Pulmonary stem cell peptide for treating pulmonary nodule as well as preparation method and application thereof

Abstract

The invention provides a lung stem cell peptide for treating pulmonary nodule as well as a preparation method and application thereof, and belongs to the technical field of biological pharmacy. The method comprises the following steps: performing induced differentiation on iPSC to obtain lung stem cells; lung stem cells are cultured and centrifuged, supernatant is collected, and a lung stem cell crude solution is obtained; mixing the lung stem cell crude liquid with protease, performing enzymolysis, centrifuging, concentrating, and performing spray drying to obtain the lung stem cell peptide for treating pulmonary nodule. By optimizing the composition of each culture medium, the iPSC is induced and differentiated into the lung stem cells, then the lung stem cell peptide is extracted and separated from the lung stem cells, and the extracted lung stem cell peptide migrates to the lung in a vein reinfusion mode, so that the generation of lung tumor nodules can be effectively inhibited, the number of the lung tumor nodules is greatly reduced, and the lung tumor nodules can be effectively inhibited. Toxic and side effects and adverse reactions are avoided.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and particularly relates to a lung stem cell peptide for treating lung nodules, a preparation method thereof, and an application thereof. Background Art

[0002] Lung nodules refer to lung lesions with a diameter generally not exceeding 3 cm, irregular shapes, and high-density shadows on imaging, which are solitary or multiple. Lung nodules have a hidden onset, accompanied by persistent chronic inflammation, and can gradually form pulmonary fibrosis, and even form lung cancer leading to death. For malignant lung nodules, or when the nodules are large, have obvious malignant characteristics and the patient's physical condition permits, surgical resection is usually considered. However, surgical resection has a high risk and a long recovery time. The drug treatment of lung nodules usually uses antibiotics, antifungal drugs, targeted therapy drugs, immunotherapy drugs, chemotherapy drugs, etc. These drugs have problems such as drug resistance, adverse reactions, affecting body functions, and poor efficacy. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a lung stem cell peptide for treating lung nodules, a preparation method thereof, and an application thereof, so as to solve the problems of drug resistance, adverse reactions, affecting body functions, and poor efficacy caused by existing drugs.

[0004] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0005] The present invention provides a preparation method of a lung stem cell peptide for treating lung nodules, comprising the following steps:

[0006] 1) Inducing and differentiating iPSCs to obtain lung stem cells;

[0007] 2) Culturing, centrifuging the lung stem cells, and collecting the supernatant to obtain a crude lung stem cell solution;

[0008] 3) Mixing and enzymolyzing the crude lung stem cell solution with protease, centrifuging, concentrating, and spray-drying to obtain a lung stem cell peptide for treating lung nodules.

[0009] Preferably, the step of inducing and differentiating in step 1) is specifically: inducing and culturing with a first induction medium on the 1st to 3rd days; inducing and culturing with a second induction medium on the 4th to 6th days; inducing and culturing with a third induction medium on the 7th to 9th days.

[0010] Preferably, the first induction medium is based on RPMI 1640 and further includes components with the following concentrations: 60 - 80 ng / mL Activin A, 12 - 16 μM LY294002, 25 - 50 ng / mL Wnt3a, 70 - 90 nM LDN193189, 20 - 50 ng / mL BMP4, 5 - 10 ng / mL FGF2, and 50 - 100 μg / mL vitamin C.

[0011] Preferably, the second induction medium is based on DMEM / F12 and further includes components with the following concentrations: 100 - 200 nM LDN193189, 10 - 30 ng / mL DMH2, 10 - 20 ng / mL FGF4, 3 - 5 μM CHIR99021, 2 - 4% N2, and 1 - 3% B27.

[0012] Preferably, the third induction medium is based on DMEM / F12 and further includes components with the following concentrations: 1 - 3 μM SM-04554, 1 - 10 μM SB-216763, 1 - 2 μM Purmorphamine, 8 - 12 μM DAPT, 0.5 - 1 μM A83-01, 80 - 120 ng / mL FGF10, 40 - 60 ng / mL FGF7, 0.5 - 2 ng / mL BMP4, 2 - 4 mM glutamine, and 0.1 - 1 μM all-trans retinoic acid.

[0013] Preferably, the rotation speed of the centrifugation in step 2) is 2000 - 3000 g, and the centrifugation time is 10 - 20 min.

[0014] Preferably, the addition amount of the protease in step 3) is 0.3 - 0.5% of the volume of the crude lung stem cell solution, the enzymatic hydrolysis temperature is 25 - 30 °C, and the enzymatic hydrolysis time is 12 - 18 h.

[0015] Preferably, the rotation speed of the centrifugation in step 3) is 8000 - 15000 g, and the centrifugation time is 3 - 5 min; the concentration method is ultrafiltration concentration, and the specification of the ultrafiltration membrane used for ultrafiltration concentration is 1000 - 500000 Da.

[0016] The present invention also provides a lung stem cell peptide for treating lung nodules prepared by the preparation method described in any one of the above.

[0017] The present invention also provides the application of the lung stem cell peptide for treating lung nodules prepared by the described preparation method or the lung stem cell peptide for treating lung nodules in the preparation of a drug for treating lung nodules.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] By optimizing the composition of each culture medium, the present invention induces the differentiation of iPSCs into lung stem cells, extracts and isolates lung stem cell peptides from the lung stem cells, and the obtained lung stem cell peptides migrate to the lungs through intravenous infusion, which can effectively inhibit the formation of lung tumor nodules, greatly reduce the number of lung tumor nodules, and have no toxic side effects and adverse reactions. Specific Embodiments

[0020] The present invention provides a method for preparing a lung stem cell peptide for treating lung nodules, comprising the following steps:

[0021] 1) Induce the differentiation of iPSCs to obtain lung stem cells;

[0022] 2) Culture the lung stem cells, centrifuge, and collect the supernatant to obtain a crude lung stem cell solution;

[0023] 3) Mix the crude lung stem cell solution with protease for enzymatic hydrolysis, centrifuge, concentrate, and spray-dry to obtain a lung stem cell peptide for treating lung nodules.

[0024] In the present invention, iPSCs are induced to differentiate into lung stem cells. The specific steps of the induction and differentiation are as follows: on the 1st to 3rd days, induction culture is carried out using a first induction medium; on the 4th to 6th days, induction culture is carried out using a second induction medium; on the 7th to 9th days, induction culture is carried out using a third induction medium. The first induction medium is based on RPMI 1640 and further includes the following components at the following concentrations: 60 - 80 ng / mL ActivinA, 12 - 16 μM LY294002, 25 - 50 ng / mL Wnt3a, 70 - 90 nM LDN193189, 20 - 50 ng / mL BMP4, 5 - 10 ng / mL FGF2, and 50 - 100 μg / mL vitamin C.

[0025] In the present invention, the concentration of Activin A is preferably 65 - 75 ng / mL; Activin A binds to Activin receptors IIA / IIB on the cell membrane, activates the SMAD2 / 3 signaling pathway, drives the expression of endoderm-specific genes. The combination of Activin A and Wnt3a mimics the Nodal signal during embryonic gastrulation, promotes the cells to exit the undifferentiated state by downregulating pluripotency markers such as OCT4 and NANOG, and induces the cells to shift from glycolysis to oxidative phosphorylation (similar to the metabolic characteristics of the embryonic endoderm).

[0026] In the present invention, the concentration of LY294002 is preferably 13 - 15 μM. LY294002 selectively inhibits PI3K (phosphatidylinositol 3-kinase), blocks Akt phosphorylation, thereby downregulating pluripotency genes, promoting cells to exit the undifferentiated state, relieving the inhibitory effect of insulin / IGF signaling, and also being able to reduce the contamination of mesoderm and ectoderm.

[0027] In the present invention, the concentration of Wnt3a is preferably 30 - 45 ng / mL; Wnt3a cooperates with Activin A to enhance the Activin A / SMAD2 / 3 signal, promotes the expression of SOX17 and FOXA2, forces cells to exit the undifferentiated state by inhibiting OCT4 and NANOG, inhibits SOX1 (ectoderm marker), and ensures endoderm specificity.

[0028] In the present invention, the concentration of LDN193189 is preferably 78 - 85 nM; LDN193189 is a highly efficient and selective BMP receptor inhibitor. It can specifically bind to the BMP receptor, prevent the BMP ligand from binding to the receptor, and then block the conduction of the BMP signal. By inhibiting the BMP signaling pathway, it can promote the differentiation of human pluripotent stem cells into definitive endoderm cells, improve the differentiation efficiency and purity, reduce the generation of other unnecessary cell types, and make the cell differentiation process more inclined to form definitive endoderm cells.

[0029] In the present invention, the concentration of BMP4 is preferably 25 - 45 ng / mL; BMP4 helps to activate the relevant signaling pathway, promotes the early differentiation of human pluripotent stem cells from the primitive state into mesoderm and endoderm, and can also act synergistically with other growth factors to initiate a series of signal transduction processes within the cell, enabling the cell to gradually acquire the characteristics of endoderm cells.

[0030] In the present invention, the concentration of FGF2 is preferably 6 - 9 ng / mL; FGF2 can bind to specific receptors on the cell surface and activate a series of signaling pathways. The activation of these signaling pathways helps to maintain the undifferentiated state and self-renewal ability of human pluripotent stem cells, ensuring that there are sufficient numbers of pluripotent stem cells for subsequent differentiation processes at the initial stage of differentiation; FGF2 can also act synergistically with other cytokines to affect gene expression and signal transduction networks within the cell, promoting the development of cells towards mesoderm and endoderm; it can also regulate the expression of some key transcription factors, promote the expression of endoderm-related genes, and inhibit the expression of ectoderm and mesoderm-related genes, thereby guiding cells to differentiate into definitive endoderm cells; it also helps to improve the survival ability of cells and reduce apoptosis; at the same time, it can also affect the migration ability of cells, enabling cells to better adjust their positions and reorganize tissues during the differentiation process, which is beneficial to the formation and development of definitive endoderm cells.

[0031] In the present invention, the concentration of the vitamin C is preferably 60-90 μg / mL; vitamin C is a strong antioxidant, which can scavenge reactive oxygen species generated intracellularly, such as superoxide anions, hydrogen peroxide, etc., and reduce the damage of oxidative stress to cells. During the differentiation of human pluripotent stem cells into definitive endoderm cells, an appropriate redox state is beneficial to maintaining the normal physiological functions and signal transduction pathways of cells, ensuring the smooth progress of the cell differentiation process; vitamin C is also involved in the hydroxylation reaction during collagen synthesis, promoting the hydroxylation of proline and lysine, thereby stabilizing the triple helix structure of collagen, contributing to the synthesis and secretion of collagen. Vitamin C indirectly affects the cell differentiation microenvironment by promoting collagen synthesis; it regulates gene expression by affecting the activities of some transcription factors and epigenetic modification enzymes, inhibits the expression of genes related to pluripotency maintenance or other germ layer differentiations, thereby promoting the differentiation of human pluripotent stem cells into definitive endoderm cells; vitamin C can also enhance the activities of certain cytokine signaling pathways, such as the Wnt and BMP signaling pathways, which helps to guide cells to differentiate into definitive endoderm cells and can improve the efficiency and quality of differentiation.

[0032] In the present invention, the second induction medium is based on DMEM / F12 as the basal medium, and further includes components with the following concentrations: 100-200 nM LDN193189, 10-30 ng / mL DMH2, 10-20 ng / mL FGF4, 3-5 μM CHIR99021, 2-4% N2, and 1-3% B27.

[0033] In the present invention, the concentration of the LDN193189 is preferably 120-180 nM; LDN193189 is a highly efficient and selective bone morphogenetic protein (BMP) receptor inhibitor. LDN193189 specifically binds to the BMP receptor, preventing the binding of BMP ligands to the receptor, thereby blocking the conduction of BMP signals, reducing the generation of other unnecessary cell types, and making the cell differentiation process more inclined to form foregut endoderm cells. By inhibiting the BMP signaling pathway, LDN193189 can affect the intracellular gene expression and signal transduction network, promote the development of definitive endoderm cells in the direction with the characteristics of foregut endoderm cells, help activate a series of genes related to the development of foregut endoderm cells, and at the same time inhibit the expression of genes related to other germ layers or non-foregut endoderm cell fates, thereby guiding cells to accurately differentiate into foregut endoderm cells and improving the efficiency and purity of differentiation.

[0034] In the present invention, the concentration of DMH2 is preferably 15 - 25 ng / mL; DMH2 is an inhibitor of the bone morphogenetic protein (BMP) signaling pathway. It can specifically block the BMP receptor and prevent the BMP ligand from binding to the receptor, thereby inhibiting the conduction of BMP signals. During the differentiation of definitive endoderm cells into foregut endoderm cells, excessive BMP signals will interfere with the differentiation of cells in the correct direction. By inhibiting this signaling pathway, DMH2 reduces the generation of other non-foregut endoderm cell types, making cell differentiation more inclined to form foregut endoderm cells. By inhibiting the BMP signaling pathway, DMH2 can affect gene expression and signal transduction networks within cells. It helps to activate genes related to the development of foregut endoderm cells, such as the expression of transcription factors like NKX2.1 and SOX2. These genes are crucial for the maintenance of the characteristics and further differentiation of foregut endoderm cells. At the same time, DMH2 can inhibit the expression of genes related to the fate of other germ layers or non-foregut endoderm cells, guiding cells to accurately differentiate into foregut endoderm cells and improving the efficiency and purity of differentiation.

[0035] In the present invention, the concentration of FGF4 is preferably 12 - 18 ng / mL; FGF4 binds to specific receptors on the surface of definitive endoderm cells and activates a series of signal transduction pathways, such as Ras-MAPK, PI3K-AKT, etc. The activation of these pathways can regulate various biological processes within cells and prepare for the differentiation of cells into foregut endoderm cells; an appropriate concentration of FGF4 can also promote the proliferation of definitive endoderm cells, increase the number of cells, and provide a basis for the subsequent differentiation to form a sufficient number of foregut endoderm cells. The FGF4 signal can affect gene expression related to the differentiation of foregut endoderm cells. It can upregulate the expression of some foregut-specific genes, such as transcription factors like SOX2 and FOXA2, and inhibit the expression of some genes related to the fate of non-foregut cells, thereby guiding cells to differentiate in the direction of foregut endoderm cells; FGF4 plays a role in signal transmission between cells, affecting the interaction and communication between cells, and helps definitive endoderm cells form an ordered foregut endoderm cell structure during differentiation, promoting the morphogenesis of the foregut tissue.

[0036] In the present invention, the concentration of CHIR99021 is preferably 3.5 - 4.5 μM; CHIR99021 inhibits the activity of GSK-3β, stabilizes β-catenin, causes it to accumulate in the cytoplasm and enter the nucleus, binds to transcription factors of the T cell factor / lymphoid enhancer factor family, activates the expression of Wnt target genes, and further activates the Wnt signaling pathway; CHIR99021 regulates the expression of a series of genes related to foregut development by activating the Wnt signal, such as upregulating the expression of transcription factors such as CDX2 and SOX2, thereby guiding the differentiation of cells in the direction of foregut embryonic cells and improving the efficiency and accuracy of differentiation; CHIR99021 acts synergistically with other factors (such as FGF4, etc.) in the culture medium to jointly regulate the signal transduction network and gene expression program in cells.

[0037] In the present invention, the concentration of N2 is preferably 2.5 - 3.5%; the components in N2 can regulate the signal pathways in cells. For example, some growth factors and hormone-like substances can bind to receptors on the cell surface, activate related signal transduction pathways, and affect the gene expression and physiological functions of cells. The regulation of these signal pathways plays a key role in guiding the correct differentiation of definitive endoderm cells into foregut embryonic cells. It can cooperate with other inducing factors to promote the expression of genes related to foregut embryonic cell differentiation and inhibit the expression of irrelevant genes, thereby guiding the transformation of cell fate.

[0038] In the present invention, the concentration of B27 is preferably 1.5 - 2.5%; the components in B27 can participate in regulating the signal pathways in cells, thereby affecting the differentiation direction of cells. It can act synergistically with other inducing factors to activate the expression of genes related to foregut embryonic cell differentiation, such as promoting the expression of some transcription factors (such as SOX2, FOXA2, etc.). These transcription factors play a key role in determining the fate and maintaining the characteristics of foregut embryonic cells. At the same time, B27 may inhibit the expression of genes related to non-foregut cell fates, guide the differentiation of definitive endoderm cells in the direction of specific foregut embryonic cells, and improve the efficiency and accuracy of differentiation. B27 helps to maintain the viability and healthy state of cells. It can provide the necessary factors for cell growth and survival, reduce cell apoptosis, and enhance the anti-damage ability of cells. B27 can regulate the composition and properties of the culture medium, bind to or remove harmful substances in the culture medium, such as free radicals, etc., reduce their damage to cells, and create a stable and favorable environment for cell growth and differentiation.

[0039] In the present invention, the third induction medium is based on DMEM / F12 and further comprises components with the following concentrations: 1-3 μM SM-04554, 1-10 μM SB-216763, 1-2 μM Purmorphamine, 8-12 μM DAPT, 0.5-1 μM A83-01, 80-120 ng / mL FGF10, 40-60 ng / mL FGF7, 0.5-2 ng / mL BMP4, 2-4 mM glutamine, and 0.1-1 μM all-trans retinoic acid.

[0040] In the present invention, the concentration of the SM-04554 is preferably 1.5-2.5 μM; SM-04554 acts on the Wnt signaling pathway to promote the differentiation of foregut endoderm cells into lung progenitor cells. During the induction of differentiation, SM-04554 helps to maintain the proliferation ability of foregut endoderm cells, increasing the cell number and providing a sufficient cell source for the formation of lung progenitor cells. At the same time, it may also have the effect of promoting cell survival, reducing apoptosis, and improving the survival rate of foregut endoderm cells during the differentiation process, thereby enhancing the induction efficiency of lung progenitor cells. SM-04554 regulates the differentiation of foregut endoderm cells into lung progenitor cells by affecting gene expression. It also interacts with transcription factors or affects the structure and function of chromatin, thereby activating genes related to lung development, such as NKX2.1, etc., while inhibiting the expression of genes related to other non-lung cell types and guiding the cells to differentiate towards lung progenitor cells.

[0041] In the present invention, the concentration of the SB-216763 is preferably 3-7 μM; during the process of inducing the differentiation of foregut endoderm into lung progenitor cells, SB-216763 activates the Wnt / β-catenin signaling pathway, helps foregut endoderm cells receive specific signal instructions, enabling them to differentiate towards lung progenitor cells rather than other cell types, and also promotes the expression of genes related to lung development while inhibiting the expression of genes related to other non-lung cell types. It may also stimulate the proliferation of foregut endoderm cells, providing a sufficient number of cells for the formation of lung progenitor cells.

[0042] In the present invention, the concentration of the Purmorphamine is preferably 1.2-1.8 μM; Purmorphamine binds to and activates the Smoothened receptor, mimics the downstream signal of SHH, activates the Gli transcription factor, promotes the expression of downstream target genes, and promotes the formation and branching of lung buds. Purmorphamine is used in combination with FGF10 / FGF7 to promote the transformation of the foregut towards the ventral lung fate, mimic the lung mesenchymal signal, induce the proliferation and branching of lung progenitor cells, and optimize the differentiation efficiency.

[0043] In the present invention, the concentration of DAPT is preferably 9 - 11 μM; DAPT inhibits the Notch signaling pathway, making it easier for foregut endoderm cells to differentiate into lung progenitor cells; it promotes the up - regulation of genes related to lung development, such as NKX2.1, SOX9, etc., and at the same time inhibits the expression of inhibitory genes related to the Notch signal, thereby promoting the differentiation of foregut endoderm cells into lung progenitor cells and promoting the formation and enrichment of lung progenitor cells.

[0044] In the present invention, the concentration of A83 - 01 is preferably 0.6 - 0.9 μM; A83 - 01 selectively inhibits the kinase activity of TGF - β receptor I, blocks the phosphorylation of downstream Smad2 / 3, thereby inhibiting TGF - β, Activin, and Nodal signal transduction, reducing apoptosis and enhancing the proliferation ability of lung progenitor cells. When combined with Purmorphamine and FGF10 / FGF2, it relieves the inhibition of TGF - β on the downstream of the SHH signal, promotes lung bud formation, mimics the lung mesenchyme - epithelium interaction, and induces branching morphogenesis.

[0045] In the present invention, the concentration of FGF10 is preferably 90 - 110 ng / mL; FGF10 acts on the FGFR2b receptor of the foregut endoderm epithelium, induces lung bud outgrowth and branching. When combined with Purmorphamine and A83 - 01, it mimics the mesenchyme - epithelium interaction, relieves the inhibition of lung differentiation, and plays an important role in maintaining the stemness of lung progenitor cells.

[0046] In the present invention, the concentration of FGF7 is preferably 45 - 55 ng / mL; FGF7 can bind to specific receptors on the surface of foregut endoderm cells, activate the intracellular signaling pathway, thereby promoting cell division and proliferation, providing a sufficient number of cells for the formation of lung progenitor cells, and helping to increase the yield of lung progenitor cells; FGF7 also has an anti - apoptotic effect, can inhibit the apoptosis of foregut endoderm cells, improve the survival rate of cells during differentiation, and participate in regulating the migration of foregut endoderm cells, enabling the cells to be correctly positioned and aggregated to form a population of lung progenitor cells, and plays an important role in maintaining the stemness of lung progenitor cells.

[0047] In the present invention, the concentration of BMP4 is preferably 0.8 - 1.5 ng / mL; BMP4 enhances the response of FGF10 to FGFR2b, and promotes the proliferation of lung progenitor cells and branching morphogenesis.

[0048] In the present invention, the concentration of glutamine is preferably 2.5 - 3.5 mM; glutamine enters the mitochondria by converting into α-ketoglutaric acid (α-KG), provides ATP and biosynthetic precursors for rapidly proliferating lung progenitor cells, promotes the production of NADPH, and counteracts oxidative stress; as a cofactor, it regulates histone / DNA demethylase and affects the chromatin open state of key genes for lung development; as a GSH precursor, it protects cells from ROS damage (lung progenitor cells are sensitive to oxidative stress), and supports the energy requirements in the early stage of lung bud budding by enhancing glycolysis.

[0049] In the present invention, the concentration of all-trans retinoic acid is preferably 0.3 - 0.7 μM; all-trans retinoic acid enhances the expression of FGFR2b, improves the responsiveness of lung progenitor cells to FGF10, inhibits intestinal epithelial differentiation caused by excessive WNT activation, maintains the purity of the lung lineage, and synergistically promotes the expansion of distal lung progenitor cells with Purmorphamine.

[0050] In the present invention, lung stem cells are cultured, centrifuged, and the supernatant is collected to obtain a crude lung stem cell solution. The rotation speed of the centrifugation is preferably 2000 - 3000 g, more preferably 2200 - 2800 g; the time of the centrifugation is preferably 10 - 20 min, more preferably 12 - 18 min.

[0051] In the present invention, the crude lung stem cell solution and protease are mixed for enzymatic hydrolysis, centrifuged, concentrated, and spray-dried to obtain lung stem cell peptides for treating lung nodules. The addition amount of the protease is preferably 0.3 - 0.5% of the volume of the crude lung stem cell solution, more preferably 0.35 - 0.45% of the volume of the crude lung stem cell solution; the temperature of the enzymatic hydrolysis is preferably 25 - 30 °C, more preferably 26 - 29 °C; the time of the enzymatic hydrolysis is preferably 12 - 18 h, more preferably 14 - 16 h; the rotation speed of the centrifugation is preferably 8000 - 15000 g, more preferably 9000 - 13000 g; the time of the centrifugation is preferably 3 - 5 min, more preferably 3.5 - 4.5 min; the concentration method is preferably ultrafiltration concentration, and the specification of the ultrafiltration membrane used for ultrafiltration concentration is preferably 1000 - 500000 Da.

[0052] The present invention also provides lung stem cell peptides for treating lung nodules prepared by the preparation method described in any one of the above.

[0053] The present invention also provides the application of the lung stem cell peptides for treating lung nodules prepared by the described preparation method or the lung stem cell peptides for treating lung nodules in the preparation of drugs for treating lung nodules.

[0054] The following combines examples to elaborate on the technical solutions provided by the present invention in detail, but they cannot be construed as limiting the protection scope of the present invention.

[0055] Example 1

[0056] When the iPSCs (purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) grew to 95% confluence, the induction of differentiation was started. From day 1 to day 3 of differentiation, the first induction medium (using RPMI 1640 as the basal medium and adding the following components at the following concentrations: 70 ng / mL Activin A, 14 μM LY294002, 40 ng / mL Wnt3a, 80 nM LDN193189, 35 ng / mL BMP4, 8 ng / mL FGF2, and 70 μg / mL vitamin C) was used for induction culture; from day 4 to day 6, the second induction medium (using DMEM / F12 as the basal medium and adding the following components at the following concentrations: 150 nM LDN193189, 20 ng / mL DMH2, 15 ng / mL FGF4, 4 μM CHIR99021, 3% N2, and 2% B27) was used for induction culture; from day 7 to day 9, the third induction medium (using DMEM / F12 as the basal medium and adding the following components at the following concentrations: 2 μM SM-04554, 5 μM SB-216763, 1.5 μM Purmorphamine, 10 μM DAPT, 0.8 μM A83-01, 100 ng / mL FGF10, 50 ng / mL FGF7, 1.5 ng / mL BMP4, 3 mM glutamine, and 0.5 μM all-trans retinoic acid) was used for induction culture to obtain lung stem cells. When the lung stem cells were cultured until the cell confluence reached 90%, they were centrifuged at 2500 g for 15 min, and the supernatant was collected to obtain the crude lung stem cell solution. The crude lung stem cell solution was mixed with a protease at 0.4% of the volume of the crude lung stem cell solution and enzymolyzed at 28 °C for 15 h, then centrifuged at 10000 g for 4 min, ultrafiltered and concentrated with a 300000 Da ultrafiltration membrane, and spray-dried to obtain lung stem cell peptides.

[0057] Example 2

[0058] When the iPSCs (purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) grew to 95% confluence, the induction of differentiation was started. From the 1st to the 3rd day of differentiation, the first induction medium (using RPMI 1640 as the basal medium and adding the following components at the following concentrations: 60 ng / mL Activin A, 12 μM LY294002, 25 ng / mL Wnt3a, 70 nM LDN193189, 20 ng / mL BMP4, 5 ng / mL FGF2, and 50 μg / mL vitamin C) was used for induction culture; from the 4th to the 6th day, the second induction medium (using DMEM / F12 as the basal medium and adding the following components at the following concentrations: 100 nM LDN193189, 10 ng / mL DMH2, 10 ng / mL FGF4, 3 μM CHIR99021, 2% N2, and 1% B27) was used for induction culture; from the 7th to the 9th day, the third induction medium (using DMEM / F12 as the basal medium and adding the following components at the following concentrations: 1 μM SM-04554, 1 μM SB-216763, 1 μM Purmorphamine, 8 μM DAPT, 0.5 μM A83-01, 80 ng / mL FGF10, 40 ng / mL FGF7, 0.5 ng / mL BMP4, 2 mM glutamine, and 0.1 μM all-trans retinoic acid) was used for induction culture to obtain lung stem cells. When the lung stem cells were cultured to a cell confluence of 90%, they were centrifuged at a speed of 2000 g for 10 min, and the supernatant was collected to obtain a crude lung stem cell solution. The crude lung stem cell solution was mixed with a protease at 0.3% of the volume of the crude lung stem cell solution and enzymolyzed at 25 °C for 12 h, then centrifuged at a speed of 8000 g for 3 min, ultrafiltered and concentrated with a 1000 Da ultrafiltration membrane, and spray-dried to obtain lung stem cell peptides.

[0059] Example 3

[0060] When the iPSCs (purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) grew to 95% confluence, the induction of differentiation was started. From day 1 to day 3 of differentiation, the first induction medium (using RPMI 1640 as the basal medium and adding the following components at the following concentrations: 80 ng / mL Activin A, 16 μM LY294002, 50 ng / mL Wnt3a, 90 nM LDN193189, 50 ng / mL BMP4, 10 ng / mL FGF2, and 100 μg / mL vitamin C) was used for induction culture; from day 4 to day 6, the second induction medium (using DMEM / F12 as the basal medium and adding the following components at the following concentrations: 200 nM LDN193189, 30 ng / mL DMH2, 20 ng / mL FGF4, 5 μM CHIR99021, 4% N2, and 3% B27) was used for induction culture; from day 7 to day 9, the third induction medium (using DMEM / F12 as the basal medium and adding the following components at the following concentrations: 3 μM SM-04554, 10 μM SB-216763, 2 μM Purmorphamine, 12 μM DAPT, 1 μM A83-01, 120 ng / mL FGF10, 60 ng / mL FGF7, 2 ng / mL BMP4, 4 mM glutamine, and 1 μM all-trans retinoic acid) was used for induction culture to obtain lung stem cells. When the lung stem cells were cultured until the cell confluence reached 90%, they were centrifuged at 3000 g for 20 min, and the supernatant was collected to obtain the crude lung stem cell solution. The crude lung stem cell solution was mixed with protease at 0.5% of the volume of the crude lung stem cell solution and enzymolyzed at 30 °C for 18 h, then centrifuged at 15000 g for 5 min, ultrafiltered and concentrated with a 500000 Da ultrafiltration membrane, and spray-dried to obtain lung stem cell peptides.

[0061] Experimental Example 1

[0062] The LLC cells (purchased from Shanghai Kanglang Biotechnology Co., Ltd.) were cultured to the logarithmic growth phase. After digestion and washing, the cell concentration was adjusted to 2.5×10 6 mL -1 , and they were injected into 6-8-week-old C57BL / 6J mice (purchased from Beijing Beiyou Biotechnology Co., Ltd.) through the tail vein (200 μL / mouse). The changes in the body weight and tumor volume of the mice were observed. When the tumor volume grew to about 50 mm 3 , they were randomly grouped and administered once a day for 12 consecutive days.

[0063] Experimental grouping: Control: Inject an equal volume of normal saline through the tail vein.

[0064] Experimental group 1: The pulmonary stem cell peptide prepared in Example 1 was dissolved in physiological saline to prepare a 3 mg / mL pulmonary stem cell peptide solution, and the 3 mg / mL concentration of the pulmonary stem cell peptide solution was injected via the tail vein at a dose of 0.1 mL / 10 g of mouse body weight.

[0065] Experimental group 2: The pulmonary stem cell peptide prepared in Example 1 was dissolved in physiological saline to prepare a 5 mg / mL pulmonary stem cell peptide solution, and the 5 mg / mL concentration of the pulmonary stem cell peptide solution was injected via the tail vein at a dose of 0.1 mL / 10 g of mouse body weight.

[0066] Experimental group 3: The pulmonary stem cell peptide prepared in Example 1 was dissolved in physiological saline to prepare a 10 mg / mL pulmonary stem cell peptide solution, and the 10 mg / mL concentration of the pulmonary stem cell peptide solution was injected via the tail vein at a dose of 0.1 mL / 10 g of mouse body weight.

[0067] Experimental group 4: The pulmonary stem cell peptide prepared in Example 2 was used at a concentration of 3 mg / mL, and the 3 mg / mL concentration of the pulmonary stem cell peptide was injected via the tail vein at a dose of 0.1 mL / 10 g of mouse body weight.

[0068] Experimental group 5: The pulmonary stem cell peptide prepared in Example 2 was dissolved in physiological saline to prepare a 5 mg / mL pulmonary stem cell peptide solution, and the 5 mg / mL concentration of the pulmonary stem cell peptide solution was injected via the tail vein at a dose of 0.1 mL / 10 g of mouse body weight.

[0069] Experimental group 6: The pulmonary stem cell peptide prepared in Example 2 was dissolved in physiological saline to prepare a 10 mg / mL pulmonary stem cell peptide solution, and the 10 mg / mL concentration of the pulmonary stem cell peptide solution was injected via the tail vein at a dose of 0.1 mL / 10 g of mouse body weight.

[0070] Experimental group 7: The pulmonary stem cell peptide prepared in Example 3 was dissolved in physiological saline to prepare a 3 mg / mL pulmonary stem cell peptide solution, and the 3 mg / mL concentration of the pulmonary stem cell peptide solution was injected via the tail vein at a dose of 0.1 mL / 10 g of mouse body weight.

[0071] Experimental group 8: The pulmonary stem cell peptide prepared in Example 3 was dissolved in physiological saline to prepare a 5 mg / mL pulmonary stem cell peptide solution, and the 5 mg / mL concentration of the pulmonary stem cell peptide solution was injected via the tail vein at a dose of 0.1 mL / 10 g of mouse body weight.

[0072] Experimental group 9: The pulmonary stem cell peptide prepared in Example 3 was dissolved in physiological saline to prepare a 10 mg / mL pulmonary stem cell peptide solution, and the 10 mg / mL concentration of the pulmonary stem cell peptide solution was injected via the tail vein at a dose of 0.1 mL / 10 g of mouse body weight.

[0073] The activities of the mice in the Control group were significantly weakened, they were listless, their water and food intakes were significantly reduced, and their hair coats were dull and lacked luster. The activities of the mice in Experimental Groups 1-9 were slightly less, their mental states were normal, their water and food intakes were significantly more than those of the mice in the Control group, their hair coats were normal, and no toxic or side effects occurred.

[0074] The lung tissues of the mice were weighed, and the number of nodules on the surface of the lung tumors was counted for evaluation.

[0075] Experimental results: As shown in Table 1.

[0076] Table 1 Number of nodules on the surface of lung tumors in different treatment groups

[0077] Number of surface nodules of lung tumor / piece Control group 30.05±1.24 Experimental group 1 14.31±0.78 Experimental group 2 10.34±0.59 Experimental group 3 13.26±1.43 Experimental group 4 16.76±1.31 Experimental group 5 15.12±0.87 Experimental group 6 16.99±0.43 Experimental group 7 17.13±1.47 Experimental group 8 16.03±1.25 Experimental group 9 16.88±0.66

[0078] As can be seen from Table 1, the number of nodules on the surface of the lung tumors in Experimental Groups 1-9 was significantly lower than that in the Control group. The lung stem cell peptides prepared in Examples 1-3 could significantly inhibit the formation of lung tumor nodules and greatly reduce the number of lung tumor nodules.

[0079] From the above examples and experimental examples, it can be seen that the present invention optimizes the composition of each culture medium, induces the differentiation of iPSCs into lung stem cells, then extracts and isolates lung stem cell peptides from the lung stem cells, and the extracted lung stem cell peptides migrate to the lungs through intravenous reinfusion, which can effectively inhibit the formation of lung tumor nodules, greatly reduce the number of lung tumor nodules, and have no toxic side effects and adverse reactions.

[0080] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of a pulmonary stem cell peptide for treating pulmonary nodules, characterized in that, It includes the following steps: 1) Induce and differentiate iPSCs to obtain lung stem cells; 2) Culture the lung stem cells, centrifuge, collect the supernatant to obtain a crude lung stem cell solution; 3) Mix the crude lung stem cell solution with protease for enzymatic hydrolysis, centrifuge, concentrate, and spray dry to obtain lung stem cell peptides for treating lung nodules.

2. The preparation method according to claim 1, wherein The specific steps of the induction and differentiation in step 1) are as follows: Use the first induction medium for induction culture on days 1 - 3; use the second induction medium for induction culture on days 4 - 6; use the third induction medium for induction culture on days 7 - 9.

3. The preparation method according to claim 2, wherein The first induction medium is based on RPMI 1640 and also includes the following components at the following concentrations: 60 - 80 ng / mL Activin A, 12 - 16 μM LY294002, 25 - 50 ng / mL Wnt3a, 70 - 90 nM LDN193189, 20 - 50 ng / mL BMP4, 5 - 10 ng / mL FGF2, and 50 - 100 μg / mL vitamin C.

4. The preparation method according to claim 2, characterized in that, The second induction medium is based on DMEM / F12 and also includes the following components at the following concentrations: 100 - 200 nM LDN193189, 10 - 30 ng / mL DMH2, 10 - 20 ng / mL FGF4, 3 - 5 μM CHIR99021, 2 - 4% N2, and 1 - 3% B27.

5. The preparation method according to claim 2, characterized in that, The third induction medium is based on DMEM / F12 and also includes the following components at the following concentrations: 1 - 3 μM SM - 04554, 1 - 10 μM SB - 216763, 1 - 2 μM Purmorphamine, 8 - 12 μM DAPT, 0.5 - 1 μM A83 - 01, 80 - 120 ng / mL FGF10, 40 - 60 ng / mL FGF7, 0.5 - 2 ng / mL BMP4, 2 - 4 mM glutamine, and 0.1 - 1 μM all - trans retinoic acid.

6. The preparation method according to claim 1, wherein In step 2), the rotation speed of the centrifugation is 2000 - 3000 g, and the time of the centrifugation is 10 - 20 min.

7. The preparation method according to claim 1, wherein, In step 3), the addition amount of the protease is 0.3 - 0.5% of the volume of the crude lung stem cell solution, the temperature of the enzymatic hydrolysis is 25 - 30 °C, and the time of the enzymatic hydrolysis is 12 - 18 h.

8. The preparation method according to claim 1, wherein In step 3), the rotation speed of the centrifugation is 8000 - 15000 g, and the time of the centrifugation is 3 - 5 min; the concentration method is ultrafiltration concentration, and the specification of the ultrafiltration membrane used for ultrafiltration concentration is 1000 - 500000 Da.

9. Lung stem cell peptides for treating lung nodules prepared by the preparation method according to any one of claims 1 - 8.

10. The application of the lung stem cell peptides for treating lung nodules prepared by the preparation method according to any one of claims 1 - 8 or the lung stem cell peptides for treating lung nodules according to claim 9 in the preparation of drugs for treating lung nodules.