CN117250353A - Application of means for regulating and controlling programmed necrosis in preparing kit for diagnosing or delaying aging of blood system - Google Patents
Application of means for regulating and controlling programmed necrosis in preparing kit for diagnosing or delaying aging of blood system Download PDFInfo
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- CN117250353A CN117250353A CN202311523907.1A CN202311523907A CN117250353A CN 117250353 A CN117250353 A CN 117250353A CN 202311523907 A CN202311523907 A CN 202311523907A CN 117250353 A CN117250353 A CN 117250353A
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Abstract
The invention relates to the field of biotechnology, in particular to application of a means for regulating and controlling programmed necrosis in preparing a kit for diagnosing or delaying aging of a blood system. The means of controlling the programmed necrosis can effectively enhance the self-renewal capacity of the hematopoietic stem cells, thereby delaying the aging of the hematopoietic system.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to application of a means for regulating and controlling programmed necrosis in preparing a kit for diagnosing or delaying aging of a blood system.
Background
A significant feature of aging individuals is a reduced capacity for tissue regeneration and repair, and tissue-specific stem cell aging is a major cause of its reduced function, such as a reduced capacity of hematopoietic stem cells to reconstitute the blood system during aging. Hematopoietic stem cells continuously update all blood cells including B cells, T cells, erythrocytes, granulocytes, natural killer cells, monocytes, macrophages, etc. throughout the life cycle of the body. Young hematopoietic stem cells have balanced ability to produce various blood cells, however, with age, hematopoietic stem cells gradually decrease in self-renewal capacity and differentiation potential. Reduced hematopoietic stem cell function is a major cause of aging of the blood system, leading to the occurrence of age-related hematopoietic changes, such as: anemia, immune aging, and the occurrence of hematological malignancies. How to younger senescent hematopoietic stem cells is currently still a major scientific challenge.
Disclosure of Invention
The present invention is directed to solving at least one of the technical problems existing in the related art. Therefore, the invention provides the application of the means for regulating and controlling the programmed necrosis in preparing the kit for diagnosing or delaying the aging of the blood system, and the means for regulating and controlling the programmed necrosis can effectively enhance the self-updating capacity of the hematopoietic stem cells so as to delay the aging of the hematopoietic fluid system.
The invention provides an application of a means for regulating and controlling programmed necrosis in preparing a kit for diagnosing or delaying aging of a blood system.
Further, the means for modulating programmed necrosis comprises: by blocking the expression of specific genes, to combat the programmed necrosis.
Further, the means for modulating programmed necrosis comprises: blocking the activity of RIPK1 kinase during programmed necrosis.
Further, the means of blocking RIPK1 kinase activity during programmed necrosis include: inhibiting the activity of RIPK1 by means of gene intervention or small molecule intervention.
Further, the means for modulating programmed necrosis comprises: blocking the activity of RIPK3 kinase activity during programmed necrosis.
Further, the means of blocking RIPK3 kinase activity during programmed necrosis include: inhibiting the activity of RIPK3 by means of gene intervention or small molecule intervention.
Further, the means for modulating programmed necrosis comprises: blocking the activity of MLKL kinase during programmed necrosis.
Further, the means of blocking MLKL kinase activity during programmed necrosis include: inhibiting MLKL activity by means of genetic intervention or small molecule intervention.
Further, the kit for diagnosing aging of the blood system comprises at least one of an antibody for detecting RIPK1 background protein and phosphorylated RIPK1, an antibody for detecting RIPK3 background protein and phosphorylated RIPK3, and an antibody for detecting MLKL background protein and phosphorylated MLKL;
the kit for delaying aging of the blood system comprises at least one of hematopoietic stem cells with RIPK 1K 45A point mutation, hematopoietic stem cells with RIPK3 knocked out and hematopoietic stem cells with MLKL knocked out.
The above technical solutions in the embodiments of the present invention have at least one of the following technical effects:
the inventor of the invention discovers that the expression quantity of the programmed necrosis pathway in the aged hematopoietic stem cells is increased, and the programmed necrosis regulation means can effectively enhance the self-updating capacity of the hematopoietic stem cells, thereby delaying the aging of the hematopoietic fluid system. The kit prepared by adopting the means of regulating and controlling the programmed necrosis can be used for diagnosing and/or delaying the aging of the blood system and is very suitable for clinical application.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the invention, and other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the detection of changes in p-MLKL, MLKL, RIPK3, RIPK1 and Caspase-3 in young and senescent hematopoietic stem cells, respectively, in example 1 of the present invention.
FIG. 2 is a graph showing the detection of Caspase-8 changes in young and senescent hematopoietic stem cells, respectively, according to example 1 of the present invention.
FIG. 3 is the proportion of cell death after 12 hours of treatment with NT, T+S+Z and T+S+Z+RIPA-56 in B6 mouse HSC of example 2 of the present invention.
FIG. 4 is a wild type mouse and Ripk1 in example 2 of the present invention K45A Proportion of cell death after 12 hours of treatment of murine KSL cells with NT, T+S+Z and T+S+Z+RIPA-56.
FIG. 5 is a wild type mouse and Ripk3 in example 2 of the present invention -/- Proportion of cell death after 12 hours of treatment of murine KSL cells with NT, T+S+Z and T+S+Z+RIPA-56.
FIG. 6 is a wild type mouse and Mlkl in example 2 of the present invention -/- Proportion of cell death after 12 hours of treatment of murine KSL cells with NT, T+S+Z and T+S+Z+RIPA-56.
FIG. 7 is a wild type HSC transplant recipient mouse and Ripk1 in example 3 of the present invention K45A Overall leukocyte reconstitution rate in peripheral blood of HSC transplant recipient mice.
FIGS. 8 to 10 are wild type HSC transplant recipient mice and Ripk1, respectively, in example 3 of the present invention K45A Proportion of donor-derived cells in B cells, T cells and myeloid cells in the peripheral blood of the recipient mice.
FIG. 11 is a graph showing the utilization of wild-type HSC and Ripk1 in example 3 of the present invention K45A Donor source (CD 45.2) in peripheral blood of recipient mice at week 12 after the first round of HSC transplantation of HSC on recipient mice + ) B cells, T cells and myeloid cells.
FIG. 12 is a graph of the present invention using wild-type HSC and Ripk1 in example 3 K45A Overall leukocyte reconstitution rate in peripheral blood of recipient mice after the second round of transplantation of recipient mice by HSCs.
FIGS. 13 to 15 are respectively the use of wild-type HSC and Ripk1 in example 3 of the present invention K45A The HSCs donor-derived cell proportion in B cells, T cells and myeloid cells in the peripheral blood of the recipient mice after the second round of transplantation.
FIG. 16 is an embodiment of the present inventionWild-type HSC and Ripk1 in example 3 K45A Donor source in peripheral blood of recipient mice at week 12 after the second round of HSC transplantation (CD 45.2 + ) Proportion of B cells, T cells and myeloid cells in the cells.
FIG. 17 is wild-type HSC and Ripk3 in example 4 of the present invention -/- 20 Overall leukocyte reconstitution rate in peripheral blood of recipient mice after HSC transplantation.
FIGS. 18 to 20 are wild-type HSC and Ripk3, respectively, in example 4 of the present invention -/- 20 Proportion of donor-derived cells in B lymphocytes, T lymphocytes and myeloid cells in the peripheral blood of recipient mice after HSC transplantation.
FIG. 21 is wild-type HSC and Ripk3 in example 4 of the present invention -/- 20 Proportion of donor-derived B cells, T cells and myeloid cells in peripheral blood of recipient mice at week 16 after HSC transplantation.
FIG. 22 is wild-type HSC and Mlkl of example 5 of the present invention -/- Overall leukocyte reconstitution rate in peripheral blood of recipient mice after the first round of hematopoietic stem cell transplantation by HSCs.
FIGS. 23 to 25 are wild-type HSC and Mlkl, respectively, in example 5 of the present invention -/- Donor-derived cell proportion in B lymphocytes, T lymphocytes and myeloid cells in the peripheral blood of recipient mice after the first round of hematopoietic stem cell transplantation.
FIG. 26 is a first round of wild-type HSC and Mlkl in example 5 of the present invention -/- The proportion of donor-derived B cells, T cells and myeloid cells in the peripheral blood of recipient mice at week 16 post-HSC transplantation.
FIG. 27 is wild-type HSC and Mlkl of example 5 of the present invention -/- Overall leukocyte reconstitution rate in peripheral blood of HSC third round of recipient mice.
FIGS. 28 to 30 are wild-type HSC and Mlkl, respectively, in example 5 of the present invention -/- Proportion of donor-derived cells in B lymphocytes, T lymphocytes and myeloid cells in peripheral blood of the recipient mice of the HSC third round of transplantation.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The following examples are illustrative of the invention but are not intended to limit the scope of the invention.
In the description of the embodiments of the present invention, it should be noted that the terms "center", "longitudinal", "lateral", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, are merely for convenience in describing the embodiments of the present invention and simplifying the description, and do not indicate or imply that the apparatus or elements referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the embodiments of the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
In describing embodiments of the present invention, it should be noted that, unless explicitly stated and limited otherwise, the terms "coupled," "coupled," and "connected" should be construed broadly, and may be either a fixed connection, a removable connection, or an integral connection, for example; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium. The specific meaning of the above terms in embodiments of the present invention will be understood in detail by those of ordinary skill in the art.
In embodiments of the invention, unless expressly specified and limited otherwise, a first feature "up" or "down" on a second feature may be that the first and second features are in direct contact, or that the first and second features are in indirect contact via an intervening medium. Moreover, a first feature being "above," "over" and "on" a second feature may be a first feature being directly above or obliquely above the second feature, or simply indicating that the first feature is level higher than the second feature. The first feature being "under", "below" and "beneath" the second feature may be the first feature being directly under or obliquely below the second feature, or simply indicating that the first feature is less level than the second feature.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the embodiments of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
The invention provides an application of a means for regulating and controlling programmed necrosis in preparing a kit for diagnosing or delaying aging of a blood system.
The inventor of the invention discovers that the expression quantity of the programmed necrosis pathway in the aged hematopoietic stem cells is increased, and the programmed necrosis regulation means can effectively enhance the self-updating capacity of the hematopoietic stem cells, thereby delaying the aging of the hematopoietic fluid system. The kit prepared by adopting the means of regulating and controlling the programmed necrosis can be used for diagnosing and/or delaying the aging of the blood system and is very suitable for clinical application.
In some embodiments of the invention, the means for modulating programmed necrosis comprises: by blocking the expression of specific genes, to combat the programmed necrosis.
The specific gene refers to a gene associated with programmed necrosis.
In some embodiments of the invention, the means for modulating programmed necrosis comprises: blocking the activity of RIPK1 kinase during programmed necrosis.
In some embodiments of the invention, the means for blocking RIPK1 kinase activity during programmed necrosis comprises: inhibiting the activity of RIPK1 by means of gene intervention or small molecule intervention. In some embodiments of the invention, the means for blocking RIPK1 kinase activity during programmed necrosis comprises: knocking out the RIPK1 gene and/or mutating the RIPK1 gene into RIPK 1K 45A.
The RIPK 1K 45A refers to a gene obtained by mutating lysine at the 45-site of the RIPK1 protein.
In some embodiments of the invention, the means for modulating programmed necrosis comprises: blocking the activity of RIPK3 kinase activity during programmed necrosis.
In some embodiments of the invention, the means for blocking RIPK3 kinase activity during programmed necrosis comprises: inhibiting the activity of RIPK3 by means of gene intervention or small molecule intervention. In some embodiments of the invention, the means for blocking RIPK3 kinase activity during programmed necrosis comprises: knocking out RIPK3 gene.
In some embodiments of the invention, the means for modulating programmed necrosis comprises: blocking the activity of MLKL kinase during programmed necrosis.
In some embodiments of the invention, the means for blocking MLKL kinase activity during programmed necrosis comprises: inhibiting MLKL activity by means of genetic intervention or small molecule intervention. In some embodiments of the invention, the means for blocking MLKL kinase activity during programmed necrosis comprises: the MLKL gene is knocked out.
In some embodiments of the invention, a kit for diagnosing aging of the blood system includes at least one of an antibody that detects a RIPK1 background protein and phosphorylates RIPK1, an antibody that detects a RIPK3 background protein and phosphorylates RIPK3, and an antibody that detects a MLKL background protein and phosphorylates MLKL.
In some embodiments of the invention, the kit for delaying aging of the blood system comprises at least one of a RIPK 1K 45A point mutated hematopoietic stem cell, a RIPK3 knocked out hematopoietic stem cell, and a MLKL knocked out hematopoietic stem cell.
In some embodiments of the invention, a kit for simultaneously diagnosing and delaying aging of the blood system comprises: detecting an antibody that phosphorylates RIPK1 and a RIPK1 background protein, and a hematopoietic stem cell having a RIPK 1K 45A point mutation; and/or detecting RIPK3 background proteins and antibodies that phosphorylate RIPK3, and hematopoietic stem cells that knocked out RIPK 3; and/or detecting antibodies to MLKL background protein and phosphorylated MLKL, and knockout of hematopoietic stem cells of MLKL.
The invention is further illustrated below in conjunction with specific examples, which are given solely for the purpose of illustration and are not to be construed as limitations of the present invention.
Examples
In the following examples, the procedure involved hematopoietic stem cell transplantation and peripheral blood analysis in mice was as follows:
1. hematopoietic stem cell transplantation
1) Recipient mice (CD 45.1/2) were prepared, odd-numbered ear tags were placed on the left ear of the mice, even-numbered ear tags were placed on the right ear, and wild-type (WT) HSCs were transplanted into the odd-numbered ear tagged mice as a control group, genotype HSCs (genotype HSCs were selected from Ripk1 K45A 、Ripk3 -/- And Mlkl -/- HSCs sorted from mice, labeled Ripk1, respectively K45A HSC、Ripk3 -/- HSC and Mlkl -/- HSC) was transplanted into even-numbered ear-tag mice as experimental groups to prevent the mice from being unable to distinguish between experimental groups and control groups after ear-tag scratching off;
2) Adding 300 mu L of bailoxin into 300 mL acidic water, and feeding the bailoxin to a receptor mouse 3-5 days in advance;
3) The recipient mice (more than 8 weeks old) were subjected to X-ray irradiation one day in advance or on the day of transplantation, each time 5 Gy in the morning and afternoon, with an interval duration of 3 hours;
4) Collecting CD45.1 mice with age of more than 8 weeks, shearing femur, preparing single cell suspension, filtering with 40 μm cell sieve, and counting living cells;
5) Sorting the same number of HSCs into 300 μl hbss+ receiving solution, typically adding 2×105 cd45.1 whole bone marrow cells (as competitors) per tube, gently vortexing and then inserting them on ice for later use, the whole bone marrow cells serving as competitors on the one hand to quantify HSC reconstitution ability and on the other hand to maintain survival of the recipient mice after lethal dose irradiation;
6) Repeatedly wiping the tail of the receptor treated in the step 3) with an alcohol cotton ball, sterilizing and exposing veins of the tail of the mouse, slowly sucking the cell suspension prepared in the step 5 by an insulin needle, and injecting the tail of the mouse by intravenous injection, wherein all cells in the storage are ensured to be transplanted into the body of the mouse;
7) After the transplantation is completed, pinching the proximal end of the rat tail, rapidly pulling out the needle to prevent the liquid from flowing out through the tail vein, adding hemostatic powder at the needle opening, and carefully pinching the rat tail and keeping for more than 10 seconds to sufficiently stop bleeding;
8) The recipient mice were placed in a cage, fed with bailoxin for 3 weeks, and then changed to normal acidic water.
2. Peripheral blood analysis of recipient mice (Linear tracking)
The donor-derived cell proportion and lineage distribution in peripheral blood were determined by flow cytometry after tail vein blood sampling at 4, 8, 12, 16 weeks after transplantation in recipient mice with bone marrow transplantation or hematopoietic stem cell transplantation, and the specific steps are as follows:
1) Collecting blood from the tail vein of a recipient mouse, collecting 30-50 mu L of peripheral blood in an EDTA (ethylenediamine tetraacetic acid) anticoagulation tube, and fully and uniformly mixing the bottom of a flick tube to prevent coagulation;
2) Adding peripheral blood into 1.5 mL erythrocyte lysate (1×ACK), reversing upside down for ten times, mixing, standing at room temperature for 5 min, and if ACK erythrocyte lysate is 4 deg.C buffer solution, and prolonging lysis time for 15 min;
3) After the completion of the lysis of erythrocytes, the mixture was centrifuged at 500 Xg at 4℃for 5 minutes, the supernatant was aspirated off, 1 mL of HBSS+ was added to resuspend the cells, and the remaining erythrocyte debris was washed off;
4) Centrifuging at 4deg.C, 500 Xg, 5 min, and discarding supernatant;
5) Add 90. Mu.L HBSS+ resuspended cells, add the antibody mixture of Table 1 for staining, and stain in ice for 30 minutes in the dark;
TABLE 1 peripheral blood analysis of recipient mice antibody mixture
6) After the staining is finished, 1 mL of HBSS+ is added to resuspend the cells, the mixture is centrifuged for 5 minutes at the temperature of 4 ℃ and 500 Xg, and the supernatant is discarded to wash free antibody;
7) Add 250. Mu.L HBSS+ resuspended cells, transfer to flow tubes, stain each tube with 5. Mu.L DAPI working fluid to differentiate dead cells;
in addition, in the following examples, flow cytometry analyzed the proportion of donor source in blood cells. CD45.2 positive cells indicate donor-derived cells, and CD45.1 positive cells indicate competitor-derived cells.
Example 1
Expression level of the programmed necrosis pathway in senescent hematopoietic stem cells
Hematopoietic stem cells were sorted from the bone marrow of young (2 months old) and aged (32 months old) mice (B6), respectively (hematopoietic stem cells were sorted by flow cell sorting), to obtain young hematopoietic stem cells and aged hematopoietic stem cells, respectively. From young and senescent hematopoietic stem cells, changes in senescent HSC (hematopoietic stem cells) were examined for p-MLKL, MLKL, RIPK3, RIPK1, caspase-3 (labeled as Casp-3 in FIG. 1) and Caspase-8 (labeled as Casp-8 in FIG. 2), and the results are shown in FIGS. 1 and 2. The essential genes MLKL, RIPK3 and RIPK1 for programmed necrosis in senescent HSCs were found to be significantly elevated.
Example 2
Relation of programmed necrosis with RIPK1-K45A, RIPK3 and MLKL
Three groups of HSCs were freshly sorted from wild-type B6 mouse bone marrow, 2000 HSCs in each group, one group was treated with t+s+z (where T is 50ng/ml tnfα (tumor necrosis factor α), S is 100 nmsmacm, Z is 20 μmzvad), another group was treated with t+s+z+ripa-56 (where T is 50ng/ml tnfα, S is 100 nmsmacm, Z is 20 μmzvad, RIPA-56 is 20 μΜ), and a third group was treated with NT (NT treatment refers to treatment with DMSO (dimethyl sulfoxide) solvent), and the cell death ratio was measured by flow cytometry after 12 hours, as shown in fig. 3, the data in fig. 3 were presented as mean ± SD.
From wild mice (labeled as WT in FIGS. 4 to 6), ripk1, respectively K45A Mice (labeled K45A in FIG. 4, ripk 1) K45A Mice were derived from the Shanghai Nutrition institute Zhang Hai laboratory, national academy of sciences, ripk3 -/- Mouse (Ripk 3) -/- Mice were derived from the Beijing institute of life sciences Wang Xiaodong laboratory) and Mlkl -/- Mouse (Mlkl) -/- Mice were derived from the bone marrow of the Beijing institute of life sciences Wang Xiaodong laboratory and three KSL cells were freshly sorted, 2000 KSL cells each, one KSL cell of each mouse was treated with T+S+Z (where T is 50ng/ml TNF alpha, S is 100 nMSmacM, Z is 20. Mu.MzVAD), a second KSL cell of each mouse was treated with T+S+Z+RIPA-56 (where T is 50ng/ml TNF alpha, S is 100 nMSmaM, Z is 20. Mu.MzVAD, RIPA-56 is 20. Mu.M), a third KSL cell of each mouse was treated with NT, and the cell death ratio was examined by flow cytometry after 12 hours, as shown in FIGS. 4 to 6, and the data in FIGS. 4 to 6 were presented as mean.+ -. SD. From the results of FIGS. 3 to 6, it can be seen that the deletions of RIPK1-K45A, RIPK3 and MLKL are effective against programmed necrosis.
In FIGS. 3 to 6, the abscissa TSZ represents the hematopoietic stem cells of mice treated with T+S+Z, and TSZ+RIPA-56 represents the hematopoietic stem cells of mice treated with T+S+Z+RIPA-56.
Example 3
Relationship between RIPK1 kinase Activity and HSC senescence
Using wild-type HSC and Ripk1 respectively K45A HSC hematopoietic Stem cell transplantation in recipient mice followed by a first round of transplantation, ripk1 K45A The overall leukocyte reconstitution rate in peripheral blood of HSC transplant recipient mice and the donor-derived cell ratios in B cells, T cells and Myeloid cells (myeloids) are shown in fig. 7 to 10, for example.
Using wild-type HSC and Ripk1 respectively K45A Donor source (CD 45.2) in peripheral blood of recipient mice at week 12 after the first round of HSC transplantation of HSC on recipient mice + ) The ratios of B cells, T cells and myeloid cells are shown in FIG. 11.
Using wild-type HSC and Ripk1 respectively K45A After a second round of transplantation of HSC to recipient mice, recipient miceThe overall leukocyte reconstitution rate in peripheral blood and the donor-derived cell ratios in B cells, T cells and myeloid cells are shown in fig. 12 to 15, for example.
Using wild-type HSC and Ripk1 respectively K45A Donor source (CD 45.2) in peripheral blood of recipient mice at week 12 after the second round of transplantation of HSCs to recipient mice + ) The ratios of B cells, T cells and myeloid cells in the cells are shown in FIG. 16.
In FIGS. 7 to 16, WT represents the group of recipient mice receiving wild-type HSC transplantation, and K45A represents the group of recipient mice receiving Ripk1 K45A The HSC-transplanted recipient mice groups of 7 mice each, the data in the figures are presented as mean ± SD. The results of fig. 7-16 show that blocking RIPK1 kinase activity in programmed necrosis delays HSC senescence.
Example 4
Relationship of RIPK3 kinase activity to HSC senescence
Using wild-type HSC and Ripk3 respectively -/- 20 Hematopoietic stem cell transplantation of recipient mice by HSC, the reconstitution rate of global leukocytes in peripheral blood of the transplanted recipient mice (CD 45.2 + ) B lymphocytes (B220) + ) T lymphocytes (CD 3) + ) And myeloid cell (Mac-1) + ) The donor-derived cell ratio is shown in, for example, FIGS. 17 to 20.
Using wild-type HSC and Ripk3 respectively -/- 20 Hematopoietic stem cell transplantation of recipient mice by HSC, donor source in peripheral blood of recipient mice at 16 weeks after transplantation (CD 45.2 + ) The ratios of B cells, T cells and myeloid cells are shown in FIG. 21.
In FIGS. 17 to 21, WT represents the group of recipient mice receiving wild-type HSC transplantation, ripk3 -/- Representative of the acceptance of Ripk3 -/- Group of recipient mice transplanted with 20HSC, 7 mice per group, data presented as mean ± SD. As can be seen from the results of fig. 17-21, blocking RIPK3 loss significantly improved HSC reconstitution ability.
Example 5
Relationship between MLKL kinase Activity and HSC senescence
Using wild-type HSC and Mlkl, respectively -/- HSC first round hematopoietic stem cell transplantation in recipient mice, and overall leukocyte reconstitution Rate (CD 45.2) in peripheral blood of recipient mice after transplantation + ) B lymphocytes (B220) + ) T lymphocytes (CD 3) + ) And myeloid cell (Mac-1) + ) The donor-derived cell ratio is shown in, for example, FIGS. 22 to 25. First round Mlkl -/- At week 16 post-HSC transplantation, donor source in recipient mice peripheral blood (CD 45.2 + ) The ratios of B cells, T cells and myeloid cells are shown in FIG. 26.
Using wild-type HSC and Mlkl, respectively -/- Overall leukocyte reconstitution rate (CD 45.2) in peripheral blood of HSC third round of transplantation recipient mice + ) B lymphocytes (B220) + ) T lymphocytes (CD 3) + ) And myeloid cell (Mac-1) + ) The donor-derived cell ratio is shown in, for example, FIGS. 27 to 30.
In FIGS. 22 to 30, WT represents the group of recipient mice receiving wild-type HSC transplantation, mlkl -/- Representative receiving Mlkl -/- The HSC-transplanted recipient mice groups of 6 mice each, data are presented as mean ± SD. As can be seen from the results of fig. 22 to 30, blocking MLKL significantly improved HSC self-renewal ability.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (9)
1. An application of a means for regulating and controlling programmed necrosis in preparing a kit for diagnosing or delaying aging of a blood system.
2. The use according to claim 1, wherein said means for modulating programmed necrosis comprises: by blocking the expression of specific genes, to combat the programmed necrosis.
3. The use according to claim 1 or 2, wherein the means for modulating programmed necrosis comprises: blocking the activity of RIPK1 kinase during programmed necrosis.
4. The use according to claim 3, wherein the means for blocking RIPK1 kinase activity during programmed necrosis comprises: inhibiting the activity of RIPK1 by means of gene intervention or small molecule intervention.
5. The use according to claim 1 or 2, wherein the means for modulating programmed necrosis comprises: blocking the activity of RIPK3 kinase activity during programmed necrosis.
6. The use according to claim 5, wherein blocking RIPK3 kinase activity during programmed necrosis comprises: inhibiting the activity of RIPK3 by means of gene intervention or small molecule intervention.
7. The use according to claim 1 or 2, wherein the means for modulating programmed necrosis comprises: blocking the activity of MLKL kinase during programmed necrosis.
8. The use according to claim 7, wherein the blocking of MLKL kinase activity during programmed necrosis comprises: inhibiting MLKL activity by means of genetic intervention or small molecule intervention.
9. The use according to claim 1 or 2, wherein the kit for diagnosing aging of the blood system comprises at least one of an antibody for detecting RIPK1 background protein and phosphorylating RIPK1, an antibody for detecting RIPK3 background protein and phosphorylating RIPK3, and an antibody for detecting MLKL background protein and phosphorylating MLKL;
the kit for delaying aging of the blood system comprises at least one of hematopoietic stem cells with RIPK 1K 45A point mutation, hematopoietic stem cells with RIPK3 knocked out and hematopoietic stem cells with MLKL knocked out.
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