CN111471652A - Application of Beclin1 in maintaining embryo hematopoiesis - Google Patents

Application of Beclin1 in maintaining embryo hematopoiesis Download PDF

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CN111471652A
CN111471652A CN202010459772.7A CN202010459772A CN111471652A CN 111471652 A CN111471652 A CN 111471652A CN 202010459772 A CN202010459772 A CN 202010459772A CN 111471652 A CN111471652 A CN 111471652A
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beclin1
hematopoietic stem
hematopoietic
progenitor cells
embryo
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王建荣
袁娜
魏雯
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Suzhou University
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Suzhou University
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Abstract

The invention discloses an application of Beclin1 in maintaining embryo hematopoiesis, in particular to the protection of the homeostasis of embryonic hematopoietic stem and progenitor cells. The invention firstly focuses on the effect of Beclin1 on hematopoietic stem progenitor cells and utilizes Beclin1f/f(ii) a The Vav-iCre mouse model proves that Beclin1 plays an important role in the process of mouse embryo hematopoiesis by means of flow technology, colony experiments, transplantation experiments and the like. The Beclin1 is proved to be capable of maintaining the normal hematopoiesis of the embryo, particularly protecting the steady state of embryonic hematopoietic stem cells and playing a role in vital importance, so that Beclin1 is expected to be applied to the maintenance of the normal hematopoiesis of the embryo in the future, particularly to the protection of the steady state of embryonic hematopoietic stem and progenitor cells, and has great significance in the maintenance of the normal hematopoiesis of the embryo.

Description

Application of Beclin1 in maintaining embryo hematopoiesis
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to application of Beclin1 in maintaining embryo hematopoiesis.
Background
Beclin1 is a 60-KDa coiled-coil protein, originally discovered as a protein that binds to the anti-apoptotic protein Bcl-2. Subsequently, Beclin1 was found to be an autophagy homologous gene of yeast atg6/vps30 in mammals, having an important role in autophagy and in inhibiting tumorigenesis. Our preliminary laboratory studies found that Beclin1 was mutated in the human leukemia cell line and in the acute phaseCD34 in patients with myeloid or acute lymphoid leukemia+CD38-The hematopoietic stem cells expressed low levels of Beclin1, suggesting that Beclin1 may play an important role in blood physiology. However, the effect of Beclin1 in the whole blood system is only reported, only the reported research objects only aim at a certain type of specific blood cells, such as platelets, CD4+ lymphocytes and the like, the used models are limited by drosophila, Beclin1 heterozygous mice and the like, and the effect of Beclin1 in the whole blood physiology is not yet clarified.
Since the discovery of Beclin1, which is a star protein, the research on the Beclin1 mainly focuses on autophagy, and Beclin1 can form a PI3KC3 complex with Atg14, UVRAG, VPS34 and the like, and plays a key role in autophagy formation and autophagy initiation. Meanwhile, with the progress of research, a plurality of proteins binding to the Beclin1 are excavated, such as Bif-1, ATM, Huntingtin, Ambra1 and the like, which indicates that the Beclin1 plays a role in apoptosis, cell cycle, neurodegenerative diseases, tumor and anti-aging, and the research is carried out by connecting the effects of the Beclin1 on autophagy. Thus, to date, many studies have shown that Beclin1, a key gene for autophagy, plays a central role in autophagy and links autophagy to related biological processes.
Atg5 and Atg7 are two important autophagy key genes and play important roles in autophagy. Studies have shown that Atg5-/-And Atg7-/-Mice can be born normally and are phenotypically normal at birth. However, after the Beclin1 is constitutively deleted in whole body, the mice die in the early embryonic development stage at E8.5 days, and the tumor generation rate of the mice with Beclin1 heterozygous deletion is increased, which indicates that the Beclin1 possibly plays more important functions besides autophagy. Beclin1 has been reported to play a role in chromosome aggregation, and our pre-laboratory studies found that nuclear-localized Beclin1 can repair DNA damage and that this pathway is independent of autophagy. In addition, in recent years, reports of the immune effect of Beclin1 have emerged, such as Beclin1 maintaining immune homeostasis and enhancing listeria resistance, Beclin1 binding cGAThe S DNA receptor regulates endogenous antibacterial immune response, and the Beclin1 is a key regulatory factor for immunotherapy of pre-B cell lymphoma. The discovery of the new functions of Beclin1 lays a certain theoretical foundation for searching new functions of Beclin1 except autophagy.
Disclosure of Invention
The invention aims to provide application of Beclin1 in maintaining embryo hematopoiesis.
In order to achieve the technical purpose and achieve the technical effect, the invention is realized by the following technical scheme:
the invention finds out the key action of the Beclin1 on the maintenance of the embryo hematopoiesis, so that the Beclin1 is expected to be applied to the maintenance of the normal hematopoiesis of the embryo, and particularly to the protection of the homeostasis of embryonic hematopoiesis stem progenitor cells.
To verify the role of Beclin1 in the hematopoietic system, the inventors of the present invention first constructed Beclin1f/fVav is a promoter specifically expressed in a hematopoietic system, and at present, Vav-iCre tool mice are recognized as tool mice for researching the hematopoietic system, and are adopted by a plurality of researches, and then the Vav-iCre tool mice are utilized to obtain Beclin1 after being hybridized with Beclin1f/f mice with L oxp locus genef/f(ii) a The Vav-iCre mouse model can realize the constitutive knockout of the Beclin1 gene in a hematopoietic system, thereby successfully establishing the mouse model of the Beclin1 constitutive deletion in the hematopoietic system.
The inventor of the invention finds no baby mouse with Beclin1 homozygous deletion in the offspring by counting the genotype frequency of the offspring mouse, and indicates that the mouse embryo dies. Whereas heterozygous deleted mice survived normally and were phenotypically indistinguishable from wild type. After further statistics of the genotype frequencies of fetal mice at different embryonic stages, it was found that there were still KO mice at 18.5 days (day before birth) of the embryonic stage, but there was some mortality, indicating that the fetal mice died at perinatal stage.
In order to further understand the change of peripheral blood image after Beclin1 deletion, the routine detection of three-classification blood discovers that Red Blood Cells (RBC), Hemoglobin (HGB) and Hematocrit (HCT) are all obviously reduced, the number of White Blood Cells (WBC), lymphocytes (lymphocytote, L YM) and platelets (patelet, P L T) is increased, and the anemia and certain inflammation of the mice are indicated.
The results all suggest that homozygous deletion of the hematopoietic system Beclin1 poses life-threatening conditions to mice, affects normal embryonic hematopoiesis of mice, and causes perinatal death and phenotypic abnormalities of mice.
Whether the hematopoietic stem progenitor cells can be normally proliferated and differentiated and whether hematopoietic reconstitution can be completed is an index for judging the functions of the hematopoietic stem progenitor cells and is also the key for detecting whether the mice can be normally hematopoietic after Beclin1 is deleted.
The invention discloses a method for judging whether hematopoietic stem progenitor cells can be proliferated and differentiated normally in vitro colony generation experiments, and the inventor discovers that after Beclin1 is deleted, the in vitro proliferation and differentiation capacity of mouse hematopoietic stem progenitor cells is completely damaged, and the in vitro proliferation and differentiation capacity is reduced.
The inventor of the invention further adopts a Rescue transplantation experiment to explore the short-range hematopoietic capacity of the hematopoietic stem progenitor cells in vivo, and the experimental result shows that the short-term hematopoietic capacity of the mouse hematopoietic stem progenitor cells is damaged after Beclin1 is deleted.
The inventor of the invention continuously utilizes competitive transplantation experiments to explore the long-term hematopoietic capacity of the hematopoietic stem progenitor cells, and the experimental result shows that the long-term hematopoietic capacity of the mouse hematopoietic stem progenitor cells is completely damaged after Beclin1 is deleted.
The above results all suggest that homozygous deletion of the hematopoietic system Beclin1 results in impaired function of mouse hematopoietic stem progenitor cells, specifically in decreased colony formation of mouse hematopoietic stem progenitor cells, impaired short term hematopoietic capacity of mouse hematopoietic stem progenitor cells, and impaired long term hematopoietic capacity of mouse hematopoietic stem progenitor cells.
The inventor of the invention stains the surface markers of the fetal liver hematopoietic progenitor cells of wild type and Beclin 1-deleted mice, and utilizes flow cytometry to analyze the proportion and the absolute quantity of various cells in a statistical manner. The experimental result shows that the absolute number of the hematopoietic stem progenitor cells is obviously increased after Beclin1 is deleted, abnormal hyperplasia is firstly carried out, and the failure of the hematopoietic stem progenitor cells is caused due to the failure of normal proliferation and differentiation.
Because Beclin1 lacks abnormal proliferation of mouse fetal liver hematopoietic stem progenitor cells, the inventor of the invention utilizes Ki67 to stain and utilizes flow cytometry to detect the proliferation condition of the mouse fetal liver hematopoietic stem progenitor cells, finds that the proportion of Ki67 positive cells is obviously increased after Beclin1 lacks, also proves the result of failure after abnormal proliferation of the mouse hematopoietic stem progenitor cells lacking in hematopoietic system Beclin1, and the hematopoietic stem progenitor cells do not have obvious apoptosis at the moment.
Hematopoietic stem cells maintain normal hematopoiesis by self-renewal and downward differentiation, while some cells are in the G0 stage, i.e., resting state, to maintain stable hematopoiesis. Although fetal liver hematopoietic stem cells are in a highly cyclic renewal process compared to adult bone marrow hematopoietic stem cells, abnormalities in the cycle affect the self-renewal and differentiation of hematopoietic stem cells, resulting in the disruption of their homeostasis.
The inventor of the invention utilizes Ki67 and DAPI double staining to detect the cycle of fetal liver hematopoietic stem progenitor cells of a mouse with Beclin1 deletion, and counts the proportion of G0, G1 and S/G2M. The experimental result shows that after Beclin1 is deleted, the phase proportion of G0 and G1 of the hematopoietic stem progenitor cells is reduced, and the phase proportion of S/G2M is obviously increased.
The cell cycle is regulated by more genes, wherein p27 and p57 belong to the CKI family, and if the genes are down-regulated, the cell cycle is accelerated, and the cell cycle is blocked by the up-regulation. The inventor of the invention detects the transcription expression level of p27 and p57 by utilizing a Qpcr technology, and finds that the hematopoietic stem-progenitor cell cycle negative regulatory factors p27 and p57 are reduced and the cell cycle is accelerated after Beclin1 is deleted, which is consistent with the cycle result.
The results all indicate that homozygous deletion of the hematopoietic system Beclin1 causes the function of the mouse hematopoietic stem progenitor cells to be abnormal, particularly causes the mouse hematopoietic stem progenitor cells to be exhausted after abnormal proliferation, and causes the cycle of the mouse hematopoietic stem progenitor cells to be abnormal.
To sum up, we found that 1) homozygous mice die in perinatal stage and have abnormal phenotype after deletion of the hematopoietic system Beclin1 biallelic genes; 2) beclin1 homozygous deletion mouse hematopoietic stem and progenitor cell dysfunction, impaired hematopoietic reconstitution ability; 3) homozygous mouse embryos are hematopoietic and hematopoietic stem and progenitor cells proliferate after the deletion of the bicell 1 bi-allele of the hematopoietic system; 4) the loss of the hematopoietic system Beclin1 interferes with the cycle of hematopoietic stem and progenitor cells.
These results are sufficient to show that the presence of Beclin1 in the hematopoietic system plays a vital role in maintaining normal hematopoiesis of the embryo, and that Beclin1 in the hematopoietic system is involved in maintaining embryonic hematopoiesis, especially in protecting embryonic hematopoietic stem and progenitor cell homeostasis.
The invention has the beneficial effects that:
the hematopoietic system is well-defined, with the most upstream cell type being hematopoietic stem progenitor cells, which are subsequently differentiated into downstream mature cells such as erythrocytes, lymphocytes, platelets, macrophages, etc. All downstream cells of the hematopoietic system are derived from the proliferation and differentiation of hematopoietic stem and progenitor cells.
The present invention was first focused on the effect of Beclin1 on hematopoietic stem progenitor cells, since the number of hematopoietic stem progenitor cells is only 104The limit of the number increases the difficulty of research, so the invention utilizes Beclin1f/f(ii) a The Vav-iCre mouse model proves that Beclin1 plays an important role in the process of mouse embryo hematopoiesis by means of flow technology, colony experiments, transplantation experiments and the like.
The Beclin1 can maintain the normal hematopoiesis of the embryo, particularly can protect the steady state of embryonic hematopoietic stem cells, and plays a vital role, so that Beclin1 is expected to be applied to the maintenance of the normal hematopoiesis of the embryo in the future, particularly to the protection of the steady state of embryonic hematopoietic stem and progenitor cells, and has great significance in the maintenance of the normal hematopoiesis of the embryo.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings. The detailed description of the present invention is given in detail by the following examples and the accompanying drawings.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the invention without limiting the invention. In the drawings:
FIG. 1 is a graph showing the result of the experiment of the present invention for studying the lethal and impaired hematopoiesis of the mouse embryo lacking the hematopoietic system Beclin1, wherein,
FIG. 1a shows the result of genotyping mouse tail in offspring;
FIG. 1b is a graph showing the results of the genotype frequencies of embryos at different stages;
FIG. 1c shows day E14.5 fetal liver CD45+The result graph of the Beclin1 expression is identified by the blood cell transcription and protein level;
FIG. 1d is a graph showing the results of E18.5 day non-hematopoietic tissue assay for Beclin1 expression;
FIG. 1E shows representative pictures of E14.5 day and E18.5 day embryos;
FIG. 1f is a graph showing statistics of fetal liver counts for E14.5 days and E18.5 days;
FIG. 1g is a graph showing statistics on E18.5 day embryo body weight;
FIG. 1H shows representative pictures of H & E staining of fetal liver at E18.5 days;
FIG. 1i is a graph showing the statistical results of the three classification tests of the peripheral blood of the E18.5 day embryo.
FIG. 2 is a graph showing the experimental results of the study of the impaired ability of the Beclin1 to proliferate and differentiate hematopoietic stem progenitor cells in vitro in the hematopoietic system.
FIG. 3 is a graph showing the results of experiments conducted in the present invention to study the impaired short-term hematopoietic capacity of mice after deletion of the hematopoietic system Beclin 1.
FIG. 4 is a graph showing the results of the experiment for studying the impaired long-term hematopoietic capacity of mice after deletion of the hematopoietic system Beclin1 in accordance with the present invention, wherein
FIG. 4a shows a schematic representation of competitive transplantation in mice;
FIG. 4b shows peripheral blood donors at 4, 8, 12, 16 weeks of transplantationCD45.2+A cell proportion result chart;
FIG. 4c shows peripheral blood B cells (B220) transplanted for 4, 8, 12, 16 weeks+) Middle CD45.2+A cell proportion result chart;
FIG. 4d shows peripheral blood T cells (CD 3) transplanted for 4, 8, 12, 16 weeks+) Middle CD45.2+A cell proportion result chart;
FIG. 4e shows peripheral blood marrow cells (Gr 1) transplanted at 4, 8, 12, 16 weeks+CD11b+) Middle CD45.2+A cell proportion result chart;
FIG. 4f shows CD45.2 in individual blood cells from bone marrow of recipient mice transplanted for 16 weeks+Cell ratio results.
FIG. 5 is a graph showing the results of the study of abnormal postproliferative failure of mouse hematopoietic stem progenitor cells deficient in the hematopoietic system, Beclin1, in which,
FIG. 5a shows a schematic representation of E14.5 day flow analysis;
FIG. 5b is a graph showing statistics of the number of hematopoietic progenitor cells in each type at day E14.5;
FIG. 5c is a graph showing the statistics of absolute numbers of hematopoietic progenitor cells at E14.5 days;
FIG. 5d shows a schematic representation of E16.5 day flow analysis;
FIG. 5E is a graph showing the statistics of the ratio of hematopoietic progenitor cells for each type at day E16.5;
FIG. 5f is a graph showing the statistics of the ratio of hematopoietic progenitor cells of each type at day E16.5.
FIG. 6 is a graph showing the results of the experiments of the present invention for examining proliferation and apoptosis of hematopoietic stem and progenitor cells deficient in the hematopoietic system, Beclin1,
FIG. 6a shows day E14.5 fetal liver hematopoietic stem progenitor Ki67+A cell proportion result chart;
FIG. 6b is a graph showing the results of apoptosis (annexin V + cell ratio) of fetal liver hematopoietic stem progenitor cells at E14.5 days.
FIG. 7 is a graph showing the statistics of the ratio of G0, G1 and S/G2M after E14.5 days of fetal liver hematopoietic stem and progenitor cell cycle detection by using Ki67 and DAPI double staining.
FIG. 8 is a graph showing the results of experiments of the present invention to study the decrease of the transcriptional levels of the hematopoietic stem progenitor cells p27 and p57 deleted from the hematopoietic system Beclin 1.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in detail below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Through various experiments, the invention discovers that Beclin1 has a key effect on maintaining embryo hematopoiesis, so that Beclin1 is expected to be applied to maintaining normal hematopoiesis of embryos, and particularly to protecting the homeostasis of embryonic hematopoiesis stem and progenitor cells. The specific experimental results are as follows:
1. hematopoietic Beclin1 deficient mice die perinatally and have an abnormal phenotype
To verify the role of Beclin1 in the hematopoietic system, the inventors of the present invention first constructed Beclin1f/fVav is a promoter specifically expressed in a hematopoietic system, and at present, a Vav-iCre tool mouse is a tool mouse generally recognized to be used for researching the hematopoietic system, and is adopted by a plurality of researches, and then the Vav-iCre tool mouse is utilized to be matched with Beclin1 with L oxp locus genef/fAfter hybridization of the mice, Beclin1 was obtainedf/f(ii) a The Vav-iCre mouse model can realize the constitutive knockout of the Beclin1 gene in a hematopoietic system.
Referring to FIGS. 1a, 1c and 1d, the inventors of the present invention identified the progeny rat tail gene and paired the Beclin1 from the transcription level, protein level using Qpcr, Western blottingf/f(ii) a Vav-cre smallThe success of the murine model establishment was verified to demonstrate that a murine model of the hematopoietic constitutive deletion Beclin1 has been successfully established.
The inventor of the invention surprisingly finds that no mice homozygous deletion of Beclin1 exist in the offspring by counting the genotype frequency of the offspring mice, and shows that the mice are dead in embryo, and the mice with heterozygous deletion can normally survive and have no difference in phenotype from the wild type. After further statistics of the genotype frequencies of fetal mice at different embryonic stages, it was found that, as shown in FIG. 1b, there were still KO mice at 18.5 days (day before birth) of the embryonic stage, but they had a certain mortality rate, indicating that the fetal mice died at perinatal stage. The major organ for embryo hematopoiesis is fetal liver, and after the Vav promoter acts on E11.5 days, E14.5 days is the period that the embryo formally enters the hematopoietic stage of fetal liver, and the frequency of each genotype of the embryo at E14.5 days accords with the genetic law, so E14.5 days also serves as the time point of later research.
Meanwhile, the inventor of the invention discovers that KO embryo is in obvious anemia by observing the appearance of the embryo, the number of fetal liver cells as hematopoietic tissues is obviously reduced by observing the appearance of the embryo, the weight of the embryo is reduced by observing the appearance of the embryo, the number of fetal liver cells as hematopoietic tissues is obviously reduced by observing the appearance of the embryo, the weight of the embryo is lightened by observing the appearance of the embryo, the fetal liver as hematopoietic tissues is subjected to ischemia and necrosis by observing the H & E staining, the fetal liver as shown in the graph 1H, in order to further understand the change of peripheral blood images after the Beclin1 is lost, the inventor of the invention discovers that Red Blood Cells (RBC), Hemoglobin (HGB) and red corpuscle (HCT) are all obviously reduced by utilizing the three categories of blood routine detection, and the white blood cells (lymphocyte.
The results all suggest that homozygous deletion of the hematopoietic system Beclin1 poses life-threatening conditions to mice, affects normal embryonic hematopoiesis of mice, and causes perinatal death and phenotypic abnormalities of mice.
2. Mouse hematopoietic stem and progenitor cell function impairment caused by deficiency of the hematopoietic system Beclin1
Whether the hematopoietic stem progenitor cells can be normally proliferated and differentiated and whether hematopoietic reconstitution can be completed is an index for judging the functions of the hematopoietic stem progenitor cells and is also the key for detecting whether the mice can be normally hematopoietic after Beclin1 is deleted.
2.1 depletion of mouse hematopoietic Stem and progenitor cell colony formation by Beclin1 in the hematopoietic System
The in vitro colony generation assay is a method for determining whether hematopoietic stem and progenitor cells are able to proliferate and differentiate normally. The inventor of the invention plants CFU on E14.5 day fetal liver cells, and discovers that after culture, as shown in figure 2, after Beclin1 is deleted, the in vitro proliferation and differentiation capacity of the mouse hematopoietic stem and progenitor cells is completely damaged, and the in vitro proliferation and differentiation capacity is reduced.
2.2 Beclin1 depletion of hematopoietic Stem progenitor cells in mice with impaired short term hematopoietic Capacity
The proliferation and differentiation capacity of the mouse hematopoietic stem progenitor cells is reduced in vitro, and the inventor of the invention further adopts a Rescue transplantation experiment to explore the short-range hematopoietic capacity of the hematopoietic stem progenitor cells in vivo. Wild type and Beclin 1-deleted E14.5 day fetal liver cells are directly transplanted into 9.0Gy X-Ray lethally irradiated CD45.1 mice through tail veins, and the survival time of the receptor mice is counted. As described with reference to FIG. 3, it was found that all of the recipient mice transplanted with KO fetal hepatocytes died within 12 days, while the recipient mice transplanted with WT fetal hepatocytes could still normally survive. The experimental results show that after Beclin1 is deleted, the short-term hematopoietic capacity of the mouse hematopoietic stem progenitor cells is damaged.
2.3 the Beclin1 deficiency of the hematopoietic System mouse hematopoietic Stem progenitor cells impaired Long-term hematopoietic Capacity
The inventors of the present invention continued to explore the long term hematopoietic capacity of hematopoietic stem and progenitor cells using competitive transplantation experiments. As described in fig. 4a, wild-type, Beclin1 heterozygous deletion, Beclin1 homozygous deletion mouse E14.5 day fetal liver cells (CD45.2) and CD45.1 mouse bone marrow cells were mixed at 1: 1, and transplanted into 9.0Gy X-Ray lethally irradiated CD45.1 receptor mice through tail vein. At 4, 8, 12, and 16 weeks after transplantation, CD45.2 was analyzed by flow cytometry for each cell type (T, B, myeloid cell) in peripheral blood of recipient mice+The ratio was measured, and as shown in FIGS. 4b to 4e, it was found that the peripheral blood of the recipient mouse in which Beclin1 was transplanted was deficient in fetal liver cellsType has almost no CD45.2+Bone marrow from recipient mice was harvested at week 16 of transplantation, stained for individual hematopoietic cell surface markers, and analyzed by flow cytometry for statistical long-range hematopoietic stem cells (L T-HSC, &lTtTtranslation = L "&gTt L &lTt/T &gTt in-Sca1+c-kit+CD48-CD150+) Short-range hematopoietic stem cells (ST-HSC, &lTtTtransformation = L "&gTt L &lTt/T &gTt in-Sca1+c-kit+CD48+CD150+) Hematopoietic stem and progenitor cells (L SK, &lTtTtransformation = L "&gTt L &lTt/T &gTt in-Sca1+c-kit+) Isoprogenitors and downstream mature cells (B, T, myeloid cell Gr 1)+CD11b+) Medium donor source CD45.2+And (4) proportion. The results are consistent with peripheral blood, and as shown in FIG. 4f, the Beclin 1-transplanted fetal liver-deficient recipient mouse bone marrow cells were almost CD 45.2-free+And (4) proportion. The experimental results show that the long-term hematopoietic capacity of the mouse hematopoietic stem progenitor cells is completely damaged after the Beclin1 deletion.
The above results all suggest that homozygous deletion of the hematopoietic system Beclin1 results in impaired function of mouse hematopoietic stem progenitor cells, specifically in decreased colony formation of mouse hematopoietic stem progenitor cells, impaired short term hematopoietic capacity of mouse hematopoietic stem progenitor cells, and impaired long term hematopoietic capacity of mouse hematopoietic stem progenitor cells.
3. Mouse hematopoietic stem and progenitor cell abnormality with deficiency of the hematopoietic system Beclin1
3.1 Beclin1 depletion of hematopoietic Stem and progenitor cells in mice in the hematopoietic System abnormally increased postnatal failure
The inventor stains surface markers of fetal liver hematopoietic progenitor cells of E14.5 days and E16.5 days of wild type and Beclin 1-deleted mice, including L T-HSC, ST-HSC, L SK, pluripotent progenitor cells (MPP, &lTtTtranslation = L "&gTt L/T &gTt in-Sca1+c-kit+CD48-CD150-) Hematopoietic progenitor cells (HPC, &lTtT transfer = 'L' &gTt L &lTt/T &gTt in-Sca1+c-kit+CD48+CD150-) And then, the proportion and the absolute quantity of various cells are statistically analyzed by utilizing flow cytometry. The results of the experiments show that the absolute numbers of hematopoietic stem and progenitor cells are obviously increased after Beclin1 is deleted and abnormal proliferation is firstly carried out, as shown in figures 5a-5c, and the abnormal proliferation and differentiation are not normally carried out, and then the absolute numbers of hematopoietic stem and progenitor cells are obviously increased after Beclin1 is deleted and shown in figures 5d-5fResulting in depletion of hematopoietic stem and progenitor cells.
3.2 the Beclin1 deletion in the hematopoietic System mouse hematopoietic Stem and progenitor cells increased in proliferation without significant apoptosis
Because Beclin1 lacks abnormal proliferation of hematopoietic stem and progenitor cells of mouse fetal liver, the inventor of the invention utilizes Ki67 to stain and utilizes flow cytometry to detect the proliferation of the fetal liver hematopoietic stem and progenitor cells, as shown in fig. 6a, finds that the proportion of Ki67 positive cells is obviously increased after Beclin1 lacks, and also proves the result in experiment 3.1, namely, the hematopoietic stem and progenitor cells of mouse hematopoietic stem and progenitor cells of hematopoietic system Beclin1 are exhausted after abnormal proliferation, as shown in fig. 6b, and the hematopoietic stem and progenitor cells do not have obvious apoptosis at the moment.
3.3 depletion of mouse hematopoietic stem and progenitor cell cycle abnormalities in the hematopoietic System Beclin1
Hematopoietic stem cells maintain normal hematopoiesis by self-renewal and downward differentiation, while some cells are in the G0 stage, i.e., resting state, to maintain stable hematopoiesis. Although fetal liver hematopoietic stem cells are in a highly cyclic renewal process compared to adult bone marrow hematopoietic stem cells, abnormalities in the cycle affect the self-renewal and differentiation of hematopoietic stem cells, resulting in the disruption of their homeostasis.
The inventor of the invention utilizes Ki67 and DAPI double staining to detect the fetal liver hematopoietic stem progenitor cell cycle of E14.5 days, and counts the proportion of G0, G1 and S/G2M. The experimental result shows that, as shown in figure 7, after Beclin1 is deleted, the phase ratio of G0 and G1 of the hematopoietic stem progenitor cells is reduced, and the phase ratio of S/G2M is obviously increased.
The cell cycle is regulated by more genes, wherein p27 and p57 belong to the CKI family, and if the genes are down-regulated, the cell cycle is accelerated, and the cell cycle is blocked by the up-regulation. The inventor of the invention detects the transcription expression level of p27 and p57 by utilizing a Qpcr technology, and finds that the negative regulatory factors p27 and p57 of the hematopoietic stem-progenitor cell cycle are reduced and the cell cycle is accelerated after Beclin1 is deleted, which is consistent with the cycle result.
The results all indicate that homozygous deletion of the hematopoietic system Beclin1 causes the function of the mouse hematopoietic stem progenitor cells to be abnormal, particularly causes the mouse hematopoietic stem progenitor cells to be exhausted after abnormal proliferation, and causes the cycle of the mouse hematopoietic stem progenitor cells to be abnormal.
In conclusion, we found that the role of Beclin1 in the hematopoietic system is embodied as follows:
1) after deletion of the hematopoietic system Beclin1 biallelic genes, homozygous mice died in perinatal stage and were phenotypically abnormal;
2) beclin1 homozygous deletion mouse hematopoietic stem and progenitor cell dysfunction, impaired hematopoietic reconstitution ability;
3) homozygous mouse embryos are hematopoietic and hematopoietic stem and progenitor cells proliferate after the deletion of the bicell 1 bi-allele of the hematopoietic system;
4) the loss of the hematopoietic system Beclin1 interferes with the cycle of hematopoietic stem and progenitor cells.
The experimental results sufficiently show that the existence of the hematopoietic system Beclin1 plays a vital role, can maintain the normal hematopoiesis of the embryo and plays a role in maintaining the key role of the hematopoiesis of the embryo, so that Beclin1 is expected to be applied to the maintenance of the normal hematopoiesis of the embryo, and is especially expected to be applied to the protection of the homeostasis of embryonic hematopoietic stem and progenitor cells.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (2)

  1. Use of Beclin1 for the maintenance of embryonic hematopoiesis.
  2. 2. The use of Beclin1 in maintaining embryo hematopoiesis according to claim 1, wherein: the presence of Beclin1 in embryonic hematopoietic stem progenitor cells may be used to preserve the homeostasis of embryonic hematopoietic stem progenitor cells.
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WO2018193393A1 (en) * 2017-04-19 2018-10-25 St. Jude Children's Research Hospital Tumor suppression by modulation of non-canonical autophagy (lap) in myeloid cells
CN110308284A (en) * 2019-07-03 2019-10-08 苏州大学 Application of the Beclin1 as the super quick albumen of radiation in body

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CN107098965A (en) * 2017-04-14 2017-08-29 深圳人仁生物医药科技有限公司 Beclin1 mutains and its preparation method and application
WO2018193393A1 (en) * 2017-04-19 2018-10-25 St. Jude Children's Research Hospital Tumor suppression by modulation of non-canonical autophagy (lap) in myeloid cells
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