CN109182270A - A kind of acusector induction abducent nerve M2 type microglia polarization method - Google Patents
A kind of acusector induction abducent nerve M2 type microglia polarization method Download PDFInfo
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Abstract
The invention discloses a kind of acusectors to induce abducent nerve M2 type microglia polarization method, is randomly divided into sham-operation group A, neurotrosis group B, acusector processing group C and AM1241 group D.4 groups of experimental animals are treated 2 weeks and carry out the expression that Western Blot detects abducent nerve microglia M1, M2 type marker molecule in the 7th day, the 14th day materials, further with immunofluorescence analysis Arginase expression the case where, and statistical difference analysis is carried out, acusector of the invention can induce the polarization of Beagle dog abducent nerve M2 type microglia to promote injuring nerve reparation.2 receptor agonist AM1241 of cannboid can play good acusector synergistic effect simultaneously, but AM1241 may not influence the up-regulation of microglia M2 phenotypic marker protein expression.
Description
Technical field
The present invention relates to nerve injury model technical field, specially a kind of acusector induces abducent nerve M2 type microglia
Polarization method.
Background technique
The abducent nerve eye movement nerve complicated as walking, adjoins many important features, such as Dorello pipe, cavernous sinus, socket of the eye
On split, and with the close relations such as internal carotid, sympathetic nerve, Gruber ligament.When intracranial infection, occupy-place, wound merge cranium
Internal pressure increases to be easy to damage often, and musculus rectus lateralis paralysis is caused eyeball esotropia occur, and outreach is limited.It is domestic at present to carry out abducent nerve
Injury repair relevant report is less.And safely and effectively repairing of neural injury mechanism is explored, it is by point of penetration of microglia
In recent years one of the hot spot in cell research field.It is considered that electroacupuncture stimulation can be obviously promoted the exhibition mind of damage in early-stage study
It is recovered.The present invention, which continues through, establishes Beagle dog abducent nerve damage model, in conjunction with 2 receptor agonist AM1241 of cannboid
Pretreatment, inquire into acusector promote abducent nerve repair whether to promote abducent nerve microglia it is related to the polarization of M2 type, progress
Related mechanism preliminary analysis.
As traditional Chinese medicine-electroacupuncture treatment by mitigating damaged cell inflammatory reaction, inhibition nerve cell apoptosis, changing
Kind metabolism microenvironment, adjusts inflammatory factor release, to promote nerve regneration, block cascade of response of inflammation.Research is main at present
It concentrates on facial nerve and sciatic nerve and injury of oculomotor nerve repairs aspect.There is document report to utilize electroacupuncture stimulation abducent nerve simultaneously
Paralysis also achieves preferable curative effect, but pierces about needle and promote the research of abducent nerve repair mechanism less.
Summary of the invention
The purpose of the present invention is to provide a kind of acusectors to induce abducent nerve M2 type microglia polarization method, on solving
State the problem of proposing in background technique.
To achieve the above object, the invention provides the following technical scheme: a kind of acusector induction small colloid of abducent nerve M2 type is thin
Born of the same parents' polarization method, the following steps are included:
A, the foundation of abducent nerve damage model；
B, electroacupuncture stimulation；
C, Western Blot detects M1, M2 developed by molecule of microglia；
D, immunofluorescence analysis；
E, it statisticallys analyze.
Preferably, in the step A specifically: inject 3%, 1mL/kg yellow Jackets vein fiber crops after preoperative 12h fasting
It is liquor-saturated, Beagle dog is placed in operating table and takes lateral position, fixed experimental animal head, ear edge caudiduct is fixed, and anesthesia is abundant
Flap turns over outward after opening cranium, through longitudinally slit at temporalis attachment, opens cranium through temporo bottom approach, bone window expands as close as possible to basis cranii
Bone window range after socket of the eye to before zygomatic arch, and along temporal bone window lower edge cut endocranium along ramp up foreign side be a wound 3.5cm ×
3.5cm；Inwardly, drawing temporal lobe in top retracts brain tissue, and the abducent nerve in deep is partially exposed before removing petrous bone；With haemostatic clamp with teeth
It squeezes folder and closes after abducent nerve 30s that tissue is poor at direct-view pincers pressure, cut off abducent nerve aixs cylinder but maintain the complete property of epilemma, with dog eye
Ball internally oblique is depending on showing modeling success；It is divided into four groups of experimental groups: sham-operation group A: only exposes and separate abducent nerve, without mind in art
It is handled through damage, skin of sewing up the incision is fettered in electroacupuncture stimulation；Damage group B: production abducent nerve damage model, in electricity
It is fettered when needle stimulus；Continuous 2 weeks progress electroacupuncture stimulations after acusector processing group C:Beagle dog abducent nerve Establishing an injured model；
AM1241 group D: abducent nerve damage+CB2R agonist AM1241, solvent 3ml/kg, which is given within continuous 2 weeks, after abducent nerve damage carries out abdomen
Chamber injection.
Preferably, acusector processing group C abducent nerve damage model sets 2 electrodes after completing respectively in the step B
Enter neural injury region two sides, pull upper palpebra inferior outward, musculus rectus lateralis is sufficiently exposed in tarsus and bulbar conjunctiva intersection, by electrode
It is inserted into musculus rectus lateralis, implantation depth about 3.0mm, spacing about 4.0mm, fixed electrode of sewing up the incision, free-end is from the eye socket back lower place
It draws and fixes, forming circuit gives electroacupuncture stimulation in postoperative continuous 2 weeks, and slightly being beated with local muscle is advisable, and continues every time
15min。
Preferably, it takes above-mentioned Beagle dog to be respectively grouped every group 6 in the step C, carries out depth with 10% chloraldurate
After anesthesia, abducent nerve segment is cut with operating scissors rapidly, is operated on cryogenic sterile ice pan, a little nerve fiber is isolated
It is placed in the spherical position of 1ml homogenizer, sufficiently shreds tissue as far as possible；The mono- Detergent Lysis liquid of 400 μ L containing PMSF is added and splits progress
It is homogenized, 12000rpm is centrifuged 10min at 4 DEG C after cracking 30min, and sorting device is taken to save in -20 DEG C；It is thin to extract small colloid
The total protein of born of the same parents, it is quantitative by NanoDrop, take 40ug albumen to carry out SDS- polyacrylamide gel electrophoresis, by albumen with constant
Electric current 30mA, electricity turn 90min and are transferred on pvdf membrane, and TBST washes film 3 times, then the speed with 60rpm of 5%TBST skimmed milk power
Degree closing 60min is added 4 DEG C of refrigerators of primary antibody and is incubated overnight, and TBST is rinsed 3 times after the completion of primary antibody is incubated for, and then moves to new hybridization bag
In, it is incubated for 60min with fluorescent marker secondary antibody room temperature, TBST is developed and taken pictures with ECL method after sufficiently cleaning, and carries out band gray scale point
Analysis.
Preferably, slice 2h is placed after each slice dries at room temperature in the step D to be put into film development box,
3 5min are washed in PBS, are then placed in mounting box, are dried substantially to moisture, using containing 5% donkey serum and 0.2%teeen-
20PBS closing, 37 DEG C are crossed 1h, are then respectively adding the anti-CD11b Identification of Monoclonal Antibodies microglia of mouse, rabbit-anti Arginase
Polyclonal antibody identifies microglia M2 type, stays overnight rear PBS for 4 DEG C and washs 3 5min, it is glimmering to add the combination of Dylight 594
The anti-mouse IgG and Dylight 488 of signal combines the anti goat igg of fluorescent marker, and 4 DEG C are protected from light incubation 2h, and incubation finishes
3 5min are washed using PBS afterwards, mounting is protected from light using anti-fluorescence decay, using confocal microscopy and takes a picture.
Preferably, for statistical analysis to data with SPSS 19.0 in the step E, microglia M1, M2
Type marker molecule expression indicates that component compares to be examined using t with mean ± standard deviation, and enumeration data uses χ2It examines, with P
< 0.05 is statistically significant for difference.
Preferably, the concentration of the primary antibody: IL-1 β is 1:200, iNOS 1:3000, Arginase 1:1000, BDNF
For 1:200.
Preferably, electroacupuncture stimulation pulse frequency is 5Hz, time-histories 0.1ms, intensity 1.0V.
Compared with prior art, the beneficial effects of the present invention are: acusector of the invention can induce Beagle dog abducent nerve
The polarization of M2 type microglia promotes injuring nerve reparation.2 receptor agonist AM1241 of cannboid can be played well simultaneously
Acusector synergistic effect, but AM1241 may not influence the up-regulation of microglia M2 phenotypic marker protein expression.
Detailed description of the invention
Fig. 1 is flow chart of the present invention；
Fig. 2 is each group labelled protein Western blot electrophoretogram in the present invention；
Fig. 3 is each group histogram of the present invention；
Fig. 4 is Arginase immunofluorescence of the present invention.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Referring to Fig. 1, the present invention provides a kind of technical solution: the invention provides the following technical scheme: a kind of acusector induces
Abducent nerve M2 type microglia polarization method, the following steps are included:
A, the foundation of abducent nerve damage model；
B, electroacupuncture stimulation；
C, Western Blot detects M1, M2 developed by molecule of microglia；
D, immunofluorescence analysis；
E, it statisticallys analyze.
In the present invention, in step A specifically: 3%, 1mL/kg Nembutal vein anesthetic are injected after preoperative 12h fasting,
Beagle dog is placed in operating table and takes lateral position, fixed experimental animal head, ear edge caudiduct is fixed, and cranium is sufficiently opened in anesthesia
Flap turns over outward afterwards, through longitudinally slit at temporalis attachment, opens cranium through temporo bottom approach, bone window expands bone window as close as possible to basis cranii
Range after socket of the eye to before zygomatic arch, and along temporal bone window lower edge cut endocranium along ramp up foreign side be a wound 3.5cm ×
3.5cm；Inwardly, drawing temporal lobe in top retracts brain tissue, and the abducent nerve in deep is partially exposed before removing petrous bone；With haemostatic clamp with teeth
It squeezes folder and closes after abducent nerve 30s that tissue is poor at direct-view pincers pressure, cut off abducent nerve aixs cylinder but maintain the complete property of epilemma, with dog eye
Ball internally oblique is depending on showing modeling success；It is divided into four groups of experimental groups: sham-operation group A: only exposes and separate abducent nerve, without mind in art
It is handled through damage, skin of sewing up the incision is fettered in electroacupuncture stimulation；Damage group B: production abducent nerve damage model, in electricity
It is fettered when needle stimulus；Continuous 2 weeks progress electroacupuncture stimulations after acusector processing group C:Beagle dog abducent nerve Establishing an injured model；
AM1241 group D: abducent nerve damage+CB2R agonist AM1241, solvent 3ml/kg, which is given within continuous 2 weeks, after abducent nerve damage carries out abdomen
Chamber injection.
In the present invention, 2 electrodes are respectively implanted by acusector processing group C abducent nerve damage model after completing in step B
Two sides at neurotrosis pull upper palpebra inferior outward, sufficiently expose musculus rectus lateralis in tarsus and bulbar conjunctiva intersection, and electrode is equal
Insertion musculus rectus lateralis, implantation depth about 3.0mm, spacing about 4.0mm, fixed electrode of sewing up the incision, free-end draw from the eye socket back lower place
It fixes out, is formed into a loop, gives electroacupuncture stimulation within postoperative continuous 2 weeks, slightly being beated with local muscle is advisable, every time lasting 15min,
Electroacupuncture stimulation pulse frequency is 5Hz, time-histories 0.1ms, intensity 1.0V.
In the present invention, above-mentioned Beagle dog is taken respectively to be grouped every group 6 in step C, carries out depth fiber crops with 10% chloraldurate
After liquor-saturated, abducent nerve segment is cut with operating scissors rapidly, is operated on cryogenic sterile ice pan, a little nerve fiber is isolated and sets
In the spherical position of 1ml homogenizer, tissue is sufficiently shredded as far as possible；It is even that 400 μ Ls mono- Detergent Lysis liquid of the addition containing PMSF splits progress
It starches, 12000rpm is centrifuged 10min at 4 DEG C after cracking 30min, and sorting device is taken to save in -20 DEG C；Extract microglia
Total protein, it is quantitative by NanoDrop, take 40ug albumen to carry out SDS- polyacrylamide gel electrophoresis, by albumen with constant electricity
30mA is flowed, electricity turns 90min and be transferred on pvdf membrane, and TBST washes film 3 times, then the speed with 60rpm of 5%TBST skimmed milk power
60min to be closed, 4 DEG C of refrigerators of primary antibody are added and are incubated overnight, the concentration of primary antibody: IL-1 β is 1:200, iNOS 1:3000,
Arginase is 1:1000, BDNF 1:200；TBST is rinsed 3 times after the completion of primary antibody is incubated for, and is then moved in new hybridization bag, and glimmering
Signal secondary antibody room temperature is incubated for 60min, and TBST is developed and taken pictures with ECL method after sufficiently cleaning, and carries out band gray analysis.
In the present invention, in step D slice is placed into 2h after each slice dries at room temperature and be put into film development box,
3 5min are washed in PBS, are then placed in mounting box, are dried substantially to moisture, using containing 5% donkey serum and 0.2%teeen-
20PBS closing, 37 DEG C are crossed 1h, are then respectively adding the anti-CD11b Identification of Monoclonal Antibodies microglia of mouse, rabbit-anti Arginase
Polyclonal antibody identifies microglia M2 type, stays overnight rear PBS for 4 DEG C and washs 3 5min, it is glimmering to add the combination of Dylight 594
The anti-mouse IgG and Dylight 488 of signal combines the anti goat igg of fluorescent marker, and 4 DEG C are protected from light incubation 2h, and incubation finishes
3 5min are washed using PBS afterwards, mounting is protected from light using anti-fluorescence decay, using confocal microscopy and takes a picture.
It is for statistical analysis to data with SPSS 19.0 in step E in the present invention, microglia M1, M2 type
Marker molecule expression indicates that component compares to be examined using t with mean ± standard deviation, and enumeration data uses χ2Examine, with P <
0.05 is statistically significant for difference.
Interpretation of result:
Compare the variation of acusector with Activated Microglia state after CB2R agonist AM1241: 4 groups of experimental animals are controlled
It treats 2 weeks and carries out Western Blot detection abducent nerve microglia M1, M2 type marker molecule in the 7th day, the 14th day materials
Expression compares.
The comparison of microglia M1 phenotype: 4 groups detect M1 specific marker proteins (iNOS, IL-1 β), C group warp
The expression of iNOS after electroacupuncture treatment reduces (P < 0.05) compared with B group；And D group through agonist AM1241 processing after compared with B group iNOS table
Up to being substantially reduced (P<0.05), while C group and D group no difference of science of statistics (P>0.05)；In addition the expression of the IL-1 β of C group, D group
Decline (P < 0.05) compared with B group, D group IL-1 β expression no difference of science of statistics compared with C group after agonist AM1241 processing
(P > 0.05), as shown in the table:
The comparison of microglia M2 phenotype: M2 specific marker proteins (Arginase, BDNF) are detected at 4 groups
Out, compared with B group, C group, D group Arginase expression increase (P < 0.05)；In addition the expression of the BDNF of C group, D group and B group ratio
More increase (P<0.05), BDNF expression is compared with C group no difference of science of statistics (P>0.05) after agonist AM1241 processing for D group, such as
Shown in following table:
It is obtained by two above table: compared with C group, D group the 7th day, the 14th day iNOS, IL-1 β after AM1241 processing
Comparison is increased slightly, but is not statistically significant (P > 0.05)；And the C group for passing through acusector processing compares D group the 7th day, the 14th
Its Arginase, BDNF comparison are increased slightly, but also without statistical significance (P > 0.05).
The expression of Activated Microglia state: the 14th day microglia M1 type marker molecule iNOS after electroacupuncture stimulation,
IL-1 β expression is reduced, and microglia M2 type marker molecule Arginase, BDNF expression increase.And D group has similar change
Change trend, the character mutation that CB2R agonist AM1241 can simulate electroacupuncture treatment microglia is horizontal, as in Figure 2-4.
In conclusion acusector of the invention can induce the polarization of Beagle dog abducent nerve M2 type microglia to promote damage
Neural restoration.2 receptor agonist AM1241 of cannboid can play good acusector synergistic effect simultaneously, but AM1241 may
The up-regulation of microglia M2 phenotypic marker protein expression is not influenced.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (8)
1. a kind of acusector induces abducent nerve M2 type microglia polarization method, it is characterised in that: the following steps are included:
A, the foundation of abducent nerve damage model；
B, electroacupuncture stimulation；
C, Western Blot detects M1, M2 developed by molecule of microglia；
D, immunofluorescence analysis；
E, it statisticallys analyze.
2. a kind of acusector according to claim 1 induces abducent nerve M2 type microglia polarization method, it is characterised in that:
In the step A specifically: inject 3%, 1mL/kg Nembutal vein anesthetic after preoperative 12h fasting, Beagle dog is set
Lateral position, fixed experimental animal head are taken in operating table, ear edge caudiduct is fixed, and flap turns over outward after cranium is sufficiently opened in anesthesia
Cranium is opened through temporo bottom approach through longitudinally slit at temporalis attachment in side, bone window as close as possible to basis cranii, expand bone window range after socket of the eye to
Before zygomatic arch, and foreign side is ramped up along temporal bone window lower edge incision endocranium edge and is a wound 3.5cm × 3.5cm；Inwardly, top is led
It draws temporal lobe to retract brain tissue, the abducent nerve in deep is partially exposed before removing petrous bone；Folder, which is squeezed, with haemostatic clamp with teeth closes abducent nerve 30s
Tissue is poor at direct-view pincers pressure afterwards, cuts off abducent nerve aixs cylinder but maintains the complete property of epilemma, is made with dog eyeball internally oblique depending on showing
Mould success；Be divided into four groups of experimental groups: sham-operation group A: only exposure separates abducent nerve, handles in art without neurotrosis, suture
Notch skin is fettered in electroacupuncture stimulation；Damage group B: production abducent nerve damage model carries out beam in electroacupuncture stimulation
It ties up；Continuous 2 weeks progress electroacupuncture stimulations after acusector processing group C:Beagle dog abducent nerve Establishing an injured model；AM1241 group D: exhibition mind
Through damage+CB2R agonist AM1241, solvent 3ml/kg is given within continuous 2 weeks after abducent nerve damage and is injected intraperitoneally.
3. a kind of acusector according to claim 1 induces abducent nerve M2 type microglia polarization method, it is characterised in that:
2 electrodes are respectively implanted two at neurotrosis by acusector processing group C abducent nerve damage model after completing in the step B
Side pulls upper palpebra inferior outward, sufficiently exposes musculus rectus lateralis in tarsus and bulbar conjunctiva intersection, electrode is inserted into musculus rectus lateralis,
Implantation depth about 3.0mm, spacing about 4.0mm, fixed electrode of sewing up the incision, free-end are drawn from the eye socket back lower place and are fixed, and are formed
Electroacupuncture stimulation is given in postoperative continuous 2 weeks in circuit, and slightly being beated with local muscle is advisable, every time lasting 15min.
4. a kind of acusector according to claim 1 induces abducent nerve M2 type microglia polarization method, it is characterised in that:
It takes above-mentioned Beagle dog to be respectively grouped every group 6 in the step C, after carrying out deep anaesthesia with 10% chloraldurate, uses hand rapidly
Art cuts abducent nerve segment, is operated on cryogenic sterile ice pan, isolates a little nerve fiber and is placed in 1ml homogenizer ball
Shape position, sufficiently shreds tissue as far as possible；400 μ Ls mono- Detergent Lysis liquid of the addition containing PMSF, which is split, to be homogenized, after cracking 30min
12000rpm is centrifuged 10min at 4 DEG C, and sorting device is taken to save in -20 DEG C；The total protein for extracting microglia, passes through
NanoDrop is quantitative, and 40ug albumen is taken to carry out SDS- polyacrylamide gel electrophoresis, and by albumen with constant current 30mA, electricity turns
90min is transferred on pvdf membrane, and TBST is washed film 3 times, and then the speed with 60rpm of 5%TBST skimmed milk power closes 60min,
4 DEG C of refrigerators of primary antibody are added to be incubated overnight, TBST is rinsed 3 times after the completion of primary antibody is incubated for, and is then moved in new hybridization bag, with fluorescent marker
Secondary antibody room temperature is incubated for 60min, and TBST is developed and taken pictures with ECL method after sufficiently cleaning, and carries out band gray analysis.
5. a kind of acusector according to claim 1 induces abducent nerve M2 type microglia polarization method, it is characterised in that:
Slice 2h is placed after each slice dries at room temperature in the step D to be put into film development box, wash 3 times in PBS
5min is then placed in mounting box, is dried substantially to moisture, is closed using containing 5% donkey serum and 0.2%teeen-20PBS, 37
DEG C 1h is crossed, is then respectively adding the anti-CD11b Identification of Monoclonal Antibodies microglia of mouse, rabbit-anti Arginase polyclonal antibody mirror
Determine microglia M2 type, stays overnight rear PBS for 4 DEG C and wash 3 5min, add Dylight 594 and combine resisting for fluorescent marker small
Mouse IgG and Dylight 488 combines the anti goat igg of fluorescent marker, and 4 DEG C are protected from light incubation 2h, is washed after incubation using PBS
3 5min are protected from light mounting using anti-fluorescence decay, using confocal microscopy and take a picture.
6. a kind of acusector according to claim 1 induces abducent nerve M2 type microglia polarization method, it is characterised in that:
It is for statistical analysis to data with SPSS 19.0 in the step E, the expression of microglia M1, M2 type marker molecule
Horizontal mean ± standard deviation indicates that component compares to be examined using t, and enumeration data uses χ2It examines, has with P < 0.05 for difference
Statistical significance.
7. a kind of acusector according to claim 4 induces abducent nerve M2 type microglia polarization method, it is characterised in that:
The concentration of the primary antibody: IL-1 β is 1:200, iNOS 1:3000, Arginase 1:1000, BDNF 1:200.
8. a kind of acusector according to claim 3 induces abducent nerve M2 type microglia polarization method, it is characterised in that:
Electroacupuncture stimulation pulse frequency is 5Hz, time-histories 0.1ms, intensity 1.0V.
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- 2018-09-14 CN CN201811073516.3A patent/CN109182270A/en active Pending
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| CN201091746Y (en) * | 2007-05-10 | 2008-07-30 | 中国人民解放军第四军医大学 | Electroacupuncture stimulation nerve injury protecting device |
| CN101332296A (en) * | 2008-06-13 | 2008-12-31 | 中国人民解放军第三军医大学第三附属医院 | Dendritic cell vaccine for promoting spinal cord injury function recovery |
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Application publication date: 20190111 |