CN105483081A - MiRNA145-5p-modified umbilical cord mesenchymal stem cell exosome and preparation and application of miRNA145-5p-modified umbilical cord mesenchymal stem cell exosome - Google Patents
MiRNA145-5p-modified umbilical cord mesenchymal stem cell exosome and preparation and application of miRNA145-5p-modified umbilical cord mesenchymal stem cell exosome Download PDFInfo
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Abstract
The invention belongs to the technical field of biology and particularly relates to a miRNA145-5p-modified umbilical cord mesenchymal stem cell exosome and preparation and application of the miRNA145-5p-modified umbilical cord mesenchymal stem cell exosome. The invention provides a preparation method of the highly-miRNA145-5p-expressed umsc-exosome (umbilical cord mesenchymal stem cell exosome) and application of various biological preparations for accelerating full-thickness skin defect healing and antagonizing scar contracture. The preparation method of the exosome includes (1), adopting fresh umbilical cords to culture human umbilical cord mesenchymal stem cells; (2) preparing mesenchymal stem cells highly expressed in miRNA145-5p; (3), storing conditional media; (4) performing exosome extraction and purification. The highly-miRNA145-5p-expressed umbilical cord mesenchymal stem cell exosome is transferred into a full-thickness wound model of rat backs by serving as a biological preparation, so that skin granulation tissue proliferation can be promoted effectively, wound healing is accelerated and scar contracture can be antagonized; novel approaches and novel methods are provided for wound treatment.
Description
Technical field
The present invention relates to biological technical field, particularly relate to microRNA145-5p, modify umbilical cord mesenchymal stem cells (humanumbilicalcordmesenchymalstemcell, hucMSC) outer in is secreted body (exosome) and is extracted the method for preparation and it suppresses scar proliferation, the application especially in joint part antagonism scar treatment at wound healing.
Background technology
Myofibroblast (myofibroblast) is a kind of cell highly differentiated by inoblast of expressing smooth muscle actin (α-SMA), it take part in processbearing astrocyte and extracellular matrix remodeling two stage (VanDeWaterL in fibrosis cascade reaction, VarneyS, TomasekJJ, MechanoregulationoftheMyofibroblastinWoundContraction, Scarring, andFibrosis:OpportunitiesforNewTherapeuticIntervention.2 013May; 2 (4): 122-141).Its major function produces strength and changes tissue tension, is the cell that wound granulation shrinks and filament contraction disease is main.These cells secrete collagen, fibronectin, somatomedin and various enzyme in a large number.When tissue sustains damage because oxidative stress, hypoxia, inflammation, apoptosis etc. stimulate, although by damaged tissue displacement being attempted to repair with extracellular matrix, but when damaging very serious or when this stimulation becomes chronicity etc., myofibroblast differentiation is then not modulated and then cause inappropriate or excessive fibrillar connective tissue to generate, and at the excessive scar proliferation that skin injury causes, the cicatricial adhesion contracture caused at joint part can think the hyperplasia of myofibroblast in the lump.Document is more had to show its existence and promote that tumor growth also has direct relation (JunkerJP
1, KratzC,
a, KratzG, echanicaltensionstimulatesthetransdifferentiationoffibro blastsintomyofibroblastsinhumanburnscars.Burns.2008Nov; 34 (7): 942-6).
In recent years, stem cell related mechanism and be applied in wound repair field and had new breakthrough.Research shows that mescenchymal stem cell (mesenchymalstemcells, MSCs) can promote the healing of skin wound and antagonism scar is excessively formed.But itself amount of survival after being implanted into new environment sharply declines in time.What it played Main Function is the microenvironment that some active factores produced by paracrine have impact on the surface of a wound, and then wound healing, suppression excessive tissue fibrosis and maintain better form (NieC, YangD, XuLeta1.Locallyadministeredadipose-derivedstemcellsacceleratewoundhealingthroughdifferentia tionandVasculogenesis [J] .CellTransplant, 2011,20 (2): 205-216).
Secrete body (exosome) outward more and more to receive publicity as the vesicles of a kind of bimolecular lipid membrane parcel from stem cells paracrine.It, by after the multivesicular body of cell and plasma membrane fusion, is discharged in born of the same parents' external environment in the mode of exocytosis.Surface is containing a large amount of and its source and the closely-related protein of function, lipid composition and nucleic acid, wherein (the SimpsonRJ based on microRNA, LiraJW, MoritzRL, et.Exosomes:proteomicinsightsanddiagnosticpotential [J] .ExpertRevProteomics, 2009,6 (3): 267-283).Secrete body cytolemma that is comparatively easy and adjacent cells outward to merge, biological active agents is optionally delivered to recipient cell, carries out information transmission at different iuntercellular, regulate intercellular intracellular signaling, play various biological function.(RecordM，SubraC,etal.Exosomesasintercellularsignalosomesandpharmacologicaleffectom[J].BiochemPharmaeol，2011，81(10)：1171—1182)。Follow-up research confirms that again the outer microRNA secreted in body still can keep higher stability under the mal-conditions such as different acid or alkali environment, anoxic, high temperature, multigelation, the effect of RNase can also be resisted, mainly microRNA can be wrapped up because secrete body outward and keep its integrity (UmezuT, OhyashikiK, KurodaM, eta1.Leukemiacelltoendothelialcellcommunicationviaexosom almiRNAs [J] .Oncogene, 2013,32 (22): 2747-55.).Therefore, mescenchymal stem cell may be by this mechanism the miRNA being conducive to wound healing and antagonism scar be passed to wound circumference and play function.
Hsa-miR-145 (miR-145, Accessionnumber:MIMAT0000437) as the microRNA that a research is relatively thorough, its report in modulate tumor propagation existing before, as Chinese patent CN201110311076.2, application publication number is CN102676516A, denomination of invention is " novelty teabag of microRNA-145 ", significantly can strengthen invasion and attack and the transfer ability of high Metastatic Colorectal Cancer cell after providing miR-145 process LAN.Its precursor sequence of miR-145 can generate two kinds of different ripe bodies and hsa-miR-145-5p (miR-145-5p under normal physiological conditions, Accessionnumber:MIMAT0000437) with hsa-miR-145-3p (miR-145-3p, Accessionnumber:MIMAT0004601).These two kinds of ripe bodies are very few at wound area research, and the miR-145-5p of one of its ripe body is in the news and can participates in regulation and control TGF-β/SMADS signal transduction pathway, and this path extensively exists in wound repair and (the reference HeldinCH that plays an important role, LandstrSmM, MoustakasA.MechanismofTGF-betasignalingtogrowtharrest, apoptosis, andepithetlial-mesenchymaltransition [J] .CurrOpinCellBiol, 2009,21:166-176).Interference TGF β/SMAD controllable inoblast alleviates excess fibrosis then to the differentiation of myofibroblast, therefore may have vital role to inoblast to the differentiation of myofibroblast to the miR-145-5p that this path has a regulating and controlling effect.
There is no relevant miR-145-5p at present and modify the outer bibliographical information secreting body; More modify the outer of umbilical cord mesenchymal stem cells secretion without miR-145-5p and secrete the relevant report of body for Wound treating field.
Summary of the invention
The object of this invention is to provide the outer of umbilical cord mesenchymal stem cells secretion modified with hsa-miR-145-5p (miR-145-5p) and secrete body and preparation method thereof; The outer body of secreting that another object of the present invention is to the umbilical cord mesenchymal stem cells secretion providing miR-145-5p to modify is accelerating union of wounded skin and is suppressing the application in scar anti-hyperproliferative agent simultaneously.
The outer body of secreting of source for mesenchymal stem cells is carried out improvement modification by this patent, the miR-145-5p that process LAN suppresses myofibroblast to be formed, exploitation becomes a kind of action character both with mescenchymal stem cell, the novel therapeutic means of its bad differentiation and tumour formation defect can be evaded again, not only promote the healing of the surface of a wound, also prevent the hyperplasia of scar, the targeted therapy for microRNA provides new thinking simultaneously.
A first aspect of the present invention, provide a kind of the outer of umbilical cord mesenchymal stem cells secretion modified with microRNA145-5p and secrete body, described outer body of secreting is secreted by the umbilical cord mesenchymal stem cells of process LAN MicroRNA145-5p.
MiR-145-5p, Accessionnumber:MIMAT0000437 in the umbilical cord mesenchymal stem cells of described process LAN MicroRNA145-5p, concrete sequence is as follows: guccaguuuucccaggaaucccu (SEQIDNO:1).
Wherein umbilical cord mesenchymal stem cells, can be selected from fresh umbilical cord cultivator umbilical cord mesenchymal stem cells, and detailed step is with reference to embodiment one.Also can be buied by ScienCell company of the U.S..
A second aspect of the present invention, provides the outer preparation method secreting body that the above-mentioned umbilical cord mesenchymal stem cells modified with microRNA145-5p is secreted.Step is as follows:
A. the Secondary Culture of umbilical cord mesenchymal stem cells;
B. the umbilical cord mesenchymal stem cells of process LAN microRNA145-5p is prepared;
C. the deposit of conditioned medium, described conditioned medium is the umbilical cord mesenchymal stem cells adding the process LAN microRNA145-5p that step B obtains in stem cell media, the supernatant liquor after 48h-72h;
D. outer extracting and the purifying secreting body.
Wherein, steps A preferably: get neonatal fresh umbilical cord, after phosphate buffered saline buffer rinses repeatedly, be soaked in L-DMEM containing 1% mycillin about 5 minutes, be cut into the tissue block that diameter is about 1.5mm size; Containing 10% foetal calf serum L-DMEM nutritive medium, 5%CO
2, 37 DEG C of saturated humidities cultivate; Until 7-10 days, afterwards visible mescenchymal stem cell climbed out of tissue, carried out Secondary Culture after removing tissue block with 0.25% tryptic digestion.
Step B is preferably: prepare high expression level miRNA145-5p mescenchymal stem cell, vitro culture.Be selected from step I) or step II):
I) the ripe body of synthetic MicroRNA145-5p, through liposome transfection the 3-6 generation mescenchymal stem cell, obtains the mescenchymal stem cell of MicroRNA145-5p high expression level; Wherein the construction process of the ripe body of microRNA145-5p is ordinary method, see reference book (work [U.S.] J. Sha nurse Brooker, Huang Peitang translates, " Molecular Cloning: A Laboratory guide ", Science Press), can be synthesized by Shanghai Ji Ma company.
II) build the recombinant vectors of the encoding gene containing MicroRNA145-5p, build the recombinant virus of the encoding gene containing MicroRNA145-5p, or build the recombinant viral vector of the encoding gene containing MicroRNA145-5p; By the recombinant vectors, the recombinant virus that obtain, or recombinant viral vector is transfected into umbilical cord mesenchymal stem cells, obtains the mescenchymal stem cell of MicroRNA145-5p high expression level.The construction process of carrier for expression of eukaryon is ordinary method, see reference book (work [U.S.] J. Sha nurse Brooker, Huang Peitang translates, " Molecular Cloning: A Laboratory guide ", Science Press), can be synthesized by Shanghai Ji Ma company.
Wherein, step I) the described concrete steps through liposome transfection are:
1) by the 3-6 arbitrary generation umbilical cord mesenchymal stem cells to be inoculated in 6 orifice plates in day before transfection and (normally to cultivate, 37 degree, 5%CO
2incubator), during transfection, cell density is about 60%.
2) lipo2000 (invitrogen company) is used to carry out transfection.Described transfection procedure is: in liposome: the ratio of microRNA=1:20 (microlitre/mole) carries out transfection to the cell growing to 60% fusion.Concrete steps are with reference to specification sheets.After transfection 48 hours, harvested cell carries out next step research.
Wherein, step II) the described structure concrete steps that contain the recombinant viral vector of the encoding gene of MicroRNA145-5p are:
1). by well-grown from steps A obtain go down to posterity after the 3-6 arbitrary generation umbilical cord mesenchymal stem cells to be inoculated into 6 orifice plates in day before transfection and (normally to cultivate, 37 degree, 5%CO
2incubator), during transfection, cell density is about 40%.
2). replace former substratum with the 2ml fresh stem cell substratum containing 6 μ g/ml polybrenes, add appropriate miRNA145-5P slow virus suspension.Hatch for 37 DEG C.
3). continue cultivation 24 hours, replace containing virulent substratum with mescenchymal stem cell substratum.
4). then 37 degree are continued cultivation 72 hours, obtain the mescenchymal stem cell (microRNA145-5p-UMSC) modified with miRNA145-5p for next step experiment.(purchased from Chinese Shanghai GenePharma company, transfecting stem cells, obtains the mescenchymal stem cell (NC-UMSC) of Nc contrast to adopt the slow virus expression system used the same method Nc.
Step C is preferably: will not add in the mescenchymal stem cell of high expression level microRNA145-5p containing the stem cell media (preparation method please refer to embodiment 2) of secreting body serum outward, collect culture supernatant after 48h-72h and be conditioned medium, as limited in culture supernatant amount of liquid, can be temporarily frozen in-80 DEG C of preservations, some amount to be collected into (about > 150ml) is in order to secrete body outside extracting;
Step D is preferably: CMC model is based on 4 DEG C of centrifugal removing cell debriss of 300 × g, 10min; The impurity such as 4 DEG C of 2000 × g, 10min centrifugal segregation dead cells; 0.22 μm of sterilised membrane filter filters removes impurity further; Obtain the exosome precipitation containing microRNA145-5p process LAN after 4 DEG C of 100000 × g, 120min ultracentrifugations, add about 200ulPBS and wash one time; 4 DEG C of 100000 × g, 120min ultracentrifugations again, can obtain the outer of umbilical cord mesenchymal stem cells source that purer concentration modified by microRNA145-5p and secrete body.
The outer body of secreting that a third aspect of the present invention provides people's umbilical cord mesenchymal stem cells source of above-mentioned microRNA145-5p is preparing wound healing medicine, prepares scar proliferation medicine and wound healing and suppresses the application in scar proliferation medicine.
The outer body of secreting of the umbilical cord mesenchymal stem cells secretion of being modified by microRNA145-5p of the present invention is used for vitro in fibroblast experiment, checks it to regulate and control fibroblastic propagation and the ability to myofibroblast differentiation.
The outer body of secreting of the umbilical cord mesenchymal stem cells secretion of being modified by microRNA145-5p of the present invention is used for rat back full thickness dermal wounds model to check its whether wound healing and antagonism scar proliferation.
The present invention the experiment proved that, the outer body of secreting of the umbilical cord mesenchymal stem cells modified with miRNA145-5p has the performance accelerated healing speed and also can suppress scar hyperplasia simultaneously, for the outer body of secreting after modified opens up new approach for clinical treatment wound.
The invention has the advantages that:
1. efficiently solve microRNA easily to degrade at born of the same parents' external environment, a difficult problem for its function cannot be played;
2. select the microRNA145-5p that has superiority at wound healing, by manual intervention, process LAN, in exosome, makes exosome more powerful and be rich in specific aim in the function in this field;
3. the outer body of secreting of process LAN miRNA145-5p is easy to-80 DEG C of preservations for a long time, still keeps due function after melting;
4. for clinical solution skin especially holostrome large defect antagonism cicatricial contracture is opened with the outer new therapy secreted body and be activeconstituents after modified.
Accompanying drawing explanation
The qualification streaming figure of Fig. 1 umbilical cord mesenchymal stem cells.
Fig. 2 A is that Nanosight detector image display the outer of source for mesenchymal stem cells secretes body; 2B is diameter range (transverse axis) and the abundance (longitudinal axis) of secreting body outside Nanosight detector display mesenchyme; 2C, for Nanosight detector measure umbilical cord mesenchymal stem cells secretion outer body of secreting mainly concentrate between 30-100nm; 2D, secrete body for WesternBlotCD81 qualification is outer.
The expression (* * P < 0.05) of the miRNA145-5p of stem cell after Fig. 3 process LAN miRNA145-5p, RT-PCR is detected the expression level of expressing mir-145-5p in umbilical cord mesenchymal stem cells after mir-145-5p;
Secrete body outside the mescenchymal stem cell of Fig. 4 process LAN miRNA145-5p and be accelerated into fibrocyte proliferation.Shown in figure A, the fluidic cell cell cycle is detected.A1, blank group; Body group (experimental group) is secreted outside A2, process LAN miRNA145-5p; A3, TGF β group (positive controls); A4, process LAN NC group; Figure B histogram is the ratio that G2 phase cell accounts for all growth cycle cells, and after visible A2 experimental group intervenes inoblast, G2 ratio is the highest, and prompting cell proliferation is the fastest; Figure C scratch experiment detects cell migration ability (48h) C1, blank group; Body group (experimental group) is secreted outside C2, process LAN miRNA145-5p; C3, TGF β group (positive controls); C4, process LAN NC group; Figure D histogram is the relative clearance measuring different time points (0h, 24h, 48h), and after visible C2 experimental group intervenes inoblast, each time point relative clearance is all minimum, is illustrated as fibrocyte transfer ability the fastest.
Secrete body outside Fig. 5 process LAN miRNA145-5p and be suppressed to fibrocyte to myofibroblast differentiation.A, westernblot detect α-SMA protein level, and secrete soma prognosis outside visible process LAN miRNA145-5p, the significant protein alpha-SMA of myofibroblast obviously declines; A1, blank group; A2, TGF β group (positive controls); Body group (experimental group) is secreted outside A3, process LAN miRNA145-5p; A4, process LAN NC group; B, RT-PCR detect myofibroblast Specific marker α-SMA expression amount and I-type collagen (CollagenI) expression amount, after visible B3 experimental group is intervened, the Specific marker α-SMA expression amount of myofibroblast and the mrna expression amount (longitudinal axis) of I-type collagen (CollagenI) are obviously lowered.B1, blank group; B2, TGF β group (positive controls); Body group (experimental group) is secreted outside B3, process LAN miRNA145-5p; B4, process LAN NC group.
Secrete body outside Fig. 6 process LAN miRNA145-5p promote nude mice back Repair of Cutaneous Full-thickness Excision and suppress cicatrization.Full-thickness defects wound model is set up at rat nude mice back, observes surface of a wound diameter afterwards, secrete body and form face diameter minimum (A) outside visible process LAN miRNA145-5p in 14 days.Observe after 25 days, outside visible process LAN miRNA145-5p, secrete body group scar minimum (B).1 blank group; Body group is secreted outside 2 process LAN miRNA145-5p; 3NC group.
Secreting body outside Fig. 7 process LAN miRNA145-5p suppresses myofibroblast to be formed.Nude mice back surface of a wound skin was in sampling in 25 days, and immunohistochemical methods detects the expression of myofibroblast specific proteins α-SMA, and it is minimum containing α-SMA to secrete body group outside visible process LAN miRNA145-5p.1 blank group; Body group is secreted outside 2 process LAN miRNA145-5p; 3NC group.N represents normal region skin histology, and W represents surface of a wound skin histology.
Embodiment
Now in conjunction with the embodiments and accompanying drawing, the present invention is described in detail, but enforcement of the present invention is not limited only to this.
Agents useful for same of the present invention and raw material all commercially maybe can be prepared by literature method.The experimental technique of unreceipted actual conditions in the following example, usually according to reference book, (show [U.S.] J. Sha nurse Brooker, Huang Peitang translates, " Molecular Cloning: A Laboratory guide ", Science Press) described in condition, or according to the condition that manufacturer advises.
Embodiment 1: the acquisition of stablizing the umbilical cord mesenchymal stem cells of high expression level MicroRNA145-5p
One, the acquisition of umbilical cord mesenchymal stem cells
Umbilical cord is from Changhai obstetrics and gynecology hospital.Original cuiture obtains mescenchymal stem cell.Obtain in the sterile petri dish or bottle that to be contained in after umbilical cord containing aseptic PBS or DMEM substratum, operate as early as possible.After phosphate buffered saline buffer rinses repeatedly, be soaked in L-DMEM (mycillin concentration is 100IU/ml) containing mycillin about 5 minutes, be cut into the tissue block that diameter is about 1.5mm size; Containing 10% foetal calf serum L-DMEM nutritive medium, 5%CO
2, 37 DEG C of saturated humidities cultivate; Until 7-10 days, afterwards visible mescenchymal stem cell climbed out of tissue, carried out Secondary Culture after removing tissue block with 0.25% tryptic digestion.Get the cell gone down to posterity in 3-6 generations and carry out subsequent operations.Umbilical cord mesenchymal stem cells qualification as shown in Figure 1.Flow cytomery result shows, and the homogeneous expression of the 4th generation Umbilical cord blood mesenchymal stem cells CD166, CD44, CD29, CD90 of cultivation, positive rate is respectively 96.4%, 97.7%, 97.1%, 96.2%, and CD45 is negative, and positive rate is 0.5%.
Two, cell transfecting
1. the ripe body of transfection synthetic MicroRNA145-5p.(wherein the ripe body of MicroRNA145-5p synthesizes in Chinese Shanghai Ji Ma company).According to the said firm's guide transfectional cell.
1) third generation umbilical cord mesenchymal stem cells is inoculated in 6 orifice plates in day before transfection (normally cultivates, 37 degree, 5%CO
2incubator), during transfection, cell density is about 60%.
2) lipo2000 (invitrogen company) is used to carry out transfection.Described transfection procedure is: in liposome: the ratio of microRNA=1:20 (microlitre/mole) carries out transfection to the cell growing to 60% fusion.Concrete steps are with reference to specification sheets.After transfection 48 hours, harvested cell carries out next step research.
2. (wherein foreign gene is miR-145-5p to transfection miRNA145-5P slow virus expression system, Accessionnumber:MIMAT0000437, purchased from Shanghai Ji Ma chemical gene Technology Co., Ltd., for the lentiviral particle of MicroRNA145-5p can be expressed), according to the said firm's guide transfectional cell.
1). by well-grown from step one obtain go down to posterity after third generation umbilical cord mesenchymal stem cells to be inoculated into 6 orifice plates in day before transfection and (normally to cultivate, 37 degree, 5%CO
2incubator), during transfection, cell density is about 40%.
2). replace former substratum with the 2ml fresh culture containing 6 μ g/ml polybrenes, add appropriate viral suspension.Hatch for 37 DEG C.
3). continue cultivation 24 hours, replace containing virulent substratum with fresh culture.
4). then 37 degree are continued cultivation 72 hours, obtain the mescenchymal stem cell (microRNA145-5p-UMSC) modified with miRNA145-5p for next step experiment.(purchased from Chinese Shanghai GenePharma company, transfecting stem cells, obtains the mescenchymal stem cell (NC-UMSC) of Nc contrast to adopt the slow virus expression system used the same method Nc.
Three, the expression of MicroRNA145-5p in cell is detected,
RT-PCR detects: utilize Trizol (Invitrogen, 15596-026) extract the total serum IgE of microRNA145-5p-UMSC and NC-UMSC of above-mentioned acquisition, take total serum IgE as template, MicroRNA145-5p primer (synthesis of Ji Ma company), carry out QRT-PCR, take U6 as internal reference (synthesis of Ji Ma company), with NC-UMSC in contrast.Result as shown in Figure 3, can be found out, with the ripe body of the MicroRNA145-5p of synthetic or with MicroRNA145-5p slow-virus transfection umbilical cord mesenchymal stem cells, all can obtain stable high expression level.Primer sequence is as follows:
MicroRNA145-5p reverse transcriptase primer:
GTCGTATCCAGTGCGAACTGTGGCGATCGGTACGGGCTACACTCGGCAATTGCACTGGATACGACAGGGA(SEQIDNO:2)
Universal PC R upstream: AGTGCGAACTGTGGCGAT (SEQIDNO:3)
PCR downstream: GTCCAGTTTTCCCAGGAATC (SEQIDNO:4)
The reverse transcriptase primer of U6: CGCTTCACGAATTTGCGTGTCAT (SEQIDNO:5)
Universal primer: CTCAAGTGTCGTGGAGTCGGCAA (SEQIDNO:6)
PCR downstream: CGCTTCACGAATTTGCGTGTCAT (SEQIDNO:7)
Outer extracting and the qualification of secreting body of embodiment 2:microRNA145-5p-UMSC
One, outer preparation of secreting body serum (exosome-free) substratum is gone
Foetal calf serum (life) is put in batches ultracentrifuge (Beckman company) carry out 4 DEG C 150000g12 hour centrifugal, secrete body to remove in foetal calf serum outer.Stem cell media will be made in proportion containing the serum secreting body outward.Obtain after this object is intended to secreting body outside purer stem cell.
Two, the extracting of body is secreted outside microRNA145-5p-UMSC
Before changing in the mescenchymal stem cell culture system of high expression level microRNA145-5p preparation go outer secrete body serum (exosome-freeFBS) substratum, culture supernatant is collected, as limited amount can be frozen in-80 DEG C of preservations temporarily after 48h-72h.To collect it after some amount (about > 150ml) in 4 DEG C of centrifugal removing cell debriss of 300 × g, 10min until conditioned medium; The impurity such as 4 DEG C of 2000 × g, 10min centrifugal segregation dead cells; 0.22 μm of sterilised membrane filter filters removes impurity further; 4 DEG C of 100000 × g, 120min ultracentrifugations obtain the exosome precipitation containing microRNA145-5p process LAN, add about 200ulPBS and wash one time; 4 DEG C of 100000 × g, 120min ultracentrifugations again, can obtain the outer of umbilical cord mesenchymal stem cells source that purer concentration modified by microRNA145-5p and secrete body.Be stored in-80 DEG C, can be preserved for a long time.
Three, the qualification of body is secreted outside microRNA145-5p-UMSC
1) Nanosight (Malvern Instr Ltd. of Britain): get 50 microliters of sample and be injected into pipe special.Fig. 2 A
2) western-blot detects the specific proteins CD81 of Exosome.
(1) with test kit, Exosome is extracted albumen, detect loading after protein content through BCA method
(2) electrophoresis, transferring film.
(3) be immersed in room temperature in 5%BSA confining liquid and slowly sway two hours.Hatch primary antibodie 4 DEG C to spend the night.
(4) suitable two are selected to resist according to primary antibodie source, by corresponding proportion dilution (1:1000 ~ 1:10000), room temperature jog two hours.
(5), after TBS washing, the colour developing of ECL luminescence reagent is used.Body Specific marker CD81 is secreted high-visible outside result.Fig. 2 B.
Embodiment 3: the outer body of secreting of the umbilical cord mesenchymal stem cells secretion of modifying with miRNA145-5p is to fibroblast proliferation and the cytology research breaking up ability of regulation and control
One, with fluidic cell cycle detection its on the impact of fibroblastic ability of cell proliferation
1) inoblast is laid in six orifice plates, until cell density 70% time, one hole add while adding TGF β cytokine outside process LAN Mir-145-5p umbilical cord mesenchymal stem cells secrete body (100ug/ml) (experimental group), a hole add while adding TGF β cytokine outside process LAN NC secrete body, a hole only adds TGF β cytokine (positive controls), a hole is not for add any intervention.
2) after 48h with 0.25% trypsin digestion cell, carry out fluidic cell cycle experimental.Result is visible: adding the inoblast secreting body outside process LAN Mir-145-5p umbilical cord mesenchymal stem cells is 20.85% in the ratio of G2 phase, adding the inoblast secreting body outside process LAN NC is 20.69% in the ratio of G2 phase, and the positive controls G2 phase is 14.84%, blank group is 14.08%.Show that the fibroblast propagation containing secreting body outside process LAN Mir-145-5p umbilical cord mesenchymal stem cells is the fastest.As shown in Fig. 4 A, B.
Two, its impact on migration of fibroblast cells ability is detected with cell scratch experiment
1) inoblast is laid in 12 orifice plates, when cell density is 90%, carries out cell cut.Clean cell 3 times with PBS, remove the cell under drawing, add serum free medium.One hole add while adding TGF β cytokine outside process LAN Mir-145-5p umbilical cord mesenchymal stem cells secrete body (100ug/ml) (experimental group), a hole add while adding TGF β cytokine outside process LAN NC secrete body, a hole only adds TGF β cytokine (positive controls), a hole is not for add any intervention.Each disposal hole respectively at once, 24h, 48h, 96h same position point under the microscope takes.Result is visible: substantially cover containing the inoblast secreting body outside process LAN Mir-145-5p umbilical cord mesenchymal stem cells cut when 48h; Blank group cut place is then still high-visible.Result shows that the outer of umbilical cord mesenchymal stem cells secretion modified with miRNA145-5p secretes the ability that body has promotion migration of fibroblast cells.As shown in Fig. 4 C, D.
Secrete body outside Fig. 5 process LAN miRNA145-5p and be suppressed to fibrocyte to myofibroblast differentiation.A, westernblot detect α-SMA protein level, and secrete soma prognosis outside visible process LAN miRNA145-5p, the significant protein alpha-SMA of myofibroblast obviously declines; A1, blank group; A2, TGF β group (positive controls); Body group (experimental group) is secreted outside A3, process LAN miRNA145-5p; A4, process LAN NC group; B, RT-PCR detect myofibroblast Specific marker α-SMA expression amount and I-type collagen (CollagenI) expression amount, after visible B3 experimental group is intervened, the Specific marker α-SMA expression amount of myofibroblast and the mrna expression amount (longitudinal axis) of I-type collagen (CollagenI) are obviously lowered.B1, blank group; B2, TGF β group (positive controls); Body group (experimental group) is secreted outside B3, process LAN miRNA145-5p; B4, process LAN NC group.
Three, detect with miRNA145-5p modify umbilical cord mesenchymal stem cells secretion outer body of secreting be suppressed to the ability of fibrocyte to myofibroblast differentiation
1.western-blot detects
1) inoblast is laid on six orifice plates, when cell density is 70%, it is processed.One hole add while adding TGF β cytokine outside process LAN Mir-145-5p umbilical cord mesenchymal stem cells secrete body (100ug/ml) (experimental group), a hole add while adding TGF β cytokine outside process LAN NC secrete body, a hole only adds TGF β cytokine (positive controls), a hole is not for add any intervention.Extract total serum IgE by Trizol method after 48h, remaining part utilizes isopropanol precipitating method to extract albumen, is placed in EP pipe by sample respectively and boils 10min.
2) loading after room temperature is returned to until sample.
3) electrophoresis, transferring film.
4) be immersed in room temperature in 5%BSA confining liquid and slowly sway two hours.α SMA (Abcam company) primary antibodie 4 DEG C of overnight incubation.
5) primary antibodie source is rabbit, therefore selects two of anti-rabbit to resist, by corresponding proportion dilution (1:200), and room temperature jog two hours.
6) wash, use the colour developing of ECL luminescence reagent.Result as shown in Figure 5A, with positive controls compared with process LAN NC group, is secreted body treatment group α-SMA and is expressed reduction outside the stem cell of process LAN microRNA145-5p.This result shows that the outer body of secreting that the umbilical cord mesenchymal stem cells modified with miRNA145-5p is secreted can be suppressed to the differentiation of fibrocyte to myofibroblast, and myofibroblast causes the factor of cicatricial contracture most critical just.
2.RT-PCR detects
1) inoblast is laid on six orifice plates, when cell density is 70%, it is processed.One hole add while adding TGF β cytokine outside process LAN Mir-145-5p umbilical cord mesenchymal stem cells secrete body (100ug/ml) (experimental group), a hole add while adding TGF β cytokine outside process LAN NC secrete body, a hole only adds TGF β cytokine (positive controls), a hole is not for add any intervention.
2) utilize Trizol (Invitrogen, 15596-026) to extract above-mentioned treatment group and control group total serum IgE after 48h, reverse transcription is cDNA.Design primer, detects the specific proteins α-SMA of myofibroblast and the expression level of extracellular matrix I-type collagen (CollagenI).GAPDH is as detection internal reference.To secrete body in contrast outside the NC of untransfected.
Primer sequence is as follows:
The primer of α-SMA is as follows:
f：GGACTCTGGGGATGGTGA(SEQIDNO:8)
r：AATGAAGGAGGGCTGGAAGA(SEQIDNO:9)
The primer of GAPDH is as follows:
f：AGTTGCGTTACACCCTTTCTTG(SEQIDNO:10)
r：GCTGTCACCTTCACCGTTCC(SEQIDNO:11)
The primer of CollagenI is as follows:
f：TGAGAGAGGGGTTGTTGGAC(SEQIDNO:12)
r：TTGAGAAGAGTTACGAGTTG(SEQIDNO:13)
Result as shown in Figure 5 B, add as seen miRNA145-5p modify umbilical cord mesenchymal stem cells secretion outer secrete body through after, α-SMA and I-type collagen (CollagenI) expression amount decline the most obvious.
Embodiment 4: the outer of the umbilical cord mesenchymal stem cells secretion of modifying with miRNA145-5p secretes the observation of curative effect that body repairs rat skin full-thickness defects model
1. skin healing situation
1) skin full-thickness defects model preparation: 12 SD rats (body weight is about 150g, male and female half and half) are divided into NC control group at random, secrete body group outside microRNA145-5p-UMSC, blank group.Often organize each four.Be positioned on operator's console with 10% chloral hydrate anesthesia rat, the circle being 1.5cm in rat back label diameter with tapping and plugging machine, sharp knife cuts transdermal to deep fascia surface along mark, creates circular skin full-thickness defects model.
2) be divided into four groups at random, often organize three.Inject at each group respectively.With the simple flap coverage of petrolatum gauze after injection, single cage is raised.
3) after treatment, 1d, 7d, 14d, 21d carry out the measurement of surface of a wound area, and continue to continue medication at subcutaneous four points of the surface of a wound.Result show: in cardinal principle, with miRNA145-5p modify outer secrete soma pre-when, the rat back surface of a wound in the 21st day close to healing, blank and NC contrast then still have slight crack.As shown in Figure 6.
2.HE dyeing and specificity α-SMA immunohistochemical staining
1) get the skin of each treatment group rat back surface of a wound and surrounding about 0.5cm healthy tissues in 21d, be fixed in 4% formaldehyde, through routine dehydration, paraffin embedding, cuts 5 μm of thick sections, and row HE dyes, specificity α-SMA histochemical staining.
2) secrete in the pre-surface of a wound of soma outside the stem cell that light Microscopic observation: miRNA145-5p modifies, the main component inoblast marshalling of healing, hair follicle structure is more ripe, and inflammatory cell infiltration is few, specific proteins α-SMA the expression amount of myofibroblast is low, and in arrangement regulation.And the myofibroblast of NC and the visible express alpha of blank group-SMA is gathered in the slight crack place of not healing, arrangement disorder, hair follicle structure is not formed.As shown in Figure 7.
This result shows that the outer body of secreting that the umbilical cord mesenchymal stem cells modified with miRNA145-5p is secreted repairs skin full-thickness defects speed obviously faster than blank and NC control group and without scar shrinkage phenomenon.
Below the preferred embodiment of the invention is illustrated, but the invention is not limited to described embodiment, those of ordinary skill in the art also can make all equivalent modification or replacement under the prerequisite without prejudice to the invention spirit, and these equivalent modification or replacement are all included in the application's claim limited range.
Claims (11)
1. the outer of umbilical cord mesenchymal stem cells secretion modified with microRNA145-5p secretes a body, and described outer body of secreting is secreted by the umbilical cord mesenchymal stem cells of process LAN microRNA145-5p.
2. the outer of umbilical cord mesenchymal stem cells secretion modified with microRNA145-5p according to claim 1 secretes body, and it is characterized in that, the sequence of described microRNA145-5p is as shown in SEQIDNO:1.
3. the outer preparation method secreting body that the umbilical cord mesenchymal stem cells modified with microRNA145-5p is as claimed in claim 1 secreted, step is as follows:
A. the Secondary Culture of umbilical cord mesenchymal stem cells;
B. the umbilical cord mesenchymal stem cells of process LAN microRNA145-5p is prepared;
C. the deposit of conditioned medium, described conditioned medium is the umbilical cord mesenchymal stem cells adding the process LAN microRNA145-5p that step B obtains in stem cell media, the supernatant liquor after 48h-72h;
D. outer extracting and the purifying secreting body.
4. according to claim 3 with the outer preparation method secreting body of the umbilical cord mesenchymal stem cells secretion of microRNA145-5p modification, it is characterized in that, wherein, steps A is: get fresh umbilical cord, after phosphate buffered saline buffer rinses repeatedly, be soaked in L-DMEM containing 1% mycillin about 5 minutes, be cut into the tissue block that diameter is about 1.5mm size; Containing 10% foetal calf serum L-DMEM nutritive medium, 5%CO
2, 37 DEG C of saturated humidities cultivate; After 7-10 day, visible umbilical cord mesenchymal stem cells climbs out of tissue, carries out Secondary Culture after removing tissue block with 0.25% tryptic digestion.
5. according to claim 3 with the outer preparation method secreting body of the umbilical cord mesenchymal stem cells secretion of microRNA145-5p modification, it is characterized in that, wherein, step B is selected from step I) or step II):
I) the ripe body of synthetic microRNA145-5p, through liposome transfection the 3-6 umbilical cord mesenchymal stem cells of arbitrary generation, obtains the umbilical cord mesenchymal stem cells of process LAN microRNA145-5p;
II) build the recombinant vectors containing microRNA145-5p encoding gene, build the recombinant virus of the encoding gene containing MicroRNA145-5p, or build the recombinant viral vector of the encoding gene containing MicroRNA145-5p; By the recombinant vectors, the recombinant virus that obtain, or recombinant viral vector is transfected into umbilical cord mesenchymal stem cells, obtains the umbilical cord mesenchymal stem cells of process LAN microRNA145-5p.
6. the outer preparation method secreting body of umbilical cord mesenchymal stem cells secretion of modifying with microRNA145-5p according to claim 5, is characterized in that, wherein, step I) described in the concrete steps through liposome transfection be:
A) by the 3-6 arbitrary generation umbilical cord mesenchymal stem cells be inoculated in 6 orifice plates in day before transfection, normally cultivate, 37 degree, 5%CO
2incubator, during transfection, cell density is 60%;
B) use lipo2000 carry out transfection, described transfection procedure is: in liposome: microRNA=1:20 microlitre/mole ratio transfection is carried out to the cell growing to 60% fusion.
7. the outer preparation method secreting body secreted with the umbilical cord mesenchymal stem cells of microRNA145-5p modification according to claim 5, is characterized in that, wherein, and step II) be:
A). by well-grown 3-6 arbitrary generation umbilical cord mesenchymal stem cells be inoculated in 6 orifice plates in day before transfection, normally cultivate, 37 degree, 5%CO
2incubator, during transfection, cell density is 40%;
B). replace former substratum by the 2ml stem cell media containing 6 μ g/ml polybrenes, add appropriate miRNA145-5P slow virus suspension; Hatch for 37 DEG C;
C). continue cultivation 24 hours, replace containing virulent substratum with mescenchymal stem cell substratum;
D). then 37 DEG C are continued cultivation 72 hours, obtain the mescenchymal stem cell microRNA145-5p-UMSC modified with miRNA145-5p.
8. according to claim 3 with the outer preparation method secreting body of the umbilical cord mesenchymal stem cells secretion of microRNA145-5p modification, it is characterized in that, step C is: by not adding in the mescenchymal stem cell of process LAN microRNA145-5p containing the stem cell media of secreting body serum outward, collecting culture supernatant and being conditioned medium after 48h-72h; If culture supernatant amount of liquid is limited, can be temporarily frozen in-80 DEG C of preservations, > 150ml to be collected into is in order to secrete body outside extracting.
9. according to claim 3 with the outer preparation method secreting body of the umbilical cord mesenchymal stem cells secretion of microRNA145-5p modification, it is characterized in that, step D is: CMC model is based on 4 DEG C of centrifugal removing cell debriss of 300 × g, 10min; The impurity such as 4 DEG C of 2000 × g, 10min centrifugal segregation dead cells; 0.22 μm of sterilised membrane filter filters removes impurity further; Obtain the exosome precipitation containing microRNA145-5p process LAN after 4 DEG C of 100000 × g, 120min ultracentrifugations, add about 200ulPBS and wash; 4 DEG C of 100000 × g, 120min ultracentrifugations again, obtain the outer of purifying and secrete body.
10. preparing the application in wound healing medicine with the outer body of secreting of the umbilical cord mesenchymal stem cells secretion of microRNA145-5p modification as claimed in claim 1 or 2 for one kind.
11. the outer of umbilical cord mesenchymal stem cells secretion modified with microRNA145-5p as claimed in claim 1 or 2 secretes the application of body in preparation suppression scar proliferation medicine.
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