CN112011509A - Separation method and application of primary microglia of rat with spinal cord injury - Google Patents
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Abstract
The invention discloses a separation method and application of primary microglia of rats with spinal cord injury, wherein rats with spinal cord injury in a short period are used as test raw materials, and microglia with considerable quantity and high purity can be obtained quickly through simple grinding, filtering and other steps; the method greatly shortens the time for extracting the microglia by the traditional method, and provides a basis for dynamically detecting the function of the microglia in vivo in real time; the method overcomes the defect that the traditional microglia purification is difficult and has high process cost, can efficiently and economically obtain the high-purity microglia, and provides a basis for the research of the microglia after spinal cord injury.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to preparation of a biological model, separation, identification and culture technology of functional cells, in particular to a separation method and application of primary microglia of a spinal cord injured rat.
Background
Spinal cord injury, which can cause loss of sensory and motor functions, is one of the diseases in which the central nervous system is seriously damaged, and imposes a heavy economic burden on society and families. Microglia are innate immune cells of the central nervous system that are activated early in spinal cord injury, not only participate in the maturation of the central nervous system, but also directly affect neuronal neogenesis after adulthood. For example, in injured optic nerves or spinal cords, implantation of new microglia or macrophages can significantly improve post-traumatic regeneration of nerve fibers, which provides a new direction for nerve regeneration studies.
At present, a great number of reports about culture methods of newborn mouse cortical microglia exist, wherein the microglia is purified mainly by culturing mixed glial cells and adopting a mild trypsinization method, a layered oscillation method, a density gradient centrifugation method or an immunomagnetic bead sorting technology, and the like, but the literature reports about the separation and purification methods of spinal microglia are few, the culture methods are mostly referred to, the period is about 10 days, and the yield and the purity are not high. In addition, the extraction method of microglia after spinal cord injury is rare, and most of the traditional microglia extraction methods are cells after culturing and proliferating for a period of time (the state is changed), so that a large number of high-purity microglia cannot be obtained in a short time, which is different from the cells in the body, and restricts the mechanism research of the microglia after spinal cord injury.
Disclosure of Invention
The technical problem to be solved is as follows: in order to overcome the defects of the prior art and obtain high-purity microglia in a short time after spinal cord injury by a simple grinding and filtering method, the invention provides a method for separating primary microglia of rats with spinal cord injury and application thereof.
The technical scheme is as follows: a method for isolating primary microglia from spinal cord injured rats, the method comprising the steps of:
step 1, preparation of spinal cord injury rat model
a. Using SD rats, measuring body weight, performing intraperitoneal anesthesia, shaving the back rat hair, spreading a towel and sterilizing with iodophor, using sterile instruments to open the skin, isolate the muscle fascia, bite the vertebral lamina and expose the spinal cord intact;
b. adopting a spinal cord impactor to strike the spinal cord, then suturing the skin, disinfecting with iodophor after operation, urinating regularly and keeping warm;
step 2, isolation and culture of microglia
c. C, killing the rat model bred for 1-14 days in the step b, taking out the spinal cord of the damaged area by using a sterile instrument after disinfection, rinsing, and placing in a DMEM/F12(HAM) complete culture medium at 0 ℃ to strip and remove the meninges and blood vessels;
d. placing the spinal cord on a filter screen, washing the spinal cord in a centrifuge tube by adopting a DMEM/F12(HAM) complete culture medium after grinding, and discarding and resuspending a supernatant after centrifuging;
e. inoculation of resuspended cells from step d, 37 ℃, 5% CO2Changing the culture solution after incubation in the incubator;
step 3, identification of microglia
f. And e, taking the cells cultured in the step e, and identifying the cells as microglia by adopting a CD11b immunofluorescence staining method.
Preferably, the T10 spinal segment is exposed intact in step a.
Preferably, the spinal cord impactor is used for impacting T10 spinal cords in the step b, the falling mass and the diameter are respectively 10g and 2.5mm, and the falling height and the falling depth are respectively 10cm and 2 mm.
Preferably, the spinal cord injury rat model after 3 days of feeding is sacrificed in step c.
Preferably, the complete medium is formulated in 89% DMEM/F12(HAM), 10% fetal bovine serum, 1% 100U/mL streptomycin.
Preferably, the size of the mesh in step d is 40 μm, and the spinal cord is ground using the tail of the syringe.
Preferably, the cells are incubated for 4h in step e and then cultured in medium.
Preferably, the purity of the microglia identified by the CD11b immunofluorescence staining method in the step f is more than 98%.
The method for separating the primary microglia of the rat with the spinal cord injury is applied to obtaining the high-purity microglia within a short time of the spinal cord injury.
Preferably, the short time is 2-3 days after spinal cord injury, and the high purity is more than 98%.
The design idea/principle of the primary microglia cell separation method for the rat with spinal cord injury provided by the invention is as follows: microglia are gathered and infiltrated into the spinal cord injury area 2-3 days after spinal cord injury, and the microglia with high quantity and high purity are obtained through a simple grinding and filtering step in the period. The microglia seed plate obtained by the method still keeps the shape of somatic cells, which provides a foundation for researching the mechanism of microglia after spinal cord injury.
Has the advantages that: (1) according to the method, rats with spinal cord injury in a short period are used as test raw materials, and microglia with considerable quantity and high purity can be obtained quickly through simple steps of grinding, filtering and the like; the method greatly shortens the time for extracting the microglia by the traditional method, and provides a basis for dynamically detecting the function of the microglia in vivo in real time; (2) the method overcomes the defect that the traditional microglia purification is difficult and has high process cost, can efficiently and economically obtain the high-purity microglia, and provides a basis for the research of the microglia after spinal cord injury.
Drawings
FIG. 1 is a view of T10 spinal cord of a rat hit by a spinal cord impactor;
FIG. 2 is a morphological feature map of microglia, wherein a is a morphological feature map under a 100-fold mirror and b is a morphological feature map under a 200-fold mirror;
FIG. 3 is a graph showing the results of immunofluorescence assay of the surface antigen CD11b of microglia, wherein a is surface antigen CD11b staining (CD11b green), b is nuclear staining (dapi blue), and c is pool (merge);
fig. 4 is a morphological feature and a histogram of the number of microglia extracted at different time periods after spinal cord injury, wherein a is normal group, i.e. spinal cord was not injured, b is 1 day after spinal cord injury, c is 3 days after spinal cord injury, d is 7 days after spinal cord injury, and e is 14 days after spinal cord injury;
figure 5 is a histogram of the number of microglia extracted at different time periods after spinal cord injury, where P <0.05 for 3d versus 7 d;
figure 6 is a graph of the morphological characteristics of microglia 3 days after spinal cord injury, where a is 1 day after plating, b is 2 days after plating, and c is 4 days after plating.
Detailed Description
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and substance of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1
1. Preparation of spinal cord injury rat model
The body weights of 180-200g SPF grade SD rats were measured and anesthetized intraperitoneally with 1% sodium pentobarbital (50 mg/kg); after anaesthesia for 5-10min, the rat was shaved off on the back, placed on a sterilized rat plate, covered with sterile gloves, draped and the skin on the back was sterilized with iodophor.
The skin is incised by taking the T10 spinous process as the incision center, the incision size is 3cm, the fascia and the dorsiflexor quadratus are separated in a blunt manner, a scalpel is tightly attached to the T9-11 vertebral body spinous process to separate the paraspinous muscle group, the T9-11 vertebral plate is exposed, then the two sides of the T9 vertebral body are clamped, a rat is lifted, the T10-11 ligament is cut off by using an elbow shear, the T10 vertebral plate is cut off along the two sides of the vertebral plate, the T10 vertebral plate is lifted upwards, and the T10 spinal cord is exposed.
A spinal cord impactor is used for impacting a T10 spinal cord, as shown in figure 1, the mass and the diameter of a falling object are 10g and 2.5mm respectively, the falling height and the falling depth are 10cm and 2mm respectively, and the tension of the two hind limbs of a rat is eliminated immediately, so that the model is successfully established, and the wound surface is disinfected after the wound is sutured layer by layer. After operation, the wound and the urethral orifice are disinfected by iodophor, and the patient is massaged to urinate and is kept warm regularly.
2. Isolation of microglia
The normal group, 1 day, 3 days, 7 days, and 14 days later, the spinal cord injury model was anesthetized, decapitated and sacrificed, the model was sterilized in 75% ethanol for 30s, the rat was placed in a sterilized clean bench, the rat was cut along the old wound with a sterile scalpel, the upper and lower vertebral bodies were cut with scissors using T10 as the center, the spinal cord of 2-3cm of the injured area was removed, and the spinal cord was rinsed with PBS and then temporarily stored in a 0 ℃ DMEM/F12(HAM) + 10% fetal bovine serum + 1% streptomycin medium in a 6-well plate.
Rapidly stripping and removing a spinal membrane and a blood vessel by using micro-forceps, replacing the culture medium, placing the spinal cord on a 40-micron filter screen by using a 1ml gun head, grinding the tissue for about 30s by using the tail part of a 20ml syringe, sucking the culture medium, washing the culture medium in a 50ml centrifuge tube, centrifuging the culture medium at 1000rpm for 5min, then removing the supernatant, and re-suspending the culture medium;
the resuspended cells were inoculated into coated 6-well plates (6-well plates were coated with 0.01% polylysine for 4h the day before, washed twice with PBS and dried for use), incubated at 37 ℃ with 5% CO2After incubation for 4h in the incubator, the cell morphology was observed by changing the medium, and it was seen under the microscope that the cell morphology was circular and grew like an adherent, as shown in fig. 2. After 3 days of spinal cord injury in rats, the number of microglia isolated was the greatest after statistical analysis, as shown in fig. 4 and 5.
3. Identification and culture of microglia
The cultured cells were taken and the microglia were identified by CD11b immunofluorescence staining. The specific operation is as follows:
the 6-well plate was removed, aspirated off the medium and washed 3 times with PBS, 5min each time, fixed with 4% paraformaldehyde for 10min, permeabilized with 0.2% Triton-100 for 10min, and washed 3 times with PBS, 5min each time. Blocking with 5% goat serum at room temperature for 60min, adding anti-CD 11b (1:300), and standing overnight at 4 deg.C. Washed 3 times with PBS for 5min each, added with secondary Alexa 488, incubated for 1h at room temperature in the dark, and washed 3 times with PBS. Dapi was counterstained and washed with PBS, and after dropping an anti-fluorescence quencher, the purity was determined to be 98% by observation under a fluorescence inverted microscope, as shown in fig. 3.
Taking the microglia cultured 3 days after the spinal cord injury, continuously culturing for 1 day, 2 days and 4 days, and observing under a microscope to find that the microglia is in a round shape and has a bigger cell body and an amoeba shape when being in the seed plate for 1 day; at 2 and 4 days on the plate, the cell branches gradually increased and the cell bodies gradually decreased, as shown in FIG. 6.
In conclusion, microglia with considerable quantity and high purity can be efficiently and economically obtained by the method in the embodiment 1, and a foundation is provided for the research of the microglia after spinal cord injury.
Claims (10)
1. A method for isolating primary microglia of a spinal cord injury rat, which is characterized by comprising the following steps:
step 1, preparation of spinal cord injury rat model
a. Using SD rats, measuring body weight, performing intraperitoneal anesthesia, shaving the back rat hair, spreading a towel and sterilizing with iodophor, using sterile instruments to open the skin, isolate the muscle fascia, bite the vertebral lamina and expose the spinal cord intact;
b. adopting a spinal cord impactor to strike the spinal cord, then suturing the skin, disinfecting with iodophor after operation, urinating regularly and keeping warm;
step 2, isolation and culture of microglia
c. C, killing the rat model bred for 1-14 days in the step b, taking out the spinal cord of the damaged area by using a sterile instrument after disinfection, rinsing, and placing in a DMEM/F12(HAM) complete culture medium at 0 ℃ to strip and remove the meninges and blood vessels;
d. placing the spinal cord on a filter screen, washing the spinal cord in a centrifuge tube by adopting a DMEM/F12(HAM) complete culture medium after grinding, and discarding and resuspending a supernatant after centrifuging;
e. inoculation of resuspended cells from step d, 37 ℃, 5% CO2Changing the culture solution after incubation in the incubator;
step 3, identification of microglia
f. And e, taking the cells cultured in the step e, and identifying the cells as microglia by adopting a CD11b immunofluorescence staining method.
2. The method for isolation of spinal cord injured rat primary microglia according to claim 1, wherein the T10 spinal segment is exposed completely in step a.
3. The method for isolating spinal cord injured rat primary microglia according to claim 1, wherein the spinal cord striker is used to strike T10 spinal cords in step b, the mass and diameter of the drop are 10g and 2.5mm, respectively, and the height and depth of the drop are 10cm and 2mm, respectively.
4. The method for isolating spinal cord-injured rat primary microglia according to claim 1, wherein in step c the spinal cord-injured rat model after feeding for 3 days is sacrificed.
5. The method for isolating spinal cord-injured rat primary microglia according to claim 1, wherein the complete medium is formulated with 89% DMEM/F12(HAM), 10% fetal bovine serum, and 1% 100U/mL streptomycin.
6. The method for isolating primary microglia in spinal cord injured rats according to claim 1, wherein the pore size of the filter net in step d is 40 μm, and the spinal cord is ground by using the tail of the syringe.
7. The method for isolating spinal cord injured rat primary microglia according to claim 1, wherein the cells are incubated for 4h in step e and then cultured in liquid change.
8. The method for isolating primary microglia in spinal cord injured rats according to claim 1, wherein the purity of the microglia is more than 98% as identified by CD11b immunofluorescence staining in step f.
9. Use of the method for isolation of spinal cord injured rat primary microglia according to any one of claims 1-8 for obtaining high purity microglia within a short time of spinal cord injury.
10. The use according to claim 9, wherein the short period of time is 2-3 days after spinal cord injury, and the high purity is 98% or more.
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| CN114561355A (en) * | 2022-01-23 | 2022-05-31 | 四川大学华西医院 | Acute and rapid separation method for spinal cord scar tissue cells |
| CN114561355B (en) * | 2022-01-23 | 2023-04-11 | 四川大学华西医院 | Acute and rapid separation method for spinal cord scar tissue cells |
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