CN105233256A - Application of erythropoietin and erythropoietin derivatives to preparation of medicine for promoting apoptotic cell clearance during disease treatment - Google Patents
Application of erythropoietin and erythropoietin derivatives to preparation of medicine for promoting apoptotic cell clearance during disease treatment Download PDFInfo
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- CN105233256A CN105233256A CN201510712877.8A CN201510712877A CN105233256A CN 105233256 A CN105233256 A CN 105233256A CN 201510712877 A CN201510712877 A CN 201510712877A CN 105233256 A CN105233256 A CN 105233256A
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Abstract
The invention belongs to the field of biological medicines and relates to application of erythropoietin and erythropoietin derivatives to preparation of the medicine for promoting apoptotic cell clearance during disease treatment. The application comprises applications to preparation of the medicine for treating diseases casued by acute and chronic inflammation, autoimmune diseases and atherosclerosis disease and promoting apoptotic cell clearance during wound healing. According to the medicine for promoting apoptotic cell clearance, an erythropoietin receptor of a macrophage can be activated to promote apoptotic cell phagocytosis and clearance by the macrophage and induce inflammation inhibition and tissue protection, the application of the medicine to treatment of inflammatory and autoimmune diseases testifies that erythropoietin and erythropoietin derivatives activate the erythropoietin receptor of the macrophage, can promote apoptotic cell phagocytosis and clearance by the macrophage in various diseases to promote rehabilitation and therefore have an important clinical application value.
Description
Technical field
The invention belongs to biomedicine field, relate to a kind of erythropoietin and derivant is preparing in disease therapy the application promoted in the medicine of apoptotic cell clearance.
Background technology
For maintaining body eubolism and function, in healthy body, there is hundreds of millions of apoptosis every day according to estimates, as immature cell in: neutrophilic granulocyte and erythrocytic old and feeble apoptosis, thymus T cells growth course removed by apoptosis, in mankind spermatozoon growth course wrong noble cells by apoptosis removal etc.; And when body inflammatory/damage also with the apoptosis of a large amount of cell, as: in histiocyte apoptosis under external force, acute inflammatory reaction process neutrophilic granulocyte due to T in the very short easy generation apoptosis of its shelf-life, immunne response and bone-marrow-derived lymphocyte the apoptosis etc. of activation-inducing that occurs.
Apoptosis is the death pathways of a kind of " safety ", and apoptotic cell cell membrane keeps complete, and cellular content does not leak outside, and does not cause damage to body.But apoptotic cell cell membrane integrity maintains and needs consumed energy; And apoptotic cell lacks due to mitochondrial function, only lean on remaining ATP energy supply, do not remove apoptotic cell in time and secondary necrosis (secondarynecrosis) easily occurs, cell membrane integrity is destroyed, release cells content.The conduct such as the HMGB1 in apoptotic cell content, uric acid (can form crystallization in born of the same parents' external environment) " endogenous danger signal " are by Toll-like receptor etc., activating macrophage, inflammatory factor, rise costimulatory molecules, the chemotactic inflammatory cell infiltrations such as induction TNF-α, IL-1 β, IL-12, cause local inflammation environment; And the proteolytic enzyme etc. of release directly can cause damaging surrounding tissue.Therefore, the apoptotic cell do not removed in time can expand local inflammation reaction, increases the weight of local inflammation damage, causes acute inflammation chronicity.The multiple autoantigen such as double-stranded DNA, histone simultaneously in apoptotic cell content is also released, can be offered by antigen presenting cell, in inflammatory environment, activate autoantigen specific lymphocytes, break immune tolerates, the Autoimmune diseases such as inducible system lupus erythematosus (SLE).
Body is removed mainly through macrophage engulfing of apoptotic cell.Macrophage engulfs removing in time to apoptotic cell, not only can prevent " secondary is downright bad " of apoptotic cell, simultaneously can suppress the inflammatory factors such as macrophages secrete TNF-α, promote the inflammation such as its secretion IL-10, TGF-β, PGE2 (ProstaglandinE2) suppress because of and the tissue repair molecule such as VEGF.Therefore, macrophage engulfing apoptotic cell, remove the apoptotic cell of local, impel the inflammation/damaging conditions of local to suppress to transform to inflammation, and promote tissue repair, making inflammation/damage location again recover stable state (homeostasis), is the key link impelling inflammation/recovering of injured.
No matter that macrophage engulfs removing obstacles to apoptotic cell, or apoptosis is too much, apoptotic cell all can be caused to assemble, generation secondary is downright bad, thus cause the chronic inflammatory disease of persistence, with multiple serious harm human health intractable, chronic disease is closely related, as atherosclerosis, chronic obstructive emphysema, systemic lupus erythematosus (sle), neurodegeneration, allergic disease etc.Although inflammation is not the inducement of these diseases usually, the low inflammatory conditions continued, impels/aggravates tissue injury to be one of common pathogenetic path of these diseases.Assemble as can be observed the apoptotic cell that causes of macrophage phagocytic function deficiency in atherosclerotic lesions district, and the necrosis of apoptotic cell secondary can aggravates inflammation damage, with lesion progress and artery thrombosis closely related.
Erythropoietin (EPO) is the most important short Hemopoietic factor of body, by activating the EPO receptor (EPOR) be expressed on erythroid cells, promote that CFU-E Differentiation is mature erythrocyte, be widely used in the anemia that treatment many reasons causes clinically.EPOR and EPO wording depth is guarded, not with other Cytokine except EPO.Due to the important pharmaceutical value of EPO; EPO is can manually generate; and carry out kinds of artificial modification, as some short peptide stretch in PEGization EPO, high-glycosylation EPO, asialoglycoprotein EPO, carbamylation EPO and EPO source, all remain the function activating EPOR.
At present by the erythropoietin receptor on erythropoietin activating macrophage, promote that the engulf removing of macrophage to apoptotic cell also not yet has research and patent disclosure.
Summary of the invention
In view of this, the object of the present invention is to provide the novelty teabag of a kind of erythropoietin and derivant thereof, namely erythropoietin and derivant thereof are preparing in disease therapy the application promoted in the medicine of apoptotic cell clearance, for in treatment various diseases, the gathering of apoptotic cell provides new treatment thoughts, additionally providing a kind of being used for the treatment of in disease promotes the medicine of apoptotic cell clearance and is that the new approaches removing regulation and control and in treatment in struvite and autoimmune disease engulfed by the medicine of target spot exploitation at apoptotic cell with erythropoietin receptor.Described medicine confirms that erythropoietin and derivant thereof have activated macrophage erythropoietin receptor in by the application in and autoimmune disease struvite in treatment, macrophage can be promoted in various diseases to engulf removing to apoptotic cell, promote the rehabilitation of disease, there is important clinical value.
For achieving the above object, technical scheme of the present invention is as follows:
The invention provides a kind of erythropoietin and derivant is preparing in disease therapy the application promoted in the medicine of apoptotic cell clearance.
Erythropoietin (EPO) is the most important short Hemopoietic factor of body, by activating the EPO receptor (EPOR) be expressed on erythroid cells, promote that CFU-E Differentiation is mature erythrocyte, be widely used in the anemia that treatment many reasons causes clinically.EPOR and EPO wording depth is guarded, not with other Cytokine except EPO.Due to the important pharmaceutical value of EPO; EPO is can manually generate; and carry out kinds of artificial modification, as some short peptide stretch in PEGization EPO, high-glycosylation EPO, asialoglycoprotein EPO, carbamylation EPO and EPO source, all remain the function activating EPOR.
Described erythropoietin and derivant thereof comprise any protein containing the natural or recombiant protein obtained by tissue, protein synthesis, cell culture that is natural or reconstitution cell with erythropoietin activity, also comprise the small-molecular peptides of its activity of reservation in mutant protein, modified protein and erythropoietin source.As carbamylation EPO and EPO parent small molecule peptide, described erythropoietin and derivant thereof all have the activity activating EPOR.
Preferred as one, described erythropoietin and derivant thereof comprise recombinant human erythropoietin, carbamylation erythropoietin and erythropoietin parent small molecule peptide.
Preferred as one, the present invention uses recombinant human erythropoietin.
Further, described erythropoietin and derivant thereof promote the application in the medicine of apoptotic cell clearance in the disease caused by preparation treatment active chronic inflammation; Or described erythropoietin and derivant thereof promote the application in the medicine of apoptotic cell clearance in preparation treatment autoimmune disease; Or described erythropoietin and derivant thereof promote the application in the medicine of apoptotic cell clearance in preparation treatment atheromatosis; Or described erythropoietin and derivant thereof promote the application in the medicine of apoptotic cell clearance in preparation treatment wound healing.
Inflammation usually can be passed through according to the course of disease and is divided into two large classes: acute inflammation (acuteinflammation) and chronic inflammatory disease (chronicinflammation).Acute inflammation onset is hurried, and the persistent period is short, only several days, and be its feature to ooze out pathological changes, inflammatory cell infiltration is based on granulocyte.The chronic inflammatory disease persistent period is longer, and the constant moon, often based on preneoplastic lesions, its inflammatory cell infiltration was then based on macrophage and lymphocyte to the several years.
Autoimmune disease is the composition generation immunoreation of immune system to its body, causes damage and diseases induced.Numerous disease is listed in autoimmune disease in succession, and originally, immune system was only to the exotic invading body under normal circumstances, as antibacterial, virus, parasite and graft etc. produce reaction, eliminates or repels these foreign bodies.Under the impact of some factor, the morphological element of body or immune system itself have occurred that some is abnormal, cause immune system to be attacked as exotic by self composition by mistake.At this time immune system can produce antibody for some compositions of body self and activated lymphocytes, and infringement destroys autologous tissue's internal organs, causes disease.Autoimmune disease is mainly reacted caused by produced a large amount of immunocyte and immunoglobulin accumulation by excessive immune, thus compromises normal structure, makes it pathological changes and functional lesion occur.
Atherosclerosis (atherosclerosis) is a kind of chronic, gradual arterial disease, and different pathogeny causes that vascular endothelial cell is impaired all can cause blood vessel subcutaneous fat deposits gradually, thus inducing chronic, progressive inflammation.The fats portion deposited in patient's tremulous pulse can block blood flow entirely or partly, and causing blood for reducing, is the Etiological of multiple cerebrovascular.Atheromatous plaque district has the infiltration of inflammation cell usually, and wherein macrophage is engulfed removing to plaque region apoptotic cell and played an important role; The apoptotic cell caused due to macrophage function obstacle is assembled.And secondary is downright bad, can incite inflammation factor storm, causing artery plaque to come off, shine into blood vessel embolism, is the lethal Etiological of atherosclerosis.
Commonly skin wound healing in wound healing, skin wound healing be one by inflammatory cell, Eponychium cell, fibroblast, vascular endothelial cell, extracellular matrix, the complex process that the various kinds of cell such as cytokine and somatomedin and medium participate in jointly.Macrophage engulfs removing to apoptosis neutrophilic granulocyte, plays an important role in skin wound healing process.In obesity or diabetics, because macrophage function is impaired, cause wound apoptotic cell to be assembled, secondary occurring downright bad, there is chronic, disunion wound in inducing chronic inflammation, is important complication that is fat or diabetics.But the cell of its promotion apoptosis of new opplication of the present invention is assembled at wound, and strengthen the therapeutical effect of engulfing promotion skin wound healing of macrophage to apoptotic cell.
Disease of the present invention is not limited in above-mentioned several, as long as to play the effect promoting apoptotic cell clearance in disease, indication disease all drops in scope.
Further, described erythropoietin and derivant thereof treat in acute and chronic peritonitis the application promoted in the medicine of apoptotic cell clearance in preparation.
Further, described erythropoietin and derivant thereof are preparing in systemic lupus erythematosus the application promoted in the medicine of apoptotic cell clearance.Systemic lupus erythematosus (sle) is autoimmune mediation, take immune inflammation as the Diffuse Connective Tissue Disease of outstanding behaviours, and generation and the apoptosis disorder of systemic lupus erythematosus (sle) are closely related.Apoptosis increases and (or) apoptotic cell clearance obstacle all can cause apoptotic cell to gather.In apoptotic process autoantigen exposure and modified, result in Autoantigen Immune originality increases, thus can cause having immunogenic autoantigen when apoptotic cell gathers and be exposed to immune system, causes the generation of autoimmune response.New opplication of the present invention can promote macrophage phagocytic apoptosis T cell, and promotes the therapeutical effect of autoimmunity systemic lupus erythematosus (sle).
Further, described erythropoietin and derivant thereof promote the application in the medicine of apoptotic cell clearance in preparation treatment autoimmune neuritis.
Further, described erythropoietin and derivant thereof promote the application in the medicine of apoptotic cell clearance in preparation treatment Autoimmune Encephalomyelitis.
Two of object of the present invention is that providing a kind of is used for the treatment of in disease the medicine promoting apoptotic cell clearance, and the active component of described medicine is containing having erythropoietin and derivant thereof.
The medicine of promotion apoptotic cell clearance provided by the invention; macrophage can be promoted to engulf removing to apoptotic cell by activating macrophage erythropoietin receptor; and incite inflammation suppresses and organization protection; confirm that medicine of the present invention can activating macrophage erythropoietin receptor in by the application in the struvite and autoimmune disease for the treatment of; macrophage can be promoted in various diseases to engulf removing to apoptotic cell, promote the rehabilitation of disease.
Further, described medicine, by the erythropoietin receptor of activating macrophage, promotes macrophage phagocytic apoptotic cell, Induced synthesis inflammation inhibitive factor and the promotion tissue repair factor; Described inflammation inhibitive factor comprises TGF-β, and the described promotion tissue repair factor comprises VEGF.
Preferred as one, the product of described medicine comprises injection.
Further, described medicine also comprises pharmaceutically acceptable carrier.
The present invention also aims to provide a kind of medicine with erythropoietin receptor being target spot is developed to engulf the application of removing regulation and control and promoting in disease therapy in apoptotic cell clearance at apoptotic cell.
Promote that macrophage engulfs removing function to apoptotic cell, the reparation of disappearing, promoting local damage tissue of active chronic inflammation can be promoted, thus promote the rehabilitation of multiple active chronic inflammation or autoimmunity relevant disease, be the novel targets of current antiinflammatory and the multiple active chronic inflammation for the treatment of or autoimmunity relevant disease.The discovery of new short macrophage phagocytic apoptotic cell molecule, can provide various diseases to treat novel targets, in biomedicine field tool significance.
Beneficial effect of the present invention is:
1. provide a kind of novelty teabag of erythropoietin, namely erythropoietin and derivant thereof are preparing in disease therapy the application promoted in the medicine of apoptotic cell clearance, in treatment various diseases, the gathering of apoptotic cell provides new treatment thoughts.
2. additionally provide to be used for the treatment of in disease and promote the medicine of apoptotic cell clearance and be that the new approaches removing regulation and control and in treatment in struvite and autoimmune disease engulfed by the medicine of target spot exploitation at apoptotic cell with erythropoietin receptor.
3. the medicine of promotion apoptotic cell clearance provided by the invention; macrophage can be promoted to engulf removing to apoptotic cell by activating macrophage EPOR; and incite inflammation suppresses and organization protection; confirm that medicine of the present invention can activating macrophage erythropoietin receptor in by the application in the struvite and autoimmune disease for the treatment of; macrophage can be promoted in various diseases to engulf removing to apoptotic cell; promote the rehabilitation of disease, there is important clinical value.
Accompanying drawing explanation
Fig. 1 is in experiment in vitro of the present invention, by flow cytometer detection F4/80
+pHrodo
+cell mass, reflection macrophage is to the schematic diagram of the phagocytic activity of apoptotic cell.
Fig. 2 is in experiment in vitro of the present invention, reflects that medicine of the present invention affects schematic diagram to macrophage inflammatory factor and tissue repair factor expression.
Fig. 3 is in experiment in vivo of the present invention, by flow cytometer detection F4/80
+pHrodo
+cell mass, reflection macrophage is to the schematic diagram of the phagocytic activity of apoptotic cell.
Fig. 4 is in experiment in vivo of the present invention, and medicine of the present invention is on the impact of macrophage inflammatory factor and tissue repair factor expression.
Fig. 5 is that medicine of the present invention is being treated in acute and chronic peritonitis, and reflection macrophage is to the schematic diagram of the phagocytic activity of apoptotic cell.
Fig. 6 is medicine of the present invention in treatment acute and chronic peritonitis, and reflection medicine affects schematic diagram to macrophage inflammatory factor and tissue repair factor expression.
Fig. 7 is that medicine of the present invention is being treated in acute and chronic peritonitis, the number of variations schematic diagram of the neutrophilic granulocyte of reflection peritonitis.
Fig. 8 be medicine of the present invention in systemic lupus erythematosus disease, the schematic diagram that there is situation of related substances in autoantibody and urine in reflection mouse blood.
Fig. 9 be medicine of the present invention in systemic lupus erythematosus disease, the schematic diagram of gathering situation of apoptosis neutrophilic granulocyte in reflection mouse blood.
Figure 10 be medicine of the present invention in systemic lupus erythematosus disease, reflection mouse kidney glomerule position counting positive cell number object schematic diagram.
Figure 11 be medicine of the present invention in systemic lupus erythematosus disease, reflection macrophage to the schematic diagram of the phagocytic activity of apoptosis T cell.
Figure 12 is in Drug therapy autoimmune neuritis of the present invention, immunity and treatment after, different pharmaceutical treatment group is relative to the schematic diagram of the curative effect of matched group.
Figure 13 is in Drug therapy autoimmune neuritis of the present invention, and reflection peripheral nervous system apoptotic cell assembles the schematic diagram of situation.
Figure 14 is that medicine of the present invention is being treated in autoimmune neuritis, and reflection macrophage is to the schematic diagram of the phagocytic activity of apoptosis T cell.
Figure 15 is that medicine of the present invention is being treated in Autoimmune Encephalomyelitis, and reflection different pharmaceutical treatment group is relative to the schematic diagram of the curative effect of matched group.
Figure 16 is that medicine of the present invention is being treated in Autoimmune Encephalomyelitis, and reflection peripheral nervous system apoptotic cell assembles the schematic diagram of situation.
Figure 17 is that medicine of the present invention is being treated in Autoimmune Encephalomyelitis, and reflection macrophage is to the schematic diagram of the phagocytic activity of apoptosis T cell.
Figure 18 is that medicine of the present invention is being treated in obesity mice wound district's apoptotic cell clearance and wound healing, and reflection different pharmaceutical treatment group is relative to the schematic diagram of the curative effect of matched group.
Figure 19 is that medicine of the present invention is being treated in obesity mice wound district's apoptotic cell clearance and wound healing, and reflection wound tissue apoptotic cell assembles the schematic diagram of situation.
Figure 20 is that medicine of the present invention is being treated in obesity mice wound district's apoptotic cell clearance and wound healing, and reflection macrophage is to the schematic diagram of the phagocytic activity of apoptosis neutrophilic granulocyte.
Figure 21 is that medicine of the present invention is being treated in atherosclerosis, and reflection different pharmaceutical treatment group is relative to the schematic diagram of the curative effect of matched group.
Figure 22 is that medicine of the present invention is being treated in atherosclerosis, and reflection artery plaque district organizes apoptotic cell to assemble the schematic diagram of situation.
Detailed description of the invention
Below with reference to accompanying drawing, the present invention is described in detail.In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with drawings and Examples, the present invention is further elaborated.The experimental technique of unreceipted actual conditions in preferred embodiment, usual conveniently condition, the such as Molecular Cloning: A Laboratory guide (third edition, J. the work such as Pehanorm Brooker, Huang Peitang etc. translate, Science Press, 2002) described in condition, or to carry out according to the condition that manufacturer advises.Illustrated embodiment is to be described content of the present invention better, but is not that content of the present invention is only limitted to illustrated embodiment.So those of ordinary skill in the art carry out nonessential improvement and adjustment according to foregoing invention content to embodiment, still belong to protection scope of the present invention.
There is the composition that erythropoietin receptor (EPOR) is active: recombinant human epo in embodiment Chinese medicine; carbamylation EPO and EPO originates little peptide molecule; all remain the function activating EPOR, medicine of the present invention is referred to as EPOR agonist in an embodiment.
Example 1EPOR agonist promotes macrophage phagocytic apoptotic cell in vitro, and Induced synthesis inflammation inhibitive factor and the short tissue repair factor
Processing by adding different EPOR agonist under macrophages in vitro condition of culture, detecting that macrophage is engulfed at apoptotic cell, impact in inflammatory factor and tissue repair cytokine secretion etc.; And knock out macrophage with EPOR and compare, thus checking EPOR agonist promotes to engulf apoptotic cell by activating macrophage EPOR in vitro, and incite inflammation suppresses and tissue repair.
1, animal: C57/BL6 mice, male, 8-10 week age, body weight 18-20 gram, be purchased from Third Military Medical University's Experimental Animal Center, normally raise in clean animal room.Macrophage EPOR conditionality knock-out mice (by this laboratory oneself prepare), 8-10 week age, body weight 18-20 gram, raise in SPF level Animal House.
2, medicine:
Treatment group: give variable concentrations recombinant human epo, the originate macrophage of little peptide molecule and In vitro culture of carbamylation EPO or EPO is hatched; Matched group: give to hatch with the normal saline for the treatment of group equivalent and the macrophage of In vitro culture.
3, test method:
Laboratory animal ether is anaesthetized, and then the concentration of lumbar injection 2 milliliters is 3% thioglycollate medium.Thioglycollate injection is after 72 hours, with 5 ml physiological saline lavation mouse peritoneals, centrifuging and taking obtains peritonitis cell, is cultivated 4 hours by above-mentioned cell as containing in the DMEM cell culture fluid of 10% hyclone, and removal suspension cell stays and is primary Turnover of Mouse Peritoneal Macrophages.Treatment group gives recombinant human epo, and originate little peptide molecule and above-mentioned macrophage of carbamylation EPO or EPO carries out hatching and process for 24 hours, and matched group is the normal saline of equivalent.
Macrophage phagocytic apoptotic cell is promoted in order to investigate EPOR agonist, get treatment group and matched group macrophage, add the apoptosis thymocyte cell incubated in vitro 1 hour of fluorescent probe pHrodo labelling, collect macrophage, adopt fluorescent labeling F4/80 antibody labeling macrophage, by flow cytometer detection F4/80
+pHrodo
+cell mass, to reflect the phagocytic activity of macrophage to apoptotic cell.Result as shown in Figure 1.
Streaming result shows, after the process of different EPOR agonist, and wild-type mice macrophage F4/80
+pHrodo
+cell number is dose-dependent remarkable increase, and show that EPOR agonist can promote macrophage phagocytic apoptotic cell, and macrophage EPOR conditionality engulfs apoptotic cell ability after knocking out significantly declines, after the process of EPOR agonist, its phagocytic activity is without change.
In order to investigate the impact of EPOR agonist on macrophage inflammatory factor and tissue repair factor expression, get the macrophage for the treatment of group and the process of matched group different time points variable concentrations, add apoptosis thymocyte cell incubated in vitro 24 hours, collect cell culture supernatant, conventional ELISA method is adopted to detect wherein inflammatory factor MCP-1, IL-6, TNF-α, the content of inflammation inhibitive factor TGF-β and tissue repair factor Ⅴ EGF.Result as shown in Figure 2.
ELISA result shows, and after different EPOR agonist treatment, inflammatory factor MCP-1, IL-6, TNF-alpha content significantly lowers, and inflammation inhibitive factor TGF-β and tissue repair factor Ⅴ EGF content significantly increase.Show that EPOR agonist can suppress macrophage inflammatory factor, promote the expression of inflammation inhibitive factor and the tissue repair factor.
Brief summary: demonstrate EPOR agonist promotion macrophage engulfing apoptotic cell by the experiment of Turnover of Mouse Peritoneal Macrophages in vitro, inflammation-inhibiting and promote the effect that reparative factor is expressed.
Example 2EPOR agonist promotes macrophage phagocytic apoptotic cell in vivo, and Induced synthesis inflammation inhibitive factor and the short tissue repair factor
After different EPOR agonist pretreatment wild-type mices or the special EPOR knock-out mice of macrophage, abdominal cavity gives fluorescently-labeled apoptotic cell, detect peritoneal macrophage engulfing apoptotic cell subsequently, and get peritoneal fluid detection inflammatory factor and the tissue repair factor, thus checking EPOR agonist promotes to engulf apoptotic cell by activating macrophage EPOR in vivo, and incite inflammation suppresses and tissue repair.
1, animal: C57/BL6 mice, male, 8-10 week age, body weight 18-20 gram, be purchased from Third Military Medical University's Experimental Animal Center, normally raise in clean animal room.Macrophage EPOR conditionality knock-out mice (by this laboratory oneself prepare), 8-10 week age, body weight 18-20 gram, raise in SPF level Animal House.
2, medicine:
Processed group: give lumbar injection recombinant human epo (5000IU/ kg body weight), carbamylation EPO (50 micrograms/kg body weight) or EPO originates little peptide molecule (20nmol/ body weight); Matched group: give the normal saline lumbar injection with treatment group equivalent.
3, test method:
Laboratory animal ether is anaesthetized; then lumbar injection recombinant human epo (5000IU/ kg body weight); carbamylation EPO (50 micrograms/kg body weight) or EPO originates little peptide molecule (20nmol/ body weight), and matched group is the normal saline of equivalent.After lumbar injection EPOR agonist, get the apoptosis thymocyte cell (10 of fluorescent probe pHrodo labelling
8) and carry out lumbar injection and carry out following different experiments.
Macrophage phagocytic apoptotic cell is promoted in order to investigate in EPOR agonist body, apoptotic cell lumbar injection 1 as a child put to death experiment mice, get processed group and matched group peritoneal lavage fluid 5 milliliters, centrifugal and collecting cell, adopt fluorescent labeling F4/80 antibody labeling macrophage, by flow cytometer detection F4/80
+pHrodo
+cell mass, to reflect the phagocytic activity of macrophage to apoptotic cell.Result as shown in Figure 3.
Streaming result shows, after the process of different EPOR agonist, and wild-type mice macrophage F4/80
+pHrodo
+cell number is dose-dependent remarkable increase, show that EPOR agonist can promote macrophage phagocytic apoptotic cell in vivo, and macrophage EPOR conditionality engulfs apoptotic cell ability after knocking out significantly declines, after the process of EPOR agonist, its phagocytic activity is without change.
In order to investigate the impact on macrophage inflammatory factor and tissue repair factor expression in EPOR agonist body, experiment mice is put to death after 24 hours at apoptotic cell lumbar injection, get processed group and matched group peritoneal lavage fluid 0.5 milliliter, supernatant is collected after centrifugal segregation cell, conventional ELISA method is adopted to detect wherein inflammatory factor MCP-1, IL-6, TNF-α, the content of inflammation inhibitive factor TGF-β and tissue repair factor Ⅴ EGF.Result as shown in Figure 4.
ELISA result shows, and after different EPOR agonist treatment, inflammatory factor MCP-1, IL-6, TNF-alpha content significantly lowers, and inflammation inhibitive factor TGF-β and tissue repair factor Ⅴ EGF content significantly increase.Show that EPOR agonist can suppress macrophage inflammatory factor in vivo, promote the expression of inflammation inhibitive factor and the tissue repair factor.
Brief summary: engulfed by Turnover of Mouse Peritoneal Macrophages in body and demonstrate EPOR agonist promotion macrophage engulfing apoptotic cell with inflammatory factor reparative factor expression experiment, the effect of inflammation-inhibiting and the expression of promotion reparative factor.
Example 3EPOR agonist, in chmice acute and chronic peritonitis, promotes macrophage engulfing apoptotic cell, and inflammation-inhibiting and promotion reparation, promote the rehabilitation of acute and chronic peritonitis.
The chmice acute peritonitis model that the zymosan A (ZymsonaA) of low dosage (1mg) induces is internationally recognized acute inflammation animal model, the a large amount of neutrophilic granulocyte of inflammation-induced and there is apoptosis, the neutrophilic granulocyte of apoptosis is removed primarily of macrophage phagocytic thus reaches disappearing of inflammation, generally within 48 hours; The mouse peritonitis model of the ZymsonaA induction of high dose (10mg) is classical chronic inflammation model, inflammation persistent induction neutrophilic granulocyte, the removing generation obstacle of apoptosis neutrophilic granulocyte thus the regression time of inflammation is extended.This experiment uses the C57/BL6 chmice acute peritonitis model of EPOR agonist treatment low dosage ZymsonaA induction, and the C57/BL6 murine chronic peritonitis model that high dose ZymsonaA induces, to verify that it is to promotion macrophage phagocytic apoptosis neutrophilic granulocyte, inflammation-inhibiting and promotion are repaired, and promote the rehabilitation of acute and chronic peritonitis.
1, animal: C57/BL6 mice, male, 8-10 week age, body weight 18-20 gram, be purchased from Third Military Medical University's Experimental Animal Center, normally raise in clean animal room.Macrophage EPOR conditionality knock-out mice (by this laboratory oneself prepare), 8-10 week age, body weight 18-20 gram, raise in SPF level Animal House.
2, medicine:
Treatment group: give recombinant human epo (5000IU/kg body weight), carbamylation EPO (50 micrograms/kg body weight) or EPO little peptide molecule (20nmol/kg body weight) of originating carries out lumbar injection; Matched group: give to carry out lumbar injection with the normal saline for the treatment of group equivalent.
3, test method:
ZymsonaA is dissolved in normal saline, makes its concentration be 1 mg/ml.Laboratory animal ether is anaesthetized, then the ZymsonaA solution of lumbar injection 1 milliliter.From after ZymsonaA injection; treatment group gives recombinant human epo (5000IU/kg body weight); carbamylation EPO (50 micrograms/kg body weight) or EPO little peptide molecule (20nmol/kg body weight) of originating carries out lumbar injection treatment, and matched group gives to carry out lumbar injection with the normal saline for the treatment of group equivalent.
Macrophage phagocytic apoptotic cell is promoted in order to investigate EPOR agonist, get the mice acute and chronic peritonitis irrigating solution 5ml of different time points, centrifugal its inflammatory cell of rear separation, first fluorescent labeling F4/80 antibody labeling macrophage is adopted, the neutrophilic granulocyte in fluorescent labeling Ly6G antibody labeled cells is adopted subsequently, by flow cytometer detection F4/80 after rupture of membranes
+ly6G
+cell mass, to reflect the phagocytic activity of macrophage to apoptosis neutrophilic granulocyte.Result as shown in Figure 5.
Streaming result shows, after different EPOR agonist treatment, and F4/80
+ly6G
+cell number significantly increases, and shows that different EPOR agonist can promote macrophage phagocytic apoptosis neutrophilic granulocyte in peritonitis.
The inflammatory factor of acute and chronic peritonitis and the impact of tissue repair factor expression is suppressed in order to investigate EPOR agonist, get the mice acute and chronic peritonitis irrigating solution 0.5ml of different time points, centrifugal rear removal inflammatory cell wherein, conventional ELISA method is adopted to detect inflammatory factor MCP-1 in peritonitis irrigating solution, IL-6, TNF-α, the content of inflammation inhibitive factor TGF-β and tissue repair factor Ⅴ EGF.Result as shown in Figure 6.
ELISA result shows, and in peritonitis, after different EPOR agonist treatment, inflammatory factor MCP-1, IL-6, TNF-alpha content significantly lowers, and inflammation inhibitive factor TGF-β and tissue repair factor Ⅴ EGF content significantly increase.Show that EPOR agonist can the inflammation-inhibiting factor, promote the expression of inflammation inhibitive factor and the tissue repair factor.
Disappearing of active chronic inflammation is promoted in order to investigate EPOR agonist, get the mice acute and chronic peritonitis irrigating solution 5ml of different time points, centrifugal its inflammatory cell of rear separation, fluorescent labeling Ly6G antibody labeling neutrophilic granulocyte is adopted after carrying out cell counting under light microscopic, by flow cytometer detection Ly6G cell mass, to reflect the number of variations of the neutrophilic granulocyte of peritonitis.Result as shown in Figure 7.
Streaming result shows, and after different EPOR agonist treatment, neutrophil numbers significantly reduces.Show that EPOR agonist promotes disappearing of active chronic inflammation.
Brief summary: by demonstrating EPOR agonist in the treatment of mice acute and chronic peritonitis promotion macrophage engulfing apoptotic cell, inflammation-inhibiting and promotion are repaired, and promote the effect that active chronic inflammation disappears.
Example 4EPOR agonist, in mouse system Erythematosus Disease, promotes macrophage engulfing apoptotic cell, and the rehabilitation of accelerating system lupus erythematosus.
This experiment uses EPOR agonist treatment C57/BL6 mouse experiment sexual system lupus erythematosus model, to verify that it is to promotion macrophage phagocytic apoptosis T cell, and promotes the therapeutical effect of autoimmunity systemic lupus erythematosus (sle).
1, animal: C57/BL6 mice, female, 6-8 week age, body weight 15-20 gram, be purchased from Third Military Medical University's Experimental Animal Center, normally raise in clean animal room.
2, medicine:
Treatment group: give recombinant human epo (5000IU/kg body weight), carbamylation EPO (50 micrograms/kg body weight) or EPO little peptide molecule (500ug/kg body weight) of originating carries out lumbar injection; Matched group: give to carry out lumbar injection with the normal saline for the treatment of group equivalent.
3, test method:
Inducing mouse disease model, to 6-8 female C57/BL6 mice single intraperitoneal injection pristane0.5 milliliter in age in week.From pristane induction after after two months, treatment group injects different EPOR agonist treatment, matched group injection normal saline.
Situation is there is in order to what investigate related substances in autoantibody in mouse blood and urine, blood and the urine of getting treatment group and control group mice are respectively measured, detect the blood urea nitrogen in the content of ANA and dsDNA in mouse blood and urine respectively, creatinine and albuminous content, operation operates according to test kit description, and result as shown in Figure 8.
The measurement result display of index of correlation in ANA and dsDNA content and urine in mouse blood, after different EPOR agonist treatment, in mouse blood, the content of ANA and dsDNA is obviously less than matched group, blood urea nitrogen in urine, creatinine and albuminous concentration also obviously reduce, the autoimmune of this prompting mice declines to some extent, and the kidney injury degree of mice also decreases.
In order to investigate the gathering situation of apoptosis neutrophilic granulocyte in blood, after treatment group and control group mice are put to death, get peripheral blood about 0.1 milliliter, adopt the neutrophilic granulocyte in fluorescent labeling LY6G antibody labeling blood, adopt fluorescently-labeled Annexin-V antibody labeling apoptotic cell, utilize flow cytomery LY6G
+annexin-V
+two positive cell group, result as shown in Figure 9.
Assembling situation to investigate apoptotic cell in Mice Body, doing TUNEL dyeing with mouse kidney, display apoptotic cell, and counting.By intracardiac perfusion after deep ether anesthetized mice, 4 DEG C of chances are cold, the phosphate buffer of 4% concentration paraformaldehyde.Left and right side kidney is quickly dismantled again, after fixedly spend the night at 4% paraformaldehyde, paraffin embedding, serial section (3 microns), through dewaxing, Kidney sections is being dyeed by TUNEL test kit, operates and operates according to test kit description.Get mouse kidney glomerule position counting positive cell number, result as shown in Figure 10.
Mouse kidney TUNEL coloration result shows, and after different EPOR agonist treatment, mouse kidney apoptotic cell number all significantly reduces, and this prompting activating macrophage EPOR can reduce the gathering of apoptotic cell in Mice Body.
Macrophage phagocytic apoptotic cell is promoted in order to investigate EPOR agonist, get mice part kidney, its mononuclearcell is separated after grinding, first fluorescent labeling CD68 antibody labeling macrophage is adopted, the T cell in fluorescent labeling CD3 antibody labeled cells is adopted subsequently, by flow cytometer detection CD68 after rupture of membranes
+cD3
+cell mass, to reflect the phagocytic activity of macrophage to apoptosis T cell.Result as shown in figure 11.
Streaming result shows, after different EPOR agonist treatment, and mouse kidney CD68
+cD3
+cell number significantly increases, and shows that EPOR agonist can promote macrophage phagocytic apoptosis T cell.
Brief summary: by the experiment in mouse experiment sexual system lupus erythematosus syndrome, demonstrates different EPOR agonist and all can promote in the organs such as mouse kidney that macrophage is to the removing of engulfing of apoptotic cell, thus the rehabilitation of accelerating system lupus erythematosus.
Example 5EPOR agonist, in rat autoimmune neuritis, promotes macrophage engulfing apoptotic cell, and promotes the rehabilitation of autoimmune neuritis.
Experimental autoimmune neuritis model is the autoimmune disease that CD4+T is cell-mediated, affect peripheral nervous system.This model is very similar to people's Guillain Barre syndrome in all many-sides such as immunopathogenesis, clinical manifestation, neuro pathology's changes, is therefore the animal model of internationally recognized Guillain Barre syndrome.This experiment uses different EPOR agonist treatment Lewis rat experimental autoimmune neuritis model, to verify that it is to promotion macrophage phagocytic apoptosis T cell, and promotes the therapeutical effect of autoimmune neuritis.
1, animal: Lewis rat, male, 8-10 week age, body weight 200-220 gram, be purchased from Third Military Medical University's Experimental Animal Center, normally raise in clean animal room.
2, medicine:
Treatment group: give recombinant human epo (5000IU/kg body weight), carbamylation EPO (50 micrograms/kg body weight) or EPO little peptide molecule (20nmol/kg body weight) of originating carries out lumbar injection; Matched group: give to carry out lumbar injection with the normal saline for the treatment of group equivalent.
3, test method:
The P2 albumen (No. 57 aminoacid-No. 81 aminoacid) of Peripheral Nerves in Rats myelin protein is carried out immunity as autoimmune antigen to experimental rat.P2 antigen is dissolved in phosphate buffer, makes its concentration be 2 mg/ml.Get the emulsifying preparation that the antigenic solution of equivalent and Freund's complete adjuvant carry out conventional method.Laboratory animal ether is anaesthetized, and then bilateral hind paw carries out the antigenic peptides-Freund's complete adjuvant emulsification preparation of each 50 microlitres of subcutaneous injection.After immunity, every day carries out the scoring of the weight of animals weighing and animal behavior.Score index is: 0 be divided into normal, 1 be divided into afterbody deliquescing, 2 to be divided into afterbody to lift, 3 be divided into position not correct voluntarily, 4 be divided into ataxia, 5 to be divided into hind leg mild paralysis, 6 to be divided into hind leg moderate to benumb, 7 be divided into hind leg severe to benumb, 8 be divided into quadriparesis, 9 to be divided into dying state, 10 to be divided into death; From the 7th after immunity day, treatment group gives the treatment of injection polypeptide small molecule.
Immunity and treatment after behavior expression as shown in figure 12, it is less and recover fast that the equal onset of rat comparing matched group different EPOR agonist treatment group is slow, the course of disease is short, the peak period order of severity reduces, body weight reduces degree, confirms that EPOR agonist treatment group is more obvious relative to the curative effect of matched group;
Assembling situation to investigate peripheral nervous system apoptotic cell, doing TUNEL dyeing with rat sciatic nerve, display apoptotic cell, and count.Cold by intracardiac perfusion after deep ether anesthetized rat 4 DEG C chance, the phosphate buffer of 4% concentration paraformaldehyde.Left and right side sciatic nerve is quickly dismantled again, after fixedly spend the night at 4% paraformaldehyde, paraffin embedding, serial section (3 microns), through dewaxing, sciatic nerve section is being dyeed by TUNEL test kit, operates and operates according to test kit description.Get each four sections of rats with bilateral sciatic nerve root and cross section, two, middle part, technology positive cell number, result as shown in figure 13.
Rat sciatic nerve TUNEL coloration result shows, and after different EPOR agonist treatment, sciatic nerve apoptotic cell number all significantly reduces, and this prompting activating macrophage EPOR can reduce the gathering of peripheral nervous apoptotic cell.
Macrophage phagocytic apoptotic cell is promoted in order to investigate EPOR agonist, get rat sciatic nerve and spinal nerve root, its mononuclearcell is separated after grinding, first fluorescent labeling CD68 antibody labeling macrophage is adopted, the T cell in fluorescent labeling CD3 antibody labeled cells is adopted subsequently, by flow cytometer detection CD68 after rupture of membranes
+cD3
+cell mass, to reflect the phagocytic activity of macrophage to apoptosis T cell.Result as shown in figure 14.
Streaming result shows, after different EPOR agonist treatment, and CD68 in peripheral nervous
+cD3
+cell number significantly increases, and shows that EPOR agonist can promote macrophage phagocytic apoptosis T cell.
Brief summary: by the experiment in rat experimental autoimmune neuritis, what demonstrate that different EPOR agonist all can promote macrophage to apoptotic cell in peripheral nervous engulfs removing, thus promotes the rehabilitation of autoimmunity peripheral nervous inflammation.
Example 6EPOR agonist, in rat Autoimmune Encephalomyelitis, promotes macrophage engulfing apoptotic cell, and promotes the rehabilitation of Autoimmune Encephalomyelitis.
Human multiple sclerosis's syndrome be a kind of CD4+Th1 cell-mediated with central nervous system's inflammatory demyelination for feature, with the autoimmune disease of limb paralysis.Experimental autoimmune encephalomyelitis is current internationally recognized multiple sclerosis syndrome animal model.This experiment uses EPOR agonist treatment Lewis rat experimental autoimmune encephalomyelitis model, to verify that it is to promotion macrophage phagocytic apoptosis T cell, and promotes the therapeutical effect of Autoimmune Encephalomyelitis.
1, animal: Male Lewis rats, 8-10 week age, body weight 170-200 gram, is purchased from Third Military Medical University's Experimental Animal Center, normally raises in clean animal room.
2, medicine:
Treatment group: give recombinant human epo (5000IU/kg body weight), carbamylation EPO (50 micrograms/kg body weight) or EPO little peptide molecule (20nmol/kg body weight) of originating carries out lumbar injection; Matched group: give to carry out lumbar injection with the normal saline for the treatment of group equivalent.
3, test method:
Using the autoimmune antigen that rat MBP68-84 (YGSLPQKSQRSQDENPV) albumen uses as immune animal, be dissolved in phosphate buffer, make its concentration be 2 mg/ml.Get the emulsifying preparation that the antigen of equivalent and Freund's complete adjuvant carry out conventional method.Laboratory animal ether is anaesthetized, and then bilateral hind paw carries out the antigenic peptides-Freund's complete adjuvant emulsification preparation of each 50 microlitres of subcutaneous injection.After immunity, every day carries out the scoring of the weight of animals weighing and animal behavior.Score index is: 0 is divided into and is divided into afterbody diminished strength, 2 to be divided into musculus caudalis power to weaken adding hind leg mild paralysis, 3 to be divided into hind leg moderate to benumb without clinical symptoms, 1,4 is divided into quadriparesis, 5 to be divided into dying state; From immunity the 6th day, treatment group injected different EPOR agonist treatment.
Immunity and treatment after behavior expression as shown in figure 15, it is less and recover fast that the equal onset of rat comparing matched group different EPOR agonist treatment group is slow, the course of disease is short, the peak period order of severity reduces, body weight reduces degree, confirms that EPOR agonist treatment group is more obvious relative to the curative effect of matched group.
Assembling situation to investigate central nervous system's apoptotic cell, doing TUNEL dyeing with rat spinal cord, display apoptotic cell, and count.Cold by intracardiac perfusion after deep ether anesthetized rat 4 DEG C chance, the phosphate buffer of 4% concentration paraformaldehyde.Spinal cord is quickly dismantled again, after fixedly spend the night at 4% paraformaldehyde, paraffin embedding, serial section (3 microns), through dewaxing, spinal cord is being dyeed by TUNEL test kit, operates and operates according to test kit description.Get different each nine sections in cross section of rat, counting positive cell number, result as shown in figure 16.
Rat spinal cord is through the display of TUNEL coloration result, and after different EPOR agonist treatment, in spinal cord, apoptotic cell number all significantly reduces, and this prompting activating macrophage EPOR can reduce the gathering of nervus centralis apoptotic cell.
Macrophage phagocytic apoptotic cell is promoted in order to investigate EPOR agonist, get rat spinal cord, after grinding, be separated its mononuclearcell, first adopt fluorescent labeling CD68 antibody labeling macrophage, the T cell in fluorescent labeling CD3 antibody labeled cells is adopted subsequently, by flow cytometer detection CD68 after rupture of membranes
+cD3
+cell mass, to reflect the phagocytic activity of macrophage to apoptosis T cell.Result as shown in figure 17.
Streaming result shows, after different EPOR agonist treatment, and CD68 in spinal cord
+cD3
+cell number significantly increases, and shows that EPOR agonist can promote macrophage phagocytic apoptosis T cell.
Brief summary: by the experiment in rat experimental autoimmune encephalomyelitis, what demonstrate that different EPOR agonist all can promote the macrophage in central nervous system to apoptotic cell engulfs removing, thus promotes the rehabilitation of Autoimmune Encephalomyelitis.
Example 7EPOR agonist, in obesity mice skin wound, promotes macrophage engulfing apoptotic cell, and promotes the healing of fat skin wound.
This experiment uses EPOR agonist treatment obesity mice skin wound incised wound model, to verify that it promotes that the cell of apoptosis is assembled at wound, and strengthens the therapeutical effect of engulfing promotion skin wound healing of macrophage to apoptotic cell.
1, animal: male C 57 BL/6 J mouse, 5-6 week age, be purchased from Third Military Medical University's Experimental Animal Center, normally raise in clean animal room.
2, medicine:
Treatment group: give recombinant human epo (5000IU/kg body weight), carbamylation EPO (50 micrograms/kg body weight) or EPO little peptide molecule (30 μ g/kg body weight) of originating carries out lumbar injection; Matched group: give to carry out lumbar injection with the normal saline for the treatment of group equivalent.
3, test method:
Be divided into 2 groups after healthy male C 57 BL/6 J mouse adaptability feeds 1 week: general food group (NC group), feed with normal feedstuff (2844kcal/kg, 4% crude fat); High fat group (HD group), feeds with high lipid food (5240kcal/kg, 34.9% crude fat).2 groups of parallel raisings of mice, freely drink water, room temperature 21-24 degree, relative humidity 40-60%, natural circadian rhythm.Within every 2 weeks, weigh, the mice that the body weight of getting high fat group group after 16 weeks is greater than the body weight 20% of general food group is high fat diet obesity-induced mice.High fat diet obesity-induced mice lumbar injection 1% Nembutal sodium solution (5 microlitres/gram) anaesthetize.Then slough mouse back fur, iodophor disinfection with 8% sodium sulfide solution, 75% ethanol takes off iodine.Finally cut with skin puncher the circular skin that diameter is 0.6 cm, leave over the wound that lower respective regions is formed.Incised wound wound the 1st day, treatment group injected different EPOR agonist treatment, and measures wound area every day, to reflect skin wound healing situation.Compare discovery with matched group, different EPOR agonist treatments all can promote skin wound healing, and result as shown in figure 18.
Organize apoptotic cell to assemble situation impact to investigate different EPOR agonist on obesity mice skin wound, we do TUNEL dyeing with skin wound, display apoptotic cell, and count.Get wound tissue's 4% paraformaldehyde fixedly to spend the night, paraffin embedding, serial section (3 microns), process dewaxing, wound tissue's TUNEL test kit dyes, and operates and operates according to test kit description.Get different each nine sections in cross section of mice, counting positive cell number, result as shown in figure 19.
Obesity mice skin wound is through the display of TUNEL coloration result, and after different EPOR agonist treatment, skin wound apoptotic cell number all significantly reduces, and this prompting EPOR agonist can reduce the gathering of apoptotic cell at skin wound.
Macrophage phagocytic apoptotic cell is promoted in order to investigate EPOR agonist, get obesity mice wound tissue, its mononuclearcell is separated after grinding, first fluorescent labeling F4/80 antibody labeling macrophage is adopted, the neutrophilic granulocyte in fluorescent labeling Ly6G antibody labeled cells is adopted subsequently, by flow cytometer detection F4/80 after rupture of membranes
+ly6G
+cell mass, to reflect the phagocytic activity of macrophage to apoptosis neutrophilic granulocyte.Result as shown in figure 20.
Streaming result shows, after different EPOR agonist treatment, and wound tissue F4/80
+ly6G
+cell number significantly increases, and shows that EPOR agonist can promote macrophage phagocytic apoptosis T cell.
Brief summary: by the experiment in obesity mice incised wound model, what demonstrate that different EPOR agonist all can promote macrophage to apoptotic cell engulfs removing, and promotes the healing of obesity mice skin wound.
Example 8EPOR agonist, in rat aorta is atherosis, promotes macrophage engulfing apoptotic cell, and promotes atherosclerotic rehabilitation.
Due to ApoE
-/-mice lacks the ability of residual grain lipoprotein removed C-VLDL and produced by chylomicrons, and just there will be atherosclerotic plaque in early days with the mice that high fat is fed, therefore the ApoE that feeds of high fat
-/-mouse model has become the basic model of studying atherosclerotic.This experiment uses different EPOR agonist treatment height fat to feed the ApoE of induction
-/-atherosclerosis Model, to verify that it promotes macrophage engulfing apoptotic cell, and promotes atherosclerotic rehabilitation.
1, animal: male ApoE
-/-mice, in 8 week age, body weight 22 ± 2g, is purchased from model organism center, Nanjing, normally raises in SPF Animal House.
2, medicine:
Treatment group: give recombinant human epo (5000IU/kg body weight), carbamylation EPO (50 micrograms/kg body weight) or EPO little peptide molecule (30 μ g/kg body weight) of originating carries out lumbar injection; Matched group: give to carry out lumbar injection with the normal saline for the treatment of group equivalent.
3, test method:
ApoE
-/-after mice (8-10 week) adaptability feeds 1 week, by ApoE
-/-mice is divided into 2 groups: general food group (NC group), feeds with normal feedstuff (2844kcal/kg, 4% crude fat); High fat group (HD group), feeds with high lipid food (5240kcal/kg, 34.9% crude fat).2 groups of parallel raisings of mice, freely drink water, room temperature 21-24 degree, relative humidity 40-60%, natural circadian rhythm.High fat group mice is further divided into treatment group and matched group, gives different EPOR agonist or contrast normal saline respectively by abdominal cavity.After feeding 12 weeks, put to death animal, get its aortic arch, row HE dyes, detect atherosclerotic lesions district area and apoptotic cell gathering situation, to verify that EPOR agonist treatment can promote macrophage engulfing apoptotic cell, and promote atherosclerotic rehabilitation.
In order to investigate different EPOR agonist to ApoE
-/-the therapeutical effect of rat aorta speckle, we do HE dyeing to aortic arch, detect EPOR agonist to the impact of atheromatous plaque damage zone area.Get aortic arch 4% paraformaldehyde fixedly to spend the night, paraffin embedding, serial section (5-7 micron), process dewaxing, aortic arch HE test kit dyes, and operates and operates according to test kit description.Get different each nine sections in cross section of mice, calculate damage zone area, result as shown in figure 21.
ApoE
-/-mouse aorta bow shows through HE coloration result, after different EPOR agonist treatment, in aortic arch, plaque region damaged area significantly reduces, and this points out different EPOR agonist can reduce the formation of atheromatous plaque, promotes atherosclerotic rehabilitation.
In order to investigate EPOR agonist to ApoE
-/-rat aorta plaque region organizes apoptotic cell to assemble situation, and we do TUNEL dyeing to aortic arch, display apoptotic cell, and counts.Get aortic arch 4% paraformaldehyde fixedly to spend the night, paraffin embedding, serial section (3 microns), process dewaxing, aortic arch TUNEL test kit dyes, and operates and operates according to test kit description.Get different each nine sections in cross section of mice, counting positive cell number, result as shown in figure 22.
ApoE
-/-mouse aorta bow is through the display of TUNEL coloration result, and after different EPOR agonist treatment, in aortic arch district, apoptotic cell number significantly reduces, and this prompting EPOR agonist can reduce the gathering of apoptotic cell in artery plaque district.
What finally illustrate is, above embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to technical scheme of the present invention or equivalent replacement, and not departing from aim and the scope of technical solution of the present invention, it all should be encompassed in the middle of right of the present invention.
Claims (10)
1. erythropoietin and derivant thereof are preparing in disease therapy the application promoted in the medicine of apoptotic cell clearance.
2. application according to claim 1, is characterized in that, described erythropoietin and derivant thereof promote the application in the medicine of apoptotic cell clearance in the disease caused by preparation treatment active chronic inflammation; Or described erythropoietin and derivant thereof promote the application in the medicine of apoptotic cell clearance in preparation treatment autoimmune disease; Or described erythropoietin and derivant thereof promote the application in the medicine of apoptotic cell clearance in preparation treatment atheromatosis; Or described erythropoietin and derivant thereof promote the application in the medicine of apoptotic cell clearance in preparation treatment wound healing.
3. application according to claim 2, is characterized in that, described erythropoietin and derivant thereof treat in acute and chronic peritonitis the application promoted in the medicine of apoptotic cell clearance in preparation.
4. application according to claim 2, is characterized in that, described erythropoietin and derivant thereof are preparing in systemic lupus erythematosus the application promoted in the medicine of apoptotic cell clearance.
5. application according to claim 2, is characterized in that, described erythropoietin and derivant thereof promote the application in the medicine of apoptotic cell clearance in preparation treatment autoimmune neuritis.
6. application according to claim 2, is characterized in that, described erythropoietin and derivant thereof promote the application in the medicine of apoptotic cell clearance in preparation treatment Autoimmune Encephalomyelitis.
7. be used for the treatment of in disease the medicine promoting apoptotic cell clearance, it is characterized in that, the active component of described medicine is containing having erythropoietin and derivant thereof.
8. medicine according to claim 7, is characterized in that, described medicine, by the erythropoietin receptor of activating macrophage, promotes macrophage phagocytic apoptotic cell, Induced synthesis inflammation inhibitive factor and the promotion tissue repair factor; Described inflammation inhibitive factor comprises TGF-β, and the described promotion tissue repair factor comprises VEGF.
9. medicine according to claim 7, is characterized in that, described medicine also comprises pharmaceutically acceptable carrier.
10. be that the application of removing regulation and control and promoting in disease therapy in apoptotic cell clearance engulfed by the medicine that target spot is developed at apoptotic cell with erythropoietin receptor.
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