CN104667294B - Purposes of the miR 216 in the medicine for promoting Osteoblast Differentiation is prepared - Google Patents
Purposes of the miR 216 in the medicine for promoting Osteoblast Differentiation is prepared Download PDFInfo
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Abstract
The present invention relates to purposes of the miR 216 in the medicine for promoting Osteoblast Differentiation is prepared.MiR 216a can promote the mescenchymal stem cell of Human Adipose Tissue-derived（hAMSCs）To Osteoblast Differentiation, alkaline phosphatase staining, alkaline phosphatase activities, Alizarin red staining and RT PCR, western blot are parsed into bone photo pass marker gene and transcription factor shows that miR 216a cause hAMSCsOsteogenic ability increase.
Description
Technical field
This area is related to regenerative medicine field, in particular to miRNA in the medicine for promoting Osteoblast Differentiation is prepared
Purposes.
Background technology
Human mesenchymal stem cell（hMSCs）With self-renewing and multilineage differentiated potential, it can divide under proper condition
Turn to bone, cartilage, fat and muscular tissue（Prockop, DJ, 1997；Phinney, DG, 2007；Pittenger, MF et al.,
1999 and Fink, T et al., 2011）.Researcher isolates MSC from marrow first（BMSCs）, and demonstrate the differentiation of its polyphyly
Ability.BMSCsIt is the physiology course of body generally existing to osteoblast differentiation.In physiological conditions, bone tissue is rendered into
The dynamic equilibrium of the bon e formation of osteocyte and the bone information of osteoclast.Once a variety of causes causes this dynamic equilibrium to destroy,
During such as the reduction of Osteoblast Differentiation ability, osteoporosis, clavicular skull hypoplasia, caput femoris necrosis, joint degeneration will be caused
The bone related diseases such as change, scoliosis.Therefore, the regulatory mechanism for disclosing mescenchymal stem cell skeletonization has extremely important clinic
Application value.
Due to being limited and sampled difficulty by originating, BMSC research and application development are slower.Later research discovery,
In addition to marrow, MSC is also widely present in the Various Tissues organs such as fat, skin, synovia, the tooth come off, amniotic fluid and placenta
（Prockop, DJ, 1997）.Research has shown that, similar to the MSC of derived from bone marrow, the MSC of Human Adipose Tissue-derived（hAMSCs）Tool
There is multi-lineage potential interdepartmental or even across germinal layer, various kinds of cell type can be divided into vivo and in vitro, such as Gegenbaur's cell, cartilage is thin
Born of the same parents, fat cell, myocyte, endothelial cell, Deiter's cells etc..In addition compared with BMSC, hAMSCsIt is sufficient with raw material,
Convenient material drawing, the advantages of in-vitro separation amplification method is simple and easy to apply, therefore enjoy researcher to favor.Current hAMSCsHave become
Research MSC self-renewings and the optimum cell model broken up and the tissue engineering seed cell for most having development potentiality.
MicroRNAs（miRNAs）The highly conserved non-coding small RNA molecular of a class, can after transcription or transcription water
Heibei provincial opera control expression of target gene（Ambros, V, 2004；Bartel, DP, 2004）.Recently, increasing evidence shows miRNAs
Being capable of regulating cell differentiation and destiny decision（Ivey, KN and D Srivastava, 2010）.At present, miR-125, miR-
133、miR-135、miR-138、miR-181、miR-206、miR-9、miR-27、miR-196a、miR-214、miR-764-5p、
The many such as miR-2861, miR-3960, miR-642a-3p, miR-141/200a and miR-17-5p~92/miR-106a
miRNAsIt is verified that participating in the regulation and control of Osteoblast Differentiation.MiR-214 transgenic mices show obvious osteogenic ability reduction
（Wang, X, 2013）.These results of study are pointed out, miRNAsIt may be played in MSC Osteoblast Differentiation regulation and control extremely important
Role.
The content of the invention
According to some embodiments, the present invention provides use of the miR-216 in the medicine for promoting Osteoblast Differentiation is prepared
On the way.
In some embodiments, miR-216 promotes the mescenchymal stem cell of Human Adipose Tissue-derived to divide to skeletonization
Change.
In some embodiments, miR-216 sequence is as shown in SEQ ID No.1.SEQ ID No.1 are
UAAUCUCAGCUGGCAACUGUGA, its accession number is miRbase Accession number MIMAT0000273.
In some embodiments, miR-216 is provided by transfection reagent；Transfection reagent is preferably liposome.
In some embodiments, transfection reagent is Lipofectamine2000.
In some embodiments, miR-216 transfection concentrations are 100nmol/L.
Brief description of the drawings
Fig. 1：hAMSCsThe ALP dyeing of 3 days after being induced through Osteogenic Induction Medium.
Fig. 2：hAMSCsThe Alizarin red staining of 12 days after being induced through Osteogenic Induction Medium.
Fig. 3 A：Transfect after NC controls, hAMSCsALP dyeing.
Fig. 3 B：Transfect after miR-216a, hAMSCsALP dyeing.
Fig. 4 A：Transfect after NC controls, hAMSCsAlizarin red staining.
Fig. 4 B：Transfect after miR-216a, hAMSCsAlizarin red staining.
Fig. 5：The activity of alkaline phosphatase after osteogenic induction.hAMSCs：HAMSC without transfections；NC：Transfection has NC pairs
According to, hAMSC by osteogenic inductions；miR-216a：Transfection has miR-216a, hAMSC by osteogenic inductions。
Embodiment
The following provide specific material and its source used in embodiment of the present invention.It is to be understood that
What these were merely exemplary, it is not intended to limitation the present invention, with the type of such as undertissue, cell, reagent and instrument, model,
Quality, property or the same or analogous material of function may be incorporated for implementing the present invention.
Embodiment 1：The separation and identification of adult fat source mescenchymal stem cell
1. adult fat tissue is derived from liposuction procedures patient（Cosmetic surgery hospital of the Chinese Academy of Medical Sciences）, signed with voluntary donor
Informed consent form is ordered, donor is the healthy women of 25-35 Sui.
2. hAMSC is separated from adipose tissuesMethod be summarized as follows：
The adipose tissue that lipsuction gathers out washes away haemocyte and arcotic, 0.2g/100mlII type glue with D-Hanks
Protoenzyme（It is purchased from：Life Technologies companies of the U.S.）1h is digested, washs 2 times with D-Hanks afterwards to remove clostridiopetidase A.
Cell is collected by centrifugation, cell is with 2 × 106/ ml density is inoculated in containing 58%（v/v）DMEM/F12+40%（v/v）
MCDB-201、2%（v/v）Hyclone, 10ng/ml EGF, 10ng/ml PDGF, 1 × Insulin-Transferrin-selenous acid
（ITS）, l × linoleic acid-bovine serum albumin(BSA), 50 μM of beta- mercaptoethanols, 2mM Glus, l00 μ g/m1 penicillin
With the stem cell medium of 100U/ml streptomycin sulphates, 37 DEG C, 5%CO2, 95% humidity incubator culture.
Liquid is changed two days later, not adherent cell is discarded, and later every 3 days half amounts change liquid.
When cell converges up to 70%~80%, 0.25g/100ml pancreatin（Gibco）Conventional digestion, cell is according to l：3 are carried out
Passage.
3.hAMSCsPhenotype：
Third generation cell is collected, PBS is washed 3 times；
Plus 0.1% saponin room temperature rupture of membranes 1 hour, PBA is washed 3 times（If detecting intracellular antigen, this step is carried out；If detection
Cell surface antigen, then directly carry out next step）；
Plus primary antibody（CD29, CD44, CD105, CD31, CD34, CD106, HLA-DR, FLK-1 monoclonal antibody, are purchased from：BD companies）,
4 DEG C are incubated 30 minutes；
Plus PBS is washed 3 times, plus FITC mark secondary antibodies（Rabbit-anti mouse, purchased from Bioisystech Co., Ltd of Zhong Shan Golden Bridge）, 4 DEG C incubate
Educate 30 minutes；
Plus PBS is washed 3 times, 4% paraformaldehyde is fixed, and 300ul PBS are resuspended；
Flow cytometer（Brand, model）Detect cell.Identified, typical AMSC phenotypes are presented in cell：
CD29+、CD44+、CD105+、FLK-1+；
CD31-、CD34-、CD106-、HLA-、DR-。
Embodiment 2：miRNAsTransfection
1. reagent：
（1）Transfection reagent is Lipofectamine2000（Invitrogen）；
（2）MiRNA to be transfecteds：
MiR-216 powder synthesizes for Life Technologies companies of the U.S.；
The unrelated sequences similar to miR-216 length are synthesized using known technology（SEQ ID No.2：
UUCUUCGAACGUGUCACGUTT）Compareed as NC.
2. miRNA to be transfectedsStoring solution is configured：
It will be equipped with miRNA to be transfectedsPipe low-speed centrifugal 5min, according to Lipofectamine2000
（Invitrogen）Specification adds Opti-MEM dissolvings miRNAs, shake the storing solution that simultaneously brief centrifugation is made into 20 μM.
3. cell prepares：
The hAMSC for taking the 3rd generation exponential phase, 70%-80% to convergesTransfected.
4. transfection process：
Dilute miRNA to be transfected respectively with Opti-MEMsAnd Lipofectamine2000, room temperature is put after gently mixing
Put 5min；
Lipofectamine2000 dilutions are slowly added dropwise in miRNAs dilutions to be transfected, gently mixed
Obtain miRNAsWith Lipofectamine2000 mixed liquors, 20min is incubated at room temperature；
Cell to be transfected is taken out from incubator, nutrient solution is suctioned out, uses DPBS liquid（Du Shi phosphate buffers）Wash 3 times, exhaust
1.5ml Opti-MEM are added after washing lotion per hole；
By the miRNA being incubateds- Lipofectamine2000 mixed liquors slowly enter transfections amount in culture dish dropwise：
miRNAsFinal concentration of 100nmol/L, jog puts incubator and continues to cultivate after mixing；
Transfect after 6h, remove transfection working solution, washed with DPBS 3 times；
Change stem cell medium；After 24h, stem cell media is removed, is washed with DPBS 3 times, subsequent replaceable skeletonization is lured
Lead culture medium.
Embodiment 3：HAMSCs is to Osteoinductive differentiation
The hAMSCs in the 3rd generation is taken respectively（Without transfection）, the cell that is obtained of embodiment 2（Transfection has miR-216 or turned
It is infected with NC controls）With 2 × 104Cell/cm2Density be inoculated in T25 blake bottles, it is adherent overnight after, observation cell reaches 70%-80%
After converging, Osteogenic Induction Medium is changed（10%FBS, 10nM dexamethasone, 0.2mM ascorbic acid are added in H-DMEM culture mediums
With 10mM beta- sodium glycero-phosphates）Continue to cultivate, half amount changes liquid once every three days.
Sampled respectively at the 0th after induction, 3,6,9 days, the expression for detecting Osteogenesis phenotype marker gene with qRT-PCR becomes
Change.
When 3 days after induction and 12 days, calcification situation and ore deposit are identified with alkaline phosphatase staining and Alizarin red staining
Change the generation of matrix.
Embodiment 4：hAMSCsTo the identification of Osteoblast Differentiation
1. alkaline phosphatase staining
1.1 experimental principle：Intracellular alkaline phosphatase hydrolyzes phosphoric acid naphthols AS-MX under the conditions of pH9.4-9.6 and produces naphthalene
Phenol, the latter is captured by diazol, generates insoluble blue precipitate.
1.2 operating procedure：
Institute's alkaline phosphatase staining kit, which is ground, using Tianjin blood carries out alkaline phosphatase staining.
Few drops of No. 1 liquid are added dropwise into the culture dish of osteogenic induction, room temperature fixes 1min, flowing water rinsing 2min；
No. 2 storing solution 10ml, plus No. 3 μ l of liquid 200 are taken, then add No. 4 pulvis 10mg, shaking is instant, is filtered immediately with filter paper,
Filtrate is working solution：
Working solution is added dropwise few drops, puts and be incubated 2h in wet box in 37 DEG C, flowing water rinses 2min；
Glycerine mounting, micro- Microscopic observation photograph.
2. Alizarin red staining
2.1 experimental principle：Alizarin red to chelate mode can form compound with calcium ion.For recognizing histiocytic calcium
Salt component.By Alizarin red staining, salmon pink deposition is produced（That is calcium tubercle）, Chinese red is dyed in extracellular matrix.Carefully
Born of the same parents are dyed to pink in itself.Calcium salt change is one of mark of bone cell proliferation differentiation and bone tissue osteogenic potential.
2.2 operating procedure：
Culture dish is rinsed 2 times with PBS, and 95% ethanol fixes 10min, deionized water rinsing 3 times；
0.1% alizarin red S（Tris-HCl, pH8.3）37 DEG C of incubation 30min；
Distilled water flushing；
Glycerine mounting, micro- Microscopic observation photograph.
3. alkaline phosphatase（ALP）Determination of activity
3.1 experimental principle：ALP and substrate pNPP reacts, and generates yellow substance, can by the absorbance determined at 405nm
To react ALP activity.
3.2 operating procedure：
Cultivate cell and remove culture medium, cold PBS is washed 2 times, plus appropriate protein lysate（RIPA lysates（In）, the green skies
Biotechnology research institute）, 30min is cracked on ice, collects cell pyrolysis liquid；
12000rpm is centrifuged 15 minutes at 4 DEG C；
5 μ l cell pyrolysis liquids are taken, 96 hole elisa Plates are added；
Plus 200 μ l alkaline phosphatase substrate pNPP（Sigma companies）；
37 DEG C of incubation 30min；
405nm detects light absorption value；
ALP activity is calculated, ALP activity is using the total protein concentration of cell pyrolysis liquid as standardization.
As a result：
1.hAMSCs（Without transfection）By the induction of Osteogenic Induction Medium, ALP stained positives after 3 days（Fig. 1）, 12
Occurs the positive calcium tubercle of Alizarin red staining after it（Fig. 2）.The result shows, hAMSCsUnder osteogenic induction condition of culture, tool
There is the ability of Osteoblast Differentiation.
2.miR-216a promotes hAMSCsOsteoblast Differentiation
（1）Fig. 3 is the result that ALP is dyed（Fig. 3 A are that NC control groups, Fig. 3 B are miR-216 groups）.Specifically, from Fig. 3 A
In it is visible, the cell of only less ratio is in ALP stained positives.Fig. 3 B show that most cells are in ALP stained positives.This difference
It is different to show miR-126 compared with NC, hAMSC can be promotedsOsteoblast Differentiation.
（2）Fig. 4 is the result of Alizarin red staining（Fig. 4 A are that NC control groups, Fig. 4 B are miR-216 groups）.Specifically, from
Visible in Fig. 4 A, small part cell is positive in Alizarin red staining.But Fig. 4 B show that most cells are positive in Alizarin red staining.
This result demonstrates again that miR-126 compared with NC, can promote hAMSCsOsteoblast Differentiation.
（3）After osteogenic induction, ALP activity increases transfect the active ratios of ALP of NC controls without osteogenic induction
hAMSCsIt is high.However, transfection has miR-216a, hAMSC after osteogenic inductionsAlkaline phosphatase activities apparently higher than turn
It is infected with the hAMSC of the osteogenic induction of the process same time of NC controlss（Fig. 5）.This result shows miR-126 compared with NC,
HAMSC can be promotedsOsteoblast Differentiation.
To sum up as a result, it was confirmed that transfecting after miR-216a, hAMSCsOsteogenic ability increase.
Bibliography：
Ambros,V.The functions of animal microRNAs.Nature2004;431:350-355.
Bartel,DP.MicroRNAs:genomics,biogenesis,mechanism,and
function.Cell2004;116:281-297.
Fink,T,JG Rasmussen,J Emmersen,et al.Adipose-derived stem cells from
the brown bear(Ursus arctos)spontaneously undergo chondrogenic and osteogenic
differentiation in vitro.Stem Cell Res2011;7:89-95.
Ivey,KN and D Srivastava.MicroRNAs as regulators of differentiation
and cell fate decisions.Cell Stem Cell2010;7:36-41.
Phinney,DG and DJ Prockop.Concise review:mesenchymal stem/multipotent
stromal cells:the state of transdifferentiation and modes of tissue repair--
current views.Stem Cells2007;25:2896-2902.
Pittenger,MF,AM Mackay,SC Beck,et al.Multilineage potential of adult
human mesenchymal stem cells.Science1999;284:143-147.
Prockop,DJ.Marrow stromal cells as stem cells for nonhematopoietic
tissues.Science1997;276:71-74.
Wang,X,B Guo,Q Li,et al.miR-214targets ATF4to inhibit bone
formation.Nat Med2013;19:93-100。
Claims (5)
- Purposes of the 1.miR-216 in the medicine for promoting Osteoblast Differentiation is prepared, wherein miR-216 nucleotide sequence such as SEQ ID Shown in No.1.
- 2. purposes according to claim 1, miR-216 promotes the mescenchymal stem cell of Human Adipose Tissue-derived to divide to skeletonization Change.
- 3. purposes according to claim 1, medicine contains transfection reagent.
- 4. purposes according to claim 3, the transfection reagent is Lipofectamine2000.
- 5. purposes according to claim 3, miR-216 transfection concentrations are 100nmol/L.
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| CN106497885B (en) * | 2016-11-16 | 2019-12-20 | 中国医学科学院基础医学研究所 | Application of miR-10b in promoting mesenchymal stem cells to differentiate into osteoblasts and inhibiting mesenchymal stem cells from differentiating into adipoblasts in vitro |
| CN106701922A (en) * | 2016-12-05 | 2017-05-24 | 边焱焱 | Application of micro RNA in preventing or treating femoral head necrosis caused by glucocorticoid |
| CN107513571B (en) * | 2017-09-30 | 2020-07-07 | 首都医科大学附属北京口腔医院 | Application of miRNA |
| CN108841947A (en) * | 2018-05-22 | 2018-11-20 | 广州中医药大学第附属医院 | A kind of urine excretion body hsa-microRNA206 and its application |
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