CN112175906B - Use of thymidine induced fibroblast transdifferentiation in the treatment of neutropenia - Google Patents
Use of thymidine induced fibroblast transdifferentiation in the treatment of neutropenia Download PDFInfo
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- CN112175906B CN112175906B CN201910603779.9A CN201910603779A CN112175906B CN 112175906 B CN112175906 B CN 112175906B CN 201910603779 A CN201910603779 A CN 201910603779A CN 112175906 B CN112175906 B CN 112175906B
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Abstract
The present invention provides the use of Thymidine (Thymidine) or a precursor or derivative thereof for inducing the transdifferentiation of fibroblasts into granulocytes. The invention also provides the use of granulocytes obtained by the invention in the treatment of (neutropenia).
Description
Technical Field
The present invention relates to the field of cell transdifferentiation. More particularly, the present invention relates to the use of Thymidine (Thymidine) or a precursor or derivative thereof for inducing the transdifferentiation of fibroblasts into granulocytes. The invention also provides the use of granulocytes obtained by the invention in the treatment of (neutropenia).
Background
Neutrophils are a type of myeloid terminal mature cells found in large numbers in the body, a type of multi-leaf nuclear leukocyte subpopulation discovered by cell staining by Paul Ehrlich et al at the end of the 19 th century, and a major effector cell population of the body against infection and inflammatory responses. Neutrophils are recruited to the site of inflammation early in inflammation to clear pathogens, playing a vital role in the clearance of infection. Abnormal development of the neutrophil lineage results in severe hematological tumors. An increase in the number of neutrophils in the blood suggests that bacterial infection is present in the body. While a reduction in the number thereof clinically presents a serious immune system deficiency.
The development of the granulocyte lineage begins with the fate decision of bone marrow Hematopoietic Stem Cells (HSCs) in both directions of differentiation to common lymphoid progenitor cells (commom lymphoid progenitor, CLP) or common myeloid progenitor cells (common myeloid progenitor, CMP). CLP can differentiate into various lymphoid precursor cells under different environmental stimuli and internal signals, eventually into mature T, B and NK lymphocytes, while CMP can differentiate into granulation-megakaryotype progenitor cells (GMP) and megakaryotype-erythroid progenitor cells (MEP). And eventually differentiate into mature monocytes, macrophages, granulocytes, DC cells, megakaryocytes and erythrocytes. Common myeloid progenitor CMP (Lin) - Scal - c-Kit + CD34 + FcγRII - FcγRIII - ) The downstream granulocyte-macrophage progenitor cells (GMP) develop into mature neutrophils via the stages of promyelocytes, and evening promyelocytes, under the action of specific growth factors and cytokines. Mature differentiation of neutrophils requires reduced self-renewal potential in precursor cells and the acquisition of expression of genes associated with specific lineage development, a process that requires precise genetic and epigenetic regulation. Transcription factors PU.1, CCAAT enhancer binding protein C/EBP, independent growth factor GFI1 and the like are all involved in regulating and controlling the fate decision process of the myeloid lineage to develop in the granulocyte direction, the low expression of the PU.1 promotes the development of neutrophils, a C/EBP alpha gene deletion mouse seriously lacks GMP progenitor cells and mature granulocytes, and GFI1 and C/EBP epsilon play an important role in the mature differentiation of promyelocytes. At the same time, epigenetic regulation plays an important regulatory role in the developmental differentiation of the granulocyte lineage. There is a paper reporting that myeloid cell development and differentiation is accompanied by a significant decrease in DNA methylation, and that TET2 expression promotes hematopoietic stem cell development to neutrophils. TET2 is involved in regulating C/EBP alpha-induced B cell reprogramming into myeloid cells. On the other hand, the lineage development of neutrophils is also highly dependent on the bone marrow microenvironmentAn external signal. Among them, granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) are the main regulatory signals for neutrophil maturation.
Neutropenia is a group of syndromes that occur due to a decrease in the absolute value of peripheral blood neutrophils. Etiology is classified as congenital or acquired, including drug, radiation, genetic, developmental defects, immunity, decreased granulopoiesis due to infection, ineffective proliferation, excessive destruction, and pseudo-granulocytopenia. Patients with severe infections associated with neutropenia may develop severe sepsis, septic shock and multiple organ failure that are significantly associated with morbidity and mortality. In the case of neutropenia, a prophylactic or therapeutic granulocyte colony stimulating factor or granulocyte macrophage colony stimulating factor may stimulate an acceleration of endogenous granulocyte recovery. However, because of the adverse effects, there is still controversy as to whether clinical benefit can be gained. In addition, donor granulocytes can be obtained from healthy donors by plasmapheresis or whole blood for use in cell therapy in patients associated with neutropenia, i.e., prophylactic infusion of concentrated white (granulocytes), to reduce the incidence of infection and sepsis. For infections associated with central granulocytopenia, the cell dose is important for the effect of granulocyte infusion. However, due to insufficient granulocyte dose, rapid loss of function after granulocyte separation, strong granulocyte antigenicity, easy allo immune reaction, and frequent mixing of a large amount of immunocompetent lymphocytes, the method can cause Graft Versus Host Disease (GVHD) after infusion of patients with low immune function, and the like, the application range is limited. The development of a method for efficiently forming neutrophils by differentiation of autologous cells through cell life operation and reinfusion is likely to be an effective path for expanding the clinical application prospect of granulocyte therapy.
Fibroblasts are the most readily available cell type in humans, as it is most abundant in human skin and also most readily cultured in vitro. The discovery of induced pluripotent stem cells (iPS cells) in 2006 uses transcription control factors related to development to interfere with the internal programming of fibroblasts, so that the fibroblasts return to a state with embryonic stem cell characteristics, thereby replacing stem cells from early embryos, avoiding ethical problems when applied clinically, solving the problem of cell genotyping, and making regenerative medicine research to break through progress. Since some genes co-expressed during the reprogramming process are associated with some differentiation lineages (e.g., neurons, epidermis, blood, etc.), this provides the possibility that fibroblasts may be directly committed to induce a particular lineage under particular conditions. Recent studies have demonstrated that fibroblasts can be transformed directly into many terminal cell types (e.g., neural cells, cardiomyocytes, macrophage-like cells, etc.). This is achieved primarily by introducing multiple additional copies of the "main switch" gene to express the proteins of the entire gene network required to activate a particular cell type. The direct conversion can avoid or bypass the links of low efficiency, long period, complex operation technology and in vivo risk of inducing the pluripotent stem cells (iPS), and has a larger application prospect in clinic. In the previous research, exogenous gene transfection is needed, so that the insertion site of the host cell genome is difficult to control during transfection, and the application risks such as mutation are brought to the genome. In 2016, a technology for directly activating natural copies already existing in a cell genome by using a genetically modified CRISPR/Cas9 gene editing technology to directly change mouse embryo fibroblasts into neurons has appeared. In recent years, there has also been a report of screening a group of small molecule compound combinations to treat cells, and reprogramming skin cells to neural stem cells or cardiomyocytes using "chemical cocktails". The chemical induction cell reprogramming method avoids gene operation and utilizes the principle of interaction between small molecules and cell endogenous factors to enable autologous cells to regain pluripotency. Compared with inserting exogenous genes, the small molecules have the advantages of simple operation, easy grasp of processing time, reduced test cost, and capability of manually adjusting concentration and combination. In the research direction of direct induction of cell fate conversion by small molecules, single-kind small molecule compounds can also realize the regulation and control of the fate conversion of lymphocytes by regulating and controlling main metabolic pathways and utilizing an epigenetic mode.
Thymidine, chinese famous chestAdenosine, CAS#:34283-30-2, molecular formula: c (C) 10 H 14 N 2 O 5 . The azidothymidine and zidovudine are mainly used as intermediates of anti-AIDS and antiviral drugs in vitro. Thymine (Thymene) is one of the bases in deoxyribonucleic acid and binds to deoxyribose to form Thymedine. Thymidine forms thymine nucleotides TMP, TDP and TTP under the catalysis of Thymidine kinase to participate in DNA synthesis. Thymidine forms thymine under the catalysis of thymine phosphorylase, is metabolized in vivo to beta-aminoisobutyric acid, and enters the tricarboxylic acid cycle or gluconeogenic metabolic pathway via succinyl CoA. Thymidine forms 5-hydroxymethyl uracil, 5-formyl uracil and the like under the catalysis of thymine 7-hydroxylase. There has been no report on the regulatory effects of Thymidine and its metabolites on granulocytic lineage fate decisions and transdifferentiation.
Disclosure of Invention
The invention seeks to explore a method for inducing fibroblasts to directly convert and differentiate into neutrophils by using a small molecular metabolite Thymidine and each possible activity modification product, thereby providing a more convenient method for treating diseases related to neutropenia. Despite the great progress that has been made in understanding the chemistry-induced cell reprogramming and metabolic regulation epigenetic direction, there remains a need to identify specific pathways and proteins that direct the transformation of activated fibroblasts as pharmaceutically acceptable targets into granulocytes, thereby providing a new approach for the treatment of neutropenia and its concomitant severe infections with autologous-derived cells. The present invention fulfills this and related needs.
Accordingly, the present invention provides the following:
1. use of Thymidine (Thymidine) or a precursor or derivative thereof (as the sole active ingredient or in combination with other active ingredients) for inducing the transformation and differentiation of fibroblasts into granulocytes (or cells having characteristics of the granulocyte lineage), wherein the precursor or derivative retains the activity of Thymidine to induce fibroblasts to granulocytes. Preferably, the fibroblasts are primary fibroblasts. The above-mentioned activity refers to an activity of inducing the transformation and differentiation of fibroblasts into granulocytes (or cells having characteristics of granulocyte lineage).
2. The use according to the above 1, wherein the thymidine or a precursor or derivative thereof has the structure of the following formula (I),
wherein:
the optional modifying groups on the ribofemale ring are independently selected from: c (C) 1 -C 12 Alkyl (e.g. C 1 -C 6 Alkyl group, C 1 -C 6 Alkylene, C 2 -C 12 Alkenyl (e.g. C 3 -C 6 Alkenyl group, C 3 -C 10 Cycloalkyl, phenyl, substituted phenyl, optionally substituted five or six membered heteroaryl, or C 6 -C 10 Aralkyl, halogen, -OH, -O-R a 、C 4 -C 12 Dienyl, C 6 -C 12 Trienyl group, C 8 -C 12 Tetraenyl, C 6 -C 10 Aryl (optionally independently selected from C 1 -C 6 Alkyl, halogen, C 1 -C 6 Substituted by one, two or three substituents of alkoxy, or hydroxy), C 1 -C 10 Alkoxy, carboxyl, cyano, C 1 -C 10 Alkanoyloxy, C 1 -C 10 Alkylthio, C 1 -C 10 Alkylsulfonyl, C 1 -C 10 Alkoxycarbonyl group, C 1 -C 10 Alkanoylamino, -S-R b 、-SO 2 R c 、-NHSO 2 R d and-NHCO 2 R e The method comprises the steps of carrying out a first treatment on the surface of the Wherein R is a 、R b 、R c 、R d And R e Is independently selected from C 1 -C 6 Alkyl, C 6 -C 10 Aryl, C 1 -C 6 C substituted with one to three groups of alkoxy, halogen, and hydroxy heteroaryl 1 -C 6 Alkyl, C 2 -C 6 Alkenyl, C 3 -C 10 Cycloalkyl, phenyl or naphthyl, said hydroxy heteroAryl contains one to five heteroatoms independently selected from sulfur, nitrogen, and oxygen.
3. The use according to 1 or 2 above, wherein the fibroblast is an embryonic fibroblast, preferably a mammalian embryonic fibroblast, such as a mouse. In a specific embodiment, the embryonic fibroblasts are primary CD45 - CD31 - CD117 - CD41 - 4 neg MEF cells. More preferably, the cells are seeded onto mitomycin treated feeder cells and then treated with Thymidine (Thymidine) or a precursor or derivative thereof, either as the sole active ingredient or in combination with other active ingredients.
4. The use according to any one of 1-3 above, wherein the granulocytes are neutrophils and express granulocyte-specific marker molecules MPO, cebp a, gr1 and CD11b and/or can form CFU-G colonies upon culture with the addition of G-CSF.
5. An in vivo or in vitro method of directly inducing transdifferentiation of mammalian fibroblasts into granulocytes, said method comprising the step of contacting said cells with Thymidine (Thymidine) or a precursor or derivative thereof as defined in any of the preceding claims.
6. (neutral) granulocytes obtainable by the use according to any of the above 1-4 or by the method according to the above 5.
7. A method of treating (neutropenia) in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of Thymidine (Thymidine) or a precursor or derivative thereof, as described in 1 or 2 above, or (neutrophil) granulocytes according to 6 above.
8. Use of Thymidine (Thymidine) or a precursor or derivative thereof as described in 1 or 2 above, or (neutrophil) granulocytes as described in 6 above as sole active ingredient, or in combination with other active ingredients, for the preparation of a medicament/kit for the treatment of (neutrophil) granulocytopenia.
9. The method according to 7 above or the use according to 8 above, wherein the (neutropenia) is neutropenia due to drug, radiation therapy, genetic, developmental defects, immunity, and/or infection.
10. The method or use according to 9 above, wherein said (neutropenia) is accompanied by sepsis, shock or multiple organ failure.
Drawings
The results shown in figures 1 a through G demonstrate that thydine induces the transdifferentiation of fibroblasts into cells with characteristics of the granulocyte lineage. Specifically, FIG. 1, A shows primary mouse sorting 4 neg MEF cells induce a transdifferentiation strategy. FIG. 1, B shows the results of CD45 antibody flow assay of cells obtained by transdifferentiation. FIG. 1, C shows the GSEA analysis of the cellular gene chip obtained by transdifferentiation. FIG. 1, D, shows fluorescent real-time quantitative PCR analysis of granulocyte marker genes. FIG. 1, E shows the results of Rayleigh staining of cells obtained by transdifferentiation. FIG. 1, F shows the result of immunofluorescent staining of cells obtained by transdifferentiation. FIG. 1, G shows the results of cell CFU-G clone formation experiments obtained by transdifferentiation.
The results shown in figures 2 a to D demonstrate that thamid-induced transdifferentiated cells iMC have granulocyte function. Specifically, FIG. 2, A shows the results of inducing ROS production by transdifferentiated cells. FIG. 2, B shows the results of chemotactic migration induced by cells obtained by the opposite transdifferentiation. FIG. 2, C, shows the results of induction of inflammatory cytokine mRNA expression by cells obtained from transdifferentiation. FIG. 2, D shows the results of cells obtained by transdifferentiation phagocytizing FITC dye-labeled E.coli bacteria in vitro.
The results shown in figures 3 a through I demonstrate that infusion of iMC in mice is effective in ameliorating neutropenia. Specifically, in fig. 3, a shows CTX-induced neutropenia model infusion iMC strategy. Figure 3, b shows that infusion of iMC in mice effectively ameliorates weight loss symptoms. Fig. 3, c shows that infusion of iMC in mice effectively ameliorates neutropenia symptoms. FIG. 3, D shows Rosa26-STOP-iDTR; lyz-Cre genetic mouse model infusion iMC strategy. Figure 3, e, shows that infusion of iMC in mice effectively ameliorates weight loss symptoms. FIG. 3, F shows that infusion of iMC in mice effectively ameliorates neutropenia symptoms. Fig. 3, g shows that the proportion of various cells in the peripheral blood of the icm mice infused repeatedly over a long period of time did not change significantly. Fig. 3, h shows that long-term multiple infusions of iMC mice did not significantly alter bone marrow and spleen. Fig. 3, i shows that long-term multiple infusions of iMC mice did not significantly alter the anatomical pathology of liver, lung, and colon sites.
Detailed Description
The inventors of the present invention found in a study relating to Mouse Embryonic Fibroblast (MEF) induced reprogramming: sorting primary CD45 - CD31 - CD117 - CD41 - 4 neg MEF cells were seeded on mitomycin treated feeder cells and induced by the addition of Thymidine for 6 days, and CD45 was observed in the culture system 9 days after the switch-over + Is similar to the appearance of hematopoietic cells. Chip analysis of the cells obtained by transdifferentiation shows that the expression of genes characteristic of fibroblasts of the transdifferentiated cells is down-regulated, while the expression of genes characteristic of granulocyte lineages is significantly up-regulated. The fluorescent real-time quantitative PCR analysis of granulocyte marker genes also proves that the genes are highly expressed in the process of transdifferentiation induction. The transdifferentiated cells were rayleigh stained and found to have granulocyte lineage precursor and mature granulocyte morphological features. Immunofluorescence staining is carried out on the transdifferentiated cells, and the cells are found to highly express marker molecules MPO, cebp alpha, gr1 and CD11b characteristic of granulocytes. The transdifferentiated cells were cultured with the addition of G-CSF on methylcellulose medium in vitro and found to form CFU-G colonies. The above results demonstrate that thamate-induced transdifferentiated cells have granulocytic lineage cell characteristics. We refer to as iMC cells.
Sorting iMC from primary mouse neutrophils (CD 11b + Gr1 + ) In vitro functional assays were performed and the results indicated that iMC cells have similar functions to primary neutrophils in generating ROS in response to stimuli. iMC cells are also capable of fMLP or PMA induced cell migration. The iMC cells may also highly express TNF alpha, IL1 beta and IL18 upon stimulation by PMA. More importantly, iMC cells are also capable of being used in vitroFITC dye-labeled E.coli bacteria can be effectively phagocytosed. The above results demonstrate that thamid-induced transdifferentiated cells iMC have granulocyte function.
Cyclophosphamide CTX mimics clinical conditions treatment of mice induces neutropenia in mice. Infusion of iMC in mice effectively ameliorates the symptoms of weight loss and neutropenia. Rosa26-STOP-iDTR; lyz-Cre genetic mouse tools can induce clearance of neutrophils in vivo following Diphtheria Toxin (DT) treatment to mimic neutropenia. Infusion of iMC in mice effectively ameliorates the symptoms of weight loss and neutropenia. Meanwhile, our results also demonstrate that the infusion of iMC into normal mice at a frequency of 1 month within 4 months, the long-term detection of the peripheral blood cell proportion of the recipient mice did not significantly change, the bone marrow and spleen did not significantly change, and the anatomical pathology of liver, lung, colon in vivo did not significantly change. Preliminary evidence has been given that long-term multiple infusions of iMC do not have the risk of inducing leukemia or solid tumors.
Thus, in a first aspect the invention provides a use of Thymidine in inducing transdifferentiation of fibroblasts, characterized in that the transdifferentiated cells induced by Thymidine have granulocytic lineage cell characteristics.
In some embodiments of the invention, primary CD45 is sorted - CD31 - CD117 - CD41 - 4 neg MEF cells were seeded on mitomycin treated feeder cells and induced by the addition of Thymidine for 6 days, and CD45 was observed in the culture system 9 days after the switch-over + Is similar to the appearance of hematopoietic cells.
In some embodiments of the invention, the cells obtained from transdifferentiation are analyzed on a chip and it is found that the expression of genes characteristic of fibroblasts of the transdifferentiated cells is down-regulated, whereas the expression of genes characteristic of granulocyte lineage is significantly up-regulated. The fluorescent real-time quantitative PCR analysis of granulocyte marker genes also proves that the genes are highly expressed in the process of transdifferentiation induction.
In other embodiments of the invention, various morphological analyses are performed on transdifferentiated cells, and Rayleigh staining is performed to find that these cells have granulocyte lineage precursor cells and mature granulocyte morphological features. Immunofluorescence staining is carried out on the transdifferentiated cells, and the cells are found to highly express marker molecules MPO, cebp alpha, gr1 and CD11b characteristic of granulocytes. The transdifferentiated cells were cultured with the addition of G-CSF on methylcellulose medium in vitro and found to form CFU-G colonies.
In a second aspect the invention provides a method of inducing direct transformation of primary fibroblasts into granulocytes, said method comprising contacting the fibroblasts with an effective amount of Thymidine and possibly each of its active modification products to induce iMC cells. And provides functional verification of transdifferentiated cells iMC in vitro and in vivo, the method comprising comparing in vitro the function of iMC cells with primary neutrophils in ROS production, chemotactic migration, inflammatory cytokine production, and phagocytic bacteria production, and administering a therapeutically effective amount of transdifferentiated cells to the neutropenia patient.
In some embodiments of the invention, the iMC cells have similar functions to primary neutrophils in generating ROS in response to stimuli. iMC cells can produce fMLP or PMA-induced chemotactic migration. The iMC cells highly express TNF alpha, IL1 beta and IL18 inflammatory cytokines under PMA stimulation. The iMC cells were able to effectively phagocytose FITC dye-labeled e.coli bacteria in vitro.
In other embodiments of the invention, cyclophosphamide CTX treatment or Rosa26-STOP-iDTR is used; lyz-Cre genetic mouse tool treatment of diphtheria toxin mimics neutropenia and confirmed that infusion of iMC in mice effectively ameliorates the symptoms of weight loss and neutropenia. Blood convention and organs such as spleen, liver, lung, colon and the like are detected in mice infused with iMC for a plurality of times for a long period, and it is confirmed that infusion of iMC does not have the risk of inducing leukemia or solid tumor.
In a third aspect, the method of any one of the above first and second aspects is that the compound that induces the conversion and differentiation of fibroblasts into granulocytes is a compound of formula (I):
in a fourth aspect, the present method relates to the compound Thymidine and possibly each of its active modification products, for inducing the transformation of fibroblasts into granulocytes.
In a fifth aspect, the present method relates to the compound thytidine and possibly each of its active modification products, inducing the transdifferentiation of fibroblasts into granulocytes, which are useful in the treatment of diseases involving neutropenia.
In any of the foregoing aspects, the thymid and each of its possible active modification products may be the same compound or different compounds.
The effective dosage of Thymidine of the invention can be adjusted correspondingly according to the cell type, the number and the like. The effective dose of the infused iMC cells of the invention can be adjusted accordingly with the mode of administration and the severity of the disease to be treated, etc. The preferred effective amount can be determined by one of ordinary skill in the art in combination with various factors. Such factors include, but are not limited to: the health condition, weight, route of administration, etc. of the patient to be treated.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
Examples
Example 1: thymidine proved to promote the transformation and differentiation of primary fibroblasts into granulocytes.
Sorting primary CD45-CD31-CD117-CD41-4neg MEF cells (see reference [1 ]) were seeded on mitomycin treated feeder cells and induced by Thymidine (Sigma, T1895, 20. Mu.M) for 6 days, and observed after 9 days of transfer, as shown in FIG. 1, A. The presence of CD45+ cells of similar hematopoietic lineage in the post-induction culture system is shown in FIGS. 1, B. The cells obtained by transdifferentiation were analyzed on chip (Roche-Nimblegen full gene expression profiling chip technical service, cat. No. 05543797001) and found that the expression of genes characteristic of fibroblasts of transdifferentiated cells was down-regulated, whereas the expression of genes characteristic of granulocyte lineage was significantly up-regulated, as shown in FIGS. 1, C. Fluorescent real-time quantitative PCR analysis of granulocyte marker genes (PCR primer sequences:
mCebpα,
f (forward primer): 5'-CAAGAACAGCAACGAGTACCG-3' (SEQ ID NO: 1), R (reverse primer): 5'-GTCACTGGTCAACTCCAGCAC-3' (SEQ ID NO: 2); the composition of mElane,
F:5’-TTGCCAGGAATTTCGTCATGT-3’(SEQ ID NO:3),
R:5’-GTTGGCGTTAATGGTAGCGGA-3’(SEQ ID NO:4)；
mLyz1,
F:5‘-GAGACCGAAGCACCGACTATG-3’(SEQ ID NO:5)，
R:5’-CGGTTTTGACATTGTGTTCGC-3’(SEQ ID NO:6)；
mMpo,
F:5’-AGTTGTGCTGAGCTGTATGGA-3’(SEQ ID NO:7),
R5'-CGGCTGCTTGAAGTAAAACAGG-3' (SEQ ID NO: 8)) demonstrated high expression of genes characteristic of the granulocyte lineage during transdifferentiation induction, as shown in FIGS. 1, D. The transdifferentiated cells were subjected to Rayleigh staining (see reference [1 ]), and these cells were found to have granulocyte lineage precursor cells and mature granulocyte morphological features, as shown in FIGS. 1, E. Immunofluorescence staining was performed on the transdifferentiated cells, and it was found that these cells highly expressed granulocyte-characteristic marker molecules MPO, cebpa, gr1 and CD11b (anti-MPO antibodies, purchased from beibezosen; anti-cebpa antibodies, purchased from Santa Cruz; anti-Gr 1 antibodies, purchased from ebioscience; anti-CD 11b antibodies, purchased from ebioscience), as shown in fig. 1, f. The transdifferentiated cells were cultured in vitro on methylcellulose medium with the addition of G-CSF, and it was found that the cells could form CFU-G colonies, as shown in FIG. 1G.
Example 2: the cells obtained by inducing the fibroblast transdifferentiation by Thymidine have granulocyte function.
Sorting imcs was functionally validated in vitro against primary mouse neutrophils (cd11b+gr1+), and the results indicated that imcs possessed similar functions to primary neutrophils in response to stimulation to produce ROS (see reference [2 ]), as shown in fig. 2, a. iMC cells were also capable of fMLP (N-Formylmethionine-phenyl-phenylalanine chemotactic tripeptide) or PMA (phorbol ester) -induced cell migration (see reference [2 ]), see fig. 2, b for steps and methods. The iMC cells also highly expressed TNF alpha, IL1 beta and IL18 under PMA stimulation, as shown in FIGS. 2, C. The iMC cells were also able to effectively phagocytose FITC dye-labeled E.coli bacteria in vitro (see references [2] and [3 ]), as shown in FIGS. 2, D.
Example 3: the implantation of transdifferentiated cells in vivo has proved to have therapeutic effect on neutropenia.
Cyclophosphamide CTX mimics the induction of neutropenia in mice (see reference [4 ] for steps and methods]) And infuse the iMC cell strategy as shown in fig. 3, a. In vivo infusion of iMC in mice (2X 10 on days 2, 4, 6 after single large dose 300mg/kg body weight intraperitoneal injection of CTX induced neutropenia, respectively 6 The infusion of iMC cells into recipient mice by tail vein injection) effectively improved the symptoms of weight loss, as shown in fig. 3, b. Infusion of iMC in mice effectively ameliorated neutropenia symptoms as shown in fig. 3, c. Rosa26-STOP-iDTR; treatment of Diphtheria Toxin (DT) induced neutropenia in mice by Lyz-Cre genetic mice (see reference [5 for steps and methods)]) And infuse iMC cell strategy (100 ng DT per mouse, once every 3 days, three total times, 2 x 10 continuously on days 11, 12, 13 after induction of neutropenia) 6 icc cells were infused into recipient mice by tail vein injection) as shown in fig. 3, d. Infusion of iMC in mice effectively ameliorated weight loss symptoms, as shown in fig. 3, e. Infusion of iMC in mice effectively ameliorated neutropenia symptoms as shown in fig. 3, f. Normal mice were infused with iMC (1 x 10) at a frequency of 1 month over 4 months 6 icc cells per mouse), the proportion of various cells in the peripheral blood of the recipient mice did not change significantly for long-term detection, as shown in fig. 3, g. Long-term multiple infusions of iMC bone marrow and spleen did not change significantly, as shown in fig. 3, h. The anatomical pathology of liver, lung and colon parts in the iMC body is not changed significantly after long-term repeated infusion, as shown in figures 3 and I.
It will be appreciated by persons skilled in the art that although the invention has been specifically described with reference to the above embodiments, the invention is not limited to these specific embodiments. Based on the methods and technical solutions taught by the present invention, those skilled in the art can make appropriate modifications or improvements without departing from the spirit of the present invention, and the equivalent embodiments thus obtained are within the scope of the present invention.
Reference to the literature
1.Batta,K.,Florkowska,M.,Kouskoff,V.&Lacaud,G.Direct reprogramming of murine fibroblasts to hematopoietic progenitor cells.Cell Rep 9,1871-1884(2014).
2.Amulic,B.,Cazalet,C.,Hayes,G.L.,Metzler,K.D.&Zychlinsky,A.Neutrophil function:from mechanisms to disease.Annu Rev Immunol 30,459-489(2012).
3.Petropoulos,M.,Karamolegkou,G.,Rosmaraki,E.&Tsakas,S.Hydrogen peroxide signals E.coli phagocytosis by human polymorphonuclear cells；up-stream and down-stream pathway.Redox Biol 6,100-105(2015).
4.Wang,S.L.,Lee,J.J.&Liao,A.T.Chemotherapy-induced neutropenia is associated with prolonged remission duration and survival time in canine lymphoma.Vet J 205,69-73(2015).
5.Wang,S.et al.Regulatory Innate Lymphoid Cells Control Innate Intestinal Inflammation.Cell 171,201-216(2017).
Claims (6)
1. Use of thymidine for inducing transdifferentiation of mammalian embryonic fibroblasts into neutrophils, said use being for non-disease treatment purposes.
2. The use of claim 1, wherein the mammal is a mouse.
3. An in vitro method of directly inducing transdifferentiation of mammalian embryonic fibroblasts into neutrophils, said method comprising the step of contacting said cells with thymidine as claimed in any one of the preceding claims.
4. Use of thymidine as the sole active ingredient or in combination with other active ingredients in the preparation of a medicament or kit for the treatment of neutropenia.
5. The use according to claim 4, wherein the neutropenia is neutropenia due to drug, radiation therapy, genetic, developmental defects, immunity, and/or infection.
6. The use of claim 5, wherein the neutropenia is accompanied by sepsis, shock, or multiple organ failure.
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| FI863971A0 (en) * | 1985-10-04 | 1986-10-01 | Chugai Pharmaceutical Co Ltd | PHARMACEUTICAL COMPOSITION ANALYZED WITH BEHINDLING AV LEUKOPENIA. |
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| CN105754935A (en) * | 2016-04-07 | 2016-07-13 | 浙江大学 | Induction medium for inducing transdifferentiation of fibroblast into adipocyte and application thereof |
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| WO2007016037A2 (en) * | 2005-07-27 | 2007-02-08 | Board Of Regents, The University Of Texas System | Methods for trans-differentiating cells |
| CN102380090A (en) * | 2010-08-31 | 2012-03-21 | 健能隆医药技术（上海）有限公司 | G-CSF (granulocyte-colony stimulating factor) dimmer applied to treat neutropenia |
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| FI863971A0 (en) * | 1985-10-04 | 1986-10-01 | Chugai Pharmaceutical Co Ltd | PHARMACEUTICAL COMPOSITION ANALYZED WITH BEHINDLING AV LEUKOPENIA. |
| CA2533926A1 (en) * | 2003-07-31 | 2005-03-24 | Endacea, Inc. | Methods and compositions for producing antigenic responses |
| CN105754935A (en) * | 2016-04-07 | 2016-07-13 | 浙江大学 | Induction medium for inducing transdifferentiation of fibroblast into adipocyte and application thereof |
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