A lung stem cell peptide for treating lung nodules and a preparation method and application thereof

Abstract

This invention provides a lung stem cell peptide for treating pulmonary nodules, its preparation method, and its application, belonging to the field of biopharmaceutical technology. The invention involves inducing iPSCs to differentiate into lung stem cells; culturing the lung stem cells, centrifuging, collecting the supernatant to obtain crude lung stem cell solution; mixing the crude lung stem cell solution with a protease for enzymatic hydrolysis, centrifuging, concentrating, and spray-drying to obtain the lung stem cell peptide for treating pulmonary nodules. This invention optimizes the composition of each culture medium to induce iPSCs to differentiate into lung stem cells, then extracts and separates lung stem cell peptides from these cells. The extracted lung stem cell peptides are intravenously reinfused into the lungs, effectively inhibiting the formation of pulmonary tumor nodules, significantly reducing the number of pulmonary tumor nodules, and without toxic side effects or adverse reactions.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, and in particular relates to a lung stem cell peptide for treating pulmonary nodules, its preparation method, and its application. Background Technology

[0002] Pulmonary nodules refer to intrapulmonary lesions that are generally no more than 3 cm in diameter, irregular in shape, and appear as high-density shadows on imaging. They can be solitary or multiple. Pulmonary nodules have an insidious onset, accompanied by persistent chronic inflammation, and can gradually develop into pulmonary fibrosis, or even lung cancer leading to death. For malignant pulmonary nodules, or nodules that are large, have obvious malignant characteristics, and where the patient's physical condition permits, surgical removal is usually considered. However, surgical removal carries significant risks and a long recovery period. Drug treatment for pulmonary nodules typically involves antibiotics, antifungals, targeted therapies, immunotherapies, and chemotherapy drugs. These drugs have problems such as drug resistance, adverse reactions, impact on bodily 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 pulmonary nodules, its preparation method and application, so as to solve the problems of drug resistance, adverse reactions, impact on bodily functions and poor efficacy of existing drugs.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides a method for preparing lung stem cell peptides for treating pulmonary nodules, comprising the following steps:

[0006] 1) Inducing iPSCs to differentiate into lung stem cells;

[0007] 2) Culture lung stem cells, centrifuge, collect the supernatant to obtain crude lung stem cell solution;

[0008] 3) Mix the crude lung stem cell liquid with protease for enzymatic hydrolysis, centrifuge, concentrate, and spray dry to obtain lung stem cell peptides for treating lung nodules.

[0009] Preferably, the induction differentiation step 1) specifically involves: induction culture using a first induction medium on days 1 to 3; induction culture using a second induction medium on days 4 to 6; and induction culture using a third induction medium on days 7 to 9.

[0010] Preferably, the first induction medium is based on RPMI 1640 and further comprises the following components at concentrations: 60–80 ng / mL Activin A, 12–16 μM LY294002, 25–50 ng / mL Wnt3a, 70–90 nM DN193189, 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 comprises the following components at 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 comprises the following components at concentrations: 1–3 μM MSM-04554, 1–10 μM SB-216763, 1–2 μM Urmorphamine, 8–12 μM DAPT, 0.5–1 μM MA83-01, 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 centrifugation speed in step 2) is 2000-3000g, and the centrifugation time is 10-20min.

[0014] Preferably, in step 3), the amount of protease added is 0.3 to 0.5% of the crude liquid volume of lung stem cells, the enzymatic hydrolysis temperature is 25 to 30°C, and the enzymatic hydrolysis time is 12 to 18 hours.

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

[0016] The present invention also provides lung stem cell peptides for treating pulmonary nodules prepared by any of the preparation methods described in any one of the inventions.

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

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

[0019] This invention optimizes the composition of various culture media to induce iPSCs to differentiate into lung stem cells, and then extracts and separates lung stem cell peptides from the lung stem cells. The extracted lung stem cell peptides are then intravenously reinfused into the lungs, which can effectively inhibit the formation of lung tumor nodules, greatly reduce the number of lung tumor nodules, and have no toxic side effects or adverse reactions. Detailed Implementation

[0020] This invention provides a method for preparing lung stem cell peptides for treating pulmonary nodules, comprising the following steps:

[0021] 1) Inducing iPSCs to differentiate into lung stem cells;

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

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

[0024] In this invention, iPSCs are induced to differentiate into lung stem cells. The specific steps of the induction differentiation are as follows: induction culture is performed using a first induction medium on days 1-3; induction culture is performed using a second induction medium on days 4-6; and induction culture is performed using a third induction medium on days 7-9. The first induction medium is based on RPMI 1640 and further includes the following components at 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.

[0025] In this 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, and drives the expression of endoderm-specific genes. When Activin A is used in combination with Wnt3a, it mimics the Nodal signaling of embryonic gastrulation, and promotes the exit of cells from the undifferentiated state by downregulating pluripotency markers such as OCT4 and NANOG, and induces cells to shift from glycolysis to oxidative phosphorylation (similar to the metabolic characteristics of embryonic endoderm).

[0026] In this 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 reducing contamination of the mesoderm and ectoderm.

[0027] In this invention, the concentration of Wnt3a is preferably 30-45 ng / mL; Wnt3a synergistically enhances Activin A / SMAD2 / 3 signaling with Activin A, promotes the expression of SOX17 and FOXA2, forces cells out of the undifferentiated state by inhibiting OCT4 and NANOG, and inhibits SOX1 (ectoderm marker) to ensure endoderm specificity.

[0028] In this invention, the concentration of LDN193189 is preferably 78-85 nM. LDN193189 is a highly efficient and selective BMP receptor inhibitor that can specifically bind to the BMP receptor, prevent the binding of BMP ligands to the receptor, and thus block the transmission of BMP signals. By inhibiting the BMP signaling pathway, it can promote the differentiation of human pluripotent stem cells into morphological endoderm cells, improve differentiation efficiency and purity, reduce the generation of other unnecessary cell types, and make the cell differentiation process more inclined to form morphological endoderm cells.

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

[0030] In this 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 maintain the undifferentiated state and self-renewal capacity of human pluripotent stem cells, ensuring a sufficient number of pluripotent stem cells for subsequent differentiation in the early stages of differentiation. FGF2 can also synergistically act with other cytokines to affect intracellular gene expression and signal transduction networks, promoting cell development towards the mesoderm and endoderm. It can also regulate the expression of some key transcription factors, promoting the expression of endoderm-related genes and inhibiting the expression of ectoderm and mesoderm-related genes, thereby guiding cells to differentiate into defined endoderm cells. It also helps improve cell viability and reduce apoptosis. At the same time, it can also affect cell migration ability, enabling cells to better adjust their position and remodel tissues during differentiation, which is beneficial to the formation and development of defined endoderm cells.

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

[0032] In this invention, the second induction medium is based on DMEM / F12 and further includes the following components at 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 this invention, the concentration of 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 BMP ligands from binding to the receptor, thereby blocking BMP signal transduction, reducing the generation of other unwanted cell types, and making cell differentiation more inclined towards foregut germ cells. By inhibiting the BMP signaling pathway, LDN193189 can affect intracellular gene expression and signal transduction networks, promoting the development of mature endoderm cells towards foregut germ cell characteristics, helping to activate a series of genes related to foregut germ cell development, while inhibiting the expression of genes related to the fate of other germ layers or non-foregut germ cells, thereby guiding cells to accurately differentiate into foregut germ cells and improving differentiation efficiency and purity.

[0034] In this invention, the concentration of DMH2 is preferably 15–25 ng / mL. DMH2 is an inhibitor of the bone morphogenetic protein (BMP) signaling pathway, which specifically blocks BMP receptors, preventing BMP ligands from binding to receptors and thus inhibiting BMP signal transduction. During the differentiation of mature endoderm cells into foregut germ cells, excessive BMP signaling can interfere with cell differentiation in the correct direction. DMH2 inhibits this signaling pathway, reducing the production of other non-foregut germ cell types and making cell differentiation more inclined to form foregut germ cells. By inhibiting the BMP signaling pathway, DMH2 can affect intracellular gene expression and signal transduction networks. It helps to activate the expression of genes related to foregut germ cell development, such as transcription factors like NKX2.1 and SOX2, which are crucial for maintaining the characteristics and further differentiation of foregut germ cells. At the same time, DMH2 can inhibit the expression of genes related to the fate of other germ layers or non-foregut germ cells, guiding cells to differentiate accurately into foregut germ cells and improving the efficiency and purity of differentiation.

[0035] In this invention, the preferred concentration of FGF4 is 12–18 ng / mL. FGF4 binds to specific receptors on the surface of morphological endoderm cells, activating a series of signal transduction pathways, such as Ras-MAPK and PI3K-AKT. Activation of these pathways regulates various intracellular biological processes, preparing cells for foregut embryonic cell differentiation. Appropriate concentrations of FGF4 can also promote the proliferation of morphological endoderm cells, increasing cell number and providing a foundation for subsequent differentiation into a sufficient number of foregut embryonic cells. FGF4 signaling can influence the expression of genes related to foregut embryonic cell differentiation. It can upregulate the expression of some foregut-specific genes, such as transcription factors SOX2 and FOXA2, and inhibit the expression of some genes related to non-foregut cell fate, thereby guiding cells towards foregut embryonic cell differentiation. FGF4 plays a role in intercellular signal transduction, influencing cell-cell interactions and communication, which helps morphological endoderm cells form an ordered foregut embryonic cell structure during differentiation, promoting foregut tissue morphogenesis.

[0036] In this invention, the concentration of CHIR99021 is preferably 3.5–4.5 μM. CHIR99021 stabilizes β-catenin by inhibiting the activity of GSK-3β, causing it to accumulate in the cytoplasm and enter the nucleus. It then binds to transcription factors of the T cell kinase / lymphocyte enhancer family, activating the expression of Wnt target genes and thus activating the Wnt signaling pathway. By activating Wnt signaling, CHIR99021 regulates the expression of a series of genes related to foregut development, such as upregulating the expression of transcription factors like CDX2 and SOX2, thereby guiding cells to differentiate towards foregut embryonic cells and improving the efficiency and accuracy of differentiation. CHIR99021 works synergistically with other factors in the culture medium (such as FGF4) to jointly regulate the intracellular signal transduction network and gene expression program.

[0037] In this invention, the concentration of N2 is preferably 2.5–3.5%. Components of N2 can regulate intracellular signaling pathways. For example, some growth factors and hormone-like substances can bind to receptors on the cell surface, activating related signal transduction pathways and affecting gene expression and physiological functions. The regulation of these signaling pathways plays a crucial role in the correct differentiation of endodermal cells into foregut germ cells. It can synergistically work with other inducing factors to promote the expression of genes related to foregut germ cell differentiation and inhibit the expression of irrelevant genes, thereby guiding the transformation of cell fate.

[0038] In this invention, the concentration of B27 is preferably 1.5–2.5%. Components of B27 can participate in regulating intracellular signaling pathways, thereby influencing cell differentiation direction. It can synergistically act with other inducing factors to activate the expression of genes related to foregut cell differentiation, such as promoting the expression of transcription factors (e.g., SOX2, FOXA2), which play a crucial role in foregut cell fate determination and characteristic maintenance. Simultaneously, B27 may inhibit the expression of genes not related to foregut cell fate, guiding mature endoderm cells to differentiate into specific foregut cell types, improving differentiation efficiency and accuracy. B27 helps maintain cell viability and health. It can provide essential factors for cell growth and survival, reduce apoptosis, and enhance cell resistance to damage. B27 can regulate the composition and properties of the culture medium, binding to or removing harmful substances such as free radicals, reducing their damage to cells and creating a stable and favorable environment for cell growth and differentiation.

[0039] In this invention, the third induction medium is based on DMEM / F12 and further includes the following components at the following concentrations: 1–3 μM SM-04554, 1–10 μM SB-216763, 1–2 μM urmorphamine, 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 this invention, the concentration of SM-04554 is preferably 1.5–2.5 μM. SM-04554 acts on the Wnt signaling pathway, promoting the differentiation of foregut embryonic cells into lung progenitor cells. During the induction of differentiation, SM-04554 helps maintain the proliferative capacity of foregut embryonic cells, increasing the cell number and providing a sufficient cell source for the formation of lung progenitor cells. Simultaneously, it may also promote cell survival, reduce apoptosis, and increase the survival rate of foregut embryonic cells during differentiation, thereby improving the induction efficiency of lung progenitor cells. SM-04554 regulates the differentiation of foregut embryonic 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, while inhibiting the expression of genes related to other non-lung cell types, guiding cells to differentiate into lung progenitor cells.

[0041] In this invention, the concentration of SB-216763 is preferably 3-7 μM. During the process of inducing foregut embryos to differentiate into lung progenitor cells, SB-216763 activates the Wnt / β-catenin signaling pathway, helping foregut embryo cells to receive specific signal instructions, causing them to differentiate into lung progenitor cells rather than other cell types. It also promotes the expression of genes related to lung development, while inhibiting the expression of genes related to other non-lung cell types. Furthermore, it may stimulate the proliferation of foregut embryo cells, providing a sufficient number of cells for the formation of lung progenitor cells.

[0042] In this invention, the concentration of Purmorphamine is preferably 1.2–1.8 μM. Purmorphamine binds to and activates the Smoothened receptor, mimicking the downstream signaling of SHH, activating the Gli transcription factor, promoting the expression of downstream target genes, and promoting the formation and branching of lung buds. When used in combination with FGF10 / FGF7, Purmorphamine promotes the conversion of foregut to ventral lung fate, mimicking lung mesenchymal signaling, and inducing lung progenitor cell proliferation and branching to optimize differentiation efficiency.

[0043] In this invention, the concentration of DAPT is preferably 9-11 μM; DAPT inhibits the Notch signaling pathway, making it easier for foregut embryonic cells to differentiate into lung progenitor cells; it promotes the upregulation of gene expression related to lung development, such as NKX2.1 and SOX9, while inhibiting the expression of repressive genes related to Notch signaling, thereby promoting the differentiation of foregut embryonic cells into lung progenitor cells and promoting the formation and enrichment of lung progenitor cells.

[0044] In this 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 signaling, reducing apoptosis and enhancing the proliferation capacity of lung progenitor cells. When used in combination with Purmorphamine and FGF10 / FGF2, it relieves the inhibition of downstream SHH signaling by TGF-β, promotes lung bud formation, simulates lung mesenchymal-epithelial interaction, and induces branching morphogenesis.

[0045] In this invention, the concentration of FGF10 is preferably 90-110 ng / mL; FGF10 acts on the FGFR2b receptor of the foregut endoderm epithelium, inducing lung bud sprouting and branching, and when used in combination with Purmorphamine and A83-01, it simulates mesenchymal-epithelial interaction, relieves the inhibition of lung differentiation, and plays an important role in maintaining the stemness of lung progenitor cells.

[0046] In this invention, the concentration of FGF7 is preferably 45-55 ng / mL. FGF7 can bind to specific receptors on the surface of foregut embryonic cells, activate intracellular signaling pathways, 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 anti-apoptotic effects, which can inhibit apoptosis of foregut embryonic cells, improve the survival rate of cells during differentiation, and participate in regulating the migration of foregut embryonic cells, enabling cells to correctly locate and aggregate, forming a lung progenitor cell population, which plays an important role in maintaining the stemness of lung progenitor cells.

[0047] In this 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 and branching morphogenesis of lung progenitor cells.

[0048] In this invention, the concentration of glutamine is preferably 2.5–3.5 mM; glutamine enters the mitochondria by being converted into α-ketoglutarate (α-KG), providing ATP and biosynthetic precursors for rapidly proliferating lung progenitor cells, promoting NADPH production, and combating oxidative stress; as a cofactor, it regulates histone / DNA demethylases, affecting the chromatin open state of key genes in lung development; as a GSH precursor, it protects cells from ROS damage (lung progenitor cells are sensitive to oxidative stress) and supports the energy needs of early lung budding by enhancing glycolysis.

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

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

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

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

[0053] The present invention also provides the lung stem cell peptide for treating pulmonary nodules prepared by the preparation method described above, or the application of the lung stem cell peptide for treating pulmonary nodules in the preparation of a drug for treating pulmonary nodules.

[0054] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0055] Example 1

[0056] When iPSCs (purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) reached 95% confluence, differentiation was induced. From day 1 to 3, the first induction medium (RPMI 1640 basal medium supplemented with the following components: 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. From day 4 to 6, the second induction medium (DMEM / F12 basal medium supplemented with the following components: 150 nM LDN193189, 20 ng / mL DMH2, 15 ng / mL FGF4, and 4 μM...) was used. Lung stem cells were induced and cultured using CHIR99021, 3% N2, and 2% B27. From day 7 to 9, the cells were induced and cultured using a third induction medium (based on DMEM / F12 with the following concentrations of added components: 2 μM MSM-04554, 5 μM SB-216763, 1.5 μM Urmorphamine, 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) to obtain lung stem cells. When the lung stem cells reached 90% confluence, they were centrifuged at 2500g for 15 min, and the supernatant was collected to obtain the crude lung stem cell solution. The crude lung stem cell solution and 0.4% of the crude lung stem cell solution were mixed and enzymatically hydrolyzed at 28°C for 15 h. Then, the mixture was centrifuged at 10000 g for 4 min, concentrated by ultrafiltration using a 300000 Da ultrafiltration membrane, and spray-dried to obtain lung stem cell peptides.

[0057] Example 2

[0058] When iPSCs (purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) reached 95% confluence, differentiation was induced. From day 1 to 3, the first induction medium (RPMI 1640 basal medium supplemented with the following components: 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. From day 4 to 6, the second induction medium (DMEM / F12 basal medium supplemented with the following components: 100 nM LDN193189, 10 ng / mL DMH2, 10 ng / mL FGF4, and 3 μM...) was used. Lung stem cells were induced and cultured using CHIR99021, 2% N2, and 1% B27. From day 7 to 9, they were induced and cultured using a third induction medium (based on DMEM / F12 with the following concentrations of added components: 1 μM MSM-04554, 1 μM SB-216763, 1 μM Urmorphamine, 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) to obtain lung stem cells. When the lung stem cells reached 90% confluence, they were centrifuged at 2000g for 10 min, and the supernatant was collected to obtain the crude lung stem cell solution. The crude lung stem cell solution and 0.3% of the crude lung stem cell solution were mixed and enzymatically hydrolyzed at 25°C for 12 h. Then, the mixture was centrifuged at 8000 g for 3 min, concentrated by ultrafiltration with a 1000 Da ultrafiltration membrane, and spray-dried to obtain lung stem cell peptides.

[0059] Example 3

[0060] When iPSCs (purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.) reached 95% confluence, differentiation was induced. From day 1 to 3, the first induction medium (RPMI 1640 basal medium supplemented with the following components: 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. From day 4 to 6, the second induction medium (DMEM / F12 basal medium supplemented with the following components: 200 nM LDN193189, 30 ng / mL DMH2, 20 ng / mL FGF4, and 5 μM...) was used. Lung stem cells were induced and cultured using CHIR99021, 4% N2, and 3% B27. From day 7 to 9, they were induced and cultured using a third induction medium (DMEM / F12 basal medium supplemented with the following components: 3 μM MSM-04554, 10 μM MSB-216763, 2 μM Urmorphamine, 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) to obtain lung stem cells. When the lung stem cells reached 90% confluence, they were centrifuged at 3000g for 20 min, and the supernatant was collected to obtain the crude lung stem cell solution. The crude lung stem cell solution and 0.5% of the crude lung stem cell solution were mixed and enzymatically hydrolyzed at 30°C for 18 hours. Then, the mixture was centrifuged at 15000g for 5 minutes, concentrated by ultrafiltration using a 500000Da ultrafiltration membrane, and spray-dried to obtain lung stem cell peptides.

[0061] Experimental Example 1

[0062] LLC cells (purchased from Shanghai Kanglang Biotechnology Co., Ltd.) were cultured to the logarithmic growth phase, and after digestion and washing, the cell concentration was adjusted to 2.5 × 10⁻⁶. 6 mL -1 The drug was injected intravenously (200 μL / mouse) into 6-8 week old C57BL / 6J mice (purchased from Beijing Beiyou Biotechnology Co., Ltd.). Changes in mouse body weight and tumor volume were observed. When the tumor volume reached 50 mm... 3 Patients were randomly assigned to groups of approximately 100 people and administered the medication once daily for 12 consecutive days.

[0063] Experimental group: Control: Injected with an equal volume of physiological saline via tail vein.

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

[0065] Experimental Group 2: The lung stem cell peptides prepared in Example 1 were dissolved in physiological saline to prepare a 5 mg / mL lung stem cell peptide solution. The 5 mg / mL lung stem cell peptide solution was injected into the tail vein at a concentration of 0.1 mL / 10 g mouse body weight.

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

[0067] Experimental group 4: The lung stem cell peptide prepared in Example 2 was injected intravenously at a concentration of 3 mg / mL, at a concentration of 0.1 mL / 10 g mouse body weight.

[0068] Experimental Group 5: The lung stem cell peptides prepared in Example 2 were dissolved in physiological saline to prepare a 5 mg / mL lung stem cell peptide solution. The 5 mg / mL lung stem cell peptide solution was injected into the tail vein at a concentration of 0.1 mL / 10 g mouse body weight.

[0069] Experimental Example 6: The lung stem cell peptides prepared in Example 2 were dissolved in physiological saline to prepare a 10 mg / mL lung stem cell peptide solution. The 10 mg / mL lung stem cell peptide solution was injected into the tail vein at a concentration of 0.1 mL / 10 g mouse body weight.

[0070] Experimental Group 7: The lung stem cell peptides prepared in Example 3 were dissolved in physiological saline to prepare a 3 mg / mL lung stem cell peptide solution. The 3 mg / mL lung stem cell peptide solution was injected into the tail vein at a concentration of 0.1 mL / 10 g mouse body weight.

[0071] Experimental Group 8: The lung stem cell peptides prepared in Example 3 were dissolved in physiological saline to prepare a 5 mg / mL lung stem cell peptide solution. The 5 mg / mL lung stem cell peptide solution was injected into the tail vein at a concentration of 0.1 mL / 10 g mouse body weight.

[0072] Experimental Group 9: The lung stem cell peptides prepared in Example 3 were dissolved in physiological saline to prepare a 10 mg / mL lung stem cell peptide solution. The 10 mg / mL lung stem cell peptide solution was injected into the tail vein at a concentration of 0.1 mL / 10 g mouse body weight.

[0073] Mice in the control group showed significantly reduced activity, lethargy, and decreased water and food intake, with dull and lackluster fur. Mice in experimental groups 1-9 showed slightly less activity, normal mental state, and significantly more water and food intake than mice in the control group. Their fur was normal, and no toxic side effects occurred.

[0074] Mouse lung tissue was collected, weighed, and the number of nodules on the surface of lung tumors was counted for evaluation.

[0075] Experimental results are shown in Table 1.

[0076] Table 1 Number of lung tumor surface nodules in different treatment groups

[0077] Number of nodules on the surface of lung tumor / individual 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 shown in Table 1, the number of lung tumor surface nodules in experimental groups 1-9 was significantly lower than that in the Control group. The lung stem cell peptides prepared in Examples 1-3 can significantly inhibit the generation of lung tumor nodules and greatly reduce the number of lung tumor nodules.

[0079] As can be seen from the above embodiments and experimental examples, the present invention optimizes the composition of each culture medium to induce iPSCs to differentiate into lung stem cells, and then extracts and separates lung stem cell peptides from lung stem cells. The extracted lung stem cell peptides are then reinfused into the lungs via intravenous infusion, which can effectively inhibit the formation of lung tumor nodules, greatly reduce the number of lung tumor nodules, and has no toxic side effects or adverse reactions.

[0080] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing lung stem cell peptides for treating pulmonary nodules, characterized in that, Includes the following steps: 1) Inducing iPSCs to differentiate into lung stem cells; 2) Culture lung stem cells, centrifuge, collect the supernatant to obtain crude lung stem cell solution; 3) Mix the crude lung stem cell liquid with protease for enzymatic hydrolysis, centrifuge, concentrate, and spray dry to obtain lung stem cell peptides for treating lung nodules; The amount of protease added is 0.3-0.5% of the crude volume of lung stem cells, the enzymatic hydrolysis temperature is 25-30℃, and the enzymatic hydrolysis time is 12-18h; Step 1) The specific steps for inducing differentiation are as follows: induction culture is performed using the first induction medium from day 1 to day 3; induction culture is performed using the second induction medium from day 4 to day 6; and induction culture is performed using the third induction medium from day 7 to day 9. 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; The second induction medium is based on DMEM / F12 and also includes the following components at the following concentrations: 100-200 nM DN193189, 10-30 ng / mL DMH2, 10-20 ng / mL FGF4, 3-5 μM CHIR99021, 2-4% N2 and 1-3% B27; 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 MA83-01, 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.

2. The preparation method according to claim 1, characterized in that, Step 2) The centrifugation speed is 2000-3000g, and the centrifugation time is 10-20min.

3. The preparation method according to claim 1, characterized in that, Step 3) The centrifugation speed is 8000-15000g, and the centrifugation time is 3-5min; the concentration method is ultrafiltration concentration, and the ultrafiltration membrane used for ultrafiltration concentration has a specification of 1000-500000Da.

4. Lung stem cell peptides for treating pulmonary nodules prepared by the preparation method according to any one of claims 1 to 3.

5. The use of the lung stem cell peptide for treating pulmonary nodules prepared by the preparation method according to any one of claims 1 to 3, or the lung stem cell peptide for treating pulmonary nodules according to claim 4, in the preparation of a medicament for treating pulmonary nodules.