CN111642464A - Animal model construction method for transplant vein restenosis research - Google Patents

Abstract

The invention discloses an animal model construction method for graft vein restenosis research, wherein a F344RG rat with severe immunodeficiency caused by CRISPR/Cas gene editing technology in an embryonic period is selected as a host, the remaining great saphenous vein of a CABG postoperative patient is selected as a graft, and a human great saphenous vein is implanted into the abdominal aorta of the F344RG rat by using a cannula method to establish a humanized rat model. The establishment method of the model is simple and convenient, can be repeated, has low rat death ratio, can complete the operation by only one person, and provides a feasible animal model scheme for the research on the aspect of the restenosis of the CABG postoperation vein bridge.

Description

Animal model construction method for transplant vein restenosis research

Technical Field

The invention relates to an animal model construction method for a transplant vein restenosis research.

Background

With the improvement of living standard of people, Coronary atherosclerotic heart disease (CHD) has become the number one killer of modern people, and the importance of the CHD is higher and higher. Clinically, the surgical treatment of CHD usually selects autologous great saphenous vein for Coronary Artery Bypass Graft (CABG), but the problem of postoperative restenosis of the graft vein is difficult to solve.

In the animal validation phase of studies of restenosis following graft vein surgery, it is often necessary to establish animal models of saphenous vein grafts. At present, rabbits are often used as experimental individuals to establish a model for the communication between jugular arteries and veins. However, the lack of reagents (such as antibodies) for rabbits, the low data in the gene bank and the slow progress of related research affect the continuity of animal model research and bring inconvenience. Most of the current animal models are connected to the arterial environment through the autologous veins of animals by means of microsurgery, which requires experimenters to have skillful microsurgery operation techniques, and is not favorable for popularization and promotion of the research. And the transplanted vein is the autologous vein of the experimental animal and not the great saphenous vein of the human, and the experimental result can be only used as an analogy reference and cannot be exactly concluded. If the vein of a common experimental animal is replaced by the great saphenous vein of a human, the influence of immunological rejection caused by species difference on the experimental result is difficult to avoid. For the above reasons, there is an urgent need to establish a humanized animal model, and the immune background of the subject cannot affect the graft.

Disclosure of Invention

The invention aims to provide an animal model construction method for graft vein restenosis research, the model construction method is simple and convenient and repeatable, the death ratio of a rat is low, and the operation can be completed by only one person, so that a feasible animal model scheme is provided for the research on the vein bridge restenosis after CABG operation.

In order to achieve the purpose, the technical scheme of the invention is to design an animal model construction method for transplant vein restenosis research, wherein a F344RG (F344-Rag 2-/-Il 2 rg-/-) rat (T, B and NK cell function deficiency) which causes severe immunodeficiency in an embryonic period by using a CRISPR/Cas gene editing technology is selected as a host to avoid immune rejection reaction, the residual great saphenous vein of a patient after CABG surgery is used as a graft, and a human great saphenous vein is implanted into the abdominal aorta of the F344RG rat by using a cannula method to establish a humanized rat model.

Preferably, a plurality of (e.g. 18) severe immunodeficiency F344RG rats are selected, the weight is 150-.

Preferably, multiple sections (such as 24 sections) of the human saphenous vein graft are selected, wherein each section is 1.8 +/-0.2 cm long, 4 +/-0.8 mm in diameter, free of redundant tissues and free of vein branches.

Preferably, F344RG rats were fasted for 24h prior to cannula surgery and fed a 5% glucose solution to complete bowel preparation to facilitate intraoperative visual field exposure.

Preferably, F344RG rats were fed IVC single cages after the cannula surgery, fasted for 12h, fed with 5% glucose solution, and after 12h, fed with glucose solution and fed with water normally; penicillin is injected into muscles with the concentration of 4 million U per 100g, and is taken once a day and is used for resisting infection continuously for three days; after operation, 20u/100g subcutaneous injection is used for three days, the medicine is taken once a day, and anticoagulation is carried out for three days continuously.

The rat model carrying human cells, tissues or organs in vivo is called a humanized rat (humanized rat), and is a chimera of human cells or tissues with rats after transplantation. It can better simulate the characteristics of human environment in some aspects, thus being an ideal animal model. The CRISPR/Cas system present in most bacteria and most archaea is the acquired immune system in which a bacterium is currently found, foreign plasmids or phages can be eliminated by them, leaving foreign gene fragments in their own genome as "memory", all known as the Clustered/Clustered signature system of constant Palindromic Repeats (Clustered Reg μ Larly interleaved Short Palindromic Repeats/CRISPR-associated proteins). Through these genomes, and using enzymes such as Cas9 as "scissors", one can accurately and efficiently edit partial genes in the organism, namely, CRISPR/Cas9 gene editing technology. The invention summarizes the past experience and tries to establish an animal model of CABG postoperation vein bridge restenosis, decides to select F344RG (F344-Rag 2-/-Il 2 rg-/-) rat (T, B and NK cell function deficiency) which causes severe immunodeficiency in embryonic stage by using CRISPR/Cas gene editing technology as a host to avoid immunological rejection reaction, uses the residual great saphenous vein of a CABG postoperation patient as a graft, and establishes a humanized rat model by implanting the human great saphenous vein into the abdominal aorta of the F344RG rat by using a cannula method. The model is simple and convenient to establish, can be repeated, has low rat death ratio, can be operated by only one person, and provides a feasible animal model scheme for the research on the aspect of CABG postoperative vein bridge restenosis.

Drawings

FIG. 1 is a diagram of a modified casing method;

FIG. 2 is a drawing of materials;

FIG. 3 is a graph of flow cytometry test results;

FIG. 4 is a graph showing the results of serum antibody level measurements;

FIG. 5 is a graph of HE staining results;

FIG. 6 is a graph of the change in the inner membrane, middle membrane + outer membrane thickness and the ratio of the two;

FIG. 7 is a graph of immunohistochemical α -SMA staining results;

FIG. 8 is a view for ultrasonically observing the inside diameter of a graft vein;

fig. 9 is a graph of mTOR pathway related protein over time.

Detailed Description

The following description of the embodiments of the present invention will be made with reference to the accompanying drawings. The following examples are only for illustrating the technical solutions of the present invention more clearly, and the protection scope of the present invention is not limited thereby.

Animal model construction method and related index detection

1.1 materials

1.1.1 Experimental animals

18 severe immunodeficiency F344RG rats were purchased from Beijing Wintoda Biotechnology Ltd, weighing 150-. IVC room feeding, experimental animal feeding and operation are all completed in the experimental animal center of Ministry of medicine of Nanchang university. The experiment was approved by the ethical committee of the first subsidiary hospital of Nanchang university.

1.1.2 grafts

24 segments of human saphenous vein graft with length of 1.8 +/-0.2 cm and diameter of 4 +/-0.8 mm are respectively taken from the residual saphenous vein of 6 CABG postoperative patients of the heart macrovascular surgery of the first subsidiary hospital of Nanchang university in 11 months in 2017 to 3 months in 2018 (informed and signed with informed consent).

1.1.3 Main Equipment instrumentation

SZ61 binocular surgical microscope: olympus et al, Japan

A western blotting apparatus: Bio-Rad Ltd

An ultracentrifuge: ThermoFisher Co

Vevo 2100 small animal ultrasound imaging platform: nippon Fuji Co Ltd

Microsurgical instruments: shanghai medical instruments Co Ltd surgical instruments factory

Leica EG1150H paraffin embedding machine: germany Leica

Leica RM2235 manual rotary paraffin slicer: germany Leica

-80 ℃ refrigerator: mitsubishi electric appliances Co Ltd

Binocular optical microscope: nikon for treating Japanese disease

Electric heating constant-heating water bath box: south China of Jiangsu province and Tonghai electric appliance factory

Fluorescence microscopy: olympus et al, Japan

1.1.4 Experimental reagent and consumable

RICTOR、PKC-α、Raptor、4EBP、

EIF4B, β -Actin mouse monoclonal antibody: abcam Corp Ltd

Rabbit anti-mouse, goat anti-mouse secondary antibodies: abcam Corp Ltd

HE staining kit: china fir Jinqiao Co Ltd

4% paraformaldehyde: beijing Solaibao Tech & ltTech & gt Ltd

α -SMA antibody: china fir Jinqiao Co Ltd

10% chloral hydrate: wuhanding high-tech Co Ltd

Western-blot gel preparation kit: philippine de SpA

Developing solution: ThermoFisher Co

Antibody dilution: philippine de SpA

Sealing liquid: philippine de SpA

Nitric acid fiber film: millipore Corp

PROLENE 4-08682G ETHICON suture: johnson company of America

PROLENE 8-0W 2777 ETHICON suture: johnson company of America

MERSILK 4-0W 501H ETHICON suture: johnson company of America

20G 1.1mm × 30mm BD Intima II PLUS venous indwelling needle: BD Co of USA

IgG, IgA, IgM detection kit: china fir Jinqiao Co Ltd

Heparin sodium: nanjing Xinbai pharmaceutical Co Ltd

Penicillin sodium for injection: north China pharmaceutical Co Ltd

1.1.5 Primary solution reagent formulation

1）PBS：

Nacl	8.00g
		Kcl	0.20g
NaH2PO4·2H2O	1.15g
		KH2PO4	0.20g
Double distilled water	1000ml

Filling into 500ml glass bottle, sterilizing with high pressure steam, cooling, and storing at 4 deg.C.

2）10%SDS:

SDS	10g
		Distilled water	90ml

3) TdT enzymeReaction solution

Equilibratin buffer	98µl
		Biotinylated nucleotides	1µl
TdT enzyme	1µl

4) 10% separation gel and 5% concentrated gel:

10% separation gel (10 ml) 5% concentrated gel (5 ml)

Ultrapure water	4.1ml	2.85ml
			30%Acr/Bis	3.3ml	0.85ml
Gel separation buffer (PH8.8)	2.5ml	-
			Concentrated gel buffer (PH6.8)	-	1.25ml
10% sodium dodecylbenzenesulfonate	100µl	50µl
			10% ammonium persulfate	50µl	25µl
TEMED	5µl	5µl

5) 5 × protein electrophoresis loading buffer:

0.5M Tris·Hcl	2.5ml
		DTT	0.39g
SDS	0.5g
		bromophenol blue	0.025g
Glycerol	2.5ml

6) 10 × electrophoretic fluid:

Tris	3.03g
		Glycine	18.8g
SDS	1.0g

dissolving in 800ml double distilled water, stirring until the solution becomes transparent, and fixing the volume to 1000 ml. It can be diluted when used.

7) 1 × electrotransformation liquid:

Tris	3.03g
		Glycinne	14.4g

dissolving with 600ml double distilled water, stirring until the solution becomes transparent, diluting with double distilled water to 800ml, and adding 200ml methanol before use.

8) TBS buffer:

Tris	2.42g
		Nacl	8.01g

double distilled water was added to 1000ml, and the pH was adjusted to 7.6.

9) TBS-T buffer:

Tween-20	1ml
		TBS	1000ml

can be used after being mixed evenly, and is prepared for use.

10) 5% milk sealing liquid:

defatted milk powder	5g
		TBST	100ml

11）10mmol/L PMSF

PMSF	0.174g
		Isopropanol (I-propanol)	100ml

After dissolution, the solution was dispensed into 1.5ml centrifuge tubes and stored at-20 ℃.

12) 1% bromophenol blue:

bromophenol blue	100mg
		ddH2O	10ml

Stirring, fixing volume and filtering.

1.2 methods

1.2.1F 344RG rat immune background assay

1.2.1.1 serum antibody detection

Add 100. mu.l negative control to wells A1 and A2, respectively;

add 100. mu.l positive control to the reaction plate A3 and A4 wells, respectively;

take 100. mu.l of the solution through a 1: 40, adding the diluted sample to be detected into a corresponding hole, and recording;

incubating at 37 ℃ for 30 min;

discarding the liquid in each hole into a waste liquid barrel, adding 250 mul of washing liquid (20 multiplied by the concentrated washing liquid in the reagent box needs to be diluted by distilled water or deionized water for use, if crystallization exists, the washing liquid needs to be dissolved firstly and then diluted) into each hole for washing the plate, and repeating for 5 times, wherein each time lasts for 30 s;

adding 100 mul of enzyme-labeled conjugate into each hole;

incubating at 37 ℃ for 30 min;

adding 100 mul of substrate solution into each hole;

incubating at 37 deg.C for 10min (color development in dark);

adding 100 mul of stop solution into each hole;

the absorbance value, i.e.the OD450 value, of each well was measured immediately at a wavelength of 450 nm.

Negative control: under normal conditions, the OD450 value of the negative control hole is less than or equal to 0.2; positive control: under normal conditions, the OD450 value of the positive control hole is more than or equal to 0.4

1.2.1.2 flow cytometric detection of lymphocyte species

Adding separating medium, blood sample and normal saline, and centrifuging at 2500r/min for 30 min.

Adding corresponding antibody, incubating in dark for 20min

Adding hemolysin, standing at room temperature for 10min

pbs washing for 1 time, and machine sorting.

1.2.2 animal experiments

1.2.2.1 Experimental groups

The 18 rats were randomly assigned to 3 groups T1, T2, T3 at the time of sacrifice on days 7, 28, and 84 after surgery. Blank control T0 was an unplanted venous segment.

1.2.2.2 graft preparation

Since the graft is a human saphenous vein, preparation of the graft is initiated from the operating room of the hospital. In the operation, the great saphenous vein of a patient is used for coronary artery bypass transplantation, when the distal end suture is completed and the proximal end suture is prepared, a doctor of a main scalpel can measure the transplanted vein and cut off redundant parts, and the redundant parts are the experimental graft. The removed great saphenous vein is soaked in heparin normal saline, the syringe with a blunt needle head is used for sucking the heparin normal saline to flush the vein lumen, and the extra fat and connective tissue outside the vein are carefully removed by using non-invasive forceps and fine scissors. Measuring and segmenting the vein, wherein each segment is about 1.8cm, and the transplanted vein meeting the requirements is ensured to have no redundant tissue and no vein branch. After the treatment was completed, the cells were placed in a container containing pre-cooled sterile PBS and placed in a refrigerated transport box and taken out of the operating room. At this point the preparation of the graft vein is complete.

1.2.2.3 surgical Environment

Is limited by immunodeficient rats, the whole operation environment is required to be as sterile as possible, and experimenters need to strictly observe the sterile operation. The operation is carried out in a clean bench with ultraviolet irradiation for 30 min.

1.2.2.4 preoperative preparation

F344RG rat was used as the receptor. Preoperative fasting for 24h and feeding of 5% glucose solution to complete the intestinal tract preparation facilitates the exposure of the visual field during surgery.

1.2.2.5 Abdominal aortic Exposure

The anesthesia was performed by intraperitoneal injection with 10% chloral hydrate at 0.4ml/100 g. After the anesthesia effect, the whole body is disinfected by iodophor for three times and fixed on a special dissecting table for rats which is paved with sterile gauze and is irradiated by ultraviolet in advance. Spreading a sterile sheet, making a median incision on abdomen, making the incision reach 1cm below xiphoid process and 1cm above urethral orifice, acutely separating skin and abdominal wall muscle, sewing a MERSILK 4-0 suture on left and right abdominal walls, and drawing towards both sides to expose operation field. At this time, heparin was injected into the tail vein at a dose of 30u/100g, and ACT was monitored for over 180 seconds. The intestinal canal is pulled out of the abdominal cavity gently by using atraumatic forceps, taking care not to damage the mesentery, and the surface is covered with gauze moistened by warm normal saline, so that the liquid loss and the heat loss are reduced. The retroperitoneum was cut open, the aorta and inferior vena cava were exposed, and the tissue between the aorta and inferior vena cava was carefully separated to free the aorta. The free length of the aorta was up to 1cm above the level of the renal arteries and down to the iliac bifurcation. And respectively blocking the blood flow of the aorta at the positions 5mm above the renal artery level and 2mm above the iliac artery bifurcation by using the atraumatic vascular clamps, and temporarily filling the aorta at the operation section with blood by first installing the distal vascular clamp and then installing the proximal vascular clamp. At this point the receptor is ready.

1.2.2.6 improved casing method

Taking a 20G BD Intima II PLUS venous indwelling needle with the diameter of 1.1mm multiplied by 30mm, the outer diameter of 1.3mm and the inner diameter of 1.1mm, drawing out an inner core, shearing 1.2cm, and immersing one end of the inner core in heparin normal saline for later use.

FIG. 1 is a schematic view of a modified casing process, as shown in FIG. 1; wherein, A and I are free of aorta and are prepared to be inserted with an indwelling needle after being blocked; B. binding and fixing the remaining needle; C. thirdly, crossing the aorta and setting the inner bracket of the indwelling needle; D. fourthly, turning over the aorta stump of the rat, and bundling up and fixing to prepare an oversleeve; E. fifthly, grafting the graft vein, and bundling and fixing; f is a schematic step diagram. According to the steps in the schematic diagram, the anterior wall of the rat aorta at the position 5mm away from the upper edge of the vascular clamp at the far end is cut by about 0.8mm by using fine scissors, the wedge-shaped end is inserted into the laceration, and the vein indwelling needle after the whole treatment is inserted into the aorta cavity. The laceration was sutured with PROLENE 8-0W 2777 ETHICON suture and the indwelling needle was moved down so that its distal end was located below the aortic suture. The aorta was tied together with the indwelling needle using MERSILK 4-0W 501H ETHICON suture at a distance of about 3mm from both ends of the indwelling needle to fix the position of the indwelling needle. The middle of the operative segment vessel is cut with fine scissors together with the aorta and the indwelling needle. At this point the two ends will retract and the two will separate by about 5-6 mm. The aortic fold at the severed end was "cuff" like and was maintained in folded state by tying with PROLENE 8-0W 2777 ETHICON suture.

Taking out the pretreated human saphenous vein, sleeving the sleeves with two folded ends into the human saphenous vein, and binding the saphenous vein and the sleeves by using MERSILK 4-0W 501H ETHICON suture to ensure that the aortic intima of the rat is in contact with the intima of the saphenous vein to form a sandwich structure: the outermost layer is a great saphenous vein, the middle part is two layers of folded rat aorta walls, and the innermost layer is a vein retention needle.

And finally, releasing the proximal end artery clamp, puncturing the hyperemic human saphenous vein by using a 1ml syringe, pumping out a small amount of residual gas in the vein, pulling out the needle head, loosening the distal end artery clamp, and slightly pressing the puncture point by using warm physiological saline gauze for 1min to stop bleeding.

1.2.2.7 finishing the operation

The intestinal tube pulled out of the body is returned, the abdominal cavity is flushed with warm normal saline, and the PROLENE 4-08682G ETHICON suture is continuously sutured to close the abdomen layer by layer. The surgical incision was sterilized with iodophor and covered with dressing. Meanwhile, observing the blood flow condition by using a Vevo 2100 small animal ultrasonic imaging platform and recording related data.

1.2.2.8 post-operative management

IVC is fed in single cage, fasting for 12h, feeding 5% glucose solution, stopping feeding glucose solution after 12h and normally feeding water. Penicillin is injected into muscle with 4 million U per 100g, and the penicillin can resist infection after being continuously used for three days. After operation, 20u/100g subcutaneous injection is carried out for three days, and anticoagulation is carried out for three days continuously.

1.2.2.9 judgment of success of surgery

After operation, the patient can be awake normally and can move freely after 24h, and the patient can be regarded as successful in the operation if the patient can normally respond to the acousto-optic stimulation. If the rats die after the operation and are not awake, hind limb or tail movement disorder, bluish purple color and low skin temperature are considered as operation failure.

1.2.3 ultrasonic examination

And selecting a 3MHz linear array probe by adopting a Vevo 2100 small animal ultrasonic imaging platform. Probed from the abdomen of the rats after the immobilization. The method is mainly used for evaluating whether the transplanted vein is unobstructed and the stenosis degree.

Grayscale ultrasound: the presence or absence of thickening of the tunica media and plaque formation were evaluated by transverse and longitudinal section examination.

Color Doppler ultrasound: the scale is adjusted to a proper level, so that the normal rat aorta section does not have aliasing, and the aliasing phenomenon prompting the blood flow acceleration is observed.

1.2.4 pathological examination of transplanted veins

1.2.4.1 tissue section staining

After the ultrasound examination, the rats were sacrificed by spondylolysis, opened abdomens, the graft veins were searched and the peripheral adherent tissues were carefully peeled off, and the graft veins were completely removed. Cleaning with physiological saline, discharging the blood remained in the blood, taking a part of vein, performing gradient cooling, freezing in a-80% refrigerator, and performing protein detection. The remaining veins were fixed in neutral buffered formalin for pathology.

1.2.4.2 Paraffin embedding

Taking out the fixed vein, and performing dehydration operation:

1) 70% ethanol for 1 hour.

2) 95% ethanol (95% ethanol/5% methanol) for 1 hour.

3) The first time the absolute ethanol is kept for 1 hour.

4) The second time of absolute ethyl alcohol for 1.5 hours.

5) And the third time is 1.5 hours of absolute ethyl alcohol.

6) Fourth time absolute ethanol for 2 hours.

7) The first xylene elution was 1 hour.

8) The second xylene elution was 1 hour.

9) The tissue is taken out and embedded in a paraffin embedding machine.

1.2.4.3 HE staining

1) Slicing: the thickness is adjusted to be 5um, and the anti-drop glass slide is fished and carefully unfolded.

2) Dewaxing: heating at 60 deg.C for 30min, melting, and dewaxing with xylene I and xylene II for 5min respectively

3) Rehydration: placing the slices into 100%, 95%, 90%, 80%, and 70% ethanol according to gradient, retaining each stage for 5min, and finally adding distilled water for 3min to complete rehydration.

4) Dyeing: staining in hematoxylin for 20min at room temperature controlled at 25 deg.C.

5) Washing with water: and (5) rinsing with a mild water flow for 15min to make the to-be-sliced become blue.

6) Differentiation: the slices were put into 1% ethanol hydrochloride solution and gently shaken for 20s, and the slices turned red.

7) Rinsing: and (5) rinsing with mild water flow for 5min to make the to-be-sliced become blue.

8) And (3) dehydration I: preparing alcohol according to gradient of 50%, 70%, 80%, placing the slices therein for 5min each

9) Counterdyeing: 0.5% eosin ethanol for 2min

10) And (II) dehydration: washing with 95% ethanol, dehydrating with anhydrous ethanol for 5min, and taking out to evaporate.

11) And (3) transparency: placing into xylene I and xylene II for 5min respectively.

12) Sealing: and (5) sealing and storing the neutral gum.

1.2.4.4 immunohistochemical SMA staining

1) The tissue sections of the patches were soaked in PBS for 10min, and after drying, the samples on the slides were enclosed with an immunohistochemical pen. Dripping 150 μ l PBS on each slide, standing for 5min, adding 150 μ l of permeable blocking solution (0.3% Triton +1% BSA +5% Donkey Serum), standing at room temperature for 1 h;

2) removing blocking liquid by suction, adding 250 mu l of PBS into each slide, standing for 5min, removing PBS by suction, and washing twice each slide;

3) adding the diluted corresponding primary antibody into each slide, and incubating for 2h at room temperature or overnight at 4 ℃;

4) pipette off primary antibody, wash 2 times per slide with PBS to avoid non-specific staining;

5) adding 100 mul of diluted corresponding secondary antibody diluent into each slide, and incubating for 1 hour at room temperature;

6) absorbing the secondary antibody by using a pipette gun, and washing twice by using PBS;

7) adding 100 mul Hoechst nuclear stain into each slide, and incubating for 20min at room temperature;

8) the blocking solution was aspirated off with a pipette and washed twice with PBS for 10min each time.

9) And (3) sucking water on the slide, adding 1-2 drops of the anti-fluorescence attenuation agent, covering the slide with a cover glass, drying in the dark for 1 hour, and taking a picture by a microscope.

1.2.5 Western-blot detection of related proteins in transplanted veins

1.2.5.1 extraction of Total protein in tissues

1) And (3) placing the reserved transplantation vein into a 1-2 ml homogenizer, and shearing the transplantation vein.

2) Adding 400ul of PMSF-containing solution, splitting in a homogenizer, and adding the transplanted vein tissue fragment for homogenizing. And then placed on ice. This was repeated several times until the tissue was crushed as much as possible.

3) Performing lysis at low temperature, transferring the lysate into a 1.5ml centrifuge tube by using a pipette after 30min, centrifuging at 12000rpm at 4 ℃ for 5min, and collecting the supernatant and subpackaging in a 0.5ml centrifuge tube.

4) Adding loading buffer, boiling water bath for 5min, and storing at-20 deg.C. Sample preparation was completed.

1.2.5.2 Western blot

1) Preparing glue: preparing SDS-PAGE separation gel with proper concentration according to the molecular weight of the target protein, pouring the separation gel into a splint, and preparing concentrated gel after gelling and solidifying after 40 min.

2) After the concentrated glue is filled, the comb is slowly inserted, so that bubbles are prevented from affecting the lane. Standing for 30min, and slowly pulling out the comb after the comb is solidified.

3) Electrophoresis: and putting the gel glass plate into an electrophoresis tank, adding the prepared electrophoresis solution, loading 20ul of sample at each hole by using a sample loading needle, and adding 5ul of rainbow marker into one hole. And (3) performing constant-voltage electrophoresis at 90v for 20min until the marker passes through the concentrated gel and is separated, continuing electrophoresis under the pressure of 120v until bromophenol blue reaches the bottom of the separation gel, and turning off the power supply.

4) Electric transfer (wet transfer method): prying off the glass, cutting the gel at the required part according to a marker, immersing the gel in the electric conversion solution, shearing the nitrocellulose membrane with the corresponding size, and soaking the nitrocellulose membrane in the electric conversion solution. Two pieces of filter paper with corresponding sizes are taken, soaked, clamped with the electric rotating plate in sequence and inserted into the electric rotating groove for electric rotation. And (5) performing constant-voltage electric rotation for 60v and 120min, and applying an ice bag to cool while performing electric rotation.

5) And (3) sealing: the membrane was removed, rinsed in TBST, immersed in blocking solution, and blocked by shaking on a shaker at room temperature for 60 min.

6) Incubating the primary antibody: the sealed membrane is taken out and put into a self-sealing bag filled with primary antibody solution, and the membrane is incubated for 1.5h by shaking in a shaking table at room temperature. Taking out, washing with TBST for 3 times, each time for 15 min.

7) Incubation of secondary antibody: the sealed membrane is taken out and put into a self-sealing bag filled with the secondary antibody solution, and the self-sealing bag is shaken by a table at room temperature for incubation for 1.5 h. Taking out, washing with TBST for 3 times, each time for 15 min.

8) Developing and exposing: mixing the above solutions at a ratio of 1:1 to make the surface of the membrane with protein contact with the mixed solution, and exposing and developing in a developer dark box. And adjusting the exposure time and keeping the picture.

1.3 statistical methods

The SPSS19.0 software is used for analyzing whether the result has statistical significance, and the measurement result is expressed by mean +/-standard deviation. The comparison among groups adopts single-factor analysis of variance, the comparison between every two groups adopts SNK test, and the difference of results is represented by P less than 0.05 and has significance.

Second, result and data analysis

2.1 animal model establishment and vein transplantation

15 of 18F 344RG rats survived to the experimental time point, and 3 died on days 3, 4 and 23 post-surgery, respectively. The lower limbs and the tail of the rest individuals move normally, and the diet and the defecation are normal. Rats were sacrificed and the material was obtained according to the planned time points (as shown in fig. 2, a indicates that the graft vein was in the recipient and B indicates that the graft vein was removed), washed with heparin normal saline and removed of the excess adherent tissue, a part of the sample was left to detect protein, and then paraffin embedding fixation, sectioning (selecting the middle section of the graft vein) and HE staining and immunohistochemical detection were performed.

2.2F 344RG rat immune background

2.2.1 flow cytometry

The flow cytometry results are shown in FIG. 3, which shows that F344RG rat helper/inducer T lymphocyte CD3+ T cell, CD4+ T cell pure subset CD45RA + and CD161a cell deletion

2.2.2 serum antibody levels

The results of the serum antibody level measurements are shown in fig. 4, and demonstrate that the levels of IgG, IgM, and IgA in the serum of F344RG rats are lower than those in F344 rats.

2.3 pathological results and measurement analysis

Thickening the vein inner membrane and the middle membrane and the outer membrane in comparison with a control group on the 7 th day after operation (220.04 +/-18.19μm vs 309.97 +/-26.44μm, P is less than 0.05; 690.58 +/-40.89μm vs 769.27 +/-38.96μm, P is less than 0.05), the venous fibrosis speed is accelerated in the 7-28 days, and the middle membrane, the outer membrane and the inner membrane are remarkably thickened; the inner membrane and the middle membrane + outer membrane thickened on day 84 compared to day 28, but the inner membrane/(middle membrane + outer membrane) ratio did not change significantly (P > 0.05). alpha-SMA expression increased from T1 until T3 was higher than in the control group.

2.3.1 HE staining results

HE stained sections, inner and middle membrane + outer membrane thickness determination using Image pro plus-6.0: using the internal elastic membrane as a boundary, the thickness from the inside to the vessel lumen is the thickness of the intima, the thickness from the outside to the vessel wall is the thickness of the media + adventitia, and each lumen measurement is averaged 10 times.

HE staining results are shown in figure 5: the magnification is 4X 10, and the dark line is the interface of the inner membrane and the middle membrane. The inner membranes of T1, T2 and T3 were thicker than those of T0 (control).

The vessel wall thickness statistics for each group are shown in the following table:

group of	Inner film (mum)	Middle and outer film (mum)	Inner membrane/(middle membrane + outer membrane) ratio
				T0	220.04±18.19	690.58±40.89	(31.94±3.07)%
T1	309.97±26.44	769.27±38.96	(40.29±2.02)%
				T2	431.68±55.55	1017.74±150.36	(43.25±7.58)%
T3	493.69±92.00	1099.68±101.16	(44.56±4.60)%

The changes of the thicknesses of the inner membrane, the middle membrane and the outer membrane and the ratio of the two are shown in FIG. 6; p < 0.05 VS control group (T0).

2.3.2 immunohistochemical staining of alpha-SMA

Immunohistochemical α -SMA staining results are shown in fig. 7, where in fig. 7, a: the distribution of alpha-SMA in the vessel wall (magnification 40 x), the brown dye material is positive alpha-SMA; the dark line is the interface of the intima and the media. B: protein expression of α -SMA in the vessel wall, GAPDH is the internal control. C: quantifying alpha-SMA; p < 0.05 vs control group (T0).

2.4 ultrasound image results

The inside diameter of the graft vein was observed by ultrasound, and as shown in fig. 8, the inside wall of the vein was observed as a long-axis section of the blood vessel, A, B, C, D being the results of imaging on days 0, 7, 28, and 84 after the T3 group graft, respectively.

2.5 Western-Blot detection of mTOR pathway-associated protein changes

The mTOR pathway related protein changes over time are shown in fig. 9; p < 0.05 vs. control group (T0).

When the material was obtained, a part of the graft vein was selected, and after peripheral tissues were cleared, western-blot analysis was performed, and as a result, it was found that Raptor, EIF4e and 4EBP expression was decreased at T1 (P < 0.05 vs. control) compared with the control group T0, and a normal level (P > 0.05) was restored at T2 and T3. Rictor and downstream protein PKC-alpha are always high expressed (T1, T2, T3 vs T0, P < 0.05).

Therefore, the establishment method of the model is simple and convenient, can be repeated, has low rat death ratio, can be operated by only one person, and provides a feasible animal model scheme for the research on the aspect of the restenosis of the CABG postoperation vein bridge.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. An animal model construction method for graft vein restenosis research is characterized in that a F344RG rat with severe immunodeficiency caused by CRISPR/Cas gene editing technology in an embryonic period is selected as a host, the residual great saphenous vein of a CABG postoperative patient is used as a graft, and a human great saphenous vein is implanted into the abdominal aorta of the F344RG rat by using a cannula method to establish a humanized rat model.

2. The method as claimed in claim 1, wherein the severe immunodeficiency F344RG rats with a weight of 150-.

3. The method for constructing an animal model for studying restenosis of graft vein according to claim 1, wherein the human saphenous vein graft is selected from multiple segments, each segment having a length of 1.8 ± 0.2cm and a diameter of 4 ± 0.8 mm.

4. The method for constructing an animal model for studying restenosis of a graft vein according to claim 1, wherein the human saphenous vein graft has no excess tissue and no branch of vein.

5. The method of claim 1, wherein F344RG rats were fasted 24h before surgery and fed with 5% glucose solution.

6. The method of constructing an animal model for transplant vein restenosis research according to claim 1, wherein F344RG rats were fed IVC single cage after surgery, fasted for 12h, fed with 5% glucose solution, and after 12h, fed with glucose solution was stopped and fed with water normally.

7. The method for constructing an animal model for studying restenosis of graft vein as claimed in claim 6, wherein F344RG rat is injected with penicillin 4 ten thousand U/100g intramuscular after operation once a day, and is used for resisting infection after three days.

8. The method for constructing an animal model for studying restenosis of a graft vein according to claim 7, wherein F344RG rat is injected subcutaneously at 20u/100g three days after operation once a day with anticoagulation for three days.

9. The method for constructing an animal model for studying restenosis of graft vein according to claim 1, wherein 18 severe immunodeficiency F344RG rats were selected.

10. The method for constructing an animal model for studying restenosis of graft vein according to claim 1, wherein 24 segments of human saphenous vein graft are selected.

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