CN105695410B - A kind of naked mole microglia cultural method - Google Patents

Abstract

The present invention relates to technical field of cell biology, in particular to a kind of naked mole microglia isolate and purify and cultural method.The present invention is comprehensive to separate from newborn naked mole cerebral cortex using a variety of culture mediums and purifies culture microglia, has found out the reasonable cultural method of the naked mole microglia of rodent mammal suitable for alternating temperature.The method of the present invention being capable of the normal naked mole microglia of a large amount of functional activities of acquisition easy, efficiently, economic, culture under hypoxia condition can guarantee that this cell is still able to maintain the biological characteristics under body state in ex vivo environment, consequently facilitating the special physiological function of naked mole microglia is directly further studied in pure in-vitro cell culture model, to provide important theoretical foundation to explore biological mechanism therein and being applied to clinical relevant.

Description

A kind of naked mole microglia cultural method

Technical field:

The present invention relates to technical field of cell biology, and in particular to a kind of cell isolate and purify and cultural method, it is special Be not related to a kind of naked mole microglia isolate and purify and cultural method.

Background technique:

Microglia (Microglia) is the important component of central nervous system immune function, in central nervous system It is played an important role in the adjusting of stable state.Microglia pays close attention to the microenvironment locating for it, will once being stimulated It activates.The study found that in central nervous systems diseases such as senile dementia, multiple sclerosis, amyotrophic lateral sclerosis side institute sclerosis The activation of microglia can be observed in disease.The activation of microglia plays important in central lesion Role, pass through the reparation after inhibiting or promote the activation of microglia to be possible to reach regulation central lesion Journey, this is repaired after also becoming regulation of scientific research personnel's research and development for microglia function to adjust central lesion A key areas.

Naked mole (Heterocephalus glaber) is that one kind is distributed in African Somalia, Kenya, Ethiopia Etc. ground wild animal, Mammalia, Rodentia, Bathyergidae, naked mole category, naked mole kind are belonged on zootaxy position. Naked mole is the rodent of the longest-lived found so far, 28 years or more reachable, about normal rats or mouse 7-9 times of service life.In the service life of its many decades, do not find that it there are the nervous system diseases such as senile dementia, Parkinson's disease Occur and is able to maintain good nervous function (Judy C.Triplett, Antonella Tramutola, Aaron Swomley,Jessime Kirk,Kelly Grimes,Kaitilyn Lewis,Miranda Orr,Karl Rodriguez, Jian Cai,Jon B.Klein,Marzia Perluigi,Rochelle Buffenstein,D.Allan Butterfield.Age-related changes in the proteostasis network in the brain of the naked mole-rat:Implications promoting healthy longevity.Biochim Biophys Acta.2015；1852(10):2213-24.).And microglia is as performance immune function important in central nervous system Cell, may monitor its brain tissue microenvironment in play an important role.But due to extremely complex in body microenvironment, It is not easy to directly observe immunoloregulation function (the Dora Brites and Adelaide of microglia in body Fernandes.Neuroinflammation and Depression:Microglia Activation,Extracellular Microvesicles and microRNA Dysregulation.Front Cell Neurosci.2015,9:476.doi: 10.3389/fncel.2015.00476.).Therefore it needs to establish in vitro naked mole microglia model and be seen with making up in body The defect examined.Currently, researcher has built up out the in vitro culture strategy of the rodents microglia such as rat, mouse (Boza-Serrano A,Reyes JF,Rey NL,Leffler H,Bousset L,Nilsson U,Brundin P, Venero JL,Burguillos MA,Deierborg T.The role of Galectin-3inα-synuclein- Induced microglial activation.Acta Neuropathol Commun.2014,2:156.), cultural method master It will be for the separation of newborn mice cortical tissue, machinery shreds and chemical method digestion, cell primary culture, strikes and hits the methods of culture bottle four A experimental procedure；Wherein cell primary culture used medium is the calf serum of DMEM culture solution and final concentration of 20%；Its Middle cell secondary culture used medium is the fetal calf serum of DMEM culture medium and final concentration of 10%.

But in the prior art no matter the microglia of mouse or rat isolate and purify it is not applicable with cultural method In naked mole, domestic and foreign literature there is no the relevant report isolated and purified in relation to naked mole microglia with cultural method at present.

Summary of the invention:

The purpose of the present invention is to provide a kind of naked mole microglia isolate and purify and cultural method, with it is convenient, It is efficient to obtain the good naked mole microglia of purity is high, state, technology is provided to establish naked mole microglia in vitro It supports, while being polarization, activation, phagocytosis, other immunoloregulation functions and the small glue of the naked mole microglia of in vitro study The screening of the therapeutic agent or target molecule of cell plastid related disease lays the foundation.

We have tried out current mouse, the microglia of rat isolates and purifies and cultural method, as a result, it has been found that can not Culture obtains the good naked mole microglia of purity is high, state, and the quantity of naked mole microglia proliferation is also less.

Feasible experimental method is not established out in culture about naked mole microglia still, we analyze its reason, Mainly: 1) naked mole is a kind of poikilotherm, and the temperature of body cell in vitro culture needs to be groped, and its non-constant machine Body metabolic rate causes its body cell in-vitro culture medium to be different from common rat or mouse；2) naked mole existence is more dark In moist environment, the culture of body cell has higher requirement to humidity；3) naked mole has been already adapted to the ring of extraneous hypoxemia Border so the oxygen concentration in its body cell in vitro culture environment is different from the animal to live in normal oxygen environment, and needs to touch Rope.

It is therefore, most important in the culture of naked mole microglia to grope to suitable temperature, humidity and oxygen concentration, And grope to suitable culture medium.

To reach this purpose, naked mole microglia of the invention isolation and purification method the following steps are included:

A, naked mole cerebral cortex cell mixing is collected:

Separation birth 1-7 Tian Luo mole brain cortical tissue, tissue is shredded, and is added after the mixing of tissue digestion liquid, will It, which is put into three gas incubators, digests；Then composite nerve cell culture medium is added and terminates digestion, it, will after centrifugation removes supernatant Cell precipitation is resuspended and blows and beats into single cell suspension and plants in culture bottle；The culture bottle is the T25 for not being coated with hypothallus Culture bottle；

The tissue digestion liquid can be conventional enzyme of proteolysis digestive juice, such as trypsase, Papain Enzyme etc. is aided with DNA enzymatic I to release cell aggregation caused by DNA fragmentation adhesion.

Preferred tissue digestion liquid: the pancreatin and final concentration 0.1mg/ml DNA enzymatic I for being 0.25% containing mass concentration Mixed liquor.

B, the culture of naked mole cerebral cortex cell mixing:

The cell mixing that step A is obtained is incubated in three gas incubators with composite nerve cell culture medium and cultivates 30-- 45 days；From after cell mixing inoculation, every method replacement composite nerve cell culture medium for taking half amount to change liquid in 10 days；

C, the separation and purifying culture of naked mole microglia:

The cell mixing isothermal vibration culture that will be cultivated in step B, 35 ± 2 DEG C of constant temperature, by the cell suspension of shake culture Being placed in the culture dish for not being coated with left-handed poly-D-lysine makes cell adherent 10 minutes or more, keeps easily adherent small colloid thin Born of the same parents are affixed on plate bottom, and it is thin to replace fresh composite nerve to remove other adherent cell conditioned mediums loosely for weak vibrations plate Continue to cultivate in born of the same parents' culture medium；

The microglia culture medium, preferably addition percentage by volume be 2%B27 Neurobasal (i.e. The volume ratio of Neurobasal:B27 is 50:1).

The composite nerve cell culture medium can be the conventional cell culture fluid of composite nerve containing serum, such as low Sugared DMEM etc..Preferred composite nerve cell culture medium is the low sugar DMEM containing 10% percentage by volume fetal calf serum.

The parameter setting of the three gas incubators are as follows: temperature is controlled at 35 ± 2 DEG C, and oxygen concentration 5%, carbon dioxide is dense Degree is 5 ± 1%, and humidity is 96 ± 2%.

Further, in step A, raw 1-7 days naked mole brains is taken out, selective separating tissue after meninx is stripped, uses microscissors Tissue shear is broken to 1mm³, it is added after the mixing of tissue digestion liquid, puts it into three gas incubators and digest.Then mixing is added Neuronal cell cultures base terminates digestion and cell precipitation is resuspended to and is blown and beaten into single cell suspension and is planted after centrifugation removes supernatant In the T25 culture bottle for not being coated with hypothallus.

Further, in step A, the tissue digestion liquid is ingredient are as follows: pancreatin mass concentration is 0.25%, DNA enzymatic I concentration For 0.1mg/ml, digestion time is to digest 30 minutes at 37 DEG C.

Further, in step A, the parameter of noncentricity is 500rpm under room temperature, 5 minutes.

Further, it in step B, is cultivated 42 days with being incubated in nerve cell mixed culture medium in three gas incubators.

Further, culture bottleneck is kept to screw in step C, during shake culture to reduce the exchange of bottle inner air and outer air.

Further, in step C, the constant-temperature table major parameter setting are as follows: temperature is 35 DEG C, revolving speed 150rpm.

Further, in step C, the shake culture time is 4 hours

Further, in step C, until when needing to microglia functional study, then 30 minutes in advance are changed to small glue Cell plastid culture is based on being cultivated in three gas incubators.

Further, in step C, the cell adherent time is 15 minutes.

Further, the parameter setting of the three gas incubator are as follows: temperature control is at 35 ± 2 DEG C, oxygen concentration 5%, dioxy Changing concentration of carbon is 5 ± 1%, and humidity is 96 ± 2%.

The beneficial effects of the present invention are: 1. it is comprehensive using a variety of culture mediums from newborn naked mole cerebral cortex separation and pure Change culture microglia, finds out the reasonable culture side of the naked mole microglia of rodent mammal suitable for alternating temperature Method.We have found that cell state can preferably be kept under the conditions of 5% oxygen concentration, and it is able to maintain growth；2. operation side Method is simple, repeatability with higher, can obtain the microglia of 98% or more purity；3. by using addition 2% B27 Neurobasal serum free medium, can effectively ensure that the proliferation and vigor of microglia, be also convenient for subsequent Pharmacology experiment research to microglia.

In conclusion the method for the present invention being capable of the normal naked moleskin of a large amount of functional activities of acquisition easy, efficiently, economic Layer neuron, the culture under hypoxia condition can guarantee that this cell is still able to maintain under body state in ex vivo environment Biological characteristics, consequently facilitating directly further studying naked mole microglia in pure in-vitro cell culture model Special physiological function, thus for explore biological mechanism therein and be applied to clinical relevant provide it is important it is theoretical according to According to.

Detailed description of the invention:

Fig. 1 is the composite nerve cell of in vitro culture；

Fig. 2 is the microglia mixed in vitro under ordinary optical microscope；Wherein A is small colloid under 10 times of common light microscopics Cell, B are microglia under 20 times of light microscopics；

Fig. 3 is that the microglia immunocytochemical stain of purification culture identifies that wherein A is Iba1, and B is Hoechst, C are the amalgamation result of Iba1 with two kinds of Hoechst dyeing pictures.

Specific embodiment:

Below in conjunction with the drawings and specific embodiments, the invention will be further described.It should be understood that following embodiment is only used for It illustrates rather than for limiting the scope of the invention.

Embodiment 1: naked mole microglia isolates and purifies and cultivates

1, experimental material

The naked mole of cleaning grade of birth 1-7 is provided by Second Military Medical University, PLA's Experimental Animal Center.It will Newborn naked mole is placed in -20 DEG C of slabs and carries out anaesthetizing to be placed in 75% ethyl alcohol impregnating 10min, then breaks end, and use penicillin 100U/ml and 0.1mg/ml streptomysin mixed liquor wipes naked mole head surface, skin of head and skull is then cut off, by brain Removing is placed in the dissection liquid of pre-cooling.

DNA enzymatic I is purchased from Solarbio Biotechnology Co., Ltd, and trypsase, mycillin mixed liquor etc. are purchased from Sigma Company, DMEM, low sugar DMEM, Australia source fetal calf serum, Neurobasal culture medium, B27 serum-free addition factor etc. are purchased from Thermo Fisher Scientific company, culture dish, T25 and T75 culture bottle are purchased from Corning company.

Composite nerve cell culture media component is that low sugar DMEM culture medium contains the fetal calf serum that volume fraction is 10%, few Prominent glial precursor cell purification culture medium is to add the Neurobasal culture medium that volume fraction is 2%B27.

2, naked mole oligodendrocyte precursor cells isolate and purify and cultural method:

1. collecting naked mole cerebral cortex mixing spongiocyte: raw 1-7 days 2, naked mole brains are taken out, after stripping meninx It obtains cortical tissue to be placed in 1ml dissection liquid, tissue shear is broken to 1mm with microscissors^3,Digestive juice is added, and (mass fraction is 0.25% pancreatin 1ml, the wherein final concentration of 0.1mg/ml of DNA enzymatic I) mix after, in 37 DEG C of digestion 30min, then with mixing It closes neuronal cell cultures base and terminates digestion, be centrifuged 5 minutes under the conditions of 500rpm to remove supernatant, then add 5ml composite nerve Cell precipitation is resuspended in cell culture medium and is carefully blown and beaten into single cell suspension (avoiding bubble from generating as far as possible).It then will be slender Born of the same parents' suspension is according to 1 × 10⁶In the T25 culture bottle for being coated with left-handed poly-D-lysine, every bottle of nutrient solution volume is density kind 5ml。

2. the culture of naked mole cerebral cortex mixing spongiocyte: will 1. obtained in cell, with composite nerve cell train It supports and is based on cultivating 42 days in three gas incubators.From after cell inoculation, half amount is taken within every 7 days to change liquid method replacement culture medium.

3. the separation and purifying culture of naked mole microglia: by the 2. middle mixing spongiocyte cultivated (such as Fig. 1 institute Show, circular is microglia) it is pressed revolving speed shake culture 4 hours of 150rpm in 35 DEG C of isothermal vibration shaking tables, concussion training Culture bottleneck is kept to screw during supporting to reduce the exchange of bottle inner air and outer air.It is careful to draw supernatant, it is centrifuged 10min in 500rpm, It abandons supernatant and cell is resuspended at slender with 5ml fresh Neurobasal/B27 (Neurobasal:B27=50:1) culture medium Born of the same parents' suspension, then according to 1 × 10⁶Density kind, and will be thin in supernatant in the culture dish or culture bottle for not being coated with hypothallus Born of the same parents plant in the culture dish for being coated with left-handed poly-D-lysine.After its is adherent, trained with Neurobasal/B27 culture medium It supports (Neurobasal:B27=50:1) 24 hours, the small glial precursor cell of purifying can be obtained, be incubated at 35 ± 2 DEG C, oxygen Concentration is 5%, and gas concentration lwevel is 5 ± 1%, in the three gas incubators that humidity is 96 ± 2%.

Embodiment 2: the identification of naked mole microglia

Using the identification of the resulting naked mole microglia of embodiment 1: using 4% paraformaldehyde in Multiplying culture Microglia in base is fixed, and the methods of combining form identification, immune refinement chemistry are identified.

1, cytomorphology is identified:

Identification method it is as described in the literature (Chung WS, Clarke LE, Wang GX, Stafford BK, Sher A, Chakraborty C,Joung J,Foo LC,Thompson A,Chen C,Smith SJ,Barres BA.Astrocytes mediate synapse elimination through MEGF10and MERTK pathways.Nature,2013,504 (7480):394-400.)。

As a result as shown in Fig. 2, under light microscopic, microglia cell space is rounded or oval, and cell space week is had no when unactivated It is with protrusion.

2, immunocytochemistry is identified:

Identification method as described in document (E,Arias C.Cholesterol-induced astrocyte activation is associated with increased amyloid precursor protein expression and processing.Glia 2015,63:2010–2022.)。

As a result as shown in figure 3, the microglia of differential velocity adherent purifying is carried out creep plate experiment, in serum free medium After culture 24 hours, using 4% paraformaldehyde, the cells are fixed, then utilizes anti-microglia surface specific antigen Iba1's Antibody carries out immunocytochemical stain.The results show that cell is able to maintain the positive (green of Iba1 in serum free medium Fluorescence).

According to above-mentioned experimental result, the naked mole microglia that the present invention isolates and purifies has typical small glial precursor Cellular morphology, it is positive in Iba1, round or ellipse structure is able to maintain when unactivated.And it is able to maintain in serum free medium Good vegetative state.

The preferred embodiment of the present invention has been described in detail above, but the invention be not limited to it is described Embodiment, those skilled in the art can also make various equivalent on the premise of not violating the inventive spirit of the present invention Variation or replacement, these equivalent variation or replacement are all included in the scope defined by the claims of the present application.

Claims (8)

1. a kind of naked mole microglia cultural method, comprising the following steps:

A, naked mole cerebral cortex cell mixing is collected:

Separation birth 1-7 Tian Luo mole brain cortical tissue, tissue is shredded, and is added after the mixing of tissue digestion liquid, is put Enter and is digested in three gas incubators；Then composite nerve cell culture medium is added and terminates digestion, after centrifugation removes supernatant, by cell Precipitating is resuspended and blows and beats into single cell suspension and plants in culture bottle；

B, the culture of naked mole cerebral cortex cell mixing:

The cell mixing that step A is obtained is based on cultivating 30--45 days in three gas incubators with composite nerve cell culture；From mixed After closing cell inoculation, every method replacement composite nerve cell culture medium for taking half amount to change liquid in 10 days；

C, the separation and purifying culture of naked mole microglia:

The culture of cell mixing isothermal vibration 4 hours will cultivated in step B, keep during shake culture culture bottleneck to screw with The exchange of bottle inner air and outer air is reduced, 35 ± 2 DEG C of constant temperature, the cell suspension of shake culture is placed in and is not coated with left-handed poly-D-lysine Culture dish in make cell adherent 10 minutes or more, remove cell conditioned medium, replace fresh composite nerve cell culture medium relaying Continuous culture；

Until when to microglia functional study, being changed within 20-40 minutes in advance microglia culture and being supported based on three air cultures It is cultivated in case；

The microglia culture medium, the Neurobasal for being 2%B27 for addition percentage by volume；

The parameter setting of the three gas incubators are as follows: temperature control is at 35 ± 2 DEG C, oxygen concentration 5%, and gas concentration lwevel is 5 ± 1%, humidity is 96 ± 2%.

2. a kind of naked mole microglia cultural method according to claim 1, which is characterized in that described in step A Tissue digestion liquid be 0.25% pancreatin of mass concentration and mixed liquor that concentration is 0.1mg/ml DNA enzymatic I.

3. a kind of naked mole microglia cultural method according to claim 1, which is characterized in that the mixing mind It is the low sugar DMEM containing 10% fetal calf serum of percentage by volume through cell culture medium.

4. a kind of naked mole microglia cultural method according to claim 1, which is characterized in that in step A, take out Raw 1-7 days naked mole brains, strip selective separating tissue after meninx, tissue shear are broken to 1mm with microscissors³, tissue is added and disappears After changing liquid mixing, puts it into three gas incubators and digest；Digestion condition is to digest 30 minutes at 37 DEG C.

5. a kind of naked mole microglia cultural method according to claim 1, which is characterized in that described in step A Parameter of noncentricity is 500rpm under room temperature, 5 minutes.

6. a kind of naked mole microglia cultural method according to claim 1, which is characterized in that in step B, with mixed Conjunction neuronal cell cultures base, which is incubated in three gas incubators, to be cultivated 42 days.

7. a kind of naked mole microglia cultural method according to claim 1, which is characterized in that in step C, cell The adherent time is 15 minutes.

8. a kind of naked mole microglia cultural method according to claim 1, which is characterized in that in step C, constant temperature Its major parameter of the constant-temperature table that shake culture uses setting are as follows: temperature is 35 DEG C, revolving speed 150rpm.