CN112522319A

CN112522319A - Fluorescent BV2 tool cell and construction method and application thereof

Info

Publication number: CN112522319A
Application number: CN202011423659.XA
Authority: CN
Inventors: 吕泽中; 詹阳
Original assignee: Shenzhen Institute of Advanced Technology of CAS
Current assignee: Shenzhen Institute of Advanced Technology of CAS
Priority date: 2020-12-08
Filing date: 2020-12-08
Publication date: 2021-03-19

Abstract

The invention discloses a fluorescent BV2 tool cell and a construction method and application thereof, wherein the construction method is characterized in that a lentivirus tool is used as a carrier, a plasmid with a fluorescent gene EYFP is integrated in the genome of a BV2 cell, and when the blue light is used for excitation, the cell can be observed to emit clear green fluorescence under a fluorescent microscope. The transgenic BV2 tool cell of the invention can autofluorescence, has high fluorescence intensity, can be conveniently observed without dyeing, and has important significance in the aspect of in vitro research on the development, behavior and function of microglia.

Description

Fluorescent BV2 tool cell and construction method and application thereof

Technical Field

The invention relates to the field of cell biology, in particular to a fluorescent BV2 tool cell and a construction method and application thereof.

Background

Microglia (Microglia) is the smallest glial cell in the Central Nervous System (CNS), distributed throughout the CNS, accounting for approximately 5% to 10% of the total glial cells. Microglia, which is an immune effector cell resident in the central nervous system, belongs to the mononuclear phagocyte family and is widely considered as a main immune effector of the central nervous system, and microglia and neurogenic inflammation mediated by the microglia play a very important role in the damage of the central nervous system and the process of outcome of diseases, and are involved in human nervous system disorder diseases such as HIV encephalopathy, Parkinson disease, Alzheimer disease, multiple sclerosis and the like.

BV2 as an immune cell in the central nervous system can not only protect neurons by phagocytosing pathogens and harmful particles in brain tissues, but also activate reactive microglia under the action of inflammatory factors, secrete inflammatory cytokines to have toxic effect on neurons, and is an important target for treating neuroinflammation and neurodegenerative diseases. The BV2 cell line is an immortalized cell line obtained by Blasi and the like in 1990 by infecting primary cultured mouse microglia with retrovirus J2 carrying oncogene v-raf/v-myc, not only is highly purified, but also basically has the morphological, phenotypic and various functional characteristics of the primary cultured microglia, is relatively easy to culture, and is the cell line which is most widely used in the world for researching the microglia in vitro.

However, the conventional BV2 cells do not have fluorescence, and the conventional observation means needs to perform immunofluorescence staining and then observe the cells, and living body observation is not easy, so that a BV2 tool cell which can perform autofluorescence and can be stably passaged is needed.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a fluorescent BV2 tool cell and a construction method and application thereof, the construction method uses lentivirus as a vector to carry a plasmid with a fluorescent gene EYFP to transfect a BV2 cell to obtain an autofluorescence BV2 tool cell, and the BV2 tool cell has important significance in the aspect of researching the development, behavior and function of microglia in vitro.

The invention provides a construction method of a fluorescent BV2 tool cell, which comprises the following steps:

(1) constructing a slow virus gene vector plasmid which is sequentially connected with a promoter and a yellow fluorescent marker gene; the promoter is any one of CMV, EF1a, iba-1, cx3cr1, CAG and TEF1, and the yellow fluorescent marker gene is EYFP;

(2) transfecting the slow virus gene vector plasmid, the pDelta 8.74 helper plasmid and the pMD2.G plasmid in the step (1) together with a packaging cell, culturing, cracking the packaging cell, purifying by using chloroform, sodium chloride and PEG8000, and taking a supernatant, wherein the supernatant contains a slow virus for infecting a BV2 cell, and the slow virus comprises a promoter and a yellow fluorescent marker gene which are sequentially connected; the transfection step of the invention does not need to carry out transfection through liposome, thus simplifying the flow of experimental operation and saving cost.

(3) Infecting BV2 cells with the lentivirus of step (2) to obtain the fluorescent BV2 tool cells. Through experimental observation, the proportion of cells capable of autofluorescence in the obtained BV2 tool cells is up to 100%, and all the cells can autofluoresce under the excitation of blue light. The BV2 tool cells obtained were stable and did not show a decrease in fluorescence with increasing passage number during passage.

Further, the lentiviral vector plasmid described in step (1) is pLenti, and the vector is shown in FIG. 2.

Further, the packaging cell in the step (2) is any one of HEK293 and 293T, AAV 293.

Further, the purification method in the step (2) comprises the following steps:

(1) collecting the cell suspension after the cell is cracked and packaged, centrifuging and discarding the supernatant, adding a cell cracking solution to resuspend cell sediment, and repeatedly freezing and thawing for cracking;

(2) after centrifugation, adding chloroform into the supernatant, centrifuging and collecting the supernatant;

(3) adding sodium chloride and PEG8000 to the supernatant respectively, and incubating;

(4) centrifuging, discarding the supernatant, and resuspending the precipitate with buffer solution;

(5) adding chloroform into the heavy suspension, centrifuging, and transferring supernatant;

(6) adding sodium chloride and PEG8000 to the supernatant respectively, and incubating;

(7) centrifuging, discarding the supernatant, and adding a buffer solution to resuspend the precipitate;

(8) adding DNA enzyme and RNA enzyme for digestion;

(9) chloroform is added for extraction, centrifugation is carried out, the upper aqueous phase is sucked out, and the upper aqueous phase is concentrated and purified lentivirus liquid.

Further, in the step (3) and the step (6), the final concentration of the sodium chloride is 0.5M, the final concentration of the PEG8000 is 8%, and the 8% is a mass-to-volume ratio.

The titer of the lentivirus obtained by the purification method can reach 10¹⁰The conditions of the above purification allow obtaining very high purity lentiviruses.

The invention also provides a fluorescent BV2 tool cell prepared by the construction method.

The invention also provides application of the fluorescent BV2 tool cell in-vitro research on development, behavior and function of microglia.

Further, the behavior of the microglia includes morphological changes of cells and phagocytosis.

In summary, compared with the prior art, the invention achieves the following technical effects:

1. the transgenic BV2 tool cell can autofluorescence, the fluorescence intensity is high, and the proportion of the autofluorescence BV2 tool cell can reach up to 100%.

2. The BV2 tool cells can be observed conveniently without staining, and the process of in vitro experiment operation is simplified.

3. The BV2 tool cell can realize the living body visualization of the BV2 cell and can be used for observing the live BV2 cell.

4. The BV2 tool cells of the present invention can be stably passaged, and the fluorescence does not decrease with the increase of the number of passages during the passage.

5. The construction method of the invention does not need liposome transfection, simplifies the flow, saves the cost, has good purification effect, and the titer of the obtained lentivirus can reach as high as 10¹⁰The purity is very high.

6. The BV2 tool cell can be accurately distinguished from other cells, and has important significance in the aspect of in vitro research on the development, behavior and function of microglia.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a schematic flow chart of the fluorescent BV2 tool cell construction method of the present invention.

FIG. 2 is a schematic diagram of a lentiviral vector plasmid carrying the EYFP fluorescent gene, in which the promoter is CMV.

FIG. 3 is a fluorescent photograph of fluorescent BV2 tool cells obtained by the construction method of the present invention stably passaged for 8 generations. The excitation light used by the fluorescence microscope has a wavelength of 473nm and is photographed under a 20-fold microscope.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, shall fall within the scope of protection of the present invention.

Lentivirus (Lentivirus) vectors are gene therapy vectors developed based on HIV-1 (human immunodeficiency virus type I). A distinction is made between retroviral vectors in general, which have the ability to infect both dividing and non-dividing cells. In the present invention, BV2 cells were transfected with a plasmid carrying the fluorescent gene EYFP using lentivirus as a vector.

In order to solve the problem that the common BV2 cell cannot be visualized and living body observation can not be realized, the invention discloses a fluorescent BV2 tool cell and a construction method and application thereof, wherein the construction method comprises the following stepsThe method comprises the steps of firstly constructing a lentiviral vector plasmid sequentially connected with a promoter and an EYFP gene, wherein the plasmid uses any one of CMV, EF1a, iba-1, cx3cr1, CAG and TEF1 as the promoter, and then is connected with the EYFP fluorescent gene. In the following examples, the CMV promoter was used as an example, and EF1a, iba-1, cx3cr1, CAG and TEF1 all induced the expression of EYFP gene, and had the same effect as that of CMV; then co-transfecting a packaging cell by using a lentivirus gene vector plasmid, a pDelta 8.74 helper plasmid and a pMD2.G plasmid, wherein the pDelta 8.74 plasmid is used for assisting transfection, the pMD2.G carries a gene for coding virus coat protein, and the packaging cell can be any one of HEK293 and 293T, AAV 293; the packaging cells are then lysed and the lentivirus purified. The steps of purifying the lentivirus need chloroform, sodium chloride and PEG8000, and the titer of the purified lentivirus can reach 10¹⁰The purification method is very efficient, and the obtained lentivirus has high purity; BV2 cells were finally infected with the obtained lentivirus, and fluorescence was observed by fluorescence microscopy 48-72 hours after infection, as shown in FIG. 1.

When excited by blue light, the BV2 tool cells prepared as described above were observed to fluoresce clearly green under a fluorescence microscope. The BV2 tool cells of the present invention did not show a decrease in fluorescence with increasing passage number during passage due to transfection with lentiviral vectors.

Example 1 construction of lentiviral vector plasmid sequentially linked to promoter and yellow fluorescent marker Gene

Design of DNA fragments for construction of vectors: the two ends of the fragment are respectively provided with PacI and EcoRI restriction enzyme sites, the two restriction enzymes PacI and EcoRI are used for carrying out enzyme digestion and then are connected with a vector plasmid to obtain a target lentiviral plasmid, the structure of the lentiviral vector plasmid is shown in figure 2, the lentiviral vector plasmid carries a CMV promoter, and then an EYFP fluorescent gene is connected.

Example 2 construction and acquisition of lentiviruses

Materials and reagents:

the whole culture medium: DMEN + 10% FBS + 1% penicillin-streptomycin double antibody

Transfection medium: DMEM + 10% FBS (without antibody)

Maintenance medium: DMEM + 2% FBS + 1% penicillin-streptomycin double antibody

Transfection reagent: calcium phosphate mammalian transfection kit (Promega corporation, E1200)

Other reagents: chloroquine (Sigma, C6628, 25g)

Plasmid: lentivirus gene vector plasmids (prepared in example 1), pDelta 8.74 helper plasmid, pmd2.g plasmid (VSVg, coat protein), 3 plasmids were co-transfected to prepare lentiviruses.

Second, construction of lentiviruses

The method comprises the following steps:

1. culturing 293T cells with a complete medium;

2.293T cells were inoculated into 20 15cm cell culture dishes to achieve a cell fusion degree of about 50%;

3. the next day transfection was started until the degree of cell fusion was more than 90%. The medium was changed 1-3h in advance and 20mL transfection medium was changed. Transfection complexes were then prepared (amount per dish):

pDelta 8.74 helper plasmid 15. mu.g

pMD2.G plasmid 15. mu.g

Lentiviral gene vector plasmid 15. mu.g

The plasmid to be transfected was mixed with 930. mu.L nuclease-free water (provided in the kit), and after thorough mixing, 140. mu.L 2M CaCl was added₂(provided within the kit); the mixture was added dropwise to about 1125. mu.L of 2 XHBS (provided in the kit) to be adjusted to the same volume as the mixture, and the 2 XHBS was shaken while adding to rapidly mix; then incubating for 30min at room temperature;

4. adding 1 per mill 25mM chloroquine solution into the cell culture medium of the culture dish before the end of the incubation;

5. after incubation, vortex again and mix, and drip the transfection complex into 293T cell culture medium, gently mix by the cross method, place at 37 deg.C, 5% CO₂After culturing for 6-8h in the incubator, changing to 20mL of maintenance medium;

6. the transfection condition can be observed under a fluorescence microscope 24h after transfection, and the cells are collected for lentivirus purification 48-72h later.

Lentiviral purification

Reagent: PEG8000(Sigma,89510), HEPES (Sigma, H3375), Tris-HCl, NaCl, CaCl₂EDTA, DNase and DNase (or nuclease), chloroform (chloroform)

The lentivirus purification comprises the following steps:

1. collecting the packaging cell suspension to a 500mL centrifuge cup, centrifuging at 4 ℃ for 30min at 3000 g; the supernatant was discarded, 2mL (20 dishes 15cm petri dish) of cell lysis solution (100mM Tris-HCl, 150mM NaCl, pH8.0) was added to resuspend the cell pellet, and four rounds of repeated freeze-thaw lysis were performed. Using dry ice-ethanol bath (-70 deg.C, or directly placing into refrigerator of-80 deg.C) and water bath of 37 deg.C, freezing and thawing for 10min each time, and vortexing briefly after each thawing to promote lysis.

After centrifugation at 12,000g for 30min at 2.4 ℃, the supernatant was transferred to a clean 1.5mL centrifuge tube (. about.4).

3. An equal volume of chloroform was added, centrifuged at 12,000g for 5min at 4 ℃ and the supernatant collected into a new 15mL centrifuge tube.

4. Adding 1/5 volumes of 3M NaCl and 40% PEG8000 solution to the supernatant respectively to final concentrations of 0.5M NaCl and 8% PEG8000(W/V), and incubating on ice for 3h or overnight;

centrifuging at 5.4 deg.C for 30min at 3200g, discarding the supernatant, and resuspending the pellet with 1.0-1.5mL of HBS (50mM HEPES, 150mM NaCl, 25mM EDTA, pH 8.0);

6. transferring the heavy suspension to a sterile 1.5mL centrifuge tube (3), adding equal volume of chloroform, centrifuging at 4 ℃ for 5min at 12,000g, and transferring the supernatant to a new 1.5mL centrifuge tube (2);

7. 1/5 volumes of 3M NaCl and 40% PEG8000 solution were added to the supernatant respectively to final concentrations of 0.5M NaCl and 8% PEG (W/V), and incubated on ice for 3h or overnight;

centrifuging at 12000g for 30min at 8.4 deg.C, discarding the supernatant, adding an appropriate volume (100-;

9. adding DNase and RNAse to a final concentration of 1 μ g/mL, and digesting at room temperature for 30 min;

10. adding equal volume of chloroform for extraction, centrifuging at 4 deg.C for 5min at 12,000g, and carefully sucking out the upper aqueous phase under aseptic condition to obtain concentrated and purified slow virus solution, which is defined as the final product. The extract is colorless clear liquid, and can be extracted again by chloroform to remove impurities such as protein;

11. the lentiviral fluid was aliquoted and stored at-80 ℃. The prepared lentivirus carries a CMV promoter, and is connected with an EYFP fluorescent gene.

EXAMPLE 3 Lentiviral transfection of BV2 cells

The lentivirus prepared in example 2 was used to transfect BV2 cells as follows:

1. adherent BV2 cells were plated at 1X 10 hours 18-24 hours prior to lentivirus transfection⁵The cells were plated in 24-well plates and cultured in high-glucose DMEM medium so that the number of cells at the time of lentivirus transfection was 2X 10⁵About hole.

2. On day 2, the original medium was replaced with 2mL of fresh medium containing 6. mu.g/mL polybrene, and an appropriate amount of lentiviral suspension at 10-100M was added. Incubation was performed at 37 ℃. The transfection complex number is the number of viruses/the number of transfected cells, the transfection efficiency is low when the transfection complex number is less than 10, and the cells can not bear more viruses and die when the transfection complex number is more than 100, and the transfection complex number is preferably 50. Polybrene acts to increase cell membrane permeability and may aid in rapid transfection.

3. After 4h 2mL fresh medium was added to dilute the polybrene.

4. The culture was continued for 12h, replacing the virus-containing medium with fresh medium.

5. And (5) continuing culturing to obtain BV2 tool cells, and performing fluorescence detection. The BV2 tool cell contains yellow fluorescent protein, and obvious fluorescent expression can be seen after 48 hours of transfection generally, and is more obvious after 72 hours. The proportion of the fluorescent cells can reach 100 percent, and the cells are frozen by liquid nitrogen or directly used for experiments after successful transfection.

Example 4 BV2 tool cells of the invention were able to auto-fluoresce and stably passaged

The tool cells prepared in example 3 are used for passage 8 generations and then fluorescence detection is carried out, blue light of 473nm is used for excitation, and the fluorescence of the BV2 tool cells is observed under a 20-fold mirror, as shown in figure 3, the fluorescence in the figure is displayed in gray scale, the cell membranes and cytoplasm of all BV2 tool cells can emit green fluorescence, the fluorescence is clear and high in brightness, and the situation that the fluorescence is weakened due to passage 8 times is avoided, so that the BV2 cells can be stably passed, the fluorescent protein cannot be lost due to passage, cells do not need to be transfected again in each experiment, and the flow of experimental operation is greatly simplified.

EXAMPLE 5 application example of BV2 tool cells of the invention

The BV2 tool cell of the invention can be used for observing the change of cell morphology in the environment of A beta/inflammatory factor in vitro. A β is amyloid β, and alzheimer's disease is thought to be caused by a β deposition. Microglia can present different forms in vitro, BV2 tool cells with different forms can be observed in figure 3, some are round, some are fusiform, and some extend out of two tentacles, because the BV2 tool cells can be directly seen under a fluorescence microscope, immunofluorescence staining is not needed, and the BV2 cell form change can be easily observed by direct living observation, and A beta or an inflammatory factor is added into a BV2 tool cell system, so that a convenient and fast means is provided for researching pathogenic mechanisms of Alzheimer's disease and other inflammations.

The BV2 tool cell of the invention can be used for observing the phagocytosis of BV2 cells to A beta/fluorescent globules and the like in vitro. The fluorescent globule is a commonly used tool for detecting phagocytosis, has different types with diameters of 0.1 μm, 0.2 μm, 1 μm and the like, characterizes the phagocytosis of microglia by calculating the number of the fluorescent globules phagocytosed in cells, and is added into a BV2 tool cell system, so that the BV2 tool cell can observe the phagocytosis process of the cells under a fluorescent microscope and count the dynamic change of the fluorescent globules phagocytosed by the cells.

The use of the BV2 tool cells of the present invention also simplifies the experimental procedures to be performed for immunofluorescent staining. If the substances phagocytized by the BV2 cells are subjected to immunofluorescence staining, the BV2 tool cells have fluorescence, and do not need to be stained, and other proteins needing to be stained can be directly subjected to immunofluorescence staining, so that a complicated set of immunofluorescence staining steps can be omitted, interference among different staining proteins is reduced due to the fact that the immunofluorescence staining is not repeatedly performed, and results are more accurate.

In conclusion, the invention discloses a fluorescent BV2 tool cell and a construction method and application thereof, wherein the construction method comprises the steps of firstly constructing a lentiviral vector plasmid sequentially connected with a promoter and an EYFP gene, wherein the plasmid uses any one of CMV, EF1a, iba-1, cx3cr1, CAG and TEF1 as the promoter, and is connected with the EYFP fluorescent gene; then co-transfecting a packaging cell by using a lentiviral vector plasmid, a pDelta 8.74 helper plasmid and a pMD2.G plasmid, wherein a liposome is not needed in the transfection process; the packaging cells are then lysed and the lentivirus purified. The titer of the purified lentivirus can reach 10¹⁰The purification method is very efficient, and the obtained lentivirus has high purity; finally, the BV2 cells are infected by the obtained lentivirus, fluorescence is observed by a fluorescence microscope 48 to 72 hours after infection, the proportion of the BV2 tool cells capable of autofluorescence can reach 100 percent, and the fluorescence intensity is still not weakened after passage 8 generations. The BV2 tool cell can be observed conveniently without staining, the flow of in vitro experiment operation is simplified, the living body visualization of the BV2 cell can be realized, and the BV2 tool cell can be used for observing the live BV2 cell. The BV2 tool cell can be accurately distinguished from other cells, and has important significance in the aspect of in vitro research on the development, behavior and function of microglia.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A method for constructing a fluorescent BV2 tool cell is characterized by comprising the following steps:

(2) transfecting the slow virus gene vector plasmid, the pDelta 8.74 helper plasmid and the pMD2.G plasmid in the step (1) together with a packaging cell, culturing, cracking the packaging cell, purifying by using chloroform, sodium chloride and PEG8000, and taking a supernatant, wherein the supernatant contains a slow virus for infecting a BV2 cell, and the slow virus comprises a promoter and a yellow fluorescent marker gene which are sequentially connected;

(3) infecting BV2 cells with the lentivirus of step (2) to obtain the fluorescent BV2 tool cells.

2. The method according to claim 1, wherein the lentiviral vector plasmid of step (1) is pLenti.

3. The method according to claim 1, wherein the packaging cell in step (2) is any one of HEK293 and 293T, AAV 293.

4. The method for constructing according to claim 1, wherein the purification method in the step (2) comprises the steps of:

(8) adding DNA enzyme and RNA enzyme for digestion;

5. The method according to claim 4, wherein the final concentration of sodium chloride in the steps (3) and (6) is 0.5M, the final concentration of PEG8000 is 8%, and the 8% is a mass-to-volume ratio.

6. A fluorescent BV2 tool cell produced by the method of any one of claims 1 to 5.

7. The use of the fluorescent BV2 tool cell of claim 6 to study microglial development, behavior and function in vitro.

8. The use according to claim 7, wherein the behaviour of microglia comprises cell morphology change and phagocytosis.