CN102166347B - New medicinal application of interleukin-12 - Google Patents
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Abstract
The invention relates to new medicinal application of interleukin-12. In the application, the interleukin-12 is applied to the preparation of medicaments for treating acute radiation sickness. Compared with the prior art, the invention has the advantages that: in the application of the interleukin-12 (interleukin-12, IL-12) to the preparation of the medicaments for treating the acute radiation sickness, the using range of the interleukin-12 is expanded, and new medicaments are provided for the treatment of the acute radiation sickness simultaneously; and results of pharmacodynamic experiments indicate that the interleukin-12 has a good effect on the acute radiation sickness.
Description
Technical field
The present invention relates to the new medicine use of a kind of interleukin 12 (interleukin-12, IL-12), its prepared application of medicine in the treatment acute radiation sickness of recombinant products of especially producing by the genetic engineering approach.
Background technology
Extensive use along with nuclear energy technology in national economy, because of reasons such as operate miss or breach of security rules, the accident that the personnel that cause are subject to the larger dose irradiation also happens occasionally.After former Soviet Union's Chernobyl accidents in 1986, the radiation accident that personnel are subject to photograph has also occurred in the Shanghai (1990) of Brazil (1987), Salvador (1989), Israel (1990), Byelorussia (1991), Russia (1997), Estonia (1994) and China and Jilin (1996) in succession.Therefore the control of acute radiation sickness is the very important link of Nuclear Accident Emergency Medical Treatment, is the key content of radiological medicine and Protective Research, is subject to various countries scholar's great attention.Acute radiation injury (ARI) often shows as Multi-system injuries, and wherein hemopoietic function of bone marrow inhibition, radiation colitis, induced lung injury are common pathological changes.It is the topmost pathology damage of ARI that hemopoietic function of bone marrow suppresses, and through radiation injury all the time, is the main cause of death.After body is subject to the fatal dose irradiation, the infection that hemopoietic function exhaustion causes, hemorrhage and metabolism disorder is murderous main cause, and therefore, except some symptomatic treatments such as infection, hemorrhage, impelling impaired hematopoietic function recovery with Hemopoietic factor is the fundamental way for the treatment of.
Development along with biological high-technology, the research of Hemopoietic factor is increasingly deep, people's hemopoietic growth factor of recombinating has been applied to hemopoietic function of bone marrow is suppressed patient's treatment as factors such as granulocyte colony-stimulating factor (G2CSF), grain giant cell colony stimulating factor (GM2CSF), erythropoietin (EPO), for new approach has been opened up in the treatment of ARI.Yet in the overwhelming majority, severe ARI patient is tiding over After acute stage, still there will be the severe complications such as hemorrhage, infection, finally cause death.Trace it to its cause, only to promote propagation and the differentiation of committed stem cell due to the hemopoietic growth factor overwhelming majority of application at present on the one hand, and very micro-to the effect of pluripotent stem cell or CFU-GM, though can cause the short-term of peripheral blood cells to raise, lack longer effect; Though can make the patient tide over what is called " bone marrow nonfunctional phase ", can not realize the reconstruction of hemopoietic function.On the other hand, if the patient's that only raises leukocyte does not reach the recovery of compromised immune system and the reconstruction of immunologic function, the patient still can be because of immunologic hypofunction or abnormal, causes the infection of a variety of causes and death.Therefore, the recovery of the reconstruction of hemopoietic function and immunologic function is two key links of acute radiation injury treatment.If the medicine of promotion can be arranged these two aspects, in the treatment acute radiation injury, will be very significant.
Summary of the invention
For the problems referred to above, the present invention discloses the application of a kind of interleukin 12 (interleukin-12, IL-12) in the medicine of preparation treatment acute radiation sickness
Technical scheme of the present invention is such: a kind of new medicine use of interleukin 12 is the application in the medicine of preparation treatment acute radiation sickness by interleukin 12.
The new medicine use of above-mentioned a kind of interleukin 12, wherein, described interleukin 12 dosage form is lyophilized formulations.
The new medicine use of above-mentioned a kind of interleukin 12, wherein, described interleukin 12 be used for the treatment of acute radiation sickness the time adopt the mode administration of subcutaneous administration.
Below research of the present invention is elaborated essence of the present invention in order better to illustrate.
Cytokine-12 (interleukin-12, IL-12) in 1989 and nineteen ninety respectively by discoveries such as Trinchieri and Gately, once called after NKSF (NKSF) and CLMF (CLMF), unified called after interleukin 12 in 1991.IL-12 is a heterologous dimer, molecular weight is 75KD, by P35 and two subunits of P40, by disulfide bond, is linked and forms, and can stimulate NK cell and T emiocytosis IFN-γ, promote the differentiation of Th1 cell, be considered to connect the bridge of inherent immunity and acquired immunity.Over nearly 20 years, the major part research of relevant IL-12 all concentrates on the immunoregulation aspect, thinks its playing an important role in antitumor, anti-infectious immunity are treated.
Along with going deep into of research, people have had more deep understanding to the biologic activity of IL-12.The Skin Cell that IL-12 causes ultraviolet has repair, mainly by the reparation to DNA, works.Recent years, find that again IL-12 also has repair to the damage of hematopoietic stem cell.1993, Jacobsen etc. tested discovery by single cell clone, and rmIL-12 (rmIL-12) and cytokine profiles have synergism, can urge collaborative other cytokine and promote Lin in mouse bone marrow cells
-sca-1
+cell is bred at double, especially can strengthen stem cell factor (SCF) and induce medullary system Lin
-sca-1
+cel l proliferation reaches more than 7 times, can also be together with colony stimulating factor (CSFs) co-induction Lin
-sca-1
+stem cells hyperplasia.When there is no rmIL-12, somatomedin seldom maybe can not stimulate high proliferative potential colony forming cell (HPP-CFCs) propagation, when adding 50ng/ml rmIL-12 to stimulate, the colony of HPP-CFCs forms all and significantly increases, and confirms that IL-12 is a kind of stem cell activation somatomedin.This is the researcher understanding on hemopoietic function to IL-12 for the first time.1994, Bellone etc. have studied the effect of hemopoietic progenitor cell in IL-12 human peripheral blood and bone marrow with mouse experiment, the rmIL-12 of discovery 1-10ng/ml concentration and steel factor (part of oncoprotein c-Kit) and IL-3 coupling can stimulate the cell clonal formation of red system, medullary system and mixing.Simultaneously they also will shift to an earlier date ready NK cell and cultivate together with CFU-GM, find that rmIL-12 suppresses forming of the hematopoietic cell clone that supported by IL-3, granulocyte-macrophage colony stimutaing factor by inducing the NK cell to produce IFN-γ and TNF-α.Therefore they think IL-12 directly the hemopoietic progenitor cell proliferation break up, two kinds of cytokines that also can produce by lymphoid cell suppress hemopoietic indirectly, have the effect of two-ways regulation.1997, Chinese scholar Wang Yi obtained by force the genetically modified cell of secretion IL-12 with carrier for expression of eukaryon, it is injected into to the mouse peritoneal that raying is processed, and finds the effect that IL-12 has hemopoietic to recover.
2007 and 2008, the Chen of American South University of California thinks that the low dose of rmIL-12 of independent use (100ng/ only) just can promote the mouse hemopoietic reconstruction, can promote three to be that hemocyte recovers, and the amount that produces IFN-γ is few, can not cause bone marrow toxicity, the inspection of gastrointestinal tract pathology is found not cause gastrointestinal is damaged.Basile is also used myelosuppressive mice after low dosage rmIL-12 treatment radiotherapy chemotherapy, finds that radiotherapy, chemotherapy give more low dosage (50ng/ only) IL-12 at twice, promote that the effect of hematopoietic function recovery is better.
Radiation-induced apoptosis is the main cause that causes tissue injury.For clear and definite which kind of cell subsets is subject to the protection of IL-12; flow cytometry analysis annexin V and cell-specific mark (Sca-1, c-kit, CD31 for Chen etc.; CD105 and alkali phosphatase) amphophilic cell, find rmIL-12 treatment group annexin V
-/ Sca-1
+cell is far away higher than matched group, and annexin V
-/ c-kit
+cell quantity is not obviously difference between rmIL-12 treatment group and matched group, and after radiation, two groups of 24h can't detect Lin
-sca-1
+c-kit
+cell, once had report to think that long-term and short-term hematopoietic stem cell is present in Lin
-sca-1
+c-kit
+in cell mass, perhaps express c-kit so rebuild the LTR HSC of bone marrow after the supposition radiation, but can't detect by the method.
Descending appearred in peripheral blood cells counting (PBCC) before this that can find to give the mice of low dosage rmIL-12 treatment after the lethal dose radiation, then bottom out soon, and control group mice drops to minimum point, dies off.After the sublethal dose radiation, low dosage rmIL-12 treatment can obviously reduce the decline of PBCC, and accelerates the recovery of PBCC.Different with the hematopoietic stimulation factor of current use, rmIL-12 can impel the complete set hemocyte that comprises leukocyte, erythrocyte, platelet to recover.
The intensity difference of lonizing radiation, the damage caused also can be different.Just can cause nerve and cerebrovascular trauma in the high dosage 1h of 100Gy left and right, show as the cental system symptom; Surpass the median dose radiation of 15Gy, within 4-6 days, will cause death because of gastrointestinal damage; 7-15Gy than the radiation of low dosage after, death occurs in 1-4 week, normally because hemopoietic function destroy and to cause.Neta finds rmIL-12 for mice (1000ng/ only) treatment of two different pedigrees, accept 12Gy and 9Gy radiation, within 4-6 days, still die from gastro-intestinal syndrome, but not the rmIL-12 treatment group accept 15Gy or more high dose just can bring out gastro-intestinal syndrome, he thinks that rmIL-12 can increase the sensitivity of gastrointestinal tract to radiation.Yet Chen also finds after the 10Gy gamma Rays by pathologic finding; the mice that low dosage rmIL-12 (100ng/ only) processes and intestinal villi, the crypts number of structures of matched group all keep complete; after accepting the 16Gy level of radiation; degree and the matched group of low dosage rmIL-12 treatment group injury of gastrointestinal tract are basic identical; irradiate rear 14 days two groups of Mouse Weights also obviously not different, therefore Chen etc. think that rmIL-12 does not demonstrate " radiosensitization reaction " under the dosage that can protect hemopoietic system.The rmIL-12 that suitable dosage is described can treat hemopoietic system damage after radiation, can not increase the sensitivity of gastrointestinal tract to radiation.
The applicant is finding normal mouse hemopoietic stem cell surface, people's neonatal umbilical cord hemocytoblast and hematopoietic stem cell (CD34 in peripheral blood after granulocyte colony-stimulating factor (G-CSF) mobilization agent injection 5 times (once a day) in research process
+) all there is an IL-12 expression of receptor, and the mescenchymal stem cell existed in the hematopoieticmicroenviron-ment of bone marrow can expression-secretion IL-12, so researcher thinks that this can directly act on stem cell, be that hematopoietic stem cell maintains normal physiological function foundation must be provided for IL-12.In addition under study for action, when mice is subject to the linear accelerator radiation damage, IL-12 can reduce the apoptosis of its stem cell, and can reduce the aberration rate of periphery blood nucleated cells, promotes in advance bone marrow hematogenesis, promotes erythrocyte, leukocyte and hematoblastic recovery early.Because the IFN-γ that IL-12 is induced may play inhibiting worry to bone marrow stem cell, in the interior experimentation of the rmIL-12 Mice Body that we carry out, do not increase the level of bone marrow IFN-gamma positive T cell, and IL-12 can also reduce the percentage rate of CD4+ and CD8+T cell in myelolymphocyte, may therefore alleviate the immunosuppressive action of T cell to bone marrow.
In sum, interleukin 12 can be by the IL-12 receptor of expressing on hematopoietic stem cell, anti-apoptotic effect while strengthening hematopoietic stem cell generation physical damnification, can promote the repair of organism when comprising that Co60 (gamma-rays) and linear accelerator (X ray) caused radiation damage etc. cause the hemopoietic system infringement simultaneously, its for the preparation of treat acute radiation sickness medicine in good application prospect is arranged.
Due to the reconstitution cell expression stability requirement of interleukin 12 (interleukin-12, IL-12), the dosage form of interleukin 12 of the present invention (interleukin-12, IL-12) is lyophilized formulations.Select the administering mode of subcutaneous administration when the treatment for acute radiation sickness, its dosage scope control is between 0.01 μ g/kg～0.2 μ g/kg.
Compared with prior art, technical scheme provided by the present invention, interleukin 12 (interleukin-12, IL-12) application in the medicine of preparation treatment acute radiation sickness, widened the scope of application of interleukin 12, the while also provides new medicine for the treatment of acute radiation sickness; And from the result of pharmacodynamic experiment, to the good effect that has for the treatment of acute radiation sickness.
The accompanying drawing explanation
Fig. 1 is the impact of IL-12 of the present invention on the mouse peripheral blood granulocyte quantity of 5.0Gy roentgenization;
Fig. 2 is the impact of IL-12 of the present invention on the peripheral blood red cell in mice quantity of 5.0Gy roentgenization;
Fig. 3 is the impact of IL-12 of the present invention on the mouse peripheral blood platelet counts of 5.0Gy roentgenization;
To be IL-12 of the present invention be subject to the impact of 30 days survival rates after the 8.0Gy roentgenization to the KM mice to Fig. 4.
The specific embodiment
1 reagent and instrument
Reagent: RPMI-1640 (GIBCO; the U.S.); hyclone (GIBCO; the U.S.); Ficoll lymphocyte separation medium (Shanghai China essence), CD3-PE, CD4-PreCP, CD8-FITC, IFN-γ-APC, Rat IgG1-APC, BFA are all purchased from BD Pharmingen company (Becton Dickinson/Phar Mingen MSA).
Instrument: flow cytometer (FACS Calibur, BD company, the U.S.), SW-CJ-2F type clean bench (SuZhou Antai Air Tech Co., Ltd.), CO2 gas incubator (SANYO, Japan), LXJ-II type centrifuge (Shanghai medical analytical instrument factory), XW-80A vortex mixer (above Industrial Co., Ltd. of Nereid section).
2 experimental techniques:
(1) bone marrow nucleated cell preparation
8 of BALB/c mouse, the cervical region dislocation is put to death, and gets the bilateral femur, puts into and adds 10% hyclone RPMI1640 complete culture solution, with syringe, repeatedly blows and beats and goes out bone marrow, with lymphocyte separation medium, isolates mononuclearcell.
(2) by (1) isolated for cell phosphate buffer (PBS) 4ml wash (1000 rev/mins, 10 minutes) 2 times, then add 400 μ l PBS re-suspended cells
(3) dyeing: two combinations of every pipe, every group of 200 μ l cells.
Cell after dyeing, under the condition of 4 ℃, keeps in Dark Place 30 minutes.
(4) again with the cell 2 times (2000 rev/mins, 5 minutes) after 400ul PBS washing dyeing
(5) every pipe adds containing machine analysis on the PBS 300 μ l of 0.1% bovine serum albumin.
Analytical method is that the minicell group with BMNC establishes an analysis.
3 results and conclusion
3.1 experimental result
Table 1 normal mice marrow hemopoietic stem cells IL-12R expresses (%) (n=11)
▲P＝0.001
3.2 conclusion: the expression of IL-12R is arranged on the normal mice marrow hemopoietic stem cells.
Embodiment 2 mouse bone marrow cells nucleated cell HSC apoptosis rate after roentgenization is expressed
Experiment purpose: observe the preventive and therapeutic action of IL-12 to the radiation damage cell
1.1 experimental technique
Select the Kunming mouse of SPF level, adopt linear accelerator 4Gy radiation damage, after the front 24h of radiation, radiation, 24h respectively is administered once; After irradiating, 4 natural gift other places are dead, detect the apoptosis rate of bone marrow HSC, and with c-kit APC labelling HSC, AnnexinVPE is that apoptosis detects index.
(1) bone marrow nucleated cell preparation
BALB/c mouse, the cervical region dislocation is put to death, get the bilateral femur under aseptic condition, cultivate with RPMI164 complete culture solution (inside adding 10% hyclone), repeatedly blow and beat punching with syringe and get bone marrow, with lymphocyte separation medium, isolate mononuclearcell, cell is washed (1000 rev/mins, 5 minutes) 2 times with PBS, add Binding Buffur (1 *) resuspended, counting cells, adjusting cell concentration is 1 * 10
6/ ml
(2) flow cytometer detection method
Dyeing: every pipe 200 μ l cells
Room temperature lucifuge 15 minutes, Binding Buffur for cell (1 *) washs (2000 rev/mins, 5 minutes) 2 times, add 300 μ l Binding Buffur (1 *) again and mix, 1 hour inner analysis, establish BMNC minicell cluster analysis HSC quantity and an apoptosis rate.
HSC：c-kit
+
HSC apoptosis rate: (c-kit
+) AnnexinV
+
3 results and conclusion
3.1 result
Apoptosis rate (%) n=10 of table 2 KM Mus through directly adding 4Gy radiation each experimental group marrow hemopoietic stem cells after four days
●P＜0.05
3.2 conclusion
The apoptosis rate of the marrow hemopoietic stem cells of rmIL-12 administration mice is starkly lower than irradiation group and the Normal group of not administration, and irradiation group simultaneously is apparently higher than Normal group.
Physically impaired prevention and the protective effect of embodiment 3IL-12 to hemopoietic system that radiation causes
1. the research of rmIL-12 to the protective effect of mice fatal dose radiation damage
1 experimental animal
60 of SPF level BALB/c mouse, 20～22g, single sex.
The test medication: negative control is sterile saline, and rmIL-12 (rmIL-12) is peprotech company product.
2 testing programs
Experimental animal is divided into 3 groups at random, 20 of every treated animals, and dosage regimen is as table 3;
Table 3 experimental animal random packet
Administering mode is subcutaneous administration, and the administration volume is 150 μ l.Radiation mode is total body radiation, and dosage is 10Gy.
Observation index: the survival rate of 4 weeks mices after radiation.
3 experimental results and conclusion
Experimental result: the 1st group of mice is all dead, 15 of second group of mouse survivals, 13 of the 3rd group of survivals.
Conclusion: rmIL-12 respectively is administered once and can obviously improves the survival rate of mice before and after the fatal dose radiation.
2. the repair of rhIL-12 to the hemopoietic system of mice non-lethal dose damage
1 materials and methods
1.1 laboratory animal
SPF level C57BL/6J mice, male, 18～22g, 120, Zhongshan University's Experimental Animal Center provides.
1.2 reagent and instrument
RmIL-12 (rmIL-12,1mg/ props up, Peprotech, the U.S.); Automatic Blood Cell Analyzer (GeniusKT-61801) and corresponding reagent are provided by Shenzhen Genius Electronics Co., Ltd.;
60the Co radiation device is provided by Guangzhou spoke sharp height energy technology company limited.
1.3 method
1.3.1 animal grouping, administration and blood sampling
By 80 C57BL/6J mices, be divided at random 4 groups, difference injecting normal saline and the basic, normal, high dosage group of rmIL-12, in Table 1,
60the Co exposure dose is 5Gy, rmIL-12 group 2h after predose 24h and irradiation of normal saline group and each dosage give normal saline and rmIL-12 each once.Each organizes after first 24 hours of mice administration and radiation the 8th, 12,16,19 days from each 60 μ l of eyeball rear vein beard blood sampling, ETDA anticoagulant, Blood cell analyzer detection hemocyte quantity.
Table 4 C57BL/6J mice group and dosage
1.3.2 the detection of hemocyte
With Automatic Blood Cell Analyzer (Genius KT-61801), detect.
1.3.3 statistical method
Application software spss13.0 is added up.
2 results
2.1 granulocytic number change
Each is organized granulocyte and within latter 8 days, all is progressively downward trend in irradiation, since the 12nd day, the middle and high dosage group of IL-12 is obviously faster than the normal saline group, and rmIL-12 high dose group granulocyte quantity is compared with low dose group with the normal saline group, and difference has statistical significance (P<0.05); After radiation the 30th day, middle and high dosage group granulocyte quantity was compared with the normal saline group, and difference has statistical significance (P<0.05), and middle and high dosage group granulocyte quantity has approached level before radiation, sees Fig. 1.
2.2 erythrocytic number change
After radiation, within first 8 days, respectively organize erythrocyte similar to level before administration, but since the 8th day, each is organized erythrocyte and starts obvious decline, from the 19th day, in, the bottom out of low dose group erythrocyte number, the most obvious with middle dosage, its erythrocyte number is compared with the normal saline group, and difference has statistical significance (P<0.05).Normal saline group and high dose group continue to descend, and the normal saline group is continued until the 19th day and minimum occurs, and the minimum of high dose group occurred at the 16th day.Each is organized the erythrocyte number variation and sees Fig. 2.
2.3 hematoblastic number change
Each organize platelet after irradiation first 12 days on a declining curve, administration is not obvious on downward trend impact, from the 16th day, high, middle dosage group platelet counts occurred ging up, the most obvious with middle dosage ascendant trend, normal saline group and low dose group continue to descend.After radiation the 16th day, middle and high dosage group platelet counts was compared variant (P<0.05) with low dose group with the normal saline group; After radiation the 19th day, middle dosage group platelet counts was compared variant (P<0.05) with low dose group with the normal saline group.Each is organized the platelet counts variation and sees Fig. 3.
3 conclusions
In this research, use low dosage IL-12, to three, be just that the recovery of hemocyte has obvious facilitation, erythrocyte and the hematoblastic range of decrease are dwindled, can reduce the situation of the hemorrhage and anoxia of body, make granulocyte, erythrocyte, platelet go up in advance simultaneously, reduce infection chance hemorrhage with continuation, promote the recovery of body.Research also shows that IL-12 can be used before raying damages prerequisite; can protect hemopoietic system; play prophylactic action, and the recovery fully of body hemopoietic system is not caused damage, in the bone marrow depression caused in the control radiation damage, great clinical value may be arranged.
The impact of embodiment 4 rhIL-12 on hematopoietic stem cell Radiation damage repair in umbilical cord blood
1 experiment material
1.1 reagent
RPMI-1640 culture medium, Hank ' s liquid, hyclone are U.S. GIBCO company product; the Ficoll lymphocyte separation medium is purchased from Shanghai China smart biotechnology company, and Annexin V apoptotic signal detection kit, antihuman CD 34-FITC are all the RPMI-1640 culture fluid containing 10% hyclone purchased from BD Pharmingen company (Becton Dickinson/Phar Mingen USA), RPMI-1640 complete culture solution.
1.2 instrument
Flow cytometer (FACS Calibur, BD company, the U.S.), SW-CJ-2F type clean bench (SuZhou Antai Air Tech Co., Ltd.), CO2 gas incubator (SANYO, Japan), LXJ-II type centrifuge (Shanghai medical analytical instrument factory), XW-80A vortex mixer (above Industrial Co., Ltd. of Nereid section), linear accelerator (Sweden's medical courses in general reach Elekta 5933).
2 experimental techniques
2.1 human umbilical cord blood mononuclear cell preparation
Extract Cord blood 20ml, anticoagulant heparin.The dilution of Hank ' s liquid equal-volume, (density: 1.077 ± 0.002) enterprising line density gradient centrifugation obtains (22 ℃ of mononuclearcells with the ratio of 1: 1, the blood of dilution slowly to be superimposed on to the Ficoll lymphocyte separation medium along tube wall, 800g * 20 minute), use Hank ' s liquid washed twice, adjusting mononuclearcell concentration with the RPMI-1640 complete culture solution is 1 * 10 again
6cell/ml.
2.2 experiment grouping and dyeing
2.2.1 experiment grouping
1. IPMI-1640 (not irradiating)
2. RPMI-1640 (4Gy single extracorporeal irradiation)
3. rhIL-120.01ng/ml is according to administration in first 24 hours (4Gy single extracorporeal irradiation)
4. rhIL-120.1ng/ml is according to administration in first 24 hours (4Gy single extracorporeal irradiation)
5. rhIL-121ng/ml is according to administration in first 24 hours (4Gy single extracorporeal irradiation)
Each sample is put to 5%CO
2, in 37 ℃ of incubators, cultivate, after irradiating, 24h detects quantity and the apoptosis rate of marrow hemopoietic stem cells with flow cytometer, with CD34FITC labelling hematopoietic stem cell, Annexin V-PE
+7AAD
+for apoptotic signal detects index; Collect and respectively to organize cell and wash (1000 turn * 5 minutes) 2 times with PBS, add Binding Buffur 200 μ l re-suspended cells.
2.2.2 dyeing
Every pipe 200 μ l cells, add following reagent: Annexin V, PE, 5 μ l; 7-AAD, 5 μ l; CD34, FITC, 5 μ l.Room temperature lucifuge 15 minutes, cell is washed (2000 turn, 5 minutes) 2 times with Binding Buffur, adds 300 μ l Binding Buffur and mixes, and establishes 1 hour inner analysis of the corresponding cell mass of door.
2.3 drug effect is observed
If door is analyzed CD34 in each experimental group
+the ratio of cell and apoptosis rate.
2.4 date processing
With
mean data, adopt the SPSS13.0 statistical software to carry out the correlation test analysis, the significance test standard is P<0.05.
3 results and conclusion
3.1 result
Table 5 rhIL-12 and Cord blood hatch In Vitro Anti apoptotic effect after 24h (n=8,
)
Annotate: *, compare P<0.05 with the irradiation model matched group.
The result of table 5 shows, cord blood CD 34
+hematopoietic stem cell apoptosis rate after linear accelerator 4.0GY extracorporeal irradiation raises, and with the normal control cell do not irradiated, significant difference (P<0.05) is relatively arranged.The CD34 of pre-irradiation 24h after the rhIL-12 of 1ng/ml is hatched
+the hematopoietic stem cell apoptosis rate is starkly lower than simple irradiation control group (P<0.05).Although the anti-apoptotic effect between the rhIL-12 experimental group of variable concentrations compares not statistically significant mutually, the apoptosis rate of hematopoietic stem cell still has the trend of reduction at the rhIL-12 low concentration.
3.2 conclusion
RhIL-12 (1ng/ml) and umbilical cord blood CD34
+after hematopoietic stem cell is hatched 24h, the apoptosis signal that the radiation damage that directly adds 4.0GY is caused rises obvious downward effect, P<0.05, and rhIL-12 is to CD34 in prompting
+hematopoietic stem cell has the anti-apoptotic effect of pre-antiradiation injury.
Embodiment 5 Kunming mouses common-size analysis of IFN-γ positive T cell in T cell and born of the same parents in myelolymphocyte after rmIL-12 stimulates
1 reagent and instrument
1.1 reagent: rmIL-12 (Peprotech; the U.S.); RPMI-1640 (GIBCO; the U.S.); hyclone (GIBCO; the U.S.), Ficoll lymphocyte separation medium (Shanghai China essence), rmIFN-γ ELISA detection kit, CD3-PE, CD4-PreCP, CD8-FITC, IFN-γ-APC, Rat IgG1-APC, BFA are all purchased from BD Pharmingen company (BectonDickinson/Phar Mingen MSA).
1.2 instrument: flow cytometer (FACS Calibur, BD company, the U.S.), SW-CJ-2F type clean bench (SuZhou Antai Air Tech Co., Ltd.), CO2 gas incubator (SANYO, Japan), LXJ-II type centrifuge (Shanghai medical analytical instrument factory), XW-80A vortex mixer (above Industrial Co., Ltd. of Nereid section).
2. experimental technique
(1) bone marrow nucleated cell preparation
The dislocation of mice cervical region is put to death, and gets the bilateral femur, with RPMI1640 complete culture solution (inside adding 10% hyclone), repeatedly blows and beats punching with syringe and gets bone marrow, with lymphocyte separation medium, isolates mononuclearcell.With 4ml RPMI1640 washed twice (1000 rev/mins, 10 minutes), with 1ml complete culture solution re-suspended cell, counting.
(2) BMNC adds albumen and turns and answer inhibitor (BFA) 10 μ g/ml In vitro culture 4 hours, mixes culture environment: 37 ℃, and 5%CO
2.
(3) cell is washed (1000 rev/mins, 5 minutes) 2 times with PBS, notices that paper using blots post-reinforcing and determines liquid, and every pipe adds 500 μ l fixation/Pereabilization, mixes, and under room temperature, keeps 20 minutes, adds rear attention and mixes.
(4) wash (2000 rev/mins, 10 minutes) 2 times with 2ml PBS, notice that paper using blots, add 400 μ l Perm/Wash
tMbuffer mixes.
(5) dyeing: two combinations of every pipe, every group of 200 μ l cells
At 4 ℃, after keeping in Dark Place 30 minutes, use washing liquid Perm/Wash
tMbuffer washes 2 times; Every pipe adds the PBS 300 μ l containing 0.1% bovine serum albumin; Upper machine analysis, establish a lymphocyte and analyze IFN-γ expression in each T cell subsets born of the same parents.
3 results and conclusion
Table 6 Kunming mouse is IFN-r expression (%) in bone marrow PBMC born of the same parents after 18 hours after rmIL-12 stimulates
☆P＜0.05；★P＜0.05
3.2 conclusion: Kunming mouse is after rmIL-12 stimulates administration, in myelolymphocyte, the percentage rate of helper T lymphocyte and cytotoxic T cell obviously reduces, and in them, IFN-γ positive rate does not all have difference in normal saline group and rmIL-12 (0.5 μ g/ only) group.
Embodiment 6 rmIL-12 are subject to survival rate and the impact of life cycle after fatal dose (8Gy) radiation gamma to the KM mice
Research purpose: inquire into rmIL-12 treatment effect to the KM mice of fatal dose (8Gy) radiation gamma induced Acute radiation injury through subcutaneous injection.
1 experiment material
1.1 laboratory animal
100 of clean level KM (Kunming kind) mices, male and female half and half, body weight 18 ± 2g, 6-8 age in week, Military Medical Science Institute's Experimental Animal Center breeding, laboratory animal credit number: SCXK (army) 2002-001.Laboratory animal is in the indoor raising of Military Medical Science Institute's zoopery, the laboratory quality certification number: SYXK (army) 2002-016.10 mices of every cage, raise the feedstuff of take specially as the mice preparation, freely drinks water.The indoor temperature of zoopery remains on 25 ℃ of left and right, and relative humidity remains on 40-70%, illumination every day 12 hours.
1.2 experiment reagent
RmIL-12 (Peprotech, the U.S.); Dilution nilestriol stock solution is provided with the radiation medicine institute by Military Medical Science Institute's radiation.
1.3 illuminate condition
Military Medical Science Institute's radiation and radiation medicine institute
60co radiation gamma source, close rate is 228.35cGy/min, irradiation time 3 minutes and 13 seconds, disposable total irradiation 8.0Gy, irradiation distance is 4 meters.
2 experimental techniques
2.1 animal grouping and medication
100 mices are randomized into normal saline and (irradiate contrast, 150 μ l/ are only), nilestriol (positive control, 0.2mg/ only), rmIL-12 low dosage (50ng/ only), middle dosage (100ng/ only), high dose (200ng/ only) are totally 5 groups, every group 20, male and female half and half.Fatal dose pre-irradiation 72h, 1h respectively are administered once, and after irradiating, be administered once every day, continuous 7 days.Mouse carotid back subcutaneous injection, the administration volume is 150 μ l/.Nilestriol diluent 0.2mg/, gavage, pre-irradiation 1h is administered once, and after irradiating, be administered once every day, continuous 6 days.
2.2 observation index
After being subject to irradiation, observe mice 2 every day, and each 30min, record hair color, activity, feed and the drinking-water of mice, and whether the situations such as diarrhoea, vomiting, death are arranged, and relatively irradiates latter 18 days mice survival rates, mean survival time and median survival time.
2.3 date processing
Data are used
mean, adopt the SPSS13.0 statistical software, with the variance analysis statistical average time-to-live, (when variance is neat, employing LSD method compares in twos, during heterogeneity of variance, adopt Tamhane and Dunnett T3 method to compare in twos), adopt relatively survival rate of the accurate probabilistic method of Fisher, and make survival analysis with Kaplan-Meier and Log-rank/Breslow, the significance test standard is P<0.05.
3 experimental results
Losing weight appears in irradiation control group mice very soon, has blood in stool, the symptom such as infection, and dies off, full group totally 20 only remaining 3 survivals of mices while finishing to the observation period; More late, degree light (dead mice makes an exception) that the similar symptom of positive controls and rmIL-12 treatment group occurs.The situation identical with positive controls is, though the administration group death time shifts to an earlier date to some extent than irradiation control group, to have reduced dead number of cases.
The rmIL-12100ng treatment group is compared with irradiation control group, and mean survival time all has statistics poor, P<0.05.Female mice rmIL-12100ng, 200ng dosage group are compared with irradiation control group, and its survival rate has significant difference (P<0.05); The average survival natural law of male mice, according to penetrating the contrast group leader, does not still have significant difference (in Table 7).
Table 7 rmIL-12 is subject to the impact of existence in 30 days after the 8.0Gy roentgenization to the KM mice
Annotate: with irradiation control group, compare, " * " P<0.05, observing time was by 30 days.
The rmIL-12 group compares with irradiation control group, and when dead number of cases reduces, the life span that rmIL-12 respectively organizes dead mice is the same with positive controls, and the shortening phenomenon is all arranged, agnogenio.Life span with the dead mice of the increase of dosage shortens (table 7) on the contrary, and the 200ng group has been compared significant difference (P<0.05) with irradiation control group, and prompting 200ng and above dosage may increase the weight of the symptom of mice.Each experimental mice death time all mainly is distributed in irradiates latter 8 to 12 days.
Table 8 rmIL-12 is on being subject to the impact of dead mice life span in 30 days after the 8.0Gy roentgenization
Annotate: PF means surviving fraction (surviving fraction=processed group dead animal mean survival time/irradiation control group dead animal mean survival time).With irradiation control group, compare, " * " P<0.05, observing time was by 30 days.
The survival rate of each rmIL-12 treatment group mice relatively, significant difference between 100ng, 200ng dosage group and matched group (P<0.01) (table 8).The demonstration of Kaplan-Meier survival analysis result, 100ng, 200ng experimental group are compared the remote effect significant difference with matched group, and the Log-rank analysis result is respectively P<0.05 and P<0.01, and the Breslow analysis result is P>0.1, shows that short-term effect is not obvious.In addition, female mice 100ng, 200ng dosage group prognosis effect are better, and the Log-rank analysis result is respectively P=0.021 and P=0.031; The Breslow analysis result is respectively P=0.076 and P=0.126, show that long-term effect is better than short-term effect, and each experimental group of male mice is compared and be there is no significant difference with irradiation control group.Above prompting: after the rmIL-12 of 100ng, 200ng dosage treatment, can improve the middle long-term survival of female KM mice., there is the difference (Fig. 4) on sex in this effect produced for rmIL-12.
Table 9 rmIL-12 is subject to the impact of 30 days survival rates after the 8.0Gy roentgenization on the KM mice
Annotate: compare " * " P<0.05, " * * " P<0.01 with irradiation control group; Observing time was by 30 days.
Respectively organize the median survival time of mice in table 10 experiment
Annotate: observing time was by 30 days.
3.2 conclusion
Originally studies show that: compare with irradiation control group, at fatal dose pre-irradiation 72h, 1h and after irradiating, within continuous 7 days, give 100ng, the 200ng/ rmIL-12 of a dosage, can obviously improve the KM mice and be subject to 8.0Gy's (close rate is 228.35cGy/min)
60survival rate after the Co radiation gamma in 30 days (P<0.05).Each organize median survival time (table 10) in conjunction with the survival analysis result be presented at 50,100,200ng/ only in the selection of three dosage, adopts 100ng, 200ng/ dosage long-term effect only better.
Claims (3)
- One kind by interleukin 12 the purposes for the preparation for the treatment of acute radiation medicine.
- 2. purposes according to claim 1, wherein the dosage form of interleukin 12 is lyophilized formulations.
- 3. purposes according to claim 1 and 2, wherein interleukin 12 adopts the mode administration of subcutaneous administration when being used for the treatment of acute radiation sickness.
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| Title |
|---|
| 王宜强等.白细胞介素12对60钴照射小鼠造血功能的影响.《中华医学杂志》.1997,第77卷(第3期),216-219. * |
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