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CN111593113A - VEGF gene binding site detection method based on novel acute rejection reaction mechanism - Google Patents

VEGF gene binding site detection method based on novel acute rejection reaction mechanism Download PDF

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CN111593113A
CN111593113A CN202010436770.6A CN202010436770A CN111593113A CN 111593113 A CN111593113 A CN 111593113A CN 202010436770 A CN202010436770 A CN 202010436770A CN 111593113 A CN111593113 A CN 111593113A
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周天保
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Ningbo Huamei Hospital University of CAS
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Abstract

The invention discloses a VEGF gene binding site detection method based on a new acute rejection reaction mechanism, which comprises the steps of taking right 8 and 9 intercostal space punctures under the guidance of B ultrasonic through liver puncture pathological biopsy and sampling to prepare a DNA dissolving solution containing VEGF gene; amplifying the DNA dissolving solution and processing the DNA dissolving solution by a PCR instrument; purifying the mixed solution to obtain a PCR product; amplifying the purified PCR product to obtain a sequencing PCR system solution, and performing cyclic heating treatment on the sequencing PCR system solution; adding 125mmol/LEDTA, 3mol/L sodium acetate and absolute ethyl alcohol into the sequencing PCR system solution, performing oscillation centrifugation, and removing a supernatant; and (3) adding high-purity formamide into the solution for shaking dissolution and performing denaturation treatment by using a qualitative PCR instrument. Finally, detecting by a sequencer to form a sequencing sequence so as to determine the binding site of the VEGF gene; the method has the advantages of effectively solving the technical problem that the binding site of the VEGF gene and the biopsy tissue can not be determined, and promoting the inhibition of the rejection reaction in the liver transplantation process.

Description

VEGF gene binding site detection method based on novel acute rejection reaction mechanism
Technical Field
The invention particularly relates to a VEGF gene binding site detection method based on a new acute rejection reaction mechanism.
Background
With the continuous development and progress of science and technology, medical technical means closely related to human health are also continuously developed and advanced, in particular to the organ transplantation technology in the medical field. Namely, the patients have organ dysfunction before operation, and organ transplantation needs to be performed through operation to improve survival rate. The development of organ transplantation brings survival hope to patients and has huge risks, organ transplantation rejection still occurs in clinic and causes considerable function loss, even the threat of infection, tumor and other series of side effects exists after the operation of a receptor, patients need to take anti-rejection medicines for a lifetime and bear heavy economic burden, and the development of organ transplantation becomes a bottleneck limiting the further development of organ transplantation, especially in the field of liver transplantation.
Statistically, among the rejection reactions occurring after liver transplantation, acute rejection reaction (acute rejection reaction ARR) is the most common type, which generally occurs within days to several months after transplantation and progresses rapidly. The mechanism of the transplantation is explained as cellular immune response in the foreign liver transplantation society at present, namely, T lymphocytes of a recipient recognize alloantigen of a donor, and the T lymphocytes are activated, proliferated and differentiated to trigger a series of immune reactions and effect mechanisms, so that the transplanted liver is finally destroyed. The theory of cellular acute rejection suggests that cytotoxic T lymphocytes of CD8+ (leukocyte differentiation antigen 8) and CD4+ (leukocyte differentiation antigen 4) are the main effector cells infiltrating and causing apoptosis of biliary epithelium, which is the most prominent target cell in liver transplantation ARR, and it suggests that it may be related to the expression of MHC class ii antigen molecules and tissue-specific antigens of bile duct.
The medical community has completed in 1997 the supplementation of the pathology of acute rejection, which is mainly manifested in three areas: inflammatory cell infiltration in the manifold area, mainly including mononuclear cells (including activated transformed lymphocytes), often mixed with a number of different neutral and eosinophilic granulocytes; (vii) infiltration of subcutaneous inflammatory cells in the veins of the tract, the terminal hepatic vein and the central vein; ③ cholangitis and degeneration and necrosis of bile duct epithelial cells.
The existing immune system of organ transplantation human body has a perfect defense mechanism to various pathogenic factors, and can attack, destroy and remove 'foreign components' such as bacteria, viruses, foreign bodies, foreign body tissues, artificial materials and the like, and the complex immunological reaction is a very important protection mechanism for human body. After the recipient has undergone allogeneic tissue or organ transplantation, the foreign tissue or organ and other transplants are recognized as a "foreign component" by the recipient's immune system, which initiates attack, destruction and removal of the transplant, and this immunological reaction is an important influencing factor of the existing transplant rejection. Graft rejection is one of the major factors affecting graft survival.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a VEGF gene binding site detection method based on a new acute rejection reaction mechanism, and the VEGF gene binding site can be accurately and rapidly detected by the method.
The technical scheme adopted by the invention for solving the technical problems is as follows: a VEGF gene binding site detection method based on a new acute rejection reaction mechanism comprises the following steps:
step 1, taking a biopsy needle to perform puncture in the right 8 and 9 intercostal spaces under the guidance of B ultrasonic through liver puncture pathological biopsy, and sampling to manufacture a DNA template;
step 2, amplifying the DNA template obtained in the step 1 to obtain a PCR amplification solution, and processing the PCR amplification solution by a PCR instrument;
step 3, sampling the solution processed in the step 2 and carrying out agarose electrophoresis observation;
step 4, when the solution in the step 3 is subjected to electrophoresis and then a strip-shaped product appears, purifying the solution obtained in the step 2 to obtain a PCR product;
step 5, preparing the purified PCR product to obtain a sequencing PCR system solution, circularly heating the sequencing PCR system solution, and refrigerating in a low-temperature environment;
step 6, adding 125mmol/LEDTA, 3mol/L sodium acetate and absolute ethyl alcohol into the sequencing PCR system solution obtained in the step 5, and performing oscillation centrifugation on the mixed solution and removing a supernatant;
and 7, injecting high-purity formamide into the solution treated in the step 6, oscillating for dissolving, and performing denaturation treatment by using a qualitative PCR instrument.
And 8, injecting sequencing glue into the solution obtained in the step 7, carrying out electrophoretic separation after the injection is finished, and detecting by a sequencer to form a sequencing sequence and a sequencing peak image.
Preferably, the amount of the recipient tissue sample sampled by the biopsy needle in step 1 is 1 mg.
Preferably, the PCR amplification solution in step 2 comprises 2 XMasterMix, the upstream primer, the downstream primer and a DNA dissolving solution, and the total amount of the PCR amplification solution is 50. mu.l.
Preferably, the agarose gel injected in step 3 has a concentration of 1-2%.
Preferably, the sequencing PCR system solution in the step 5 comprises PCR products, BigDye Buffer, sequencing primers and sterile purified water, and the total amount of the sequencing PCR system solution is 20 μ l.
Preferably, the heating process in step 5 is as follows: firstly heating for 1 minute in a 96 ℃ environment, heating for 10 seconds in a 96 ℃ environment, heating for 5 seconds in a 50 ℃ environment, heating for 4 seconds in a 60 ℃ environment, circulating for 25 times, and finally preserving heat in a 4 ℃ environment.
Preferably, the high-purity Formamide in the step 7 is 10 mu l of Hi-Di Formamide.
Preferably, the sequence glue added in the step 8 is 2 μ l.
Preferably, the upstream primer sequence is: 5'-CAT GCA GAT TAT GCG GAT CAA-3', the sequence of the downstream primer is: 5'-TTT GTT GTG CTG TAG GAA GCT CA-3' are provided.
Compared with the prior art, the invention has the advantages that the sampling after liver transplantation can be quickly and accurately realized by taking the right 8 and 9 costal spaces for puncture and sampling under the guidance of the B ultrasonic through a large amount of clinical realization, thereby reducing the phenomenon of multiple times of failure of liver puncture biopsy, improving the success rate of the liver puncture biopsy and reducing the secondary puncture injury possibly suffered by a patient. Meanwhile, the binding site sequencing of the VEGF gene can be carried out through the steps shown by the invention, so that the binding sites of the VEGF gene are specifically and clinically determined to be bound with the liver biopsy tissue, and the rejection reaction in the liver transplantation operation is caused. The specific determination of the binding site between the VEGF gene and the liver biopsy tissue effectively solves the technical problem that the binding site between the VEGF gene and the biopsy tissue can not be determined in the field. Meanwhile, after the combination site between the VEGF gene and the liver biopsy tissue is specifically determined, the rejection inhibition in the liver transplantation process can be effectively promoted, so that the survival rate of patients is improved, and the economic burden of the patients is relieved.
Detailed Description
The present invention is described in further detail below by way of specific examples.
Clinical researches show that the acute rejection of liver transplantation can be divided into two types, one type is the traditional cellular acute rejection; the other is a new theory of acute rejection of liver transplantation, namely, the acute rejection of body fluid. The cellular liver transplantation acute rejection is mainly a T cell-mediated cellular immune response type acute rejection; the humoral acute rejection of liver transplantation is mainly mediated by antibodies, i.e., based on the B cell-mediated acute rejection of the humoral immune response type.
Various classifications can be made on the basis of the function of cytokines involved in the acute rejection process of liver transplantation, and among them, the growth factors that directly induce the division, proliferation and migration of vascular endothelial cells are mainly VEGF (vascular endothelial growth factor), which is closely associated with inflammatory cells and angiogenesis. The reconstruction of blood vessels is a main characteristic of inflammation, angiogenesis is a common characteristic of various pathophysiological processes including wound repair, immune response, inflammation and the like, and inflammatory cells release angiogenesis promoting factors to stimulate new capillary angiogenesis and mitosis of endothelium in vivo. Inflammatory mediators such as prostaglandin E1, prostaglandin E2, interleukin-1, interleukin-6, interleukin-8, nitric oxide, tumor necrosis factor-alpha and tumor necrosis factor-beta, and the like, have direct or indirect angiogenic effects. Thus, there is a correlation between inflammatory cells and angiogenesis, and many drugs that block inflammatory responses, such as steroids, block angiogenesis. Moreover, prostaglandin E2, inflammatory mediators such as interleukin-1 and interleukin-6 and hypoxia can induce VEGF expression.
Studies have shown that the mutual coordination between VEGF and cytokines responsible for inflammation may play a role in regulating vascular growth in different pathological states, including cancer. VEGF plays a role in local leukocyte trafficking in allograft transplantation, and the anti-VEGF method can effectively inhibit the development of ARR, but the anti-VEGF method cannot inhibit the activation of T lymphocytes in vivo, but only inhibits the expression of various endothelial cell adhesion molecules including IP 10 and chemo-activin in vivo of the graft, so that VEGF acts on inflammatory mediators and plays an important role in allograft ARR. It was also shown that VEGF was locally produced after the transplantation was completed.
The mechanism of the humoral immune response type acute rejection mediated by the B cell is explained as that the VEGF in the liver of a recipient is expressed on the surface of non-parenchymal cells such as vascular epithelium and the like, platelet is activated through cytokine-adhesion molecule linkage reaction, B lymphocyte is induced and differentiated to generate and convert various antibodies, macrophage, neutrophil granulocytes and the like generate directional movement, alloantigen is recognized, histiocyte in the liver is killed, tissue necrosis is caused, and finally transplanted liver is destroyed. And the polymorphism of the VEGF gene between-1154 to-2578 sites is associated with the increased expression of VEGF and the risk of transplant rejection. Therefore, VEGF not only has promotion effect in the immune rejection process of transplanted liver, but also is an early acting factor, which binding sites of the gene in liver biopsy pathological biopsy tissues are related to the increased risk of acute liver allograft rejection can be determined by determining the binding sites of VEGF gene, and the accurate detection of the binding sites of VEGF gene can have profound influence on the rejection reaction in the organ transplantation process.
The role of the VEGF gene in liver transplantation can be determined by:
firstly, an organ transplantation detection test is carried out by making and grouping animal models:
(1) the establishment of an inbred line DA (RT1a) - > AUG (RT1c) orthotopic liver transplantation acute rejection reaction model adopts a two-cuff method to carry out rat orthotopic liver transplantation;
(2) the experiment is divided into two groups: (ii) DA (RT1a) - > AUG (RT1c) inter-rat transplantation as an allograft acute rejection group (acute allograft rejection OLT, OLTR, n = 40); ② transplanting DA-DA rats into a syngeneic transplantation control group (syngeneic OLT, OLTS, n = 40); in each group, 10 rats were observed for survival and were divided into subgroups on days 1, 3, 7 after transplantation, and 10 rats in each subgroup were used for further experiments. Evaluating transplanted liver function by monitoring survival and biochemical or serological indexes of a rat of a recipient, and determining rejection by histology;
(3) the DA (RT1a) - > AUG (RT1c) model of acute rejection in situ liver transplantation is divided into seven groups according to the different drugs for the receptor, and each group of 12 rats comprises A. methylprednisolone, B. methylprednisolone + Kjekto, C. methylprednisolone + SMATAI, D. methylprednisolone + Kjekto + SMATAI, E.VEG monoclonal antibody, F. endostatin, G.FK506+ Cydoceptor (as a control group), and is injected into the body of the receptor rat via the abdominal cavity. According to different time after transplantation, each of the seven groups is divided into subgroups of 1, 3 and 7 days after transplantation, and each subgroup is provided with 4 rats;
(4) the levels of VEGF, IL-2, IL-8, HLA antigens, lgA, lgG, lgD, lgM, lgE in the serum of the recipient were measured by ELISA. DA (RT1a) - > AUG (RT1c) rejection model each subgroup of receptor rats are respectively bled from inferior vena cava for standby before liver transplantation, then killed according to the predetermined days 1, 3 and 7 after the liver transplantation, and are respectively bled from inferior vena cava for 2ml, and the level of VEGF, IL-2, IL-8, HLA antigen, lgA, lgG, lgD, lgM and lgE in the serum before and after the liver transplantation operation of each subgroup of receptor rats in a double antibody sandwich ELISA method is respectively detected;
(5) detecting VEGF by immunohistochemistry; detecting the expression of various indexes such as CD 68(+) macrophages, CD3(+) Tcells, CD19(+) B cells, CD56(+) NK cells, CD115(+) monocytes, CD4, CD8, CD31, CD44, CD45, CCR1, CCR3, CCR5, CXCR3 and the like in the liver and spleen before and after liver transplantation in various subgroups of receptors of a DA (RT1a) - > AUG (RT1c) rejection model by flow cytometry;
(6) the relation between the expression of VEGF before and after liver transplantation operation and the relation between macrophage, monocyte, T cell, B cell, NK cell, new blood vessel and inflammatory mediator after the non-blocking VEGF effect and the blocking VEGF effect are determined through immunohistochemical and flow cytometry detection, the mutual effect of VEGF on the mediation of inflammatory cell infiltration and angiogenesis in a manifold area is determined, and the VEGF further acts on the B cell and plays a promoting role in liver transplantation acute rejection reaction instead of acting on the T cell;
(7) the expression of various indexes such as VEGF, eotaxin, IP-10, lymphotactin, MCP-1, ICAM-1, VCAM-1 and the like in the liver and spleen before and after the liver transplantation operation in various subgroup receptors of a DA (RT1a) - > AUG (RT1c) rejection model is respectively detected by a quantitative RT-PCR method, and the mutual relations between the expression of VEGF before and after the liver transplantation operation and various inflammatory cells and mediators are further determined from the molecular gene level after the action of the unblocked VEGF and the action of the blocked VEGF are further determined, so that the antibody-B cell mediated humoral immune response caused by the interaction between inflammatory cell infiltration in a convergent area and angiogenesis and the overlapping of time is further determined to cause the acute rejection reaction of the in-situ liver transplantation.
Selecting, grouping, detecting and carrying out liver biopsy through clinical liver transplantation cases:
(1) selecting 30 clinical liver transplantation cases which do not have acute rejection at random and 30 clinical liver transplantation cases which have acute rejection proved by pathology, and detecting the levels of VEGF, IL-2, IL-8, HLA antigen, lgA, lgG, lgD, lgM and lgE in the serum of 60 liver transplantation receptors by an ELISA method before operation;
(2) measuring the level of VEGF in the serum of a receptor after liver transplantation, namely respectively detecting the levels of VEGF, IL-2, IL-8, HLA antigen, lgA, lgG, lgD, lgM and lgE in the serum of the receptor of 60 cases of liver transplantation at 1, 3 and 7 days after the liver transplantation by using an ELISA method, and then comparing the levels in the VEGF serum of the receptor of two liver transplantation cases before and after the operation pairwise;
(3) grouping 30 cases of the receptors which have acute rejection are divided into five groups according to the difference of the drugs for the receptors, each 6 cases of each group are A, Methylpredene, B, Methylpredene + Kjest + Syrmet, C, Methylpredene + Kjest, D, Methylpredene + Syrmet, E.FK506+ Cellcept (control group), the using method is oral administration and intravenous drip, and the specific dosage is as the instruction. Detecting the levels of VEGFIL-2, IL-8, HLA antigen, lgA, lgG, lgD, lgM and lgE in the serum of a receptor which has undergone acute rejection reaction and is taken before and after the administration for 1, 3 and 7 days by an ELISA method, and comparing the obtained values with each other to determine whether each group has an effect of eliminating the acute rejection reaction of liver transplantation after VEGF is blocked;
(4) and (3) liver biopsy, namely performing living liver biopsy on the liver supplies 1 day before and after the administration of the medicine for each group of receptors respectively, and taking the right 8 and 9 intercostal spaces for puncture under the guidance of B ultrasonic by adopting a special quantitative liver biopsy needle. The total biopsy tissue is divided into 5 parts, and one part is used for pathological HE staining and is used for diagnosing whether acute rejection reaction occurs. The other four parts are respectively used for RT-PCR, flow cytometry, immunohistochemistry and PCR-DNA sequencing detection;
(5) PCR-DNA sequencing directly detects the binding sites of VEGF gene in liver biopsy, thereby determining which binding sites of VEGF gene in liver biopsy are associated with increased risk of acute liver allograft rejection.
The experimental protocol adopted in the above experimental procedure was explained:
detection by an ELISA method:
taking blood samples, and establishing a standard curve according to the use instruction, wherein 8 standard holes are arranged, 100ul of sample diluent is added into each hole, and the 8 th hole is a blank control hole; and then loading the sample, mixing uniformly, washing the plate, measuring an absorbance value at the position of 492nm, calculating after subtracting a blank value from all OD values, drawing a graph on semilog paper by using the OD value of the standard substance, drawing a standard curve, and checking the content of the corresponding rat cytokine on the graph according to the OD value of the sample.
Immunohistochemistry, flow cytometry and quantitative RT-PCR detection steps:
before liver transplantation, a small amount of tissues are taken out from the liver and the spleen of each subgroup of receptor rats respectively for standby, and after the receptor rats are killed after the liver transplantation, a small amount of tissues for the liver and the spleen are taken out for standby;
a immunohistochemistry (EnVision method):
cutting the paraffin embedded tissue into thin slices with the thickness of 4um, mounting the thin slices on a glass slide, dyeing the thin slices with a hematoxylin lining, and bluing the thin slices with hot water; after drying, the resin is sealed, and the immunohistochemical result is quantified by adopting a CMIAS true color medical image analysis system. And (5) judging the VEGF positive result, wherein the VEGF positive cells are brown yellow particles or brown particles and are distributed in cytoplasm. The specific operation method comprises randomly selecting 10 positive cell most dense areas under 100 times of visual field, converting to CMIAS true color medical image analyzer under 400 times of visual field, counting positive tumor cells on screen, and multiplying by 1.6 to obtain cell number/mm 2;
b, flow cytometry:
(1) lavage the prepared liver tissue from the receptor rat before and after the liver transplantation operation with 0.05% collagenase to prepare single cell suspension, and then the single cell suspension is cultured in a plastic culture dish after the middle layer is sucked and the same amount of culture medium is added by using a Percoll equal density gradient centrifugation method (25% and 50% Percoll), 40C, 800g and 30 Min;
(2) placing the prepared spleen tissue taken out from a receptor rat before and after the liver transplantation into a culture dish containing 5ml of Hanks liquid, placing the spleen on a stainless steel mesh (100 or 200 meshes) by adopting a steel mesh grinding method, lightly grinding and pressing the spleen by using a syringe needle core, cracking red blood cells by using Tris-ammonia chloride, washing the red blood cells by using RPMI1640 for three times, and obtaining a cell suspension, namely the spleen cells; then the method is carried out according to the conventional operation steps.
c quantitative RT-PCR:
(1) total RNA extraction: weighing rat liver and spleen tissue preserved by ultralow temperature liquid nitrogen on an electronic balance (estimated to be 100mg), transferring the rat liver and spleen tissue into a mortar precooled by liquid nitrogen, and grinding the tissue by using a pestle, wherein the liquid nitrogen is continuously added until the rat liver and spleen tissue is ground into powder; measuring OD value, and detecting RNA concentration by an ultraviolet spectrophotometer (A260/A280);
(2) reverse transcription reaction: sequentially adding 1ul sample total RNA, MgCl22.2ul, 1ul 10 XT buffer solution, RNase freedH203.75ul, dNTP mix 1ul and 0.25 ul RNase inhibitor; 0.5 ul of reverse transcriptase and 0.5 ul of OligodT-Adaptor primer form a 10ul reaction system; completing reverse transcription reaction at 30 deg.C for 10min, 42 deg.C for 10min, 99 deg.C for 5min, and 5 deg.C for 5 min;
(3) and (3) PCR: VEGF upstream primer: 5'-ACC TCA CCA AAG CCA GCA CA-3', downstream primer: 5 'one GGCATG GTG GTGGTG ACA TGG TT-3' (i.e. oligonucleotide sequence), the length of the amplified product fragment is 536bp, and beta-actin is internal reference; 2min at 94 ℃ for 1 cycle; 30S at 94 ℃, 30S at 55 ℃, 1min at 72 ℃ and 35 cycles;
(4) then, the detection was carried out by a quantitative PCR instrument 7900 type.
From the above test scheme and test steps, it can be seen that some binding sites of VEGF gene in liver biopsy tissue are related to the increased risk of acute liver allograft rejection, and further verify the new theory of acute rejection of liver transplantation, namely acute rejection of body fluid. The specific binding site of VEGF is determined so as to facilitate the later phase to resist the production of rejection drugs and solve the rejection reaction after organ transplantation.
The specific binding site of the VEGF gene can be determined by the following steps:
step 1, taking the right 8 and 9 intercostal spaces of a receptor to puncture under the guidance of B ultrasonic by adopting a biopsy needle, and taking a liver tissue sample to prepare a DNA template; 1mg or less than 1mg of recipient liver tissue obtained by liver biopsy was placed in a 1.5ml centrifuge tube. DNA in the sample is extracted using a tissue DNA extraction kit. The amount of tissue used for purification is determined according to the condition and form of the sample. If minced prior to proteinase K treatment, up to 25mg of tissue may also be used for purification. Sample treatment: add freshly prepared 50-100ul incubation buffer/proteinase K solution. Incubate at 56 ℃ for 2 hours to overnight. Most tissues were completely digested within two hours. The incubated sample tube was removed and two volumes of lysis buffer in the DNAIQTM system were added to 7ul of DNAIQTM resin in the DNAIQTM system in complete suspension. Incubation was performed for 5 minutes at room temperature after 3 seconds of high speed vortex shaking. Then, the tube was placed on a magnetic separation stand to perform magnetic separation by high-speed vortex oscillation for 2 seconds. All solutions were removed and 100 μ l of the prepared lysis buffer was added, the tubes were removed from the magnetic separation rack and vortexed at high speed for 2 seconds. The tube was placed back on the magnetic separation rack and all the lysate was removed. 100 μ l of the formulated 1 x wash solution was added, the tube removed from the magnetic separation rack and vortexed at high speed for 2 seconds. The tube was returned to the magnetic separation rack and all the wash solution was removed. And to ensure that all liquid is removed after the last wash. The lid was opened and the tube was placed on a magnetic separation rack and allowed to air dry for 5 minutes. According to the amount of the test material used for DNA extraction, 25-100 mul of eluent is added, and the small elution volume is helpful for obtaining DNA with higher final concentration. The tube cap was capped and vortexed at high speed for 2 seconds. Incubation was carried out at 65 ℃ for 5 minutes. The incubated tube was removed and vortexed at high speed for 2 seconds. Immediately placed on a magnetic separation rack. The DNA solution was carefully transferred to the selected container. The obtained DNA was dissolved in 50. mu.l of TE Buffer, and the extracted DNA containing the VEGF gene was used as a DNA template.
Step 2, amplifying the DNA dissolving solution obtained in the step 1 to obtain a PCR amplification solution, and processing the PCR amplification solution by a PCR instrument; the extracted VEGF region was PCR amplified using a commercial 2 Xsuper Taq PCR MasterMix. The PCR reaction system was 50. mu.l. The method specifically comprises the following steps: 25. mu.l of 2 XMASTERMix, 5. mu.l of the forward primer, 5. mu.l of the reverse primer and 15. mu.l of the DNA lysis solution. The working environment of the PCR instrument is as follows: firstly heating for 2-5 minutes in an environment of 95 ℃, heating for 30 seconds in an environment of 96 ℃, heating for 30 seconds in an environment of 58 ℃ and heating for 1 minute in an environment of 72 ℃ in sequence, circulating for 40 times, and finally preserving heat in an environment of 72 ℃.
Step 3, sampling the solution processed in the step 2, namely adding 1-2% agarose gel into 5 mul of PCR amplification solution and carrying out electrophoresis observation;
step 4, when the solution in the step 3 is subjected to electrophoresis and then a strip-shaped product appears, purifying the PCR amplification solution obtained in the step 2 to obtain a PCR product;
step 5, amplifying the purified PCR product to obtain a sequencing PCR system solution, circularly heating the sequencing PCR system solution, and refrigerating the sequencing PCR system solution in a low-temperature environment; the mixed solution is heated circularly, namely the mixed solution is heated in an environment of 96 ℃ for 1 minute, sequentially heated in an environment of 96 ℃ for 10 seconds, 50 ℃ for 5 seconds and 60 ℃ for 4 seconds and circulated for 25 times, and finally the mixed solution is kept warm in an environment of 4 ℃.
Step 6, adding 2 mul of 125mmol/LEDTA, 3mol/L sodium acetate and absolute ethyl alcohol into the sequencing PCR system solution obtained in the step 5, carrying out oscillation centrifugation on the mixed solution, and removing the supernatant;
and 7, injecting high-purity formamide into the solution treated in the step 6, oscillating for dissolution, performing denaturation treatment through a qualitative PCR instrument, namely adding 2 mul 125mmol/LEDTA and 2 mul 3mol/L sodium acetate (pH5.2) into the solution obtained in the step 6 to the bottom of the tube, adding 50 mul 100% absolute ethyl alcohol, tightly covering the tube cover, oscillating for a short time, and placing for 15 minutes in a dark place at room temperature. The supernatant was removed by centrifugation at 12000rpm for 30 minutes at 4 ℃. Then adding 150 mul precooled 70% ethanol, centrifuging for 10 minutes at the rotating speed of 12000rpm at the temperature of 4 ℃, removing supernatant, placing for 15-30 minutes in a dark place at room temperature,
and adding 10 mu l of Hi-Di Formamide, oscillating for a short time to dissolve DNA, and centrifuging for a short time to completely centrifuge the liquid on the tube wall to the tube bottom. The dissolved sample was denatured at 95 ℃ for 5 minutes in a qualitative PCR instrument, rapidly cooled in ice for 4 minutes, and subjected to electrophoresis.
And 8, filling sequencing glue into the solution obtained in the step 7, carrying out electrophoretic separation after filling, detecting by a sequencer to form a sequencing sequence and a sequencing peak image, and determining the binding site of the VEGF gene. Namely, the denatured sequencing product to be detected is filled into a 96-well plate matched with a gene analyzer, and analysis and data collection are carried out through gene analysis after the cover is closed. The sequencing result is automatically stored in a preset position of a gene analyzer, so that the binding site of the VEGF gene is determined. Solves the technical problem that the VEGF gene binding site can not be accurately detected in the medical field at present, and provides for the subsequent intervention of reducing rejection reaction on patients.
The above-mentioned embodiments are only preferred embodiments of the present invention, and the description thereof is more specific and detailed, but not to be construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

Claims (9)

1. A VEGF gene binding site detection method based on a new acute rejection reaction mechanism is characterized by comprising the following steps:
step 1, taking a biopsy needle to perform puncture in the right 8 and 9 intercostal spaces under the guidance of B ultrasonic through liver puncture pathological biopsy, and sampling to manufacture a DNA template;
step 2, amplifying the DNA template obtained in the step 1 to obtain a PCR amplification solution, and processing the PCR amplification solution by a PCR instrument;
step 3, sampling the solution processed in the step 2 and carrying out agarose electrophoresis observation;
step 4, when the solution in the step 3 is subjected to electrophoresis and then a strip-shaped product appears, purifying the solution obtained in the step 2 to obtain a PCR product;
step 5, preparing the purified PCR product to obtain a sequencing PCR system solution, circularly heating the sequencing PCR system solution, and refrigerating in a low-temperature environment;
step 6, adding 125mmol/LEDTA, 3mol/L sodium acetate and absolute ethyl alcohol into the sequencing PCR system solution obtained in the step 5, carrying out oscillation centrifugation on the mixed solution, and removing a supernatant;
step 7, injecting high-purity formamide into the solution treated in the step 6, oscillating for dissolving, and performing denaturation treatment by using a qualitative PCR instrument;
and 8, injecting sequencing glue into the solution obtained in the step 7 for electrophoretic separation, detecting by a sequencer to form a sequencing sequence and a sequencing peak image, and determining the binding site of the VEGF gene.
2. The method for detecting VEGF gene binding site based on the novel acute rejection mechanism of claim 1, wherein the amount of the recipient tissue sample sampled by the biopsy needle in step 1 is 1 mg.
3. The method for detecting VEGF gene binding site based on the novel acute rejection mechanism of claim 1, wherein the PCR amplification solution of step 2 comprises 2 xMasterMix, the upstream primer, the downstream primer and a DNA lysis solution, and the total amount of the PCR amplification solution is 50 μ l.
4. The method for detecting VEGF gene binding site based on the novel mechanism of acute rejection according to claim 1, wherein the agarose gel applied in step 3 is at a concentration of 1-2%.
5. The method for detecting VEGF gene binding site based on the novel mechanism of acute rejection of claim 1, wherein the sequencing PCR system solution of step 5 comprises PCR product, BigDye Buffer, sequencing primer and sterile purified water, and the total amount of the sequencing PCR system solution is 20 μ l.
6. The method for detecting VEGF gene binding site based on the novel acute rejection mechanism of claim 1, wherein the heating process in step 5 is as follows: firstly heating for 1 minute in a 96 ℃ environment, heating for 10 seconds in a 96 ℃ environment, heating for 5 seconds in a 50 ℃ environment, heating for 4 seconds in a 60 ℃ environment, circulating for 25 times, and finally preserving heat in a 4 ℃ environment.
7. The method for detecting VEGF gene binding sites based on a new acute rejection mechanism according to claim 1, wherein the high purity Formamide in the step 7 is Hi-Di Formamide of 10 μ l.
8. The method for detecting the VEGF gene binding site based on the new acute rejection mechanism according to claim 1, wherein the sequencing gel added in the step 8 is 2 μ l.
9. The method for detecting VEGF gene binding site based on the novel acute rejection mechanism of claim 3, wherein the sequence of the upstream primer is as follows: 5'-CATGCA GAT TAT GCG GAT CAA-3', the sequence of the downstream primer is: 5'-TTT GTT GTG CTG TAG GAA GCT CA-3' are provided.
CN202010436770.6A 2020-05-21 2020-05-21 VEGF gene binding site detection method based on novel acute rejection reaction mechanism Pending CN111593113A (en)

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