CN105078948A - Application of P2X7 receptor inhibitor - Google Patents
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Abstract
The invention discloses application of a P2X7 receptor inhibitor in the preparation of drugs for treating radiation brain injuries. Through experimental deep research on pathogenesis of radiation brain injuries and effect of a P2X7 receptor upon occurrence and development of the radiation brain injuries, discovered is that radioactive rays activating microglial cells by the P2X7 receptor cause following inflammatory response, the P2X7 receptor specific inhibitor BBG is effective in inhibiting this response, and nerve cells are thereby protected; the inhibitor is applicable to the treatment of radiation brain injuries.
Description
Technical field
The present invention relates to the purposes of P2X7 acceptor inhibitor, be specifically related to the purposes of P2X7 acceptor inhibitor in the medicine of preparation radiotherapy brain injury.
Background technology
Brain Radiation Injury refers to that cerebral tissue is subject to radiation exposure, and cause under many factors synergy neuron generation degeneration, necrosis and cause central nervous system disease.After being common in the head and neck Malignant and benign lesions radiotherapy of former or secondary, also see radiation injury in commercial production.According to statistics, the incidence rate of radiotherapy tissues following MCAO in rats necrosis can up to 28.5%, and radiation tissues following MCAO in rats is downright bad, and once be formed, the course of disease is irreversible, not only has a strong impact on life in patients, brings heavy burden and significant damage also to the family of patient and society.At present for the research of Brain Radiation Injury, mainly concentrate on radiation tissues following MCAO in rats pathological change and Imageology two aspects, lack its pathogenetic discussion, and traditional medication effect is limited, specify the pathogeny of Brain Radiation Injury and actively to find effective treatment means be key issue urgently to be resolved hurrily at present.
Research finds, in the damage of central nervous system, microglia plays a part key as the intrinsic cell with immunocompetence and phagocytic activity, by the release cells factor as pro-inflammatory cytokine, active oxygen vehicle, protease and complement protein, participate in generation and the progress of multiple sacred disease, comprise Alzheimer, parkinson disease, multiple sclerosis, acquired immune deficiency syndrome (AIDS).Microglia plays a part in pivot system chronic immunity diseases associated with inflammation dual in these; cascade of response of inflammation is amplified on the one hand by release pro-inflammatory cytokine; mediation neurocyte degenerative change, plays the function protecting central nervous system to damaging cells chemotactic, engulf etc. on the other hand.RE and these chronic inflammation diseases have similarity in clinical manifestation and Radiologic imaging.Also observe lonizing radiation in experiment and can activate microglia, make cell by the branched ameba sample changing activation into of tranquillization.Radiate COX-2, TNF-α, IL-6mRNA and correlative protein expression level in postactivated microglia to increase, after finding radiation in zoopery simultaneously, nerve fiber is chaotic, improper arrangement, neuronal quantity reduces, neuronic branch disappears, and prompting Activated Microglia take part in postradiation brain injury, can cause neuronic damage by release Inflammatory substances.Therefore, the inflammatory reaction that microglial activation causes may play an important role in RE.
Inflammatory reaction is usually with in cell or the rising of Extracellular ATP level.The ATP acting on purinoceptor normally can derive from neuron, astrocyte and microglia.Under pathological conditions, as mechanical damage or metabolite stimulate, the change of inflammatory reaction or cell injury or cell plasma environment, the release of ATP can be stimulated.Notice in experiment, as the purinoceptor of sensor in the diseases associated with inflammation that radiotherapy causes, vital effect may be played in the activation of the generation development of RE and microglia.The purinoceptor that microglia is expressed is divided into two classes, and ion channel receptor (P2XReceptor, P2XR) and g protein coupled receptor (P2YReceptor, P2YR), all by mediate outside-in cell Ca
2+flowing play a role.P2X7 receptor (P2X7R), one special in P2X receptor family, fastens high expressed in Monocytes/Macrophages, microglia also has the wide expression of P2X7 receptor.Have been reported both at home and abroad and propose it at central nervous system injury and diseases associated with inflammation as Alzheimer, all play an important role in spinal cord injury and neuropathic pain.There is no bibliographical information, whether P2X7 receptor can mediate the inflammatory reaction participating in RE and cause.
Summary of the invention
The object of the invention is to overcome weak point that prior art exists and provide the novelty teabag of P2X7 acceptor inhibitor, being specifically related to the purposes of P2X7 acceptor inhibitor in the medicine of preparation radiotherapy brain injury.
For achieving the above object, the technical scheme taked: the purposes of P2X7 acceptor inhibitor in the medicine of preparation radiotherapy brain injury.
Preferably, described P2X7 acceptor inhibitor is BBG.The English full name of BBG of the present invention is brilliantblueG, and Chinese is light blue G.
Principle of the present invention is: during the brain injury caused at lonizing radiation develops, the microglia of Radiation active has played pivotal role.Lonizing radiation by P2X7 receptor activation microglia, thus discharge a large amount of inflammatory mediators and inflammatory factor, as IL-6, COX-2, TNF – α.Interaction between these cytokines can regulate and control the growth of neuron and neurogliocyte, differentiation and activation, causes the formation of brain injury.And the release of these pro-inflammatory cytokines, can block by the inhibitor B BG of P2X7 receptor-specific.
Experiment of the present invention shows, postradiation microglia can be become from the long projection " branched " time static " amoebiform " of short projection, raises P2X7 receptor and the mRNA of inflammatory factor IL-6, COX-2, TNF α and the expression of protein level.The pretreatment of BBG can suppress the activation of microglia; reduce P2X7 receptor and inflammatory factor IL-6; COX-2; the mRNA of TNF α and the expression of protein level; and alleviate postradiation nerve injury, thus neuroprotective unit and cerebral tissue, therefore; P2X7 receptor modulators microglia plays an important role in Brain Radiation Injury process, and BBG is a kind of feasible brain injury neuroprotective.
Beneficial effect of the present invention is: the invention provides the purposes of P2X7 acceptor inhibitor in the medicine of preparation radiotherapy brain injury; the present invention furthers investigate the pathogenesis of Brain Radiation Injury by experiment and P2X7 receptor, in Brain Radiation Injury, developing effect occurs; find that lonizing radiation cause follow-up inflammatory reaction by P2X7 receptor activation microglia; and the specific inhibitor BBG of P2X7 receptor effectively can suppress this reaction; and then neuroprotective unit, can be used for radiotherapy brain injury.
Accompanying drawing explanation
Fig. 1 is the RT-PCR electrophoretogram of various P2X expression of receptor after in vitro pre-irradiation in embodiments of the invention 1;
Fig. 2 is the contrast block diagram of various P2X expression of receptor after in vitro pre-irradiation in embodiments of the invention 1;
Fig. 3 is metamorphosis figure after in vitro microglia BV2 pre-irradiation in embodiments of the invention 1;
Fig. 4 is after in embodiments of the invention 1, in vitro microglia BV2 irradiates and the block diagram of the activation rate of preincubate BBG;
Fig. 5 is the change block diagram that in embodiments of the invention 1, in vitro P2X7 receptor participates in the mRNA level in-site of irradiating rear P2X7 receptor;
Fig. 6 is the change block diagram that in embodiments of the invention 1, in vitro P2X7 receptor participates in the mRNA level in-site of irradiating rear inflammatory factor COX-2;
Fig. 7 is the change block diagram that in embodiments of the invention 1, in vitro P2X7 receptor participates in the mRNA level in-site of irradiating rear inflammatory factor IL-6;
Fig. 8 is the change block diagram that in embodiments of the invention 1, in vitro P2X7 receptor participates in the mRNA level in-site of irradiating rear inflammatory factor TNF-a;
Fig. 9 is that in embodiments of the invention 1, in vitro P2X7 receptor participates in the protein electrophoresis figure that rear P2X7 receptor and inflammatory factor are irradiated in regulation and control, wherein R24 represents latter 24 hours of irradiation, R48 represents latter 48 hours of irradiation, B24 represents that BBG pretreatment is irradiated latter 24 hours, and B48 represents that BBG pretreatment is irradiated latter 48 hours;
Figure 10 is the change block diagram that in embodiments of the invention 1, in vitro P2X7 receptor participates in irradiating rear P2X7 protein level;
Figure 11 is that in embodiments of the invention 1, in vitro P2X7 receptor participates in irradiating the change block diagram that rear BV2 discharges inflammatory factor COX-2 protein level;
Figure 12 is that in embodiments of the invention 2, in animal body, P2X7 receptor participates in irradiating the change block diagram of rear P2X7 acceptor gene level;
Figure 13 is that in embodiments of the invention 2, in animal body, P2X7 receptor participates in irradiating the change block diagram of rear proinflammatory inflammation factor IL-6 gene level;
Figure 14 is that in embodiments of the invention 2, in animal body, P2X7 receptor participates in irradiating the change block diagram of rear proinflammatory inflammation factor COX-2 gene level;
Figure 15 is that in embodiments of the invention 2, in animal body, P2X7 receptor participates in irradiating the change block diagram of rear proinflammatory inflammation factor TNF-a gene level;
Figure 16 is that in embodiments of the invention 2, in animal body, P2X7 receptor participates in the protein electrophoresis figure that rear P2X7 receptor and inflammatory factor are irradiated in regulation and control;
Figure 17 is that in embodiments of the invention 2, in animal body, P2X7 receptor participates in irradiating the change block diagram of rear P2X7 protein level;
Figure 18 is that in embodiments of the invention 2, in animal body, P2X7 receptor participates in irradiating the change block diagram that rear BV2 discharges inflammatory factor COX-2 protein level;
Figure 19 is metamorphosis figure after microglia pre-irradiation in animal body in embodiments of the invention 2;
Figure 20 is after in embodiments of the invention 2, in animal body, microglia irradiates and the block diagram of the activation rate of preincubate BBG.
Detailed description of the invention
For better the object, technical solutions and advantages of the present invention being described, below in conjunction with specific embodiment, the invention will be further described.
Embodiment 1: under ex vivo, P2X7 receptor activates the effect in microglia and proinflammatory inflammation factor dispose procedure at lonizing radiation.
(1) cell culture and preparation: go down to posterity microglia strain BV2 in containing concentration expressed in percentage by volume be 10% hyclone, mass percentage concentration be cultivate in the DMEM culture medium of dual anti-(penicillin, the streptomycin) of 1%, be the CO of 5% in concentration expressed in percentage by volume
2, under 37 DEG C of conditions, cultivate in incubator with culture bottle, within two days, change liquid once.When going down to posterity, mass percentage concentration is 0.25% pancreatin (be 0.02%EDTA containing mass percentage concentration) digestion at every turn, is inoculated in new culture bottle with uniform density.By cell according to 2 × 10
5/ ml density is inoculated in 6 orifice plates, and 24 orifice plate inoculum densities are 1 × 10
4/ ml, 37 DEG C, concentration expressed in percentage by volume is 5%CO
2under condition, cultivate after 24 hours in incubator, change serum-free medium and cultivate 4 hours.
(2) experiment grouping and cell irradiation: cell is divided into three groups: matched group, irradiation group, medical preconditioning group, irradiation group and medical preconditioning group are further divided into irradiates latter 24 hours, 48 hours two subgroups.Add normal saline 17ul at pre-irradiation 60min respectively to matched group, irradiation group, add BBG17ul (BBG:100nmol/L) to medical preconditioning group.Ready cell is positioned on radiation therapy bed, culture bottle covers the Corii Sus domestica that 1cm is thick, ray is enable better to reach the cell face of culture bottle, adjustment radiation comes from 1m place bottom culture bottle, single fraction, absorbed dose rate 6MeV/min, absorb accumulated dose 10Gy, the cell of matched group brings radiotherapy indoor into, but irradiates under being not interposing at radioactive source, gets cell conditioned medium liquid and detects for Q-PCR and Westernblot.
(3) Trizol reagent one-step extraction extracts total serum IgE, reverse transcription synthesis cDNA.Reaction condition is 30 DEG C of 5min, 42 DEG C of 60min, 90 DEG C 15 seconds.CDNA PremixTaq carries out polymerase chain reaction PCR.
(4) expression of RT-PCR half-quantitative detection cell P2X1 receptor (P2X1R), P2X2 receptor (P2X2R), P2X3 receptor (P2X3R), P2X4 receptor (P2X4R), P2X5 receptor (P2X5R), P2X6 receptor (P2X6R), P2X7 receptor (P2X7R), its RT-PCR primer is as shown in table 1, wherein Fprimer is forward primer, and Rprimer is primer anyway.
Table 1RT-PCR primer
The amplification condition of PCR:
1. Beta-actin:94 DEG C of 3min, 30 circulations (94 DEG C of 30sec, 58 DEG C of 30sec, 72 DEG C of 1min), 72 DEG C of 10min.
2. P2X1 receptor, P2X2 receptor, P2X3 receptor, P2X5 receptor, P2X6 receptor: 94 DEG C of 3min, 30 circulations (94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 1min), 72 DEG C of 10min.
3. P2X4 receptor, P2X7 receptor: 94 DEG C of 3min, 30 circulations (94 DEG C of 30sec, 56 DEG C of 30sec, 72 DEG C of 1min), 72 DEG C of 10min.
PCR primer is separated with 2% agarose gel electrophoresis containing TBE, the imaging of UVP gel imaging system.
(5) with the expression of the gene of Q-PCR technology for detection P2X7 receptor and inflammatory factor COX-2, IL-6, TNF-a, its Q-PCR primer is as shown in table 2, and wherein Fprimer is forward primer, and Rprimer is primer anyway.
Table 2Q-PCR primer
Reaction condition
Stage1 denaturation (95 DEG C, 30sec)
Stage2PCR reacts (95 DEG C of 5sec, 60 DEG C of 20sec, 40cycles)
Stage3 melt curve analysis analyzes (95 DEG C of 0sec, 65 DEG C of 20sec, 95 DEG C of 0sec)
(6) Western-Blot detects the expression of BV2 cell P2X7 receptor and COX-2 albumen.
Get total protein 100 DEG C of degeneration 10min, add 5 × protein electrophoresis sample-loading buffer according to 1:4 volume ratio, mixing loading, and add protein molecular weight pre-dyed Marker electrophoresis (concentrated glue 30min, 50V together; Separation gel 100min, 120V, arrive bottom separation gel to bromophenol blue); Electricity transferring film, constant current 120mA, 180min, visible pre-dyed Marker goes on pvdf membrane; TBST washes film, 5min × 2 time; Confining liquid room temperature closes 1h, adds primary antibodie (P2X7: by volume for 1:350 adds; The primary antibodie of COX-2: by volume for 1:500 adds; The primary antibodie of GAPDH: by volume for 1:500 adds) hatch, take out after 4 DEG C of shaken overnight, wash film with TBST, 10min × 3 time; Add two anti-(by volume for 1:15000 adds) of HRP labelling, incubated at room 1h; TBST washes film, 10min × 3 time; Darkroom exposes: the A liquid of ECL test kit and B liquid are mixed for 1:1 by volume, drop on film, expose in darkroom.
(7) immunofluorescence observes the change of P2X7 expression of receptor and form after BV2 cell irradiation.(, for labelling microglia, P2X7 (green fluorescence) is for labelling P2X7 receptor for cd11b (red fluorescence).)
Within after cell irradiation the 24th hour, remove culture fluid, PBS cleaning once, is the paraformaldehyde fixed cell of 4% by concentration expressed in percentage by volume, 30min.0.01MPBS cleans, 5min × 3 time.Concentration expressed in percentage by volume is the TritonX-1001ml permeable membrane 30min of 0.3%.Again with 0.01MPBS cleaning, 5min × 3 time.Concentration expressed in percentage by volume is the Normal Goat Serum confining liquid 1ml of 10%, and room temperature closes 60min.0.01MPBS cleans, 5min × 3 time.Add primary antibodie CD11b (by volume for 1:40 adds), P2X7 (by volume for 1:50 adds) is hatched, and 4 degree, spends the night.0.01MPBS cleans, each 5min × 3 time.Cy3 mark fluorescent two anti-(by volume for 1:32 adds) is hatched, room temperature 60min.0.01MPBS cleans, 5min × 3 time.Observation of cell form under inverted fluorescence microscope.
As shown in Figures 1 to 4, under ex vivo, microglia BV2's result is activated after radiation, and P2X7 receptor and inflammatory factor all raise at gene and protein expression.Not only can suppress the activation of radioactive microglia after BBG pretreatment, the expression of P2X7 receptor and inflammatory factor can also be suppressed in various degree.
As shown in Figure 1: the first row: matched group purinoceptor (P2X1 receptor, P2X2 receptor, P2X3 receptor, P2X4 receptor, P2X5 receptor, P2X6 receptor, P2X7 receptor); Second row: irradiation group purinoceptor, irradiates latter 24 hours.The purinoceptor P2X1 receptor of microglia BV2, P2X3 receptor, P2X4 receptor and P2X7 receptor have expression after pre-irradiation, but in radiation latter 24 hours, only there are P2X3 receptor and P2X7 expression of receptor to have rising.As can be seen from Figure 2, P2X7 expression of receptor amount is in latter 24 hours of radiation (0.75 ± 0.03), and compared with normal group (0.39 ± 0.01) obviously raises, and is close to 2 times into pre-irradiation; P2X3 receptor expresses identical trend with P2X7 receptor after pre-irradiation, and its expression is in latter 24 hours of radiation (0.31 ± 0.01), and compared with normal group (0.09 ± 0.01) also obviously raises, and is 3 times of pre-irradiation.GeneToolsfromSyngene analyzes band brightness value, carries out statistical analysis, has significant difference, P<0.05.P2X1 receptor and the change of P2X4 receptor after pre-irradiation do not reach statistical significance.
As shown in Figure 3: A, D, G are the cd11b of normal group (microscope magnification is 400 times), P2X7 respectively, overlap; B, E, H are the cd11b of irradiation group (microscope magnification is 400 times), P2X7 respectively, overlap; C, F, I are the cd11b of pharmaceutical intervention group (microscope magnification is 400 times), P2X7 respectively, overlap; Wherein overlap and refer to cd11b and P2X7 labelling altogether.Get microglia and within 24 hours, fixedly do cellular immunofluorescence after irradiation.Typical immunofluorescence results shows in matched group, and the BV2 of tranquillization state is the growth of multi-branched shape, and cell space is less.Irradiate (10Gy) after 24 hours, the tranquillization state of less by cell space, the branched projection of BV2 change that cell space circle is large, projection is short and small into, activated state in " ameba " sample; Meanwhile, the microglia quantity of P2X7 labelling comparatively matched group increases, P<0.05.After pharmaceutical intervention, the BV2 cells switch that great majority activate returns branched tranquillization state, and the BV2 quantity comparatively irradiation group of P2X7 labelling reduces, P<0.05.
As shown in Figure 4: with times visual field, fluorescence microscope × 200 for counting criteria, choose 5 visuals field respectively in different disposal group and do mean and standard deviation, calculate the activation rate of BV2 cell in different processed group.In matched group, the activation rate of BV2 is (1.86 ± 0.51) %, and in irradiation group, the activation rate of BV2 is (21.6 ± 2.41) %, and in pharmaceutical intervention group, the activation rate of BV2 is (9.14 ± 0.56) %.Result shows that P2X7 receptor participates in the change of form and quantity after mediation microglia BV2 pre-irradiation.
As shown in Fig. 5,6,7,8, irradiate the change causing the mRNA level in-site of P2X7 receptor and inflammatory factor COX-2, IL-6 and TNF-a.The mRNA level in-site of P2X7 receptor after irradiation 24 hours comparatively matched group raise (2.84 ± 0.59) doubly (P<0.05), after preincubate BBG intervenes, comparatively irradiate and can lower P2X7 receptor 23.2% in latter 24 hours.P2X7 receptor raises after irradiation and reaches maximum for 48 hours, and comparatively matched group raises (3.48 ± 0.23) doubly (P<0.05).Now, the effect that preincubate BBG intervenes is also the most remarkable, comparatively irradiates and can lower 36.1% (P<0.05) in latter 48 hours.The change discharging inflammatory factor level before and after BV2 cell irradiation is as follows: the mRNA level in-site of COX-2 after irradiation 24 hours comparatively matched group raise (4.82 ± 0.86 times) (P<0.05), but BBG preincubate can not suppress it to express.It irradiates latter 48 hours comparatively matched groups rising (21.75 ± 2.37 times) (P<0.05), BBG preincubate significantly can suppress the expression of now COX-2, comparatively irradiates and lowers 34.8% (P<0.05) in latter 48 hours.The mRNA level in-site of IL-6 after irradiation 24 hours comparatively matched group raise (5.02 ± 0.78) doubly (P<0.05), it can not be suppressed after BBG preincubate to express.Irradiate latter 48 hours comparatively matched groups rising (33.34 ± 3.01 times) (P<0.05), preincubate BBG can significantly suppress it to express, and comparatively irradiates and lowers 47.3% (P<0.05) in latter 48 hours.The mRNA of TNF-a after irradiation 24 hours comparatively matched group raise (3.25 ± 0.58) doubly (P<0.05), BBG preincubate can not suppress it to express.Irradiate latter 48 hours comparatively matched group raise 4.55 ± 0.67 times (P<0.05), preincubate BBG can significantly suppress it to express, comparatively irradiate latter 48 hours downward 49.8% (P<0.05).
In sum, irradiate the P2X7 receptor of rear microglia BV2 and proinflammatory inflammation factor COX-2, the mRNA of IL-6 and TNF-a all raises after irradiation for 24 hours and 48 hours, and time positive correlation after elevated-levels and irradiation, preincubate BBG can suppress the gene level of P2X7 receptor after irradiation for 24 hours, but can not lower the gene level of each inflammatory factor.But the gene level of 48 hours P2X7 receptors and each inflammatory factor all significantly can be suppressed by BBG after irradiation.Infer thus, P2X7 receptor participates in the change that mediation microglia BV2 irradiates rear inflammatory factor gene level.
As shown in Fig. 9,10,11, P2X7 receptor protein level is 24 hours (1.22 ± 0.28) comparatively matched group (1.06 ± 0.14) increase after irradiation, but does not reach statistical significance (P>0.05).At this time point, BBG pretreatment can not lower P2X7 receptor protein level.But, P2X7 receptor protein level after irradiation 48 hours (1.49 ± 0.07) comparatively matched group (1.06 ± 0.14) obviously raise, and significantly can be suppressed by BBG, its P2X7 receptor protein level (0.73 ± 0.08) is comparatively irradiated latter 48 hours (1.49 ± 0.07) and is obviously reduced, and has statistical significance (P<0.05).The change of inflammatory factor COX-2 protein level, has identical trend with the change of P2X7 acceptor levels.COX-2 albumen is 24 hours (0.85 ± 0.18) comparatively matched group (0.55 ± 0.13) rising after irradiation, and at this time point, BBG pretreatment can not lower COX-2 protein level.Irradiate latter 48 hours (2.01 ± 0.19) comparatively matched group (0.55 ± 0.13) obviously raise, now, BBG pretreatment significantly can lower the expression of P2X7 receptor protein, and COX-2 protein level (0.96 ± 0.19) comparatively irradiates latter 48 hours (2.01 ± 0.19) and obviously declines.
Can infer thus, after microglia BV2 irradiates, the protein level of P2X7 receptor and inflammatory factor COX-2 raises, and 48 hours after irradiation, BBG pretreatment can significantly lower its protein expression level.
Embodiment 2: in animal experiment, P2X7 receptor activates the effect in microglia and proinflammatory inflammation factor dispose procedure at lonizing radiation.
(1) experiment grouping: get healthy adult male C 57 BL/6 J mouse 63 and be divided into three groups at random: matched group, irradiation group, irradiation+medical preconditioning group.Irradiation group and pharmaceutical intervention group are further divided into irradiates latter 3 days, 7 days four subgroups.Wherein matched group 10, two irradiate each 10 of subgroups, each 10 of two medical preconditioning subgroups.
(2) animal is irradiated: pre-irradiation is first chloral hydrate (200mg/kg) intraperitoneal injection of anesthesia of 10% by concentration expressed in percentage by volume, then mice ventricumbent position is fixed on a horizontal plank, be spaced 5cm between two mices, the head of every mice keeps point-blank.Then mice is placed in radiation therapy bed, is covered in part beyond mouse head with the Corii Sus domestica of 1cm thickness.Radioactive source distance mouse head is regulated to be about 100cm.Regulate radial extent in: after eyes after corner of the eyes line between ears line, left and right dew is empty, and area is about 2*3cm
2, depth of shine is 2cm.Radiation adopts single fraction irradiation, illuminate condition: with 6Mev/min, and accumulated dose is: 30Gy/ only.Matched group brings radiotherapy room into, but radiotherapy under not being in radioactive source, and animal was pre-irradiation 60 minutes, and pharmaceutical intervention group lumbar injection BBG1mg (50mg/kg.d. is only), injects 7 days afterwards more continuously.Matched group and irradiation group give the normal saline lumbar injection of consubstantiality accumulated amount.
(3) performance of mice radiation rear behavioristics is observed: observation mice irradiates rear autonomic activities situation, the feed water yield, hair and body weight change.
(4) draw materials: after radiation the 3rd day respectively, 7th day time point, the 7th day after irradiation, with 10% chloral hydrate (200mg/kg) intraperitoneal injection of anesthesia, open breast chamber, fully free exposure heart, puncture in left ventricle with 7 number sword-shaped needles, in right auricle, place cuts off right atrium, then through left ventricle ice normal saline drop heart perfusion, until mouse liver gill edge, after the peripheral circulation places such as lip lose color, about 15-20ml is slowly poured into again stiff to mouse systemic with 4% paraformaldehyde, broken end is opened cranium and is got brain, with 4% paraformaldehyde, cerebral tissue is stored in 4 degree of refrigerator overnight, re-use 10%, 20%, 30% gradient sucrose liquid serial dehydration process, when being sunken to bottom vessel to mice at every turn, use the sucrose liquid of higher concentration instead.Get 0.5cm cerebral tissue specimen OCT before and after optic chiasma and embed rear row frozen section.Slice thickness is 10um.Section is stored in-30 degree refrigerators for subsequent use.
(5) Q-PCR method and westernblot detect the expression of Mice brain tissues P2X7 receptor, COX-2, TNF-α, IL-6, beta-actinmRNA.The method that Trizol method extracts total serum IgE, reverse transcription synthesis cDNA and Q-PCR detects is as described in amputated body parts.Western-Blot detects the expression of Mice brain tissues P2X7 receptor and COX-2 albumen.Histogenic immunity fluorescence method is observed and is irradiated rear mouse brain cortex microglia P2X7 expression of receptor change and Activated Microglia situation.
Result is as shown in Figure 12 to Figure 20, and in zoopery: in the Brain Radiation Injury model of mice, P2X7 receptor is activating microglia and promoting all to play important effect in the dispose procedure of inflammatory factor.BBG can obviously suppress mice to irradiate the activation rate of cerebral tissue No microglial and the release of inflammatory factor.
As shown in Figure 12,13,14,15, the change of the mRNA level in-site of P2X7 receptor and inflammatory factor COX-2, IL-6 and TNF-a after pre-irradiation.The mRNA level in-site of P2X7 receptor after irradiation 3 days comparatively matched group raise (3.21 ± 0.25 times) (P<0.05), after BBG pretreatment, effectively can lower P2X7 receptor 32.4% (P<0.05).The mRNA of P2X7 receptor reaches maximum in 7 days after irradiation, and comparatively matched group raises (4.91 ± 0.59 times) (P<0.05).14.7% (P<0.05) can be lowered after BBG pretreatment.The change of inflammatory factor mRNA level in-site is also as shown in Figure 5: the mRNA of COX-2 after irradiation 3 days comparatively matched group raise (5.93 ± 0.64 times) (P<0.05), 23.4% (P<0.05) can be suppressed after BBG pretreatment.After irradiation 7 days comparatively matched group raise (7.15 ± 0.15 times) (P<0.05), 29.4% (P<0.05) can be suppressed after BBG pretreatment.The mRNA of IL-6 after irradiation 3 days comparatively matched group raise (3.71 ± 0.52 times) (P<0.05), 31.6% (P>0.05) can be suppressed after BBG pretreatment.After irradiation 7 days comparatively matched group raise (7.37 ± 0.33) doubly (P<0.05).32.1% (P<0.05) can be suppressed after BBG pretreatment.The mRNA of TNF-a after irradiation 3 days comparatively matched group raise (3.58 ± 0.62 times) (P<0.05), BBG pretreatment can not suppress it to express.After irradiation 7 days comparatively matched group raise (5.12 ± 0.49 times) (P<0.05), 45.7% (P<0.05) can be suppressed after BBG pretreatment.
In sum, in body, irradiate the rising of the mRNA of rear P2X7 receptor and proinflammatory inflammation factor COX-2, IL-6 and TNF-a, and elevated-levels and time are proportionate.After BBG pretreatment, 3 days after irradiation, the mRNA level in-site of P2X7 receptor and inflammatory factor COX-2, IL-6 all can be suppressed.7 days after irradiation, the mRNA level in-site of P2X7 receptor and proinflammatory inflammation factor COX-2, IL-6 and TNF-a also can be suppressed by BBG, and inhibition is more remarkable.Can infer thus, the change of proinflammatory inflammation factor mRNA level in-site after mice P2X7 receptor also participates in mediating pre-irradiation.
As shown in Figure 16,17,18, P2X7 receptor protein level after irradiation 3 days comparatively matched group raise (P<0.05), after BBG pretreatment, effectively can lower P2X7 receptor protein level (P<0.05).P2X7 receptor protein level after irradiation 7d reaches maximum, and comparatively matched group raises (P<0.05).Can lower (P<0.05) after BBG pretreatment.Inflammatory factor COX-2 protein level after irradiation 3 days comparatively matched group raise (P<0.05), after BBG pretreatment express decline (P<0.05).After irradiation 7 days comparatively matched group raise (P<0.05), can suppress (P<0.05) after BBG pretreatment.Can infer thus, the change of proinflammatory inflammation factor protein level after mice P2X7 receptor also participates in mediating pre-irradiation.
As shown in figure 19: A, D, G are the iba1 of normal group (microscope magnification is 400 times), P2X7 respectively, overlap; B, E, H are the iba1 of irradiation group (microscope magnification is 400 times), P2X7 respectively, overlap; C, F, I are the iba1 of BBG intervention group (microscope magnification is 400 times), P2X7 respectively, overlap; Wherein overlap and refer to ibal and P2X7 labelling altogether.Get mouse brain after irradiation 7 days frozen sections do histogenic immunity fluorescence.As shown in Figure 2-5, result shows the tranquillization state that the microglia of matched group cortex is mostly branched, and only the microglia of minority by P2X7 institute labelling.Irradiation causes microglial activation, and cell space becomes large and becomes the activated state of justifying into " ameba " sample, and meanwhile, the microglia number of P2X7 labelling comparatively matched group increases (P<0.05).But in pharmaceutical intervention group, most of microglia is converted back into branched tranquillization state, and the microglia number comparatively irradiation group of P2X7 labelling reduces (P<0.05).
As shown in figure 20: with times visual field, fluorescence microscope × 200 for counting criteria, choose 5 visuals field respectively in different disposal group and do mean and standard deviation, calculate the activated microglia rate of P2X7 labelling in different processed group.The activation rate of matched group No microglial is (2.1 ± 0.67) %, and the activation rate of irradiation group No microglial is (37.2 ± 3.9) %, and the activation rate of pharmaceutical intervention group No microglial is (15.4 ± 0.4) %.Result shows, mice P2X7 receptor also participates in mediating the change of form and quantity after microglia pre-irradiation.
The present invention is from vitro and in body two, utilize Protocols in Molecular Biology, inquire into the effect of P2X7 receptor in lonizing radiation activation microglia and proinflammatory inflammation factor dispose procedure, infer that P2X7 receptor may occur in Brain Radiation Injury and play an important role in development.And confirm that BBG can suppress the activation of microglia and the release of inflammatory factor effectively, may Brain Radiation Injury be used for the treatment of.
Finally to should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although be explained in detail the present invention with reference to preferred embodiment; those of ordinary skill in the art is to be understood that; can modify to technical scheme of the present invention or equivalent replacement, and not depart from essence and the scope of technical solution of the present invention.
Claims (2)
- The purposes of 1.P2X7 acceptor inhibitor in the medicine of preparation radiotherapy brain injury.
- 2. purposes according to claim 1, described P2X7 acceptor inhibitor is BBG.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110157668A (en) * | 2019-05-30 | 2019-08-23 | 潍坊医学院 | The effect that CD39 internalization regulation activates P2X7R |
| CN113930436A (en) * | 2021-10-28 | 2022-01-14 | 吉林正业生物制品股份有限公司 | Double-standard-curve rabies virus positive standard plasmid and application thereof in quantitative detection of rabies virus |
-
2015
- 2015-06-04 CN CN201510300864.XA patent/CN105078948A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 唐亚梅等: "P2X7介导小胶质细胞旁分泌功能在放射性脑损伤发病机制中的研究", 《中华医学会第十七次全国神经病学学术会议论文汇编（下）》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110157668A (en) * | 2019-05-30 | 2019-08-23 | 潍坊医学院 | The effect that CD39 internalization regulation activates P2X7R |
| CN110157668B (en) * | 2019-05-30 | 2023-07-14 | 潍坊医学院 | Effect of CD39 internalization modulation on P2X7R activation |
| CN113930436A (en) * | 2021-10-28 | 2022-01-14 | 吉林正业生物制品股份有限公司 | Double-standard-curve rabies virus positive standard plasmid and application thereof in quantitative detection of rabies virus |
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