Application of in vitro senescent cell model of mouse bone marrow hematopoietic stem / progenitor cells

Abstract

The present invention relates to the fields of biotechnology and medicine, and specifically to the application of an in vitro senescent cell model of mouse bone marrow hematopoietic stem / progenitor cells. The in vitro senescent cell model of mouse bone marrow hematopoietic stem / progenitor cells can be used to establish a pathological model of myelodysplastic syndrome and screen drugs for treating myelodysplastic syndrome. The present invention has found that HSPC senescence plays an important role in the pathogenesis of MDS. The changes in HSPC senescence are qualitatively similar to those of MDS.

Description

Technical Field

[0001] The invention relates to the fields of biotechnology and medicine, and to the application of an in vitro senescent cell model of mouse bone marrow hematopoietic stem / progenitor cells. Background Art

[0002] Although blood is a self-renewing tissue, hematopoiesis is still not immune to aging. Hematopoietic aging is manifested in the increase of malignant myeloid blood diseases (such as myelodysplastic syndrome, leukemia, etc.), body aging, immune dysfunction, etc.

[0003] Therefore, the development of drugs that delay hematopoietic aging has important theoretical significance and application value for the prevention and treatment of related diseases. The blood system of mammals is composed of various types of mature blood cells, such as red blood cells, T cells, B cells, etc. However, most of these blood cells are short-lived and need to be replaced by hematopoietic progenitor cells (HPCs) and eventually hematopoietic stem cells (HSCs). Previous studies have also shown that the aging of hematopoietic stem / progenitor cells is the primary factor in the occurrence of hematopoietic aging in the body.

[0004] The hematopoietic stem / progenitor cell aging model is an important platform for the study of hematopoietic aging. So far, the in vitro hematopoietic stem / progenitor cell aging models reported in the literature mainly include the following: the aging hematopoietic stem / progenitor cell model of aging animals, the aging hematopoietic stem / progenitor cell model induced by busulfan in vitro, the aging hematopoietic stem / progenitor cell model caused by radiation, and the aging hematopoietic stem / progenitor cell model induced by t-BHP in vitro. However, these methods all have limitations to varying degrees, such as cumbersome operation steps or poor aging effects.

[0005] In the previous work, the inventor established a cell model that can quickly and easily obtain a large number of aged HSPCs (patent application number: 201711157564.6, publication number CN107858330A) and disclosed a method for establishing an in vitro aging cell model of mouse bone marrow hematopoietic stem / progenitor cells, which can quickly obtain a large number of aged in vitro hematopoietic stem / progenitor cells. This model is a cell model that can quickly and easily obtain a large number of aged HSPCs, and it can even be said to be an in vitro HSPCs aging model that accelerates the aging of HSPCs in vivo to a certain extent.

[0006] Research suggests that among various blood diseases, including myelodysplastic syndromes (MDS), acquired aplastic anemia (AA), pure red cell aplasia (PRCA), acquired amegakaryocytic thrombocytopenia (AATP), paroxysmal nocturnal hemoglobinuria (PNH), etc., the disease most associated with hematopoietic aging may be MDS.

[0007] MDS is a group of acquired, heterogeneous, myeloid clonal blood diseases. Its basic lesion is the abnormal development of HSC / HSPC, which leads to ineffective hematopoiesis and high risk of transformation to malignant blood diseases such as acute myeloid leukemia (AML). MDS is highly heterogeneous, with great differences in clinical manifestations and a poor overall prognosis. Its pathogenic mechanism is complex and has not yet been clarified. Clinical data show that the age of onset of MDS is on the rise, with an average age of 77 years. However, the pathogenesis of MDS is complex and has not yet been clarified.

[0008] If a cell model can be found to establish a pathological model of myelodysplastic syndrome, it can be used to study the disease mechanism and develop and screen related drugs. Summary of the invention

[0009] The present invention aims to provide a cytopathological model of MDS, and the in vitro senescent cell model of mouse bone marrow hematopoietic stem / progenitor cells (HSPC) can be used to establish a pathological model of myelodysplastic syndrome.

[0010] CFU-Mix and SA-β-Gal were used to evaluate HSPC aging. It was found that the SA-gal positive rate was significantly increased, the number of CFU-Mix colonies was significantly reduced, and the expression of P21 mRNA, which is closely related to aging, was significantly increased, whether in the HSPC of the MDS patient group or in the HSPC of the mouse aging model. "Aging" was a term significantly enriched in the RNA-seq GO analysis, and "Cellular Senescence" was a pathway significantly enriched in the RNA-seq KEGG analysis. Therefore, this mouse hematopoietic stem cell aging cell model can be used to establish a pathological model of myelodysplastic syndrome and can be used to screen drugs for the treatment of myelodysplastic syndrome. Therefore, this model can be used as a pathological model of MDS.

[0011] Technical solution of the present invention:

[0012] The in vitro senescent cell model of mouse bone marrow hematopoietic stem / progenitor cells can be used to establish a pathological model of myelodysplastic syndrome.

[0013] The in vitro senescent cell model of mouse bone marrow hematopoietic stem / progenitor cells can be used to screen drugs for the treatment of myelodysplastic syndrome.

[0014] Preferably, the method for establishing an in vitro senescent cell model of mouse bone marrow hematopoietic stem / progenitor cells, such as CN107858330A, comprises the following steps:

[0015] Mouse bone marrow cells are subjected to lineage negative enrichment and CD117 positive screening to separate and obtain mouse bone marrow stem / progenitor cells, wherein hematopoietic progenitor cells are the main ones; the lineage negative enrichment is CD5, CD45R / B220, CD11b, Anti-Gr-1, 7-4 and Ter-119 negative enrichment; and the cells are cultured for 5 to 10 days in a stem cell culture medium containing IL3, IL6 and SCF.

[0016] In the stem cell culture medium, the content of IL3 is 8-20 ng / mL, the content of IL6 is 8-20 ng / mL, and the content of SCF is 25-60 ng / mL.

[0017] Preferably, in the stem cell culture medium, the content of IL3 is 9-12 ng / mL, the content of IL6 is 9-12 ng / mL, and the content of SCF is 27-35 ng / mL.

[0018] During the culture process, the stem cell culture medium is replaced every 2 to 3 days. The stem cell culture medium is a serum-free culture medium.

[0019] The stem cell culture medium also contains streptomycin and penicillin.

[0020] Clinical data show that the age of onset of MDS is on the rise, with the median age of 77 years. However, the pathogenesis of MDS is complex and has not yet been clarified. We have clarified the role of HSPC aging in the pathogenesis of MDS, and combined the HSPCs of MDS patients in this study. The results showed that the HSPC SA-gal positivity rate of MDS patients was significantly increased, and the number of CFU-Mix colonies was significantly reduced. The expression of P21 mRNA, which is closely related to aging, was significantly increased in both the MDS group and the mouse hematopoietic stem / progenitor cell aging model group. RNA-seq data showed that aging was one of the "Terms" with significant differences in GO analysis, and the cellular aging pathway was one of the "Pathways" with significant differences in KEGG analysis. This is the same as the performance of the in vitro aging model of mouse hematopoietic stem / progenitor cells.

[0021] Therefore, the mouse HSPC aging cell model has the same aging mechanism as the HSPC cells of MDS patients. The present invention uses the mouse hematopoietic stem and progenitor cell aging model as an MDS pathological model, which can be used to explore the aging mechanism of hematopoietic stem and progenitor cells in the blood disease MDS, in order to provide experimental support for the clinical development of drugs for the prevention and treatment of hematopoietic aging-related blood diseases MDS. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Representative bone marrow smear of a patient with MDS

[0023] Figure 2 SA-β-gal staining results of normal group HSPC cells, MDS group HSPC cells and mouse hematopoietic stem and progenitor cell senescence cell model HSPC cells

[0024] Figure 3 CFU-Mix test results of normal group HSPC cells, MDS group HSPC cells and mouse hematopoietic stem and progenitor cell senescence cell model HSPC cells

[0025] Figure 4 GO analysis bar chart of RNA-seq in MDS patients shows 25 terms enriched in significantly differentially expressed genes

[0026] Figure 5 Bubble chart for GO analysis in RNA-seq of MDS patients

[0027] Figure 6 25 pathways enriched in KEGG analysis bar graph of significantly differentially expressed genes in RNA-seq of MDS patients

[0028] Figure 7 Bubble chart for KEGG analysis in RNA-seq of MDS patients

[0029] Figure 8 Changes in the cellular senescence pathway in HSPCs of MDS patients

[0030] Fig. 9 Joint analysis of RNA-Seq data of MDS patients and normal subjects (in vivo) (MvsN), and RNA-Seq data of mouse hematopoietic stem / progenitor cell in vitro aging model (Aging Model hspc) and young hspc (in vitro) (YoungvsD8): 9A is the number of overlapping genes, 9B is the number of overlapping GO Terms, and 9C is the number of overlapping KEGG pathways DETAILED DESCRIPTION

[0031] Example 1

[0032] Representative bone marrow smears of MDS patients Figure 1As shown (Swiss stain, 10×100× oil), the results showed: 1. Bone marrow nucleated cells proliferated significantly; 2. Granulocyte proliferation was obvious, primitive granulocytes accounted for 16.0%, the proportion of neutrophils increased, and the developmental morphology was abnormal, such as double-layer tumors, vacuoles, and imbalanced development of the nucleus and cytoplasm; 3. Erythrocyte proliferation decreased, and abnormal developmental morphology such as nuclear budding was observed. MDS patients have peripheral blood cell reduction for more than 4 months, excluding other diseases such as infection and immunity that can cause blood cell reduction and developmental abnormalities. The abnormal proportions of granulocytes and erythrocytes in the bone marrow are both above 10%; the diagnosis is MDS (EB-2).

[0033] CFU-Mix and SA-β-Gal were used to evaluate HSPC aging. The HSPCs of MDS patients were studied and it was found that the Gal positive rate of HSPCs of MDS patients increased significantly and the number of CFU-Mix colonies decreased significantly, which is consistent with the mouse hematopoietic stem / progenitor cell aging model established by the CN107858330A method. Figure 2 and Figure 3 shown.

[0034] SA-β-gal is a marker of senescence and can produce blue staining in the cytoplasm of senescent cells. Our results showed that the proportion of SA-β-gal-positive cells in the MDS group was significantly higher than that in the control group, P<0.01, P<0.05. Blue-stained cells (senescent cells) were larger than non-senescent cells, and most of them showed morphological abnormalities, which was consistent with the results of the mouse hematopoietic stem / progenitor cell senescence model (after 8 days of culture).

[0035] The CFU-Mix assay is used to understand the frequency and growth characteristics of hematopoietic progenitor cells at different stages of development. Individual progenitor cells, called colony forming units (CFUs), proliferate and differentiate to form discrete cell populations or colonies containing identifiable progeny. Each colony is derived from a single ancestor. Therefore, the number and type of colonies counted in the CFU assay provides information about the frequency and type of progenitor cells in the original cell population and their ability to proliferate and differentiate. Figure 3 It can be seen that the cells in the MDS patient group did not form colonies compared to the colonies formed by normal HSPCs, which were clearly visible to the naked eye, indicating that the colony formation ability and multidirectional differentiation ability of HSPCs were significantly reduced. This suggests that the proliferation and differentiation ability of HSPCs in the MDS group was significantly lower than that in the control group, which is also consistent with the results of the mouse hematopoietic stem / progenitor cell aging model.

[0036] Example 2 RNAomics experiment

[0037] The RNA-Seq data of MDS patients vs normal subjects (denoted as MvsN) and the RNA-Seq data of aging model HSPCs vs young HSPCs (in vitro) (denoted as Youngvs8D) were jointly analyzed.

[0038] The analysis found that the aging pathway was a significantly different pathway enriched in the differentially expressed genes in normal HSPCs and HSPCs of MDS patients, whether in the GO database or the KEGG database.

[0039] RNA-seq analysis of MDS patients showed that aging was one of the terms with significant differences in GO analysis (e.g. Figure 4 , 5 ), the cellular senescence pathway is one of the "Pathways" with significant differences in KEGG analysis (such as Figure 6 , 7 The results of the top 25 genes with different expressions in the Aging Term and Cellular Senescence pathways are shown in Tables 1 and 2. The GO analysis bar chart and bubble chart of RNA-seq in MDS patients are shown in Figure 4 and Figure 5 , which are the 25 terms enriched by significantly differentially expressed genes, among which Aging is located. The bar graph and bubble graph of KEGG analysis in RNA-seq of MDS patients are shown in Figure 6 and Figure 7 , which are the 25 pathways enriched with significantly differentially expressed genes, among which the Cellular Senescence pathway is located. Figure 8 shown.

[0040] Tab.1 Top25 differently expressed genes in GO aging Term

[0041] Table 1 The 25 most significantly expressed genes in the GO aging stage

[0042]

[0043]

[0044]

[0045] Tab.2 Top25 differently expressed genes in KEGG cellular senescencepathway

[0046] Table 2 The 25 most significantly expressed genes in the KEGG cell senescence pathway

[0047]

[0048]

[0049] We combined the RNA-Seq data of MDS (M) and normal cells (in vivo) (N) (MvsN), as well as the RNA-Seq data of the mouse hematopoietic stem / progenitor cell in vitro aging model (Aging Model HSPC) (D8) and young HSPC (in vitro) (Young) cultured for 8 days (YoungvsD8). The joint analysis results showed that the number of overlapping genes was 1444 ( Fig. 9 A), the number of overlapping Go Terms is 412 ( Fig. 9 B), the number of overlapping KEGG pathways is 22 ( Fig. 9 C). The expression of P21 mRNA, which is closely related to aging, was significantly increased in both the MDS group and the aging model group (P<0.05), as shown in Table 3 and Fig. 9 . The P53 signaling pathway was a pathway with significantly enriched differential genes in both the MDS group and the aging model group (P<0.05), as shown in Table 4. Therefore, our in vivo and in vitro studies consistently showed that the mRNA expression of P21, which is closely related to aging, was significantly increased. Studies have shown that the upregulation of p21 is the main response to induce cell senescence. P53 inhibits aging by inhibiting the expression of P53 downstream target gene p21 / CDKN1A.

[0050] Table.3 Comparison of P21 mRNA expression in RNA-seq analysis

[0051] Table 3 Comparison of P21 mRNA expression in RNA-seq analysis

[0052]

[0053] Table.4 Comparison of P53 Pathway in RNA-seq analysis

[0054] Table 4 Comparison of P53 pathways analyzed by RNA-seq

[0055]

Claims

1. Application of an in vitro senescent cell model of mouse bone marrow hematopoietic stem / progenitor cells in establishing a pathological model of EB2 myelodysplastic syndrome, characterized in that: The method for establishing an in vitro senescent cell model of mouse bone marrow hematopoietic stem / progenitor cells comprises the following steps: Mouse bone marrow cells are enriched by lineage negative, and then screened by CD117 positive, to separate and obtain mouse bone marrow stem / progenitor cells; the lineage negative enrichment is CD5, CD45R / B220, CD11b, Anti-Gr-1, 7-4 and Ter-119 negative enrichment; Culture the cells in stem cell culture medium containing IL3, IL6, and SCF for 5 to 10 days; In the stem cell culture medium, the content of IL3 is 9-12 ng / mL, the content of IL6 is 9-12 ng / mL, and the content of SCF is 27-35 ng / mL.

2. The use according to claim 1, characterized in that: During the culture process, the stem cell culture medium was replaced every 2 to 3 days.

3. The use according to claim 1, characterized in that: The stem cell culture medium is a serum-free culture medium.

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