CN111265550A

CN111265550A - Stem cell factor liposome for repairing damaged tissues and preparation method thereof

Info

Publication number: CN111265550A
Application number: CN202010278708.9A
Authority: CN
Inventors: 邢杰; 乔彦良; 苗昱; 韩爽; 李朝阳; 马广斌; 邹海涛; 李广森
Original assignee: Shandong Sinder Technology Co ltd; Shandong Xinda Gene Technology Co Ltd; Qingdao Xindi Cell Biotechnology Development Co Ltd
Current assignee: Shandong Sinder Technology Co ltd; Shandong Xinda Gene Technology Co Ltd; Qingdao Xindi Cell Biotechnology Development Co Ltd
Priority date: 2020-04-10
Filing date: 2020-04-10
Publication date: 2020-06-12

Abstract

The invention discloses a preparation method of stem cell factor liposome for repairing damaged tissues, which comprises the following steps: step 1, separating and culturing canine adipose-derived mesenchymal stem cells; step 2, digesting and crushing the cultured canine adipose mesenchymal stem cells, and centrifuging, filtering and ultrafiltering to obtain a stem cell extracting solution; and 3, mixing the prepared liposome with the stem cell extracting solution, carrying out ultrasonic treatment to obtain stem cell factor liposome suspension, adding sodium propionate, mixing uniformly, and filtering to obtain a stem cell factor liposome solution. The stem cell factor liposome prepared by the method is a pure natural component, is safe to apply, has no toxic or side effect, no drug residue, high activity, small clinical dosage and quick response.

Description

Stem cell factor liposome for repairing damaged tissues and preparation method thereof

Technical Field

The invention belongs to the technical field of stem cells, and particularly relates to a stem cell factor liposome for repairing damaged tissues and a preparation method thereof.

Background

Adipose-derived mesenchymal stem cells are a cell population with self-renewal and multidirectional differentiation potential in adipose tissues, and have multidirectional differentiation capacity and strong in vitro proliferation capacity due to convenient acquisition, so that the role of the adipose-derived mesenchymal stem cells in the processes of wound healing and tissue repair is increasingly emphasized by researchers in recent years. Adipose-derived mesenchymal stem cells can actively migrate to a tissue damage part to participate in immune regulation and tissue damage repair, wherein the paracrine action of adipose-derived mesenchymal stem cells can generate a large amount of soluble stem cell factors to promote the repair of tissue damage, and the adipose-derived mesenchymal stem cells are generally considered as a main mechanism for participating in the repair of tissue damage. The adipose-derived stem cell factor comprises interleukin, stem cell growth factor, vascular endothelial cell growth factor, fibroblast growth factor, stromal cell derived factor, placenta growth factor, insulin-like growth factor, survivin, granulocyte-macrophage colony stimulating factor and other active substances. Many substances are transported in the form of vesicles such as exosomes, but the membrane structure is very easy to break in the extraction process, so that the absorption effect is adversely affected to a certain extent.

Liposomes are bilayer, completely closed multilamellar vesicles formed from lipids such as phospholipids, and have a structure similar to that of cells, and the liposome membrane is similar to the constituent components of cell membranes, and can be inserted into the lipid layer of cell membranes to release aqueous phase encapsulated substances into cells, and can be adsorbed on the cell surface and then separated from the cells only under the action of proteolytic enzyme, so that the concentration of the encapsulated substances around the cells is increased, and the encapsulated substances can slowly permeate into the cells. And contact of the liposome with the cell causes an increase in liposome permeability, the liposome encapsulate is "contact released". The thus released encapsulates form high concentrations in the vicinity of the cell membrane.

The general stem cell factor preparation mostly depends on active absorption of cells and concentration-dependent passive absorption, so the absorption efficiency is not high, and the preparation of the stem cell factor preparation with high absorption efficiency and good treatment effect is a problem which needs to be solved urgently at present.

Accordingly, further developments and improvements are still needed in the art.

Disclosure of Invention

Aiming at various defects in the prior art and solving the problems, the stem cell factor liposome for repairing the damaged tissue and the preparation method thereof are provided, and the method has the advantages of high activity, small clinical dosage, quick response and toxic and side effects.

In order to achieve the purpose, the invention provides the following technical scheme:

a preparation method of stem cell factor liposome for repairing damaged tissues comprises the following steps:

step 1, separating and culturing canine adipose-derived mesenchymal stem cells;

step 2, digesting and crushing the cultured canine adipose-derived mesenchymal stem cells, and centrifuging, filtering and ultrafiltering to obtain a stem cell extracting solution;

and 3, mixing the prepared liposome with the stem cell extracting solution, carrying out ultrasonic treatment to obtain stem cell factor liposome suspension, adding sodium propionate, mixing uniformly, and filtering to obtain a stem cell factor liposome solution.

Preferably, the specific steps for isolating the canine adipose-derived mesenchymal stem cells in the step 1 comprise:

obtaining fresh mesenteric fat from young dogs, adding 2% double-resistant DPBS buffer solution for soaking and cleaning, removing blood vessels and fascia in adipose tissues, cutting into small pieces, adding trypsin digestive juice, centrifuging at 3000r/min after shaking digestion to remove supernatant and floating fat, then uniformly mixing and cleaning sedimentary layer cells by using DPBS at room temperature, centrifuging at 3000r/min again to remove supernatant, and resuspending sedimentary cells by using MEM culture solution containing 10% FBS, 100U/ml penicillin and 100U/ml streptomycin.

Preferably, the activity of the separated canine adipose-derived mesenchymal stem cells is detected, and the living cell ratio is more than or equal to 95%.

Preferably, the canine adipose-derived mesenchymal stem cells are cultured in MEM culture medium containing 10% FBS, 100U/ml penicillin and 100U/ml streptomycin for propagation and subculture.

Preferably, the step 2 specifically includes:

digesting the canine fat source mesenchymal stem cells by trypsin, centrifuging at 3000r/min to collect the stem cells, uniformly mixing and cleaning the cells of a precipitation layer by using room-temperature DPBS, centrifuging at 3000r/min again to remove supernatant, suspending the separated cells in sterile physiological saline, ultrasonically crushing the cells by ice bath, 12000r/min, centrifuging at 4 ℃, taking the supernatant and discarding the precipitate, filtering and sterilizing the supernatant by using a 0.22 mu m filter membrane, filtering and concentrating by using a 5k ultrafiltration membrane, regulating the protein concentration by using the physiological saline, filtering by using the 0.22 mu m filter membrane and then storing at 4 ℃, wherein the conditions of ultrasonically crushing the cells by ice bath are as follows: the ultrasound intensity was 40% for 3 seconds with 5 seconds intervals for 8 sonications.

Preferably, the liposome preparation method specifically comprises:

weighing 0.4g of soybean phospholipid, 0.05g of cholesterol and 0.1g of tween 80, dissolving in 10mL of absolute ethanol, performing ultrasonic treatment to fully dissolve the soybean phospholipid, the cholesterol and the tween 80, introducing the mixture into 10mL of 10% ammonium sulfate solution under the oscillation of a water bath shaker at 40-60 ℃, performing reduced pressure evaporation at 60 ℃ on the organic solvent, and dialyzing in DPBS with pH7.0 for 24 hours to form the liposome.

Preferably, the preparation method of the stem cell factor liposome specifically comprises the following steps:

slowly adding the obtained stem cell extractive solution into the prepared stem cell extractive solution

Slowly adding 20mL of the stem cell extract obtained in step (4) into the prepared liposome under shaking of a water bath shaker at 40-60 deg.C, and continuing to shake for 30min at 40-60 deg.C; then placing the stem cell factor into an ultrasonic cell crusher to carry out ice bath low-temperature ultrasonic treatment, so that the stem cell factor is fully and uniformly fused into the liposome, thereby obtaining stem cell factor liposome suspension; adding 0.5-1% of sodium propionate according to the mass ratio, fully and uniformly mixing, and respectively and sequentially extruding through microporous filter membranes with the aperture of 0.45 mu m and the aperture of 0.22 mu m to obtain the stem cell factor liposome solution.

The stem cell factor liposome is obtained by the preparation method, comprises liposome and stem cell factors encapsulated in the liposome, wherein the stem cell factors are one or more of interleukin, stem cell growth factors, vascular endothelial cell growth factors, fibroblast growth factors, stromal cell derived factors, placenta growth factors, insulin growth factors, survivin or granulocyte-macrophage colony stimulating factors.

Has the advantages that:

the invention provides a preparation method of stem cell factor liposome for repairing damaged tissues, which can improve the absorption rate of stem cell factors, protect the activity of the stem cell factors, reduce the loss of effective components and effectively improve the utilization rate of the effective components.

The stem cell factor liposome for repairing damaged tissues is prepared from pure natural components, is safe to apply, free of toxic and side effects, free of drug residues, high in activity, small in clinical dosage and quick in effect.

Drawings

FIG. 1 is a graph showing the effect of temperature of a water-domain shaker on the encapsulation efficiency of liposomes in a method for preparing stem cell factor liposomes for repairing damaged tissues according to an embodiment of the present invention;

FIG. 2 is a graph showing the effect of temperature of a water-region shaking bed on the drug loading of liposomes in a method for preparing stem cell factor liposomes for repairing damaged tissues according to an embodiment of the present invention;

FIG. 3 is a graph showing the effect of a stem cell factor liposome on cell proliferation to repair damaged tissues in an embodiment of the present invention.

Detailed Description

In order to make the technical solutions of the present invention better understood, the following description of the technical solutions of the present invention with reference to the accompanying drawings of the present invention is made clearly and completely, and other similar embodiments obtained by a person of ordinary skill in the art without any creative effort based on the embodiments in the present application shall fall within the protection scope of the present application. In addition, directional terms such as "upper", "lower", "left", "right", etc. in the following embodiments are directions with reference to the drawings only, and thus, the directional terms are used for illustrating the present invention and not for limiting the present invention.

The following are specific embodiments of the present invention:

specific example 1:

obtaining fresh mesenteric fat from healthy young dogs, transferring the mesenteric fat into a 50ml sterile centrifuge tube, and soaking and cleaning the mesenteric fat for three times by using 2% double-antibody DPBS buffer solution; purifying the cleaned adipose tissues, and removing blood vessels and fascia in the adipose tissues to obtain high-purity adipose tissues; cutting high purity adipose tissue into 1mm³Transferring the small blocks into a 50ml sterile centrifuge tube, adding 10ml of 0.25% trypsin digestion solution, and performing shake digestion at 37 ℃ for 30 minutes; after centrifuging the digested adipose tissue at 3000r/min for 10 minutes, the supernatant was removed, and 5ml of MEM culture medium containing 10% FBS, 100U/ml penicillin and 100U/ml streptomycin was added to the removed supernatant to resuspend the precipitated cells, thereby obtaining a resuspended cell fluid.

For separationAdding an equal volume of 0.4% trypan blue solution into the discharged heavy suspension cell liquid; detecting the proportion of living cells of the mixed substances by a Cellometer-Mini full-automatic cell counter; if the proportion of the living cells is less than 95%, unqualified cells are required to be re-separated, if the proportion of the living cells is more than or equal to 95%, qualified cells are qualified, and the cell density of qualified re-suspended cell liquid is adjusted to 1 x 10 by using 5ml of MEM culture liquid containing 10% FBS, 100U/ml penicillin and 100U/ml streptomycin⁵Per ml; culturing 5ml of the cells with adjusted cell density, inoculating the cells into a T25 cell culture bottle, transferring into an incubator, and setting the incubator at 37 ℃ and 5% CO₂Culturing under saturated humidity condition; after culturing for 48 hours, changing the culture solution, taking out other kinds of cells which cannot adhere to the wall, and then changing the MEM culture solution every 72 hours; when the cells grew to 80% confluence, they were digested with 0.25% trypsin for 2min and then stopped with MEM; and (3) repeatedly blowing the digested substances evenly to prepare cell suspension, carrying out passage according to the ratio of 1:3, repeating the operation for repeated passage amplification and collection, and culturing sufficient canine adipose-derived mesenchymal stem cells.

Digesting the fat source mesenchymal stem cells after proliferation and subculture by using 0.25% trypsin, and centrifuging at 3000r/min for 10min to collect the stem cells; uniformly mixing and cleaning the cells of the precipitation layer by using 10ml of room-temperature DPBS buffer solution, centrifuging at 3000r/min for 10 minutes, taking out supernatant, and repeating for three times; resuspending the cells in sterile physiological saline, and then carrying out ice-bath ultrasonic cell disruption on the cells resuspended in the physiological saline, wherein the specific method is to use ultrasonic with the intensity of 40% for 3 s/time and 8 times in total, and the interval is 5s each time; centrifuging the crushed cells at 12000r/min for 10min, controlling the whole centrifugation process at 4 ℃, and then taking supernatant; filtering the obtained supernatant with 0.22 μm filter membrane for sterilization, and filtering with 5k ultrafiltration membrane for concentration; after concentration, the protein concentration was measured by BCA kit, and the protein concentration was adjusted to 300. mu.g/ml with physiological saline, and then the stem cell extract was filtered through a 0.22 μm filter and stored at 4 ℃.

Weighing 0.4g of soybean phospholipid, 0.05g of cholesterol and 0.1g of Tween 80, dissolving in 10mL of absolute ethyl alcohol, and performing ultrasonic treatment to fully dissolve the soybean phospholipid; introducing the mixture of soybean phospholipid, cholesterol and tween 80 into 10mL of 10% ammonium sulfate solution under the oscillation of a water bath shaker at 40 ℃, and then placing the mixture into a round-bottom flask; the solution was evaporated at 60 ℃ under reduced pressure to remove the organic solvent and dialyzed against DPBS at pH7.0 for 24h to form liposomes.

Placing the dialyzed liposome into a 40 ℃ water bath shaker for oscillation, then slowly adding 20ml of the prepared stem cell extracting solution, and after the addition is finished, continuing to keep the 40 ℃ for oscillation for 30 min; performing ice-bath ultrasonic treatment by using an ultrasonic crusher, wherein the ultrasonic conditions are that the ultrasonic intensity is 40%, the ultrasonic time is 3s, the interval is 5s, and the total action time is 20min, so that the stem cell factor is fully and uniformly fused into the liposome, and a stem cell factor liposome suspension is obtained; adding 0.5-1% sodium propionate according to the mass ratio, fully and uniformly mixing, then sequentially extruding the mixture through microporous filter membranes with the aperture of 0.45 mu m and the aperture of 0.22 mu m, repeating the steps for three times respectively to obtain the stem cell factor liposome solution, and storing the stem cell factor liposome solution at 4 ℃.

Specific example 2:

the same parts of this embodiment as those of embodiment 1 are not described again, but the differences are that

Weighing 0.4g of soybean phospholipid, 0.05g of cholesterol and 0.1g of Tween 80, dissolving in 10mL of absolute ethyl alcohol, and performing ultrasonic treatment to fully dissolve the soybean phospholipid; introducing a mixture of soybean phospholipid, cholesterol and tween 80 into 10mL of 10% ammonium sulfate solution under the oscillation of a water bath shaker at 50 ℃, and then placing the mixture into a round-bottom flask; the solution was evaporated at 50 ℃ under reduced pressure to remove the organic solvent and dialyzed against DPBS at pH7.0 for 24h to form liposomes.

Placing the dialyzed liposome into a water bath shaker at 50 deg.C for oscillation, slowly adding 20ml of the prepared stem cell extractive solution, and continuing to maintain 50 deg.C for oscillation for 30min after the addition is completed; performing ice-bath ultrasonic treatment by using an ultrasonic crusher, wherein the ultrasonic conditions are that the ultrasonic intensity is 40%, the ultrasonic time is 3s, the interval is 5s, and the total action time is 20min, so that the stem cell factor is fully and uniformly fused into the liposome to obtain stem cell factor liposome suspension; adding 0.5-1% sodium propionate according to the mass ratio, fully and uniformly mixing, then sequentially extruding the mixture through microporous filter membranes with the aperture of 0.45 mu m and the aperture of 0.22 mu m, repeating the steps for three times respectively to obtain the stem cell factor liposome solution, and storing the stem cell factor liposome solution at 4 ℃.

Specific example 3:

the same parts of this embodiment as those of embodiment 1 are not described again, but the differences are as follows:

weighing 0.4g of soybean phospholipid, 0.05g of cholesterol and 0.1g of Tween 80, dissolving in 10mL of absolute ethyl alcohol, and performing ultrasonic treatment to fully dissolve the soybean phospholipid; introducing the mixture of soybean phospholipid, cholesterol and tween 80 into 10mL of 10% ammonium sulfate solution under the oscillation of a water bath shaker at 60 ℃, and then placing the mixture into a round-bottom flask; the solution was evaporated at 60 ℃ under reduced pressure to remove the organic solvent and dialyzed against DPBS at pH7.0 for 24h to form liposomes.

Placing the dialyzed liposome into a water bath shaker at 60 deg.C for oscillation, slowly adding 20ml of the prepared stem cell extractive solution, and continuing to maintain the oscillation at 60 deg.C for 30min after the addition is completed; performing ice-bath ultrasonic treatment by using an ultrasonic crusher, wherein the ultrasonic conditions are that the ultrasonic intensity is 40%, the ultrasonic time is 3s, the interval is 5s, and the total action time is 20min, so that the stem cell factor is fully and uniformly fused into the liposome to obtain stem cell factor liposome suspension; adding 0.5-1% sodium propionate according to the mass ratio, fully and uniformly mixing, then sequentially extruding the mixture through microporous filter membranes with the aperture of 0.45 mu m and the aperture of 0.22 mu m, repeating the steps for three times respectively to obtain the stem cell factor liposome solution, and storing the stem cell factor liposome solution at 4 ℃.

Specific example 4:

weighing 0.4g of soybean phospholipid, 0.05g of cholesterol and 0.1g of Tween 80, dissolving in 10mL of absolute ethyl alcohol, and performing ultrasonic treatment to fully dissolve the soybean phospholipid; introducing the mixture of soybean phospholipid, cholesterol and tween 80 into 10mL of 10% ammonium sulfate solution under the oscillation of a water bath shaker at 30 ℃, and then placing the mixture into a round-bottom flask; the solution was evaporated at 30 ℃ under reduced pressure to remove the organic solvent and dialyzed against DPBS at pH7.0 for 24h to form liposomes.

Placing the dialyzed liposome into a water bath shaker at 30 ℃ for oscillation, slowly adding 20ml of the prepared stem cell extracting solution, and continuing to keep oscillating at 30 ℃ for 30min after the addition is finished; performing ice-bath ultrasonic treatment by using an ultrasonic crusher, wherein the ultrasonic conditions are that the ultrasonic intensity is 40%, the ultrasonic time is 3s, the interval is 5s, and the total action time is 20min, so that the stem cell factor is fully and uniformly fused into the liposome to obtain stem cell factor liposome suspension; adding 0.5-1% sodium propionate according to the mass ratio, fully and uniformly mixing, then sequentially extruding the mixture through microporous filter membranes with the aperture of 0.45 mu m and the aperture of 0.22 mu m, repeating the steps for three times respectively to obtain the stem cell factor liposome solution, and storing the stem cell factor liposome solution at 4 ℃.

Specific example 5:

weighing 0.4g of soybean phospholipid, 0.05g of cholesterol and 0.1g of Tween 80, dissolving in 10mL of absolute ethyl alcohol, and performing ultrasonic treatment to fully dissolve the soybean phospholipid; introducing the mixture of soybean phospholipid, cholesterol and tween 80 into 10mL of 10% ammonium sulfate solution under the oscillation of a water bath shaker at 70 ℃, and then placing the mixture into a round-bottom flask; the solution was evaporated at 70 ℃ under reduced pressure to remove the organic solvent and dialyzed against DPBS at pH7.0 for 24h to form liposomes.

Placing the dialyzed liposome into a 70 ℃ water bath shaker for oscillation, then slowly adding 20ml of the prepared stem cell extracting solution, and after the addition is finished, continuing to keep the 70 ℃ for oscillation for 30 min; performing ice-bath ultrasonic treatment by using an ultrasonic crusher, wherein the ultrasonic conditions are that the ultrasonic intensity is 40%, the ultrasonic time is 3s, the interval is 5s, and the total action time is 20min, so that the stem cell factor is fully and uniformly fused into the liposome to obtain stem cell factor liposome suspension; adding 0.5-1% sodium propionate according to the mass ratio, fully and uniformly mixing, then sequentially extruding the mixture through microporous filter membranes with the aperture of 0.45 mu m and the aperture of 0.22 mu m, repeating the steps for three times respectively to obtain the stem cell factor liposome solution, and storing the stem cell factor liposome solution at 4 ℃.

The effect of the temperature of the water-area shaking table on the encapsulation efficiency of the liposomes in examples 1 to 5 is shown in FIG. 1, and it can be seen by comparing that the encapsulation efficiency of the liposomes to the stem cell factor is the highest when the temperature of the water-area shaking table is 50 ℃, and the encapsulation efficiency is decreased to different degrees when the temperature is increased or decreased.

The effect of the temperature of the water-bath shaking table on the drug loading of the liposomes in examples 1 to 5 is shown in fig. 2, and the drug loading of the liposomes on the stem cell factor is the largest when the temperature of the water-bath shaking table is 50 ℃ in comparison, and the drug loading is reduced to different degrees when the temperature is increased or decreased.

Specific example 6:

damaged tissue repair effect experiment:

selecting 12 beagle dogs of 3 years old, randomly dividing into three groups, wherein the first group is a control group, and treating with normal saline; the second group is a normal treatment group, which is treated with iodine tincture; the third group is a stem cell treatment group, and the canine adipose-derived mesenchymal stem cell factor liposome is used.

Intravenous anesthesia is carried out on all experimental dogs with 3% sodium pentobarbital, shaving and disinfection are carried out on the back, a circular wound surface with the diameter of 2cm is manufactured on the back through the whole skin layer in an operation, the wound surface does not touch muscles, corresponding therapeutic agents are smeared on the wound surface twice in the morning and at night every day according to groups, the wound is protected by a paste type sterile dressing, the size of the wound surface is measured 4, 8, 12 and 20 days after treatment, and the area of the wound surface is measured by an image analyzer.

The healing rate is represented by (area of original wound surface-area of remaining wound surface)/area of original wound surface, and the table is the treatment result of percentage of healing area after dog skin injury.

Table one: percent area healed after dog skin injury

Group of	4 days	8 days	12 days	20 days
					Control group	26±1.9	33±6.0	31±7.6	69±4.3
Normal treatment group	29±3.1	38±1.3	53±2.0	77±5.8
					Stem cell treatment group	30±1.2	48±4.7	68±3.5	90±0.6

The experimental result shows that the healing speed of the wound surface is obviously increased after the wound surface is smeared with the canine adipose-derived mesenchymal stem cell factor liposome. The difference between the control group and the stem cell treatment group is obvious at the 8 th day, and the stem cell treatment group is healed at the 20 th day, while the wound surface of the normal treatment group is not completely healed. The dog fat source mesenchymal stem cell factor liposome can effectively promote the skin repair of the damaged part of the dog, and has good clinical application prospect.

Specific example 7:

effect of stem cell factor liposomes on cell proliferation:

taking 4-6-week-old ICR mice, carrying out conventional cervical dislocation and sacrifice, taking out spleens of the mice aseptically, placing the spleens in a dish containing Hank's solution, and gently washing and stripping surrounding connective tissues; wetting a 200-mesh cell sieve with a small amount of Hank's solution, and then transferring the spleen into the cell sieve; shearing spleen with scissors, grinding gently with grinder to make spleen cell penetrate cell sieve and enter Hank's solution, adding small amount of Hank's solution, and blowing with pipette for several times to obtain single cell suspension; taking a proper amount of spleen lymphocyte suspension, centrifuging for 8min at 1500r/min, discarding the supernatant, adding an isovolumetric erythrocyte lysate, centrifuging for 8min at 1500r/min, likewise discarding the supernatant, collecting the precipitate, and repeatedly washing for 2 times by Hank's to obtain the required spleen lymphocyte; cell count, cell density adjusted to 4.0 × 106/mL with RPMI1640 complete medium; adding the cell suspension into a 96-well cell plate, wherein each well is 100 mu L, each well is respectively added with 80 mu L of stem cell factor liposome prepared by a 50 ℃ water bath shaker, each concentration of stem cell factor liposome and blank liposome is 8-well repeated, and a blank control hole is additionally arranged; then culturing for 44h at 37 ℃ under 5% CO2, taking out each well, adding 30 mu L of MTT into each well, continuously culturing for 4h, absorbing supernatant, adding 100 mu L of lysate DMSO into each well, placing the cell plate in a micro-oscillator, shaking for 5min to completely dissolve the precipitate, and detecting the A570 value on an enzyme linked immunosorbent assay to be used as an index of cell proliferation.

The effect of the stem cell factor liposome on cell proliferation shown in fig. 3 can be used to show that the stem cell factor liposome has significant effect on cell proliferation.

The present invention has been described in detail, and it should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.

Claims

1. A preparation method of stem cell factor liposome for repairing damaged tissues is characterized by comprising the following steps:

step 1, separating and culturing canine adipose-derived mesenchymal stem cells;

step 2, digesting and crushing the cultured canine adipose mesenchymal stem cells, and centrifuging, filtering and ultrafiltering to obtain a stem cell extracting solution;

2. The method for preparing the stem cell factor liposome for repairing damaged tissues as claimed in claim 1, wherein the specific steps of isolating the canine adipose-derived mesenchymal stem cells in the step 1 comprise:

3. The method for preparing the stem cell factor liposome solution for repairing damaged tissues as claimed in claim 2, wherein the activity of the isolated canine adipose-derived mesenchymal stem cells is detected, and the living cell content is more than or equal to 95%.

4. The method for preparing the stem cell factor liposome solution for repairing damaged tissue according to claim 2, wherein sufficient canine adipose derived mesenchymal stem cells are cultured by proliferation and subculture in MEM culture medium containing 10% FBS, 100U/ml penicillin and 100U/ml streptomycin.

5. The method for preparing the stem cell factor liposome solution for repairing damaged tissues as claimed in claim 1, wherein the step 2 comprises:

digesting the canine fat source mesenchymal stem cells by trypsin, centrifuging at 3000r/min to collect the stem cells, uniformly mixing and cleaning the cells of a precipitation layer by using DPBS at room temperature, centrifuging at 3000r/min again to remove supernatant, suspending the separated cells in sterile physiological saline, carrying out ice bath ultrasonic cell disruption, centrifuging at 12000r/min, centrifuging at 4 ℃, taking supernatant and discarding precipitates, filtering and sterilizing the supernatant by using a 0.22 mu m filter membrane, filtering and concentrating by using a 5k ultrafiltration membrane, regulating the protein concentration by using physiological saline, and storing at 4 ℃ after filtering by using a 0.22 mu m filter membrane.

6. The method for preparing the stem cell factor liposome solution for repairing damaged tissues as claimed in claim 5, wherein the conditions for ultrasonic cell disruption in ice bath are as follows: the ultrasound intensity was 40% for 3 seconds with 5 seconds intervals for 8 sonications.

7. The method for preparing the stem cell factor liposome solution for repairing damaged tissue according to claim 1, wherein the method for preparing liposome comprises:

weighing 0.4g of soybean phospholipid, 0.05g of cholesterol and 0.1g of tween 80, dissolving in 10mL of absolute ethanol, performing ultrasonic treatment to fully dissolve the soybean phospholipid, the cholesterol and the tween 80, introducing the mixture into 10mL of 10% ammonium sulfate solution under the oscillation of a water bath shaker at 40-60 ℃, performing reduced pressure evaporation at 60 ℃ on the organic solvent, and dialyzing in DPBS with the pH of 7.0 for 24 hours to form the liposome.

8. The method for preparing the stem cell factor liposome solution for repairing damaged tissue according to claim 1, wherein the step 3 of preparing the stem cell factor liposome specifically comprises:

9. A stem cell factor liposome for repairing damaged tissues, which is prepared by the method according to any one of claims 1 to 8.

10. The stem cell factor liposome for repairing damaged tissue of claim 9, wherein the stem cell factor liposome comprises liposome and stem cell factor encapsulated in the liposome, and the stem cell factor is one or more of interleukin, stem cell growth factor, vascular endothelial cell growth factor, fibroblast growth factor, stromal cell derived factor, placental growth factor, insulin growth factor, survivin or granulocyte-macrophage colony stimulating factor.