WO2022179445A1

WO2022179445A1 - Chemical micromolecule composition for chemically inducing fibroblasts to be reprogrammed into lung stem cells, and use thereof

Info

Publication number: WO2022179445A1
Application number: PCT/CN2022/076814
Authority: WO
Inventors: 张培霖
Original assignee: 海门雨霖细胞科技有限责任公司
Priority date: 2021-02-23
Filing date: 2022-02-18
Publication date: 2022-09-01
Also published as: CN114958726A

Abstract

Provided are a chemical micromolecule composition for chemically inducing fibroblasts to be reprogrammed into lung stem cells, and the use thereof. The induction of the composition does not require the introduction or use of any exogenous genes/transcription factors/MicroRNA genes, and cytokines/growth factors. The composition has a variety of uses. Further provided is an interfering RNA and chemical micromolecule mixed induction composition, comprising: siGSK3β, siG9a and a tretinoin compound; or shGSK3β, shG9a and a tretinoin compound. The interfering RNA and chemical micromolecule mixed induction composition also has the function of inducing fibroblasts to be reprogrammed into lung stem cells.

Description

Chemical small molecule composition for chemically induced fibroblast reprogramming into lung stem cells and its application

technical field

The present invention belongs to the intersection of cell biology, stem cell biology (cell reprogramming), molecular biology, medicine and pharmacy; miRNA) gene, and its RNA, protein or polypeptide; nor use any exogenous cytokines or growth factors, only chemical small molecules (referred to as small molecules) are combined to chemically induce fibroblasts to reprogram into lung stem cells. Compositions and their uses.

Background technique

Lung injury and chronic lung disease, except for lung transplantation, most of the current treatments can only relieve the symptoms of patients, but cannot achieve the effect of cure. Due to the limitations of donor sources and surgical techniques, the research focus of lung disease treatment has shifted to adult stem cell and embryonic stem cell transplantation for the treatment of lung diseases, as well as related research on lung regenerative medicine. Adult stem cells have attracted more attention because they can avoid the ethical, sampling, and immune rejection issues faced by embryonic stem cells. In particular, the new coronavirus is a global pandemic, and the acute respiratory distress syndrome (ARDS) caused by it has led to the death of a large number of patients. In addition to ARDS, pulmonary fibrosis from various causes is the leading cause of death worldwide. Both ARDS and pulmonary fibrosis lack effective treatments. Stem cell therapy, which can be used for acute and chronic lung diseases, is a promising treatment modality.

Pluripotent stem cells (PSCs) are a class of pluripotent cells with self-replication ability and multi-differentiation potential. Under certain conditions, they can differentiate into a variety of functional cells. Stem cells are classified according to their differentiation potential: stem cells with strong differentiation potential include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs); stem cells with weak or limited differentiation potential are adult stem cells Or tissue stem cells, including pluripotent stem cells with weak differentiation potential, such as mesenchymal stem cells (MSCs), and unipotent stem cells that can only differentiate into certain types of tissue functional cells, such as lung stem cells (lung stem cells).

Since adult stem cells or tissue stem cells can avoid the ethical issues faced by embryonic stem cells and induced pluripotent stem cells, the risk of carcinogenicity in vivo transplantation, and the risk of immune rejection, etc., have been studied more in the research of regenerative medicine of the lung and its clinical application. of attention.

Lung stem cells refer to adult or tissue stem cells that can continuously self-renew and differentiate into functional lung tissue cells under specific conditions. Due to the complexity of the lung structure, it is currently believed that lung injury depends on the differentiation, renewal and repair of lung tissue stem cells in different anatomical parts of the lung. Studies have shown that there is no single lung tissue stem cell in the lung. On the contrary, different lung stem cells exist in different anatomical parts of the lung tissue, mainly including: basal cells (BCs), which express the specific marker Trp63 (p63 ) and keratin 5 (Krt5); distal airway stem cells (DASCs; hereafter referred to as airway stem cells) that also express Trp63 (p63) and keratin 5 (Krt5); bronchioles expressing specific markers SPC and CC10 Alveolar stem cells (bronchoalveolar stem cells, BASCs; hereinafter referred to as alveolar stem cells); alveolar type 2 cells (AT2Cs) expressing the specific marker SPC. These types of lung tissue stem cells are important for maintaining lung homeostasis, function, and promoting lung injury repair. Lung stem cells are currently considered to be unipotent stem cells, which can only be directly differentiated into functional cells of lung tissue. Among them, lung basal cells can be extracted from human trachea and bronchi, expanded and cultured in vitro, and then transplanted to treat ARDS and pulmonary fibrosis (fibroblasts do not have this function). Although this method has been proved to be an effective treatment method and method, however, the method of obtaining materials is relatively complicated, and the number of obtained lung stem cells is limited, which can only be obtained from allogeneic organs, which cannot meet the needs of acute respiratory tract injury. For chronic pulmonary fibrosis, there are still questions about whether a sufficient number of lung basal cells can be obtained, and their allogeneic stem cells can lead to immune rejection. Therefore, research and development of new sources of lung stem cells is still an important problem to be solved.

Directed differentiation of stem cells is one of the ways to obtain lung stem cells. Stepwise differentiation of human pluripotent stem cells (hPSCs), including directed induced human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) into lung stem cells is another source of lung stem cells. hPSCS can differentiate into proximal airway cells and distal lung epithelial cells. However, the application of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSC) to direct differentiation of lung stem cells is still ethical and the residual hESCs and hiPSCs have potential tumorigenic risks, which limit hESCs and hiPSCs and their directed differentiation. cell applications.

Mesenchymal stem cells (MSCs) with weak differentiation potential belong to adult stem cells, and it has not been reported that they can differentiate into pulmonary functional cells, but they can secrete several immunoregulatory factors through transplantation of MSCs. Transplantation of MSCs has been reported in many cases, whether it can improve the distress syndrome caused by acute respiratory injury or the improvement of pulmonary fibrosis. It has also been reported that MSCs can be transformed under different internal environmental conditions. Most of the COVID-19 patients reported worldwide died of cytokine storm syndrome (CSS). CSS is the cause of ARDS and multiple organ failure caused by the rapid production of various inflammatory factors after infection with viruses or bacteria. However, more research is needed to determine whether MSCs are a safe and effective source of transplanted cells for ameliorating acute respiratory injury or pulmonary fibrosis. Therefore, obtaining enough lung stem cells for transplantable applications through other approaches or means such as cell reprogramming is a major requirement to improve the health of patients with lung diseases.

Cell reprogramming is the conversion of cells from one type to another by introducing exogenous transcription factors (genes), or small chemical molecules, growth factors, cytokines, and other inducing factors, targeting The process of inducing the regulation of specific cellular signaling pathways or epigenetic changes that enable the transformation of one type of cell into another. The so-called epigenetic refers to the DNA sequence, structure does not change, but gene expression has undergone heritable changes. Induced cell reprogramming includes: (1) reprogramming of induced pluripotent stem cells in which differentiated cells are reversed and restored to a pluripotent or totipotent state; (2) direct transformation from one differentiated cell type to another without going through the pluripotent stem cell stage. Direct reprogramming of cells of a differentiated cell type (aka: transdifferentiation, lineage reprogramming).

Chemically induced cell reprogramming: without introducing or using any exogenous genes/transcription factors/MicroRNA (miRNA) genes, and their RNAs, proteins and polypeptides; and exogenous cytokines or growth factors and other inducing factors; only use The targeted induction of chemical small molecules regulates changes in cell signaling pathways and epigenetic modifications. On the premise of not changing cell structure genes, only the gene expression profile of cells is changed, and the process of transforming one cell into another. Chemically induced cell reprogramming includes: (1) chemically induced pluripotent stem cell reprogramming (Hongkui Deng et al., Science. 341, 651-4, 2013); (2) chemically induced direct reprogramming of cells (transdifferentiation, lineage reprogramming) (Li X et al, Cell Stem Cell; 17(2): 195-203, 2015; Hu W et al, Cell Stem Cell. 17(2): 204-212, 2015).

The present invention only uses chemical small molecule composition to induce fibroblast reprogramming into lung stem cells, which belongs to adult stem cell reprogramming. Reprogramming is abbreviated as "lung stem cell reprogramming" or "reprogramming".

Since 2006, Shinya Yamanaka introduced four transcription factors (genes) Oct4 (also known as Oct, Oct3, or OCT-3 polypeptide expression factor, etc.), Sox2, Klf4 and c-Myc induction combined in differentiated somatic cells In recent years, since iPS cells were obtained by reprogramming induced pluripotent stem cells, due to the exogenous introduction of transcription factors/genes, the stability of the original gene sequence of the cell has been destroyed, which can lead to the risk of mutation and carcinogenesis. Therefore, the reprogramming method is constantly improving, gradually changing to only 1-2 exogenous transcription factors for induction, combining the use of MicroRNA (miRNA) genes and their RNAs, protein polypeptides, as well as chemical small molecules, cytokines, growth factors It has been developed to use only chemical small molecule combinations to induce reprogramming of differentiated cells into iPS cells (CN201010296987.8. Deng Hongkui et al.). However, only using chemical small molecules, chemically induced fibroblasts to reprogram into lung tissue stem cells have not been reported in any literature so far.

Because chemical small molecules (referred to as small molecules) are targeted molecular compounds, they can target and induce regulation of specific signaling pathways and epigenetics, so that one cell can be transformed into another cell; and it has good druggability, low cost, and stability. It has good performance and simple operation, so it is the best candidate for replacing exogenous transcription factors.

Based on this, the present inventors explored to induce fibroblast reprogramming into lung stem cells (including BASCs, AT2Cs, BCs, DASCs, etc.) using only chemical small molecule compositions.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a kind of inducing factor without introducing or using any exogenous gene/transcription factor/MicroRNA (miRNA) gene, and its RNA, protein or polypeptide; and exogenous cytokine or growth factor; only Using chemical small molecule GSK3β inhibitors, G9a histone methyltransferase (G9aHMT) inhibitors, and retinoic acid compounds, chemically induced reprogramming of fibroblasts into lung stem cells (including BASCs, AT2Cs, BCs, DASCs, etc. ) of small molecule compositions and their applications. The chemical small molecule composition for chemically inducing the reprogramming of fibroblasts into lung stem cells according to the present invention includes chemical small molecules: GSK3β inhibitor, G9aHMT inhibitor, retinoic acid compound; or on this basis, the small molecule combination In addition, chemical small molecule TGFβ inhibitors can also be added.

The inventors have found through a large number of experimental studies that although the three chemical small molecule GSK3β inhibitors, G9aHMT inhibitors, retinoic acid compounds, and TGFβ inhibitors that can be added, which constitute the small molecule composition, do not induce induction when they exist alone. The function of reprogramming fibroblasts into lung stem cells; and the combination of GSK3β inhibitor and G9aHMT inhibitor; the combination of GSK3β inhibitor and TGFβ inhibitor also does not have the function of inducing fibroblast reprogramming into lung stem cells; but GSK3β inhibition The chemical small-molecule composition composed of the agent, G9aHMT inhibitor, and retinoic acid compounds has the effect of inducing the reprogramming of fibroblasts into lung stem cells.

The uses of the chemical small molecule composition include: reprogramming fibroblasts into lung stem cells to prepare transformed lung stem cells to provide a source of lung stem cells for clinical and scientific research; it can also be used as a small molecule pharmaceutical composition for preparing The effect of inducing in situ fibroblasts to reprogram lung stem cells in vivo, promoting the repair of lung injury and improving and relieving pulmonary fibrosis drugs, prodrugs, and pharmaceutical compositions; or, preparation of reagents for inducing fibroblasts to reprogram into lung stem cells Or culture medium; Or, chemically induced fibroblasts are reprogrammed and transformed into pulmonary stem cells, which are further prepared into cell preparations, cell active ingredient preparations or drugs for transplantation to treat lung injury and improve and alleviate pulmonary fibrosis; or based on transformation Bioactive drugs or preparations prepared from active ingredients such as bioactive substances, exosomes, and bioactive vesicles extracted from lung stem cells.

In addition, the present invention also derives and develops a kind of down-regulating agent, such as interfering RNA (interfering RNA, iRNA), including small interfering RNA (Small interfering RNA, siRNA) or short hairpin RNA (short hairpin RNA, hRNA) and chemical small The mixed induction composition of molecular composition: siGSK3β, siG9a, retinoic acid compounds; or shGSK3β, shG9a, retinoic acid compounds, also has the function of inducing the reprogramming of fibroblasts into lung stem cells. It can also be developed and prepared as a mixed induction composition of interfering RNA and small molecules, or its pharmaceutical composition/drug/prodrug/drug preparation. It also belongs to the protection scope of the present invention.

Advancement and innovation of the present invention: The present invention is the first to provide a kind of gene, RNA, protein or polypeptide without introducing or using any exogenous gene/transcription factor/MicroRNA (miRNA) gene, and its RNA, protein or polypeptide; and exogenous cytokine or growth factor Factors and other inducing factors, using only chemical small molecule GSK3β inhibitors, G9aHMT inhibitors, retinoic acid compounds, chemically induced fibroblasts to reprogram into lung stem cells (mainly BASCs, AT2Cs, BCs, DASCs) small molecules The composition and its application; its advanced and innovative advantages are as follows:

(1) Lung stem cells prepared by using the small molecule inducing composition of the present invention belong to unipotent stem cells, which avoids the risk of carcinogenesis caused by reprogramming into induced pluripotent stem cells (iPSCs); (2) The small molecule inducing composition of the present invention induces The fibroblasts can be derived from the patient's own, so the lung stem cells obtained by reprogramming have individual characteristics and have two major advantages: one is easier to enter clinical applications; The risk of immune rejection caused by stem cell transplantation; (3) the small molecule inducing composition of the present invention and its application do not introduce or use any exogenous gene/transcription factor/MicroRNA gene during the reprogramming process; Exogenous genes cause new carcinogenic risks, which is safer and more reliable; and does not use any exogenous cytokines or growth factors, the cost is lower, and the operation is simpler; therefore, it can provide sufficient quantity and quality for clinical and scientific research. Lung stem cell source; (4) The chemical small molecule composition of the present invention is a pharmaceutical composition, which can promote the repair of lung injury and improve and alleviate lung fibrosis by inducing the reprogramming of fibroblasts into lung stem cells in vivo. It is also easy to be further developed or prepared into corresponding drugs, prodrugs, pharmaceutical compositions, etc.; (5) Although the chemical small molecule GSK3β inhibitors, G9aHMT inhibitors, retinoic acid compounds that make up the small molecule composition, and can be added The TGFβ inhibitor alone does not have the function of inducing the reprogramming of fibroblasts into lung stem cells; and the combination of GSK3β inhibitor and G9aHMT inhibitor; the combination of GSK3β inhibitor and TGFβ inhibitor does not have the ability to induce fibroblast reprogramming. The function of reprogramming fibroblasts into lung stem cells; however, the chemical small molecule composition of the present invention has the overall function of inducing the reprogramming of fibroblasts into lung stem cells; (6) the chemical small molecules are targeted small molecule compounds , stable in nature, easy to control the time, dose and combination of action, stable and reliable effect, good druggability; (7) small chemical molecules are based on their common targeting, induction and regulation of a specific cell signaling pathway and its epigenetic changes The unique function classification and naming of the chemical small molecules within the category, the differences or differences in the type, structure, and physicochemical properties of chemical small molecules within the category do not affect the performance of their unique functions, so it is convenient for the screening and optimization of small molecule composition components; (8) The small molecule inducing composition of the present invention has various uses, including: in vitro chemically inducing the reprogramming of fibroblasts into lung stem cells, to prepare lung stem cells, and providing a source of lung stem cells for clinical treatment or scientific research; or, adding a cell-based culture Base/physiological saline/organic solvent, prepared into reprogramming medium or reagent; or based on lung stem cells obtained by induction and transformation, to prepare lung stem cell drugs or preparations for clinical transplantation treatment or repairing lung injury and improving and relieving pulmonary fibrosis ; Or prepare bioactive drugs or preparations based on bioactive substances, exosomes, bioactive vesicles and other active ingredients extracted from lung stem cells obtained by inducing and transforming them; or, based on in-situ chemical induction of small molecule compositions The effect of reprogramming fibroblasts into lung stem cells is used to prepare pharmaceutical compositions/drugs/prodrugs/drug preparations for promoting the repair of lung injury and improving and relieving pulmonary fibrosis.

In the first aspect of the present invention, there is provided an application or use of a chemical small molecule composition in chemically inducing fibroblasts to reprogram into lung stem cells, wherein the chemical small molecule composition comprises: GSK3β inhibitor, G9aHMT inhibitor , retinoic acid compounds; or, the chemical small molecule combination only consists of GSK3β inhibitors, G9aHMT inhibitors, retinoic acid compounds; wherein, in the process of inducing fibroblasts to reprogram into lung stem cells, do not import Or do not use any exogenous gene/transcription factor/MicroRNA (miRNA) gene, and its RNA, protein or polypeptide; and various exogenous cytokines or growth factors; or, the chemical small molecule composition does not contain Any exogenous gene/transcription factor/MicroRNA (miRNA) gene, or its transcribed RNA, or its translated protein and polypeptide; and various exogenous cytokines or growth factors.

In a preferred example, in the application or use of the small molecule composition, the chemical small molecule composition can further include: TGFβ inhibitor; or, the chemical small molecule composition can also be inhibited only by GSK3β It is composed of G9aHMT inhibitors, retinoids and TGFβ inhibitors.

In a preferred example, the application or use of the small molecule composition includes:

The application of the small molecule composition for chemically inducing the reprogramming of fibroblasts into lung stem cells includes: the use of the small molecule composition for chemically inducing the reprogramming of fibroblasts into lung stem cells in vitro to prepare lung stem cells for clinical treatment or Scientific research provides lung stem cell source; or, for the preparation of pharmaceutical compositions/drugs/drug precursors/preparations for in vivo chemical induction of in situ reprogramming of fibroblasts into lung stem cells to promote repair of lung injury and improve and alleviate pulmonary fibrosis ; Or used to add cell basal medium/physiological saline/organic solvent to prepare a culture medium or preparation, reagent for chemically induced fibroblast reprogramming into lung stem cells; or based on the lung stem cells obtained by the induced reprogramming transformation, to prepare Pulmonary stem cell drugs or preparations for clinical transplantation to treat or repair lung injury and improve and relieve pulmonary fibrosis; or based on the lung stem cells induced and transformed, the active ingredients such as bioactive substances, exosomes, and bioactive vesicles are extracted, Thereby, a biologically active drug or formulation is prepared.

In a preferred example, the chemical small molecule composition for chemically inducing fibroblasts to reprogram into lung stem cells includes: a GSK3β inhibitor, a G9aHMT inhibitor and a retinoic acid compound; or, it is only It is composed of GSK3β inhibitor, G9aHMT inhibitor and retinoic acid compound; the chemical small molecule composition does not contain any biological inducing factor, or the chemical small molecule composition does not induce fibroblasts to reprogram into lung stem cells during the process. Introduce or not use any exogenous gene/transcription factor/MicroRNA (miRNA) gene, and its RNA, protein or polypeptide; and various exogenous cytokines or growth factors.

In a preferred example, the chemical small molecule composition can further include: TGFβ inhibitor; or, the chemical small molecule composition can also be inhibited only by GSK3β inhibitor, G9aHMT inhibitor, retinoids and TGFβ agent composition.

In a preferred example, the GSK3β inhibitor in any of the chemical small molecule compositions refers to the general term for inhibitors that can target and inhibit the GSK3β signaling pathway, preferably including but not limited to: CHIR-99021, BIO , LiCl, IM-12, TWS119, 1-Azakenpaullone, CHIR-98014, Tideglusib, AR-A014418, LY2090314, SB216763, AZD1080, other GSK3β small molecule inhibitors that target and induce inhibition of GSK3β signaling pathway, or their equivalents Pharmaceutical products, analogs, isomers, salts, hydrates or precursors, or a combination thereof; more preferably it comprises a GSK3β inhibitor CHIR-99021, LiCl, BIO, LY2090314.

In another preferred example, the G9aHMT inhibitor in any of the chemical small molecule compositions refers to the general term for inhibitors that can target and inhibit G9aHMT, preferably including but not limited to: BIX01294, UNC0638, A- 366, UNC0631, BRD4770, UNC0224, UNC0646, UNC0642, UNC0321, BRD4770, HKMTI-1-247, HKMTI-1-248, CPUY074020, DCG066, other small molecule inhibitors of G9aHMT targeting G9aHMT, or their equivalents A pharmaceutical product, analog, isomer, salt, hydrate or precursor, or a combination thereof; more preferably it comprises a G9aHMT inhibitor BIX01294 (or BIX-01294), UNC0638 or UNC0642.

In another preferred example, the retinoic acid compound in any of the chemical small molecule compositions refers to the ability to target and specifically bind to retinoic acid response elements (RARE), thereby targeting induction Compounds that modulate the RA signaling pathway; preferably, it includes but is not limited to: retinoic acid (RA), 13-cis retinoic acid, 9-cis retinoic acid, UAB7, UAB8, TTNPB, 3-methyl- TTN PB, AM80, AM580, CD437, Targretin, LGD1069, isotretinoin, viaminate, etretinic acid, etretinate, tazarotene, adapalene, other vitamins that target and induce regulation of RA signaling pathway Formic acid compounds; or their equivalent pharmaceutical preparations, analogs, salts, hydrates, precursors, or combinations thereof; more preferably, it includes retinoic acid (Retinoic acid, RA), 13-cis-vitamin Formic acid or 9-cis retinoic acid.

In another preferred embodiment, the TGFβ inhibitor in any of the chemical small molecule compositions refers to the general term for inhibitors that can target and inhibit the TGFβ signaling pathway, preferably, it includes but is not limited to: SB431542, A83- 01. SB525334, LY2109761, RepSox, SD-208, GW788388, SB505124, EW-7197, Galunisertib, other TGFβ small molecule inhibitors that induce inhibition of TGFβ signaling pathway, or their equivalent pharmaceutical products, analogs, isomers , a salt, a hydrate or a precursor, or a combination thereof; more preferably it comprises a TGFβ inhibitor SB431542, A83-01, RepSox or LY2109761.

In a preferred example, the chemical small molecule composition, wherein the chemical small molecule GSK3β inhibitor, G9aHMT inhibitor, and retinoic acid compounds are:

GSK3β inhibitor: 5-80 parts by weight, preferably 10-70 parts by weight; or the final concentration in solution state is 0.1-20 μM/mM, preferably 0.5-15 μM/mM (preferably, according to the molecular weight of GSK3β inhibitor Calculate the molar concentration, use μM concentration unit when the molecular weight is large; use mM concentration unit when the molecular weight of individual small molecules is small);

G9aHMT inhibitor: 0.1-50 parts by weight, preferably 0.5-40 parts by weight; or the final concentration in solution state is 0.01-20 μM, preferably 0.05-10 μM;

Retinoic acid compound: 0.05-20 parts by weight; preferably 0.15-15 parts by weight; or the final concentration in solution state is 0.1-20 μM; preferably 0.5-10 μM.

In a preferred example, the chemical small molecule composition, wherein the optional chemical small molecule TGFβ inhibitor: 0.1-50 parts by weight, preferably 0.5-40 parts by weight; or the final concentration in the solution state is 0.01- 20 μM, preferably 0.05-10 μM.

In a preferred embodiment, the GSK3β inhibitor, G9aHMT inhibitor, and retinoic acid compound in the small molecule composition are in a ratio by weight: (5-80):(0.1-50):(0.05-20); or The molar concentration ratio in solution state is (0.1-20):(0.01-20):(0.1-20).

In a preferred example, the ratio of GSK3β inhibitor, G9aHMT inhibitor, retinoic acid compound and TGFβ inhibitor in the composition is: (5-80):(0.1-50):(0.05-20):( 0.1-50); or the molar concentration ratio in solution state is (0.1-20):(0.01-20):(0.1-20):(0.01-20).

In a preferred example, the components of the chemical small molecule composition are in proportion by weight, the GSK3β inhibitor (such as CHIR99021, LiCl, BIO or Ly2090314), G9aHMT inhibitor (such as BIX01294, UNC0638 or UNC0642 ), retinoids (such as retinoic acid, 13-cis retinoic acid or 9-cis retinoic acid), TGFβ inhibitors (such as SB431542, A83-01 or RepSox) are (5-80): (0.1-50 ): (0.05-20): (0.1-50); preferably (10-70): (0.5-40): (0.15-15): (0.5-40); or the molar concentration ratio in the solution state is (0.1-20):(0.01-20):(0.1-20):(0.01-20); preferably (0.5-15):(0.05-10):(0.5-10):(0.05-10 ).

In another preferred embodiment, the above-mentioned small molecule composition is a pharmaceutical composition or preparation, which may further comprise a pharmaceutically acceptable carrier or excipient; Carriers or excipients such as saline, buffers, or additions include cell basal medium.

In another preferred embodiment, the small molecule GSK3β inhibitor, G9aHMT inhibitor, retinoic acid compound, and the added TGFβ inhibitor account for 0.01-99.9% of the total weight of the composition; more preferably 50% ～99.9%; 0.01～50% in solution state, such as 0.01%, 1%, 5%, 10%, 20%, 30%, etc.

The weight unit of above-mentioned weight parts ratio can be: any weight unit such as kilogram (kg), milligram (mg), microgram (μg); the molar unit of molar concentration ratio can be: mole (M), millimolar (mM), Any unit of molarity such as micromolar (μM).

In another preferred example, when the small molecule composition is applied to large animals and patients, the effective dosage (including solid or solution state) for large animals or humans is converted by corresponding professional conversion formulas according to the dosage used by small animals. It is a well-known technique in the art to convert the dosage according to the dosage of animals or estimate the dosage of human. For example, the calculation can be performed according to the Meeh-Rubner formula: Meeh-Rubner formula: A=k×(W2/3)/10,000. In the formula, A is the body surface area, calculated in m2; W is the body weight, calculated in g; K is a constant, which varies with animal species. Generally speaking, mice and rats are 9.1, guinea pigs are 9.8, rabbits are 10.1, cats are 9.9, and dogs 11.2, Monkey 11.8, Human 10.6. It should be understood that, depending on the composition or drug and the situation of the subject being treated, the conversion of the dose given may vary according to the evaluation of an experienced professional or pharmacist.

In another preferred embodiment, the small molecule composition is a reprogramming pharmaceutical composition or formulation, which further includes a pharmaceutically acceptable carrier or excipient, preferably, the carrier or excipient includes (but without limitation) one or more selected from the group consisting of: water, saline, phosphate buffer or other aqueous solvents; DMSO (dimethyl sulfoxide), glycerol and ethanol or other organic solvents; microspheres, liposomes, Microemulsion or polymer surfactant; colloidal drug delivery system or polymer drug delivery system; or preservatives, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, pH buffer substances, adhesives, fillers agents, lubricants or other pharmaceutical excipients.

In another preferred example, the pharmaceutical dosage forms that can be prepared from the chemical small molecule composition include (but are not limited to): solid dosage forms, including (but not limited to): powder, powder, tablet, pill, capsule, slow Release, controlled immediate release; liquid dosage forms, including (but not limited to): injections, infusions, suspensions, or other liquid dosage forms; gas dosage forms; or semi-solid dosage forms.

In another preferred embodiment, the chemical small molecule composition is a pharmaceutical composition, and a pharmaceutically acceptable carrier or excipient can be added to prepare the effect of chemically inducing the reprogramming of in situ fibroblasts into lung stem cells in vivo, A pharmaceutical composition, prodrug, drug or preparation for promoting repair of lung injury and improving and relieving pulmonary fibrosis.

In another preferred example, the small molecule composition can be prepared as a reprogramming preparation for chemically inducing fibroblasts to reprogram into lung stem cells by adding organic solvents, physiological saline, or other carriers or excipients or reagent;

In another preferred embodiment, any of the small molecule compositions can be prepared by adding a cell basal medium to prepare a reprogramming medium for chemically inducing fibroblasts to reprogram into lung stem cells.

In another aspect of the present invention, there is provided a kit or kit for chemically inducing the reprogramming of fibroblasts into lung stem cells, comprising: the chemical small molecule composition described above; based on the small molecule combination A pharmaceutically acceptable carrier or excipient is added to the drug to prepare the obtained pharmaceutical composition for in vivo chemical induction of in situ fibroblast reprogramming into lung stem cells, promoting the repair of lung injury and improving and relieving pulmonary fibrosis. , drug precursors, drugs or preparations; adding organic solvents, physiological saline, buffers, cell basal medium and other carriers or excipients or basal nutrient solutions, the prepared induced fibroblasts are reprogrammed into reprogramming culture of lung stem cells bases, reagents and preparations; based on the pulmonary stem cells obtained by chemically induced reprogramming of fibroblasts, further prepare lung stem cell drugs or cell preparations for clinical transplantation treatment or repair of lung injury and improvement and relief of pulmonary fibrosis; and Lung stem cells obtained by induced transformation, active ingredients such as extracted biologically active substances, exosomes, biologically active vesicles, or biologically active drugs or preparations prepared therefrom.

The kit or kit of the present invention does not include: any exogenous gene/transcription factor/MicroRNA (miRNA) gene for introduction into fibroblasts, or added to the small molecule composition or transdifferentiation medium, and its components such as RNA, protein or polypeptide; and various exogenous cytokines or growth factors; or its combination with gene carrier.

According to any one of the preceding aspects, the fibroblasts include: human fibroblasts or mammalian fibroblasts; preferably, including but not limited to human or mammalian: skin fibroblasts, liver fibroblasts (liver fibroblasts) stellate/shaped cells (hepatic stellate cells, HSCs), lung fibroblasts, renal fibroblasts, pancreatic fibroblasts, and fibroblasts of other tissues or organs of humans or mammals; preferably skin fibroblasts .

According to any one of the preceding aspects, the transformed lung stem cells obtained by inducing fibroblast reprogramming include: alveolar type 2 cells (AT2Cs), bronchoalveolar stem cells (BASCs), basal cells ( basal cells, BCs), distal airway stem cells (DASCs), or their mixture (hybrid cells).

In another aspect of the present invention, there is provided a method for reprogramming fibroblasts into lung stem cells without introducing or using any exogenous gene/transcription factor/MicroRNA (miRNA) gene, and RNA, protein or polypeptide thereof and other inducing factors; also do not use exogenous cytokines or growth factors; only use chemical small molecules to chemically induce the reprogramming of fibroblasts into lung stem cells (including BASCs, AT2Cs, BCs, DASCs) The method comprises: applying the above Any of the chemical small molecule compositions, a method for inducing fibroblast reprogramming into lung stem cells in vitro to prepare lung stem cells;

Or the method is a method for preparing a small-molecule pharmaceutical composition, a drug precursor, and a drug that induces the reprogramming of fibroblasts into lung stem cells in situ in vivo, promotes the repair of lung damage, and improves and alleviates pulmonary fibrosis; Or a method for preparing a small molecule composition for inducing the reprogramming of fibroblasts into lung stem cells; or a method for preparing a culture medium, reagent, or preparation for inducing the reprogramming of fibroblasts into lung stem cells.

In another aspect of the present invention, the chemical small molecule composition of the present invention is characterized in that the interfering RNA having the same inhibition target gene as the chemical small molecule GSK3β inhibitor in the composition: siGSK3β/shGSK3β, It also has the function of inducing and inhibiting GSK3β; the interfering RNA with the same inhibition target gene as the chemical small molecule G9aHMT inhibitor in the composition: siG9a/shG9a, also has the function of inducing and inhibiting G9aHMT;

In another preferred example, the chemical small molecule composition and the interfering RNA are characterized in that the mixed induction combination consisting of: siGSK3β, siG9a, and retinoic acid compounds; or shGSK3β, shG9a, and retinoic acid compounds compound, which has the same function of inducing fibroblast reprogramming into lung stem cell as the chemical small molecule composition;

In another preferred embodiment, the mixed induction composition is characterized in that the siGSK3β/shGSK3β, siG9a/shG9a belong to 20-25 base pairs, and a vector or a vector delivery system is required to be introduced into cells. The carrier or carrier delivery system includes but is not limited to: viral carrier delivery system, non-viral carrier delivery system, such as: chemical modification delivery system, microinjection delivery system, non-viral nanocarrier delivery system.

The present invention provides a method for inducing the reprogramming of fibroblasts into lung stem cells to prepare lung stem cells, and a method for preparing a reprogramming medium or a pharmaceutical composition/reagent and preparation. The specific experimental steps include:

(1) Concentrate reagent preparation: According to any of the aforementioned small molecule compositions, each component is dissolved in an organic solvent or an aqueous solvent to prepare a concentrate reagent (ranging from 1:50 to 1:10,000) ; Preferably, the organic solvent includes dimethyl sulfoxide; preferably, the aqueous solvent includes: water, physiological saline, and phosphate buffer;

(2) Obtaining the reprogramming medium for fibroblast reprogramming lung stem cells: Dilute the concentrate reagent in step (1) into the cell basal medium containing 5-20% calf serum (so that the The concentration conforms to the final concentration defined in any of the aforementioned small molecule compositions) to obtain a reprogramming medium; wherein, the percentage of each component of the medium can also fluctuate by 50%; preferably up and down Float 30%; better float 20%, such as 10%, 5%;

(3) Induce the reprogramming of fibroblasts into lung stem cells (mainly BASCs, AT2Cs, BCs, DASCs) culture: Suspend and plate fibroblasts in cell basal medium containing 5-20% calf serum, and place the cells in the cells. After adhering, it was changed to the reprogramming medium of step (2), cultured at 37°C, and the medium was changed every 2-4 days; passage was once every 3-15 days.

(4) The weight ratio of each component of the small molecule combination is formulated into a concentrated solution of 100 mg/ml according to the weight ratio of kilograms: → Dilute into 0.9% NaCl physiological saline (50 μl/mouse) and mix, and then prepare Obtaining a reprogramming aerosol reagent for chemically induced in situ reprogramming of fibroblasts into lung stem cells in vivo.

(5) Subculture of induced fibroblast-reprogrammed lung stem cells: discard the original culture medium, wash with PBS once, add cell digestion solution to digest cells, 37°C for 1-5 minutes, stop cell digestion, centrifuge, discard supernatant, and put Cell pellets were resuspended and plated at 1:1-1:3 passages. The transdifferentiation medium of step (2) was applied, and cultured according to the above method, and the medium was changed every 2-4 days. Digestive solutions used include trypsin, EDTA, Acutase, TrypleE, etc. Passage once every 3-15 days.

(6) Harvest of transformed lung stem cells: After the above experimental steps (4), (5) reprogramming lung stem cells culture and subculture for 2-4 weeks, fibroblasts can be reprogrammed into lung stem cells (transformed lung stem cells obtained). , including ciBASCs, ciAT2Cs, ciBSCs, ciDASCs, etc. with substantially the same shape, or a mixture thereof).

The above method does not introduce or use exogenous genes/transcription factors/MicroRNA genes, and various exogenous RNAs, proteins or polypeptides and other inducing factors; nor does it add or use any exogenous cytokines or growth factors; only chemical A method of inducing the reprogramming of fibroblasts into lung stem cells with small molecule compositions.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

Figure 1. Morphological comparison of lung stem cells transformed by small molecule chemical-induced fibroblasts (mice). Comparison of the morphology of fibroblasts (MF) reprogrammed and transformed lung stem cells (ciAT2Cs) with primary alveolar type II cells (pAT2) and MF cells induced by chemical small molecule compositions; Figure 1 shows that small molecule chemically induced fibroblast reprogramming The morphology of programmed transformed lung stem cells (ciAT2Cs) was the same or similar to that of primary alveolar type II cells (pAT2Cs); it was completely different from the morphology of MF induced initiating cells.

Figure 2. Chemically induced transformed alveolar type II cells (ciAT2Cs) have a lamellar structure unique to AT2Cs. Figure 2 shows that the reprogrammed and transformed lung stem cells have a lamellar structure unique to alveolar type II cells (AT2Cs) (electron microscope observation results), confirming that the small molecule composition chemically induces fibroblast reprogramming and transformed lung stem cells to be alveolar II type cells (AT2Cs).

Figure 3. Lung stem cell-like cells transformed by chemically induced fibroblasts (mice) were subjected to immunostaining of SPC, a specific marker of AT2Cs, and nuclear staining (DAPI). Figure 3 shows that the reprogrammed transformed lung stem cell-like cells were positive for alveolar type II cell-specific marker SPC immunofluorescence staining. The experimental results showed that the small molecule composition induced fibroblasts to reprogram the transformed lung stem cells, and the immunofluorescence staining of SPC-specific markers was positive. Combined with the same or similar shape as pAT2Cs (Example 2, Figure 1), and the unique lamellar structure of AT2Cs (Example 3, Figure 2), it further indicates that the chemically induced and transformed lung stem cells belong to alveolar type II cells (AT2Cs). , namely chemically induced transformed alveolar type II cells (ciAT2Cs).

Figure 4. The gene expression comparison of specific markers (SPC, CC10) of alveolar stem cells (BASCs) in reprogrammed and transformed lung stem cell-like cells. Figure 4 shows that the alveolar stem cell (BASCs)-specific marker genes SPC and CC10 of the lung stem cell-like cells obtained in the treatment group were up-regulated; the epithelial cell-specific marker gene E-cad was also up-regulated. The expressions of fibroblast-related genes Vimentin, αSMA and Timp were down-regulated. It is indicated that the fibroblasts induced by the small molecule composition have undergone MET transformation and are reprogrammed into lung stem cells, and the obtained lung stem cell-like cells belong to alveolar stem cells, namely chemically induced transformed alveolar stem cells (ciBASCs).

Figure 5. Immunostaining for specific markers (SPC, CC10) and nuclear staining (DAPI) of alveolar stem cells (BASCs) in reprogrammed and transformed lung stem cell-like cells. Figure 5 shows that the lung stem cell-like cells obtained by induction and transformation were positive for immunostaining of SPC and CC10, the specific markers of BASCs. The results further indicate that the pulmonary stem cells obtained by the reprogramming and transformation of fibroblasts induced by the small molecule composition belong to alveolar stem cells, namely chemically induced and transformed alveolar stem cells (ciBASCs).

Figure 6. The reprogrammed and transformed lung stem cell-like cells were subjected to immunostaining and nuclear staining (DAPI) for the specific markers of lung basal cells or lung airway stem cells, p63 and Krt5. Figure 5 shows that the lung stem cell-like cells obtained by induction and transformation were positive for conventional immunostaining of lung basal cells (BCs) or lung airway stem cells (DASCs) specific markers p63 and Krt5. It is indicated that the lung stem cell-like cells obtained by the reprogramming and transformation of fibroblasts induced by the small molecule composition belong to lung basal cells or lung airway stem cells; that is, chemically induced and transformed lung basal cells (ciBCs) or chemically induced and transformed lung airway stem cells (ciDASCs) ).

Figure 7. Immunostaining of specific markers (SPC, CC10) and nuclear staining (DAPI) for alveolar stem cells (BASCs) of reprogrammed and transformed lung stem cell-like cells. Figure 7 shows that the lung stem cells obtained by inducing the transformation of human fibroblasts were positive for immunostaining of SPC and CC10, the specific markers of BASCs. It is indicated that the pulmonary stem cells obtained by the reprogramming and transformation of human fibroblasts induced by the small molecule composition belong to alveolar stem cells (BASCs), namely chemically induced and transformed alveolar stem cells (ciBASCs).

Figure 8. Immunostaining and nuclear staining (DAPI) of the reprogrammed and transformed human lung stem cell-like cells for the specific markers p63 and Krt5 of lung basal cells or lung airway stem cells. Figure 8 shows that the human lung stem cell-like cells obtained by chemically induced transformation were positive for conventional immunostaining of lung basal cells (BSCs) or lung airway stem cells (DASCs) specific markers p63 and Krt5. It is fully demonstrated that the lung stem cell-like cells obtained by the reprogramming and transformation of human fibroblasts induced by the small molecule composition belong to lung basal cells or lung airway stem cells; that is, chemically induced and transformed lung basal cells (ciBCs) or lung airway stem cells (ciDASCs) ).

Figure 9. The effect of chemically induced in situ fibroblast reprogramming into lung stem cells by aerosol inhalation reagent 1 promotes repair of lung injury and improves and relieves lung fibrosis. Figure 9, the immunohistochemical staining results of the treatment group (Treat) and the control group (Control) showed that the lung tissue of the control group was consolidated without normal alveolar structure; the activated lung fibroblast marker αSMA (brown staining) was in the control group. The expression of αSMA was high in the lung tissue of the mice in the small molecule treatment group (Treat), while the expression of αSMA was not significantly reduced in the lung tissue of the mice in the small molecule treatment group (Treat); the lung tissue and alveolar structure basically returned to normal. It is indicated that the reprogramming effect of the reprogramming aerosol inhalation agent has the effect of promoting the repair of lung injury and improving and relieving pulmonary fibrosis.

Figure 10. The effect of chemically induced in situ fibroblast reprogramming into lung stem cells by aerosol inhalation reagent 2 to promote the repair of lung injury and improve and alleviate lung fibrosis. Figure 10 shows that the immunohistochemical staining results of the treatment group (Treat) and the control group (Control) showed that the lung tissue of the control group was consolidated without normal alveolar structure; the activated lung fibroblast marker αSMA (brown staining) was in the control group. The expression of αSMA was high in the lung tissue of the mice in the small molecule treatment group; αSMA was not expressed in the lung tissue of the small molecule treatment group (Treat), or the expression was significantly reduced; the lung tissue and alveolar structure basically returned to normal. It is indicated that the reprogramming effect of the reprogramming aerosol inhalation agent has the effect of promoting the repair of lung injury and improving and relieving pulmonary fibrosis.

Figure 11. Reprogramming transformed lung stem cell transplantation to treat lung injury and improve and alleviate lung fibrosis test. Figure 11 shows that the immunohistochemical staining results of the treatment group (Treat) transplanted with transformed lung stem cells and the control group (Control) transplanted with fibroblasts showed that the lung tissue of the mice in the control group was consolidated without normal alveolar structure; activated lung fibers The cell marker αSMA (brown staining) was highly expressed in the lung tissue of the mice in the control group; while the lung tissue of the mice in the transformed lung stem cell transplantation group did not express αSMA, or the expression was significantly reduced; the lung tissue and alveolar structure basically returned to normal. It is shown that the chemically induced reprogramming and transformed lung stem cells have the effect of transplanting treatment of lung injury and improving and relieving pulmonary fibrosis. It is shown that the cells transformed by the reprogramming of fibroblasts induced by the combination of small molecules are lung stem cells, which have the effect of treating lung injury and improving and relieving pulmonary fibrosis, while fibroblasts do not have such functional effects.

Fig. 12. Comparison of cell transformation (morphological comparison) independently induced by each single small molecule component of chemical small molecule composition 6 (GSK3β inhibitor, G9aHMT inhibitor, retinoid, TGFβ inhibitor). According to the figure, the control group MF on the left side of the figure is compared with the small molecule treatment groups on the right side of the figure. When each single small molecule component in the small molecule composition exists independently, there is no chemically induced fibroblast reprogramming into lung stem cells. The functional effect of fibroblasts was basically unchanged before and after its induction treatment, and the morphology of cells in the treatment group was still similar to fibroblasts, and there was no similarity with the morphology of lung stem cells.

Detailed ways

After in-depth research, the inventors have revealed a chemically induced fibroblast reprogramming into lung stem cells with only chemical small molecule compositions (GSK3β inhibitor, G9aHMT inhibitor, retinoic acid compounds) as the core inducing factor components applications and compositions thereof. Using the chemical small molecule inducing composition of the present invention, no exogenous gene/transcription factor/MicroRNA (miRNA) gene, and its RNA, protein, polypeptide, and various exogenous cells are introduced or used in the reprogramming process. factor or growth factor. The chemical small molecule composition can be mainly used to induce the reprogramming of fibroblasts into lung stem cells to prepare transformed lung stem cells (including BASCs, AT2Cs, BCs, DASCs with basically the same morphology), and to provide a source of lung stem cells for clinical treatment or scientific research ; It can also be used as a small molecule pharmaceutical composition, adding a pharmaceutically acceptable carrier or excipient to prepare the reprogramming effect of chemically inducing in situ fibroblast reprogramming into lung stem cells, promoting the repair of lung injury and improving and small molecule pharmaceutical compositions, prodrugs, drugs or preparations for relieving pulmonary fibrosis; or adding cell basal medium; or carriers or excipients such as aqueous or organic solvents, physiological saline, buffers, etc., to prepare induced fibrogenesis Reprogramming medium or reagent for reprogramming cells into lung stem cells; or based on the induced and transformed lung stem cells, to prepare lung stem cell drugs or cell preparations for clinical transplantation; or based on the biological activity extracted from lung stem cells obtained by its induced transformation Bioactive drugs or preparations prepared from active ingredients such as substances, exosomes, and biologically active vesicles.

Basic mechanism

The reprogramming small molecule composition, reprogramming technology and method of the present invention belong to chemically induced lung stem cell reprogramming; only chemically induced fibroblasts are reprogrammed into lung stem cells (adult or tissue stem cells) using a combination of chemical small molecules.

The present invention is only through the small molecule components of each chemical small molecule composition: GSK3β inhibitor, G9aHMT inhibitor, and retinoic acid compounds, by each exerting its unique induction and regulation of specific signaling pathways or epigenetic functions, so as to jointly play a role Coordination; targeted induction and inhibition of GSK3β signaling pathway, G9aHMT epigenetic enzyme in fibroblasts, and regulation of retinoic acid (RA) signaling pathway, so that the above-mentioned related signaling pathways and epigenetic changes in fibroblasts are coordinated; Small molecule compositions generate new overall functional effects: induction and regulation of fibroblasts to form new signaling pathways and epigenetic changes, enabling fibroblasts to undergo stem cell reprogramming, transforming their gene expression profiles, and inducing fibroblasts to reprogram into Lung tissue stem cells. The chemical small molecule composition induces the overall function of reprogramming fibroblasts into lung stem cells, which is a function that any single small molecule in the composition does not have when it exists independently.

Chemical small molecule regulation characteristics: chemical small molecules used in the field of stem cell research are generally classified and named according to their common functions of targeting, inducing and regulating specific cell signaling pathways or epigenetic enzymes (the classification and naming work is organized by chemical scientists. It is usually done in collaboration with biological scientists), usually its class name is the specific function of targeting and inducing regulation of specific signaling pathways or epigenetic modification enzymes; for example, all small molecules in the GSK3β inhibitor class have the ability to target the inhibition of GSK3β signaling pathway. common functional characteristics. However, differences in chemical structure, physical or chemical properties between small molecules in the same class do not affect the performance of their common unique functions; among small molecule inhibitors in the same class, there are effective doses, activity levels, and effects. There are differences in degree, but there is no essential difference in the specific functions that induce and regulate specific signaling pathways or epigenetic enzyme activities. The mechanism of chemically inducing fibroblasts to reprogram into lung stem cells of the present invention utilizes the unique regulation of specific cell signaling pathways or epigenetic functions of each small molecule in the small molecule combination. Therefore, based on the particularity of chemical small molecules, the chemical small molecules in the same category have basically the same functions as individual components; as the participating components of the composition, their functions in the organic whole of the composition are also basically the same. The same, but there is a difference in the degree of efficacy, but there is no qualitative difference. This is common knowledge well known to those skilled in the art.

Therefore, in the specific embodiments of the present specification, for each type of small molecule, only 2-3 representative chemical small molecules are exemplified to form a representative small molecule composition, and a representative experiment is performed. It is not necessary to exhaustively enumerate chemical small molecules within each class, and it is not possible to exhaustively enumerate combinations of small molecules in the limited space of this specification, and it is common practice and common sense to provide representative experimental evidence.

Therefore, GSK3β inhibitors, G9aHMT inhibitors, retinoic acid compounds, and TGFβ inhibitors, respectively, contain targeted induction and regulation of the same specific cell signaling pathway or the same epigenetic modification enzyme, as well as their functional activities and effects. The different combinations formed by many small molecule compounds within the same class with consistent effects can be expected to induce the reprogramming of fibroblasts into lung stem cells to varying degrees. Therefore, small molecule compounds in the same class that induce and regulate the same signaling pathway or the same epigenetic enzyme target, and have the same functional effect, and the composition can induce and regulate the reprogramming of fibroblasts into Small molecule combinations of lung stem cells all fall within the protection scope of the present invention.

On the other hand, fibroblasts, also known as fibroblasts, are the main cellular components of loose connective tissue and are differentiated from mesenchymal cells in the embryonic stage. Fibroblasts can be divided into fibroblasts and fibroblasts according to different functional activity states; fibroblasts have vigorous functional activities, cytoplasm is basophilic, and have obvious protein synthesis and secretion activities; They are called fibroblasts; under certain conditions, the two can be transformed into each other. Fibroblasts come in different types and are found in various tissues or organs in the body; they have different names and properties in different tissues or organs, including: skin fibroblasts, liver fibroblasts (hepatic stellate cells), lung Fibroblasts, pancreatic fibroblasts, and fibroblasts in other tissues or organs.

The fibroblasts induced by the method or the small molecule composition of the present invention, that is, the aforementioned fibroblasts include: human fibroblasts or mammalian fibroblasts; preferably, including but not limited to human or mammalian fibroblasts : Skin fibroblasts, liver fibroblasts (hepatic stellate cells), lung fibroblasts, kidney fibroblasts, pancreatic fibroblasts, and fibroblasts from other tissues or organs of humans or mammals. More preferably skin fibroblasts.

In addition, according to the target gene of each chemical small molecule inhibitor in the small molecule composition, the siRNA/shRNA with the same induction inhibition target is designed and constructed, and the corresponding small molecule inhibitor has the same target induction inhibition Function. For example, the interfering RNA with the same inhibitory target gene as the chemical small molecule GSK3β inhibitor in the composition: siGSK3β/shGSK3β, also has the function of inducing and inhibiting GSK3β; and the chemical small molecule G9aHMT inhibitor in the composition has the same function of inducing and inhibiting GSK3β. Interfering RNA of the same inhibitory target gene: siG9a/shG9a, also has the function of inducing and inhibiting G9aHMT;

Therefore, a mixed induction composition consisting of siGSK3β, siG9a, and retinoic acid compounds (or: shGSK3β, shG9a, and retinoic acid compounds) has the same effect as the chemical small molecule composition in inducing the reprogramming of fibroblasts into lung stem cells Function; it can also be developed and prepared as a mixed induction composition of interfering RNA and small molecules, or its pharmaceutical composition/drug/prodrug/drug preparation. This is a mechanism well known to those skilled in the art.

Pharmaceutical composition and its application

Based on extensive research, the inventors first proposed a chemical small molecule composition for chemically inducing fibroblasts to reprogram into lung stem cells. The composition is only composed of small chemical molecules: GSK3β inhibitor, G9aHMT inhibitor, and retinoic acid compounds as active components; and on the basis of the small molecule composition, chemical small molecule TGFβ inhibitor can also be added together.

The above-mentioned small molecule composition is a pharmaceutical composition, which can be used to chemically induce the reprogramming effect of in situ fibroblast reprogramming into lung stem cells in vivo, promote the repair of lung injury and improve and alleviate pulmonary fibrosis. , prodrugs, drugs or preparations. Therefore, drug carriers or excipients can be added to develop and prepare corresponding innovative drugs, prodrugs, and pharmaceutical preparations, which should also be included in the present invention.

It should be understood that among chemical small molecules within the same class, only the effective dose, activity size, and effect are different, but there is no essential difference in the function of inducing, inhibiting or regulating specific signaling pathways or epigenetic enzymes. Therefore, in addition to the specific and representative chemical small molecule GSK3β inhibitors (CHIR-99021, LiCl, BIO, LY2090314) listed in the examples of the present invention, and other GSK3β small molecule inhibitors that target and inhibit the GSK3β cell signaling pathway, The same technical effect can also be achieved and should also be included in the present invention.

In addition to the specific, representative G9aHMT inhibitors (BIX01294, UNC0638 or UNC0642) listed in the examples of the present invention, other chemical small-molecule G9aHMT inhibitors that target, induce and inhibit G9aHMT can also achieve the same technical effect. should be included in the present invention.

Similarly, in addition to the specific and representative retinoic acid compounds (RA, 13-cis retinoic acid or 9-cis retinoic acid) listed in the examples of the present invention, other targeted induction regulation and activation of other RA signaling pathways Chemical small-molecule retinoic acid compounds, which can also achieve the same technical effect, should also be included in the present invention.

Similarly, in addition to the specific and representative TGFβ inhibitors (SB431542, A83-01 or RepSox) listed in the examples of the present invention, other chemical small molecule TGFβ inhibitors targeting the inhibition of TGFβ signaling pathway can also achieve the same The technical effect should also be included in the present invention.

As used herein, the terms "comprising" or "including" include "comprising," "consisting essentially of," and "consisting of."

As used herein, the term "consisting essentially of" means that the composition may contain, in addition to essential ingredients or essential components, minor amounts of minor ingredients and/or impurities that do not affect the active ingredient. For example, sweeteners may be included to improve taste, antioxidants to prevent oxidation, and other pharmaceutical additives, carriers, and excipients commonly used in the art. In the present invention, "comprising GSK3β inhibitor, G9aHMT inhibitor, retinoic acid compound" or "consisting of GSK3β inhibitor, G9aHMT inhibitor, retinoic acid compound" includes "substantially consisting of GSK3β inhibitor, G9aHMT inhibitor" “It is mainly composed of GSK3β inhibitors, G9aHMT inhibitors, and retinoic acid compounds as active ingredients”, “with GSK3β inhibitors, G9aHMT inhibitors, and retinoic acid compounds as the only active ingredients”, “ Basically use GSK3β inhibitors, G9aHMT inhibitors, and retinoids as active ingredients".

As used herein, the term "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and animals without undue adverse side effects (such as toxicity, irritation and allergy), ie, a substance with a reasonable benefit to risk ratio; Such as pharmaceutical carriers or excipients commonly used in the art.

As used herein, the term "effective amount" refers to an amount that produces function or activity in humans or animals and is acceptable to humans or animals.

As used herein, the term "pharmaceutically acceptable carrier or excipient", wherein a carrier refers to a system capable of altering the manner in which a drug enters the human body and its distribution in the body, controlling the rate of drug release, and delivering the drug to targeted organs; The pharmaceutical carrier itself is not an essential active ingredient and is not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art and include, but are not limited to: water, saline, phosphate buffered saline and other aqueous solvents; DMSO (dimethyl sulfoxide), glycerol and ethanol and other organic solvents; microspheres, Liposomes, microemulsions, polymer surfactants; colloidal drug loading systems, new polymer drug loading systems, new drug carriers, and other pharmaceutical carriers; wherein excipients refer to substances other than the main drug in pharmaceutical preparations. Additives, also known as excipients. Such as binders, fillers, disintegrants, lubricants in tablets; wine, vinegar, concoction, etc. in traditional Chinese medicine pills; matrix parts in semi-solid preparations ointments and creams; preservatives in liquid preparations, Antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. can all be called excipients.

The general requirements for excipients are stable in nature, no incompatibility with the main drug, no side effects, no influence on the curative effect, not easy to deform, crack, mildew, moth-eaten at room temperature, harmless to the human body, no physiological effect, no It has chemical or physical effects with the main drug, and does not affect the content determination of the main drug. A full discussion of pharmaceutically acceptable carriers or excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

As used herein, the pharmaceutical dosage form in the term "pharmaceutical dosage form from which the composition can be prepared" refers to: a pharmaceutical application form prepared to meet the needs of treatment or prevention, called a pharmaceutical dosage form;

As used herein, "parts by weight" or "parts by weight" are used interchangeably, and the parts by weight can be any fixed weight expressed in micrograms, milligrams, grams or kilograms (eg, 1ug, 1mg, 1g, 2g, 5g, or kg, etc.). For example, a composition consisting of 1 part by weight of component a and 9 parts by weight of component b may be 1 gram of component a + 9 grams of component b, or 10 grams of component a + 90 grams of component b etc. composition. In the composition, the percentage content of a certain component=(weight parts of this component/sum of weight parts of all components)×100%. Therefore, in the composition composed of 1 part by weight of component a and 9 parts by weight of component b, the content of component a is 10%, and the content of component b is 90%.

In addition, in the solution state, the above-mentioned "parts by weight" can also be converted into "number of moles"; "parts by weight ratio" can also be converted into "molar concentration ratio". The weight unit of the weight part ratio can be: any weight unit such as kilogram (kg), milligram (mg), microgram (ug); the molar unit of the molar concentration ratio can be: mole (M), millimolar (mM) , micromolar (μM) and other units of molarity.

In the chemical small molecule composition: GSK3β inhibitor, G9aHMT inhibitor, retinoic acid compound, TGFβ inhibitor, in proportion by weight: (5-80): (0.1-50): (0.05-20): (0.1 -50); preferably (10-70):(0.5-40):(0.15-15):(0.5-40); or the molar concentration ratio in solution state is (0.1-20):(0.01-20 ):(0.1-20):(0.01-20); preferably (0.5-15):(0.05-10):(0.5-10):(0.05-10) exists.

For example, in the composition, the components and weight parts included are as shown in Table 1 or molar concentration as shown in Table 2 (solution state).

Table 1. Weight ratio (weight unit: kg, mg, μg...)

Table 2. Molar concentration ratio (molar unit: M, mM, μM...)

The formulation ranges in Tables 1 and 2 can be used as a guideline. It will be understood, however, that when used in the development and manufacture of pharmaceutical compositions, the effective dosage of the composition employed may vary with the mode of administration and the physical condition of the patient with the disease to be treated, or the severity of the disease. Moreover, when used in vivo, "weight/kg (body weight)" is usually used as the dosage unit; when the small molecule composition is applied to large animals and patients with liver disease, the dosage of small animals is calculated by the corresponding professional conversion formula. The effective dose for large animals or humans (including dose conversion in solid or solution state).

It should also be understood that due to the large number of small molecule members of each type of chemical small molecule, and the large differences in the effective dose and activity of each small molecule member, although the inventors have long studied chemical small molecule compositions to induce and regulate stem cell directional differentiation. and cell reprogramming, a large number of experiments have been done, but the examples in the description cannot be all exemplified. , typically demonstrated by several small molecule inhibitors such as CHIR-99021, LiCl, BIO, etc.) used concentration in the experiment; therefore, the reasonable concentration range outlined in the claims of the present invention naturally includes, but is not limited to, the implementation of For each category in the example, the concentration range used in a specific representative small molecule experiment; this basic and simple reason can be understood by those skilled in the art.

For example, among the small-molecule compounds that are GSK3β inhibitors, the molecular weights vary greatly. In the present invention, when "parts by weight" and "molar concentration" are used to define the concentration/amount, the molar concentration and dosage range of compounds with different molecular weights may be very different. Therefore, in a preferred embodiment of the present invention, the molar concentration is calculated according to the molecular weight of the GSK3β inhibitor, and when the molecular weight is large, the concentration unit of μM is used; when the individual molecular weight is small, the concentration unit of mM is used.

As used in the present invention, the GSK3β inhibitor refers to the general term of small molecule inhibitors that can target and inhibit the cellular GSK3β signaling pathway, including but not limited to: CHIR-99021, BIO, LiCl, IM-12, TWS119, Ly2090314, 1-Azakenpaullone, CHIR-98014, Tideglusib, AR-A014418, SB216763, AZD1080, other GSK3β small molecule inhibitors or small molecule compounds that induce the same target to inhibit GSK3β signaling pathway; preferably GSK3β inhibitors CHIR-99021, LiCl , BIO, LY2090314.

As a preferred mode of the present invention, the GSK3β inhibitor is CHIR-99021, whose alias is CT99021; its molecular structure is shown in the following formula (I):

The chemical small molecule G9aHMT inhibitor refers to the general term of chemical small molecule inhibitors that can target and inhibit G9aHMT, including but not limited to: BIX01294, UNC0638, A-366, UNC0631, BRD4770, UNC0224, UNC0646, UNC0642, UNC0321 , BRD4770, HKMTI-1-247, HKMTI-1-248, CPUY074020, DCG066, other G9aHMT small molecule inhibitors or small molecule compounds that induce the same target to inhibit G9aHMT; preferably G9aHMT inhibitors BIX01294, UNC0638 or UNC0642.

As a preferred mode of the present invention, the G9aHMT inhibitor is BIX01294 (or BIX-01294); its molecular structure is shown in the following formula (II):

The chemical small molecule TGFβ inhibitor refers to the general term of chemical small molecule inhibitors that can inhibit the TGFβ signaling pathway in cells, including but not limited to: SB431542, A83-01, SB525334, LY2109761, RepSox, SD-208, GW788388 , SB505124, EW-7197, Galunisertib, other small molecule TGFβ inhibitors or small molecule compounds that induce the same target to inhibit TGFβ signaling pathway; preferably TGFβ inhibitors SB431542, A83-01 or RepSox.

As a preferred mode of the present invention, the chemical small molecule TGFβ inhibitor is small molecule SB431542 (or called SB-431542); its molecular structure is shown in the following formula (III):

As used in the present invention, the retinoids compound can target and specifically bind to retinoic acid response elements (RARE), thereby regulating the RA signaling pathway and the transcriptional activity of specific nuclear genes, Differentiation-inducing agents that promote cell differentiation; including but not limited to: retinoic acid (RA), alias: all trans retinoic acid (ATRA); 13-cis retinoic acid (13-cis retinoic acid) , 13-CRA), 9-cis-retinoic acid (9-CRA), UAB7, UAB8, TTNPB, 3-methyl-TTN PB, AM80, AM580, CD437, Targretin, LGD1069, induced Other retinoic acid compounds with the same target regulating RA signaling pathway; or their equivalent pharmaceutical products, analogs and/or their salts, hydrates or precursors, or their combinations; preferably retinoic acid (Retinoic acid) , RA), 13-cis retinoic acid or 9-cis retinoic acid.

Retinoids have the functions of regulating cell proliferation, differentiation and physiological apoptosis (apo ptosi s), because they can activate the corresponding retinoie acid receptor (RAR) and retinoic acid X nuclear receptors. Retinoid x receptor (RXR) protein, by specifically binding to retinoic acid response elements (RARE), induces and regulates the transcriptional activity of RA signaling pathway and specific nuclear genes, and promotes cell differentiation. Many of the retinoic acid compounds and their isomeric derivatives have the same or similar functions, so they have become an important class of differentiation-inducing agents.

As a preferred mode of the present invention, the retinoic acid (RA), alias: all trans retinoic acid (ATRA), retinoic acid, retinoic acid, retinoic acid, retinoic acid, All-trans retinoic acid and retinoic acid, the molecular structure of which is shown in the following formula (V):

The present invention also includes compounds, pharmaceutical preparations, analogs and/or their salts, hydrates or precursors equivalent to the above-mentioned small molecule compounds I, II, III, IV; and also includes their naturally occurring and artificially synthesized compounds.

The analogs of the small molecule compounds include, but are not limited to: isomers and racemates of the small molecule compounds. Compounds have one or more asymmetric centers. Accordingly, these compounds may exist as racemic mixtures, individual enantiomers, individual diastereomers, mixtures of diastereomers, cis or trans isomers.

Described "salt" includes but is not limited to: (1) salts formed with the following inorganic acids: such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, etc.; (2) salts formed with the following organic acids, such as acetic acid, oxalic acid, succinic acid acid, tartaric acid, methanesulfonic acid, maleic acid, or arginine, etc. Other salts include salts with alkali or alkaline earth metals such as sodium, potassium, calcium or magnesium, and the like.

The "precursor of a compound" refers to a compound that can be converted into any one of the above compounds in a culture medium, or in animals, or in humans, after being administered or treated by an appropriate method, or the above-mentioned compounds. A salt or solution of a compound of any compound.

The chemical small molecule composition of the present invention is a pharmaceutical composition for inducing the reprogramming of fibroblasts into lung stem cells, which can be added with drug carriers and excipients, and is developed and prepared for in vivo chemical induction of in situ fibroblast reprogramming into lung stem cells. The effect of lung stem cells, promoting the repair of lung damage and improving and relieving pulmonary fibrosis small molecule pharmaceutical composition/prodrug/drug/preparation; therefore also include: pharmaceutically acceptable carrier or excipient; preferably, The carrier or excipient includes one or more selected from the group consisting of water, saline, phosphate buffer or other aqueous solvents; DMSO, glycerol and ethanol or other organic solvents; microspheres, liposomes, microemulsions Or polymer surfactants; colloidal drug delivery systems or polymer drug delivery systems; preservatives, antioxidants, flavoring agents, fragrances, cosolvents, emulsifiers, pH buffer substances, adhesives, fillers, lubricants or other pharmaceutical excipients.

The dosage form of the chemical small molecule composition of the present invention is not particularly limited, and can be any dosage form suitable for human or mammals; the dosage forms that can be prepared include: powder, powder, tablet, pill, capsule, sustained release injections, infusions, suspensions and other liquid dosage forms; and gas dosage forms, semi-solid dosage forms and other dosage forms. Preferably, the dosage form may be, but is not limited to, solid dosage forms such as powder, granule, capsule, sustained release agent, tablet, or liquid dosage form such as injection, infusion, solution, and suspension.

The preparation method of the small molecule composition of the present invention is determined according to the dosage form to be prepared and the route of administration. Those skilled in the art can use the conventional preparation method of the pharmaceutical composition after referring to the combination and ratio provided by the present invention. The chemical small molecule composition of the present invention can be prepared.

It should be understood that small chemical molecules are classified and named according to their common functions of inducing and regulating specific cell signaling pathways or epigenetic enzymes. Among small molecule inhibitors in the same category, only the effective dose and activity There are differences in size and degree of effect, but there is no essential difference in the specific functions of inducing and regulating specific signaling pathways or epigenetic enzyme activities. Therefore, based on the above-mentioned characteristics of the chemical small molecule method of the present invention and the limitation of the length of this specification, in the examples of the present specification, for each type of small molecule, only 2-3 representative chemical small molecules are exemplified, A representative small molecule composition was composed and a representative experiment was performed. However, those skilled in the art can deduce or expect any other small molecule combination form of the present invention to have the same effect according to the disclosure of the present specification, as well as their well-known practices and common sense.

The inventors have confirmed for the first time that the small molecule composition of the present invention is a pharmaceutical composition, which can be used to prepare the reprogramming effect of in situ induction of fibroblasts into lung stem cells in vivo, promote the repair of lung injury and improve and alleviate lung fibrosis pharmaceutical compositions, prodrugs, and drugs.

It should be understood that the small molecule composition has been further developed and perfected, and it should have similar effects on the treatment of fibrotic diseases in other tissues and organs; drug carriers or excipients can be added to develop and prepare corresponding innovative drugs or prodrugs. , so it should also be included in the present invention.

When used to promote repair of lung damage and improve and alleviate pulmonary fibrosis, as well as fibrotic disease of other organs or tissues, the effective dose of the composition used may vary with the mode of administration and the type of fibrotic disease to be treated and the severity of the disease . The specific situation is determined according to the individual situation of the subject, which is within the judgment of a skilled physician or pharmacist.

In the present invention, the fibroblasts include human fibroblasts or mammalian fibroblasts; preferably, including but not limited to human or mammalian: skin fibroblasts, liver fibroblasts (hepatic stellate cells) ), lung fibroblasts, kidney fibroblasts, pancreatic fibroblasts, and fibroblasts of other tissues or organs of humans or mammals; more preferably skin fibroblasts.

The lung stem cells obtained by reprogramming any of the induced fibroblasts include: alveolar type 2 cells (AT2Cs), bronchioalveolar stem cells (BASCs), basal cells (BCs), distant Terminal airway stem cells (DASCs), or mixtures thereof (hybrid cells).

It should be understood that, based on the induction and transformation of lung stem cells by any of the small molecule compositions, a lung stem cell drug or preparation for clinical transplantation to treat or repair lung injury and improve and alleviate pulmonary fibrosis is prepared; or based on its chemically induced reprogramming transformation Bioactive drugs or preparations prepared from the biologically active substances, exosomes, biologically active vesicles and other active ingredients extracted from the obtained lung stem cells. should also be included in the present invention.

It should be understood that the mixed induction composition consisting of siGSK3β, siG9a, retinoids (or: shGSK3β, shG9a, retinoids) has the same effect as the chemical small molecule composition in inducing fibroblast reprogramming into the lung Stem cell function; it can also be developed and prepared as a mixed induction composition of interfering RNA and small molecules, or its pharmaceutical composition/drug/prodrug/drug preparation. This is the knowledge that can be understood and well known to those skilled in the art. The same should be included in the present invention.

Since the siGSK3β/shGSK3β, siG9a/shG9a belong to 20-25 base pairs, a vector or a vector delivery system is required for introduction into cells. The carrier or carrier delivery system includes but is not limited to: viral carrier delivery system, non-viral carrier delivery system, such as: biochemical modification delivery system, microinjection delivery system, non-viral nanocarrier delivery system.

Preparation of reprogramming medium and reprogramming aerosol inhalation reagent

The present invention also provides a small molecule composition that can be added with carriers/excipients/basal nutrient solution such as organic solvent, or physiological saline, or buffer, or cell basal medium to prepare chemically induced fibroblasts to reprogram into lung stem cells Reprogramming medium or reagents; reprogramming medium or reprogramming inhalation reagent for inducing fibroblasts to reprogram into lung stem cells, including BASCs, AT2Cs, BCs, DASCs, etc.

According to the final concentration formula of the chemical small molecule composition provided by the present invention, the small molecule composition with a specific final concentration is selected for preparation. As a preferred mode of the present invention, the different components in the specific small molecule composition are respectively dissolved in DMSO (dimethyl sulfoxide) or other organic solvents or aqueous solvents according to the different properties and different solubility of the solutes. Then, according to the final concentration requirements of the specific small molecule composition, each small molecule organic solution concentrate reagent was diluted and added into the cell base containing 10% calf serum. In the medium (DMEM), the reprogramming medium can be obtained. The reprogramming medium or reprogramming process does not use or add any exogenous genes/transcription factors/MicroRNA genes, as well as their RNAs, proteins and polypeptides; nor add or use any exogenous cytokines or growth factors. . Wherein, the percentage content of each component of the medium can also fluctuate by 50%; preferably by 30%; more preferably by 20%, such as 10%, 5%. Unless otherwise stated, percentages are in v/v.

As a preferred mode of the present invention, the cell basal medium includes but is not limited to: DMEM/F12, MEM, DMEM, F12, IMDM, RPMI1640, Neuronal basal or Fischers, etc., all of which are commercially available commodities.

As a preferred mode of the present invention, the aforementioned reprogramming medium can also be prepared using a serum-free medium if there are special requirements. The "serum-free medium" refers to a cell culture medium that does not contain serum but contains various nutrients (such as growth factors, tissue extracts, etc.) that support cell proliferation and biological responses. A cell culture medium consisting of adding various cytokines or growth factors other than serum to the cell basal medium.

As a preferred mode of the present invention, the serum-free medium containing various cytokines or growth factors includes but is not limited to: ITS, N2, B27, etc., all of which can be self-prepared or commercially available products.

It should be understood that those skilled in the art are familiar with the preparation or purchase route of the described cell basal medium or serum-free medium, therefore, the cell basal medium or serum-free medium is not limited to those exemplified in the present invention.

1. As a preferred mode of the present invention, a method for preparing or preparing a described "reprogramming medium" is provided:

(1) Put ① GSK3β inhibitor (such as CHIR99021): the final concentration is 0.1-20 μM/mM; the preferred amount is: 0.5-15 μM/mM; (individual GSK3β inhibitors with smaller molecular weight such as LCI use the concentration in mM); ② G9aHMT The final concentration of inhibitor (such as BIX01294) is 0.01-20 μM; the preferred amount is: 0.05-10 μM; ③ Retinoic acid compound: the final concentration is 0.1-20 μM; the preferred amount is 0.5-10 μM. Or add ④TGFβ inhibitor: the final concentration is 0.01-20 μM, and the preferred amount is: 0.05-10 μM. Dissolve in DMSO (dimethyl sulfoxide) or other organic solvent or aqueous solvent according to the above concentrations to prepare a concentrated reagent (ranging from 1:50 to 1:10,000). Then the concentrate reagent of each component of the small molecule composition for chemically inducing fibroblasts to be reprogrammed into lung stem cells of the present invention can be obtained;

(2) The above-mentioned small molecules are respectively processed through: dissolving and preparing the concentrate reagent → diluting into the cell basal medium → mixing, and then the "reprogramming medium" for chemically induced fibroblasts to be reprogrammed into lung stem cells can be prepared and obtained.

2. As a preferred mode of the present invention, there is also provided a preparation method of a reprogramming aerosol inhalation reagent for chemically inducing the reprogramming of in situ fibroblasts into lung stem cells in vivo:

The above ① the weight ratio of the combination of small molecules is formulated into a concentrated solution of 100 mg/ml according to the weight ratio of kilograms: → Diluted into 0.9% NaCl physiological saline (50 μl/mouse) and mixed, the chemical can be prepared. A reprogramming nebulized agent for inducing in situ reprogramming of fibroblasts into lung stem cells in vivo.

As a preferred mode of the present invention, the composition of each small molecule in any of the aforementioned compositions is calculated according to kilogram body weight, and the corresponding dosage is calculated, and dissolved in a physiological saline (0.9% NaCl) solution to obtain a reprogramming mist for experimental animals. Chemical inhalation reagent.

3. As a preferred mode of the present invention, the present invention also discloses a chemical small molecule composition for chemically inducing fibroblasts to reprogram into lung stem cells in vitro to prepare lung stem cells (BASCs, AT2Cs, BCs, DASCs) method, The method steps include:

(1) Concentrate reagent preparation: according to the composition described in any of the present invention, according to the aforementioned reprogramming medium preparation method, the components of the small molecule composition are respectively prepared as concentrate reagents;

(2) Reprogramming medium acquisition: Dilute the concentrate reagent in step (1) into cell basal medium (such as DMEM) containing 5-20% calf serum (so that the concentration of each component conforms to the previous The concentration defined in the described composition) to obtain a reprogramming medium;

(3) Induction of fibroblast reprogramming into lung stem cell culture: fibroblasts were placed in cell basal medium (DMEM) containing 5-20% calf serum; suspended and plated, and the original medium was discarded after the cells adhered , change the reprogramming medium of step (2), culture at 37°C, change the medium every 2-4 days; passage once every 3-15 days.

(4) Subculture: discard the original culture medium, wash once with PBS, add cell digestion solution to digest the cells, 37°C for 1-5 minutes, stop cell digestion, centrifuge, discard the supernatant, and resuspend the cell pellet at a ratio of 1:1 -1:3 passaging plate. Culture according to steps (2) and (3) of the experimental procedure, and change the medium every 2-4 days. Digestive solutions used include trypsin, EDTA, Acutase, TrypleE, etc. Passage once every 3-15 days.

(5) Harvesting reprogrammed lung stem cells: After the above experimental steps (3) and (4) reprogramming culture and subculture for 2-4 weeks, transformed lung stem cells (including ciBASCs, ciAT2Cs, ciBASCs, ciAT2Cs, ciBCs, ciDASCs); the lung stem cells can be used for related scientific research experiments.

It should be noted that the obtained transformed lung stem cells include basically the same or similar morphology: BASCs, AT2Cs, BCs, DASCs or mixtures thereof. Which one of the lung tissue stem cells is dominant has a certain correlation with the type and proportion of small molecule components, as well as changes in culture time and culture environment. In any case, the examples of the present invention have undoubtedly confirmed that all lung stem cells are obtained by induced reprogramming.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that in these examples, only 2-3 representative small molecules within each category of chemical small molecules are selected to form representative small molecule compositions, and representative experiments are carried out according to the reprogramming experimental methods and steps disclosed earlier in the specification, It is used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples are usually based on conventional conditions, such as [America] J.S. Bonifessnon et al; Zhang Jingbo, Fang Jin, Wang Haijie et al. Protocols in Cell Biology] (2007); or as suggested by the manufacturer.

Example 1. Small molecule composition for inducing fibroblast reprogramming into lung stem cells (abbreviation: reprogramming small molecule composition), and preparation or preparation of reprogramming medium and reprogramming inhalation reagent

Design the following small molecule composition, add the cell basal medium to prepare a reprogramming medium, and formulate at a molar concentration or a concentration by weight (the following reprogramming medium is at a molar concentration; the reprogramming aerosol inhalation reagent is at a concentration by weight); Or the small molecule composition is dissolved in 0.9% (w/v) NaCl to prepare a reprogramming aerosol inhalation reagent, and it is administered according to mg/kg body weight:

1. The formulation of the reprogramming small molecule composition

(1) Reprogramming Small Molecule Composition 1

GSK3β inhibitor CHIR-99021: final concentration 6 μM;

G9aHMT inhibitor BIX01294: final concentration 3 μM;

Retinoic acid RA: final concentration 5 μM;

(2) Reprogramming Small Molecule Composition 2

GSK3β inhibitor BIO: final concentration 2 μM;

G9aHMT inhibitor UNC0638: final concentration 5 μM;

Retinoic acid RA: final concentration 2 μM;

(3) Reprogramming Small Molecule Composition 3

GSK3β inhibitor Ly2090314: final concentration 1 μM;

G9aHMT inhibitor UNC0642: final concentration 2.5 μM;

Retinoic acid RA: final concentration 3 μM;

(4) Reprogramming Small Molecule Composition 4

GSK3β inhibitor LiCl: final concentration 4mM;

G9aHMT inhibitor BIX01294: final concentration 2 μM;

Retinoic acid RA: final concentration 2 μM;

(5) Reprogramming Small Molecule Composition 5

GSK3β inhibitor Ly2090314: final concentration 0.5 μM;

G9aHMT inhibitor UNC0642: final concentration 5 μM;

Retinoic acid compound 9-cis-retinoic acid: final concentration 2 μM;

TGFβ inhibitor SB431542: final concentration 2 μM;

(6) Reprogramming Small Molecule Composition 6

GSK3β inhibitor CHIR99021: final concentration 3 μM;;

G9aHMT inhibitor BIX01294: final concentration 2 μM;

Retinoic acid RA: final concentration 2.5 μM;

TGFβ inhibitor RepSox: final concentration 0.8 μM;

(7) Reprogramming Small Molecule Composition 7

GSK3β inhibitor Ly2090314: final concentration 1.5 μM;

G9aHMT inhibitor BIX01294: final concentration 0.5 μM;

Retinoic acid RA: final concentration 5 μM;

(8) Reprogramming small molecule composition 8 (preparation of aerosol inhalation reagent)

GSK3β inhibitor CHIR99021: 1mg/kg;

G9aHMT inhibitor UNC0642: 0.3mg/kg;

Retinoic acid compound RA: 1mg/kg;

(9) Reprogramming small molecule composition 9 (preparation of aerosol inhalation reagent)

GSK3β inhibitor BIO: 0.5mg/kg;

G9aHMT inhibitor BIX01294: 0.5mg/kg;

Retinoic acid compound 13-cis retinoic acid: 0.5mg/kg;

TGFβ inhibitor A83-01: 0.5mg/kg;

(10) Reprogramming Small Molecule Composition 10

GSK3β inhibitor LiCl: final concentration 15mM;

G9aHMT inhibitor UNC0638: final concentration 10 μM;

Retinoic acid RA: final concentration 5 μM.

The small molecule components of each specific small molecule composition can be first dissolved in DMSO to prepare a concentrate reagent by referring to the above-mentioned steps of "Preparation or Preparation of Reprogramming Medium".

2. Preparation of reprogramming medium

The DMSO concentrate reagents of the above-mentioned reprogramming small molecule compositions 1 to 7 and 10 are prepared according to the steps of the aforementioned "reprogramming medium preparation method", and the selected cell basal medium is DMEM, which is added with 10% calf serum, Reprogramming medium 1 to 7, 10 can be obtained (that is, the components and final concentrations of reprogramming medium 1 and composition 1 are the same, the components and final concentrations of medium 2 and composition 2 are the same, ..., medium 10 The same components and final concentrations as in Composition 10).

3. Preparation of Reprogramming Nebulized Inhalation Reagent

The DMSO concentrated solution of the reprogramming small molecule compositions 8 and 9 is dissolved in 0.9% NaCl solution, and is prepared according to "the preparation method of the reprogramming nebulized inhalation reagent" (1) the proportion by weight of the small molecule combination is proportioned by kilogram body weight. , respectively prepared into 100 mg/ml concentrated solution, then diluted and mixed in 0.9% NaCl saline (50 μl saline/mouse) to become "Reprogramming Nebulized Inhalation Reagents 1 and 2 (with Small Molecule Composition 8, The components of 9 are formulated in the same kilogram body weight)".

4. Preparation of lung stem cell suspension or preparation for transplantation

Collect lung stem cells transformed by chemically induced fibroblast reprogramming; use normal saline to prepare lung stem cell suspension or preparation (0.5×10 ⁶ cells/50ul).

Example 2. Comparative experiment on the morphology of lung stem cells transformed by chemically induced fibroblasts (mice) in reprogramming medium 1

The mouse fibroblasts used in subsequent examples were derived from C57/BL6 mouse skin fibroblasts (MF).

Use reprogramming medium 1 to chemically induce mouse fibroblasts to reprogram into lung stem cells, and the experimental culture method for preparing lung stem cells refers to the previous "chemically induced fibroblasts to reprogram into lung stem cells in vitro to prepare lung stem cells (BASCs, AT2Cs)". , BCs, DASCs)": dilute the concentrate reagent into DMEM containing 10% calf serum, so that the concentration of each component conforms to the concentration defined in "Reprogramming Small Molecule Composition 1" described above ) to obtain reprogramming medium 1. Fibroblasts (MF) were added to the medium, suspended, plated, replaced with fresh reprogramming medium after cells adhered, cultured at 37°C, and the medium was changed every 3 days; passaged once every 7 days. Subculture: discard the original culture medium, wash once with PBS, add cell digestion solution to digest cells, 37 °C for 3 minutes, stop cell digestion, centrifuge, discard the supernatant, resuspend the cell pellet, and plate at a rate of 1:2; Change the medium every 3 days.

Lung stem cells were obtained after the above-mentioned culture and subculture for 3 weeks. The lung stem cells (ciAT2Cs) obtained by the reprogramming transformation were compared with the primary alveolar type II cells (pAT2Cs) isolated from mice and MF. The results are shown in Figure 1.

Figure 1 shows that the lung stem cells obtained by chemically induced reprogramming have the same or similar morphology as primary alveolar type II cells (pAT2Cs, where "p" stands for "primary"), which is completely different from the MF morphology of the induced initial cells. Therefore, the small molecule composition of the present invention successfully induces fibroblasts to reprogram the transformed lung stem cells, which belong to alveolar type II cells. The inventors abbreviated the transformed alveolar type II cells obtained by chemically induced reprogramming as "ciAT2Cs", where "ci" stands for "chemically induced transformation".

Example 3. Reprogramming medium 2 chemically induced fibroblasts (mice) to reprogram and transform lung stem cells

Experimental steps: use reprogramming medium 2 to chemically induce mouse fibroblasts to reprogram into lung stem cells, and the experimental culture method for preparing lung stem cells is the same as Example 2; the lung stem cells obtained by reprogramming and transformation are prepared according to conventional methods and detected by electron microscopy , the electron microscope pictures of the lamellar structure unique to AT2Cs were obtained, and the results are shown in Figure 2.

Figure 2 shows that the reprogrammed and transformed lung stem cells have a lamellar structure unique to alveolar type II cells (AT2Cs) (electron microscope observation results), confirming that the small molecule composition chemically induces the reprogrammed and transformed lung stem cells to be alveolar type II cells, That is ciAT2Cs.

Example 4. Reprogramming medium 3 chemically induced fibroblasts (mice) to reprogram and transform lung stem cell-like cells for immunostaining and nuclear staining of SPC, a specific marker of AT2Cs

Experimental steps: use reprogramming medium 3 to chemically induce mouse fibroblasts to reprogram into lung stem cells, and the experimental culture method for preparing lung stem cells is the same as Example 2; Specific marker SPC antibody routine immunofluorescence staining; and routine nuclear staining. The experimental results are shown in Figure 3.

Figure 3 shows that the small molecule composition induces fibroblasts (mice) to reprogram the transformed lung stem cell-like cells, and the immunofluorescence staining of SPC, a specific marker of alveolar type II cells, is positive. The experimental results show that the small molecule composition induces fibroblasts to reprogram and transform lung stem cells into alveolar type II cells (ciAT2Cs).

Combined with the chemically induced lung stem cells of the present invention, the morphology is the same as or similar to that of pAT2Cs (Example 2, Figure 1), and it has a lamellar structure unique to AT2Cs (Example 3, Figure 2) and other indicators, indicating that the chemically induced reprogramming transformation The main lung stem cells are alveolar type II cells (AT2Cs), namely ciAT2Cs.

Example 5. Gene expression comparison experiment of specific markers (SPC, CC10) of alveolar stem cells (BASCs) in lung stem cell-like cells transformed by chemical induction of mouse fibroblasts (MF)

Experimental steps: use reprogramming medium 4 to chemically induce mouse fibroblasts to reprogram into lung stem cells, and the experimental culture method and steps for preparing lung stem cells are the same as those in Example 2; The RNA of the reprogrammed and transformed lung stem cell-like cells was subjected to qRT-PCR to detect the expression levels of specific markers (SPC, CC10) of alveolar stem cells (BASCs). The experimental results are shown in Figure 4.

Figure 4 shows that the BASCs-specific marker genes SPC and CC10 of the lung stem cell-like cells obtained in the treatment group were up-regulated; the epithelial cell-specific marker gene E-cad was also up-regulated. The expressions of fibroblast-related genes Vimentin, αSMA and Timp were down-regulated. It is shown that the fibroblasts induced by the small molecule composition have been reprogrammed and transformed into lung stem cells, and the transformed lung stem cell-like cells obtained are confirmed to be alveolar stem cells (BASCs) from the level of gene expression. The inventors referred to the chemically induced transformed alveolar stem cells as "ciBASCs" for short.

Example 6. Immunostaining and nuclear staining experiments for specific markers (SPC, CC10) of alveolar stem cells (BASCs) in lung stem cell-like cells transformed by chemical induction of mouse fibroblasts (MF)

Experimental steps: use reprogramming medium 5 to chemically induce mouse fibroblasts to reprogram into lung stem cells, and the experimental culture method for preparing lung stem cells is the same as Example 2; the lung stem cell-like cells obtained by induction and transformation are used for alveolar stem cells (BASCs) The specific markers (SPC, CC10) antibody routine immunofluorescence staining, and routine immunofluorescence staining of the nucleus, the results are shown in Figure 5.

Figure 5 shows that the lung stem cells obtained by induction and transformation were positive for immunostaining of specific markers SPC and CC10 of alveolar stem cells (BASCs). It is indicated that the pulmonary stem cells obtained by chemically induced fibroblast reprogramming transformation by the small molecule composition are alveolar stem cells (BASCs), namely chemically induced reprogramming and transformed alveolar stem cells, referred to as "ciBASCs".

Example 7. Immunostaining and nuclear staining experiments of lung basal cells or lung airway stem cells specific markers p63 and Krt5 in lung stem cell-like cells transformed by chemical induction of mouse fibroblasts (MF)

Experimental steps: use reprogramming medium 6 to chemically induce mouse fibroblasts to reprogram into lung stem cells, and the experimental culture method for preparing lung stem cells is the same as Example 2; ) or pulmonary airway stem cells (DASCs) specific markers p63 and Krt5 routine immunostaining and routine nuclear staining, the experimental results are shown in Figure 6.

Figure 6 shows that the lung stem cell-like cells obtained by induction and transformation were positive for conventional immunostaining of lung basal cells or lung airway stem cell specific markers p63 and Krt5. The results fully show that the small molecule composition induces the reprogramming of fibroblasts into lung stem cells, and further presents the unique marker characteristics of lung basal cells or lung airway stem cells; it shows that the chemically induced and reprogrammed lung stem cells are chemical Induced transformed lung basal cells (BCs) or pulmonary airway stem cells (DASCs), abbreviated as ciBCs or ciDASCs, respectively.

Example 8. The chemical small molecule composition induces human fibroblasts (HF) to reprogram and transform lung stem cell-like cells to carry out immunostaining and nuclear staining experiments for specific markers (SPC, CC10) of alveolar stem cells (BASCs)

Experimental steps: use reprogramming medium 7 to chemically induce human fibroblasts to reprogram into lung stem cells, and the experimental culture method for preparing lung stem cells is the same as Example 2; the lung stem cell-like cells obtained by induction and transformation are subjected to alveolar stem cells (BASCs). Specific marker (SPC, CC10) antibody routine immunostaining, and routine nuclear staining, the results are shown in Figure 7.

Figure 7 shows that the lung stem cells obtained by induction and transformation were positive for SPC and CC10 immunostaining, the specific markers of BASCs. It is shown that the pulmonary stem cells obtained by the reprogramming and transformation of human fibroblasts induced by the small molecule composition are alveolar stem cells (BASCs), namely chemically induced and transformed alveolar stem cells (ciBASCs).

Example 9. Immunostaining and nuclear staining experiments for lung basal cells or lung airway stem cells specific markers p63 and Krt5 in lung stem cell-like cells transformed by chemically induced human fibroblasts (HF)

Experimental steps: use reprogramming medium 10 to chemically induce human fibroblasts to reprogram into lung stem cells, and the experimental culture method for preparing lung stem cells is the same as Example 2; Or pulmonary airway stem cells (DASCs) specific markers p63 and Krt5 routine immunostaining and nuclear routine staining, the experimental results are shown in Figure 8.

Figure 8 shows that the lung stem cell-like cells obtained by induction and transformation were positive for conventional immunostaining of lung basal cells (BCs) or lung airway stem cells (DASCs) specific markers p63 and Krt5. It is fully demonstrated that the lung stem cell-like cells obtained by the reprogramming and transformation of human fibroblasts induced by the small molecule composition are lung basal cells or lung airway stem cells; namely, ciBCs or ciDASCs.

Example 10. Inhalation reagent 1 chemically induces in situ fibroblast reprogramming into lung stem cell effect, promotes repair of lung injury and improves and alleviates pulmonary fibrosis

In this example, the use of aerosol inhalation reagent 1 (prepared by small molecule combination 8, see Example 1) chemically induces the reprogramming of in situ fibroblasts into lung stem cells in vivo, and observes whether it promotes repair of lung damage and improves and relieves lung fibrosis.

Experimental operation steps:

1. Making the mouse model of fibrosis induced by lung injury: 18 male C57/BL6 mice of 4-5 weeks old were used to prepare the mouse model of pulmonary fibrosis induced by lung injury with bleomycin (BLM).

A single dose (aerosol inhalation reagent 1) was administered via endotracheal intubation at a dose of 5 mg/kg. After a single dose of BLM, the acute inflammatory response lasted for 8 days, and the inflammation turned to pulmonary fibrosis on the 9th day. After 28 or 35 days, tissue matrix deposition appeared, showing fibrotic changes. On the 10th day of modeling, a mouse was dissected, and the lung tissue was fixed and sliced, and routine immunohistochemical staining was performed to confirm that the mouse lung injury-induced pulmonary fibrosis model was successfully created;

2. 16 mice with lung injury-induced pulmonary fibrosis models were randomly divided into two groups, the control group and the treatment group;

3. Take the aerosol inhalation reagent 1 (prepared with small molecule combination 8), and make the animal model of the treatment group inhale the small molecule aerosol inhalation reagent 1 through the aerosol inhaler; use the same ingredients except that no small molecule composition is added. Control Nebulized Inhalation Reagent", a control group animal model was treated by a nebulized inhaler.

4. In the 4th week of treatment, the mouse models of the control group and the treatment group were dissected respectively, and fixed sections of lung tissue were taken for routine immunohistochemical staining.

The experimental results showed that the lung injury and pulmonary fibrosis of the mice in the treatment group were significantly alleviated, and the lung tissue structure basically returned to normal.

The immunohistochemical staining results of the most representative sections in the control group and the treatment group were taken for comparison, and the results are shown in Figure 9.

Figure 9 shows that the lung tissue of the control group (Control) mice was consolidated without normal alveolar structure; the activated pulmonary fibroblast marker αSMA (brown staining) was highly expressed in the lung tissue of the control group mice; while the treatment group (Treat) mice The lung tissue did not express αSMA, or the expression was significantly reduced; the lung tissue and alveolar structure basically returned to normal. It is indicated that the reprogramming effect of the small molecule reprogramming aerosol inhalation agent has the effect of promoting the repair of lung injury and improving and relieving pulmonary fibrosis.

Example 11. Nebulized Inhalation Reagent 2 Chemically induces the reprogramming of in situ fibroblasts into lung stem cells in vivo, promotes the repair of lung injury and improves and alleviates pulmonary fibrosis

In this example, the use of aerosol inhalation reagent 2 (prepared by small molecule combination 9, see Example 1) chemically induces the reprogramming of in situ fibroblasts into lung stem cells in vivo, and observes whether it promotes repair of lung damage and improves and relieves lung fibrosis. The experimental operation steps are the same as those in Example 10.

The experimental results showed that compared with the control group, the lung injury and pulmonary fibrosis of the mice in the treatment group were significantly reduced, and the lung tissue structure basically returned to normal. The immunohistochemical staining results of the most representative sections in the control group and the treatment group were taken for comparison, and the results are shown in Figure 10.

Figure 10 shows that the lung tissue of the control group (Control) mice was consolidated and lost the normal alveolar structure; the activated lung fibroblast marker αSMA (brown staining) was highly expressed in the lung tissue of the control group mice; while the treatment group (Treat) mice The lung tissue did not express αSMA, or the expression was significantly reduced; the lung tissue and alveolar structure basically returned to normal. It is indicated that the reprogramming effect of the small molecule reprogramming aerosol inhalation agent has the effect of promoting the repair of lung injury and improving and relieving pulmonary fibrosis.

Example 12. Transplantation of reprogrammed pulmonary stem cells to treat lung injury and improve and alleviate pulmonary fibrosis test

In this example, the pulmonary stem cells obtained by the reprogramming and transformation of fibroblasts induced by chemical small molecules were transplanted into an animal model of pulmonary fibrosis caused by lung injury, and the improvement or alleviation effect of the treatment of pulmonary fibrosis caused by lung injury was observed.

1. Use reprogramming medium 10 to chemically induce mouse fibroblasts to reprogram into lung stem cells, and the experimental culture method for preparing lung stem cells is the same as Example 2; collect reprogrammed and transformed lung stem cells; use normal saline to prepare lung stem cell suspension Or preparation (0.5×10 ⁶ cells/50ul/only)

2. The preparation of the fibrosis mouse animal model caused by lung injury was the same as in Example 10; 16 mice were randomly divided into 2 groups, the control group and the treatment group;

3. Each animal model of the disease in the treatment group was transplanted with 50ul of the transformed lung stem cell saline suspension (0.5×10 ⁶ cells/50ul/animal) through pulmonary tracheal intubation at a time; while each animal model of the disease in the control group was in the same way as the pulmonary trachea A single injection of 50ul mouse fibroblast physiological saline suspension (0.5×10 ⁶ cells/50ul/cell);

4. Treated for 30 days; the mice models of the control group and the treatment group were dissected respectively, and the fixed sections of the lung tissue were taken for routine immunohistochemical staining.

The experimental results showed that compared with the control group, the lung injury and pulmonary fibrosis of the mice in the treatment group were significantly reduced, and the lung tissue structure basically returned to normal. The immunohistochemical staining results of the most representative sections in the control group and the treatment group were taken for comparison, and the results are shown in Figure 11.

Figure 11 shows that the lung tissue of the control group (Control) mice was consolidated without normal alveolar structure; the activated lung fibroblast marker αSMA (brown staining) was highly expressed in the lung tissue of the control group mice; while the treatment group (Treat) mice The lung tissue did not express αSMA, or the expression was significantly reduced; the lung tissue and alveolar structure of the mice basically returned to normal. It shows that the cells transformed by the reprogramming of fibroblasts induced by the combination of small molecules are lung stem cells, which have the effect of treating lung injury and improving and alleviating lung fibrosis; fibroblasts do not have the effect of treating lung injury and improving and alleviating lung fibrosis. Effect.

Example 13. Comparative test of cell transformation independently induced by each single small molecule component of chemical small molecule composition 6

In this example, a comparative test of the independent induction of fibroblast transformation of each single small molecule component (GSK3β inhibitor/G9aHMT inhibitor/retinoic acid compound/TGFβ inhibitor) of chemical small molecule composition 6 was performed. The experimental steps are as follows:

(1) Preparation of GSK3β inhibitor CHIR99021, G9aHMT inhibitor BIX01294, TGFβ inhibitor SB431542, retinoic acid compound RA, and preparation of medium with various small molecule components: GSK3β inhibitor CHIR99021, G9aHMT inhibitor BIX01294, retinoic acid (RA) ), TGFβ inhibitor SB431542, each individual small molecule component was prepared with reference to the concentration ratio of small molecule composition 6 and the preparation method of the reprogramming medium to obtain a single small molecule component medium.

(2) The steps of the culture method for inducing the reprogramming of fibroblasts into lung stem cells are the same as those in Example 2; the cells used are mouse fibroblasts (MF), which are the same as those in Example 2.

(3) Compare the morphology of the cells obtained in the treatment group (Treat) and the fibroblasts (MF) in the control group (Control). The only difference in the culture conditions of the treatment groups was that no chemical small molecule components were added). The experimental results are shown in Figure 12.

Figure 12 shows that the control group MF on the left side of the figure compared with the small molecule treatment groups on the right side of the figure, each single small molecule component in the small molecule composition did not induce reprogramming of fibroblasts into lung stem cells, fibroblasts Before and after the induction treatment, there was basically no change in the morphology. The morphology of the cells in the treatment group was still similar to fibroblasts, and there was no similarity with the morphology of lung stem cells.

The experimental results show that the individual chemical small molecule components of the chemical small molecule composition of the present invention: GSK3β inhibitor, G9aHMT inhibitor, retinoic acid compound, and TGFβ inhibitor, when they exist alone, do not have the ability to induce fibroblast remodeling. Programmed for the function of lung stem cells.

To sum up, in the above embodiment, only 3-4 representative small molecules in each category of chemical small molecules are selected to form representative small molecule compositions, and representative experiments are carried out according to the reprogramming experimental methods and steps disclosed earlier in the specification, Predictable and representative results were obtained for the induction of reprogramming of fibroblasts into lung stem cells. It should be noted that the inventors have shown through existing experimental studies that the obtained transformed lung stem cells include basically the same or similar morphology: BASCs, AT2Cs, BCs, DASCs; or a mixture thereof. Which of these transformed lung tissue stem cells is dominant has a certain correlation with the type and proportion of small molecule components, as well as changes in culture time and culture environment. In any case, the examples of the present invention have undoubtedly confirmed that all lung stem cells are obtained by induced reprogramming.

The chemical small molecule composition of the present invention induces the reprogramming of fibroblasts into the chemical small molecule composition of lung stem cells and its application has the following beneficial effects:

1. The chemical small molecule composition of the present invention and its application do not introduce or use any exogenous gene/exogenous transcription factor/MicroRNA (miRNA) gene during the reprogramming process; nor use any exogenous cytokine or Growth factors; chemically induced reprogramming of fibroblasts into lung stem cells using only an induction composition consisting of small chemical molecules. The prepared transformed lung stem cells can be applied to clinical lung stem cell transplantation therapy, which not only avoids the cancer risk of stem cell transplantation by introducing foreign genes, but also overcomes the pathogenic risk of clinical transplantation of induced pluripotent stem cells to reprogram iPS cells.

2. The chemical small molecule composition has a variety of uses: (1) used to induce the reprogramming of fibroblasts into lung stem cells, to prepare lung stem cells, and to provide a source of lung stem cells for clinical treatment or scientific research; The effect of chemically induced in situ fibroblast reprogramming into lung stem cells, promoting the repair of lung injury and improving and relieving pulmonary fibrosis small molecule pharmaceutical composition/prodrug/drug; (3) for the preparation of chemically induced fibroblasts Preparations, reagents or culture media for reprogramming into lung stem cells; (4) Lung stem cells obtained by chemical induction and transformation can be used to prepare cell drugs or cell preparations for clinical transplantation therapy or for promoting the repair of lung injury and improving and relieving lung fibrosis ; The extracted bioactive substances, exosomes, bioactive vesicles and other active ingredients are prepared into bioactive drugs or preparations.

3. The prepared reprogrammed lung stem cells can be obtained from the patient's own fibroblasts. The transformed lung stem cells have individual characteristics, are easy to enter into clinical applications, and minimize or avoid the risk of immune rejection caused by allogeneic lung stem cell transplantation;

4. The reprogramming method for preparing lung stem cells has simple steps, easy operation, low cost, and easy clinical transformation and application.

5. It has the characteristics of conventional culture, short cycle, suitable for mass production and easy industrialization.

6. The mixed induction composition of interfering RNA and chemical small molecule of the present invention: siGSK3β, siG9a, retinoic acid compounds; or the mixed induction composition of shGSK3β, shG9a, and retinoic acid compounds, which also has the ability to induce fibroblasts to reprogram into lungs. function of stem cells. Among them, siRNA does not insert into the genome of the cell structure, does not change the genetic structure of the cell, and also avoids the new risk of carcinogenesis caused by the introduction of foreign genes. Compared with the introduction of various transcription factors to induce cell reprogramming, the method is simple to operate, has better reprogramming effect, has no carcinogenic risk, is safer and more reliable, and is easy to apply in clinic.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

Application of a chemical small molecule composition in chemically inducing fibroblasts to reprogram into lung stem cells, wherein the chemical small molecule composition comprises: a GSK3β inhibitor, a G9aHMT inhibitor and a retinoic acid compound; or, the chemical The small molecule composition consists only of GSK3β inhibitors, G9aHMT inhibitors and retinoids;

Wherein, in the process of inducing fibroblasts to reprogram into lung stem cells, no exogenous genes/transcription factors/MicroRNA genes, RNAs, proteins, polypeptides thereof, and exogenous cytokines or growth factors are introduced or used. .
The application according to claim 1, characterized in that, the chemical small molecule composition can further comprise: a TGFβ inhibitor; Composed of retinoids and TGFβ inhibitors.
The application according to claim 1, wherein the application of the chemical small molecule composition further comprises:

It is used to chemically induce the reprogramming of fibroblasts into lung stem cells in vitro to prepare lung stem cells and provide a source of lung stem cells for clinical treatment or scientific research; or,

A pharmaceutical composition/drug/prodrug/drug preparation for the preparation of chemically induced in situ reprogramming of fibroblasts into lung stem cells, promoting repair of lung injury and improving and relieving pulmonary fibrosis; or,

For adding cell basal medium/physiological saline/organic solvent to prepare reprogramming medium or reagents or preparations; or,

Based on the pulmonary stem cells obtained from the induced transformation, prepare pulmonary stem cell drugs or pulmonary stem cell preparations for clinical transplantation to treat or repair lung injury and improve and relieve pulmonary fibrosis; or,

Bioactive drugs or preparations are prepared based on the lung stem cells obtained by the induced transformation, and the active ingredients such as bioactive substances, exosomes, and bioactive vesicles are extracted.
A chemical small molecule composition for chemically inducing fibroblast reprogramming into lung stem cells, comprising: GSK3β inhibitor, G9aHMT inhibitor and retinoic acid compound; Retinoic acid compounds; the chemical small molecule composition does not contain biological inducing factors, or the chemical small molecule composition does not introduce or use any exogenous gene/transcription during the process of inducing fibroblast reprogramming into lung stem cells Factor/MicroRNA genes, and their RNAs, proteins, polypeptides; and exogenous cytokines or growth factors.
The chemical small molecule composition according to claim 4, characterized in that, the chemical small molecule composition can further comprise: a TGFβ inhibitor; G9aHMT inhibitors, retinoids and TGFβ inhibitors.
The application according to any one of claims 1 to 3 or the chemical small molecule composition according to any one of claims 4 to 5, wherein the GSK3β inhibitor is an inhibitor capable of targeting and inhibiting the GSK3β signaling pathway; Preferably, it includes: CHIR-99021, LiCl, BIO, Ly2090314, IM-12, TWS119, 1-Azakenpaullone, CHIR-98014, Tideglusib, AR-A014418, SB216763, AZD1080, targeting and inducing other GSK3β that inhibit the GSK3β signaling pathway Small molecule inhibitors, or their equivalent pharmaceutical preparations, analogs, isomers, salts, hydrates or precursors, or combinations thereof; more preferably, it includes a GSK3β inhibitor CHIR-99021, LiCl, BIO or LY2090314;

The G9aHMT inhibitor is an inhibitor capable of targeting G9aHMT; preferably it includes: BIX01294, UNC0638, A-366, UNC0631, BRD4770, UNC0224, UNC0646, UNC0642, UNC0321, BRD4770, HKMTI-1-247, HKMTI -1-248, CPUY074020, DCG066, other small molecule inhibitors of G9aHMT targeting G9aHMT, or their equivalent pharmaceutical preparations, analogs, isomers, salts, hydrates or precursors, or combinations thereof; more Preferably it comprises selected from: G9aHMT inhibitor BIX01294, UNC0638 or UNC0642;

The retinoic acid compound is a small chemical molecule that can specifically bind to a retinoic acid response factor, thereby targeting, inducing and regulating the RA signaling pathway; preferably, it includes: retinoic acid, 13-cis retinoic acid or 9-cis retinoic acid Retinoic acid, UAB7, UAB8, TTNPB, 3-methyl-TTN PB, AM80, AM580, CD437, Targretin, LGD1069, other retinoic acid compounds that target the induction and regulation of RA signaling pathway, or their equivalent pharmaceutical products , an analog, a salt, a hydrate, a precursor, or a combination thereof; more preferably, it comprises a retinoic acid, 13-cis retinoic acid, or 9-cis retinoic acid; or

Optionally, the TGFβ inhibitor is an inhibitor that can target and inhibit the TGFβ signaling pathway; preferably, it includes: SB431542, A83-01, SB525334, LY2109761, RepSox, SD-208, GW788388, SB505124, EW-7197 , Galunisertib, other TGFβ small molecule inhibitors that target and induce inhibition of TGFβ signaling pathway, or their equivalent pharmaceutical preparations, analogs, isomers, salts, hydrates, precursors, or combinations thereof; more preferably its Including selected from: SB431542, A83-01 or RepSox.
The application according to any one of claims 1 to 3 or the chemical small molecule composition according to any one of claims 4 to 5, wherein the small molecule composition comprises:

GSK3β inhibitor: 5-80 parts by weight; or final concentration in solution: 0.1-20 μM/mM;

G9aHMT inhibitor: 0.1-50 parts by weight; or final concentration in solution: 0.01-20 μM;

Retinoic acid compound: 0.05-20 parts by weight; or final concentration in solution state: 0.1-20 μM; or

Optionally, TGFβ inhibitor: 0.1-50 parts by weight; or final concentration in solution state: 0.01-20 μM.
The application according to any one of claims 1 to 3 or the chemical small molecule composition according to any one of claims 4 to 5, wherein the small molecule composition:

According to the weight ratio, the GSK3β inhibitor, G9aHMT inhibitor, and retinoic acid compounds are (5-80): (0.1-50): (0.05-20); or the molar concentration ratio in the solution state is (0.1-20): (0.01-20): (0.1-20); or

According to the weight ratio, GSK3β inhibitor, G9aHMT inhibitor, retinoic acid compound and TGFβ inhibitor are (5-80):(0.1-50):(0.05-20):(0.1-50); or in solution state The molar concentration ratio was (0.1-20):(0.01-20):(0.1-20):(0.01-20).
The application according to any one of claims 1 to 3 or the chemical small molecule composition according to any one of claims 4 to 5, wherein the small molecule composition is a pharmaceutical composition or preparation, further comprising: A pharmaceutically acceptable carrier or excipient; preferably, the carrier or excipient includes one or more selected from the group consisting of water, saline, phosphate buffer or other aqueous solvents; DMSO, glycerol and Ethanol or other organic solvents; microspheres, liposomes, microemulsions or polymer surfactants; colloidal drug delivery systems or polymer drug delivery systems; preservatives, antioxidants, flavoring agents, fragrances, cosolvents , emulsifiers, pH buffer substances, adhesives, fillers, lubricants or other pharmaceutical excipients; or, the pharmaceutical dosage forms that can be prepared from the chemical small molecule composition include: solid dosage forms, including: powder, powder, tablet doses, pills, capsules, sustained-release preparations, controlled-release preparations, or other solid dosage forms; liquid dosage forms, including: injections, infusions, suspensions, or other liquid dosage forms; gas dosage forms; or semi-solid dosage forms; or The small molecule composition is a reprogramming preparation or reagent, and preferably further includes an organic solvent, physiological saline, or other carriers or excipients.
The application according to any one of claims 1 to 3 or the chemical small molecule composition according to any one of claims 4 to 5, wherein the small molecule composition can be supplemented with a cell basal medium to be prepared for Reprogramming medium for chemically inducing fibroblasts to reprogram into lung stem cells; or adding organic solvent, physiological saline, prepared as a reagent or preparation for chemically inducing fibroblasts to reprogramming into lung stem cells; or adding pharmaceutically acceptable Carriers or excipients for preparing pharmaceutical compositions, prodrugs, drugs or preparations for chemically inducing in situ fibroblast reprogramming into lung stem cells, promoting repair of lung injury and improving and relieving pulmonary fibrosis.
A kit or kit for chemically induced reprogramming of fibroblasts into lung stem cells, comprising:

The chemical small molecule composition according to any one of claims 4 to 10; a pharmaceutical composition and a drug precursor obtained by adding a pharmaceutically acceptable carrier or excipient based on the small molecule composition; adding an organic solvent, physiological saline, Buffer or cell basal medium type carriers or excipients, reprogramming medium or reagents prepared;

Lung stem cells prepared by induction and transformation based on the chemical small molecule composition; preparation of lung stem cell drugs or cell preparations for clinical transplantation based on the obtained lung stem cells; body, bioactive vesicles and other active ingredients, or bioactive drugs or preparations prepared from them;

Wherein, the kit does not include any exogenous gene/transcription factor/MicroRNA gene, and its RNA, protein, polypeptide; and exogenous cytokine or growth factor.
The application according to any one of claims 1 to 3, the chemical small molecule composition according to any one of claims 4 to 5, or the kit or kit according to claim 11, wherein the fibroblasts Including human or mammalian fibroblasts; preferably, including but not limited to human or mammalian: skin fibroblasts, liver fibroblasts, lung fibroblasts, kidney fibroblasts, pancreatic fibroblasts, or Fibroblasts of other tissues or organs of humans or mammals; more preferably skin fibroblasts; or,

The lung stem cells obtained by any of the chemically induced fibroblast reprogramming include: alveolar type II cells (AT2Cs), bronchioloalveolar stem cells (BASCs), basal cells (BCs), distal airway stem cells (DASCs), or their mixture or mixed cells.
The chemical small molecule composition of claim 4, wherein the interfering RNA with the same inhibition target gene as the chemical small molecule GSK3β inhibitor in the composition: siGSK3β/shGSK3β, also has the same ability to induce and inhibit GSK3β Function: The interfering RNA with the same inhibition target gene as the chemical small molecule G9aHMT inhibitor in the composition: siG9a/shG9a, also has the function of inducing and inhibiting G9aHMT.
The chemical small molecule composition according to claim 4 and the interfering RNA according to claim 13, characterized in that, the mixed induction combination consisting of: siGSK3β, siG9a, and retinoic acid compounds; or shGSK3β, shG9a, and retinoic acid compounds It has the same function of inducing fibroblast reprogramming into lung stem cell as the chemical small molecule composition.
The mixed induction composition of claim 14, wherein the siGSK3β/shGSK3β, siG9a/shG9a belong to 20-25 base pairs, and a carrier or a carrier delivery system is required to be introduced into cells; The systems include but are not limited to: viral vector delivery systems, non-viral vector delivery systems, such as: biochemical modification delivery systems, microinjection delivery systems, and non-viral nanocarrier delivery systems.