CN105483081B - MiRNA145-5p modifies excretion body and its preparation and application of umbilical cord mesenchymal stem cells secretion - Google Patents
MiRNA145-5p modifies excretion body and its preparation and application of umbilical cord mesenchymal stem cells secretion Download PDFInfo
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Abstract
The invention belongs to field of biotechnology, and in particular to miRNA145-5p modifies excretion body and its preparation and application of umbilical cord mesenchymal stem cells secretion.The present invention provides the applications in the preparation method of excretion body (umsc-exosome) secreted by the umbilical cord mesenchymal stem cells of high expression microRNA145-5p and its healing of acceleration skin the full-thickness defects simultaneously various biological agents of antagonism cicatricial contracture.Excretion body of the invention is to be prepared by the following method: (1) using fresh umbilical cord culture people's umbilical cord mesenchymal stem cells；(2) mescenchymal stem cell of the high expression miRNA145-5p of preparation；(3) deposit of conditioned medium；(4) extracting and purifying of excretion body.The excretion body in the human umbilical cord mesenchymal stem cells source of high expression microRNA145-5p of the present invention is transferred in rat back holostrome wound model as biological agent can effectively facilitate skin granulation tissue hyperplasia, accelerate Wound healing speed and energy antagonism cicatricial contracture.The present invention provides new tool and method for Wound treating.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to microRNA145-5p, modification umbilical cord mesenchymal stem cells
Excretion body (exosome) in (human umbilical cordmesenchymal stem cell, hucMSC) extracts preparation
Method and its in wound healing and inhibit scar proliferation, the especially application in joint part antagonism scar treatment.
Background technique
Myofibroblast (myofibroblast) be it is a kind of expression smooth muscle actin (α-SMA) by fiber
The cell that cell height differentiates, it takes part in fiber in fibrosis cascade reaction and is formed and two ranks of extracellular matrix remodeling
Section (Van De Water L, Varney S, Tomasek JJ, Mechanoregulation of the Myofibroblast
in Wound Contraction,Scarring,and Fibrosis:Opportunities for NewTherapeu tic
Intervention.2013May；2(4):122-141).Its major function is to generate strength and change tissue tension, is wound
Face granulation is shunk and the main cell of filament contraction disease.These cells largely secrete collagen, fibronectin, growth because
Sub and various enzymes.When tissue is damaged due to the stimulation such as oxidative stress, hypoxia, inflammation, apoptosis, although passing through
When being replaced with extracellular matrix by injury tissue and attempt to repair, but damaging very serious or when the stimulation becomes chronicity
Deng, myofibroblast differentiation is not then modulated and then inappropriate or excessive fibrous connective tissue is caused to generate, and in skin
Excessive scar proliferation caused by damaging, the cicatricial adhesion contracture caused by joint part can think myofibroblast together
Hyperplasia.More there is document to show its presence and tumour growth is promoted also to have direct relation (Junker JP1,Kratz C,A,Kratz G,echanical tension stimulates the transdifferentiation of
fibroblasts into myofibroblasts in huma n burn scars.Burns.2008Nov；34(7):942-
6)。
In recent years, it stem cell related mechanism and applies and has new breakthrough in wound repair field.Research shows that mesenchyma
Stem cell (mesenchymal stem cells, MSCs) can promote healing and the excessive shape of antagonism scar of skin wound
At.But itself amount of survival after being implanted into new environment sharply declines at any time.What it played main function is by side point
The some active factors for secreting generation affect the microenvironment of the surface of a wound, and then wound healing, inhibition excessive tissue fibrosis are simultaneously
And maintain more good form (Nie C, Yang D, Xu L et a1.Locally administered adipose-
derived stem cells accelerate wound healing through differentiation and
Vasculogenesis [J] .Cell Transplant, 2011,20 (2): 205-216).
Vesicles of the excretion body (exosome) as a kind of bimolecular lipid membrane package from stem cells paracrine
More and more attention has been paid to.After it is by the multivesicular body and plasma membrane fusion of cell, it is discharged into extracellular environment in a manner of exocytosis.Surface
Containing protein largely closely related with its source and function, lipid components and nucleic acid, wherein based on microRNA
(Simpson RJ, Lira JW, Moritz RL, et.Exosomes:proteomic insights and diagnostic
Potential [J] .Expert Rev Proteomics, 2009,6 (3): 267-283).Excretion body is easier to and adjacent cells
Cell membrane merges, and biological active agents are selectively delivered to recipient cell, carries out information biography in different iuntercellulars
It passs, adjusts intercellular signal transduction, play multiple biological function.(Record M, Subra C, et al.Exosomes
as intercellular signalosomes and pharmacological effectom[J].Biochem
Pharmaeol, 2011,81 (10): 1171-1182).Subsequent research confirms the microRNA in excretion body in different acid again
Higher stability is still able to maintain under the mal-conditions such as alkali environment, anoxic, high temperature, multigelation, moreover it is possible to the effect of RNase is fought,
Be primarily due to excretion body microRNA can be wrapped up and keep its integrality (Umezu T, Ohyashiki K, Kuroda M,
et a1.Leukemia cell to endothelial cell communication via exosomal miRNAs[J]
.Oncogene, 2013,32 (22): 2747-55.).Therefore, mescenchymal stem cell may be by this mechanism and be beneficial to create
The miRNA of face healing and antagonism scar is transferred to wound circumference and functions.
Hsa-miR-145 (miR-145, Accession number:MIMAT0000437) is relatively saturating as a research
Thorough microRNA has its report in terms of modulate tumor proliferation, such as Chinese patent CN201110311076.2, Shen before
Please publication No. be CN102676516A, it is entitled " new application of microRNA-145 ", provide miR-145 be overexpressed after
Invasion and the transfer ability of high Metastatic Colorectal Cancer cell can be significantly increased.Its precursor sequence of miR-145 is in normal physiological conditions
Under can generate two different maturation body i.e. hsa-miR-145-5p (miR-145-5p, Accession number:
MIMAT0000437) with hsa-miR-145-3p (miR-145-3p, Accession number:MIMAT0004601).This two
Kind of mature body is very few in wound area research, and the miR-145-5p of one of its mature body be reported can participate in regulation TGF-β/
SMADS signal transduction pathway, and the access is widely present in terms of wound repair and (the bibliography Heldin that plays an important role
CH, LandstrSm M, Moustakas A.Mechanism of TGF-beta signaling to growth arrest,
Apoptosis, and epithetlial-mesenchymal transition [J] .Curr Opin Cell Biol, 2009,
21:166-176).Differentiation of the controllable fibroblast of TGF β/SMAD to myofibroblast is interfered then to mitigate excess fibrous
Change, therefore there is the miR-145-5p of regulating and controlling effect may have to fibroblast to the differentiation of myofibroblast the access
It plays an important role.
It there is no the document report of related miR-145-5p modification excretion body at present；It modifies between umbilical cord and fills there are no miR-145-5p
The excretion body of matter stem cell secretion is used for the relevant report in Wound treating field.
Summary of the invention
The object of the present invention is to provide the umbilical cord mesenchymal stem cells modified with hsa-miR-145-5p (miR-145-5p)
Excretion body of secretion and preparation method thereof；Another object of the present invention is to provide the umbilical cord mesenchyma of miR-145-5p modification is dry
The excretion body of cell secretion is accelerating union of wounded skin and is inhibiting the application in scar anti-hyperproliferative agent simultaneously.
The excretion body of source for mesenchymal stem cells is carried out improvement modification by this patent, is overexpressed and is inhibited myofibroblast shape
At miR-145-5p, be developed into it is a kind of not only with the action character of mescenchymal stem cell, but also can evade its bad differentiation and
Tumour forms the novel therapeutic means of defect, not only promotes the healing of the surface of a wound, while also preventing the hyperplasia of scar, is
The targeted therapy of microRNA provides new thinking.
The first aspect of the present invention provides a kind of umbilical cord mesenchymal stem cells secretion with microRNA145-5p modification
Excretion body, the excretion body be by be overexpressed MicroRNA145-5p umbilical cord mesenchymal stem cells secrete.
MiR-145-5p, Accession in the umbilical cord mesenchymal stem cells of the overexpression MicroRNA145-5p
Number:MIMAT0000437, particular sequence are as follows: guccaguuuucccaggaaucccu (SEQ ID NO:1).
Wherein umbilical cord mesenchymal stem cells can be selected from fresh umbilical cord culture people's umbilical cord mesenchymal stem cells, detailed step reference
Embodiment one.It can also be bought by ScienCell company, the U.S..
The second aspect of the present invention provides the above-mentioned umbilical cord mesenchymal stem cells secretion with microRNA145-5p modification
Excretion body preparation method.Steps are as follows:
A. the secondary culture of umbilical cord mesenchymal stem cells；
B. the umbilical cord mesenchymal stem cells for being overexpressed microRNA145-5p are prepared；
C. the deposit of conditioned medium, the conditioned medium are the mistake that step B is added in stem cell media and obtains
Express the umbilical cord mesenchymal stem cells of microRNA145-5p, the supernatant after 48h-72h；
D. the extracting and purifying of excretion body.
Wherein, step A is preferably: neonatal fresh umbilical cord being taken to be soaked in after phosphate buffer repeated flushing
L-DMEM containing 1% mycillin about 5 minutes, it is cut into the tissue block of diameter about 1.5mm size；Containing 10% fetal calf serum L-
DMEM nutrient solution, 5%CO2, 37 DEG C of saturated humidity cultures；Visible mescenchymal stem cell climbs out of tissue, removal group after 7-10 days
It knits and secondary culture is carried out with 0.25% trypsin digestion after block.
Step B is preferably: the high expression miRNA145-5p mescenchymal stem cell of preparation, in vitro culture.Selected from step I) or
Person's step II):
I) the mature body of artificial synthesized MicroRNA145-5p is obtained through liposome transfection the 3-6th generation mescenchymal stem cell
Obtain the highly expressed mescenchymal stem cell of MicroRNA 145-5p；Wherein the construction method of microRNA145-5p maturation body is normal
Rule method, reference can be made to reference book (writes [beauty] J. Sha's nurse Brooker, Huang Peitang is translated, " Molecular Cloning:A Laboratory guide ", scientific publication
Society), by Shanghai, Ji Ma company synthesizes
II) recombinant vector of the encoding gene containing MicroRNA145-5p is constructed, building contains MicroRNA145-5p
Encoding gene recombinant virus, or building the encoding gene containing MicroRNA145-5p recombinant viral vector；It will obtain
Recombinant vector, recombinant virus or recombinant viral vector be transfected into umbilical cord mesenchymal stem cells, obtain MicroRNA145-5p high
The mescenchymal stem cell of expression.The construction method of carrier for expression of eukaryon is conventional method, reference can be made to reference book (writes [beauty] J. Sha's nurse
Brooker, Huang Peitang are translated, " Molecular Cloning:A Laboratory guide ", Science Press), by Shanghai, Ji Ma company is synthesized.
Wherein, step I) specific steps through liposome transfection are as follows:
1) the 3-6th any generation umbilical cord mesenchymal stem cells are inoculated into 6 orifice plates in the day before transfection and (are normally cultivated, 37
Degree, 5%CO2Incubator), cell density is 60% or so when transfection.
2) it is transfected using lipo2000 (invitrogen company).The transfection procedure are as follows: press liposome:
The ratio of microRNA=1:20 (microlitre/mole) transfects the cell for growing to 60% fusion.Specific steps reference
Specification.Harvest cell carries out the research of next step after transfection 48 hours.
Wherein, step II) it is described building the encoding gene containing MicroRNA145-5p recombinant viral vector it is specific
Step are as follows:
1) by well-grown the 3-6th any generation umbilical cord mesenchymal stem cells after the passage that step A is obtained in turn
Dye the previous day, which is inoculated into 6 orifice plates, (normally cultivates, 37 degree, 5%CO2Incubator), cell density is 40% or so when transfection.
2) replaces former culture medium with the 2ml fresh stem cell culture medium containing 6 μ g/ml polybrenes, is added appropriate
MiRNA145-5P slow virus suspension.37 DEG C of incubations.
3) continues culture 24 hours, contains virulent culture medium with the replacement of mescenchymal stem cell culture medium.
4) then. continues culture 72 hours for 37 degree, obtains for next step experiment to fill between miRNA145-5p modification
Matter stem cell (microRNA145-5p-UMSC).The slow virus expression system of Nc (is purchased from by Chinese Shanghai using same method
GenePharma company, transfecting stem cells obtain the mescenchymal stem cell (NC-UMSC) of Nc control.
Step C is preferably: the stem cell media (preparation method please refers to embodiment 2) without excretion body serum is added
In the mescenchymal stem cell for entering high expression microRNA145-5p, it is conditioned medium that culture supernatant is collected after 48h-72h,
If culture supernatant amount of liquid is limited, it can temporarily freeze and be saved in -80 DEG C, wait collect to certain amount (about > 150ml) to take out
Mention excretion body；
Step D is preferably: CMC model is based on 4 DEG C of 300 × g, and 10min is centrifuged off cell fragment；4 DEG C of 2000 × g,
The impurity such as 10min centrifugation removal dead cell；0.22 μm of sterilised membrane filter filtering further removes impurity；4 DEG C of 100000 × g,
The exosome precipitating being overexpressed containing microRNA145-5p is obtained after 120min ultracentrifugation, and about 200ulPBS washing is added
One time；4 DEG C of 100000 × g again, 120min ultracentrifugation, the umbilical cord that available purer concentration is modified by microRNA145-5p
The excretion body of source for mesenchymal stem cells.
The third aspect of the present invention provides the excretion in people's umbilical cord mesenchymal stem cells source of above-mentioned microRNA145-5p
Body is preparing wound healing drug, prepares scar proliferation drug and wound healing and inhibits in scar proliferation drug
Using.
The excretion body for the umbilical cord mesenchymal stem cells secretion that microRNA145-5p of the present invention is modified is used for body
Outer fibroblast experiment, examines it to regulate and control fibroblastic proliferation and the ability to myofibroblast differentiation.
The excretion body for the umbilical cord mesenchymal stem cells secretion that microRNA145-5p of the present invention is modified is used for greatly
Mouse back full thickness dermal wounds model with examine its whether wound healing and antagonism scar proliferation.
The present invention the experiment proved that, with miRNA145-5p modification umbilical cord mesenchymal stem cells excretion body have accelerate
Healing speed and the performance that can inhibit scar hyperplasia simultaneously are that the excretion body after modified is created for clinical treatment
Wound opens up new approach.
The present invention has the advantages that
It is easy degradation in extracellular environment 1. efficiently solving microRNA, the problem of its function can not be played；
2. selection is in the advantageous microRNA145-5p of wound healing, by manual intervention, it is overexpressed in exosome,
Keep exosome more powerful in the function in this field and rich in specific aim；
3. the excretion body for being overexpressed miRNA145-5p is easy to -80 DEG C of long-term preservations, due function is still kept after melting；
4. being the clinical skin especially holostrome large defect and the unlatching of antagonism cicatricial contracture of solving with outer after modified
Secrete the new treatment that body is active constituent.
Detailed description of the invention
The identification streaming figure of Fig. 1 umbilical cord mesenchymal stem cells.
Fig. 2 A is the excretion body that Nanosight detector image shows source for mesenchymal stem cells；2B is Nanosight
Detector shows the diameter range (horizontal axis) and abundance (longitudinal axis) of mesenchyma excretion body；2C, it is measured for Nanosight detector
The excretion body of umbilical cord mesenchymal stem cells secretion is concentrated mainly between 30-100nm；2D, it reflects for Western Blot CD81
Determine excretion body.
The expression (* * P < 0.05) of the miRNA145-5p of stem cell, RT-PCR after Fig. 3 overexpression miRNA145-5p
Detect the expression of mir-145-5p in umbilical cord mesenchymal stem cells after being overexpressed mir-145-5p；
The mescenchymal stem cell excretion body that Fig. 4 is overexpressed miRNA145-5p accelerates fibroblast proliferation.Scheme stream shown in A
The detection of formula cell cycle.A1, blank control group；A2, it is overexpressed miRNA145-5p excretion body group (experimental group)；A3, TGF
β group (positive controls)；A4, it is overexpressed NC group；Figure B histogram is the ratio that G2 phase cell accounts for all growth cycle cells, can
After seeing that A2 experimental group intervenes fibroblast, G2 ratio highest prompts cell Proliferation most fast；Figure C scratch experiment detection cell moves
Shifting ability (48h) C1, blank control group；C2, it is overexpressed miRNA145-5p excretion body group (experimental group)；C3, TGF β group are (positive
Control group)；C4, it is overexpressed NC group；Figure D histogram be the relative clearance of measurement different time points (0h, for 24 hours, 48h), it is seen that C2
Various time points relative clearance is minimum after experimental group intervenes fibroblast, illustrates that fibroblast transfer ability is most fast.
Fig. 5 is overexpressed miRNA145-5p excretion body and inhibits fibroblast to myofibroblast differentiation.A,western
Blot detect α-SMA protein level, it is seen that be overexpressed the soma prognosis of miRNA145-5p excretion, myofibroblast it is significant
Protein alpha-SMA is decreased obviously；A1, blank control group；A2, TGF β group (positive controls)；A3, it is overexpressed outside miRNA145-5p
Secrete body group (experimental group)；A4, it is overexpressed NC group；B, RT-PCR detects myofibroblast Specific marker α-SMA expression quantity
And I-type collagen (Collagen I) expression quantity, it is seen that after B3 experimental group is intervened, the specificity marker of myofibroblast
The mrna expression amount (longitudinal axis) of object α-SMA expression quantity and I-type collagen (Collagen I) is obviously lowered.B1, blank pair
According to group；B2, TGF β group (positive controls)；B3, it is overexpressed miRNA145-5p excretion body group (experimental group)；B4, it is overexpressed NC
Group.
Fig. 6 is overexpressed miRNA145-5p excretion body and promotes nude mice back Repair of Cutaneous Full-thickness Excision and inhibit scar
It is formed.Full-thickness defects wound model is established at rat nude mice back, and surface of a wound diameter is observed after 14 days, it is seen that is overexpressed
MiRNA145-5p excretion body group surface of a wound diameter is minimum (A).It is observed after 25 days, it is seen that be overexpressed miRNA145-5p excretion body group scar
Trace minimum (B).1 blank control group；2 are overexpressed miRNA145-5p excretion body group；3NC group.
Fig. 7 is overexpressed miRNA145-5p excretion body and myofibroblast is inhibited to be formed.Nude mice back surface of a wound skin was in 25 days
Sampling, immunohistochemistry detect the expression of myofibroblast specific proteins α-SMA, it is seen that are overexpressed miRNA145-5p excretion
Body group-SMA containing α is minimum.1 blank control group；2 are overexpressed miRNA145-5p excretion body group；3NC group.N represents normal region skin
Skin tissue, W represent surface of a wound skin histology.
Specific embodiment
Now in conjunction with embodiment and attached drawing, the present invention is described in detail, but implementation of the invention is not limited only to this.
The reagents and materials used in the present invention are commercially available or can prepare by literature method.Tool is not specified in the following example
The experimental method of concrete conditions in the establishment of a specific crime usually (writes [beauty] J. Sha's nurse Brooker, Huang Peitang is translated, and " Molecular Cloning: A Laboratory refers to according to reference book
South ", Science Press) described in condition, or according to the normal condition proposed by manufacturer.
Embodiment 1: stablize the acquisition of the umbilical cord mesenchymal stem cells of high expression MicroRNA145-5p
One, the acquisition of umbilical cord mesenchymal stem cells
Umbilical cord comes from Changhai obstetrics and gynecology hospital.Originally culture obtains mescenchymal stem cell.It is mounted in after acquisition umbilical cord and is contained
Have in sterile petri dish or the bottle of sterile PBS DMEM culture medium, operates as early as possible.After phosphate buffer repeated flushing,
L-DMEM (mycillin concentration is 100IU/ml) about 5 minutes containing mycillin are soaked in, it is big to be cut into diameter about 1.5mm
Small tissue block；Containing 10% fetal calf serum L-DMEM nutrient solution, 5%CO2, 37 DEG C of saturated humidity cultures；It is visible after 7-10 days
Mescenchymal stem cell climbs out of tissue, removes and carries out secondary culture with 0.25% trypsin digestion after tissue block.Take passage 3-
The cell in 6 generations carries out subsequent operation.Umbilical cord mesenchymal stem cells identification is as shown in Figure 1.Flow cytomery is the results show that training
Feeding uniform expression CD166, CD44, CD29, CD90 of the 4th generation Umbilical cord blood mesenchymal stem cells, positive rate is respectively 96.4%,
97.7%, 97.1%, 96.2%, and CD45 is negative, positive rate 0.5%.
Two, cell transfecting
1. transfecting the mature body of artificial synthesized MicroRNA145-5p.(the wherein mature body synthesis of MicroRNA145-5p
In Chinese Shanghai Ji Ma company).Cell is transfected according to the said firm's guide.
1) third generation umbilical cord mesenchymal stem cells are inoculated into 6 orifice plates in the day before transfection and (are normally cultivated, 37 degree, 5%
CO2Incubator), cell density is 60% or so when transfection.
2) it is transfected using lipo2000 (invitrogen company).The transfection procedure are as follows: press liposome:
The ratio of microRNA=1:20 (microlitre/mole) transfects the cell for growing to 60% fusion.Specific steps reference
Specification.Harvest cell carries out the research of next step after transfection 48 hours.
2. transfecting miRNA145-5P slow virus expression system, (wherein foreign gene is miR-145-5p, Accession
Number:MIMAT0000437 is purchased from Shanghai Ji Ma chemical gene Technology Co., Ltd., for that can express MicroRNA145-5p's
Lentiviral particle), cell is transfected according to the said firm's guide.
1) is previous in transfecting by the third generation umbilical cord mesenchymal stem cells after well-grown passage obtained from step 1
It, which is inoculated into 6 orifice plates, (normally cultivates, 37 degree, 5%CO2Incubator), cell density is 40% or so when transfection.
2) replaces former culture medium with the 2ml fresh culture containing 6 μ g/ml polybrenes, and appropriate viral suspension is added.37
DEG C be incubated for.
3) continues culture 24 hours, contains virulent culture medium with fresh culture replacement.
4) then. continues culture 72 hours for 37 degree, obtains for next step experiment to fill between miRNA145-5p modification
Matter stem cell (microRNA145-5p-UMSC).The slow virus expression system of Nc (is purchased from by Chinese Shanghai using same method
GenePharma company, transfecting stem cells obtain the mescenchymal stem cell (NC-UMSC) of Nc control.
Three, the expression of MicroRNA145-5p in cell is detected,
RT-PCR detection: the microRNA145- of above-mentioned acquisition is extracted using Trizol (Invitrogen, 15596-026)
The total serum IgE of 5p-UMSC and NC-UMSC, using total serum IgE as template, MicroRNA145-5p primer (synthesis of Ji Ma company) is carried out
QRT-PCR, using U6 as internal reference (synthesis of Ji Ma company), using NC-UMSC as control.As a result fig. 3, it is shown that with
Artificial synthesized MicroRNA145-5p maturation body or with MicroRNA145-5p slow-virus transfection umbilical cord mesenchymal stem cells,
Stable high expression can be obtained.Primer sequence is as follows:
MicroRNA145-5p reverse transcriptase primer:
GTCGTATCCAGTGCGAACTGTGGCGATCGGTACGGGCTACACTCGGCAATTGCACTGGATACGACAGG
GA(SEQ ID NO:2)
The general upstream PCR: AGTGCGAACTGTGGCGAT (SEQ ID NO:3)
The downstream PCR: GTCCAGTTTTCCCAGGAATC (SEQ ID NO:4)
The reverse transcriptase primer of U6: CGCTTCACGAATTTGCGTGTCAT (SEQ ID NO:5)
Universal primer: CTCAAGTGTCGTGGAGTCGGCAA (SEQ ID NO:6)
The downstream PCR: CGCTTCACGAATTTGCGTGTCAT (SEQ ID NO:7)
The extracting and identification of the excretion body of embodiment 2:microRNA145-5p-UMSC
One, the preparation of excretion body serum (exosome-free) culture medium is gone
By fetal calf serum (life) be put into batches ultracentrifuge (Bake Mann) carry out 4 DEG C of 150000g 12 hours from
The heart, to remove the excretion body in fetal calf serum.Serum without excretion body is made into stem cell media in proportion.This purpose meaning
Purer stem cell excretion body is being obtained for after.
Two, the extracting of microRNA145-5p-UMSC excretion body
What is prepared before replacing in the mescenchymal stem cell cultivating system of high expression microRNA145-5p removes excretion body
Serum (exosome-free FBS) culture medium collects culture supernatant after 48h -72h, as limited amount can temporarily freeze in -80
DEG C save.It is centrifuged off carefully in 4 DEG C of 300 × g, 10min after conditioned medium is collected into certain amount (about > 150ml)
Born of the same parents' fragment；The impurity such as 4 DEG C of 2000 × g, 10min centrifugation removal dead cell；0.22 μm of sterilised membrane filter filtering further removes impurity；
4 DEG C of 100000 × g, 120min ultracentrifugation obtain the exosome precipitating being overexpressed containing microRNA145-5p, are added about
200ulPBS is washed one time；Purer concentration can be obtained by microRNA145- in 4 DEG C of 100000 × g again, 120min ultracentrifugation
The excretion body in the umbilical cord mesenchymal stem cells source of 5p modification.- 80 DEG C are stored in, it can long-term preservation.
Three, the identification of microRNA145-5p-UMSC excretion body
1) Nanosight (Malvern Instr Ltd., Britain): 50 microlitres of sample injections is taken to enter pipe special.Fig. 2A
2) western-blot detects the specific proteins CD81 of Exosome.
(1) Exosome is extracted into albumen with kit, the loading after BCA method detects protein content
(2) electrophoresis, transferring film.
(3) room temperature in 5%BSA confining liquid is immersed in slowly to sway two hours.It is incubated for 4 DEG C of primary antibody overnight.
(4) suitable secondary antibody is selected according to primary antibody source, dilutes (1:1000~1:10000) by corresponding proportion, room temperature is light
It shakes two hours.
(5) it after TBS washing, is developed the color using ECL luminescence reagent.As a result excretion body Specific marker CD81 is high-visible.
Fig. 2 B.
Embodiment 3: fibroblast is increased with the excretion body of the umbilical cord mesenchymal stem cells secretion of miRNA145-5p modification
Grow and break up the cytology research of ability of regulation and control
One, with its influence to fibroblastic ability of cell proliferation of fluidic cell cycle detection
1) fibroblast is laid in six orifice plates, when cell density is 70%, TGF β cell factor is added in a hole
It is added simultaneously and is overexpressed Mir-145-5p umbilical cord mesenchymal stem cells excretion body (100ug/ml) (experimental group), TGF β is added in a hole
Addition is overexpressed NC excretion body while cell factor, TGF β cell factor (positive controls) is only added in a hole, and a hole is not
Add any intervention.
2) fluidic cell cycle experimental is carried out with 0.25% trypsin digestion cell after 48h.As a result visible: to be added and be overexpressed
Ratio of the fibroblast of Mir-145-5p umbilical cord mesenchymal stem cells excretion body in the G2 phase is 20.85%, is added and is overexpressed
Ratio of the fibroblast of NC excretion body in the G2 phase is 20.69%, and the positive controls G2 phase is 14.84%, blank control
Group is 14.08%.Show most fast containing the fibroblast proliferation for being overexpressed Mir-145-5p umbilical cord mesenchymal stem cells excretion body.Such as
Shown in Fig. 4 A, B.
Two, its influence to migration of fibroblast cells ability is detected with cell scratch experiment
1) fibroblast is laid in 12 orifice plates, when cell density is 90%, carries out cell scratch.It is cleaned with PBS
Cell 3 times, serum free medium is added in the cell under removal stroke.One hole is added while TGF β cell factor is added and is overexpressed
While TGF β cell factor is added in Mir-145-5p umbilical cord mesenchymal stem cells excretion body (100ug/ml) (experimental group), a hole
Addition is overexpressed NC excretion body, TGF β cell factor (positive controls) is only added in a hole, and a hole is that any intervention is not added.Each
Handle hole respectively at once, for 24 hours, the same position point of 48h, 96h under the microscope shot.As a result visible: to contain table
Up to Mir-145-5p umbilical cord mesenchymal stem cells excretion body fibroblast in 48h scratch covered substantially；And blank pair
According to then still high-visible at group scratch.The result shows that with the outer of the umbilical cord mesenchymal stem cells secretion of miRNA145-5p modification
Secreting body has the ability for promoting migration of fibroblast cells.As shown in Fig. 4 C, D.
Fig. 5 is overexpressed miRNA145-5p excretion body and inhibits fibroblast to myofibroblast differentiation.A,western
Blot detect α-SMA protein level, it is seen that be overexpressed the soma prognosis of miRNA145-5p excretion, myofibroblast it is significant
Protein alpha-SMA is decreased obviously；A1, blank control group；A2, TGF β group (positive controls)；A3, it is overexpressed outside miRNA145-5p
Secrete body group (experimental group)；A4, it is overexpressed NC group；B, RT-PCR detects myofibroblast Specific marker α-SMA expression quantity
And I-type collagen (Collagen I) expression quantity, it is seen that after B3 experimental group is intervened, the specificity marker of myofibroblast
The mrna expression amount (longitudinal axis) of object α-SMA expression quantity and I-type collagen (Collagen I) is obviously lowered.B1, blank pair
According to group；B2, TGF β group (positive controls)；B3, it is overexpressed miRNA145-5p excretion body group (experimental group)；B4, it is overexpressed NC
Group.
Three, the excretion body for detecting the umbilical cord mesenchymal stem cells secretion modified with miRNA145-5p inhibits fibroblast
To the ability of myofibroblast differentiation
1.western-blot detection
1) fibroblast is laid on six orifice plates, it is handled when cell density is 70%.TGF β is added in one hole
It is added while cell factor and is overexpressed Mir-145-5p umbilical cord mesenchymal stem cells excretion body (100ug/ml) (experimental group), one
It is added while TGF β cell factor is added in hole and is overexpressed NC excretion body, TGF β cell factor (positive control is only added in a hole
Group), a hole is that any intervention is not added.Total serum IgE is extracted with Trizol method after 48h, remaining part utilizes isopropanol precipitating method
Albumen is extracted, sample is placed in EP pipe boils 10min respectively.
2) loading after sample is restored to room temperature.
3) electrophoresis, transferring film.
4) room temperature in 5%BSA confining liquid is immersed in slowly to sway two hours.4 DEG C of primary antibody of α SMA (Abcam company) was incubated for
Night.
5) primary antibody source is rabbit, therefore selects the secondary antibody of anti-rabbit, dilutes (1:200) by corresponding proportion, room temperature jog two hours.
6) it washs, is developed the color using ECL luminescence reagent.As a result as shown in Figure 5A and positive controls and overexpression NC group phase
Than the stem cell excretion body processing group α-SMA expression for being overexpressed microRNA145-5p reduces.This result shows that with
The excretion body of the umbilical cord mesenchymal stem cells secretion of miRNA145-5p modification can inhibit fibroblast to myofibroblast
Differentiation, and myofibroblast is exactly the factor for leading to cicatricial contracture most critical.
2.RT-PCR detection
1) fibroblast is laid on six orifice plates, it is handled when cell density is 70%.TGF β is added in one hole
It is added while cell factor and is overexpressed Mir-145-5p umbilical cord mesenchymal stem cells excretion body (100ug/ml) (experimental group), one
It is added while TGF β cell factor is added in hole and is overexpressed NC excretion body, TGF β cell factor (positive control is only added in a hole
Group), a hole is that any intervention is not added.
2) above-mentioned processing group and control group total serum IgE are extracted using Trizol (Invitrogen, 15596-026) after 48h, it is inverse
It is transcribed into cDNA.Design primer detects the specific proteins α-SMA and extracellular matrix type i collagen egg of myofibroblast
The expression of white (Collagen I).GAPDH is as detection internal reference.Using the NC excretion body of untransfected as control.
Primer sequence is as follows:
The primer of α-SMA is as follows:
F:GGACTCTGGGGATGGTGA (SEQ ID NO:8)
R:AATGAAGGAGGGCTGGAAGA (SEQ ID NO:9)
The primer of GAPDH is as follows:
F:AGTTGCGTTACACCCTTTCTTG (SEQ ID NO:10)
R:GCTGTCACCTTCACCGTTCC (SEQ ID NO:11)
The primer of Collagen I is as follows:
F:TGAGAGAGGGGTTGTTGGAC (SEQ ID NO:12)
R:TTGAGAAGAGTTACGAGTTG (SEQ ID NO:13)
As a result as shown in Figure 5 B, it is seen that the excretion body of the umbilical cord mesenchymal stem cells secretion of miRNA145-5p modification is added
After, α-SMA and the decline of I-type collagen (Collagen I) expression quantity are the most obvious.
Embodiment 4: it is complete that rat skin is repaired with the excretion body of the umbilical cord mesenchymal stem cells secretion of miRNA145-5p modification
The observation of curative effect of layer defect model
1. skin healing situation
1) prepared by skin full-thickness defects model: 12 SD rats (weight is about 150g, half male and half female) are randomly divided into NC
Control group, microRNA145-5p-UMSC excretion body group, blank control group.Every group each four.It is big with 10% chloral hydrate anesthesia
Mouse is simultaneously placed on station, in rat back label diameter is the circle of 1.5cm with puncher, sharp knife cuts through skin along label
To deep fascia surface, round skin full-thickness defects model is created
2) four groups, every group three are randomly divided into.It is injected respectively in each group.It is simple with petrolatum gauze after injection
Flap coverage, single cage raising.
3) treat after 1d, 7d, 14d, 21d carry out surface of a wound area measurement, and subcutaneous four points of the surface of a wound continue for
Medicine.As the result is shown: in substantially with the excretion soma of miRNA145-5p modification it is pre- in the case where, the rat back surface of a wound is in the 21st
It has been approached healing, and blank control and NC control then still have slight crack.As shown in Figure 6.
2.HE dyeing and specificity α-SMA immunohistochemical staining
1) skin that each processing group rat back surface of a wound and surrounding about 0.5cm normal tissue are taken in 21d, is fixed in
In 4% formaldehyde, through conventional dehydration, paraffin embedding cuts 5 μ m-thicks slice, row HE dyeing, specificity α-SMA histochemical staining.
2) light microscopic observation: in the pre- surface of a wound of the stem cell excretion soma of miRNA145-5p modification, the main component of healing
Fibroblast marshalling, hair follicle structure is more mature, and inflammatory cell infiltration is few, the specific proteins α-of myofibroblast
SMA expression quantity is low, and is in arrangement regulation.And the myofibroblast of NC and the visible express alpha-SMA of blank control group are gathered in not
Disorganized at the slight crack of healing, hair follicle structure is not formed.As shown in Figure 7.
This is the result shows that repair skin holostrome with the excretion body of the umbilical cord mesenchymal stem cells secretion of miRNA145-5p modification
Defect speed is significantly faster than that blank control and NC control group and without scar shrinkage phenomenon.
The preferred embodiment of the present invention has been described in detail above, but the invention be not limited to it is described
Embodiment, those skilled in the art can also make various equivalent on the premise of not violating the inventive spirit of the present invention
Variation or replacement, these equivalent variation or replacement are all included in the scope defined by the claims of the present application.
Claims (6)
1. described with the preparation method of the excretion body of the umbilical cord mesenchymal stem cells secretion of microRNA145-5p modification
As shown in SEQ ID NO:1, excretion body fills the sequence of microRNA145-5p between the umbilical cord by being overexpressed microRNA145-5p
Matter stem cell secretion, includes the following steps:
A. the secondary culture of umbilical cord mesenchymal stem cells；
B. the umbilical cord mesenchymal stem cells for being overexpressed microRNA145-5p are prepared, step I is selected from) or step II):
I) the mature body of artificial synthesized microRNA145-5p, through any generation umbilical cord mesenchymal stem cells of liposome transfection the 3rd ~ 6,
Obtain the umbilical cord mesenchymal stem cells for being overexpressed microRNA145-5p；
II) recombinant vector containing microRNA145-5p encoding gene, coding of the building containing microRNA145-5p are constructed
The recombinant virus of gene, or the recombinant viral vector of encoding gene of the building containing microRNA145-5p；By the recombination of acquisition
Carrier, recombinant virus or recombinant viral vector are transfected into umbilical cord mesenchymal stem cells, obtain and are overexpressed microRNA145-5p's
Umbilical cord mesenchymal stem cells；
C. the deposit of conditioned medium
It is filled between the overexpression microRNA145-5p that addition step B is obtained into the stem cell media without excretion body serum
Matter stem cell, collecting culture supernatant after 48h-72h is conditioned medium；It collects after conditioned medium to > 150mL, uses
In extracting excretion body；
D. the extracting and purifying of excretion body
CMC model is based on 4 DEG C, and 300 × g, 10min is centrifuged off cell fragment；4 DEG C, 2000 × g, 10min centrifugation removal are dead
The impurity such as cell；0.22 μm of sterilised membrane filter filtering further removes impurity；4 DEG C, 100000 × g, after 120min ultracentrifugation
To the exosome precipitating being overexpressed containing microRNA145-5p, 200uL PBS washing is added；4 DEG C again, 100000 × g,
120min ultracentrifugation to get purifying excretion body.
2. the excretion body of the umbilical cord mesenchymal stem cells secretion according to claim 1 with microRNA145-5p modification
Preparation method, which is characterized in that wherein, step A are as follows: take fresh umbilical cord, after phosphate buffer repeated flushing, be soaked in and contain
There is the L-DMEM5 minute of 1% mycillin, is cut into the tissue block of diameter 1.5mm size；Containing 10% fetal calf serum L-DMEM nutrition
Liquid, 5%CO2, 37 DEG C of saturated humidity cultures；Umbilical cord mesenchymal stem cells climb out of tissue after 7-10 days, remove after tissue block with
0.25% trypsin digestion carries out secondary culture.
3. the excretion body of the umbilical cord mesenchymal stem cells secretion according to claim 1 with microRNA145-5p modification
Preparation method, which is characterized in that wherein, step I) described in the specific steps through liposome transfection are as follows:
A) the 3rd ~ 6 any generation umbilical cord mesenchymal stem cells are inoculated into 6 orifice plates in the day before transfection, it is normal to cultivate, 37 DEG C, 5%
CO2Incubator, cell density is 60% when transfection；
B) transfected using lipo2000, the transfection procedure are as follows: by liposome: microRNA=1:20 microlitres/is rubbed
Your ratio transfects the cell for growing to 60% fusion.
4. the excretion body of the umbilical cord mesenchymal stem cells secretion according to claim 1 with microRNA145-5p modification
Preparation method, which is characterized in that wherein, step II) are as follows:
A) the well-grown 3rd ~ 6 any generation umbilical cord mesenchymal stem cells are inoculated into 6 orifice plates by the day before transfection, normally
Culture, 37 DEG C, 5%CO2Incubator, cell density is 40% when transfection；
B) replaces former culture medium with the 2ml stem cell media containing 6 μ g/ml polybrenes, and appropriate microRNA145- is added
5p slow virus suspension；37 DEG C of incubations；
C) continues culture 24 hours, contains virulent culture medium with the replacement of mescenchymal stem cell culture medium；
D) then. continues culture 72 hours for 37 DEG C, obtains the mescenchymal stem cell modified with microRNA145-5p
microRNA145-5p-UMSC。
5. a kind of as described in claim 1 with the excretion body of the umbilical cord mesenchymal stem cells secretion of microRNA145-5p modification
Preparing the application in wound healing drug.
6. a kind of as described in claim 1 with the excretion body of the umbilical cord mesenchymal stem cells secretion of microRNA145-5p modification
Inhibit the application in scar proliferation drug in preparation.
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