CN110408691A - Marker EGR-1 relevant to prediction after liver transplantation rejection and its application - Google Patents

Marker EGR-1 relevant to prediction after liver transplantation rejection and its application Download PDF

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CN110408691A
CN110408691A CN201910735605.8A CN201910735605A CN110408691A CN 110408691 A CN110408691 A CN 110408691A CN 201910735605 A CN201910735605 A CN 201910735605A CN 110408691 A CN110408691 A CN 110408691A
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egr
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刘景丰
陈丽红
朱珠
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Mengchao Hepatobiliary Hospital Of Fujian Medical University (fuzhou Hospital For Infectious Diseases)
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Abstract

The present invention relates to field of medical molecular biology, marker EGR-1 and its application more particularly to prediction after liver transplantation rejection.It is capable of the generation of early warning acute rejection after liver transplantation reaction using marker of the invention, there is preferable directive significance to clinical early diagnosis and therapy acute rejection.

Description

Marker EGR-1 relevant to prediction after liver transplantation rejection and its application
Technical field
The present invention relates to field of medical molecular biology, more particularly to the mark with prediction after liver transplantation rejection Object EGR-1 and its application.
Background technique
The whole world is every year because cirrhosis and PLC mortality number are about 1,340,000 people according to the statistics of the World Health Organization.China is B-mode Hepatitis virus carrier is more, therefore liver cancer incidence is also far beyond American-European countries's height.Generation has been carried out by Starzl professor et al. from 1963 Since boundary's the first mankind's Analyze of Orthotopic Liver Transplantation, as the update of transplanting correlation theory, the perfect of crossmatch technology, organ are protected It deposits the improvement of method and the whole of surgical procedure technical ability improves, liver transfer operation has become the terminals phase such as treatment cirrhosis, liver cancer at present The preferred treatment method of hepatopathy.But due to for liver it is in short supply and transplanting after rejection problem seriously limit its extensively carry out.
Rejection is broadly divided into hyperacute rejection, acute rejection and chronic rejection after transplanting.It is wherein anxious Property rejection be liver transfer operation immunological rejection most common type, be the most important complication of after liver transplantation, at the same be also cause Chronic rejection and most important risk factor is lost for liver function.Ischemical reperfusion injury is that orthotopic liver transplantation is inevitably asked Topic, the innate immune response caused by multifactor, the non-antigen that it causes is to cause liver transplantation early primary nonfunctional and liver After transplanting the main reason for graft rejection.Although existing a large amount of novel immunosuppressive drugs are applied to clinic, due to its target Tropism is poor, administration time is long, side effect is big and part transplant patient's inferior clinical symptom compares concealment leads to treatment not in time, therefore Very satisfactory therapeutic effect is not obtained.For liver function once losing, receptor certainly will face liver retransplantation treatment.This Not only aggravate for liver status in short supply but also more to make receptor's body and mind by huge injury and increase financial burden.Therefore subtract as far as possible Light ischemical reperfusion injury and effectively prevent, treat acute rejection even inducing immune tolerance will be for a long time liver transfer operation and its The focus on research direction of its organ transplant circle.
Summary of the invention
In order to solve the above-mentioned technical problem, inventor be surprised to find that in tissue or serum EGR-1 transcriptional level and/or EGR-1 protein level can be as the marker of prediction after liver transplantation rejection, thereby completing the present invention.
One aspect of the present invention provides the detection reagent of EGR-1 gene transcript and/or the detection reagent of EGR-1 albumen exists Prepare the application in the kit for predicting object after liver transplantation rejection.
Early gowth response factor -1 (Early Growth Response-1, EGR-1) be also known as NGFI-A, Zif268, Krox24, TIS8 etc. are a typical Cys2.His2 type zinc finger transcription factors, identification that can be specific and combination downstream target Gene promoter area is rich in the region GC and in a variety of stimulants (such as growth factor, proinflammatory cytokine, lipopolysaccharides, anoxic, ischemic Or reperfusion injury, ultraviolet light etc.) stimulation under quick and of short duration activation.
In some embodiments of the present invention, the detection reagent of the EGR-1 gene transcript include can be special Property EGR-1 gene transcript primer combination.
In some specific embodiments of the invention, the transcription product includes being obtained by EGR-1mRNA reverse transcription cDNA。
In some specific embodiments of the invention, the primer sets for capableing of specificity EGR-1 gene transcript Conjunction includes having the upstream primer of nucleotide sequence shown in SEQ ID NO.1 and/or with nucleotides sequence shown in SEQ IDNO.2 The downstream primer of column.
In some embodiments of the present invention, the detection reagent of the EGR-1 albumen be Mass Spectrometric Identification reagent, antibody or Its antigen-binding fragment.
In some specific embodiments of the invention, the antibody is sheep anti mouse EGR-1 antibody.
The second aspect of the present invention provides a kind of kit for predicting object after liver transplantation rejection, the kit The detection reagent of detection reagent and/or EGR-1 albumen including EGR-1 gene transcript.
In some embodiments of the present invention, the kit include EGR-1 gene transcript detection reagent and It does not include the detection reagent of EGR-1 albumen.
In some embodiments of the present invention, the detection reagent of the EGR-1 gene transcript include can be special Property EGR-1 gene transcript primer combination.
In some specific embodiments of the invention, the transcription product includes being obtained by EGR-1mRNA reverse transcription cDNA。
In some specific embodiments of the invention, the primer sets for capableing of specificity EGR-1 gene transcript Conjunction includes having the upstream primer of nucleotide sequence shown in SEQ ID NO.1 and/or with nucleotides sequence shown in SEQ ID NO.2 The downstream primer of column.
Further, the kit further includes the detection reagent of IL-6, IL-10 gene transcript.
In some embodiments of the present invention, the detection reagent of the IL-6 gene transcript include can specificity The primer of IL-6 gene transcript combines.In certain preferred embodiments of the invention, the primer combination includes having The upstream primer of nucleotide sequence shown in SEQ ID NO.3 and/or downstream with nucleotide sequence shown in SEQ ID NO.4 are drawn Object.
In some embodiments of the present invention, the detection reagent of the IL-10 gene transcript include can be special Property IL-10 gene transcript primer combination.In certain preferred embodiments of the invention, the primer combination includes tool There are the upstream primer of nucleotide sequence shown in SEQ ID NO.5 and/or the downstream with nucleotide sequence shown in SEQ ID NO.6 Primer.
Further, the kit further includes the detection reagent of reference gene 18S rRNA gene transcript.
In some embodiments of the present invention, the detection reagent of the 18S rRNA gene transcript include can be special The primer of anisotropic 18S rRNA gene transcript combines.In certain preferred embodiments of the invention, the primer combination Including the upstream primer with nucleotide sequence shown in SEQ ID NO.7 and/or there is nucleotide sequence shown in SEQ ID NO.8 Downstream primer.
Still further, the kit further includes that RNA provides reagent.
Again further, the kit further includes reverse transcription reagents.
In some embodiments of the present invention, the kit includes the detection reagent of EGR-1 albumen without including The detection reagent of EGR-1 gene transcript.
In some embodiments of the present invention, the detection reagent of the EGR-1 albumen be Mass Spectrometric Identification reagent, antibody or Its antigen-binding fragment.
In some specific embodiments of the invention, the antibody is sheep anti mouse EGR-1 antibody.
Further, the kit further includes the detection reagent of IL-1 β, IFN-γ.
Still further, the kit further includes that whole-cell protein extracts reagent.
In some embodiments of the present invention, the kit had both included the detection reagent of EGR-1 gene transcript It again include the detection reagent of EGR-1 albumen.
Beneficial effects of the present invention
It is capable of the generation of early warning acute rejection after liver transplantation reaction using marker of the invention, clinic is early diagnosed There is preferable directive significance with treatment acute rejection.
Detailed description of the invention
Fig. 1 shows the postoperative 10th day gross specimen situation of each group Rat Liver Transplantation.
Fig. 2 shows the postoperative hepatic tissue HE dyeing (10 × 20) of Rat Liver Transplantation.A, b, c, d, e are respectively allotransplantation The postoperative 6h, 12h of group, for 24 hours, 5d, 10d hepatic tissue；F, g, h, i, j be respectively isograft group of postoperative 6h, 12h, for 24 hours, 5d, 10d hepatic tissue；K is the postoperative 6h hepatic tissue of sham-operation group.
Fig. 3 shows the postoperative hepatic tissue EGR-1mRNA expression of Rat Liver Transplantation.* it indicates to do evil through another person with same time point Art group (Sham) compares, p < 0.05；# indicates allotransplantation group (Allograft) and isograft group of same time point (Syngraft) compare, p < 0.05.
Fig. 4 shows the postoperative hepatic tissue IL-6mRNA expression of Rat Liver Transplantation.* it indicates and same time point sham-operation Group (Sham) compares, p < 0.05；# indicates allotransplantation group (Allograft) and isograft group of same time point (Syngraft) compare, p < 0.05.
Fig. 5 shows the postoperative hepatic tissue IL-10mRNA expression of Rat Liver Transplantation.* it indicates to do evil through another person with same time point Art group (Sham) compares, p < 0.05；# indicates allotransplantation group (Allograft) and isograft group of same time point (Syngraft) compare, p < 0.05.
Fig. 6 shows EGR-1 albumen WB testing result in transplanting hepatic tissue.A figure is the postoperative EGR-1 interior for 24 hours of each group rat And corresponding GAPDH WB developing result: wherein A, D, G are followed successively by sham-operation group (Sham) 6h, 12h, for 24 hours；B, E, H are followed successively by Isograft group of (Syngraft) 6h, 12h, for 24 hours；C, F, I be followed successively by allotransplantation group (Allograft) 6h, 12h,24h.B figure is each group rat postoperative 5d, 10d EGR-1 and corresponding GAPDH WB developing result: wherein J, M distinguish For sham-operation group 5d, 10d；K, N is respectively isograft group of (Syngraft) 5d, 10d；L, O is respectively that allogene moves Plant group (Allograft) 5d, 10d.C figure is after each group EGR-1 gray value is normalized with corresponding GAPDH gray value as a result, * table Show compared with same time point sham-operation group (Sham), p < 0.05；# indicates allotransplantation group (Allograft) and with for the moment Between put isograft group (Syngraft) and compare, p < 0.05.GAPDH albumen is internal reference albumen.
Fig. 7 shows EGR-1 protein I HC testing result in transplanting hepatic tissue.A figure is EGR-1IHC result (10 × 20), Wherein a, b, c, d, e be followed successively by allotransplantation group (Allograft) 6h, 12h, for 24 hours, 5d, 10d, f, g, h, i, j are followed successively by Isograft (Syngraft) 6h, 12h, for 24 hours, 5d, 10d, k be sham-operation group (Sham) 6h；B figure is liver cell IHC Positive findings score, and * refers to compared with same time sham-operation group (Sham), P < 0.05；C figure is EGR-1 positive lymphocyte ratio , * refers to compared with same time sham-operation group (Sham), P < 0.05；# refers to allotransplantation group (Allograft) and with for the moment Between isograft (Syngraft) compare, P < 0.05.
Fig. 8 shows EGR-1 content in the postoperative serum of Rat Liver Transplantation.* it indicates compared with same time point sham-operation group, p<0.05；# indicate allotransplantation group (Allograft) compared with same time point isograft (Syngraft), p < 0.05。
Specific embodiment
In order to which the technical problems, technical solutions and beneficial effects solved by the present invention is more clearly understood, below in conjunction with Embodiment, the present invention will be described in further detail.
Embodiment
Following example is used herein to demonstration the preferred embodiments of the invention.Those skilled in the art, it will be appreciated that under State the technology disclosed in example represent inventor discovery can be used for implementing technology of the invention, therefore can be considered as implementation this The preferred embodiment of invention.But those skilled in the art should be understood that specific reality disclosed herein according to this specification Many modifications can be made by applying example, still can be obtained identical or similar as a result, rather than away from the spirit or scope of the present invention.
Unless otherwise defined, the term of all technologies as used herein and science, and the technology in fields of the present invention Personnel institute is normally understood equivalent in meaning, and being disclosed reference and their materials of reference will all be incorporated.
Those skilled in the art will recognize or just will appreciate that by routine test many described here Invention specific embodiment many equivalent technologies.These will equally be comprised in claims.
Experimental method in following embodiments is unless otherwise specified conventional method.Instrument as used in the following examples Device equipment is unless otherwise specified laboratory routine instrument device；Test material as used in the following examples, such as without spy Different explanation, is to be commercially available from routine biochemistry reagent shop.
Experimental method in following embodiments, unless otherwise specified, experiment gained measurement data mean+SD (mean ± SD) is indicated, carries out statistical analysis to result using SPSS20.0 software.Two comparison among groups of numerical value are used Student's t test method；Compare two-by-two between multiple groups using one-way analysis of variance (ANOVA).P < 0.05 is considered to have Statistical significance.
The building of 1 Rat Liver Transplantation acute rejection model of embodiment
1. experimental animal
(all experimental rats are purchased from Beijing dimension tonneau China for healthy inbred strais male cleaning grade Lewis rat and BN rat Experimental animal Co., Ltd), 200~250g of weight, confession, recipient body weight be poor≤and 10%, random pair.
Experimental group: isograft group (syngraft), n=15.Establishing by BN rat is donor, BN rat is receptor Model of orthotopic liver；Allotransplantation group (allograft), n=25.Establish by Lewis rat donor, BN rat be by The model of orthotopic liver of body；Sham-operation group (sham), 5.BN rat only switchs abdomen processing.
2. the building of Rat Liver Transplantation acute rejection model
2.1 Preoperative Method
1) 12~15h of rat pre-operative anxiety can't help drinking, and operation carries out under common clean conditions；
2) 4 DEG C of perfusion liquids, for liver test tube of hepari liquid, save liquid preparation；
3) operating table and instrument include the preparation of conduit, needlework etc..
The operation of 2.2 donors
1) donor uses yellow Jackets 50mg/kg intraperitoneal injection of anesthesia；
2) preserved skin sterilizes, and does abdominal transverse incision, bustle is placed under In Rat Lumbar, sufficiently exposes upper abdomen；
3) detachment falciform ligament and deltoid ligament, nearly liver side ligature left inferior phrenic veins；
4) liver is gently opened upwards and protects covering liver with physiological saline gauze, cut lesser omentum, dissociate shape of tail Leaf ligatures liver oesophagus ramus communicans close to liver；
5) intestinal tube is turned over to left-external side and is covered with physiological saline gauze；
6) exposure porta hepatis: free stomach latarget's vein, the ligation of 8-0 silk thread, blunt separation portal vein surrounding tissue to spleen Below vein；Free proper hepatic artery, reserved 5-0 silk thread do not ligature；Upper section in choledochus is separated, away from left and right and caudate lobe Antetheca cuts an angle at common hepatic duct meet about 1cm, is inserted into inner support tube, and the ligation of 5-0 silk thread is fixed and reserves a silk ligature, It does not cut；
7) close to the free coeliac trunk of liver shape of tail blade root, band line is not ligatured；
8) sufficiently free infrahepatic vena cava isolates vena renalis dextra, abuts lower cavity of resorption to left and right common iliac vein crotch Vein ligation vena renalis dextra；
9) dissociate common iliac vein to crotch lower section 1cm, dissociate abdominal aorta；
10) physiological saline of 4 DEG C of 100U containing heparin of 1mL is injected below common iliac vein crotch；4 DEG C of 20mL will be equipped with The syringe of perfusion liquid is fixed in infusion pump, is adjusted rate of flooding 150mL/h and is fixed after being inserted into abdominal aorta, ligatures at this time Reserved silk thread above coeliac trunk；
11) it extracts 1mL heparin injections needle and expands pinprick while cutting off the diaphram by xiphoid-process and enter thoracic cavity, cut chest actively Arteries and veins；
12) to shorten liver isolated time, oversleeve production is carried out in perfusion, will be infused pump speed after injecting 10mL liquid Degree is adjusted to 75mL/h, during which constantly pours the physiological saline of some pre-coolings in liver surface；
13) the specific manufacturing process of oversleeve: first detachment vena renalis dextra, and it is close to common iliac vein crotch detachment common iliac vein； Lower inferior caval vein oversleeve handle (direction for paying attention to sleeve shank) is fixed with blood vessel clip；Lower inferior caval vein is passed through into distal end from casing center Overturning covering oversleeve, is ligatured with 5-0 silk thread and is fixed；Detachment stomach latarget's vein, same method make portal vein oversleeve；
14) (oversleeve production also has been approached completion after general perfusion) after liver perfusion, liver is in the colour of loess at this time Color cuts off liver superior and inferior vena cava against diaphram, and holding notch as far as possible is smooth, the left vena phrenica of detachment, right liver week ligament, left liver Zhou Ren Band, esophageal venous plexus, stomach latarget's vein and free nipple leaf out；
15) ligation and detachment right side adrenal veins clump and lumbar veins, detachment ligature proper hepatic artery, take out liver and are placed in 4 DEG C of preservation liquid save.
The operation of 2.3 receptors
1) the preoperative 30min intramuscular injection atropine 0.1mg/kg of receptor, ether inhalation anesthesia；
2) preserved skin sterilizes, and median incision places bustle under In Rat Lumbar, makes small drag hook by oneself to two side-lining arcus costarums, only into abdomen Blood clamps fixed xiphoid-process and cephalad traction, sufficiently exposes surgical field of view；
3) detachment falciform ligament ligatures left inferior phrenic veins, and liver is gently pushed open to the left, the right liver week ligament of detachment；
4) dissociate nipple leaf, former to ligature right side adrenal veins clump and lumbar veins from liver；
5) liver is gently opened upwards and protects covering cephalad traction with physiological saline gauze, cavity of resorption is quiet under the liver that dissociates Arteries and veins is to vena renalis dextra and retains right kidney arteriovenous；
6) intestinal tube is turned over to left-external side abdomen and protects covering, exposure porta hepatis with physiological saline gauze, the liver that dissociates is consolidated There are artery ligation two sides and cut, dissociate choledochus, ligatures two sides as close to Left And Right Hepatic Duct meet and cuts, by door Vein dissociates to latarget's vein；
7) lesser omentum is cut, dissociate caudate lobe；
8) liver is gently shelled to the right, the left liver week ligament of detachment, ligatures liver oesophagus ramus communicans far from liver；
9) liver is put back in situ, carefully passes through liver superior and inferior vena cava rear wall with curved tweezer, with ensure its opisthodetic ligament from Trunk snap is net；
10) after ensuring that rat anesthesia depth is moderate, etherization is withdrawn, successively blocks infrahepatic vena cava with blood vessel clip And portal vein.Trans-portal vein or so crotch injects 2-3mL physiological saline to drive blood in liver, and most of liver is in the colour of loess at this time Color；
11) with vascular occlusion clamp will (attention avoids hyper-traction big every flesh influence receptor together with part diaphram clamp Mouse autonomous respiration), liver superior and inferior vena cava is blocked, and entirely cut off liver superior and inferior vena cava as far as possible close to liver (rear wall can be appropriate Reservation part hepatic tissue, to ensure to have sufficient length conducive to tube wall suture), be close to liver and cut cavity of resorption under portal vein and liver Vein (antetheca can moderately retain part caudate lobe), so far receptor liver separates completely with surrounding tissue completely, cuts each blood vessel The ligation broken ends of fractured bone removes it；
12) it takes and is placed in receptor that (attention sets position, liver offset occurs after avoiding suture, makes upper and lower cavity caliber for liver Become smaller), first suture confession, receptor liver superior and inferior vena cava right corner, right-side cavity knot outside, cut the band needle broken ends of fractured bone, then suture confession, receptor liver Superior and inferior vena cava left comer, continuously sutures rear wall and antetheca after knotting, wouldn't tighten in suture antetheca, with normal saline flushing blood vessel After bubble is discharged in chamber, suture is tightened, is knotted outside chamber with left comer line tail；
13) blood vessel clip at of short duration release portal vein, to release the high blood coagulation of a little portal vein, normal saline flushing lumen is used Micro- artery forceps clamps donor portal vein oversleeve (it is ensured that blood vessel is without torsion), and Xiang Shouti portal vein is close, will insert for liver casing It is intracavitary to enter receptor portal vein, the ligation of 5-0 silk thread is fixed；
14) blood vessel clip at portal vein is removed, portal venous flow is opened, when seeing infrahepatic vena cava has blood outflow, It can remove vascular occlusion clamp, terminate without the liver phase.Liver softly is stirred, makes each leaf is intact reset, be well perfused.Visible liver at this time It reddens rapidly；
15) of short duration release receptor infrahepatic vena cava artery clamp releases a little high blood coagulation, cavity of resorption under normal saline flushing liver Vein lumen, will be intracavitary for liver inferior caval vein casing insertion receptor inferior caval vein, and the ligation of 5-0 silk thread is fixed, and artery herein is removed Press from both sides open Inferior Vena Cava Blood Flow；
16) angular cut is cut at common bile duct ligation, appears lumen, receptor lumen will be inserted into for hepatic duct inner support tube Interior, the ligation of 5-0 silk thread is fixed, and the ligature is tensed with the root knot binding for reserving at hepatic duct and is knotted；
17) intraperitoneal foreign matter is removed, each internal organs position is restored, checks that whether there is or not bleeding, liver superior and inferior vena cava sutures in abdominal cavity Whether there is or not leakage blood, and whether each pipeline is unobstructed, with antibiotic normal saline flushing abdominal cavity, close abdomen, 40,000 units of Penicillin of intramuscular injection.
2.4 post surgery treatment
1) postoperative heat lamp rewarming when actively walking about, withdraws heat lamp to the drying of receptor mouse fur and mental restoration；By receptor mouse Room temperature control is placed between 22 DEG C -24 DEG C of animal feeding, single cage is fed；
2) postoperative 5% glucose water that can give allows it freely to suck.It is postoperative can be put into for 24 hours feed by its freedom into Food.The record rat state of mind, activity condition daily, skin of pinna xanthochromia degree, stool and urine color, rat cadavers corpse as early as possible Inspection；
3) postoperative no longer to give the measures such as antibiotic and vein fluid infusion.
It is completely awake in cipient rats generally after surgery 30min, it can free water in 1h.
Isograft group and the postoperative rat diet of sham-operation group be normal, weight is without significant change, skin and mucosa, stool and urine Color is normal, and the state of mind is good.Occurring feed from allotransplantation group the 5th day to reduce, mucocutaneous micro- Huang, urine color are slightly yellow, Activity is opposite to be reduced.Rat body weight is begun to decline from 7th day, and skin and mucosa xanthochromia, urine color are also deepened, and fur is unglazed in a jumble Pool, the state of mind is poor, and autonomic activities are reduced, and weakens to extraneous stimulate the reaction.
2.5 sample disposal
In the postoperative 6h, 12h of post-transplantation, for 24 hours, 5d, 10d it is random in Syngraft group and Allograft group respectively Cipient rats each 3 are selected to draw materials.
1) etherization, rat are fixed on operating table (anesthesia is unsuitable excessively, very few to prevent blood sampling volume)；
2) it sterilizes, cross recess is into abdomen；
3) intestinal tube is removed to right side, sufficiently to expose infrahepatic vena cava, free infrahepatic vena cava is to covering area under control；
4) it is inserted into blood taking needle, and is fixed with artery clamp, blood taking needle is inserted into biochemical tube；
5) blood sampling is placed in 4 DEG C of centrifuges of pre-cooling, 2000rpm, is centrifuged 10min, and supernatant packing is taken to save and -80 DEG C Refrigerator；
6) liver is adhered tissue with surrounding as far as possible to be stripped clean, and is close to liver detachment blood vessel and bile duct, rapidly by liver group It knits and is placed on trash ice, hepatic tissue is drawn materials according to its anatomy leaflet.It is divided into: lobus sinister, left secondary leaf, lobus dexter, right accessory lobes, cream Head lobe, caudate lobe, every leaf are divided into two parts of processing, and portion is placed in 4% formalin after carrying out respective markers, and portion is placed in -80 DEG C It saves.All experiments take same region of anatomy hepatic tissue to be compared.
7) two parts of progress different disposal preservations will be also divided to by body-centered, right lung, spleen, left kidney, right kidney simultaneously.
Substantially draw materials situation:
1) sham-operation group, isograft group and the postoperative 6h, 12h of allotransplantation group, liver is full good for 24 hours, Color is scarlet, without obvious extravasated blood, three groups of liver naked eyes no significant differences；
2) postoperative 5d, 10d liver of sham-operation group, spleen are normal, and abdominal cavity is without being adhered；
3) isograft group it is equal with the postoperative 5d liver of allotransplantation group and surrounding tissue (diaphram, stomach and intestine, nethike embrane) It is adhered, can be allowed to separate with cotton swab blunt separation.Two groups of liver volumes are big compared with sham-operation group, and allotransplantation group ratio is same Gene transplant group slightly increases.Three groups of spleens have no significant change；
4) it is adhered significantly with the postoperative 10d liver of allotransplantation group and surrounding for isograft group, it need to be by microscissors Make its separation.Allotransplantation group liver volume and spleen volume are significantly greater than isograft group and sham-operation group is (homogenic Transplantation group liver is more bigger than sham-operation group, Spleen Size no significant difference).Allotransplantation group liver diffuses enlargement, edge circle Blunt, section is coarse, the visible mottled change red and white diffused, individual visible focal downright bad (as shown in Figure 1).
3. pathology of hepar is observed
3.1 tissues are fixed
Take 6h, 12h after transplanting, for 24 hours, 5d, 10d liver organization, be soaked in 4% formaldehyde fixer, wait organize it is fixed after, press Following steps carry out serial dehydration, transparency of organization and waxdip and embedding treatment:
The production of 3.2 paraffin sections
1) slicer is mounted on the holding on cutter holder of tool apron, adjusts the gradient of slicer；
2) tuning drive gear of inching gear is adjusted to the reading for requiring slice thickness；
3) paraffin embedded tissues for crossing precooling treatment, which are placed in, holds on wax stone platform, makes wax stone close to blade, forms right angle with blade, Modify wax stone section；
4) 4 μm of slice thickness, serial section or interruption serial section；
5) first extended piece in cold water, then being moved in warm water with slide is fully deployed it as far as possible, lose gauffer (but Also can not place for a long time, and crack slice) piece (should make tissue adhension 1/3 under slide at) is fished out afterwards；
6) 65 DEG C of baking 20min.
3.3HE dyeing
L) I 10min of dimethylbenzene；
2) II 10min of dimethylbenzene；
3) III 10min of dimethylbenzene；
4) 100%, 95%, 85%, 70% each 2min of alcohol；
5) haematoxylin dyeing 3-5min, after flowing water rinses 1min, l% hydrochloride alcohol (70% alcohol) breaks up the several seconds. Under the microscope, if color is shallower can to carry out haematoxylin dyeing again, if relatively deep can extend hydrochloric acid divergaence time, until nucleus and core Until interior chromatin is clear, then flowing water rinses 10min (blue effect is returned in flowing water flushing)；
6) 0.5% eosin stain dyes 1-5min；
7) through 70%, 85%, 95%, 100% each 2min of dehydration of alcohol；
8) I 10min of dimethylbenzene；
9) II 10min of dimethylbenzene；
10) mounting: electric wind dries up the moisture on slide, and appropriate neutral gum, covered is added dropwise.
3.4 assessment hepatic injury degree
1) postoperative 6h, 12h, Suzuki ' the s pathology of cipient rats hepatic tissue application Ischemia-reperfusion Injury in Rat are commented for 24 hours Minute mark standard is scored (as shown in table 1).
Suzuki ' the s histological scores standard of 1 Ischemia-reperfusion Injury in Rat of table
2) postoperative 5d, 10d cipient rats hepatic tissue rejection histological scores standard application Banff marking scheme carries out It is 9 points that activity index (RAI) total mark is repelled in scoring, and 0~2 point is set to no repulsion, and 3 points sexually revise to have a common boundary, and 4~5 points are light Degree repels, and 6~7 points are repelled for moderate, and 8~9 points are repelled (as shown in table 2) for severe.
2 acute rejection Banff rank scores scheme of table: rejection activity index
Histological findings (as shown in Figure 2):
1) the postoperative 6h, 12h of sham-operation group, for 24 hours, under each time point mirror of 5d, 10d performance without significant difference: lobuli hepatis knot Structure is clear, and liver cell and sinusoidal endothelial cells are normal, the visible a small amount of lymphocytic infiltration in part portal area, but without neutrophil leucocyte, Without expansion, gallbladder tube structure is normal for portal area.
2) the obvious oedema of liver cell around the visible central vein of isograft group of postoperative 6h, with a small amount of necrosis, in sinus Chrotoplast obvious tumefaction, it is seen that more monocyte and neutrophil infiltration, portal area is without significant change；Postoperative 12h liver The slight oedema of cell, sinus endothelia swelling, partial region hepatic sinusoid narrow or disappear, and monocyte and neutral grain infiltration increase, and converge Area under control is also without significant change；Postoperative for 24 hours liver cell and sinusoidal endothelial cells have without obvious tumefaction, still a small amount of neutrophil leucocyte and Monocyte infiltration；Portal area and a small amount of chronic inflammatory cell infiltration fibre of central vein are shown as under postoperative 5d and 10d mirror Hyperblastosis is tieed up, no Combination inflammatory cell infiltration is not belonging to acute rejection model without intravenous dermatitis and bile duct injury Farmland.
3) it allotransplantation group postoperative 6h, 12h and is showed under mirror under the mirror at corresponding with isograft group time point for 24 hours Show similar, no significant difference.5d is shown in that there are the Combination inflammatory cell including neutrophil leucocyte in most of portal areas Infiltration, most of bile duct have cell infiltration, have lymphocyte and neutrophil infiltration under nearly all portal vein inner membrance, Part is 6-7 points with necrosis of liver cells by vein, RAI scoring, is moderate acute rejection；The visible all headers of 10d The a large amount of Combination inflammatory cell infiltrations of Qu Douyou, and involve liver parenchyma around.Nearly all bile duct visible inflammatory cell infiltration, part Bile duct official jargon is destroyed, broken with a large amount of Combination lymphocytic infiltrations and surrounding liver parenchyma under tunica intima of vein, and RAI scoring is 8- 9 points, be severe acute rejection.
4) sight under sham-operation group, isograft group and the postoperative 6h, 12h of allotransplantation group, mirror for 24 hours is carried out Suzuki ' s histological scores, as shown in table 3: postoperative each time point allotransplantation group and isograft group of ischemia-reperfusion It damages total score no difference of science of statistics (P > 0.05)；Postoperative each time point allotransplantation group and isograft group of ischemia-reperfusion It is high (P < 0.05) compared with sham-operation group ischemical reperfusion injury total score to damage total score.
Suzuki ' the s histological scores of 3 Ischemia-reperfusion Injury in Rat of table
AIt indicates: compared with isograft group, P > 0.05；BIt indicates: compared with sham-operation group, P < 0.05.
5) it carries out RAI and comments to being seen under the mirror of sham-operation group, isograft group and allotransplantation group postoperative 5d, 10d Point, as shown in table 4: isograft group of postoperative each time point is scored no difference of science of statistics (P > 0.05) with sham-operation group RAI；Art Each time point allotransplantation group RAI scores respectively compared with isograft group and sham-operation group RAI scoring, compared with two groups afterwards High (P < 0.05).RAI of the RAI scoring higher than 5d of allotransplantation group 10d scores (P < 0.05).
4 liver transplantation tissue rejection reaction Ba nff of table, 97 histological scores
AIt indicates: compared with isograft group, P < 0.05；BIt indicates: compared with sham-operation group, P < 0.05.
4 Liver function grades
4.1 glutamic-pyruvic transaminase (ALT) detection
4.1.1 each group Serum ALT detects
1) 20 μ L glutamic-pyruvic transaminase matrix liquids are added in sample well and control wells；
2) 5 μ L samples are added in sample well, inhale repeatedly and play mixing, control wells are not added；
3) 37 DEG C of water-bath 30min；
4) 20 μ L 2,4-dinitrophenylhydrazine liquid are added in each hole；
5) 5 μ L samples are added in control wells, inhale repeatedly and play mixing, sample well is not added；
6) 37 DEG C of water-bath 20min；
7) 200 μ L 0.4mol/L NaOH, 96 orifice plate of jog is added in every hole；
8) it is incubated at room temperature 15min, 510nm surveys OD value.
4.1.2ALT standard curve operates
1) 6 holes are selected in 96 orifice plates, respectively label 0,1,2,3,4,5；
2) every hole is separately added into 5 μ L of 0.1mol/L phosphate buffer；
3) 2 μm of 0 μ L of ol/mL Sodium Pyruvate titer, 2 μ L, 4 μ L, 6 μ L, 8 μ L, 10 μ L are sequentially added；
4) 20 μ L of Matrix buffer, 18 μ L, 16 μ L, 14 μ L, 12 μ L, 10 μ L are sequentially added；
5) (note: pipette tips are protruded into hole board bottom to every hole addition 20 μ L of 2-4 dinitrophenylhydrazine liquid by one standard of every every absorption in hole It in portion's liquid, inhales repeatedly and plays mixing)；
6) 37 DEG C of water-bath 20min；
7) 200 μ L 0.4mol/L NaOH, 96 orifice plate of jog is added in every hole；
8) it is incubated at room temperature 15min, 510nm surveys OD value.
4.2 glutamic-oxalacetic transamineases (AST) detection
4.2.1 each group serum AST detects
1) 20 μ L glutamic-oxalacetic transaminease matrix liquids are added in sample well and control wells；
2) 5 μ L samples are added in sample well, inhale repeatedly and play mixing, control wells are not added；
3) 37 DEG C of water-bath 30min；
4) 20 μ L 2,4-dinitrophenylhydrazine liquid are added in each hole；
5) 5 μ L samples are added in control wells, inhale repeatedly and play mixing, sample well is not added；
6) 37 DEG C of water-bath 20min；
7) 200 μ L 0.4mol/L NaOH, 96 orifice plate of jog is added in every hole；
8) it is incubated at room temperature 15min, 510nm surveys OD value.
4.2.2AST standard curve operates
1) 6 holes are selected in 96 orifice plates, respectively label 0,1,2,3,4；
2) every hole is separately added into 5 μ L of 0.1mol/L phosphate buffer；
3) 2 μm of 0 μ L of ol/mL Sodium Pyruvate titer, 2 μ L, 4 μ L, 6 μ L, 8 μ L are sequentially added；
4) 20 μ L of Matrix buffer, 18 μ L, 16 μ L, 14 μ L, 12 μ L are sequentially added；
5) 20 μ L of 2-4 dinitrophenylhydrazine liquid is added in every hole；
6) after mixing, 37 DEG C of water-bath 20min；
7) 200 μ L 0.4mol/L NaOH, 96 orifice plate of jog is added in every hole；
8) it is incubated at room temperature 15min, 510nm surveys OD value.
4.3 results calculate and judgement
1) all OD values should all subtract row calculating (i.e. absolute OD value) again after blank value；
It 2) is ordinate, ALT standard items absolute value with standard items enzyme activity Ka Menshi unit 0,28,57,97,150,200 OD is that abscissa draws ALT standard curve；It is ordinate, AST with standard items enzyme activity Ka Menshi unit 0,24,61,114,190 Standard items absolute value OD is that abscissa draws AST standard curve；
3) go out the value of the ALT and AST of respective sample according to sample OD value technology.
Liver function (ALT, AST) testing result:
ALT, AST are primarily present in liver cell endochylema in body.When liver cell is damaged, intracytoplasmic ALT with AST is just released into blood, therefore ALT and AST is in the extent of damage that can reflect liver cell to a certain degree.
The postoperative different time points Serum ALT of each group, AST testing result are as shown in table 5: postoperative 6h, 12h, for 24 hours serum The horizontal allotransplantation group of ALT and AST, isograft group of equal 6h highest, are then gradually reduced, and two groups of each time point values are equal More same time sham-operation group is high (P < 0.5), but no significant difference (P > 0.05) to each other；Postoperative 5d, allotransplantation group ALT and AST is above sham-operation group (P < 0.5), but no significant difference (P > 0.05) compared with isograft group；Postoperative 10d, allotransplantation group ALT and AST are above isograft group and sham-operation group (P < 0.5).
The postoperative each time point Serum ALT of 5 each group rat implantation of table, the active variation (unit U/L) of AST
AIt indicates: compared with sham-operation group, p < 0.05；BIt indicates: compared with homogenic group, p > 0.05；CIt indicates: with allogene Group compares, p < 0.05.
The present embodiment the result shows that, inbred strais Lewis rat → BN rat orthotopic liver transplantation acute rejection model can be used for Ischemical reperfusion injury and acute rejection of liver transplantation are studied, for stable rat orthotopic liver transplantation acute rejection model.
Effect of 2 EGR-1 of embodiment after predicting orthotopic liver transplantation in rats in acute rejection
1. zoological specimens
Embodiment 1 model after freeze the postoperative 6h, 12h of each group in -80 DEG C of refrigerators, for 24 hours, the same region of anatomy of 5d, 10d Hepatic tissue and packing serum.
2. method
EGR-1, IL-6, IL-10 gene expression in 2.1 qPCR detection transplanting hepatic tissue
Utilize TransZolTMKit provides each sample total serum IgE to specifications, and reverse transcription is after carrying out integrity analysis CDNA is used for follow-up test.
1) design of primers: entering the official website NCBI, carries out design of primers, selects best primer through primer blast.EGR- 1, the primer sequence such as table 6 of IL-6, IL-10,18S rRNA (internal reference):
6 primer sequence of table
2) qPCR reaction system is as shown in table 7:
7 qPCR reaction system of table
3) qPCR amplification reaction condition: entering PCR cycle after 95 DEG C of initial denaturation 2min, PCR cycle reaction condition is 95 DEG C, 10s；60 DEG C, 30s；72 DEG C, 30s, 40 circulations；Entering melting curve after the completion of amplification and detects program, reaction condition is 95 DEG C, 15s；60 DEG C, 1min；95 DEG C, 15s；60 DEG C, 15s；
4) each sample does 3 multiple holes, obtains the relative quantification Ct value of sample.
Using 2-△△CtThe relative quantitative assay of method progress data.
EGR-1 mRNA expression is as shown in Figure 3: it is postoperative for 24 hours in, allotransplantation group, isograft group in art 6h expression quantity highest afterwards is higher than sham-operation group (p < 0.05), is then gradually decreased to normal level, same time point is to each other No difference of science of statistics (p > 0.05)；The expression quantity of the postoperative 10d of allotransplantation group is higher than 5d (p < 0.05), and the two is equal Isograft group of more same time point and sham-operation group height (p < 0.05)；Isograft group of 5d and 10d expression quantity Indifference (p > 0.05) compared with sham-operation group.Show that EGR-1 participates in the acute row after early stage ischemical reperfusion injury and transplanting Reprimand reaction, and its expression quantity also rises with the increase of repulsion intensity.
IL-6 mRNA expression is as shown in Figure 4: the postoperative 6h, 12h of allotransplantation group, for 24 hours, 10d be above artificial hand Art group (P < 0.5), postoperative 10d are higher than isograft group (P < 0.5)；Isograft group of postoperative 6h, 12h are higher than vacation Operation group (P < 0.5)；Allotransplantation group is postoperative interior in 6h expression quantity highest for 24 hours with isograft group, then gradually Decline.
IL-10 mRNA expression is as shown in Figure 5: allotransplantation group postoperative 12h, 5d, 10d are above sham-operation Group (P < 0.5), postoperative 5d, 10d are higher than isograft group (P < 0.5)；Isograft group of postoperative 12h, it is higher than for 24 hours Sham-operation group (P < 0.5).
2.2 Western-blot detect EGR-1 protein level
2.2.1 the preparation of whole-cell protein sample
The whole-cell protein of each tissue samples is extracted, and detects protein concentration using BCA method.Use ddH2O is to surveyed various kinds Product concentration is adjusted, and keeps concentration between each sample identical.It is mixed with 5 × albumen loading buffer (being diluted to 1 ×), 100 DEG C, 10min is boiled, -80 DEG C of preservations after packing.
2.2.2 electrophoresis
2.2.2.1 PAGE gel is prepared
1) it after cleaning Bio-Rad glass plate and stripping fork, dries, is mounted on on glue frame；
2) 12%SDS-PAGE separation gel: ddH is prepared according to the following recipe2O 2.0mL, 30%Acr-Bis (29:1) 4mL, 1mol/L Tris-HCl (pH 8.8) 3.8mL, 10% ammonium persulfate 0.1mL, 10%SDS0.1mL, TEMED 0.004mL is mixed immediately；
3) separation gel is filled into the gap among two pieces of glass plates rapidly, until away from about 3.0cm high at the top of glass plate Stop perfusion when spending；
4) ddH is carefully used2O covering, room temperature is disposed vertically about 30-60min (can be in separation gel after the completion of separation gel polymerization With ddH2O intersection is shown in a clearly refractive power line)；
5) ddH of covering is outwelled2O, the liquid put on venting separation gel as far as possible, is blotted with blotting paper；
6) 5%SDS-PAGE being prepared according to the following recipe, glue: ddH is concentrated2O 2.7mL, 30%Acr-Bis (29:1) 0.67mL, 1mol/L Tris-HCl (pH 6.8) 0.5mL, 10% ammonium persulfate 0.04mL, 10%SDS0.04mL, TEMED 0.004mL is mixed immediately；
7) concentration glue is directly poured on the separation gel having polymerize, until at the top of glass plate；
8) it is inserted perpendicularly into stripping fork (bubble cannot be mixed into) immediately, room temperature is disposed vertically 30-40min；
9) it is carefully removed from comb, washs loading slot with distilled water.
2.2.2.2 loading and electrophoresis
1) loading: gel is fixed in electrophoretic apparatus, and 1 × running buffer is added, and excludes bubble；According to predetermined Sequentially each sample loading total protein concentration is 30 μ g, and a reserved hole adds 5 μ L pre-dyed albumen marker, the sample cell of remaining not loading It is middle that isometric 1 × loading buffer is added (loading time is short as far as possible, avoids sample diffusion and edge effect)；
2) electrophoresis: powering on, 80V constant pressure, after sample enters separation gel, adjusts voltage to 120V, until bromophenol blue Dyestuff arrives at separation gel bottom about 1cm distance, disconnects power supply.
2.2.3 transferring film
1) it wears gloves, unloads lower glass plate, gently pried open in the flowing water of mitigation, cut concentration glue, take out separation gel simultaneously It is dipped in 1 × transferring film liquid；
2) NC film, the filter paper cut by foam-rubber cushion and in advance is soaked in 1 × transferring film liquid；
3) open transferring film folder, black is put on one side in transferring film liquid, be sequentially placed into from the bottom up foam-rubber cushion, filter paper, gel, NC film, filter paper, foam-rubber cushion, covering transferring film folder fine flour and clipping clip (will drive bubble away with sleaker when spreading each layer, especially It is centainly cannot there are bubbles between glue and film)；
4) transferring film is folded up and sets in electric transferring film slot (for black flour to cathode, fine flour is to anode), the transferring film buffer of pre-cooling is added, 100V constant pressure transferring film in ice bath, time about 60min (about 1min of every 1kDa molecular weight transferring film time).
2.2.4 closing
After transferring film, NC film is taken out, is cut centered on the molecular weight present position of destination protein；1×TBST Cleaning 5 minutes, removes TBST；It is added in the confining liquid prepared in advance, room temperature closes 2h or 4 DEG C of closing overnight on shaking table.
2.2.5 primary antibody is incubated for
1 × TBST is rinsed film 3 times, each 10min；Film immerses in primary antibody dilution, is placed in incubation at room temperature 2h or 4 on shaking table DEG C be incubated overnight.
2.2.6 secondary antibody is incubated for
Primary antibody is recycled, is cleaned 3 times with 1 × TBST, each 10min；Film is immersed in the secondary antibody after dilution, room temperature on shaking table It is incubated for 1h or 4 DEG C of overnight incubation.
2.2.7 Protein Detection
1) secondary antibody is recycled, is cleaned 3 times with 1 × TBST, each 10min；
2) West Pico Stable peroxide solution and the West Pico in albumen development liquid kit Lumonol/Enhancer Solution carries out 1:1 on demand in 15ml centrifuge tube and mixes in equal volume；
3) the NC memebrane protein after above-mentioned incubation is shifted up, is placed in the sealing camera bellows in gel imaging system, Suitable mixing developer solution is added dropwise, reacts at room temperature 1min；
4) exposure program is set, is exposed in darkroom X egative film；
5) the suitable picture of selection exposure analyzes the gray value of band with Image J software.
It extracts the postoperative different time points hepatic tissue total protein of each group and carries out WB detection, as a result as shown in Figure 6: allotransplantation Group and isograft group of EGR-1 expression quantity are down to normal level in interior for 24 hours in postoperative 6h highest (P < 0.05).Different base Because transplantation group and isograft group of postoperative 5d, 10d EGR-1 expression quantity are above sham-operation group (P < 0.05), and allogene Isograft group of transplantation group more same time point high (P < 0.05).
2.3IHC
1) tissue is fixed, embeds, the same second part of paraffin section step, using adherency glass slide when fishing out piece；
2) it dewaxes: glass slide being successively put into dimethylbenzene 10min；Dimethylbenzene 10min；Dimethylbenzene 10min；100% alcohol 2min；100% alcohol 2min；95% alcohol 2min；80% alcohol 2min；70% alcohol 2min；
3) flowing water rinses 2min, is put into the container for repairing liquid equipped with sodium citrate；Container and its intra slice, lemon Sour sodium repairs liquid and is put into water proof progress Pressure method in pressure cooker together, until starting timing 3min after the continuous jet of pressure cooker, so After power off, cooled to room temperature；
4) flowing water is washed 2 times with PBS again after rinsing 5min, each 3min；
5) PBS around tissue is dried, 3%H is added dropwise2O2, 37 DEG C of incubation 10min, then 3 times are washed with PBS, every time 3min；
6) PBS around tissue is dried, 37 DEG C of incubators of confining liquid close 30min；
7) confining liquid that tissue is put is got rid of, the serum around tissue is dried with blotting paper, is added dropwise 4 DEG C of primary antibody dilution and incubates It educates overnight, control experiment is just in the tissue of control plus PBS；
8) refrigerator is first placed at room temperature for 30min after taking out, then is washed 3 times with PBS, each 3min；
9) PBS around tissue is dried, is added dropwise polymer intensifier (reagent A), 20min in 37 DEG C of incubators, then washed with PBS 3 times, each 3min；
10) it dries the PBS around tissue, is added dropwise the anti-mouse of enzyme mark/anti-rabbit polymer (reagent B), 30min in 37 DEG C of incubators, It is washed 3 times with PBS again, each 3min；
11) PBS around tissue is dried, now (configuration of color developing agent: in 850 μ L water plus 1 drop develops the color with color developing agent for dropwise addition Agent A, shakes up, then plus 1 drop color developing agent B, shake up, then plus 1 drop color developing agent C, shake up.A:DAB buffer；B:DAB substrate；C: DAB chromogen), the display time is determined under the microscope；
12) slide is soaked in hematoxylin after flowing water flushing color development stopping and is dyed, determine dyeing time under the microscope；
13) flowing water, which rinses, terminates dyeing, and glass slide is successively put into -95% alcohol -100% of -80% alcohol of 70% alcohol - 100% alcohol of alcohol-dimethylbenzene-dimethylbenzene-dimethylbenzene.Each reagent standing time is 2min；
14) neutral gum is added dropwise, covered is dried in vent cabinet, under the microscope.
The observation discovery of IHC result: postoperative EGR-1 positive expression interior for 24 hours sees wounded hepatocytes nucleus, endothelial cell Core, Kupffer nucleus, and the inflammatory cell core of activation, wherein liver cell nuclear staining power can be observed significantly with shifting It plants Post surgery duration and extends the obvious decrease of dyeing；Postoperative 5d and 10d EGR-1 is primarily targeted in lymphocyte.Therefore to scarce Blood Reperfusion injury injures acute cellular rejection EGR-1 positive findings statistics and uses different artificial counting methods.Each group 6h, 12h, Each sample hepatic tissue chooses 5 high power field of view (i.e. 10 × 40 times) for 24 hours, and the positive liver for counting IHC label accurate positioning is thin Born of the same parents account for the ratio of liver cell and score, and karyon positive cell number accounts for 25% or less 1 point of note of liver cell number, 25%-50% note 2 Point, 50-75% remembers 3 points, 75% or more 4 points of note；Staining power (±) is 1 point, and (+) is 2 points, and (++) is 3 points, finally calculates two The product of kind label；The positive lymphocyte that each group 5d, 10d only carry out IHC label accurate positioning accounts for total lymphocyte Percentage is counted.
IHC expression of results is as shown in Figure 7 A: negative or weak sun is presented in each time point liver cell of sham-operation group, lymphocyte Property be (±)；Allotransplantation group and isograft group of 6h IHC positive cell be mainly around centrally located vein liver it is thin Born of the same parents, intensity (++)；Allotransplantation group and isograft group of 12hIHC positive cell intensity (+)；Allotransplantation group and Isograft group for 24 hours (±) similar to sham-operation group result.Fig. 7 B shows postoperative allotransplantation group interior for 24 hours and homogenic shifting Plant group EGR-1 positive expression result is then gradually decreased to normal level in postoperative 6h highest (P < 0.05).Fig. 7 C shows that allogene moves Plant group 5d and 10d IHC positive cell number is higher than isograft group of same time point and sham-operation group (P < 0.05), and the 10d is also higher than 5d (P < 0.05).
2.4 ELISA detect serum EGR-1, IL-1 β, IFN-γ expression
1) it extracts and gets out test serum and required reagent and working concentration standard items；
2) standard items working solution is added sequentially in first two columns hole, the working solution of each concentration adds two holes, every hole side by side 100μL.Sample to be tested and Sample dilution are added to other holes, every 100 μ L of hole by after 1:1 dilution proportion；
3) ELISA Plate overlay film, 37 DEG C of incubation 90min are given；
4) liquid is discarded, dries, does not wash.100 μ L of biotinylated antibody working solution is added in every hole, mixes, and ELISA Plate adds Upper overlay film, 37 DEG C of incubation 1h；
5) liquid in most hole is got rid of, every hole adds 350 μ L of cleaning solution, impregnates 1-2min, gets rid of the liquid in ELISA Plate, in thickness It is patted dry on blotting paper.Repeat this clear plate step 3 time；
6) the enzyme 100 μ L of conjugate working solution in every hole, in addition overlay film, 37 DEG C of incubation 30min；
7) liquid in hole is discarded, is dried, board-washing 5 times, method is the same as step 5；
8) every hole adds 90 μ L of substrate solution, and overlay film, 37 DEG C are protected from light incubation 15min or so；
9) 50 μ L of terminate liquid is added in every hole, terminates reaction；
10) use microplate reader in the optical density (OD value) in each hole of 450nm wavelength measurement immediately.
As a result as shown in Figure 8: postoperative interior for 24 hours, allotransplantation group and isograft group of three time points are above together One time sham-operation group (P < 0.05), two groups of after surgery 12h reach peak (P < 0.05)；Postoperative 5d and 10d, it is different EGR-1 content is above isograft group and sham-operation group (P < 0.05) in gene transplant group serum, and 10d content is higher than 5d (P < 0.05)；Isograft group of 5d and 10d content are also higher than sham-operation group (P < 0.05).
Each group rat different time points -1 β expression of serum IL is as shown in table 8: postoperative each time point allotransplantation group And isograft class value is obviously higher than sham-operation group (P < 0.01)；Postoperative 6h, 12h, for 24 hours allotransplantation group and same base Because between transplantation group and no difference of science of statistics (P > 0.05), two groups reach high level in 12h, then decline.Postoperative 5d, 10d More isograft group of allotransplantation group expression high, difference is statistically significant (P < 0.05).
Postoperative -1 β expression (unit pg/mL) of serum IL of 8 each group rat of table
AIt indicates: compared with sham-operation group, p < 0.05；BIt indicates: compared with isograft group, p > 0.05；
CIt indicates: compared with isograft group, p < 0.05；
Each group rat different time points serum I FN- γ expression is as shown in table 9: postoperative each time point allotransplantation Group and isograft class value are above sham-operation group (P < 0.05).Postoperative 6h, 12h, for 24 hours allotransplantation group and same base Because between transplantation group and no difference of science of statistics (P > 0.05)；The more homogenic shifting of postoperative 5d, 10d allotransplantation group expression Plant group is high, and difference is statistically significant (P < 0.05), and allotransplantation group 10d expression is higher than 5d, and difference has system Meter learns meaning (P < 0.05).
The postoperative serum I FN- γ expression (unit pg/ml) of 9 each group rat of table
AIt indicates: compared with sham-operation group, p < 0.05；BIt indicates: compared with isograft group, p > 0.05；
CIt indicates: compared with isograft group, p < 0.05；
The present embodiment passes through IL-6, IL-10mRNA expression in detection post-transplantation different time points liver tissues of rats And the content of IL-1 β in blood, IFN-γ, to observe different times i.e. ischemical reperfusion injury phase and acute row after rat implantation Inflammatory activity situation in reprimand phase body.
Through this embodiment, it can be deduced that draw a conclusion:
1) EGR-1 is enabled briefly in liver transfer operation ischemia-reperfusion early stage, participates in ischemical reperfusion injury.Liver cell EGR-1 ImmunohistochemistryResults Results degree of strength can be used as hepatocellular injury degree judgment criteria；
2) EGR-1 participates in acute rejection of liver transplantation reaction, and its expression quantity and acute cellular rejection degree are positively correlated.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.
Sequence table
<110>Medical University Of Fujian Meng Chao liver and gallbladder hospital (Fuzhou City Infectious Disease Hospital)
<120>marker EGR-1 relevant to prediction after liver transplantation rejection and its application
<130> XY-2019-1-W-065
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
aacaacccta cgagcacctg 20
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
aaaggggttc aggccacaaa 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
agcgatgatg cactgtcaga 20
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
tagcacacta ggtttgccga 20
<210> 5
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
ttgaaccacc cggcatctac 20
<210> 6
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
ccaaggagtt gctcccgtta 20
<210> 7
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gccatgcatg tctaagtacg c 21
<210> 8
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
ccgtcggcat gtattagctc 20

Claims (10)

  1. The detection reagent of 1.EGR-1 gene transcript and/or the detection reagent of EGR-1 albumen are being prepared for predicting object liver Application in the kit of post-transplantation rejection.
  2. 2. application according to claim 1, which is characterized in that the detection reagent of the EGR-1 gene transcript includes It is capable of the primer combination of specificity EGR-1 gene transcript.
  3. 3. application according to claim 2, which is characterized in that the transcription product includes being obtained by EGR-1 mRNA reverse transcription The cDNA arrived.
  4. 4. application according to claim 2 or 3, which is characterized in that the specificity EGR-1 gene transcript of capableing of Primer combination includes having the upstream primer of nucleotide sequence shown in SEQ ID NO. 1 and/or having shown in SEQ ID NO. 2 The downstream primer of nucleotide sequence.
  5. 5. application according to claim 1, which is characterized in that the detection reagent of the EGR-1 albumen is Mass Spectrometric Identification examination Agent, antibody or its antigen-binding fragment.
  6. 6. purposes according to claim 6, which is characterized in that the antibody is sheep anti mouse EGR-1 antibody.
  7. 7. a kind of kit for predicting object after liver transplantation rejection, which is characterized in that the kit EGR-1 gene turns Record the detection reagent of product and/or the detection reagent of EGR-1 albumen.
  8. 8. kit according to claim 7, which is characterized in that the detection reagent packet of the EGR-1 gene transcript Include the primer combination for capableing of specificity EGR-1 gene transcript.
  9. 9. kit according to claim 8, which is characterized in that the specificity EGR-1 gene transcript of capableing of Primer combination includes having the upstream primer of nucleotide sequence shown in SEQ ID NO. 1 and/or having shown in SEQ ID NO. 2 The downstream primer of nucleotide sequence.
  10. 10. kit according to claim 8, which is characterized in that the detection reagent of the EGR-1 albumen is Mass Spectrometric Identification Reagent, antibody or its antigen-binding fragment.
CN201910735605.8A 2019-08-09 2019-08-09 Marker EGR-1 relevant to prediction after liver transplantation rejection and its application Pending CN110408691A (en)

Priority Applications (1)

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CN201910735605.8A CN110408691A (en) 2019-08-09 2019-08-09 Marker EGR-1 relevant to prediction after liver transplantation rejection and its application

Publications (1)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000045771A2 (en) * 1999-02-05 2000-08-10 Daniel Mercola Suppression of transformation of cells by the transcription factor egr
US6969704B1 (en) * 2000-08-25 2005-11-29 The Trustees Of Columbia University In The City Of New York Methods for suppressing early growth response—1protein (Egr-1) to reduce vascular injury in a subject

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000045771A2 (en) * 1999-02-05 2000-08-10 Daniel Mercola Suppression of transformation of cells by the transcription factor egr
US6969704B1 (en) * 2000-08-25 2005-11-29 The Trustees Of Columbia University In The City Of New York Methods for suppressing early growth response—1protein (Egr-1) to reduce vascular injury in a subject

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
任祖海等: "terlipressin对肝大部切除术后早期小体积肝功能保护机制的研究", 《中国普通外科杂志》 *
游伟等: "一氧化氮促释放剂在保护大鼠减体积肝移植后缺血再灌注损伤中的作用", 《南京医科大学学报(自然科学版)》 *
马跃峰等: "大鼠小体积肝移植物再灌注早期EGR-1的表达及其意义", 《中国普通外科杂志》 *

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