Long-acting stem cell as well as preparation method and application thereof

Abstract

The invention discloses a long-acting stem cell as well as a preparation method and application thereof. The long-acting stem cell is obtained by introducing an exogenous polypeptide and connecting a stem cell synergist to the surface of the stem cell by utilizing a receptor-ligand recognition effect, the stem cell synergist is a nano-liposome; the long-acting stem cell has high survival efficiency and strong differentiation and proliferation capacity in a disease microenvironment, can effectively load a drug for improving the activity of the stem cell on a cell membrane, and realizes improvement of the in-vivo repair activity of the stem cell drug and enhancement of the survival ability of the stem cell on the premise of maintaining the integrity of the membrane structure of the stem cell. And the preparation method is simple to operate, is convenient and practical, and has a great prospect in practical application, and can be used for treating different injury-related diseases such as fibrosis lesions of liver, lung, pancreas and the like.

Description

Technical Field

[0001] The present invention relates to a long-acting stem cell, and also relates to a preparation method and application of the long-acting stem cell. Background Art

[0002] Stem cells are a type of primitive cells that have pathological targeting, tissue repair, and immune regulation functions. However, due to the influence of cytokines (such as lipopolysaccharide, interleukin, extracellular DNA) or toxic substances (such as β-amyloid protein), stem cell vitality is reduced, thereby inducing cell apoptosis, causing related stem cell drug research to be extremely delayed.

[0003] In order to improve the cell viability and cell function of stem cells, the current methods mainly include the following pretreatment or engineering transformation of stem cells: (1) Pretreatment of stem cells usually refers to culturing stem cells in certain conditioned culture media in vitro, which usually contains exogenously added cytokines (such as IFN-γ, TNF-α, interleukins, growth factors), chemical drugs (such as ginsenosides, vitamins, amino acids), or cells (such as platelets); (2) Pretreatment of stem cells also includes culturing under certain external environments or stimuli, such as hypoxic environments, mechanical stimulation, etc.; (3) Regulating the protein expression of stem cells through genetic modification can enhance the engraftment rate and anti-apoptosis ability of stem cells; (4) Stem cells are doped with hydrogels.

[0004] However, strategies for in vitro pretreatment or regulation of the culture environment may cause the engineered stem cells cultured in vitro to have changed their original cell characteristics, genetically modified stem cell drugs are prone to changes or loss of genetic information, and hydrogel-encapsulated stem cells are difficult to transplant or release biological factors in the body after transplantation. Therefore, it is of great significance to construct an engineering modification technology that maintains the cell characteristics of stem cell drugs themselves and even improves their therapeutic effects. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a long-acting stem cell with high survival efficiency and strong differentiation ability in a disease microenvironment, and also to provide a preparation method and application of the above-mentioned long-acting stem cell.

[0006] Technical solution: The long-acting stem cells described in the present invention are obtained by introducing exogenous polypeptides and connecting stem cell enhancers to the surface of stem cells by utilizing receptor-ligand recognition; the stem cell enhancer is a nanoliposome; the polypeptide chain of the exogenous polypeptide contains one or more of the following functional sequences R1-3:

[0007] R1: a sequence that undergoes receptor-ligand recognition with specific receptors on the surface of stem cells;

[0008] R2: sequence that is fragmented in response to cytokines in vivo;

[0009] R3: Cysteine ​​residue located at the C-terminus of the peptide chain.

[0010] Wherein, the stem cells are in vitro stem cells, preferably mesenchymal stem cells, and the nanoliposomes are nanoliposomes encapsulating nitric oxide donor drugs.

[0011] Among them, in the functional sequence, R1 undergoes receptor-ligand recognition with CD44 or CD105 on the surface of stem cells.

[0012] Among them, in the functional sequence, the cytokine that responds to R2 is a reactive oxygen molecule or a matrix metalloproteinase.

[0013] Wherein, the polypeptide chain is a CD44 connecting peptide (A6 peptide) or a CD105 connecting peptide.

[0014] The method for preparing the long-acting stem cells comprises the following steps:

[0015] (1) preparing nanoliposomes and adding distearoylphosphatidylethanolamine-polyethylene glycol-maleimide (DSPE-PEG-MAL), incubating the prepared nanoliposomes with polypeptide chains, and ultrafiltration to obtain nanoliposomes with polypeptide-modified surfaces;

[0016] (2) Nanoliposomes or polypeptide chains with polypeptide modifications on their surfaces are incubated together with stem cells, and after centrifugation, they are resuspended in culture medium to obtain long-acting stem cells.

[0017] Wherein, in step (1), the nanoliposome is a nitric oxide donor liposome, which is prepared by a lipid membrane material and a nitric oxide donor, and the molar ratio of the nanoliposome to the polypeptide chain is 1:1 to 1:3.

[0018] Among them, step (1) specifically comprises taking a lipid membrane material and a nitric oxide donor, adding an organic solvent to mix and dissolve, hydrating after rotary evaporation, breaking the liposomes, and removing the free nitric oxide donor by centrifugation to obtain nanoliposomes; incubating the nanoliposomes with polypeptide chains to carry out Michael addition reaction, and removing the free peptide by centrifugation to obtain nanoliposomes with polypeptide surface modification.

[0019] Specifically, step (2) comprises inoculating stem cells in a culture dish, and when the cell confluence reaches 60-80%, adding nanoliposomes modified with a polypeptide on the surface to the dish, incubating at 37° C. for 0.5-2 h, then digesting and collecting the cells, and removing the unbound liposomes in the dish by centrifugation to obtain polypeptide-modified long-acting stem cells; more preferably, the cell confluence reaches 70%, and the nanoliposomes are incubated at 37° C. for 2 h.

[0020] The above-mentioned long-acting stem cells can also be used in the preparation of anti-fibrosis drugs.

[0021] Principle of the invention: The long-acting stem cells of the present invention respond to the active oxygen in the microenvironment on the surface of the stem cells, and release a protective layer of nitric oxide gas while consuming the active oxygen. The nitric oxide gas acts on the stem cells to promote the expression of related proteins and genes for anti-apoptosis, proliferation and differentiation, and improves its effect in the microenvironment. The selected connection sites are positive receptors specifically expressed on the surface of the stem cell membrane, such as CD44 and CD105, and the cell endocytosis ability mediated by this type of positive receptor is weak, and the binding of ligand peptides (such as A6 peptides, etc.) will not affect the main function of the stem cells. Therefore, such polypeptides can be connected to nitric oxide donor liposomes and incubated with stem cells to obtain the long-acting stem cells of the present invention. This connection is on the surface of the stem cell drug. Under the premise of not affecting the characteristics of the cell itself, the strategy of microenvironment-specific drug release has better safety and stability than the existing engineering transformation technology; therefore, the long-acting stem cells of the present invention can effectively load the drug that enhances the activity of stem cells on the cell membrane, and under the premise of maintaining the integrity of the stem cell membrane structure, realize the improvement of the repair activity of the stem cell drug in vivo, enhance the survival ability of stem cells in vivo, and thus be used to treat different injury-related diseases such as fibrotic lesions of the liver, lungs, and pancreas.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The long-acting stem cells of the present invention have high survival efficiency in the disease microenvironment and strong differentiation and proliferation ability, which can increase the survival and proliferation of stem cells by about 3-5 times; (2) The preparation method is simple to operate, convenient and practical, and has great prospects in practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the process for preparing the long-acting stem cells of the present invention;

[0024] Figure 2 This is a morphological diagram of primary stem cells in Example 1;

[0025] Figure 3 This is a diagram for identifying stem cell surface markers in Example 1;

[0026] Figure 4 This is the grafting diagram of A6 peptide in Example 1;

[0027] Figure 5 This is a fluorescence image of the A6 peptide connecting stem cells and nanoformulations in Example 1;

[0028] Figure 6 This is a graph showing the retention time of A6 peptide linking stem cells and nanoformulations in Example 1;

[0029] Figure 7This is a flow cytometry image of the connection between stem cells and nanoformulations by A6 peptide in Example 2;

[0030] Figure 8 This is a flow cytometry image of CD105 connecting peptide connecting stem cells and nanoformulations in Example 2;

[0031] Fig. 9 is the oxidative stress clearance diagram of the synergist in Example 3;

[0032] Fig.10 This is the MTT graph of stem cell survival in Example 3;

[0033] Fig.11 This is a comparison chart of stem cell survival and apoptosis in Example 3;

[0034] Fig.12 This is a diagram of stem cell proliferation in Example 3;

[0035] Fig.13 This is a diagram showing the repairing effect of long-acting stem cells on damaged endothelial cells in Example 4;

[0036] Fig.14 This is a diagram showing the repairing effect of long-acting stem cells on damaged acinar cells in Example 4; DETAILED DESCRIPTION

[0037] The technical scheme of the present invention is further described below in conjunction with the examples. The test materials used in the examples can all be purchased through conventional channels.

[0038] Among them, the stem cells are rat mesenchymal stem cells, and the extraction method thereof includes the following steps: male SD rats are killed by cervical dislocation and immersed in 75% ethanol for 10 minutes. Subsequently, in a sterile clean bench, the fur and muscle tissue are cut in sequence with surgical scissors, and the adipose tissue around the epididymis is removed with tweezers and immersed in saline for cleaning. Subsequently, the adipose tissue is placed in the lid of a culture dish, quickly chopped and transferred to a 10mL EP tube, and after adding an equal volume of saline, collagenase is added and mixed, sealed, and digested at 37°C for 30 minutes. After the digestion is terminated, an FBS solution equal to the volume of collagenase is added to terminate the digestion, centrifuged at 1600rpm for 5min to collect the bottom precipitate, 2mL of saline is added, resuspended, passed through a 200-mesh sieve, insoluble matter is filtered out, transferred to a new EP tube, and centrifuged at 1600rpm for 5min to collect the bottom precipitate. Subsequently, 3mL of red blood cell lysis buffer is added, lysed at room temperature for 2min, 3mL of saline is added, and the bottom precipitate is collected by centrifugation at 1600rpm for 5min, and repeated 3 times. Discard the supernatant, add DMEM low-glucose medium containing 10% FBS and transfer to a T25 culture flask for cell culture. After the cells adhere to the wall, discard the supernatant, add fresh medium, and observe the cell morphology under an inverted microscope and record the image of primary stem cells, such as Figure 2 shown.

[0039] Flow cytometry was used to detect the expression of MSCs cell surface antigens CD29, CD44, CD90 and CD45. 5 The cell density of each well was inoculated in a 6-well plate. When the cell confluence reached 80%, fluorescent antibodies of CD44, 90, 29, and 45 were added. After incubation, the supernatant was discarded and the single cell suspension was collected in the dark for flow cytometry detection. As shown in Figures A, B, C, and D, the surface of the extracted MSCs expressed positive CD29, CD44, and CD90, and negative CD45, indicating the successful extraction of MSCs.

[0040] Example 1

[0041] The long-acting stem cells of the present invention are long-acting stem cells modified with A6 peptide, and the preparation method comprises the following steps:

[0042] (1) Accurately weigh the lipid membrane material and drug (12.00 mg HSPC, 2.96 mg cholesterol, 1.88 mg DSPE-PEG-Mal and 1.07 mg nitric oxide donor) into a round-bottom flask, then add 3 mL of chloroform solution and mix and dissolve. Remove the organic solvent by rotary evaporation at 45°C, wait for a uniform film to form on the bottom of the flask, add 3 mL of normal saline, and hydrate at 37°C and 20 rpm / min for 30 min. Finally, collect the liquid in the flask and use an ultrasonic disruptor at 30% power in an ice bath to break the liposomes. After 10 min, a liposome solution with a light blue opalescence is obtained. Remove the free nitric oxide donor by low-speed centrifugation (4°C, 3000 rpm, 5 min) and ultrafiltration centrifugation (room temperature, 3000 rpm, 20 min) to obtain nitric oxide donor liposomes;

[0043] 3.00 mg of A6 peptide (SEQ ID NO. 1: KPSSPPEECGPLGIAGQC) was added to the nitric oxide donor liposome solution, stirred to dissolve, and stirred overnight at 4°C for Michael addition reaction. Subsequently, ultra-high speed centrifugation (4°C, 10 w g, 1 h) was performed to remove free A6 peptide, and the bottom precipitate was resuspended with physiological saline to obtain nitric oxide donor liposomes anchored with A6 peptide. H NMR was used for connection characterization, such as Figure 4 As shown, lipsome-A6 has the characteristic peaks of A6 peptide, indicating that A6 peptide is successfully anchored on the surface of nitric oxide donor liposomes.

[0044] (2) The nitric oxide donor liposomes were anchored to CD44 on the surface of the stem cell membrane through the receptor-ligand recognition of the A6 peptide. 5The cells in each well were seeded in a culture dish at a density of 100 μg / cm2. When the cell confluence reached 70%, liposomes anchored with A6 peptide were added to the dish and incubated at 37°C for 2 h. The cells were then digested and collected, and the unbound liposomes in the dish were removed by low-speed centrifugation at 1000 rpm for 5 min to obtain long-acting stem cells modified with A6 peptide. The prepared stem cells were characterized using CLSM, as shown in Figure 2. Figure 5 As shown in Figure 2, the DiO loaded by the liposomes showed obvious co-localization with the MSCs cell membrane, indicating that the A6 peptide-modified liposomes were successfully anchored on the surface of the MSCs cell membrane. The prepared stem cells were characterized by flow cytometry, as shown in Figure 2. Figure 6 and 7 As shown, the A6 peptide-modified liposomes could remain stable on the surface of MSCs for more than four days, showing good stability.

[0045] Example 2

[0046] The long-acting stem cells of the present invention are long-acting stem cells modified with CD105 peptide, and the preparation method comprises the following steps:

[0047] (1) The rat mesenchymal stem cells were extracted and the nitric oxide donor liposomes were prepared in the same manner as in Example 1. 3.00 mg of CD105 connecting peptide (SEQ ID NO.2: LRVPVHHVHKHACGPLGIAGQC) was added to the obtained nitric oxide donor liposome solution, stirred to dissolve, and stirred overnight at 4°C for Michael addition reaction. Subsequently, ultrahigh speed centrifugation (4°C, 10 w g, 1 h) was performed to remove the free CD105 connecting peptide, and the bottom precipitate was resuspended with physiological saline to obtain nitric oxide donor liposomes anchored with CD105 connecting peptide.

[0048] (2) The nitric oxide donor liposomes were anchored to CD105 on the surface of the stem cell membrane through the receptor-ligand recognition of CD105 peptide. 5 The cells in each well were seeded in a culture dish at a density of 100 μg / cm2. When the cell confluence reached 70%, liposomes anchored with CD105 peptide were added to the dish and incubated at 37°C for 2 h. The cells were then digested and collected, and the unbound liposomes in the dish were removed by low-speed centrifugation at 1000 rpm for 5 min to obtain long-acting stem cells modified with CD105-linked peptide. The prepared stem cells were characterized using flow cytometry, such as Figure 8 As shown, it was shown that CD105 peptide successfully anchored nitric oxide donor liposomes on the surface of MSCs membrane.

[0049] Example 3

[0050] Verification of the survival ability of long-acting stem cells:

[0051] 1. Oxidative stress removal by synergists:

[0052] Pancreatic stellate cells were cultured at 1×10 5 The cells were inoculated at a density of 100 cells / mL in a glass-bottom culture dish, and then the cells were intervened with a serum-free medium containing 200 μM hydrogen peroxide. After overnight incubation, the medium was discarded, and a serum-free medium containing an enhancer was added. After 4 h, the cells were placed under a laser confocal microscope to collect fluorescence images of the cells. Fig. 9 As shown, with the increase of the concentration of the enhancer, the degree of oxidative stress in PSCs gradually decreased.

[0053] 2. Stem cell survival after peptide-linked synergist liposomes:

[0054] (I) MTT method

[0055] MSCs were plated at 5 × 10 3 The cell density was inoculated in a 96-well plate. When the cell confluence reached 80%, the culture medium was discarded and 200 μM hydrogen peroxide solution was added to damage the cells. After 12 hours, the supernatant was discarded. 100 μL of nitric oxide donor liposomes containing 0.2, 0.4, 0.6, 1, 2, 10, 30, 60, 100 and 200 μM were added. After incubation for 24 hours, 48 ​​hours and 72 hours, the drug solution was discarded, 200 μL of 5 mg / mL MTT solution was added to each well, and the cells were incubated at 37 ° C for 4 hours in the dark. The MTT solution was discarded, 150 μL of DMSO was added to each well, and the cells were shaken at 120 rpm on a decolorizing shaker for 10 minutes to dissolve the methyl guanidine. The cells were placed in a microplate reader in the dark to detect the cell absorbance at 570 nm, and the OD value was recorded. The untreated cells were used as the control. The subsequent drug treatment steps are the same as above. The calculation formula is as follows:

[0056] Cell survival rate (%) = (OD value of treatment group - OD value of blank group) / (OD value of control group - OD value of blank group)

[0057] The results are as follows Fig.10 As shown, under the stimulation of high concentration of hydrogen peroxide, MSCs with surface-anchored liposomes still have a high survival rate.

[0058] (ii) Live / dead cell double staining

[0059] Stem cells were collected at 8×10 4The cells were seeded at a density of 100 μL in a 24-well plate. When the cell confluence reached 80%, the culture medium was discarded and 200 μM hydrogen peroxide solution was added to damage the cells. After 12 hours, the supernatant was discarded and free nitric oxide donors, a mixed group of stem cells and nitric oxide donor liposomes, and a stem cell and nitric oxide donor liposome polypeptide connection group were added. After incubation for 24 hours, the drug solution was discarded and 250 μL of Calcein AM / PI detection working solution was added. The cells were incubated at 37°C in the dark for 30 minutes and then placed under an inverted fluorescence microscope to record the image. Calcein AM is green fluorescence, and Ex / Em is 494 / 517 nm; PI is red fluorescence, and Ex / Em is 535 / 617 nm. The results are shown in Fig.11 As shown, MSCs with surface-anchored nitric oxide donor liposomes showed the highest survival rate.

[0060] 3. Stem cell proliferation ability after peptides are linked to synergist liposomes:

[0061] MSCs were cultured at 2×10 5 The cell density was inoculated in a 6-well plate. When the cell confluence reached 80%, the culture medium was discarded and 200 μM hydrogen peroxide solution was added to damage the cells. After 12 hours, the supernatant was discarded, and free nitric oxide donors, a mixed group of stem cells and nitric oxide donor liposomes, and a stem cell and nitric oxide donor liposome polypeptide connection group were added. After incubation for 4 hours, the drug solution was discarded, 10 μM EdU solution was added, and incubated for 4 hours (about 10% of the MSCs proliferation cycle). The supernatant was discarded, and 4% paraformaldehyde solution was added and fixed at room temperature for 20 minutes. Washing solution was added for washing, and then permeabilization with permeabilization solution for 15 minutes, and PBS was washed three times. Subsequently, Click reaction solution was added and incubated at room temperature in the dark for 30 minutes. After washing with PBS three times, Hoechst 33342 was used for nuclear staining, and finally the image was recorded under an inverted fluorescence microscope. The results are shown as follows. Fig.12 As shown, MSCs with surface-anchored nitric oxide donor liposomes retained the strongest proliferation capacity.

[0062] Example 4

[0063] Verification of the ability of long-acting stem cells to function:

[0064] 1. Long-acting stem cells’ repairing effect on damaged endothelial cells:

[0065] (I) Tube Formation Experiment

[0066] 24 hours before the experiment, the complete medium in the culture dish was replaced with a medium containing only 0.2% FBS to starve the cells. Subsequently, 50 μL of Matrigel solution was added to the 96-well plate and placed in a 37°C cell culture incubator for 30 minutes. After the colloid solidified, serum-free medium, nitric oxide donor liposomes, stem cell supernatant, and a mixed solution of nitric oxide donor liposomes and stem cell supernatant were added to the wells in sequence. Subsequently, 2×10 4 The endothelial cells were pretreated with 50 μL of 200 μM oxidative stress solution at a cell density of 1.5 μg / mL and images were collected under an inverted microscope at 0 h and 2 days. Fig.13 As shown in A, endothelial cells treated with stem cell supernatant and nitric oxide donor liposomes showed a clear tendency to form vascular structures.

[0067] (II) Western blotting

[0068] Endothelial cells were cultured at 2×10 5 The cells were inoculated in a 6-well plate at a density of 100 μg / mL. When the cell confluence reached 80%, the culture medium was discarded and 200 μM hydrogen peroxide solution was added to damage the cells. After 12 hours, serum-free culture medium, nitric oxide donor liposomes, stem cell supernatant, and a mixed solution of nitric oxide donor liposomes and stem cell supernatant were added. After 24 hours of treatment, the cells were washed three times with sterile pre-cooled PBS, and cell proteins were collected for Western blotting. The results are shown in Fig.13 As shown in B, the expression of angiogenesis-related pathway proteins in endothelial cells treated with a mixed solution of nitric oxide donor liposomes and stem cell supernatant was significantly increased.

[0069] 2. The repair effect of long-acting stem cells on damaged acinar cells:

[0070] An in vitro stem cell differentiation model was established by Transwell, and acinar cells were co-cultured with stem cells. The experiment was divided into 6 groups: (1) untreated stem cells, (2) untreated acinar cells, (3) stem cells co-cultured without TGF-β, (4) long-term stem cells co-cultured without TGF-β, (5) stem cells co-cultured with TGF-β, and (6) long-term stem cells co-cultured with TGF-β. 100 μL of acinar cells were cultured at 5×10 4 The cells were seeded at a density of 8×10 4 The stem cells / long-acting stem cells were inoculated on a cell slide at a cell density of 10%, and a culture medium containing 10% FBS (with or without TGF-β at a concentration of 50 ng / mL) was added for cell culture. After 72 hours, the concentration of cell secreted enzymes in the supernatant was measured. The results were as follows: Fig.14As shown, under the stimulation of TGF-β, PACs co-cultured with long-term stem cells retained the strongest secretory capacity.

[0071] Therefore, the long-acting stem cells of the present invention can effectively load drugs that enhance stem cell activity onto the cell membrane, and while maintaining the integrity of the stem cell membrane structure, achieve the enhancement of the in vivo repair activity of stem cell drugs, enhance the survival ability of stem cells in the body, have high survival efficiency and strong differentiation and proliferation ability in the disease microenvironment, and increase stem cell survival and proliferation by about 3-5 times, thereby being used to treat different injury-related diseases such as fibrotic lesions of the liver, lungs, and pancreas.

Claims

1. A long-acting stem cell, characterized in that: The long-acting stem cells are obtained by introducing exogenous polypeptides and connecting stem cell enhancers to the surface of stem cells by using receptor-ligand recognition; the stem cell enhancer is a nanoliposome; the polypeptide chain of the exogenous polypeptide contains one or more of the following functional sequences R1-3: R1: a sequence that undergoes receptor-ligand recognition with specific receptors on the surface of stem cells; R2: sequence that is fragmented in response to cytokines in vivo; R3: Cysteine ​​residue located at the C-terminus of the peptide chain.

2. The long-acting stem cell according to claim 1, characterized in that: The stem cells are in vitro stem cells, and the nanoliposomes are nanoliposomes encapsulating nitric oxide donor drugs.

3. The long-acting stem cell according to claim 1, characterized in that: In the functional sequence, R1 undergoes receptor-ligand recognition with CD44 or CD105 on the surface of stem cells.

4. The long-acting stem cell according to claim 1, characterized in that: In the functional sequence, the cytokine that responds to R2 is a reactive oxygen molecule or a matrix metalloproteinase.

5. The long-acting stem cell according to claim 1, characterized in that: The polypeptide chain is A6 peptide or CD105 connecting peptide.

6. A method for preparing the long-acting stem cells according to claim 1, characterized in that: The following steps are involved: (1) preparing nanoliposomes, incubating with polypeptide chains, and ultrafiltration to obtain nanoliposomes with polypeptide-modified surfaces; (2) The nanoliposomes modified with peptides on the surface are anchored on the surface of the stem cell membrane through the receptor-ligand recognition effect of the peptide, and the nanoliposomes modified with peptides on the surface or the polypeptide chains are incubated with the stem cells, and the cells are resuspended in culture medium after centrifugation to obtain long-acting stem cells.

7. The method according to claim 6, characterized in that: In step (1), the nanoliposome is a nitric oxide donor liposome, which is prepared by a lipid membrane material and a nitric oxide donor, and the molar ratio of the nanoliposome to the polypeptide chain is 1:1 to 1:

3.

8. The method according to claim 6, characterized in that: Step (1) specifically comprises taking a lipid membrane material and a nitric oxide donor, adding an organic solvent to mix and dissolve them, hydrating them after rotary evaporation, breaking the liposomes, and removing the free nitric oxide donor by centrifugation to obtain nanoliposomes; incubating the nanoliposomes with polypeptide chains to perform Michael addition reaction, and removing the free peptides by centrifugation to obtain nanoliposomes with polypeptide surfaces.

9. The method according to claim 6, characterized in that: Step (2) is specifically as follows: the stem cells are inoculated in a culture dish, and when the cell confluence reaches 60-80%, nanoliposomes with surface modified with polypeptides are added to the dish, incubated for 0.5-2 hours, and then the cells are digested and collected, and the unbound liposomes in the dish are removed by centrifugation to obtain polypeptide-modified long-acting stem cells.

10. Use of the long-acting stem cell drug according to claim 1 in the preparation of anti-fibrosis drugs.

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