CN102370967A

CN102370967A - Novel application of IL-1Ra (interleukin-1 receptor antagonist) and tumor treatment medicinal composition kit thereof

Info

Publication number: CN102370967A
Application number: CN201010253070XA
Authority: CN
Inventors: 韩伟; 俞雁; 于鸿晶
Original assignee: SHANGHAI WUYANG BIOMEDICAL TECHNOLOGY Co Ltd; Shanghai Jiaotong University
Current assignee: SHANGHAI WUYANG BIOMEDICAL TECHNOLOGY Co Ltd; Shanghai Jiaotong University
Priority date: 2010-08-13
Filing date: 2010-08-13
Publication date: 2012-03-14

Abstract

The invention belongs to the technical field of biological medicines, in particular relates to novel application of IL-1Ra (interleukin-1 receptor antagonist), and discloses application of the IL-1Ra protein (shown in the specification) in preparing a medicament for treating or protecting thymic lymphocyte injury or peripheral T lymphopenia caused by radiotherapy and/or chemotherapeutic drugs. The protein can effectively reduce, treat or prevent acute thymus injury and/or immunological function repression side effect caused by radiotherapy and/or chemotherapeutic drugs, and is expected to be a novel choice for clinical tumor treatment in the future.

Description

The new purposes of IL-1Ra and anti-tumor medicine Combined drug box thereof

Technical field

The invention belongs to the biological medicine technology field, be specifically related to new purposes and the anti-tumor medicine Combined drug box thereof of IL-1Ra.

Background technology

In recent years, along with the progress of medical science, modern humans's average life rises to 80 years old, and the sickness rate of tumor obviously increases.Tumor and cardiovascular and cerebrovascular disease become two big killers of human health, and the high incidence of tumor and high fatality rate become the key factor that threatens human health.

Along with to the tumor invasion Study on Mechanism, people have had further understanding to tumor.Research shows that tumor cell can migrate to other position by former position of tumor for growth abnormal cell out of control, if can not control this diffusion of tumor, tumor will be invaded the vitals of human body, cause the organ failure, finally cause death.The common method of treating tumor at present clinically has surgery operating removing tumor, radiotherapy, chemotherapy and Chinese traditional treatment etc.In view of tumor is a kind of disease of general, rely on the therapeutic effect of tumor resection often not ideal enough merely.Show when tumour patient is made a definite diagnosis, only have the patient about 1/3rd to suit to cure through operation according to interrelated data.Be in the cancer middle and advanced stage when most humans is made a definite diagnosis, need be aided with radiotherapy with (or) chemotherapy, so chemicotherapy is the important means of current treating malignant tumor and prevention of recurrence.But chemicotherapy " is failed to differentiate between the enemy and ourselves ", and pair cell lacks selectivity, in the kill tumor cell, kills and wounds normal cell equally, though thereby chemicotherapy be the important method of oncotherapy, but still have serious toxic and side effects [4].

Radiotherapy with (or) major side effects of chemotherapy is that immunologic function suppresses and the bone marrow hematogenesis inhibition; Most of chemotherapeutics; (5-azacytidine or5-azacitidine 5-Aza) waits chemotherapeutics can cause the acute injury of thymus immune organ and the decline of periphery immunologic function, and this has directly limited the application of chemotherapy like cyclophosphamide, 5-fluorouracil, 5-azacytidine; And patient is under the state of immunologic function disappearance after the chemotherapy, and causing infecting probability increases.If patient can't recover the immunologic function of normal level after chemotherapy, patient suffers from various immune diseases, virus and the infection of antibacterial and the probability of cancer return can increase greatly so.

Thymus is the important immune organ of body, is the cytocerastic place of T.Thymus T CFU-GM derives from bone marrow, and its surface marker is CD3 ^-CD4 ^-CD8 ^-(triple negative TN), has the ability that is divided into T cell, BMDC or natural killer cell, and it has experienced the complex differentiation process from cortex to the medullary substance in thymus.The TN cell in thymus with the complicated groups of cells phase-splitting effect of microenvironment, obtain the CD3 molecule, become CD4 ^-CD8 ^-Jack to jack adapter property (double-negative, DN) cell.TN cell and DN cell all belong to early stage pre-T cell.Subsequently, the DN cell is passing through DN I (CD44 successively ⁺CD25 ^-), DN II (CD44 ⁺CD25 ⁺), DN III (CD44 ^-CD25 ⁺), DN IV (CD44 ^-CD25 ^-) four-stage grows and to be CD4, the two positive cell (CD4 of CD8 ⁺CD8 ⁺Double-positive, DP).DP cell in the cortex is through TXi Baoshouti (T-cell antigen receptor) gene rearrangement; Accomplish positive the selection (positive selection, PS) with negative the selection (negative selection, NS); Grow and be the single positive of CD4 or the single positive (single-positive of CD8; SP) naive T cell is transported to periphery, as helper T cell or killer T cell performance immunologic function.

" interleukin-1 receptor antagonist " (interleukin-1 receptor antagonist IL-1Ra), belongs to the IL-1 family member with IL-1 α, IL-1 β three jointly.IL-1Ra is IL-1 α, the common antagonist of IL-1 β, and it can combine with its receptor IL-1RI by emulative inhibition IL-1, is arrested in the biological activity of expressing IL-1 in a plurality of tissues and the organ.IL-1Ra is used for diseases such as rheumatoid arthritis, septicemia clinically.The IL-1Ra of people and mice has 77% homology, and the biological action of IL-1Ra does not have species specificity.The difference of the IL-1Ra molecular weight that different cells produce is mainly by due to the degree of glycosylation difference.Confirm that glycosylation does not have influence (R Daig, G Rogler, E Aschenbrenner, et al.Gut2000 (46): 350-358) to the IL-1Ra BA.

Summary of the invention

Technical problem to be solved by this invention is to solve immunologic function inhibition side-effect problem in tumour radiotherapy and/or the chemotherapy process.

For this reason, the invention discloses a kind of (a) as follows or albumen (b) preparation be used for treatment or protection radiotherapy with (or) the thymus acute injury that causes of chemotherapeutics, thymic lymphocytes damage and (or) purposes of the medicine that reduces of peripheral T lymphocyte:

(a) its aminoacid sequence such as the described albumen of SEQ ID NO.1;

(b) at least with (a) in aminoacid sequence 70% homology and have radiotherapy with (or) the thymus acute injury that causes of chemotherapeutics, thymic lymphocytes damage and (or) peripheral T lymphocyte reduces active albumen.

In certain embodiments, said thymic lymphocytes are thymus T cell, and thymus T cell can be DN T cell, DP T cell, CD4 SP T cell or CD8 SP T cell.Said peripheral T lymphocyte is CD4 ⁺CD45RA ⁺T cell and CD8 ⁺CD45RA ⁺The T cell.

In one embodiment, the albumen of said (b) is aminoacid sequence such as the described albumen of SEQ ID NO.2.

In some embodiments, said chemotherapeutics can be selected from alkylating agent class medicine, antimetabolitas, antibiotics, plant medicine thing, hormone medicine, metal complex or protein drug, wherein is preferably antimetabolitas.

On the other hand, the invention discloses a kind of pharmaceutical composition, it contains as the above-mentioned albumen of active component and pharmaceutically acceptable carrier or excipient.

In some embodiments, the preparation of said pharmaceutical composition is an intestinal external administration preparation, and said intestinal external administration preparation can be injection or injectable sterile powder.

On the other hand, the invention discloses a kind of anti-tumor medicine Combined drug box, it comprises the preparation of tumor chemotherapeutic drug preparation and pharmaceutical composition according to the invention.

On the other hand, the invention discloses above-mentioned albumen and can treat as active component or prevent radiotherapy or/and the thymus acute injury that chemotherapeutics causes and/or immunologic function suppress the method for side effect, this method comprises the above-mentioned albumen that gives individual effective dose.

On the other hand, the invention also discloses a kind of method of treating tumor, this method is included in radiotherapy or/and before the chemotherapeutics, give the above-mentioned albumen of individual effective dose.

Proteic new purposes according to the invention can reduce treatment effectively or prevent radiotherapy or/and thymus acute injury that chemotherapeutics causes and/or immunologic function suppress side effect, possibly new selection is provided for the treatment of clinical tumor from now on.

Description of drawings

Fig. 1: T cells whose development process sketch map in the thymus;

Fig. 2: recombinant human interleukin--1's receptor antagonist (recombinant human interleukin-1 receptorantagonist, the protective effect of the injury of thymus gland that rHuIL-1Ra) the 5-Aza chemotherapy is caused;

Fig. 3: the H&E colored graph (low power lens) of different time mouse thymus after the 5-Aza chemotherapy;

Fig. 4: the 7th day mouse thymus section H&E colored graph (high power lens) after the 5-Aza chemotherapy;

The protective effect that the thymus index that Fig. 5: rHuIL-1Ra causes the 5-Aza chemotherapy damages;

The protective effect that the thymocyte cell sum that Fig. 6: rHuIL-1Ra causes the 5-Aza chemotherapy damages;

Fig. 7: the detection of thymocyte proliferation;

The specific embodiment

At this paper, (interleukin-1 receptor antagonist IL-1Ra), belongs to the IL-1 family member with IL-1 α, IL-1 β three to term said " interleukin-1 receptor antagonist " jointly.IL-1Ra is IL-1 α, the common antagonist of IL-1 β, and ability antagonism IL-1 combines with its receptor interleukin-1 receptor I's (IL-1RI), and suppresses the BA of IL-1.Aminoacid sequence such as the SEQ IDNO.1 of the IL-1Ra of people's mature form (hIL-1Ra) are said, and the IL-1Ra of people and mice has 77% homology, and the biological action of IL-1Ra does not have species specificity.The difference of the IL-1Ra molecular weight that different cells produce is mainly by due to the degree of glycosylation difference.Confirm, glycosylation to the IL-1Ra BA do not have influence (R Daig, G Rogler, E Aschenbrenner, et al.Gut 2000 (46): 350-358).

Said protein of the present invention preferably this material is hIL-1Ra protein and mutant thereof; Its functional activity fragment or its analog; The proteinic carrier that homologue and coding with high homology comprises the aminoacid sequence that SEQ ID NO.1 describes is dna vector (plasmid or virus) for example.Functional activity fragment or analog can form through one or more amino acid residue that adds, inserts, modifies, replaces or lack in the above listed aminoacid sequence.

Term " analog " also comprises precursor and other functional equivalent or the analogies of chimeric protein, fusion rotein, anti-idiotype antibody, above-claimed cpd.Also comprise the conjugated protein active compound body of Simulation with I L-1Ra.

Term " mutant " is meant the mutant of aminoacid sequence such as the said hIL-1Ra of SEQ ID NO.1.Than natural IL-1Ra albumen, this mutant is compared with their wild types, has enhanced activity and/or altered stereospecificity.The aminoacid sequence mutant of native protein can be through introducing suitable nucleotide variation or preparing through external synthetic required polypeptide in nucleotide of the present invention.These mutants comprise, for example lack, insert or replace the residue in this aminoacid sequence.Can through disappearance, insert and the combination of replacement to obtain final construct, final protein product is provided.

Proteic percent homology is analyzed (GCG program) by GAP (Needleman and Wunsh, 1970) and is confirmed, parameter gap creation penalty=5 wherein, gap extension penalty=0.3.When the sequence length of being analyzed was at least 15 aminoacid, GAP just analyzed and tests in 15 the amino acid whose zones that are at least of two sequences of participating in test.More preferably, when the sequence length of being analyzed was at least 50 aminoacid, GAP just analyzed and tests in 50 the amino acid whose zones that are at least of two sequences of participating in test.More preferably, when the sequence length of being analyzed was at least 100 aminoacid, GAP just analyzed and tests in 100 the amino acid whose zones that are at least of two sequences of participating in test.More preferably, when the sequence length of being analyzed was at least 250 aminoacid, GAP just analyzed and tests in 250 the amino acid whose zones that are at least of two sequences of participating in test.Even more preferably, when the sequence length of being analyzed was at least 500 aminoacid, GAP just analyzed and tests in 500 the amino acid whose zones that are at least of two sequences of participating in test.

The aspect that the present invention relates to also comprises IL-1Ra albumen analog; Carrying out different modifications between their synthesis stages or after synthetic; For example, through biotinylation, benzylization, glycosylation, acetylation, phosphorylation, by known protection/blocking groups derivatization, proteoclastic dissection, be connected to antibody molecule or other cell ligand is first-class.These modifications can be used for increasing proteic stability of IL-1RA of the present invention and/or biological activity.

Pharmaceutical composition

Be used for the present invention or contain proteic pharmaceutical composition according to the invention.Usually; When pharmaceutical composition of the present invention is used for such use; Said albumen can be processed the pharmaceutical dosage form of different way of administration with one or more pharmaceutically acceptable carriers or mixed with excipients, like tablet, capsule, powder, granule, syrup, solution, oral liquid, spirit, tincture, aerosol, powder spray, injection, injectable sterile powder, suppository etc.

" pharmaceutically acceptable " composition is to be applicable to people and/or animal and not have the material that excessive bad side reaction (like toxicity, stimulation and allergy) promptly has rational benefit/risk ratio." pharmaceutically acceptable carrier " is acceptable solvent, suspending agent or the excipient pharmaceutically or on the food that is used for fusant albumen of the present invention is sent to the animal or human.Carrier can be a liquid or solid.

That albumen of the present invention can pass through is oral, intravenous, intramuscular or subcutaneous route administration.

But the dosage form of oral administration administration is in the above-mentioned dosage form: tablet, capsule, powder, granule, syrup, solution, spirit.Solid-state carrier comprises: starch, lactose, calcium hydrogen phosphate, microcrystalline Cellulose, sucrose, kaolin, micropowder silica gel, Pulvis Talci, low-substituted hydroxypropyl cellulose, carboxymethyl starch sodium, polyvinylpyrrolidone.And liquid carrier comprises: sterilized water, ethanol, Polyethylene Glycol, nonionic surfactant and edible oil (like Semen Maydis oil, Oleum Arachidis hypogaeae semen and Oleum sesami).Normally used adjuvant comprises in the process of pharmaceutical compositions: flavoring agent, coloring agent, antiseptic (like oxybenzene alkyl butyl ester, sodium benzoate, sorbic acid) and antioxidant (like vitamin E, vitamin C, sodium pyrosulfite and dibenzylatiooluene).

The dosage form that can be used for injection administration in the above-mentioned dosage form comprises: injection, injectable sterile powder, they are that medicine and one or more pharmaceutically acceptable mixed with excipients are processed the form for drug administration by injection.Solvent comprises: sterilized water, ethanol, glycerol, propylene glycol, Polyethylene Glycol.In addition, also need add antibacterial (like benzyl alcohol, butyl hydroxybenzoate, thimerosal), isoosmotic adjusting agent (like sodium chloride, glucose), suspending agent (like sodium carboxymethyl cellulose, methylcellulose), solubilizing agent (tween 80, lecithin), antioxidant (like vitamin E, vitamin C, sodium pyrosulfite) and filler (like lactose, mannitol).

See that from the position that is easy to prepare preferred pharmaceutical composition is a solid-state composition, especially lyophilized injectable powder with administration.

Pharmaceutical composition of the present invention can prepare according to the method that the pharmaceutical manufacturing of knowing and generally acknowledge requires.Pharmaceutical composition is suitable to comprise protein of the present invention and pharmaceutically acceptable carrier, and is suitably unit dosage form.Pharmaceutical composition of the present invention can comprise the protein of the present invention of prodrug forms, and this prodrug can change into the activity form of thing of the present invention at receiver's host intracellular metabolite.

The invention discloses a kind of anti-tumor medicine Combined drug box, it comprises the preparation of tumor chemotherapeutic drug preparation and pharmaceutical composition according to the invention.

Purposes

Disclose said albumen and can treat as active component or prevent radiotherapy or/and the thymus acute injury that chemotherapeutics causes and/or immunologic function suppress the method for side effect, this method comprises above-mentioned (a) that give patient's effective dose or (b) described albumen.

In some embodiments, said method can be through administering mode, radiotherapy administering mode before the chemotherapeutics administration, gives above-mentioned (a) of patient's effective dose or (b) described albumen.

" effective dose " or " therapeutic dose " all is meant the amount that is enough to produce curative effect.Effective dose can divide one or multiple dosing.Usually, effective dose is enough to relax, improve, stablize, slow down or postpone further developing of disease.

The effective dose of used active component can and wait that the order of severity of treating disease changes with mode of administration.As far as most of large mammal, the accumulated dose that imposes effective ingredient every day is about 0.01-1000mg.Usually, the scope of adult's clinical administration amount is 0.01-200mg/ day, is preferably 0.05-100mg/ day.

In some embodiments, said chemotherapeutics can be selected from alkylating agent class medicine, antimetabolitas, antibiotics, plant medicine thing, hormone medicine, metal complex or protein drug, wherein is preferably antimetabolitas.Antimetabolite drugs selected from doxifluridine 5 - doxifluridine, fluorouracil glycosides, propylthiouracil, butyl fluorouracil, tegafur dual 5 - fluoro pyrimidine alcohol, sodium methyl mercaptopurine, mercapto purine microphone 6 - mercaptopurine and 6 - amino-purine hydrochloride, glycine azathioprine, sulfur guanine, oncolytic methotrexate, hydrazine sulfate, Wei Kang alcohol, Yasi quinones, Finland can Loening, iso gifted methadone, endostatin , chlorine flexor phosphate, disodium phosphate, chlorine flexor ring leucine, to investigate citrate Lin, A yellow citric acid to investigate forest, Berry Farquhar, Austria celecoxib allopurinol, Australia Mallet, bromine Pakistani acid (sodium) , 100 lied fixed, Broxuridine, fluorouracil hexylamine 10 - ethyl-go nitrogen aminopterin, methotrexate fluorine, carbon methotrexate, 5,10 - nitrogen double to tetrahydrofolate, A full neopterin, Ding mercaptopurine, dink he amides, bladder aza guanosine, carmofur, uracil tegafur, amyl dilute indole, sulfur if the star, UFT, methoxy Bo because, formyl dissolved sarcoma Su, ammonia (base) neopterin, aminopterin sodium 8 - azaguanine, dimethylamine adenosine (nitro) azathioprine, uracil, mercapto A uracil, azaserine, raltitrexed , raltitrexed accounted hydrochloride nolatrexed, sophoridine, folinic acid, methyl tetrahydro high folic acid, zoledronic acid, temozolomide, bicalutamide, asparaginase, left leucovorin, leucovorin, Kun to Spartak, triazine benzamide, Qu Mai Kete, tramadol, chlorine Bobby acid and 5 - diazo uracil, piracetam, topotecan, topotecan hydrochloride, A cytarabine, cyclocylidine, hydroxy guanidine and 5 - fluorouracil nucleoside and 5 - fluorouracil nucleoside, nucleoside fluorouracil, glycerol citrus base, A barrier can be scattered, isoxazole acetic acid, ammonia Glutethimide , ammonia naphthalene Sofitel, chlorine cytidine, A he exemestane, azacytidine, anti-tumor acid, deoxy-azacytidine, dexrazoxane, Craig Stowe, Knies he acids, Kela Li Ping, carat Libin , Gallo capecitabine, gemcitabine, capecitabine Iba, enocitabine, Anxi gemcitabine, decitabine, fluorine gemcitabine, capecitabine, enocitabine, imidazole gemcitabine, Crane non-Lu , Kara amide, carbazole amides, Kabbah naphthoquinone, azobenzene bromopropylamine, curcumin, curcumin diketones, ketones song sand, Acamprosate, Adams poured Guning, phenylephrine Adams poured Guning, naphthalene urea ammonium phosphate chloride to Te Kali phosphate fluoride Dana glycosides, fluorobenzyl chlorthalidone, Garcinia acid, goserelin, nitrogen Citrus Ridge, Haynie Bai Ruining, inosine dialdehyde, chlorobenzene ammonia pyridine, dibromo-mannitol , dibromo-dulcitol, Corti Persia, the benefits if the blue fine, multi-Pan, America Luoge Rui, C m hydrazone, fans care quinones, mitotane, Fazha La Marina, fludarabine, cladribine , amyl glucoside, benzene to Ning, benzene to the United States special, phosphorus Azeri ethyl amine, horse hair Nepal azole, polyolefin Rainey acid, folic aspartic acid, folic three glutamic acid, purine m tongue poise, Filippo adenosine, hyperbolic Qin, split tuck polysaccharides, bromine fennel sodium acrylate, benzyl ammonium azo, Qu Ding sulfonester, triethylene melamine, three quinone imine, Qu Xi Ruibin, phosphoric song Xirui Bin, Lei Su A vine , triptorelin, nine Buloh azole, UFT, vitamin B-17, Weimaining, z-aza-adenosine, cytidine Tashi, according horses piperidine, A Monuo period Addo to new , Ake Ronin, Alano new, Ami anthraquinone, anastrozole, Ana Xi Rong, A past that one, Aspen indole awake, acivicin, atenolol Wei pyridine, Addo raloxifene, cancer can naphthalene Ngong research Geneen, a Ludaboxin, anti-tumor ketone, anti-tumor ketone A-10, Asha Fang, asparagus fine, mustard indole acid, Berry Farquhar (sodium), (hydrochloric acid) must Sang-gun, granisetron Joan, tropisetron, dacarbazine, ondansetron, thymosin, tramadol, imatinib mesylate, diclofenac, thalidomide, prop fluorine death star, toremifene, Ann ambroxol, lappaconitine, Snipes and other reasons, thymosin, flutamide, imine, amine benzene oxide, new quinoline, N-dimethyl formamide, prison up to azole, anthracene methylthioureido, Austria Xi Shu Lun, oxidation Lycorine, parked Fen Saer, parked Zeni Pu fixed, amyl will Rolle, snails chloropropanol, protoanemonin, Jia-generation cellular, fixed mine for Nibu, raw Suo Lade, Su Tao neomycin , eggplant soft ester base, imine quinone, Sri Lanka for benzene pyrimidine, Taimo Zorro, Taiwan Duoxi Long, New Orinoco sulfur, ammonium nitrate acridine acridine or safety of one or more, one of the most preferred 5 - azacytidine.

The chemotherapeutics dosage is according to known effective dose administration, is generally intravenous injection, each 1-2mg/kg, 1 time/day as 5-azacytidine is clinical.

On the other hand, the invention also discloses a kind of method of treating tumor, this method is included in radiotherapy or/and before the chemotherapeutics, give the above-mentioned albumen of individual effective dose.Pharmaceutical composition of the present invention also can with other tumor therapy Combined application, as simultaneously, sequential or use respectively.Pharmaceutical composition of the present invention can include other active function thing.

Below in conjunction with specific embodiment, further illustrate the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.

Among the embodiment, " rHuIL-1Ra ", " IL-1Ra " are same substance, are preceding text indication " hIL-1Ra ".

Materials and methods

Main agents

5-azacytidine (5-Aza, Sigma company), formalin solution (4% paraformaldehyde), cell membrane penetrating dose of (0.1%Triton x-100/0.1% sodium citrate/PBS pH value 7.2～7.4), BrdU (Sigma company)

Laboratory animal

SPF level BALB/c mouse, 4 weeks, male, this dish gram Experimental Animal Center of Shanghai.

Experimental technique

1,5-azacytidine (5-Aza) lumbar injection:

1) aseptic disposable 1mL syringe is used in the mouse peritoneal injection.

2) right hand is held syringe during lumbar injection, the little finger of toe of left hand and the nameless tail of catching mice, and other three fingers are caught the cervical region of mice, make the head of mice downward.From abdominal part one side inserting needle, behind the about 1cm of ventrimeson direction inserting needle, inject medicine during inserting needle, slowly extracted syringe needle, and slightly rotate syringe needle, prevented leakage.

3) the 5-Aza ID is 10mg/kg, 30mg/kg, 100mg/kg, and the administration volume is 0.1-0.2mL.

2, the obtaining and weighing of mouse thymus:

1) thymus is positioned at the thoracic cavity anterior mediastinum, near heart, is canescence, divides left and right two leaves.

2) disconnected neck is put to death mice, drops in 75% ethanol that fills 100mL to soak into, and after bringing out mice being faced upward, turning over cranes one on the antiseptic gauze that is laid on the super-clean bench in the sterilization medical tray puts to death mice.

3) carefully pinched the skin of mice thoracic cavity portion with the ophthalmology tweezer, carefully cut off skin with eye scissors, and separate skin and muscular tissue.Under the barrier film of mice thoracic cavity, upwards cut off breastbone, open the thoracic cavity.

4) push heart gently aside, note not puncturing heart or blood vessel, expose thymus, thymus and tissue are on every side cut, place clean glass culture dish.

5) wash thymic tissue gently with aseptic PBS, on toilet paper, inhale blood and other liquid that goes to the thymus surface, exhaustion as far as possible gently.

6) carefully remove thymus surperficial connective tissue and lymphoid tissue with eye scissors, after the removal totally, weigh as far as possible with the rigorous analysis balance.

3, thymus index:

Heavy (the mg)/body weight (g) of thymus index=thymus

4, thymocyte cell sum:

1) thymus is placed on the 200 clean eye mesh screens.The culture dish that screen cloth underlying one is clean.

2) add 1-2mL 10%FBS/RPMI 1640 culture medium to screen cloth

3) pintle with glass syringe grinds thymus gently on screen cloth, until no visible block thymic tissue.

4) contain 10%FBS/RPMI 1640 culture medium flushing screen cloth with 3-5mL, rinse well as far as possible, avoid that residual thymocyte cell is arranged on the screen cloth.10%FBS/RPMI 1640 culture medium flow in the culture dish of screen cloth below.

5) the thymocyte cell lapping liquid in the collection culture dish adds in the aseptic centrifuge tube of 15mL.

6) 1600rpm is centrifugal 5 minutes.

7) 0.5%FBS/PBS with pre-cooling washs once.

8) cell is resuspended among the 2mL 5mM EDTA/PBS, if any block float, as far as possible evenly with the piping and druming of rifle head, if can not, then discard.

9) get the resuspended liquid of 10 μ L thymocyte cells, add in the preprepared cytometry instrument dilution, mixing turns upside down.Detect with blood-counter system.

5, thymic tissue is fixed:

PBS flushing thymus with pre-cooling; Thymus after the flushing is put into freshly prepared 4% paraformaldehyde fixative, and wherein the fixative volume should be greater than 10 times thymus volume; Room temperature is fixed, fixing 24 hours at least; Every 1-2 week is changed fixative, but room temperature is preserved for a long time.

6, the thymus T cell subsets propagation method of inspection

As above 4 prepare the resuspended liquid of thymocyte cells; After adding PE-CD4 and PE-Cy5-CD8 antibody carry out the surface antigen labelling; With 2% paraformaldehyde fixed cell, 0.1% Triton X-100/0.1% sodium citrate is changed cell membrane thoroughly, and suitable concentration DNase I unties the dna double chain;

Analyze through forward angle (FSC) and direction finding angle scattered light (SSC) earlier behind the thymocyte cell FITC-BrdU antibody staining, remove cytoadherence and cell debris, analyze the cell mass of choosing.FITC-BrdU antibody staining visible cell is proliferative cell (BrdU feminine gender, BrdU not ^-) and proliferative cell (the BrdU positive, BrdU ⁺) distinguish obviously, select BrdU ⁺/ SSC establishes a delimitation proliferative cell, through analyzing the FL2-H and the FL3-H dual pathways simultaneously, detects the distribution of thymocyte cell CD4 and CD8 surface antigen.

In the following example, matched group (NS or control) is the mice group of intraperitoneal injection of saline, and administration group (rHuIL-1Ra or IL-1Ra) is the mice group of lumbar injection rHuIL-1Ra.

Embodiment 1.5-Aza chemotherapy is to the protection to thymus of the damage of thymus and rHuIL-1Ra preventive administration

Injected 3mg/kg/ days rHuIL-1Ra in preceding 5 days continuously in chemotherapy; Lumbar injection 100mg/kg 5-Aza (being designated as the 0th day) in the protein medicine-feeding 24 hours the last time; The 0th day (injection 5-Aza before), got matched group and administration group mouse thymus in 1.5 days, 3 days, 7 days, 11 days, 14 days respectively, observation thymus index and the painted variation of thymus pathological section H&E.

Experimental result shows that 5-Aza is obvious to the toxic and side effects of mouse thymus, and causes the injury of thymus gland that chemotherapy causes and the process of reparation.After the chemotherapy the 1.5th day, 3 days, 7 days, matched group (NS) mice and administration group (rHuIL-1Ra) mouse thymus index sharply descended.Before the 3rd day control group mice thymus index is chemotherapy 13.20% of level; The administration group is 22.86% of the preceding level of chemotherapy, and is higher by 82.22% than matched group thymus index, is significantly higher than matched group, and (two tail T check, n=4-6) to have statistical significance.At the 7th day; Matched group and administration group mouse thymus index are reduced to minimum point, show the 7th day for chemotherapy causes the most serious toxic and side effects time point, and the control group mice thymus index is 8.94% of the preceding level of chemotherapy; The administration group is 20.09%; Higher by 136.1% than matched group, (two tail T check, n=4-6) to be significantly higher than matched group.Mouse thymus begins rapid recovery after the 7th day.At the 14th day, the control group mice thymus index rose to 70.43% of the preceding level of chemotherapy, and the administration group returns to 64.93% of the preceding level of chemotherapy, and thymus index does not have significant difference (Fig. 2) between administration group and matched group.

Thymus HE stained to the 0th day, 1.5 days, 7 days, 11 days and 14 days after the chemotherapy is analyzed,

The result shows

(1) before chemotherapy, thymic tissue cortex, medullary substance are complete, and cortex is thick and with medullary substance demarcation line is clearly arranged.Matched group and administration group do not have significant difference (Fig. 3).

(2) chemotherapy the 1.5th day and the 7th day, the thymus volume, cortex, medullary substance area reduce, and matched group (NS) thymic cortex medullary substance boundary is fuzzy, and administration group (rHuIL-1Ra) thymic cortex medullary substance boundary is clear.This shows that the thymic cortex medullary substance is impaired in 5-Aza injection back, and rHuIL-1Ra to the thymus in the chemotherapy played protective effect (Fig. 3, Fig. 4).

(3) after chemotherapy the 11st day and the 14th day, complete through sections observation thymic tissue skin medullary substance, area and thickness returned to level before the chemotherapy gradually, and chest gland skin medullary substance boundary is clear, shows that thymus recovers (Fig. 3) gradually.

Through 5-Aza injection back mouse thymus index and thymus section H&E colored graph; Can know that after the 5-Aza chemotherapy 0 to 7 day is the acute injury phase of mouse thymus; Sharply reduce at damage phase thymic weight, thymus index descends, and wherein the 7th day is the most serious time point of injury of thymus gland.The rHuIL-1Ra preventive administration can reduce chemotherapy to the exponential minimizing of mouse thymus in the 3rd day damage phase, the 7th day of mouse thymus; And can alleviate the histopathologic damage of mouse thymus at the 7th day, show that rHuIL-1Ra can alleviate the mouse thymus damage that the 5-Aza chemotherapy causes.

Embodiment 2.rHuIL-1Ra preventive administration has been protected injury of thymus gland and has been promoted its reparation

Laboratory animal be 4 age in week the BALB/c male mice, normal saline matched group and rHuIL-1Ra protein medicine-feeding group are established in experiment.The dosage of rHuIL-1Ra is 3mg/kg, and in chemotherapy preceding 5 days per 12 hours injection once, injection system is a subcutaneous injection, and is matched group with injection with the mice that volume does not have thermal source normal saline (NS).Inject in the protein 24 h abdominal cavity disposable injection 5-Aza 100mg/kg the last time.The 0th day (before the injection 5-Aza), the 7th day, the dead a collection of mice in the 11st natural gift other places, detect the variation of mouse thymus index, thymocyte cell sum, thymocyte cell subgroup ratio, thymocyte proliferation and peripheral blood T cells.

2.1 rHuIL-1Ra is to the effect of thymus index and thymocyte cell sum

The medication of experimental design and mice is seen the foregoing description 2 descriptions.After chemotherapy the 0th day, 7 days, 11 days are got 5～6 of matched group, administration group mices respectively; Put to death the back and separate thymus; The preparation thymus cell suspension; RHuIL-1Ra is consistent with the experimental result of embodiment 1 to the protective effect of thymus index and thymocyte cell sum: after (1) chemotherapy the 7th day, administration group mouse thymus index was significantly higher than matched group (Fig. 5); (2) after the chemotherapy the 7th day, rHuIL-1Ra administration group mouse chest cell sum is significantly higher than matched group, and was higher by 131.11% than matched group.The 11st day thymus convalescent period, rHuIL-1Ra administration group thymocyte cell sum is significantly higher than matched group, than matched group high by 28.03% (Fig. 6).

2.2 rHuIL-1Ra is to the effect of thymus T cell subsets

Test method:

1) medication of experimental design and mice is seen the foregoing description 2 descriptions.Got 5～6 of matched group, administration group mices respectively in the 0th day, 7 days, 11 days after chemotherapy, and put to death the back and separate thymus, preparation thymus cell suspension, counting cells concentration.Prepare cell according to following table:

Table .1 thymus T cell subsets surface antigen CD4, CD8 ratio test sample

2) the centrifugal 5min of 2400rmp, collecting cell.

3) supernatant is abandoned in suction, adds 50 μ L PBS/0.5%FBS, and fully mixing makes cell resuspended.

4) divide into groups per sample, add corresponding antibodies respectively; 4 ℃ of lucifuges are hatched 30min.

6) add 200 μ L PBS/0.5%FBS washing.

7) the centrifugal 5min of 2400rmp, collecting cell.

8) abandon supernatant, add 500 μ LPBS/0.5%FBS re-suspended cells, 4 ℃ keep in Dark Place in the immigration streaming pipe, go up machine testing in the 24h.

9) statistical analysis

A) flow cytometer detects FITC and PE-Cy5 coloration result, with Cell Quest software analysis, calculates thymocyte cell DN, DP, CD8 SP, CD4SP cell proportion.

B) result carries out two tail T checks and variance analysis, and for having significant difference, * * represents p＜0.01 with p＜0.05, for difference extremely remarkable.

Result of the test:

With surface of cell membrane antibody CD4, CD8 thymocyte cell is divided into DN (CD4 ^-CD8 ^-), DP (CD4 ⁺CD8 ⁺), CD4SP (CD4 ⁺CD8 ^-) and CD8SP (CD4 ^-CD8 ⁺) four subgroups.CellQuest software analysis flow cytometer detects data, and the result is as shown in table 2.The mice number of elements that the N representative detects.

RHuIL-1Ra is to the effect of thymus T subsets distribution in the table 2.5-Aza chemotherapy

The result shows:

According to above experimental result, rHuIL-1Ra albumen preventive administration can change the ratio of mouse thymus T cell subsets before chemotherapy and after the chemotherapy.

(1) rHuIL-1Ra albumen preventive administration has significantly reduced the ratio (p＜0.05) of mouse thymus DN cell before chemotherapy.Because the DN cell belongs to early stage pre-T cell in the T cells whose development, have the ability that further is divided into DP, CD4SP, CD8SP cell subset.Thereby this presentation of results rHuIL-1Ra has suppressed the quantity that the propagation of DN cell has reduced the DN cell subset.Repressed DN cell reduces the sensitivity of chemotherapy, thereby protected DN cell just can continue to be divided into CD4SP and CD8SP cell subset after chemotherapy, and the recovery of immunologic function is played an important role.

(2) after chemotherapy the 7th day, the ratio of rHuIL-1Ra protein groups mice DN cell subset was compared no significant difference with matched group, and DP cell subset ratio is significantly higher than matched group, showed that the DN cell differentiation is that the propagation of DP cytosis or DP cell self increases.Simultaneously, CD4SP and CD8SP cell subset in the administration group thymus significantly are lower than matched group.After the T cell development is divided into CD4SP and CD8SP cell subset in the thymus; As sophisticated T cell delivery and settle down to periphery; There are two kinds of possibilities in the minimizing of CD4SP and CD8SP cell in the thymus, and one of which is the minimizing that CD4SP and CD8SP cell self generate; Its two generation for CD4SP and CD8SP does not reduce; But got into the periphery immune system, and be the minimizing which kind of reason has caused administration group CD4SP and CD8SP cell actually, this need verify the detection of peripheral blood T cells quantity.

(3) recover the impaired back of thymus after chemotherapy the 11st day, DN, DP, CD4SP, the CD8SP cell subset of matched group and protein medicine-feeding group mice there are no significant difference.

2.3 rHuIL-1Ra is to the effect of thymocyte proliferation

Test method:

The medication of experimental design and mice is seen the foregoing description 2 descriptions.After chemotherapy the 0th day, 7 days, 11 days are got 5～6 of matched group, administration group mices respectively.Mouse peritoneal injection 100mg/kg BrdU solution (10mg/mL), injection at twice, each 1.5h at interval; Inject for the first time BrdU and crane one after 3 hours and put to death mice, get thymus and detect.With proliferating cells ratio in the flow cytometry mouse thymus.Cell analysis process and BrdU detect sketch map and see Fig. 7 (detection of thymocyte proliferation.(A) thymocyte cell forward angle (FSC) is analyzed with lateral angle (SSC), and circle door R1 removes cell debris and adhesion.(B) FL-1H and SSC analyze, and circle door R2 analyzes the BrdU+ cell proportion.(C) the BrdU+ cell proportion is analyzed block diagram).

Result of the test:

RHuIL-1Ra is as shown in table 3 to the effect of thymocyte proliferation.With two tail T check comparative statistics differences, the result is with average (Mean) ± standard error (SEM) expression in the table.*p＜0.05，**p＜0.01。The mice number of elements that the N representative detects.

Table 3.rHuIL-1Ra is to the effect of thymocyte proliferation

The result shows:

Above presentation of results, behind the injection rHuIL-1Ra, matched group thymocyte proliferation ratio was 14.58% in the 0th day, administration group thymocyte proliferation ratio is 10.54%, be 73.0% of matched group, and the statistical difference heteropole is remarkable.Administration group mouse thymus BrdU positive cell (BrdU ⁺) number is lower than normal mouse, shows that rHuIL-1Ra has suppressed the propagation of normal mouse thymocyte cell, this result and rHuIL-1Ra are consistent to the result in normal mouse chest cell cycle.

Caused injury of thymus gland the most serious the 7th day in chemotherapy, matched group thymus carefully propagation ratio is 12.73%, and administration group thymocyte proliferation ratio is 15.59%, and is higher by 22.5% than matched group, has statistical significance.At the 11st day, matched group thymocyte proliferation ratio was 12.74%, and administration group thymocyte proliferation ratio is 17.86%, and is higher by 40.2% than matched group, and the statistical difference heteropole is remarkable.The 7th day and the 11st day, administration group mouse thymus BrdU ⁺Cell number is higher than matched group, is illustrated in the 5-Aza chemotherapy process, and the preventive administration of rHuIL-1Ra can promote injury of thymus gland phase and convalescent cell proliferation, the recovery after the promotion thymus chemotherapy.

2.4 rHuIL-1Ra is to the effect of thymus T cell subsets propagation

Test method:

The medication of experimental design and mice is seen the foregoing description 2 descriptions.Got 5～6 of matched group, administration group mices respectively, mouse peritoneal injection 100mg/kg BrdU solution (10mg/mL), injection at twice, each 1.5 hours at interval in the 0th day, 7 days, 11 days after chemotherapy; Inject for the first time BrdU and crane one after 3 hours and put to death mice, get thymus and detect.The proliferative cell ratio of thymus DN, DP, CD4SP, CD8SP cell subset in the flow cytometry mouse thymus.Thymocyte cell is removed cell debris through the analysis of forward angle (FSC) and direction finding angle scattered light (SSC) during analysis, and the analysis FL2-H and the FL3-H dual pathways, and thymocyte cell is divided into CD4SP, CD8SP, DP and DN subgroup according to the distribution of CD4 and CD8 surface antigen.Each subgroup is analyzed the FL1-H passage, and wherein the cell of FL1-H passage high expressed is BrdU+ cell (proliferative cell).

Result of the test:

RHuIL-1Ra is as shown in table 4 to the effect of thymus DN, DP, CD4SP, CD8SP cell subset propagation.The result is with average (Mean) ± standard error (SEM) expression in the table.Two tail T check comparative statistics differences, * p＜0.05, * * p＜0.01.The mice number of elements that the N representative detects.

Table 4.rHuIL-1Ra is to the effect of thymus T cell subsets propagation

72.0％。Can know that from The above results rHuIL-1Ra is inhibited to the propagation of normal mouse chest cell.

(2) after the chemotherapy the 7th day, consistent with the result of matched group thymocyte proliferation ratio, different T subgroup propagation ratios all significantly descended before comparing chemotherapy.And the administration group changes not quite before comparing chemotherapy.RHuIL-1Ra administration group DN cell subset BrdU ⁺Cell proportion is significantly higher than matched group, is 189.4% of matched group; CD8SP cell subset BrdU ⁺Cell proportion is significantly higher than matched group, is 219.0% of matched group.This presentation of results IL-1Ra has effectively protected the propagation of thymocyte cell.

(3) after the chemotherapy the 11st day, each T cell subsets BrdU of rHuIL-1Ra administration group ⁺Cell proportion all is higher than matched group, and the statistical difference heteropole is remarkable.Wherein the DN cell subset is 166.7% of a matched group, and the DP cell subset is 147.6% of a matched group, and the statistical difference heteropole is remarkable; The CD4SP cell subset is 156.1% of a matched group, and statistical discrepancy is remarkable; 179.5% of CD8SP cell subset matched group, the statistical difference heteropole is remarkable.This presentation of results IL-1Ra has promoted that effectively thymocyte cell recovers the propagation in stage after chemotherapy.

According to The above results, preventive administration rHuIL-1Ra before chemotherapy can protect the mouse thymus damage that chemotherapy causes, and promotes the propagation of mouse chest cell in thymus convalescent period.This part experimental result shows; In the middle of each subgroup of thymus T cell; RHuIL-1Ra can suppress the propagation of normal mouse DN, DPT cell subsets; Be that rHuIL-1Ra mainly has inhibitory action to the propagation of breaking up early stage T cell, this inhibition reduces the damage of 5-Aza chemotherapy to thymus DN, DP cell subset.The most serious the 7th day of injury of thymus gland; RHuIL-1Ra administration group mouse chest cell propagation ratio is significantly higher than matched group; Show that rHuIL-1Ra has promoted the propagation of injury of thymus gland phase thymocyte cell to a great extent, thymus differentiation T cell ability is had protective effect.At thymus convalescent the 11st day, each T cell subsets propagation ratio of rHuIL-1Ra administration group all was significantly higher than matched group, explains that rHuIL-1Ra has facilitation to thymus function and the immune recovery of periphery.

2.5 the rHuIL-1Ra preventive administration is to the effect of peripheral blood T cells

Test method:

The medication of experimental design and mice is seen the foregoing description 2 descriptions.Got 5～6 of matched group, administration group mices respectively, and plucked eyeball with elbow ophthalmology tweezer and get blood in the 0th day, 7 days, 11 days after chemotherapy; Rapidly the mice drop of blood is gone into to have added in advance in the EP pipe of sodium citrate anticoagulant, rock repeatedly and make mice peripheral blood and anticoagulant mix homogeneously; Get 10 μ L, 3% glacial acetic acid and split red back with blood counting chamber counting cells number.Can cell be adjusted to suitable concn behind the counting and carry out the periphery blood T cell test experience; In the ammonium chloride solution splitting erythrocyte of ratio adding pre-cooling in 1: 9, mixing, 10 minutes on ice, centrifugal back was with the resuspended mixing of 1mL PBS.Cell is adjusted to respective concentration; 4 ℃ of lucifuges are hatched 10min; Take out centrifuge tube, according to 1: 2 (NH ₄Cl: ratio PBS/0.5%FBS) adds the abundant mixing of PBS/0.5%FBS; Centrifugal, room temperature, 1600rpm, 5min; Topple over supernatant, absorb residual liquid, add 1mL PBS/0.5%FBS liquid, fully mixing in absorbent paper; Draw 95 μ L cell suspension, add 5 μ L, 3% glacial acetic acid (19: 1) mixing counting cells concentration; The cell suspension room temperature is centrifugal, 2400rpm, 5min; Abandon supernatant, resuspended with 1.2mL PBS/0.5%FBS liquid.Prepare sample cell according to following table.

Table 5 peripheral blood CD4 ⁺CD45RA ⁺, CD8 ⁺CD45RA ⁺T cell proportion test sample

Divide into groups per sample, add corresponding antibodies respectively; 4 ℃ of lucifuges are hatched 30min; With 200 μ lPBS/0.5%FBS washing once; Add 500 μ l PBS/0.5%FBS re-suspended cells, 4 ℃ keep in Dark Place in the immigration streaming pipe, go up machine testing in the 24h.

Statistical analysis:

A) flow cytometer detects FITC, PE-Cy5, PE coloration result, with Cell Quest software analysis, calculates PBC CD4 ⁺CD45RA ⁺, CD8 ⁺CD45RA ⁺The T cell proportion.

B) result carries out two tail T checks and variance analysis

Result of the test:

CD45RA is the important cells surface marker of difference T cells and memory T cell, CD45RA ⁺Cell is that thymus generates, not contacted antigenic T cell, so CD4 in the peripheral blood ⁺CD45RA ⁺With CD8 ⁺CD45RA ⁺The variation of (being T cells) cell proportion is an important indicator of estimating thymus T cell output function.

RHuIL-1Ra is as shown in table 6 to the exercising result of peripheral blood T cells.The result is with average (Mean) ± standard error (SEM) expression in the table.Two tail T check comparative statistics differences, * p＜0.05, * * p＜0.01.The mice number of elements that the N representative detects.

Table 6.rHuIL-1Ra is to CD4 ⁺CD45RA ⁺With CD8 ⁺CD45RA ⁺The effect of T cell

The result shows:

(1) injection rHuIL-1Ra the administration group at the 0th day, CD4 ⁺CD45RA ⁺The T cell is lower than matched group, is 49.30% of matched group.CD8 ⁺CD45RA ⁺The T cell is lower than matched group, is 38.80% of matched group; Has statistical significance through two tail T checks.

(2) after chemotherapy the 7th day, administration group mice CD4 ⁺CD45RA ⁺T cell and CD8 ⁺CD45RA ⁺T is significantly higher than matched group, is respectively 202.9% and 228.0% of matched group, has statistical significance through two tail T checks.

(3) after chemotherapy the 11st day, CD4 ⁺CD45RA ⁺The T cell is significantly higher than matched group, is 340.0% of matched group, and two tail T checks have statistical significance.CD8 ⁺CD45RA ⁺T cell meansigma methods also is higher than matched group, is 304.5% of matched group.

The The above results presentation of results, rHuIL-1Ra can reduce CD4 in the normal mouse peripheral blood ⁺CD45RA ⁺With CD8 ⁺CD45RA ⁺The quantity of T cells.Yet; Owing to do not find that in the lymphopoietic detection of thymus T rHuIL-1Ra can suppress the propagation of CD4SP T cell significantly, thus in the peripheral blood CD4 T cells minimizing maybe because the migration that rHuIL-1Ra has influenced CD4SP T cell in the thymus with settle down.

The 7th day data show that the ratio of CD4 and CD8 T cells sharply increases in the peripheral blood after chemotherapy, and this possibly be because the peripheral blood after chemotherapy sum sharply reduces the apparent increase of the T cells that causes.This has also reflected animal body when chemotherapy, because the effect of chemotherapeutics causes the immune immunocyte of secondary such as peripheral blood sharply to reduce, the periphery immune system almost is in the state of " paralysis ".At this moment, to the immune damage of periphery, the stress of body can impel the output of one-level immune organ increase T cell, thus the disappearance of T cell in the balance periphery immune system.Between matched group and administration group, the increase of administration group CD4, CD8 T cells all is significantly higher than matched group.This explanation rHuIL-1Ra is divided into two aspects to the protective effect of thymus; One of which; RHuIL-1Ra protection also promoted after the chemotherapy propagation of DN, DP cell in the thymus, its two, the rHuIL-1Ra protection has also increased CD4SP in the thymus, the CD8SP hypotype cell migration to periphery.

Among the 11st day the result; Administration group CD4, CD8 T cells number are higher than matched group; Be respectively 3.4 times and 3 times of matched group; In conjunction with the detection to CD4SP, CD8SP cell proliferation in the thymus, rHuIL-1Ra had both increased the propagation of CD4SP, CD8SP cell in the thymus, had also increased the quantity of CD4, CD8 T cells in the peripheral blood.Thymus on the cycle of recovery after the chemotherapy and periphery immune system have been played the effect that promotes immune restoration.

Discuss: rHuIL-1Ra can play a protective role to thymus in the 5-Aza chemotherapy.The preventive usage of rHuIL-1Ra can suppress the propagation of thymus earlier T cell, reduces its sensitivity to chemotherapeutics, and promotes the propagation of each T cell subsets of thymus in thymus convalescent period.The detection of peripheral blood T cells shows that rHuIL-1Ra can promote the immune recovery of periphery through promoting T cell proliferation and migration.

Scope of the present invention does not receive the restriction of said specific embodiments, and said embodiment also comprises the method and the component of functional equivalent only as the single example of illustrating various aspects of the present invention in the scope of the invention.In fact, except content as herein described, those skilled in the art can easily grasp multiple improvement of the present invention with reference to the description and the accompanying drawing of preceding text.Said improvement also falls within the scope of appended claims.Every piece of list of references that preceding text are mentioned is listed this paper in as a reference all in full.

Claims

One kind as follows (a) or albumen (b) preparation be used for treatment or protection radiotherapy with (or) the thymus acute injury that causes of chemotherapeutics, thymic lymphocytes damage and (or) purposes of the medicine that reduces of peripheral T lymphocyte:

(a) its aminoacid sequence such as the described albumen of SEQ ID NO.1;

(b) at least with (a) in aminoacid sequence 70% homology and have prevention or treatment radiotherapy with (or) the thymus acute injury that causes of chemotherapeutics, thymic lymphocytes damage and (or) peripheral T lymphocyte reduces active albumen.
2. purposes as claimed in claim 1 is characterized in that said thymic lymphocytes are thymus T cell.
3. purposes as claimed in claim 1 is characterized in that said peripheral T lymphocyte is CD4 ⁺CD45RA ⁺The T cell with (or) CD8 ⁺CD45RA ⁺The T cell.
4. purposes as claimed in claim 1, it is characterized in that said thymus T cell can be DN T cell, DPT cell, CD4SPT cell and (or) the CD8SPT cell.
5. purposes as claimed in claim 1, the albumen that it is characterized in that said (b) are aminoacid sequence such as the described albumen of SEQ ID NO.2.
6. purposes as claimed in claim 1 is characterized in that said chemotherapeutics can be selected from alkylating agent class medicine, antimetabolitas, antibiotics, plant medicine thing, hormone medicine, metal complex or protein drug.
7. purposes as claimed in claim 1 is characterized in that said chemotherapeutics is an antimetabolitas.
As claimed in claim 1, characterized in that the anti-metabolic drug is selected doxifluridine, 5 - doxifluridine, fluorouracil glycosides, propylthiouracil, butyl fluorouracil, tegafur bis pyridine and 5 - fluoro pyrimidine alcohol, sodium methyl mercaptopurine, thiol microphone purine and 6 - mercaptopurine and 6 - amino-purine hydrochloride, glycine azathioprine, sulfur guanine, oncolytic methotrexate, hydrazine sulfate, Wei Kang alcohol, Yasi quinone, Finland can Loening, iso gifted methadone, endostatin, chlorine flexor phosphate, disodium phosphate, chlorine flexor ring leucine, to investigate citrate Lin, A yellow citric acid to investigate forest, Berry Farquhar, Austrian celecoxib allopurinol, Australia Mallet, bromine Pakistani acid (sodium), 100 lied fixed, Broxuridine, fluorouracil hexylamine 10 - ethyl-go nitrogen aminopterin, methotrexate fluorine, carbon A aminopterin, 5,10 - nitrogen double to tetrahydrofolate, A full neopterin, Ding mercaptopurine, dink he amides, bladder aza guanosine, carmofur, uracil, tegafur, amyl dilute indole, sulfur If the star, UFT, methoxy Bo because, formyl dissolved sarcoma, ammonia (base) methotrexate, aminopterin sodium 8 - azaguanine, dimethylamine adenosine (nitro) azathioprine, uracil, mercapto A uracil, azaserine, raltitrexed, raltitrexed accounted hydrochloride nolatrexed, sophoridine, folinic acid, methyl tetrahydro high folic acid, zoledronic acid, temozolomide , bicalutamide, asparaginase, left leucovorin, leucovorin, Kun to Spartak, triazine benzamide, Qu Mai Kete, tramadol, chlorine Bobby acid and 5 - diazo uracil, Piracetam, topotecan, topotecan hydrochloride, cytarabine, cyclocylidine, hydroxy guanidine and 5 - fluorouracil nucleoside and 5 - fluorouracil nucleoside, nucleoside fluorouracil, glycerol citrus base , A barrier can be scattered, isoxazole acetic acid, ammonia Glutethimide, naphthalene Sofitel ammonia, chlorine cytidine, A he exemestane, azacytidine, anti-tumor acid, deoxy-azacytidine, dexrazoxane, Craig Stowe, Knies he acids, Kela Li Ping, carat Libin, Gallo capecitabine, gemcitabine, capecitabine Iba, enocitabine, Anxi gemcitabine, decitabine, fluorine gemcitabine, capecitabine gemcitabine, enocitabine, imidazole gemcitabine, Crane non-Lu, Kara amide, carbazole amides, Kabbah naphthoquinone, azobenzene bromopropylamine, curcumin, curcumin diketones, ketones song sand, Acamprosate, Adams Guning splash, splash Guning deoxyribonucleic Adams, naphthalene urea ammonium phosphate, chlorinated to Te Kali phosphate fluoride Dana glycosides, fluorobenzyl chlorthalidone, Garcinia acid, goserelin, nitrogen Citrus Ridge, Haynie Bai Ruining, inosine dialdehyde, chlorobenzene ammonia pyridine, dibromo-mannitol, dibromo-dulcitol, Corti Persia, the benefits if the blue fine, multi-Pan, America Luoge Rui, C m hydrazone, fans care quinones, m Tuotan, Fazha La Marina, fludarabine, cladribine, amyl glucoside, benzene to Ning, benzene to the United States special, phosphorus Azeri ethyl amine, horse hair Nepal azole, polyolefin Rainey acid, aspartic acid butterfly acid, folic three glutamic acid, purine m tongue poise, Filippo adenosine, hyperbolic Qin, split tuck polysaccharides, bromine fennel sodium acrylate, benzyl ammonium azo, Qu Ding sulfonester, triethylene melamine, three quinone imine Qu Xi Ruibin, phosphoric song Xirui Bin, Lei palmatine A, triptorelin, nine Buloh azole, UFT, vitamin B-17, Weimaining, z-aza-adenosine, Tashi cell glycosides, according horses piperidine, A Monuo period Addo to new, Ake Ronin, Alano new, Ami anthraquinone, anastrozole, Ana Xi Rong, A past that one, Aspen indole awake, Assi Auschwitz, atenolol Wei pyridine, Addo raloxifene, cancer can naphthalene, expensive research Geneen, a Ludabuxin, anti-tumor ketone, anti-tumor ketone A-10, Asha Fang, asparagus fine, acetic acid, mustard, white Swiss Farquhar (sodium), (hydrochloric acid) must Sang-gun, granisetron, tropisetron, dacarbazine, ondansetron, thymosin, tramadol, imatinib mesylate, diclofenac, Thalidomide Trust fluorine death star, toremifene, Ann ambroxol, lappaconitine, Snipes and other reasons, thymosin, flutamide, imine, amine benzene oxide, new quinoline, N-methyl- formamide, prison up to azole, anthracene methylthioureido, Austria Xi Shu Lun, oxidative Lycorine, parked Fen Saer, parked Zeni Pu set, e will Rolle, snails chloropropanol, protoanemonin, Kayo cell Ray scheduled for Nibu, raw Suo Lade, vegetarian Road neomycin, eggplant soft ester base, imine quinone, Sri Lanka for benzene pyrimidine, Taimo Zorro, Taiwan Duoxi Long, sulfur Orie New, ammonium nitrate Ah An piperidine or acridine in one or more of.
9. purposes as claimed in claim 1 is characterized in that said antimetabolitas is a 5-azacytidine.
10. anti-tumor medicine Combined drug box, it comprises that tumor chemotherapeutic drug preparation and active component are the preparation of the said proteic pharmaceutical composition of claim 1.