DEG C, 5%CO2Under conditions of 95% air Separating digesting 12h；
(3) after centrifugal for the umbilical cord tissue block after separating digesting, supernatant is abandoned, suspension cell subsequently, fills between isolated umbilical cord Matter stem cell.
Further, the cell separation liquid by volume percentages in described step (2) includes: DMEM/F12 culture fluid accounts for 80～90% volume ratio, hyclone accounts for 10～20% volume ratio, and II Collagenase Type accounts for 0.01～0.1% volume ratio and hyalomitome Acid enzyme accounts for 0.05～0.1% volume ratio.
Further, cell separation liquid by volume percentages, DMEM/F12 culture fluid accounts for 80% volume ratio, hyclone Accounting for 19.94% volume ratio, II Collagenase Type accounts for 0.01% volume ratio and hyaluronidase accounts for 0.05% volume ratio.
Further, oncostatinM of the present invention coordinates mescenchymal stem cell to have in preparation treatment bone injury medicine There is important effect.Described bone injury disease refers to osteoporosis, fracture, Cranial defect and bone wound etc..
Compared with prior art, the invention have the benefit that
(1) the invention provides the new application of cytokine oncostatinM, i.e. promote that Derived from Mesenchymal Stem Cells is that skeletonization is thin The effect of born of the same parents.Described oncostatinM can raise Osteoblast Differentiation mark ALP, Runx2, OPN and OCN at mRNA, protein level Express and calcium ion deposition, promote that mescenchymal stem cell is to osteoblast differentiation.
(2) oncostatinM can be used as the rush cell differentiation of cellular replacement therapy bone injury disease and reconstruction osseous tissue Agent.Cellular replacement therapy bone injury disease is currently under the experimentation stage (containing clinical and experimental study), it is contemplated that promote cell The introducing of differentiation agent oncostatinM will assist in the research in this field, for exploitation preparation treatment osteoporosis, fracture, Cranial defect, bone The medicine of the diseases such as wound provides material base.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing In having technology to describe, the required accompanying drawing used is briefly described.
Fig. 1 shows that oncostatinM promotes the 7th day alkaline phosphatase staining result of umbilical cord mesenchymal stem cells Osteoblast Differentiation, its In, (a), (b), (c), (d) and (e) are respectively comparative example 1, comparative example 2, embodiment 3, embodiment 4, the alkaline phosphorus of embodiment 5 Acid enzyme coloration result.
Fig. 2 shows that oncostatinM promotes the 14th day alkaline phosphatase staining result of umbilical cord mesenchymal stem cells Osteoblast Differentiation, its In, (a), (b), (c), (d) and (e) are respectively comparative example 1, comparative example 2, embodiment 3, embodiment 4, the alkaline phosphorus of embodiment 5 Acid enzyme coloration result.
Fig. 3 show oncostatinM promote the 21st day Alizarin red staining result of umbilical cord mesenchymal stem cells Osteoblast Differentiation, wherein, A (), (b), (c), (d) and (e) are respectively comparative example 1, comparative example 2, embodiment 3, embodiment 4, the Alizarin red staining of embodiment 5 Result.
Fig. 4 shows that oncostatinM promotes the immunocytochemical stain method inspection in the 14th day of umbilical cord mesenchymal stem cells Osteoblast Differentiation Surveying osteopontin and Bone Gla protein, wherein, (a), (b) and (c) are respectively comparative example 1, comparative example 2, embodiment 5 immunocytochemistry Staining detection osteopontin and the testing result of Bone Gla protein.
Fig. 5 shows that oncostatinM promotes umbilical cord mesenchymal stem cells Osteoblast Differentiation 3 days, 7 days, 14 days, 21 days real-time quantitatives RT-PCR detects Osteoblast Differentiation transcription factor Runx2, Collagen I level, and wherein, (a) is to comparative example 1, comparative example 2, reality Execute example 3, embodiment 4, embodiment 5 respectively 3 days, 7 days, 14 days, 21 days time Runx2 genetic fragment is carried out real-time quantitative RT- PCR detection analysis result, (b) be comparative example 1, comparative example 2, embodiment 3, embodiment 4, embodiment 5 respectively 3 days, 7 days, 14 days, 21 days time Collagen I genetic fragment carried out the analysis result of real-time quantitative RT-PCR detection.
Fig. 6 shows that oncostatinM promotes that umbilical cord mesenchymal stem cells Osteoblast Differentiation uses Western blot detection for 14 days Osteopontin and BGP level, wherein, (a), (b), (c), (d) and (e) be respectively comparative example 1, comparative example 2, embodiment 3, Embodiment 4, the Western blot detection osteopontin of embodiment 5 and BGP level result.
Detailed description of the invention
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but those skilled in the art will Understanding, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.In embodiment unreceipted specifically Condition person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or instrument unreceipted production firm person, be Can be by the commercially available conventional products bought and obtain.
Preparing of embodiment 1 umbilical cord mesenchymal stem cells
(1) umbilical cord being put into alcohol-pickled sterilization, be rinsed with PBS subsequently, the umbilical cord after rinsing separates Umbilical vein and umbilical artery, then become the piece of tissue of length about 5mm by umbilical cord scissors；
(2) in the umbilical cord tissue block sheared, cell separation liquid is added, at 37 DEG C, 5%CO2Under conditions of 95% air Separating digesting 12h；
(3) the umbilical cord block after separating digesting is centrifuged 10min with 1500rpm, abandons supernatant, with containing 10% hyclone DMEM/F12 fluid medium suspension cell, isolated umbilical cord mesenchymal stem cells, cell content is 5 × 104cells/ml。
Embodiment 2 umbilical cord mesenchymal stem cells cultivates amplification procedure
(1) original cuiture: by the umbilical cord mesenchymal stem cells that separated with 5 × 104The density of cells/ml is seeded in 37 DEG C, the CO of 5%2, 95% air gas phase under cultivate after 48h and change liquid, first rinse gently by PBS solution 3 times, to remove major part Non-attached cell, change basic culture solution, 37 DEG C, the CO of 5%2, 95% air conditions under continue cultivate.
Heretofore described basic culture solution refers to the DMEM/F12 culture fluid containing 10% hyclone.
(2) Secondary Culture refers to pass on when umbilical cord mesenchymal stem cells grows to account for culture dish bottom surface 80%, first Sucking old culture fluid, PBS rinses 2 times, bounces back to most cells kytoplasm with the digestion of cell separation liquid, will be from cultivation When ware diapire comes off, add basic culture solution and terminate digestion, blow and beat gently with suction pipe and make single cell suspension, with the ratio of 1:3 Cell is seeded in another culture dish continuation cultivate.
(3) frozen refer to when the umbilical cord mesenchymal stem cells after Secondary Culture grows to account for culture dish bottom surface 80%, Digesting and collect cell suspension, 1000rpm is centrifuged 5min, abandons supernatant, with 5 × 106The ratio of cells/ml adds precooling Liquid storage, fully mixes in the cryopreservation tube of rearmounted pre-cooling.By 4 DEG C of 30min ,-20 DEG C of 30min ,-80 DEG C of programs overnight are frozen Deposit.Finally it is placed in liquid nitrogen container preservation.Separated in time recovery cell, observation of cell growing state is answered after Long-term Cryopreservation.
(4) recovery refers to take out cryopreservation tube from liquid nitrogen container, puts in 37 DEG C of water-baths rapidly and quickly rocks, until frozen stock solution It is completely dissolved, rewarming will be completed in 1-2min.Then sucking-off cell suspension inoculation is in culture dish, adds basic culture solution, In 37 DEG C, 5%CO2, 95% air gas phase under cultivate, within second day, change liquid.
The preparation of comparative example 1 basic culture solution group (Normal)
By umbilical cord mesenchymal stem cells with 2 × 104cells/cm2Density is inoculated in 6 well culture plates, then by culture plate The culture fluid of the DMEM/F12 being placed in the hyclone containing 10% carries out cultivating 7 to 21 days.
The preparation of comparative example 2 chemical reagent induction liquid group (Control)
By umbilical cord mesenchymal stem cells with 2 × 104cells/cm2Density is inoculated in 6 well culture plates, then by culture plate The culture fluid of the DMEM/F12 being placed in the hyclone containing 10% and Osteoblast Differentiation chemical inducer carries out cultivating 7 to 21 days.
Above-mentioned Osteoblast Differentiation chemical inducer includes: dexamethasone 100nmol/L, sodium β-glycerophosphate 10mmol/L, anti- Bad hematic acid 2 phosphate 50mg/l.
The preparation of embodiment 3 oncostatinM low dose group (OSM-Low)
By umbilical cord mesenchymal stem cells with 2 × 104cells/cm2Density is inoculated in 6 well culture plates, then by culture plate It is placed in the culture fluid of the DMEM/F12 of the hyclone containing 10%, Osteoblast Differentiation chemical inducer and 0.1ng/ml oncostatinM Carry out cultivating 7 to 21 days.
Above-mentioned Osteoblast Differentiation chemical inducer includes: dexamethasone 100nmol/L, sodium β-glycerophosphate 10mmol/L, anti- Bad hematic acid 2 phosphate 50mg/l.
The preparation of dosage group (OSM-Mid) in embodiment 4 oncostatinM
By umbilical cord mesenchymal stem cells with 2 × 104cells/cm2Density is inoculated in 6 well culture plates, then by culture plate The culture fluid of the DMEM/F12 being placed in the hyclone containing 10%, Osteoblast Differentiation chemical inducer and 1ng/ml carries out cultivating 7 To 21 days.
Above-mentioned Osteoblast Differentiation chemical inducer includes: dexamethasone 100nmol/L, sodium β-glycerophosphate 10mmol/L, anti- Bad hematic acid 2 phosphate 50mg/l.
The preparation of embodiment 5 oncostatinM high dose group (OSM-High)
By umbilical cord mesenchymal stem cells with 2 × 104cells/cm2Density is inoculated in 6 well culture plates, then by culture plate The culture fluid of the DMEM/F12 being placed in the hyclone containing 10%, Osteoblast Differentiation chemical inducer and 10ng/ml carries out cultivating 7 To 21 days.
Above-mentioned Osteoblast Differentiation chemical inducer includes: dexamethasone 100nmol/L, sodium β-glycerophosphate 10mmol/L, anti- Bad hematic acid 2 phosphate 50mg/l.
Alkali phosphatase detection is analyzed
For showing that oncostatinM of the present invention is to promoting that Derived from Mesenchymal Stem Cells is osteoblastic effect, by above-mentioned enforcement Example 2～5 and comparative example 1～2 carry out alkali phosphatase analysis, and result of the test is as shown in table 1 below:
Experimental technique: alkali phosphatase analysis uses ALP staining kit, is operated by test kit description.
(1) sucking the culture fluid in Tissue Culture Plate, every hole adds 400 μ l and clears up liquid, washed cell surface.
(2) sucking cleaning liquid, every hole adds 400 μ l fixatives, covers whole growing surface.At room temperature hatch 2min, Carefully suck fixative.Every hole adds 400 μ l and clears up liquid, washes paint cell surface, cleans secondary altogether.
(3) plus 200 μ l dyeing liquors, covering whole cell surface, temperature hatches 15 to 30min in lower darkroom, or until can See blueness；Carefully suck dyeing liquor, add 400 μ l and clear up liquid, incubated at room temperature 2min, after sucking cleaning liquid, add 100 μ l Cleaning liquid, basis of microscopic observation, take pictures.
Table 1: alkali phosphatase analysis result
Alkali phosphatase (ALP) is Osteoblast Differentiation early sign thing.ALP coloration result shows: osteogenic induction the 4th day, Normal group has no that bluish violet dyes；Control group and OSM-Low, Mid, High group all can detect that shallower bluish violet sun Property dyeing, show in cell the expression of existing ALP.Osteogenic induction the 7th day, Normal group has no that bluish violet dyes；Remaining each group All can detect that stronger bluish violet positive staining, show that in cell, ALP expression increases.Osteogenic induction the 14th day, except Normal Outside group, remaining is respectively organized and all shows that bluish violet dyes, and OSM-Low, Mid, High group is significantly stronger than Control group, and along with OSM agent Measure the biggest color the deepest.Show that OSM inducing mesenchymal stem cell Osteoblast Differentiation not only has time dependence but also have dose-dependant Property.(see Fig. 1,2)
Calcium ion deposition detection is analyzed
For showing that oncostatinM of the present invention is to promoting that Derived from Mesenchymal Stem Cells is osteoblastic effect, by above-mentioned enforcement Example 2～5 and comparative example 1～2 carry out the method for Alizarin red staining and carry out calcium ion deposition detection analysis.
Experimental technique: calcium ion deposition detection uses Alizarin red staining test kit, operates by test kit description.By each group Cell induction discarded culture fluid after 21 days, washed 2 times with PBS, and distilled water flushing after 1h fixed by 70% ice ethanol, inhaled suitable Amount 2%AR-S solution is completely soaked sample, dyes and rock culture plate gently under room temperature, inhales and abandon dye liquor, with double steamings after 10 minutes Water clean 5 times, then with PBS rock rinsing 15 minutes with remove non-specific binding alizarin red S, light after natural drying Learning basis of microscopic observation and take pictures, positive findings is that calcium knuckle areas takes on a red color.
Test result analysis: in addition to Normal group has no that salmon pink Mineral nodules occurs, all see orange for remaining each group Color Mineral nodules, and along with the increase of oncostatinM dosage, the quantity of salmon pink Mineral nodules gradually increases.OncostatinM skeletonization lures Lead group inducing mesenchymal stem cell Osteoblast Differentiation effect and be better than Control group, show that oncostatinM has promotion cellular matrix ore deposit Change effect.Osteogenic induction 21 days, carries out Alizarin red staining, in addition to Normal group has no that salmon pink Mineral nodules occurs, remaining All seeing more salmon pink Mineral nodules for each group, oncostatinM-Low, Mid, High group salmon pink Mineral nodules quantity are the most In 14 days, the most significantly more than Control group, show that oncostatinM inducing mesenchymal stem cell Osteoblast Differentiation had both had time-dependent Property has again dose dependent, can strengthen the Osteoblast Differentiation efficiency of chemical inducer.(see Fig. 3)
Immunohistochemical kit detection is analyzed
For showing that oncostatinM of the present invention is to promoting that Derived from Mesenchymal Stem Cells is osteoblastic effect, by above-mentioned enforcement Example 2～5 and comparative example 1～2 application immunohistochemical kit detection Osteoblast Differentiation marker protein OPN and OCN.
Experimental technique: hatch 10min, permeation cell film in 0.25%Triton X-100 solution.PH7.4TBS rinses 5min×3.3% glacial acetic acid room temperature treatment 30min is with closing cell's endogenous enzyme.PH7.4TBS rinses 5min × 3.Dropping 5% BSA confining liquid, room temperature is closed 20min, is inhaled and abandon surplus liquid, do not wash.(1:100 is dilute for dropping rabbit anti-human OPN, OCN polyclonal antibody Release), 4 DEG C of overnight incubation.PH7.4TBS rinses 5min × 3.PBS replaces one anti-as negative control.Dropping biotinylated goat Anti-rabbit IgG, hatches 20min for 37 DEG C.PH7.4TBS rinses 5min × 3.Dropping reagent SABC-AP (streptavidin-alkaline phosphatase Enzyme), hatch 20min for 37 DEG C.PH7.4TBS rinses 5min × 3, and washing is once.Dropping developer BCIP/NBT, hatches 10 for 37 DEG C ～30min.Response time, distilled water wash is controlled under microscope.Inverted microscope is observed, photograph.
Interpretation: osteopontin (OPN) and Bone Gla protein (OCN) are the late-stage markers things of Osteoblast Differentiation.In order to bright Really OSM has effect to the differentiation of MSCs late osteogenic, and we induce the expression of the 14th day detection OPN and OCN at OSM.Normal group Not observing positive staining, remaining each group all there is sepia positive staining, and along with the increase of OSM dosage, positive staining is thin Born of the same parents' increasing number.The dyeing of OSM osteogenic induction group is better than Control group, shows that OSM can be enhanced to self-bone grafting differentiation agent Differentiation efficiency, and there is dose dependent.(see Fig. 4)
Western blot detection is analyzed
For showing that oncostatinM of the present invention is to promoting that Derived from Mesenchymal Stem Cells is osteoblastic effect, by above-mentioned enforcement Example 2～5 identifies oncostatinM inducing umbilical cord mesenchymal stem Osteoblast Differentiation with comparative example 1～2 application Western blot.
Experimental technique: when each group is induced 14 days, cell cracks, and cell lysate 12000g is centrifuged 10min, heating 5min and makes Albuminous degeneration, carry out electrophoresis with the SDS-PAGE glue of 10%, be then transferred on pvdf membrane, containing 5% defatted milk powder PBST closes 1h for 4 DEG C.By an anti-overnight incubation of 1:1000 dilution, add the two of HRP labelling and resist, photograph.
Interpretation: corresponding protein band does not occurs in Normal group, remaining each group all there is destination protein band, and Along with the increase of oncostatinM dosage, destination protein amount increases.OncostatinM osteogenic induction group destination protein amount is higher than Control Group, shows that oncostatinM can be enhanced to the differentiation efficiency of self-bone grafting differentiation agent, and has dose dependent.(see Fig. 6)
Real-time quantitative RT-PCR is identified
For showing that oncostatinM of the present invention, to promoting that Derived from Mesenchymal Stem Cells is osteoblastic effect, uses and determines in real time Amount RT-PCR method identifies oncostatinM inducing umbilical cord mesenchymal stem Osteoblast Differentiation.
Experimental technique: by resuspended for the mescenchymal stem cell culture fluid that digests, be centrifuged, wash, adjusting cell density is 5 ×104Cells/ml inoculates 10cm plate, and every plate adds 10ml cell suspension, is placed in 37 DEG C, 5%CO2Incubator is cultivated 24h. Join in the induction broth of embodiment 2～5 and comparative example 1～2 after cell is the most adherent, process cell.Trizol method Never isogeneous induction group cell extracts total serum IgE, application real-time PCR kit amplification Runx2, Collagen I gene sheet Section.Amplified production carries out 1% agarose gel electrophoresis, sees whether to obtain the genetic fragment of expection size.
Interpretation: the gene expression of Normal group has no and extends in time and occur substantially to change.Remaining each group The gene expression of Runx2, Collagen I all changes with induction time change.It is the highest that oncostatinM respectively organizes gene expression amount In Control group, show that oncostatinM and chemical inducer there occurs collaborative thorn when mescenchymal stem cell is carried out osteogenic induction Swash effect.The gene expression of Runx2 peaks when inducing the 7th day, hereafter expression (see Fig. 5) on a declining curve.
Although illustrate and describing the present invention with specific embodiment, but it will be appreciated that without departing substantially from the present invention's May be made that in the case of spirit and scope many other change and amendment.It is, therefore, intended that in the following claims Including all such changes and modifications belonged in the scope of the invention.

Claims (10)

1. oncostatinM application in the product that preparation promotes derived mesenchymal stem cells in vitro directed differentiation to be osteocyte.
Apply the most as claimed in claim 1, it is characterised in that the mescenchymal stem cell obtained by Isolation and culture is inoculated into It is added with in the basal medium of oncostatinM and chemical inducer and carries out cultivation and obtain osteoblast, described incubation time 6～21 My god.
Apply the most as claimed in claim 2, it is characterised in that the inoculum density of mescenchymal stem cell is 2 × 104cells/cm2 ～5 × 104cells/cm2。
Apply the most as claimed in claim 2, it is characterised in that described basal medium includes hyclone and DMEM/F12 training Nutrient solution, wherein, described DMEM/F12 culture fluid accounts for 80～90% volume ratio, and hyclone accounts for 10～20% volume ratio.
Apply the most as claimed in claim 2, it is characterised in that described oncostatinM concentration in basal medium be 0.1～ 10ng/mL。
Apply the most as claimed in claim 2, it is characterised in that described chemical inducer is mainly by dexamethasone, β-glycerol phosphorus Acid sodium and the combination of ascorbic acid 2 phosphate are formed, and wherein, the concentration of described dexamethasone is 90～110nmol/L, described β-sweet The concentration of oleophosphoric acid sodium is 9～11mmol/L, and the described phosphatic concentration of ascorbic acid 2 is 40～60mg/L.
Apply the most as claimed in claim 1, it is characterised in that described mescenchymal stem cell is umbilical cord mesenchymal stem cells.
Apply the most as claimed in claim 7, it is characterised in that described umbilical cord mesenchymal stem cells prepares as follows: (1) by umbilical cord soaking disinfection, it is rinsed with buffer subsequently, the umbilical cord separation umbilical vein after rinsing and umbilical artery, then Umbilical cord is cut into umbilical cord tissue block；
(2) in the umbilical cord tissue block sheared, cell separation liquid is added, at 37 DEG C, 5%CO2Separate with under conditions of 95% air Digestion 12h；
(3) abandoning supernatant after centrifugal for the umbilical cord tissue block after separating digesting, suspension cell subsequently, isolated umbilical cord mesenchyma is done Cell.
Apply the most as claimed in claim 8, it is characterised in that the cell separation liquid by volume percentage ratio in described step (2) Meter includes: DMEM/F12 culture fluid accounts for 80～90% volume ratio, and hyclone accounts for 10～20% volume ratio, and II Collagenase Type accounts for 0.01% volume ratio and hyaluronidase account for 0.05% volume ratio.
10. oncostatinM coordinates mescenchymal stem cell application in preparation treatment bone injury medicine.
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