CN112126660A - Preparation method of animal model with high expression of gene transfected in lung tissue through intratracheal way - Google Patents
Preparation method of animal model with high expression of gene transfected in lung tissue through intratracheal way Download PDFInfo
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Abstract
The invention discloses a preparation method of an animal model with high expression of genes transfected by an endotracheal way in lung tissues. Which comprises the following steps: (1) constructing a recombinant virus vector carrying the vascular endothelial growth factor A, and adjusting the virus titer to 0.5-2 multiplied by 1011PFU/mL; (2) anesthetizing animal, suspending and fixing, irradiating neck, opening oral cavity, inserting tracheal catheter, and injecting virus via tracheal catheterAnd (4) putting the animal into the lung tissue of the animal, then connecting a breathing machine until the animal is awake, and finally removing the tracheal catheter. The application constructs an animal model for realizing the target transfection of the recombinant adenovirus vector in the lung tissue of a newborn rat through an intratracheal approach, the adenovirus vector is transfected in the trachea, the virus amount is proper, the transfection rate of other organs except the lung is low, and the transfection targeting property is improved.
Description
Technical Field
The invention belongs to the technical field of animal model preparation, and particularly relates to a preparation method of an animal model with high expression of genes transfected by an endotracheal way in lung tissues.
Background
The gene intervention technology is a hot technology for the basic research of clinical diseases at present, and can achieve the purpose of improving or inhibiting the expression of a target gene in tissues or cells through gene transfection, thereby having important significance for further researching the function of the target gene in the pathogenesis of related diseases. The adenovirus vector is an important medium of gene intervention technology, an adenovirus shuttle plasmid carrying a target gene and a packaging plasmid carrying an adenovirus genome co-transfect HEK293 cells, recombinant adenovirus is generated under the action of a Cre/loxP recombinase system, cells in a division stage and a non-division stage are infected, the target gene is expressed, and the adenovirus genome is not integrated into a host cell chromosome and cannot be copied, so that the adenovirus vector is a safe and effective gene vector.
Neonatal rats are important model animals for the basic study of neonatal disease. The establishment of a gene intervention model of respiratory diseases of newborn rats usually requires the integration of a target gene or an interfering gene fragment thereof into the lung tissues of the newborn rats, an adenovirus vector carrying the target gene is a commonly used transfection medium at present, and a key technology for transfecting the adenovirus vector into the lungs is the method. The adenovirus transfected by the tail vein can reach the lung, so that a target gene is expressed in lung tissues, however, the mass of a newborn rat is small, the tail vein is slender, the puncture is difficult, the blood volume of the tail vein is very small, the blood return is not obvious, whether the puncture is successful can be judged by injecting a small amount of medicine, once the puncture is unsuccessful, the puncture is repeated, the medicine is injected repeatedly, tail edema can be caused, the puncture difficulty is further increased, the puncture success rate is reduced, the medicine dosage is difficult to control and the medicine is wasted due to repeated puncture, therefore, in order to improve the puncture success rate, researchers always need to wait for the newborn rat to start an experiment after reaching a certain physical mass, and the experiment process is limited. The intratracheal administration is another important way for reaching the medicine to the lung, however, no report is found about the targeting transfection of the recombinant adenovirus vector in the lung tissue of a newborn rat through the intratracheal way.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a preparation method of an animal model with high expression of genes transfected by an endotracheal way in lung tissues, and the animal model with the targeting transfection of recombinant adenovirus vectors in the lung tissues of newborn rats by the endotracheal way can be constructed.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
a method for preparing an animal model in which a gene transfected through an endotracheal route is highly expressed in lung tissue, comprising the steps of:
(1) constructing a recombinant virus vector carrying the vascular endothelial growth factor A, and adjusting the virus titer to 0.5-2 multiplied by 1011PFU/mL；
(2) The animal is anesthetized and then suspended and fixed, the neck of the animal is irradiated, the oral cavity of the animal is opened, then the tracheal catheter is inserted, the virus is injected into the lung tissue of the animal through the tracheal catheter, then the animal is connected to a breathing machine until the animal is awake, and finally the tracheal catheter is pulled out.
Further, the recombinant viral vector is a recombinant adenovirus vector which comprises a vector plasmid GV314 with the element sequence of CMV-MCS-3FLAG-SV40-EGFP and an auxiliary packaging plasmid.
Further, the construction process of the recombinant viral vector is as follows:
(1) VEGF-A overexpression adenovirus vector construction: the vector name is GV314 (element sequence: CMV-MCS-3FLAG-SV40-EGFP), the vector is linearized by BamHI/AgeI enzyme digestion, the target gene humanized VEGF-A is amplified by a PCR method, and amplification primers are shown as follows;
VEGFA(4907-1)-P1:AGGTCGACTCTAGAGGATCCCGCCACCATGAACTTTCTGCTGTCTTGG；(SEQ ID NO.1)
VEGFA(4907-1)-P2:TCCTTGTAGTCCATACCCCGCCTCGGCTTGTCAC(SEQ ID NO.2)；
(2) adenovirus packaging and detection
The main process is that the shuttle plasmid of adenovirus carrying exogenous gene and the auxiliary packaging plasmid pBHGlox delta E1,3Cre carrying most genome of adenovirus (E1/E3 deletion) transfect HEK293 cell together to generate non-replicative recombinant adenovirus carrying exogenous gene. HEK293 cells were digested with 0.25% trypsin and cultured until cell density reached 50-60% for transfection. 5 mu g of each of the adenovirus shuttle plasmid and the auxiliary packaging plasmid is uniformly mixed with a DMEM medium, and the mixture and the cationic liposome form a compound to be incubated at room temperature. The complex is transfected into HEK293 cells, the culture is continued, and the virus is collected when most cells are diseased. The HEK293 cells in good growth state are transferred into a T25 cell culture flask, viruses are added, and the cells are purified, collected and stored after 2 times of amplification. Detecting the physical state, sterile state and virus titer of the purified virus, wherein the virus titer is 1 multiplied by 1011(PFU/ml)。
Further, the preparation process of the tracheal catheter comprises the following steps:
taking a 4.5F scalp needle, removing the extension tube, connecting the front end of the needle core with a PE50 catheter, and extending about 1.5-2.0 cm beyond the needle core; the front end of a needle core of the 1.9F central venous catheter is intercepted and inserted into a scalp needle (the insertion depth is shorter than the front end of a PE50 catheter), so that on one hand, the length of the tracheal catheter is prolonged, the operation is convenient, on the other hand, the hardness of the front end of the PE50 catheter is increased, the tracheal catheter is convenient to insert into the trachea of a newborn rat, and the tracheal catheter is soaked in 75% alcohol for standby after being prepared, as shown in figure 2.
Further, the animal model is a neonatal rat model or an adult rat model.
Further, the specific process of the step (2) is as follows:
(1) 20-30g of new rats for model building are taken, ketamine (100mg/kg) and xylazine (10mg/kg) are used for intraperitoneal anesthesia, and after the anesthesia is effective, the trachea intubation is performed.
(2) Hanging the front incisor and clinging the back of the newborn rat to the tripod support to fix the newborn rat, irradiating the neck of the newborn rat by the LED lamp facing the head and the face of the newborn rat, measuring the body surface distance from the front incisor to the throat by using the tracheal catheter, and marking.
(3) The operator faces the back of a newborn rat, pulls out the tongue from the left mouth corner of the rat to the outer upper side with the left hand, a light spot with the size of about 2mm can be seen in the center of the throat, the light spot can be opened and closed along with respiratory motion, namely, a glottis part, the operator holds the self-made tracheal catheter with the right hand, inserts the glottis to the catheter mark part, and fixes the tracheal catheter.
(4) Each newborn rat needs to be transfected with adenovirus 2X 108PFU, because the prepared adenovirus vector has a titer of 1X 1011(PFU/ml), 2. mu.L of the adenovirus transfection stock solution was aspirated by a pipette, diluted to 25. mu.L with PBS, and injected into an endotracheal tube.
(5) Placing the newborn rat in a horizontal position, connecting a small animal respirator to promote the medicine to be dispersed into the lung and maintain stable respiration, and withdrawing the machine after the newborn rat is anesthetized and conscious, and removing the tracheal catheter.
(6) After the new rat transfects the recombinant adenovirus for 24 hours, tissues such as lung, liver, spleen, kidney and the like are extracted, frozen sections are made, paraffin sections of lung tissues are made, and the transfection effect is identified.
The invention has the beneficial effects that:
the tongue body is pulled out from the left oral angle of the newborn rat to the outside upper side, the glottis can be exposed by irradiating the neck part through the LED lamp, the virus transfection liquid can be injected after the tracheal catheter is inserted into the glottis without using a special laryngoscope, the operation method is simple, the operation success rate is high, and the tracheal catheter is a self-made catheter, so the cost is low, and the method has good practicability. The influence of the mass of the newborn rat on the diameter of the laryngeal opening is small, but the influence of the mass of the newborn rat on the diameter of the tail vein is large, so that the success rate of trachea intubation is obviously higher than that of tail vein puncture when the mass of the newborn rat is small, the influence of the mass of the newborn rat on the technology is relatively smaller than that of the tail vein puncture, and the experimental process is accelerated; in addition, the tracheal catheter is self-made, the operation is convenient, the cost is low, the neck is irradiated by the LED lamp without the help of a special laryngoscope, the experiment cost is reduced, and the economic practicability is good.
In addition, by injecting virus transfection liquid into the trachea, the technology provides proper virus amount, the virus vector can directly reach alveolar epithelium and diffuse to parts such as pulmonary vessels through paracrine and blood circulation, the target gene is transfected in the lung tissues in a targeted manner, and the transfection effect is improved.
The application has the advantages that the adenovirus vector is transfected in the trachea, the virus amount is appropriate, the transfection efficiency of other organs except the lung is low, and the transfection targeting property is improved. In addition, the recombinant adenovirus vector carrying VEGF-A is transfected in the trachea, and the immunohistochemical result of the lung tissue of the newborn rat is extracted to show that the expression of VEGF-A is increased, and the immunohistochemical result also shows that VEGF-A can be positioned in the endothelium of pulmonary vessels, thereby further verifying the effectiveness of the technology. Also provides a certain reference for the basic research method in the aspect of neonatal respiratory disease genes.
Drawings
FIG. 1 is an electrophoretogram of PCR amplification products of VEGF-A;
FIG. 2 is a photograph of an endotracheal tube;
FIG. 3 shows the results of frozen section examination of lung, liver, spleen and kidney tissues;
FIG. 4 shows the results of immunohistochemical detection of lung tissue.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.
1. Experimental animals: healthy and clean Wistar newborn rats aged 7-10 days and 20-30g in body mass are tested in laboratory of laboratory zoology of Xinjiang medical university. The newborn rat and the mother rat are bred together, the temperature of an animal house is 25 +/-1 ℃, and the relative humidity is 40-60%.
2. Experimental reagents and instrumentation: a PCR amplification kit; a plasmid extraction kit; a humanized VEGF-A primer; a restriction enzyme; a VEGF-A monoclonal antibody; 75% alcohol; a PCR instrument; sequencing the positive clone; a voltage-stabilizing DNA electrophoresis apparatus; a small animal ventilator; laser confocal fluorescence microscopy; an optical microscope; 4.5F scalp needle; a PE50 catheter; 1.9F central venous catheter core; an LED lamp; and (4) a liquid transferring gun.
EXAMPLE 1 construction of recombinant adenovirus vectors carrying VEGF-A
1. VEGF-A overexpression adenovirus vector construction: the vector name is GV314 (element sequence: CMV-MCS-3FLAG-SV40-EGFP), the BamHI/AgeI enzyme digestion is adopted to linearize the vector, the target gene humanized VEGF-A is amplified by a PCR method, the PCR reaction system is shown in Table 1, the amplification product is shown in figure 1, and the specific sequence of the amplification primer is shown as follows:
VEGFA(4907-1)-P1:AGGTCGACTCTAGAGGATCCCGCCACCATGAACTTTCTGCTGTCTTGG；(SEQ ID NO.1)
VEGFA(4907-1)-P2:TCCTTGTAGTCCATACCCCGCCTCGGCTTGTCAC(SEQ ID NO.2)。
TABLE 1 reaction System
| Reagent | Volume (μ L) |
| ddH2O | 32.5 |
| 5× |
10 |
| dNTP Mix(2.5mM each) | 4 |
| Upstream amplification primer (10. mu.M) | 1 |
| Downstream amplification primer (10. mu.M) | 1 |
| Form panel1(10ng/μL) | 1 |
| PrimeSTAR HS DNA polymerase | 0.5 |
| Total | 50 |
Wherein the template source is plasmid abstinent bacteria liquid. The dosage of the plasmid template is generally less than 200ng, the dosage of the bacterial liquid template is generally 1 mu L, and the optimal dosage refers to the instruction for using PrimeSTAR HS DNA polymerase.
The reaction procedure is as follows: pre-denaturation at 98 ℃ for 5 min; denaturation at 98 ℃ for 10 s; annealing at 55 ℃ for 10 s; extension at 72 ℃ for 90 s; for 30 cycles, final extension at 72 ℃ for 8min, and storage at 4 ℃.
As shown in figure 1, a reaction system prepared by the PCR amplification product and a linearized adenovirus expression vector is subjected to recombination reaction to realize the in vitro cyclization of the linearized vector and a target gene segment, the recombined product is transformed into competent cells, a proper amount of bacterial liquid is inoculated to a plate for culture, a single clone on the plate is selected for PCR identification, and the positive clone is subjected to sequencing and result analysis. And carrying out amplification culture and extraction on the correct clone bacterial liquid to obtain the high-purity adenovirus shuttle plasmid carrying the VEGF-A gene.
2. Adenovirus packaging and detection: the main process is that the shuttle plasmid of adenovirus carrying exogenous gene and the auxiliary packaging plasmid pBHGlox delta E1,3Cre carrying most genome of adenovirus (E1/E3 deletion) transfect HEK293 cell together to generate non-replicative recombinant adenovirus carrying exogenous gene. HEK293 cells were digested with 0.25% trypsin and cultured until cell density reached 50-60% for transfection. Adenovirus shuttle plasmid and helper packaging plasmid were each 5. mu.lg, mixing the mixture with a DMEM medium uniformly, and incubating the mixture with the cationic liposome to form a compound at room temperature. The complex is transfected into HEK293 cells, the culture is continued, and the virus is collected when most cells are diseased. The HEK293 cells in good growth state are transferred into a T25 cell culture flask, viruses are added, and the cells are purified, collected and stored after 2 times of amplification. Detecting the physical state, sterile state and virus titer of the purified virus, wherein the virus titer is 1 multiplied by 1011(PFU/ml)。
EXAMPLE 2 self-made endotracheal tube
Taking a 4.5F scalp needle, removing the extension tube, connecting the front end of the needle core with a PE50 catheter, and extending about 1.5-2.0 cm beyond the needle core; the front end of a needle core of the 1.9F central venous catheter is intercepted and inserted into a scalp needle (the insertion depth is shorter than the front end of a PE50 catheter), so that on one hand, the length of the tracheal catheter is prolonged, the operation is convenient, on the other hand, the hardness of the front end of the PE50 catheter is increased, the tracheal catheter is convenient to insert into the trachea of a newborn rat, and the tracheal catheter is soaked in 75% alcohol for standby after being prepared, as shown in figure 2.
Example 3 construction of rat model
1. Setting parameters of a small animal breathing machine: the respiratory rate is 90-100 times/min, and the tidal volume is 4-6ml/kg for standby.
2. 20-30g of new rats for model building are taken, ketamine (100mg/kg) and xylazine (10mg/kg) are used for intraperitoneal anesthesia, and after the anesthesia is effective, the trachea intubation is performed.
3. Hanging the front incisor and clinging the back of the newborn rat to the tripod support to fix the newborn rat, irradiating the neck of the newborn rat by the LED lamp facing the head and the face of the newborn rat, measuring the body surface distance from the front incisor to the throat by using the tracheal catheter, and marking.
4. The operator faces the back of a newborn rat, pulls out the tongue from the left mouth corner of the rat to the outer upper side with the left hand, a light spot with the size of about 2mm can be seen in the center of the throat, the light spot can be opened and closed along with respiratory motion, namely, a glottis part, the operator holds the self-made tracheal catheter with the right hand, inserts the glottis to the catheter mark part, and fixes the tracheal catheter.
5. Each newborn rat needs to be transfected with adenovirus 2X 108PFU, due to the preparation of adenovirus vector titer of 1X 1011(PFU/ml), so pipetting was used2 μ L of adenovirus transfection stock solution was aspirated by a gun, diluted to 25 μ L with PBS, and injected into an endotracheal tube.
6. Placing the newborn rat in a horizontal position, connecting a small animal respirator to promote the medicine to be dispersed into the lung and maintain stable respiration, and withdrawing the machine after the newborn rat is anesthetized and conscious, and removing the tracheal catheter.
7. After the new rat transfects the recombinant adenovirus for 24 hours, tissues such as lung, liver, spleen, kidney and the like are extracted, frozen sections are made, paraffin sections of lung tissues are made, and the transfection effect is identified.
Example 4 identification of adenovirus vector transfection targeting
After anesthetizing a newborn rat, extracting tissues such as lung, liver, spleen, kidney and the like, washing with PBS, soaking in 4% paraformaldehyde for 4 hours, dehydrating with sucrose and distilled water overnight, embedding with OCT (optical coherence tomography) fixing solution, and preparing a frozen section. The expression intensity of the green fluorescent protein EGFP carried by the adenovirus in each tissue is observed under a fluorescent microscope, and the result shows that the expression intensity of the green fluorescent protein EGFP in the lung tissue is obviously higher than that of other tissues, which indicates that the technology can enable the adenovirus vector to be transfected to the lung tissue in a targeted manner (see figure 3).
Example 5 identification of expression of the protein of interest VEGF-A in Lung tissue
The right upper lung tissue specimen was left, fixed with 4% paraformaldehyde for 1 week, and paraffin sections were routinely prepared for storage (transfection group), and then neonatal rats injected with the empty adenovirus vector were used as a control group. The method comprises the following steps of section dewaxing, cleaning, citric acid heat antigen restoration, room temperature cooling, PBS flushing, serum sealing, adding VEGF-A antibody, keeping the temperature at 4 ℃ overnight, adding a secondary antibody, observing the expression condition of VEGF-A in lung tissues under an optical microscope after staining (brown particles are positive expression), wherein the result shows that the expression of VEGF-A in lung tissues is increased, and the technology can enable target protein to be efficiently expressed in the lung tissues of newborn rats (see figure 4, A in figure 4 is a microscopic examination map of a control group and a transfection group, and B is an expression quantity detection result of VEGF-A in the control group and the transfection group).
Sequence listing
<110> Sinkiang university of medicine
<120> a method for preparing an animal model in which a gene transfected through an endotracheal route is highly expressed in lung tissue
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 48
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
aggtcgactc tagaggatcc cgccaccatg aactttctgc tgtcttgg 48
<210> 2
<211> 34
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tccttgtagt ccataccccg cctcggcttg tcac 34
Claims (5)
1. A method for preparing an animal model in which a gene transfected through an endotracheal route is highly expressed in lung tissue, comprising the steps of:
(1) constructing a recombinant virus vector carrying the vascular endothelial growth factor A, and adjusting the virus titer to 0.5-2 multiplied by 1011PFU/mL；
(2) The animal is anesthetized and then suspended and fixed, the neck of the animal is irradiated, the oral cavity of the animal is opened, then the tracheal catheter is inserted, the virus is injected into the lung tissue of the animal through the tracheal catheter, then the animal is connected to a breathing machine until the animal is awake, and finally the tracheal catheter is pulled out.
2. The method for preparing an animal model in which a gene transfected by an endotracheal route is highly expressed in lung tissue according to claim 1, wherein the recombinant viral vector is a recombinant adenovirus vector comprising a vector plasmid GV314 having the element sequence CMV-MCS-3FLAG-SV40-EGFP, and a helper packaging plasmid.
3. The method for preparing an animal model in which a gene transfected through an endotracheal route is highly expressed in lung tissue according to claim 1, wherein the endotracheal tube is prepared by:
taking a 4.5F scalp needle, removing the extension tube, connecting the front end of the needle core with a PE50 catheter, and enabling the catheter to exceed the needle core by 1.5-2.0 cm after connection; then the front end of the needle core of the central venous catheter of 1.9F is cut out and inserted into the scalp needle, and the insertion depth does not exceed the front end of the PE50 catheter.
4. The method for preparing an animal model in which a gene transfected by an endotracheal route is highly expressed in lung tissue according to claim 1, wherein the animal model is a neonatal rat model or an adult rat model.
5. The method for preparing an animal model in which a gene transfected by an endotracheal route is highly expressed in lung tissue according to claim 1 or 4, characterized in that the specific process of the step (2) is:
(1) after the rats are anesthetized in the abdominal cavity, the rats are fixed on a triangular bracket in a front door tooth suspension and back clinging mode, and then the neck of the rats is irradiated by an LED lamp;
(2) pulling out the tongue body of the rat from the left mouth corner, inserting a tracheal catheter into the glottis part, injecting recombinant virus through the tracheal catheter by using a pipette gun, connecting a respirator with the injection amount of 25 mu L/rat, the connection frequency of 90-100 times/minute and the tidal volume of 4-6mL/kg, removing the tracheal catheter after the rat is awake.
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