CN106215170A - Interleukin Ⅲ 6 α treats in preparation, diagnoses or prevent the purposes in medication for treating pyemia - Google Patents

Abstract

The invention provides interleukin Ⅲ 6 α in preparation treatment, diagnose or prevent the application in medication for treating pyemia, pyemia complexity regulated and control network searches out new treatment, diagnoses, prevents target spot, pyemic preventing and treating is brought new breakthrough.

Description

Interleukin Ⅲ 6 α treats in preparation, diagnoses or prevent the purposes in medication for treating pyemia

Technical field

The present invention relates to technical field of pharmaceutical biotechnology, particularly to the medicinal usage that IL-36 α is new.

Background technology

Pyemia (Sepsis) is the systemic inflammatory response syndrome (systemic caused by infection Inflammatory response syndrome, SIRS), be wound, burn, suffer a shock, the clinical critically ill patients such as infection One of severe complication, is the main cause of death of nosocomial infection.Pyemia is generally infected by antibacterial and causes, it is possible to have true Bacterium, parasitic infection cause.The course advancement of pyemia is mainly four parts: nonspecific infection, systemic inflammatory response is comprehensive Levy, pyemia, serious symptom pyemia；As can not get controlling timely and effectively, shock may be entered, organ failure, even It is dead.Owing to sepsis breaks with tremendous force, disease progression is rapid, causes multiple organ dysfunction syndrome further, and case fatality rate is high, is faced Bed treatment work brings extreme difficulties, and WHO, will in order to improve various circles of society to the concern of pyemia and understanding Annual JIUYUE 13 is set to world's septicemia.

Pyemia mechanism is sufficiently complex, with a series of physiology such as infection, inflammation, immunity, blood coagulation and histologic lesions Pathological process all has correlative connection.The inflammatory factor infecting release relate to whole body by neuro-endocrino immunological regulation system Various kinds of cell and multiple system, form the network of bulky complex of knowing clearly, and cascading is amplified, and restricts each other, therefore clinical Diagnosis and treatment are the most difficult.Current Therapeutic Method is to give effective antibacterial therapy, and combines change each organ systems comprehensive Treatment, but poor effect, mortality rate is the highest.According to statistics, the whole world has 18,000,000 people's morbidities, the common cognition meeting of people every year Think that cancer or acquired immune deficiency syndrome (AIDS) are the most terrified, but hardly realize that the death that septicemia causes every year has exceeded carcinoma of prostate, pulmonary carcinoma and HIV/ The summation of AIDS quantity.In the U.S., sepsis patient mortality rate accounts for the 17% of inpatient.At China ICU, serious symptom pyemia Incidence rate be 8.68%, mortality rate is up to 44.7%.

Pyemia onset is anxious, and the course of disease is critical, and mortality rate is the highest, and medical expense is higher, and medical resource consumes big, to people Class health causes grave danger, has a strong impact on the quality of life of patient and household.If patient the most correctly diagnoses and accepts effectively Antibiotic therapy, mortality risk will gradually rise, often postpone one hour, mortality rate increase by 7.6%.Thus, it could be seen that find fast The survival rate tool improving patient is of great significance by speed diagnosis, the novel targets of effectively treatment.

Space interleukin is to be produced and act on a type cytokines of various kinds of cell by various kinds of cell, due to initially Being to be produced by leukocyte and play a role between leukocyte, so thus gaining the name, now continuing to use the most always.Interleukin English is Interleukin, is abbreviated as IL.In the research process to immunne response, send out in the cells and supernatant of mitogenstimulated Having showed many and had bioactive molecule, the activity that researcher respectively records with oneself is named, and the more than ten years report nearly hundred Plant the factor.Comparing research by Protocols in Molecular Biology later to find, the most many factors with biological activity name are real It is that there is polyphenic same substance on border.In order to avoid the confusion of name, Second Committee world lymphokine workshop in 1979 Discussing the cytokine Uniform Name of leukocyte interphase interaction during immunne response is interleukin, add after title Ah Arabic numbers is numbered to show difference, such as IL-1, IL-2 ..., the newly determined factor is named successively.

Interleukin is very important cytokine family, and the family having been found that up to now has reached 38；They Immunocyte maturation, activate, breed and immunomodulating etc. a series of during all play a role, they also participate in machine in addition The multiple physiology of body and pathological reaction.

IL-1 is also known as lymphocyte stimulating factor, cell derived: mainly produced by the monocytes/macrophages activated.Exist Form: IL-1 α and IL-1 β.Principal biological function (1) locally low concentration-immunomodulating: the collaborative APC of stimulation and T cell are lived Change, promote B cell proliferation and secretory antibody.(2) a large amount of generation-endocrine effects: induced liver acute phase protein synthesizes；Cause Heating and cachexia.IL-2 is also known as SCIF, TCGF.Cell derived: mainly produced by T cell.Model of action: with Autocrine and paracrine mode play effect.Principal biological function (1) activating T cell, promotes that cytokine produces；(2) thorn Swashing NK cell proliferation, strengthen NK killing activity and produce cytokine, induction LAK cell produces；(3) promote B cell proliferation and Secretory antibody；(4) activating macrophage.IL-4 cell derived: mainly produced by Th2 cell, mastocyte and basophilia, granulocyte Raw.Major function (1) promotees B cell proliferation, differentiation；(2) induction IgG1 and IgE produces；(3) promote that Th0 cell divides to Th2 cell Change；(4) suppression Th1 cell activation and secrete cytokines；(5) collaborative IL-3 stimulates proliferation of mast cells etc..IL-6 is anti-human in vain Cytokine, cell derived is mainly produced by mononuclear phagocyte, Th2 cell, vascular endothelial cell, fibroblast.Mainly Function: (1) stimulates activating B cell propagation, secretory antibody；(2) T cell propagation and CTL activation are stimulated；(3) cell cultured supernatant is closed Become acute phase protein, participate in inflammatory reaction；(4) blood cell development is promoted.IL-10 cell derived is mainly Th2 cell and monokaryon Macrophage produces.Major function: (1) suppression Pro-inflammatory mediator produces.(2) suppression MHC-II quasi-molecule and B-7 molecule The cytokine such as expression.(3) suppression T cell synthesis IL-2, IFN-γ.(4) B cell differentiation and proliferation can be promoted.IL-12 cell Source: mainly produced by monocytes/macrophages, B cell.Major function: (1) is activated and strengthens NK cell killing activity and IFN- γ (2) promotes that Th0 breaks up to Th1 cell, secretes IL-2, IFN-γ.(3) CD8+CTL cell killing activity is strengthened；(4) can assist LAK cell is induced with IL-2；(5) suppression Th0 cell synthesizes to the differentiation of Th2 cell and IgE.

Interleukin Ⅲ 6 α (Interleukin-36 α, IL-36 α) be the most newfound IL-1 cell because of The newcomer of Zijia race, be found also has IL-36 β, IL-36 γ, is referred to as IL-36 simultaneously.IL-36 mainly comprises: IL-36 α (IL-1F6), IL-36 β (IL-1F8), IL-36 γ (IL-1F9), IL-36Ra receptor antagonist, IL-37, also have by IL-36R Receptor dimer with IL-1RAcP composition.The various kinds of cell such as IL-36 α can be by mononuclear cell, T lymphocyte, horn cell are produced Raw, it is mainly distributed in skin, lungs, joint, intestinal, kidney and brain.IL-36 α is mainly combined by receptor corresponding to it Rear activation mitogen activated protein kinase (MAPK) and nuclear factor B (NF-κ B) signal transduction pathway are at inherent immunity and adaptability Immunity plays an important role.

Summary of the invention

It is an object of the invention in pyemia complexity regulated and control network, search out new treatment, diagnose, prevent target Point, brings new breakthrough to pyemic preventing and treating.

It is an object of the invention to be realized by following measures:

Interleukin Ⅲ 6 α (Interleukin-36 α, IL-36 α) treats in preparation, diagnoses or prevent medication for treating pyemia In application.

Interleukin Ⅲ 6 α (Interleukin-36 α, IL-36 α) answering in preparation regulation macrophage function medicine With.

Interleukin Ⅲ 6 α (Interleukin-36 α, IL-36 α) treats in preparation, diagnoses or prevent medication for treating pyemia In application, dosage is 1-1000 μ g/kg body weight.

The invention provides interleukin Ⅲ 6 α (IL-36 α) pyemia treatment, diagnose, prevent in application, Give ectogenic IL-36 α and sepsis can be had obvious therapeutic action, be remarkably improved its survival rate.

Present invention discover that and give ectogenic IL-36 α albumen, the ability of body bacteria removal can be significantly improved, and make The multiple organ inflammation damnification degree such as liver lung kidney alleviate.

Accompanying drawing explanation

Fig. 1: IL-36 α in animal pyemia model, IL-36 β, IL-36 γ test experience:

Fig. 2: survival rate test experience

The bacterial loads experiment of Fig. 3: pyemia animal model: A figure is 24 hours, and B figure is 48 hours

The pathology detection of Fig. 4: each organ injury

Fig. 5: the IL-36 α impact on the Phagocytosis function of mouse macrophage

Fig. 6: CLP modeling process figure

Detailed description of the invention

The following stated is the preferred embodiments of the present invention, but the present invention is not limited to following example.Described method such as without Special instruction is conventional method.Described raw material the most all can obtain from open commercial sources.

Embodiment 1

The foundation (Cecal Ligation paracentesis Cecal ligation andpuncture, CLP) of sepsis animal model

Laboratory animal: experiment mice used is male, and body weight is 18-22g, about 6-8 week old.Raise in Medical University Of Chongqing Experimental Animal Center SPF (Specific Pathogen Free) level laboratory.It is dynamic that mice used by this experiment is the experiment of SPF level Thing.Wild type C57BL/6 mice (Wild type, WT) is bought from Beijing HFK Bio-Technology Co., Ltd..

Method: grow up C57 mice, with the pentobarbital sodium intraperitoneal injection of anesthesia mice of 100 microlitres 1.5%, is fixed on behaviour Make plate, shave off abdominal part hair with Pet electric clipper, skin of sterilizing, the otch of 1cm length is done in abdomen center, cecal ligation, with No. 26 Syringe needle punctures, such as Fig. 5.Finally sew up a wound, skin of sterilizing.(with specific reference to: Daniel Rittirsch, Peter A Ward, et al.Immunodesign of experimental sepsis by cecal ligation and puncture.Nat Protoc.2009；4 (1): 31-36).This modeling method is the classical modeling side of sepsis animal model Method, and this modeling method is to study at present pyemic goldstandard animal model.

IL-36 α in animal sepsis models, IL-36 β, IL-36 γ test experience

After mice is built pyemia model, respectively 6 hours, 12 hours, 24 hours these three time points take lung tissue, Extracting its RNA, reverse transcription is cDNA, carries out quantitative PCR, measures IL-36 α, and the expression of IL-36 β, IL-36 γ, such as Fig. 1-A； Mice is built pyemia model, take after 24 hours cardiac blood, lung, peritoneal lavage fluid (Peritoneal lavagefluid, Etc. PLF) specimen processes, and measures the expression of IL-36 α, IL-36 γ by ELISA kit (being purchased from MyBioSourse company) Amount, such as Fig. 1-B.Can draw from result, IL-36 α expresses notable rising in the animal body of pyemia.

Survival rate is tested:

Mice is built sepsis model, and the big portion of caecum ligatures, and makees severe sepsis model, gives experimental group little after skin of sewing it up Mus lumbar injection 500ng recombined small-mouse IL-36 α albumen (being purchased from the R&D company of the U.S.), equal to control group mice lumbar injection The aseptic PBS of volume, the Survival of observed and recorded mice, observe every day twice, Continuous Observation 2 weeks, the most dead to mice Till.Shown in result such as Fig. 2 (A), the mouse survival rate of injection rIL-36 α group is 57.1%, the mouse survival rate of injection PBS group It is 14.3%, it is seen that rIL-36 α group survival rate is apparently higher than PBS group, and energy long-term surviving, illustrates that IL-36 α is conducive to mice to exist Existence in pyemia.

Mice is cooked severe sepsis model, (is purchased from the U.S. to the IL-36 Alpha antibodies of experimental mice lumbar injection 1 μ g R&D company), to the IgG of control group mice lumbar injection equal volume, the Survival of observed and recorded mice, observe two every day Secondary, Continuous Observation 2 weeks, to mice no longer death.Shown in result such as Fig. 2 (B), the experimental mice survival rate of injection of antibodies Being decreased obviously, and significantly lower than the survival rate of matched group, reverse instruction IL-36 α is conducive to mice life in pyemia Deposit.

The mensuration of bacterial loads:

Experimental group lumbar injection rIL-36 α after mouse modeling, the aseptic PBS of matched group lumbar injection, 24 hours and 48 little Take peritoneal lavage fluid, cardiac blood and spleen time after respectively, after peritoneal lavage fluid and blood are done 10 times of serial dilutions, take 100 microlitre pavings Plate, spleen is first homogenized, and takes 100 microlitre bed boards after then doing 10 times of serial dilutions, counts on blood agar plate after 18-24 hour Clump count.

Result as it is shown on figure 3, compared with matched group, IL-36 α group after setting up pyemia model, its cardiac blood, spleen Lower with the bacterial loads in peritoneal lavage fluid, body bacteria removal ability is higher, illustrates that IL-36 α is conducive to the removing of antibacterial.

The pathology detection of each organ injury:

After mouse modeling, experimental group lumbar injection rIL-36 α, the aseptic PBS of matched group lumbar injection, at 6 hours and 24 By putting to death after mouse anesthesia after hour, take lung, liver,spleen,kidney, in 4% paraformaldehyde, fix 24--48 hour, take off respectively Water, waxdip, embed, cut into slices, after HE dyes under microscope microscopy.Result as shown in Figure 4, compared with IL-36 α experimental group, The each organ injury of control group mice is the most heavier, and from increased inflammation obvious seen from pathological section, inflammatory cell infiltration, albumen is scorching Property ooze out, organize congestion and edema, the performance such as necrocytosis.Biochemical indicator ALT and AST can reflect hepatic injury, ALT's and AST Raise prompting hepatocyte injury serious.

After mouse modeling, experimental group lumbar injection rIL-36 α, the aseptic PBS of matched group lumbar injection, at 6 hours and 24 By putting to death after mouse anesthesia after hour, take lung, liver,spleen,kidney, in 4% paraformaldehyde, fix 24--48 hour, take off respectively Water, waxdip, embed, cut into slices, then with TUNEL method (DAB colour developing) detection apoptosis.Result is as shown in Figure 4, real with IL-36 α Testing group to compare, control group mice hepatocellular apoptosis becomes apparent from, and 24 hours less than 6 time apoptosis degree more serious, IL-36 is described α can alleviate the pyemia infringement to organ.

The IL-36 α impact on the Phagocytosis function of mouse macrophage

To mouse peritoneal injection paraffin with inducing macrophage, take peritoneal macrophage after 3-5 days and cultivate, treat that cell is complete After portion is adherent, process group cell adds IL-36 α recombiant protein, and matched group PBS compares.37 degree, that 5%CO2 cultivates 24 is little Shi Hou, two groups of cells all add the same amount of Pseudomonas aeruginosa inactivated with FITC fluorescein labelling.Hatch again After 30 minutes, discard cell conditioned medium, wash cell with PBS, afterwards mounting, with fluorescence microscope and take pictures.Result such as Fig. 5 (A), shown in, the macrophage that processed by IL-36 α recombiant protein is more antibacterial, explanation than matched group macrophage phagocytic IL-36 α can improve the phagocytic function of macrophage.With IL-36 β, IL-36 γ be experiment contrast group and find no raising huge Phagocytal phagocytic function and the effect of sterilizing function, do not have therapeutic effect to sepsis.

To mouse peritoneal injection paraffin with inducing macrophage, take peritoneal macrophage after 3-5 days and cultivate, spread two respectively Individual plate, after cell is the most adherent, process group cell adds IL-36 α recombiant protein, and matched group PBS compares.37 degree, After 5%CO2 cultivates 24 hours, in two groups of cells, add the same amount of Pseudomonas aeruginosa not inactivated, abandon after 30 minutes Remove all cells supernatant.A collection of cell does 10 times of gradient dilutions after being cracked by Triton, be laid on blood plate, and another batch of cell continues After cultivating 90 minutes, crack with Triton, after 10 times of gradient dilutions, be laid on blood plate.Blood plate 37 degree, 5%CO2 cultivates Overnight, second day enumeration of bacterial colonies.By the difference of the quantity of two batches of intracellular antibacterials, this experiment reflects that macrophage gulps down Bite antibacterial and kill the effect of antibacterial.As shown in Fig. 5 (B), compared with matched group, IL-36 α process after cell can swallow and Kill more antibacterial, illustrate that IL-36 α can improve phagocytic function and the sterilizing function of macrophage.Macrophage sterilization energy The potential mechanism that power strengthens is relevant with MARCO, and MARCO full name is macrophage receptor with collagenous Structure, by MARCO gene code, is a member of scavenger receptor family.IL-36a may be by strengthening MARCO Expression, thus strengthen the function of macrophage.

Claims (3)

1. interleukin Ⅲ 6 α treats in preparation, diagnoses or prevent the application in medication for treating pyemia.

2. interleukin Ⅲ 6 α application in preparation regulation macrophage function medicine.

3. interleukin Ⅲ 6 α treats in preparation, diagnoses or prevent the application in medication for treating pyemia, and dosage is 1-1000 μ g/kg Body weight.