Application of kukoamine A, protective liquid containing kukoamine A and application of protective liquid

Abstract

The invention discloses application of kukoamine A, protective liquid containing kukoamine A and application of the protective liquid. The protective liquid containing kukoamine A is composed of the following components: 8-12% of kukoamine A; the human platelet lysate accounts for 4-6%; the content of the low molecular weight heparin sodium is 0.005%-0.02%; the antibacterial peptide LL-37 accounts for 0.00005 percent to 0.0002 percent; the balance is normal saline. The invention discloses application of a protective solution containing kukoamine A in preparation of a medicine for promoting human placenta-derived mesenchymal stem cells to inhibit pulmonary fibrosis. The invention also discloses an application of the protective liquid containing kukoamine A in preparation of a medicine for improving the multiplication capacity of human placenta mesenchymal stem cells. The kukoamine A has multiple biological activities such as oxidation resistance, inflammation resistance and apoptosis resistance, can protect the mesenchymal stem cells from being influenced by adverse factors in an in-vivo pathological microenvironment, and improves the survival rate of the mesenchymal stem cells.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and more specifically, relates to an application of lycopodiella citrate, a protective solution containing lycopodiella citrate and an application thereof. Background Art

[0002] The human body contains many different cell types that form tissues and organs with specific functions.

[0003] Mesenchymal stem cells (MSCs) are a type of cell with important biological properties and broad application prospects. Mesenchymal stem cells are widely available and can be obtained from a variety of tissues such as bone marrow, fat, and umbilical cord. At the same time, mesenchymal stem cells also have the potential to differentiate into all cell types of the three germ layers, which means that mesenchymal stem cells can differentiate into a variety of cell types such as bone cells, chondrocytes, muscle cells, and fat cells under appropriate conditions, providing possibilities for tissue repair and regeneration.

[0004] Compared with traditional treatments, mesenchymal stem cells have shown extraordinary potential in regulating the function of the immune system. Mesenchymal stem cells can accurately inhibit the excessive proliferation of T cells and the overactivity of natural killer cells (such as NK cells), thereby cleverly balancing and restoring the normal functioning of the immune system. This characteristic enables mesenchymal stem cells to show unprecedented therapeutic effects in the treatment of autoimmune diseases, such as the difficult systemic lupus erythematosus, bringing new treatment hope to patients.

[0005] Although mesenchymal stem cells have shown extraordinary potential in treating diseases. However, when mesenchymal stem cells are introduced into the patient's body, they will be affected by a variety of pathological microenvironmental factors (such as blood oxygen conditions, inflammatory environment, nutritional and metabolic status, cell-cell interactions, matrix and microstructure, etc.). These complex microenvironmental factors may cause the survival rate of mesenchymal stem cells in the body to be significantly reduced, their differentiation potential is weakened, and their functions are severely limited. Therefore, when mesenchymal stem cells are introduced into the patient's body, how to protect the survival rate and activity of mesenchymal stem cells becomes a technical problem that needs to be solved urgently.

[0006] Lycium barbarum is an active ingredient extracted from the dried root bark of Lycium barbarum or Lycium barbarum, a plant of the Solanaceae family. Lycium barbarum has been shown to have multiple biological activities, including lowering blood pressure, anti-oxidation, anti-inflammatory, neuroprotective, anti-tumor, lowering blood sugar and regulating blood lipids. Summary of the invention

[0007] Purpose of the invention: In view of the above-mentioned deficiencies in the prior art, the present invention discloses the application of Lycopodiella ciliata, a protective solution containing Lycopodiella ciliata and its application.

[0008] Technical solution: Application of lycopodine in the preparation of a medicament for improving the ability of mesenchymal stem cells to cope with pathological microenvironment in vivo.

[0009] Furthermore, the use of lycopodine as the only active ingredient in the preparation of a medicament for improving the ability of mesenchymal stem cells to cope with the pathological microenvironment in the body.

[0010] The protective solution containing lycopodin is composed of the following components in percentage by mass:

[0011] Lycopodiol accounts for 8% to 12%;

[0012] Human platelet lysate accounts for 4% to 6%;

[0013] Low molecular weight heparin sodium accounts for 0.005% to 0.02%;

[0014] The antimicrobial peptide LL-37 accounts for 0.00005% to 0.0002%;

[0015] The balance is saline.

[0016] Further, physostigmine accounts for 10%;

[0017] Human platelet lysate 5%;

[0018] Low molecular weight heparin sodium accounts for 0.01%;

[0019] The antimicrobial peptide LL-37 accounted for 0.0001%;

[0020] The balance is saline.

[0021] Furthermore, the enzyme activity of the low molecular weight heparin sodium is at least 100U.

[0022] Use of any of the above-mentioned protective solutions containing phytoserotonin in the preparation of a drug for promoting human placenta-derived mesenchymal stem cells to inhibit pulmonary fibrosis.

[0023] Use of any one of the above-mentioned protective solutions containing phytoserotonin in the preparation of a drug for improving the proliferation ability of human placental mesenchymal stem cells.

[0024] The functions of the various ingredients of the protective solution containing lycopodiol:

[0025] 1. Lycium barbarum: Lycium barbarum is a natural compound extracted from Lycium barbarum, which has multiple biological activities such as antioxidant, anti-inflammatory, and anti-apoptotic. In the protective solution, Lycium barbarum can effectively reduce the oxidative stress damage faced by mesenchymal stem cells in the pathological microenvironment in the body, inhibit inflammatory response, reduce cell apoptosis, and thus improve the survival rate and function of mesenchymal stem cells.

[0026] 2. Human platelet lysate: Human platelet lysate is an additive that replaces bovine serum. Human platelet lysate can be obtained by simply freezing and thawing platelet products. It is rich in a variety of growth factors released from platelets, including platelet-derived growth factor, transforming growth factor-1, insulin-like growth factor and basic fibroblast growth factor.

[0027] 3. Low molecular weight heparin sodium: Low molecular weight heparin sodium is a low molecular weight aminoglucan sulfate, which is produced by controlled nitrous acid depolymerization of heparin sodium prepared from porcine intestinal mucosa. Low molecular weight heparin sodium enhances the ability to inhibit coagulation factor Xa, which is relatively greater than its ability to prolong coagulation time. Heparin sodium has less effect on platelet function and platelet adhesion than heparin, so it has only a small effect on primary hemostasis. 1 / 2 The bioavailability is 90% and the pharmacokinetics are basically non-dose dependent.

[0028] 4. Antimicrobial peptide LL-37: Named Cathelicidin LL-37, its mature peptide consists of 37 amino acid residues, starting with two leucine residues. In addition to its antimicrobial properties, antimicrobial LL-37 also has the ability to induce a variety of biological effects, including regulating immunity, angiogenesis, wound healing, tumor growth, as well as inflammation and osteoporosis.

[0029] 5. Normal saline: Normal saline is a 0.9% sodium chloride aqueous solution. Because its osmotic pressure value is roughly the same as that of normal human plasma and tissue fluid, it can be used for fluid replacement (it will not reduce or increase the sodium ion concentration in the normal human body) and other medical purposes. It is also often used for in vitro culture of living tissues and cells.

[0030] Compared with traditional genetic engineering technology, Lycopodiella citrate has significant advantages in modifying mesenchymal stem cells. It can endow mesenchymal stem cells with antioxidant and anti-inflammatory capabilities, which helps mesenchymal stem cells survive and function better in complex pathological microenvironments. In addition, Lycopodiella citrate can also improve the adaptability of mesenchymal stem cells to pathological microenvironments, thereby further expanding its application potential in disease treatment.

[0031] Beneficial effects: The application of Lycopodiella ciliata, the protective liquid containing Lycopodiella ciliata and the application thereof disclosed in the present invention have the following beneficial effects:

[0032] 1. Lycopodiol has multiple biological activities such as antioxidant, anti-inflammatory, and anti-apoptotic, which can protect mesenchymal stem cells from the adverse factors in the pathological microenvironment in the body and improve the survival rate and functional activity of mesenchymal stem cells;

[0033] 2. The application method of the present invention can provide new ideas for the application of mesenchymal stem cells in the field of regenerative medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a morphological observation diagram of serum-free culture of human placenta-derived mesenchymal stem cells of the present invention.

[0035] Figure 2 The figure is a schematic diagram of the flow cytometry method for detecting surface markers of serum-free cultured human placenta-derived mesenchymal stem cells according to the present invention.

[0036] Figure 3A and Figure 3B This is a schematic diagram of the analysis of differentiation potential of mesenchymal stem cells derived from the fetal side of human placenta in serum-free culture according to the present invention.

[0037] Figure 4 This is a schematic diagram showing that the protective solution containing phytomycin of the present invention promotes human placenta-derived mesenchymal stem cells to inhibit pulmonary fibrosis.

[0038] Figure 5 Schematic diagram of the inhibitory effect of the protective solution containing phycoerythrin of the present invention on fPMSCs on collagen deposition in a mouse pulmonary fibrosis model.

[0039] Figure 6 The schematic diagram shows that the protective solution containing phytosporin of the present invention improves the proliferation ability of human placental mesenchymal stem cells.

[0040] Figure 7 The figure is a schematic diagram showing that the protective solution containing phytosterol of the present invention enhances the viability of human placental mesenchymal stem cells in the pathological microenvironment of pulmonary fibrosis.

[0041] Figure 8 The figure is a schematic diagram showing that the protective solution containing phytoserotonin of the present invention significantly inhibits the apoptosis of human placental mesenchymal stem cells in the pathological microenvironment of pulmonary fibrosis. DETAILED DESCRIPTION

[0042] The specific embodiments of the present invention are described in detail below.

[0043] The "range" disclosed in the present invention is defined in the form of a lower limit and an upper limit, and a given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of the particular range. The range defined in this way can be inclusive or exclusive of the end values, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a range. For example, if a range of 10 to 50 is listed for a specific parameter, it is understood that the range of 10 to 40 and 20 to 50 is also expected. In addition, if the minimum range values ​​1 and 2 are listed, and if the maximum range values ​​3, 4 and 5 are listed, the following ranges can all be expected: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4 and 2 to 5. In this application, unless otherwise specified, the range of values ​​"a to b" represents an abbreviation of any real number combination between a and b, where a and b are both real numbers. For example, the range of values ​​"0 to 5" means that all real numbers between "0 to 5" have been fully listed in this article, and "0 to 5" is only an abbreviation of these numerical combinations.

[0044] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0045] Unless otherwise specified, all technical features and optional technical features of this application can be combined with each other to form a new technical solution.

[0046] If there is no special explanation, all steps of the present application can be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), which means that step (c) may be added to the method in any order. For example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0047] If there is no special explanation, the "include" and "comprising" mentioned in this application represent open-ended or closed-ended expressions. For example, the "include" and "comprising" may represent that other components not listed may also be included or only the listed components may be included or only the listed components may be included.

[0048] Unless otherwise specified, the reaction is carried out at room temperature and pressure.

[0049] Unless otherwise specified, all parts or percentages are by weight.

[0050] In the present invention, all substances used are known substances and can be purchased or synthesized by known methods.

[0051] In the present invention, the devices or equipment used are all conventional devices or equipment known in the art and are commercially available.

[0052] In this application, the preparation method of the protective solution containing lycopodiella citrate is as follows:

[0053] The formulated amounts of lycopodiella citrate, human platelet lysate, low molecular weight heparin sodium and antimicrobial peptide LL-37 are sequentially added into physiological saline, and the mixture is stirred evenly to obtain a protective solution containing lycopodiella citrate.

[0054] In this application, unless otherwise specified, the protective solution containing lycopodiol used in the examples is the protective solution containing lycopodiol in Example 1. That is:

[0055] The protective solution containing lycopodin is composed of the following components in percentage by mass:

[0056] Lycopodiella oxyphylla accounts for 10%;

[0057] Human platelet lysate 5%;

[0058] Low molecular weight heparin sodium accounts for 0.01%;

[0059] The antimicrobial peptide LL-37 accounted for 0.0001%;

[0060] The balance is saline.

[0061] Experimental conditions and methods

[0062] 1. Isolation, culture and identification of mesenchymal stem cells:

[0063] (1) Isolation and serum-free culture of mesenchymal stem cells: Placental tissue was collected from healthy full-term pregnant women. Tissue about 0.5 cm thick was taken from the side of the placenta and cut into 1 mm 3 After the fragments were fully rinsed with HBSS, 1g / L Collagenase A and 1μg / mL DNaseⅠ were added and digested at 37℃ water bath for 2.0h. The cells in the supernatant were collected and transferred to serum-free medium (STEMCELL Technologies) and cultured in a 37℃, 5% carbon dioxide incubator. The medium was changed for the first time after 24h, and the cells were passaged at a ratio of 1:3 when they were 80% to 90% confluent.

[0064] (2) Identification of specific markers of mesenchymal stem cells:

[0065] Mesenchymal stem cells were prepared into a single cell suspension and 1×10 6Fluorescently labeled mAbs were added into flow cytometer tubes containing 10 cells: IgG2a-FITC, IgG1-PE, CD14-FITC, CD34-FITC, CD45-FITC, CD73-PE, CD90-FITC, CD105-PE, HLA-DR-FITC and isotype control antibodies to a final volume of 100 μL. The tubes were protected from light and allowed to stand at room temperature for 20 min. After rinsing with PBS, flow cytometry was performed.

[0066] (3) Identification of mesenchymal stem cell differentiation potential:

[0067] According to 1×10 5 Mesenchymal stem cells were seeded per well in a 6-well plate. When the cells grew to about 80% confluence, the original culture medium was discarded, and adipocyte induction medium (high-glucose DMEM medium containing 10% FBS, 1 nM dexamethasone, 0.2 mM indomethacin, 0.5 mM 3-isobutyl-1-methylxanthine and 10 mg / L insulin) and osteoblast induction medium (high-glucose DMEM containing 10% FBS, 0.1 nM dexamethasone, 10 mM sodium glycerophosphate and 0.5 mM ascorbic acid) were added, respectively. The cells were cultured in a cell culture incubator at 37°C and 5% CO2 with saturated humidity, and the medium was changed twice a week. After 2 to 3 weeks of culture, the cells were stained with Oil Red O and Alizarin Red, respectively, and observed under an inverted microscope.

[0068] 2. Efficacy evaluation of protective solution containing lycopodiella citrate and its composition and proportion

[0069] (1) Treatment of mesenchymal stem cells with protective solution:

[0070] Normally grown mesenchymal stem cells of the second generation were selected and digested with trypsin at a density of 6000 to 8000 cells / cm 2 Inoculate in a culture dish. When the cells grow to 60-70% confluence, add protective solution to the cell culture medium at a dilution ratio of 1:100, continue culturing for 24 hours, digest with trypsin and collect the cells for later use.

[0071] The protective solution containing lycopodin is composed of the following components in percentage by mass:

[0072] Lycopodiella oxyphylla accounts for 10%;

[0073] Human platelet lysate 5%;

[0074] Low molecular weight heparin sodium accounts for 0.01%;

[0075] The antimicrobial peptide LL-37 accounted for 0.0001%;

[0076] The balance is saline.

[0077] (2) Animal grouping, preparation of mouse pulmonary fibrosis model and mesenchymal stem cell injection:

[0078] Forty male C57BL / 6J mice aged 6 weeks were randomly divided into bleomycin group, mesenchymal stem cell treatment group, protective solution intervention mesenchymal stem cell group, and negative control group, with 10 mice in each group. After intraperitoneal injection of 10g / L sodium pentobarbital, 50μL bleomycin (1g / L) was injected into the bleomycin group, mesenchymal stem cell treatment group, and protective solution intervention mesenchymal stem cell group to prepare pulmonary fibrosis model, and 50μL PBS was injected into the negative control group. Mesenchymal stem cells were labeled with Dil Stain from ThermoFisher, and 200μL of 5×10 5 The PBS solution of 10 cells was added to the pulmonary fibrosis model mice in the mesenchymal stem cell treatment group and the protective fluid intervention mesenchymal stem cell group.

[0079] (3) Evaluation of the effect of protective fluid on the treatment of pulmonary fibrosis by mesenchymal stem cells:

[0080] On the 7th day after modeling, 5 mice in each experimental group were taken, and lung frozen sections were prepared. The number and distribution of mesenchymal stem cells in the lung tissue were observed under fluorescence confocal microscopy to determine the role of protective fluid in the pathological microenvironment of mesenchymal stem cells against pulmonary fibrosis. On the 21st day after modeling, 5 mice in each experimental group were taken, and paraffin sections of the lungs of each group of mice were prepared. HE and Masson staining were performed to observe the deposition of collagen in the lung tissue.

[0081] (4) Protective fluid improves the proliferation ability of mesenchymal stem cells:

[0082] Normally grown mesenchymal stem cells of the second generation were selected, digested with trypsin, and seeded in 96-well plates at 1000 cells / well. At the same time, protective solution was added at a ratio of 1:100. CCK8 was used to detect the proliferation of cells at 24h, 48h, 72h, 96h and 120h, and cells without protective solution were used as the control group.

[0083] (5) Protective fluid improves the survival of mesenchymal stem cells in the pathological microenvironment of pulmonary fibrosis

[0084] The mouse pulmonary fibrosis model was established using bleomycin, and the alveolar lavage fluid (BALF) was collected on the 7th day after modeling. The mesenchymal stem cells digested with trypsin were inoculated in a 96-well plate at 2000 cells / well, and protective solution and BALF were added at a ratio of 1:100. CCK8 was used to detect the proliferation of cells after 72 hours of culture, and cells without protective solution were used as the control group.

[0085] (6) Protective fluid inhibits apoptosis of mesenchymal stem cells in the pathological microenvironment of pulmonary fibrosis

[0086] The mesenchymal stem cells digested with trypsin were inoculated in a 35 mm culture dish. When the cells grew to 60-70%, protective solution and BALF were added to the cells at a ratio of 1:100. After 24 hours, the apoptosis of the cells was detected using the Annexin V & Propidium Iodide (PI) apoptosis kit, and the cells without protective solution were used as the control group.

[0087] Example 1

[0088] Application of lycopodin in the preparation of a medicament for improving the ability of mesenchymal stem cells to cope with pathological microenvironment in vivo.

[0089] Furthermore, the use of lycopodine as the only active ingredient in the preparation of a medicament for improving the ability of mesenchymal stem cells to cope with the pathological microenvironment in the body.

[0090] The protective solution containing lycopodin is composed of the following components in percentage by mass:

[0091] Lycopodiella oxyphylla accounts for 10%;

[0092] Human platelet lysate 5%;

[0093] Low molecular weight heparin sodium accounts for 0.01%;

[0094] The antimicrobial peptide LL-37 accounted for 0.0001%;

[0095] The balance is saline.

[0096] Furthermore, the enzyme activity of the low molecular weight heparin sodium is 100U.

[0097] Example 2

[0098] Application of lycopodin in the preparation of a medicament for improving the ability of mesenchymal stem cells to cope with pathological microenvironment in vivo.

[0099] Furthermore, the use of lycopodine as the only active ingredient in the preparation of a medicament for improving the ability of mesenchymal stem cells to cope with the pathological microenvironment in the body.

[0100] The protective solution containing lycopodin is composed of the following components in percentage by mass:

[0101] Lycopodiella oxyphylla accounts for 8%;

[0102] Human platelet lysate accounted for 4%;

[0103] Low molecular weight heparin sodium accounts for 0.005%;

[0104] The antimicrobial peptide LL-37 accounted for 0.00005%;

[0105] The balance is saline.

[0106] Furthermore, the enzyme activity of the low molecular weight heparin sodium is 105U.

[0107] Example 3

[0108] Application of lycopodin in the preparation of a medicament for improving the ability of mesenchymal stem cells to cope with pathological microenvironment in vivo.

[0109] Furthermore, the use of lycopodine as the only active ingredient in the preparation of a medicament for improving the ability of mesenchymal stem cells to cope with the pathological microenvironment in the body.

[0110] The protective solution containing lycopodin is composed of the following components in percentage by mass:

[0111] Lycopodiella oxyphylla accounts for 12%;

[0112] Human platelet lysate accounted for 6%;

[0113] Low molecular weight heparin sodium accounts for 0.02%;

[0114] The antimicrobial peptide LL-37 accounted for 0.0002%;

[0115] The balance is saline.

[0116] Furthermore, the enzyme activity of the low molecular weight heparin sodium is 120U.

[0117] Example 4

[0118] After obtaining the placental tissue of a healthy full-term pregnant woman, a portion of the placenta with a thickness of about 0.5 cm was selected and cut into 1 mm 3 The fragments were thoroughly rinsed with HBSS, followed by addition of 1g / L Collagenase A and 1μg / mL DNaseⅠ, and digested in a 37°C constant temperature water bath for 2 hours. The cells in the digested supernatant were collected and transferred to serum-free medium (purchased from STEMCELL Technologies) and cultured in an incubator at 37°C and 5% carbon dioxide concentration. The first medium change was performed after 24 hours of culture, and when the cell fusion reached 80% to 90%, the cells were passaged at a ratio of 1:3. Human placenta-derived mesenchymal stem cells were cultured to the third generation in serum-free medium.

[0119] Figure 1 4 is a morphological observation diagram of serum-free cultured human placenta-derived mesenchymal stem cells of the present invention. Figure 1As shown, the morphology of the serum-free cultured human placenta-derived mesenchymal stem cells of the present invention is uniform, adherent to the wall and presents a spindle-shaped vortex growth trend, which is consistent with the typical morphological characteristics of mesenchymal stem cells (magnification 40×).

[0120] Example 5

[0121] Mesenchymal stem cells were made into single cell suspension, and according to the relevant instructions, 1×10 6 Monoclonal antibodies labeled with fluorescent groups, including IgG2a-FITC, IgG1-PE, CD14-FITC, CD34-FITC, CD45-FITC, CD73-PE, CD90-FITC, CD105-PE, HLA-DR-FITC and corresponding isotype control antibodies, were added to the flow cytometer of each cell, so that the final reaction volume reached 100 μL. After the addition, it was placed in a light-proof environment and allowed to stand at room temperature for 20 minutes, then rinsed with PBS, and then detected and analyzed by flow cytometry. The specific results are shown in the figure. Figure 2 As shown, it was found that human placenta-derived mesenchymal stem cells (hfPMSC) cultured in serum-free culture showed positive expression of CD73, CD90 and CD105, while the marker molecules of hematopoietic cells CD14, CD34, CD45 and HLA-DR were negatively expressed, indicating that it has the typical surface marker molecular characteristics of mesenchymal stem cells.

[0122] Example 6

[0123] Take an appropriate amount of mesenchymal stem cells, 1×10 5 The cells were inoculated at a density of 100 cells / well in a 6-well culture plate, and the original culture medium was removed when the cells grew to about 80% confluence. Subsequently, adipocyte induction culture medium (its composition is a high-glucose DMEM culture medium containing 10% fetal bovine serum, 1nM dexamethasone, 0.2mM indomethacin, 0.5mM 3-isobutyl-1-methylxanthine and 10mg / L insulin) and osteoblast induction culture medium (high-glucose DMEM containing 10% fetal bovine serum, 0.1nM dexamethasone, 10mM sodium glycerophosphate and 0.5mM ascorbic acid) were added, and the culture plate was placed in a cell culture incubator at 37°C, 5% carbon dioxide and saturated humidity. The culture medium was replaced twice a week during the period. After continuous culture for 2 to 3 weeks, Oil Red O and Alizarin Red were used for staining, and observed under an inverted microscope.

[0124] After induction, mesenchymal stem cells from the fetal side of the human placenta can form bright red lipid droplets (such as Figure 3B as shown) and red calcium nodular deposits (as Figure 3AThis indicates that the isolated mesenchymal stem cells derived from the fetal side of the human placenta have the potential for multidirectional differentiation, which is consistent with the typical characteristics of mesenchymal stem cells.

[0125] Example 7

[0126] The protective solution containing physostigmine in Example 1 promotes human placenta-derived mesenchymal stem cells to inhibit pulmonary fibrosis.

[0127] The second generation of mesenchymal stem cells in a normal growth state were selected, digested with trypsin, and then inoculated into a culture dish at a density of 6,000 to 8,000 cells per square centimeter. When the cells grew to a confluence of 60% to 70%, the protective solution was added to the cell culture medium at a dilution ratio of 1:100, and then cultured for 24 hours. After that, the cells were digested with trypsin again, and the obtained cells were collected for later use.

[0128] HE staining analysis was used to evaluate the therapeutic effects of human placenta-derived mesenchymal stem cells and mesenchymal stem cells treated with protective fluid after bleomycin treatment for 21 days. In this process, normal mice and mice treated with bleomycin for 21 days were set as control groups (magnification of 40×) for comparative observation and analysis. The results showed that protective fluid can significantly enhance the effect of mesenchymal stem cells in improving pulmonary fibrosis.

[0129] Figure 4 Middle: Control means normal mice; BLM means bleomycin-treated mice; fPMSCs means human placenta-derived mesenchymal stem cells; fPMSCs means human placenta-derived mesenchymal stem cells treated with protective solution.

[0130] The Masson staining method was used to analyze the effects of human placenta-derived mesenchymal stem cells and mesenchymal stem cells treated with protective solution on collagen deposition in a mouse pulmonary fibrosis model after 21 days of bleomycin treatment. During the entire experiment, normal mice and mice treated with bleomycin for 21 days were set as control groups (observation magnification was 400×) to more intuitively and accurately compare and evaluate the differences in the effects of cells on collagen deposition under different treatment conditions. Figure 5 As shown, the protective solution containing phycoerythrin in Example 1 enhances the inhibitory effect of fPMSCs on collagen deposition in the mouse pulmonary fibrosis model.

[0131] Figure 5 Middle: Control means normal mice; BLM means bleomycin-treated mice; fPMSCs means human placenta-derived mesenchymal stem cells; fPMSCs means human placenta-derived mesenchymal stem cells treated with protective solution.

[0132] Example 8

[0133] Normally grown mesenchymal stem cells of the second generation were selected, digested with trypsin, and seeded in a 96-well plate at 1000 cells / well. At the same time, protective solution was added at a ratio of 1:100. CCK8 was used to detect the proliferation of cells at 24h, 48h, 72h, 96h and 120h, and cells without protective solution were used as the control group. Figure 6 As shown, from Figure 6 It can be seen that the protective solution containing phytosporin in Example 1 can better maintain the long-term culture of mesenchymal stem cells in vitro and maintain a high proliferation capacity.

[0134] Example 9

[0135] The mouse pulmonary fibrosis model was established using bleomycin, and the alveolar lavage fluid (BALF) was collected on the 7th day after modeling. The mesenchymal stem cells digested with trypsin were seeded in a 96-well plate at 2000 cells / well, and the protective solution and BALF were added at a ratio of 1:100.

[0136] CCK8 was used to detect the proliferation of cells after 72 hours of culture, and the cells without protection solution were used as the normal control group.

[0137] Figure 7 The figure is a schematic diagram showing that the protective solution containing phytosporin of the present invention enhances the viability of human placental mesenchymal stem cells in the pathological microenvironment of pulmonary fibrosis. Figure 7 It can be seen from the results that the addition of the protective solution containing physostigmine in Example 1 can significantly improve the survival rate of human placental mesenchymal stem cells in bronchoalveolar lavage of pulmonary fibrosis model mice.

[0138] Example 10

[0139] The mesenchymal stem cells digested with trypsin were seeded in a 35 mm culture dish, and when the cells grew to 60-70%, the cells were treated with protective solution and BALF.

[0140] The mouse pulmonary fibrosis model was established using bleomycin, and the alveolar lavage fluid (BALF) was collected on the 7th day after modeling. Human placental mesenchymal stem cells were seeded in a 35 mm culture dish, and when the cell confluence reached 60-70%, protective solution and / or BALF were added at a ratio of 1:100. Dead Cell Apoptosis Kit with Annexin V Alexa Fluor TM The 488&Propidium Iodide (PI) kit was used to detect cell apoptosis after 24 hours of culture, and the cells without protection solution were used as the normal control group.

[0141] Figure 8The figure is a schematic diagram showing that the protective solution containing phytosporin of the present invention significantly inhibits the apoptosis of human placental mesenchymal stem cells in the pathological microenvironment of pulmonary fibrosis. Figure 8 It can be seen that compared with the control group (Q2=19.6%), the alveolar lavage of the pulmonary fibrosis model mice to which the protective solution containing physostigmine of the present invention (Q2=8.23%) was added can significantly inhibit cell apoptosis.

[0142] The present invention uses the natural drug extract sarcosin as the main component of the protective solution, replacing existing methods such as gene editing or chemical drug treatment, and uses pure natural substances to intervene in mesenchymal stem cells, so that they can better adapt to the complex pathological microenvironment in the body and improve their survival ability and therapeutic potential.

[0143] The above describes the embodiments of the present invention in detail. However, the present invention is not limited to the above embodiments, and various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. The application of lycopodine in the preparation of a medicament for improving the ability of mesenchymal stem cells to cope with the pathological microenvironment in the body.

2. The application of lycopodine as the only active ingredient in the preparation of a drug for improving the ability of mesenchymal stem cells to cope with the pathological microenvironment in the body.

3. A protective solution containing lycopodin, characterized in that: In terms of mass percentage, it is composed of the following components: Lycopodiol accounts for 8% to 12%; Human platelet lysate accounts for 4% to 6%; Low molecular weight heparin sodium accounts for 0.005% to 0.02%; The antimicrobial peptide LL-37 accounts for 0.00005% to 0.0002%; The balance is saline.

4. The protective solution containing lycopodine as claimed in claim 3, characterized in that It is composed of the following components: Lycopodiella oxyphylla accounts for 10%; Human platelet lysate 5%; Low molecular weight heparin sodium accounts for 0.01%; The antimicrobial peptide LL-37 accounted for 0.0001%; The balance is saline.

5. The protective solution containing lycopodine as claimed in claim 4, characterized in that The enzyme activity of the low molecular weight heparin sodium is at least 100U.

6. Use of the protective solution containing physostigmine according to any one of claims 3 to 5 in the preparation of a drug for promoting human placenta-derived mesenchymal stem cells to inhibit pulmonary fibrosis.

7. Use of the protective solution containing phycopodiol as claimed in any one of claims 3 to 5 in the preparation of a drug for improving the proliferation ability of human placental mesenchymal stem cells.

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