CN107446884A - Human umbilical cord mesenchymal stem cells membrane granule and its preparation and application - Google Patents
Human umbilical cord mesenchymal stem cells membrane granule and its preparation and application Download PDFInfo
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Abstract
The present invention relates to human umbilical cord mesenchymal stem cells membrane granule and its preparation and application；The cell membrane particles surface expression human umbilical cord mesenchymal stem cells surface marker, wrapping biological macromolecular and organelle；The macromolecular includes the mRNA products of the human umbilical cord mesenchymal stem cells dryness gene；The organelle includes mitochondria, proteasome, lysosome and autophagosome；The cell membrane particles do not have nucleus, nucleus DNA and nucleoprotein, without viral glycoprotein VSV G.Filter membrane of the present invention through mechanical presses by 3 μm of apertures, form 15 μm of cell membrane particles, particle is bigger, the delivering to large biological molecule and organelle and can deliver large biological molecule and organelle to liver and spleen in vitro, have repair to senile cell and damaging cells.The human umbilical cord mesenchymal stem cells membrane granule of the present invention is due to without VSV G, avoiding the generation of the immune response for virus protein；Also core DNA is free of, therefore the probability of inducing target cell canceration there's almost no.
Description
Technical field
The present invention relates to synthetic biology technology, more particularly to human umbilical cord mesenchymal stem cells membrane granule and its preparation and
Transmit the application in large biological molecule and organelle.
Background technology
Recently, a controversial mitochondria replaces the approval that therapy obtains British government.This method is based on nuclear transfer skill
Art, for treating the infertile problem of advanced age women, it can also be used to solve to be stranded caused by the lethal Mitochondrial DNA Mutation of ovum carrying
Border.In addition, the disease related to aging, such as atherosclerosis, diabetes B, old dull disease and cancer, generally tool
There are many defects.Research shows, the accumulation of lipid, Amyloid deposition, misfolded protein endoplasmic reticulum delay,
It can all cause the generation and deterioration of diseases of aging with the proteasome, autophagosome and mitochondria of damage.At present, there is no effective
The mechanism and measure that measure can directly be repaired to endochylema and organelle failure.
It is a kind of to express the cell membrane particles for melting memebrane protein and its preparation and apply (application number：201510655854.8) report
A kind of technology that cell membrane particles are prepared by mechanical presses, except nucleus, all cell membranes and cytoplasmic components, such as egg
In vain, RNA and organelle, are included in cell membrane particles.Briefly, cell membrane particles are as the stoning of same miniaturization
Cell.Importantly, cell membrane particles and mitochondrial deletion type cell Rho0's merges the recovery for causing ability of cell proliferation.
Human umbilical cord mesenchymal stem cells are the precursors of pluripotency, easily can obtain from fetal cord and
Cultured and amplified in vitro.The product of mark such as Oct4, Nanog and SOX2 gene of these cells expression stem cell, Telomerase
Activity can be also detected.In addition, these cells do not express immune costimulatory molecules and II type HLA compounds, I types HLA's
Expression is also very low, and this causes human umbilical cord mesenchymal stem cells to turn into preferable heterologous cells and be used for regenerative medicine and immune tune
Control treatment.
Stem cell is had been reported that there may be canceration, therefore the misgivings to stem cell biological security are so that the clinic of stem cell
Using being severely limited.Therefore, human umbilical cord mesenchymal stem cells membrane granule is prepared, and establishes a kind of big point of efficiently transmission biology
The method of son and organelle, it is of great importance for treating a variety of degenerative diseases.
The content of the invention
The first object of the present invention is to provide human umbilical cord mesenchymal stem cells membrane granule, and large biological molecule and thin can be achieved
Effective transmission of born of the same parents' device.
In order to solve the above-mentioned technical problem, the present invention adopts the following technical scheme that：
A kind of human umbilical cord mesenchymal stem cells membrane granule, the cell membrane particles surface expression human umbilical cord mesenchymal stem cells
Surface marker, wrapping biological macromolecular and organelle；The macromolecular includes the human umbilical cord mesenchymal stem cells dryness base
The mRNA products of cause；The organelle includes mitochondria, proteasome, lysosome and autophagosome；The cell membrane particles do not have
There are nucleus, nucleus DNA and nucleoprotein.Mitochondria can be effectively wrapped up, and is delivered in target cell and plays function.
Further, the human umbilical cord mesenchymal stem cells cell membrane particles size is 1 μm~5 μm.
The second object of the present invention is the preparation method for providing human umbilical cord mesenchymal stem cells membrane granule, including following step
Suddenly：
(1) human umbilical cord mesenchymal stem cells are cultivated under aseptic condition；
(2) umbilical cord mesenchymal stem cells are digested with pancreatin, centrifuges 3min, cell precipitation, will with 3mL nutrient solution in bottom
The umbilical cord mesenchymal stem cells of precipitation are resuspended；
(3) cell suspension obtained by step (2) is placed in syringe, and syringe is installed to the syringe needle for having disposed polycarbonate membrane
In formula filter, promote needle tubing cell suspension is released from filter to other while, acquisition umbilical cord mesenchymal stem cells
Membrane granule.
Step (2) is more beneficial for preparing membrane granule using the more other volumes of nutrient solution of 3mL volume such as 0.5mL.
Hyclone, 1% penicillin and streptomysin of the nutrient solution for DMEM nutrient solutions addition 10% in step (2).
Pancreatin is a kind of protease, by protein degradation on location, makes iuntercellular junction and cell-culture dish
Locate protein degradation, at this moment cell is due to turning into spherical under the tension force effect of therein cytoskeleton, so that cell separates.
Further, the step (1) comprises the following steps：
(11) umbilical cord is rinsed with after amnion removal red blood cell, Wharton ' s jelly to be cut into 3-4cm fritter with PBS,
Blood vessel is rejected, then is cut into smaller fragment, and is transferred to culture plate, back-off culture 25-35 minutes are until tissue can be attached to training
Support on plate；
(12) DMEM in high glucose nutrient solution is carefully added, in 37 DEG C of 5%CO2Humidified incubator culture, change culture within every two days
Liquid, it can see that within 5-7 days that fibrous human umbilical cord mesenchymal stem cells occur；
(13) human umbilical cord mesenchymal stem cells in 5-8 generations are used for subsequent treatment.
Further, the polycarbonate membrane pore size is 3 μm；The filter filters for 25mm film changeables needle-based
Device.It is 3 μm of circle to be covered with diameter above polycarbonate membrane, advantageously forms that form is complete, cell membrane of uniform size
Grain.
The third object of the present invention is to provide human umbilical cord mesenchymal stem cells membrane granule and is transmitting large biological molecule and thin
Application in born of the same parents' device.Purpose large biological molecule and organelle are delivered in the cell of large biological molecule and organelle defect.People
Umbilical cord mesenchymal stem cells membrane granule can effectively wrap up mitochondria, and be delivered in target cell and play function.Can be mesh
Large biological molecule and organelle be delivered in the cell of large biological molecule and organelle defect, recover the normal function of cell.
Further, including the delivering to large biological molecule and organelle in vitro and big point of biology is delivered liver and spleen
Son and organelle.
Compared with prior art, the present invention is that (a kind of express has melted the cell membrane particles and its system of memebrane protein having invented
Standby and application, number of patent application：201510655854.8) on improvement；The present invention relates to human umbilical cord mesenchymal stem cells film
Grain and its preparation and application；Human umbilical cord mesenchymal stem cells have youth, Immune privilege and multipotency；Filled between people's umbilical cord
Matter stem cell also has good film amalgamation.Therefore the human umbilical cord mesenchymal stem cells membrane granule that prepared by the present invention contains year
Light material, include the RNA and protein product of dryness gene, and intact organelle, such as mitochondria, proteasome, lysosome
And autophagosome, there is repair to senile cell and damaging cells；Human umbilical cord mesenchymal stem cells membrane granule is without promoting melting property
Viral glycoprotein VSV-G, avoid the generation of the immune response for virus protein；Human umbilical cord mesenchymal stem cells membrane granule
Probability without core DNA, therefore inducing target cell canceration there's almost no.Large biological molecule and cell are not only realized in vitro
The delivering of device, the delivering to liver and spleen is also achieved by mouse peritoneal injection.This not only for research large biological molecule and
The related disease of organelle defect is laid a good foundation, and significant for research cell nucleocytoplasmic relation.
Brief description of the drawings
Fig. 1 is the preparation method schematic diagram of the present inventor's umbilical cord mesenchymal stem cells membrane granule：
Fig. 2 is the phenogram of the present inventor's umbilical cord mesenchymal stem cells；
Fig. 3 is the phenogram of the present inventor's umbilical cord mesenchymal stem cells membrane granule；
The stem cell that Fig. 4 is included by the present inventor's umbilical cord mesenchymal stem cells membrane granule and human umbilical cord mesenchymal stem cells
The mRNA expression figures of dryness gene；
Fig. 5 is excretion body caused by the present inventor's umbilical cord mesenchymal stem cells membrane granule and human umbilical cord mesenchymal stem cells
(Exosome) size is schemed with volume vs；
Fig. 6 is that the fluorescence that the present inventor's umbilical cord mesenchymal stem cells membrane granule includes mitochondria, lysosome and autophagosome shows
Micro- figure；
Fig. 7 is that the present inventor's umbilical cord mesenchymal stem cells membrane granule recovers the common of mitochondrial deletion cell 143B/Rho0
Optical microscope and number of cells statistical chart；
Fig. 8 is mitochondrial membrane of the 143B/Rho0 cells of the present invention in human umbilical cord mesenchymal stem cells membrane granule before and after the processing
Current potential comparison diagram；
Fig. 9 is the DiI fluorochrome label figures of the present inventor's umbilical cord mesenchymal stem cells membrane granule；
Figure 10 is liver after DiI fluorescence labelings human umbilical cord mesenchymal stem cells membrane granule of the present invention injects mouse peritoneal 12h
And the fluorescence microscopy figure of spleen.
Embodiment
To make the object, technical solutions and advantages of the present invention clearer, the present invention is made into one below in conjunction with accompanying drawing
It is described in detail on step ground.
Embodiment 1
The preparation method of human umbilical cord mesenchymal stem cells membrane granule, is mainly included the following steps that：
(1) caesarean birth health full term umbilical cord from Medical College of Shantou University gynemetrics of the second affiliated hospital and obtains with amnion
Family members agree to and the approval of Ethics Committee of school；
(2) after washing off red blood cell with PBS, Wharton ' s jelly are cut into 3-4cm fritter, reject blood vessel, then
Smaller fragment is cut into, and is transferred to 10cm culture plates, back-off culture can be attached on culture plate for 30 minutes up to organizing；
(3) carefully addition 5ml DMEM in high glucose (10% hyclone, 100mg/ml penicillin, 100mg/ml streptomysins) training
Nutrient solution, in 37 degree of 5%CO2 humidified incubator cultures, change nutrient solution within every two days, can see that within 5-7 days that fibrous cell occurs；
(4) cell in 5-8 generations is used to test；
(5) umbilical cord mesenchymal stem cells are digested with pancreatin, centrifuges 3min, cell precipitation, will with 3mL nutrient solution in bottom
The umbilical cord mesenchymal stem cells of precipitation are resuspended；
(6) cell suspension obtained by step (5) is placed in syringe, and syringe is installed to the syringe needle for having disposed polycarbonate membrane
In formula filter, promote needle tubing cell suspension is released from filter to other while, acquisition umbilical cord mesenchymal stem cells
Membrane granule, as shown in Figure 1.
Filter membrane of the umbilical cord mesenchymal stem cells through mechanical presses by 3 μm of apertures, 1-5 μm of cell membrane particles are formed, its
In comprising the organelle such as RNA, albumen, lysosome, mitochondria, autophagosome, but acellular core.
Embodiment 2
2x10 is collected according to the method for embodiment 16Human umbilical cord mesenchymal stem cells, be resuspended in 200 μ l nutrient solution, so
After add 1：50 dilution PE fluorescence labelings antibody, including CD13, CD29, CD44, CD73, HLA-ABC, CD45, CD117,
CD31, CD34 and HLA-DR antibody, 4 DEG C of incubation 1-2h, are fixed with 1% paraformaldehyde, are then divided with flow cytometer
Analysis.Analysis result is as shown in Fig. 2 the typical undifferentiated cell table of human umbilical cord mesenchymal stem cells expression that embodiment 1 is cultivated
Face labelled protein, such as CD13, CD29, CD44, CD73 and HLA-ABC, the coded markings albumen broken up without expression cell, such as
CD45, CD117, CD31, CD34 and HLA-DR.
Embodiment 3
Collect 2x106Human umbilical cord mesenchymal stem cells, be resuspended in 200 μ l nutrient solution, then add 1：50 dilutions
The antibody of PE fluorescence labelings, including CD13, CD29, CD44, CD73, HLA-ABC, CD45, CD117, CD31, CD34 and HLA-DR
Antibody, 4 DEG C incubation 2h, clean 3 times, be then resuspended in 3mL nutrient solutions.The polycarbonate membrane that pore size is 3 μm is disposed
In 25mm film changeable syringe-driven filters, and filter is screwed, cell suspension is drawn with 10ml syringe, firmly thin
Born of the same parents from filter shift onto it is other while, collected with 15ml centrifuge tube.
Obtained human umbilical cord mesenchymal stem cells membrane granule is collected, 1000g centrifugation 10min, supernatant is lightly drawn, leaves
General 200 μ l nutrient solution, and centrifuge tube is gently shaken, obtain the human umbilical cord mesenchymal stem cells film that size is about 1-5 μm
Grain.Gained human umbilical cord mesenchymal stem cells membrane granule is placed on to 35-mm copolymerization Jiao's culture plate, stands 30min, then with sharp
Light Laser Scanning Confocal Microscope detects.As a result as shown in figure 3, cell membrane particles are such as human umbilical cord mesenchymal stem cells, expression CD13,
CD29, CD44, CD73 and HLA-ABC, do not express CD45, CD117, CD31, CD34 and HLA-DR.
The membrane granule of umbilical cord mesenchymal stem cells and stem cell preparation will be collected, extract total serum IgE with Trizol reagents, use
RT-PCR technology expands corresponding gene.As a result as shown in figure 4, stem cell membrane particle is similar with stem cell, stem cell is all contained
The mRNA products of dryness gene.
The excretion body of stem cell membrane particle and stem cell secretion is collected, is then detected with laser nano particle instrument.As a result such as
Shown in Fig. 5, stem cell membrane particle has a significant peak at 1-5 μm, and excretion body then without；The film of 1-5 μ m diameters simultaneously
Volume ratio is more than 50% shared by particle.
Embodiment 4
The organelle that human umbilical cord mesenchymal stem cells membrane granule includes characterizes.
Human umbilical cord mesenchymal stem cells in 37 DEG C with fluorochrome label 30min-1h, including with 1 μM of MitoTracker
Labeled mitochondria, lysosome is marked with 1 μM of LysoTracker, collect cell and the extruded film side used by above-described embodiment 1
Cell membrane particles obtained from formula, observed under Laser Scanning Confocal Microscope.In addition, by the 20 unlabelled human umbilical cord mesenchymals of μ l
Stem cell membrane grain blobs add on the cover slip, dry 1h at room temperature, are fixed with 4% paraformaldehyde, perforated with 1%Triton-X,
Add 1：The LC3b antibody of 100 dilutions, it is the mark of autophagosome, is incubated at room temperature 2h, cleaning, then adds FITC marks
Secondary antibody is incubated 1h, is detected after cleaning with Laser Scanning Confocal Microscope.As a result as shown in fig. 6, being wrapped in umbilical cord mesenchymal stem cells membrane granule
Containing mitochondria, lysosome and autophagosome.
Embodiment 5
Human umbilical cord mesenchymal stem cells membrane granule transmits the monitoring and functional verification of mitochondria.
Human umbilical cord mesenchymal stem cells membrane granule is obtained using the extruded film of above-described embodiment 1, and is added to 1.5x105Line grain
In the 143B/Rho0 cells of volume defect.Change liquid per 24h and rejoin membrane granule, 24h, 48h, 72h collect cell and use blood cell
Tally carries out the statistics of cell quantity.143B/Rho0 is typical mitochondrial defects type cell, it is necessary to extra in nutrient solution
Add uridine (Uridine, Uri.) and pyruvic acid.By 143B/Rho0 cell culture in 96 orifice plates, nutrient solution addition guanosine and third
Ketone acid, cell attachment after 24h, condition of culture in setting 3：It is not added with the cell membrane particles of guanosine/be not added with umbilical cord mesenchymal stem cells
(PMVs), add guanosine but be not added with PMVs, be not added with guanosine but supplement PMVs.Take pictures after 48h, then disappeared with pancreatin under the microscope
Change, collect cell, counted with haemocyte ware, dead cell is excluded with trypan blue.From figure 7 it can be seen that filled between addition people's umbilical cord
After matter stem cell membrane particle, even if no longer adding Uridine, 143B/Rho0 cells still can show people's umbilical cord with normal growth
Mescenchymal stem cell membrane granule can transmit mitochondria and reply the multiplication capacity of mitochondrial defects type cell.
After adding membrane granule culture 3 days, dyed with 5 μ g/ml JC-1 lines (mitochondrial membrane potential staining reagent), it is red
Fluorescence is strong and the weak mitochondrial membrane potential that represents of green fluorescence is high, otherwise it is low to represent mitochondrial membrane potential.From figure 8, it is seen that add
After blooming particle, the mitochondrial membrane potential of 143B/Rho0 cells significantly raises.The visible membrane granule of experimental result can be successfully entered target
Cell, and after entrance cell, membrane granule can successfully discharge mitochondria, and mitochondria can replicate in target cell.It is above-mentioned
As a result demonstrate human umbilical cord mesenchymal stem cells membrane granule and can effectively transmit mitochondria and concurrently wave biological function.
Embodiment 6
Human umbilical cord mesenchymal stem cells membrane granule delivers in the material of mouse liver and spleen.
With 1 μ g/ml CM-DiI (red fluorescence dyestuff of mark cell membrane) on 37 DEG C of incubated cell 30min, utilization
The human umbilical cord mesenchymal stem cells membrane granule that the extruded film of embodiment 1 obtains CM-DiI marks is stated, cell membrane particles as shown in Figure 9 are
Fluorescent red is marked as by DiI, by intraperitoneal injection to Balb/c mouse, after 12 hours, mouse is put to death, obtains liver and spleen
It is dirty, detected under fluorescence microscope, it is seen that substantial amounts of membrane granule is by liver cell and spleen cell endocytosis, DiI as shown in Figure 10
The PMVs of mark can be very good to blend with liver cell and spleen cell, and PMVs institutes can be transferred to by prompting comprising material
Liver and spleen cell.The above results demonstrate human umbilical cord mesenchymal stem cells membrane granule can effectively transmit life in Mice Body
Thing material.
The above disclosed right for being only presently preferred embodiments of the present invention, the present invention can not being limited with this certainly
Scope, therefore the equivalent variations made according to the claims in the present invention, still belong to the scope that the present invention is covered.
Claims (7)
1. human umbilical cord mesenchymal stem cells membrane granule, it is characterised in that the cell membrane particles surface expression human umbilical cord mesenchymal
Stem cell surface marker, wrapping biological macromolecular and organelle；The macromolecular includes the human umbilical cord mesenchymal stem cells
The mRNA products of dryness gene；The organelle includes mitochondria, proteasome, lysosome and autophagosome；The cell membrane
Grain does not have nucleus, nucleus DNA and nucleoprotein.
2. human umbilical cord mesenchymal stem cells membrane granule according to claim 1, it is characterised in that the human umbilical cord mesenchymal
Stem cells membrane granule size is 1 μm~5 μm.
A kind of 3. preparation method of human umbilical cord mesenchymal stem cells membrane granule as claimed in claim 1 or 2, it is characterised in that
Comprise the following steps：
(1) human umbilical cord mesenchymal stem cells are cultivated under aseptic condition；
(2) human umbilical cord mesenchymal stem cells cultivated with pancreatin digestion step (1), 3min is centrifuged, by umbilical cord mesenchymal stem cells
It is suspended in 3mL complete culture solution；
(3) umbilical cord mesenchymal stem cells to be suspended obtained by step (2) are placed in syringe, and syringe is installed to and has disposed poly- carbonic acid
In the syringe-driven filter of ester film, promote needle tubing cell suspension is released from filter to other while, between acquisition umbilical cord
Mesenchymal stem cells membrane granule.
4. the preparation method of human umbilical cord mesenchymal stem cells membrane granule according to claim 3, it is characterised in that the step
Suddenly (1) comprises the following steps：
(11) umbilical cord is rinsed with after amnion removal red blood cell, Wharton ' s jelly to be cut into 3-4cm fritter, are rejected with PBS
Blood vessel, then smaller fragment is cut into, and culture plate is transferred to, back-off culture 25-35 minutes are until tissue can be attached to culture plate
On；
(12) DMEM in high glucose nutrient solution is carefully added, in 37 DEG C of 5%CO2Humidified incubator culture, change nutrient solution, 5-7 within every two days
It can see that fibrous human umbilical cord mesenchymal stem cells occur；
(13) human umbilical cord mesenchymal stem cells in 5-8 generations are used for subsequent treatment.
5. the preparation method of human umbilical cord mesenchymal stem cells membrane granule according to claim 3, it is characterised in that described poly-
Carbonic ester membrane aperture size is 3 μm；The filter is 25mm film changeable syringe-driven filters.
6. a kind of human umbilical cord mesenchymal stem cells membrane granule as claimed in claim 1 or 2 is transmitting large biological molecule and organelle
In application.
7. a kind of human umbilical cord mesenchymal stem cells membrane granule as claimed in claim 6 is in large biological molecule and organelle is transmitted
Using, it is characterised in that big point of biology is delivered including the delivering to large biological molecule and organelle in vitro and to liver and spleen
Son and organelle.
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| WO2022228223A1 (en) * | 2021-04-26 | 2022-11-03 | 重庆理工大学 | Engineered mitochondria and preparation method therefor |
| CN115433708A (en) * | 2022-08-22 | 2022-12-06 | 浙江大学 | Biomaterial delivered by specific cell-targeted metabolic system and preparation method and application thereof |
| CN115505554A (en) * | 2022-08-22 | 2022-12-23 | 浙江大学 | Cell membrane based biological material for cross-species cell component delivery and preparation method thereof |
| CN117487737A (en) * | 2023-12-26 | 2024-02-02 | 汕头大学 | Fe-containing material 3 O 4 Enucleated cells of nanoparticles, preparation thereof and application thereof in anti-aging |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022228223A1 (en) * | 2021-04-26 | 2022-11-03 | 重庆理工大学 | Engineered mitochondria and preparation method therefor |
| CN115433708A (en) * | 2022-08-22 | 2022-12-06 | 浙江大学 | Biomaterial delivered by specific cell-targeted metabolic system and preparation method and application thereof |
| CN115505554A (en) * | 2022-08-22 | 2022-12-23 | 浙江大学 | Cell membrane based biological material for cross-species cell component delivery and preparation method thereof |
| WO2024040806A1 (en) * | 2022-08-22 | 2024-02-29 | 浙江大学 | Biological material for delivering specific cell-targeted metabolic system, preparation method therefor, and use thereof |
| CN117487737A (en) * | 2023-12-26 | 2024-02-02 | 汕头大学 | Fe-containing material 3 O 4 Enucleated cells of nanoparticles, preparation thereof and application thereof in anti-aging |
| CN117487737B (en) * | 2023-12-26 | 2024-04-12 | 汕头大学 | Fe-containing material 3 O 4 Enucleated cells of nanoparticles, preparation thereof and application thereof in anti-aging |
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Application publication date: 20171208 |