CN114181955A - Construction experiment method of vascular endothelial cell PDHA1 gene specific knock-out mouse - Google Patents

Construction experiment method of vascular endothelial cell PDHA1 gene specific knock-out mouse Download PDF

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CN114181955A
CN114181955A CN202111388529.1A CN202111388529A CN114181955A CN 114181955 A CN114181955 A CN 114181955A CN 202111388529 A CN202111388529 A CN 202111388529A CN 114181955 A CN114181955 A CN 114181955A
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pdha1
mouse
mice
vascular endothelial
gene
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杨安宁
孙岳
姜怡邓
刘志宏
郝玮
宝瑞
刘耀阳
王秋实
刘太阳
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Ningxia Medical University
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Abstract

The invention relates to a construction experiment method of a vascular endothelial cell PDHA1 gene specific knock-out mouse, which specifically comprises the following steps: 1) breeding sufficient amount of PDHA1-Tek-creERT2(f/f,+)The mice are combined with 2 male mice and 4 female mice, and the pregnancy of the female mice is determined when the female mice have the vaginal embolus; 2) and (3) identifying the mouse gene: 3) detecting the protein expression level in the lung tissue of the mouse by using Western blot; 4) detecting mRNA expression level in mouse lung tissue by real-time quantitative PCR; 5) mouse lung tissue immunofluorescence VE-cadherin and PDHA1 co-staining. The invention has the advantages that: the scheme is reasonable in design, can research the energy metabolism relationship between diseases and endothelial cells, verifies the expression of speed-limiting enzymes HK2 and IDH of glycolysis and oxidative phosphorylation, confirms the change of energy metabolism of vascular endothelial cells, and has good popularization prospect and medical economic value.

Description

Construction experiment method of vascular endothelial cell PDHA1 gene specific knock-out mouse
Technical Field
The invention relates to a construction experimental method of a vascular endothelial cell PDHA1 gene specific knock-out mouse.
Background
Energy metabolism of vascular Endothelial Cells (ECs) is related to many diseases, and research has shown that during the occurrence and development of Atherosclerosis, oxidative stress of the vascular endothelial cells causes oxidative metabolism imbalance, high-level ROS secretion can reduce the production of NO and hydrogen sulfide, and cause enhanced inflammatory response of the ECs, increased leukocyte adhesion, platelet aggregation, formation of malignant cycle, and aggravation of the progression of Atherosclerosis (AS), and CoQ10 is one of important components of the mitochondrial respiratory chain and can promote metabolism to play a role in the prevention and treatment of AS. HE et al found that by knocking out Sirt3 gene, oxygen consumption of EC was increased, but angiogenesis ability was decreased, and myocardial cell function was lost due to hypoxia, so that glycolytic pathway of EC function was closely related to myocardial function. The vascular endothelial cells promote the polarization of macrophages to M2 type through glycolysis pathway, and improve the muscle regeneration capability. Whereas upon knockout of PFKFB3, macrophages were polarized to M1 type and muscle regeneration was reduced. Therefore, the respiratory metabolic mode of the vascular endothelial cells is closely related to the occurrence and development of diseases. Although vascular endothelial cells are metabolized mainly by glycolysis, aerobic phosphorylation mainly by mitochondrial respiration is not eliminated in endothelial cells. The PDHA1 gene is a vector gene encoding an important subunit of pyruvate dehydrogenase 1(PDHE1), is a pivotal junction connecting glycolysis and tricarboxylic acid cycle, and is a key gene for energy regulation. Studies with Weiqiong et al show that the lack of PDHA1 gene can cause Leigh syndrome, and cause lactic acid to accumulate in the neuromuscular system to cause related diseases. PDHA1 is involved in various diseases and tumorigenesis. Sirt3 exerts an anti-Warburg effect by inhibiting the occurrence of cholangiocarcinoma tumors through the HIF1 α/PDK1/PDHA1 pathway. At present, researches on PDHA1 gene and tumor are mostly limited to cell and molecular level, while the construction technology of a vascular endothelial cell specific knockout mouse is mature, but the knockout of the PDHA1 gene in vascular endothelial cells is firstly constructed, and the expression of the PDHA1 gene in vascular endothelial cell related diseases is analyzed at the whole animal level to map the effect of energy metabolism on different diseases. The method is a commonly used technology for constructing a conditional gene knockout mouse by changing the energy metabolism mode of endothelial cells to possibly influence the occurrence and development of diseases, wherein the Cre recombinase comprises 2, one is the conditional gene knockout from the gestation period and is generally expressed as Cre recombinase; the other is an inducible recombinase, generally designated as CreERT2 recombinase, CreERT2 is a fusion protein of Cre recombinase and estrogen receptor, in which the ER ligand is capable of binding to tamoxifen, mediating CreERT2 recombinase from the cytoplasm into the nucleus, excising the DNA sequence between the 2 LoxP sites in the same orientation, so that the knock-out or activation of a particular targeted gene at a particular time and space becomes controllable. In order to meet the controllability of the experiment, a CreERT2 recombinase system is adopted for endothelial cell PDHA1 specific knockout. We intervene contemporaneously with the modelling intervention by simultaneously effecting knock-out contemporaneous through Tamoxifen (Tamoxifen) induction. Establishing an endothelial cell conditional knockout mouse of the PDHA1 gene is an important way for researching the relation between endothelial cell respiratory metabolism and diseases. To date, PDHA1 gene studies have mostly focused on tumors and PDHA 1-specific mutant diseases, but have not been reported in others. Since vascular endothelial cells function as organs, energy metabolism of vascular endothelial cells may be involved in various organ diseases. The study shows that the gavage can relieve the development of silico-pulmonary fibrosis, the HGF gene knockout of vascular endothelial cells has a promoting effect on the development of fibrosis, the breeding and identification of a mouse with vascular endothelial cell specific knockout of the PDHA1 gene provide experimental basis for the subsequent study of related mechanisms of vascular endothelial cell PDHA1 gene and pulmonary fibrosis, and a new idea is provided for the study of other diseases caused by energy metabolism of vascular endothelial cells.
Disclosure of Invention
The invention aims to provide a construction experimental method for a vascular endothelial cell PDHA1 gene specific knock-out mouse, so as to solve the problems in the background technology.
In order to solve the technical problems, the technical scheme provided by the invention is as follows: the construction experiment method of the vascular endothelial cell PDHA1 gene specific knock-out mouse specifically comprises the following steps:
1) breeding sufficient amount of PDHA1-Tek-creERT2(f/f,+)The 2 male mice and 4 female mice are combined in a cage by the mouse, the female mice are determined to be pregnant when the female mice have the vaginal embolus, the birth and the birth number of the mice are observed after the birth of the newborn mice, and the mice take ear tags as marks after the mice are born for three weeks, and the toes are cut for gene identification; carrying out intraperitoneal injection on tamoxifen 20mg/mL at the weight dose of 75mg/kg at the age of 6 weeks of newborn mice, injecting 1 time every 24 hours, continuously injecting for 5 days, and taking materials at the age of 8 weeks after injection; carrying out lung lobe perfusion 10% OCT on the right lower lobe of the lung tissue to carry out frozen tissue embedding, and directly putting the rest lung lobes at-80 ℃;
2) and (3) identifying the mouse gene:
2a) preparation of 10 × MGB solution: taking 1 clean centrifuge tube of 50mL, adding 0.05mol of Tris crystal into the centrifuge tube, dissolving the Tris crystal in 50mL of deionized water, measuring the pH value of the centrifuge tube, and adjusting the pH value of the centrifuge tube to 8.8; another 15mL clean centrifuge tube was added 0.02mL (NH)4)2SO4And 10mL of deionized water until completely dissolved; another 15mL clean centrifuge tube was added with 0.01mL MgCl2 and 10mL deionized water; after the solution was completely prepared, 6.7mL of Tris solution was placed in a clean centrifuge tube and 830. mu.L of (NH) was added4)2SO4Solution, 650. mu.L of MgCl2Solution and 1.82mL of ddH2Fully and uniformly mixing O;
2b) preparation of Lysismix: uniformly mixing prepared 1mL of 10 XMGB, 50 mu L of 100% TritonX, 100 mu L of beta-mercaptoethanol and 8.4mL of ddH 2O;
2c) mouse toe digestion to extract DNA: taking the prepared and calculated Lysismix, adding proteinase K into the Lysismix, adding 3 mu L of proteinase K into every 200 mu LLysimix, preparing the mixture on site, adding 120 mu L of the prepared solution into an EP tube containing toes of a mouse, carrying out water bath at 55 ℃ for overnight lysis, carrying out denaturation at 95 ℃ for 10min, then centrifuging at 11000r/min for 5min, and taking the supernatant;
2d) DNA replication and gene identification: respectively adding 10 mu L of Novovozam blue enzyme, 0.3 mu L of upstream primer and 0.3 mu L of downstream primer and 0.59 mu L of extracted DNA into an eight-connected tube of the PCR; pre-denaturing at 95 ℃ for 5min, 30s at 95 ℃, 30s at 60 ℃, 42s at 72 ℃, 40 cycles, preserving at 72 ℃ for 5min, and 16 ℃, then performing DNA amplification, performing 1% agarose gel electrophoresis on the amplified DNA, and exposing and observing DNA bands;
3) western blot detection of protein expression levels in mouse lung tissues: aseptically taking lung tissues of a mouse, shearing the lung tissues by using scissors, adding a phosphatase inhibitor, a protease inhibitor, PMSF and a lysine Buffer which are well proportioned, fully breaking histiocytes by using a high-shear dispersion emulsifying machine, and extracting the total protein of the lung tissues of the mouse; adding Loading buffer and ultrapure water into part of total protein to dilute the protein to 3 mu g/mL, and denaturing at 95 ℃ for 5 min; taking 10 mu L of denatured protein sample, converting the denatured protein sample into 120V after the constant voltage of 10% SDS-PAGE gel electrophoresis is 80V; transferring the protein of the gel onto a PVDF membrane, performing constant pressure 15V, 75min, sealing with 5% skimmed milk powder prepared by PBST buffer solution for 2h, and adding 1:1000 dilution PDHA1, 1:1000 dilution HK2, 1: shaking table incubating IDH diluted by 500 and GAPDH primary anti-dilution solution diluted by 1: 5000 overnight at 4 deg.C, and washing with PBST buffer solution for 3 times, each time for 10 min; incubating horseradish peroxidase-labeled secondary antibody for 2h at room temperature, and eluting with PBST buffer solution for 3 times, each time for 10 min; ECL chemical luminescence method development, exposure time is adjusted according to the intensity of a luminescence signal, and Image Lab measures a gray value;
4) real-time quantitative PCR detection of mRNA expression levels in mouse lung tissues: collecting lung tissues of a mouse into a 1.5mL EP tube, shearing the lung tissues by using scissors, adding about 1mL of lysis solution RZ into the EP tube, adding lysis solution RZ1mL into each 50-100mg of tissue samples, and homogenizing in a homogenizer; centrifuging at 4 ℃ and 12000r/min for 5min, and taking supernatant to a new RNA ase-free EP tube; adding 200 μ L chloroform, shaking, standing at room temperature for 3min, centrifuging at 4 deg.C 12000r/min for 5min, eluting the aqueous phase to obtain RNA, and storing; measuring A260/A280 value by using an ultraviolet spectrophotometer, selecting RNA with A260/A280 ratio between 1.8-2.1 for reverse transcription, synthesizing cDNA as a template for q-PCR reaction, wherein the reaction system is 20 mu L, TB Gerrn Premix Ex Taq II (2) is 10 mu L, the upstream primer and the downstream primer are 0.8 mu L respectively, the cDNA template is 2 mu L, and the rest is filled with sterilized water; the reaction conditions are 10s at 95 ℃, 30s at 60 ℃ and 42s at 72 ℃, the total is 40 cycles, and the preheating at 95 ℃ is carried out for 5min before the reaction starts; 3 replicate wells per sample;
5) mouse lung tissue immunofluorescence VE-cadherin and PDHA1 co-staining: taking out the frozen section of the mouse lung from-80 ℃, putting the section into a wet cassette to restore to room temperature and dry the tissue surface, putting the section into PBS (0.01 mol/L), rinsing the section for 5min by a shaking table, and reducing the self background; soaking and covering with 4% paraformaldehyde for 25min, and rinsing with PBS for 5min for 3 times; perforating 0.3% triton-100 cell membrane for 20min, and rinsing with PBS for 5min for 3 times; sealing with volume fraction of 5% fetal calf serum for 30min, adding 1: 200VE-cadherin and PDHA1 primary antibody, sealing at 4 deg.C overnight; the next day, the cassette was taken out and returned to room temperature, rinsed with PBS 3 times, incubated with secondary antibody in dark, incubated at 37 ℃ for 1h, and rinsed with PBS 3 times; adding DAPI, incubating for 3min in dark, and rinsing with PBS for 3 times; and (3) dropwise adding an anti-fluorescence quencher, covering the glass slide, sealing the glass slide by using nail polish, and observing the glass slide by using a Zeiss LSM800 laser confocal microscope.
As a preferred embodiment, the primer sequence of PDHA1 is
F:TGTGACCTTCATCGGCTAGAA,R:TGATCCGCCTTTAGCTCCATC。
Preferably, the HK2 primer sequence is
F:ATGATCGCCTGCTTATTCACG；R:CGCCTAGAAATCTCCAGAAGGG。
As a preferred embodiment, the IDH primer sequence is
F:GTGGGCGTCAAGTGTGCTA；R:CCACCCAGAATGTTTCGGATG。
The invention has the advantages that: the scheme is reasonable in design, can research the energy metabolism relationship between diseases and EC, verifies the expression of speed-limiting enzymes HK2 and IDH of glycolysis and oxidative phosphorylation, confirms the change of energy metabolism of vascular endothelial cells, and has good popularization prospect and medical economic value.
Drawings
FIG. 1 is a diagram showing the results of gene identification in a newborn mouse, wherein a is an electron micrograph and b is a histogram.
Drawing notes: lanes 1, 2, 4, 5, 7, 8 are PDHA1(iΔEC/iΔEC)Mouse, lane 3 PDHA1(iΔEC/-)A mouse.
FIG. 2 is PDHA1(iΔEC/iΔEC)Conditional knockout mice and PDHA1(iΔEC/-)A comparison graph of the expression level of the mouse PDHA1 protein, wherein c is an electron microscope graph and d is a histogram.
Drawing notes: and PDHA1(iΔEC/-)Group comparison of<0.01。
FIG. 3 is PDHA1(iΔEC/iΔEC)Conditional knockout mice and PDHA1(iΔEC/-)Mouse PDHA1 mRNA expression levels are plotted against histograms.
Drawing notes: and PDHA1(iΔEC/-)Group comparison of<0.05。
FIG. 4 is PDHA1(iΔEC/iΔEC)Conditional knockout mice and PDHA1(iΔEC/-)Mouse HK2 and IDH1 protein and mRNA expression level.
Drawing notes: and PDHA1(iΔEC/-)Group comparison, P<0.05。
FIG. 5 is a graph showing the immunofluorescence assay for the expression of PDHA1 protein (magnification:. times.400) in vascular endothelial cells.
Drawing notes: VE-Cadherin localized vascular endothelial cells and expressed PDHA1 in vascular endothelial cells, and co-localization of both showed expression of PDHA1 in vascular endothelial cells.
Detailed Description
The invention is illustrated below by means of specific examples, without being restricted thereto.
Examples
The construction experiment method of the vascular endothelial cell PDHA1 gene specific knock-out mouse specifically comprises the following steps:
1) breeding sufficient amount of PDHA1-Tek-creERT2(f/f,+)The mice are combined with the 2 male mice and the 4 female mice, the pregnancy of the female mice is determined when the female mice have the vaginal embolus, and the birth of the mice are observed after the birth of the newborn miceThe quantity, after the mouse is born for three weeks, the mouse takes an ear tag as a mark, and carries out gene identification by cutting toes; carrying out intraperitoneal injection on tamoxifen 20mg/mL at the weight dose of 75mg/kg at the age of 6 weeks of newborn mice, injecting 1 time every 24 hours, continuously injecting for 5 days, and taking materials at the age of 8 weeks after injection; carrying out lung lobe perfusion 10% OCT on the right lower lobe of the lung tissue to carry out frozen tissue embedding, and directly putting the rest lung lobes at-80 ℃;
2) and (3) identifying the mouse gene:
2a) preparation of 10 × MGB solution: taking 1 clean centrifuge tube of 50mL, adding 0.05mol of Tris crystal into the centrifuge tube, dissolving the Tris crystal in 50mL of deionized water, measuring the pH value of the centrifuge tube, and adjusting the pH value of the centrifuge tube to 8.8; another 15mL clean centrifuge tube was added 0.02mL (NH)4)2SO4And 10mL of deionized water until completely dissolved; another 15mL clean centrifuge tube was added with 0.01mL MgCl2 and 10mL deionized water; after the solution was completely prepared, 6.7mL of Tris solution was placed in a clean centrifuge tube and 830. mu.L of (NH) was added4)2SO4Solution, 650. mu.L of MgCl2Solution and 1.82mL of ddH2Fully and uniformly mixing O;
2b) preparation of Lysismix: uniformly mixing prepared 1mL of 10 XMGB, 50 mu L of 100% TritonX, 100 mu L of beta-mercaptoethanol and 8.4mL of ddH 2O;
2c) mouse toe digestion to extract DNA: taking the prepared and calculated Lysismix, adding proteinase K into the Lysismix, adding 3 mu L of proteinase K into every 200 mu LLysimix, preparing the mixture on site, adding 120 mu L of the prepared solution into an EP tube containing toes of a mouse, carrying out water bath at 55 ℃ for overnight lysis, carrying out denaturation at 95 ℃ for 10min, then centrifuging at 11000r/min for 5min, and taking the supernatant;
2d) DNA replication and gene identification: respectively adding 10 mu L of Novovozam blue enzyme, 0.3 mu L of upstream primer and 0.3 mu L of downstream primer and 0.59 mu L of extracted DNA into an eight-connected tube of the PCR; pre-denaturing at 95 ℃ for 5min, 30s at 95 ℃, 30s at 60 ℃, 42s at 72 ℃, 40 cycles, preserving at 72 ℃ for 5min, and 16 ℃, then performing DNA amplification, performing 1% agarose gel electrophoresis on the amplified DNA, and exposing and observing DNA bands;
3) western blot detection of protein expression levels in mouse lung tissues: aseptically taking lung tissues of a mouse, shearing the lung tissues by using scissors, adding a phosphatase inhibitor, a protease inhibitor, PMSF and a lysine Buffer which are well proportioned, fully breaking histiocytes by using a high-shear dispersion emulsifying machine, and extracting the total protein of the lung tissues of the mouse; adding Loading buffer and ultrapure water into part of total protein to dilute the protein to 3 mu g/mL, and denaturing at 95 ℃ for 5 min; taking 10 mu L of denatured protein sample, converting the denatured protein sample into 120V after the constant voltage of 10% SDS-PAGE gel electrophoresis is 80V; transferring the protein of the gel onto a PVDF membrane, performing constant pressure 15V, 75min, sealing with 5% skimmed milk powder prepared by PBST buffer solution for 2h, and adding 1:1000 dilution PDHA1, 1:1000 dilution HK2, 1: shaking table incubating IDH diluted by 500 and GAPDH primary anti-dilution solution diluted by 1: 5000 overnight at 4 deg.C, and washing with PBST buffer solution for 3 times, each time for 10 min; incubating horseradish peroxidase-labeled secondary antibody for 2h at room temperature, and eluting with PBST buffer solution for 3 times, each time for 10 min; ECL chemical luminescence method development, exposure time is adjusted according to the intensity of a luminescence signal, and Image Lab measures a gray value;
4) real-time quantitative PCR detection of mRNA expression levels in mouse lung tissues: collecting lung tissues of a mouse into a 1.5mL EP tube, shearing the lung tissues by using scissors, adding about 1mL of lysis solution RZ into the EP tube, adding lysis solution RZ1mL into each 50-100mg of tissue samples, and homogenizing in a homogenizer; centrifuging at 4 ℃ and 12000r/min for 5min, and taking supernatant to a new RNA ase-free EP tube; adding 200 μ L chloroform, shaking, standing at room temperature for 3min, centrifuging at 4 deg.C 12000r/min for 5min, eluting the aqueous phase to obtain RNA, and storing; measuring A260/A280 value by using an ultraviolet spectrophotometer, selecting RNA with A260/A280 ratio between 1.8-2.1 for reverse transcription, synthesizing cDNA as a template for q-PCR reaction, wherein the reaction system is 20 mu L, TB Gerrn Premix Ex Taq II (2) is 10 mu L, the upstream primer and the downstream primer are 0.8 mu L respectively, the cDNA template is 2 mu L, and the rest is filled with sterilized water; the reaction conditions are 10s at 95 ℃, 30s at 60 ℃ and 42s at 72 ℃, the total is 40 cycles, and the preheating at 95 ℃ is carried out for 5min before the reaction starts; 3 replicate wells per sample;
5) mouse lung tissue immunofluorescence VE-cadherin and PDHA1 co-staining: taking out the frozen section of the mouse lung from-80 ℃, putting the section into a wet cassette to restore to room temperature and dry the tissue surface, putting the section into PBS (0.01 mol/L), rinsing the section for 5min by a shaking table, and reducing the self background; soaking and covering with 4% paraformaldehyde for 25min, and rinsing with PBS for 5min for 3 times; perforating 0.3% triton-100 cell membrane for 20min, and rinsing with PBS for 5min for 3 times; sealing with volume fraction of 5% fetal calf serum for 30min, adding 1: 200VE-cadherin and PDHA1 primary antibody, sealing at 4 deg.C overnight; the next day, the cassette was taken out and returned to room temperature, rinsed with PBS 3 times, incubated with secondary antibody in dark, incubated at 37 ℃ for 1h, and rinsed with PBS 3 times; adding DAPI, incubating for 3min in dark, and rinsing with PBS for 3 times; and (3) dropwise adding an anti-fluorescence quencher, covering the glass slide, sealing the glass slide by using nail polish, and observing the glass slide by using a Zeiss LSM800 laser confocal microscope.
As a preferred embodiment of this example, the primer sequence of PDHA1 is
F:TGTGACCTTCATCGGCTAGAA,R:TGATCCGCCTTTAGCTCCATC。
As a preferred embodiment of this example, the HK2 primer sequence is
F:ATGATCGCCTGCTTATTCACG；R:CGCCTAGAAATCTCCAGAAGGG。
As a preferred embodiment of this example, the IDH primer sequence is
F:GTGGGCGTCAAGTGTGCTA；R:CCACCCAGAATGTTTCGGATG。
The main observation indexes are as follows: (r) PDHA1(iΔEC/iΔEC)Mouse and PDHA1(iΔEC/-)Body length and body mass of the mouse; ② PCR gene identification result; ③ the lung PDHA1 protein expression of the mouse; immunofluorescence expression of mouse vascular endothelial cell PDHA 1; mouse vascular endothelial cell HK2 and IDH protein and mRNA expression.
Statistical analysis: statistical analysis is carried out on all data by SPSS 24.0 software, the measured data are expressed by mean +/-standard deviation (X +/-S), the comparison among groups adopts t test, and the difference with P <0.05 has significance.
The experimental results are as follows:
results of gene identification in mice PDHA1(i Δ EC/i Δ EC) progeny mice were obtained by mating PDHA1(i Δ EC/i Δ EC) conditional knockout mice with PDHA1(i Δ EC/-) mice. PCR amplification creERT2 gene detection progeny mice, as shown in FIG. 1, agarose gel electrophoresis was used for gene identification, 334bp was the target band size, and the presence of a band at the position of 300-400bp was confirmed to be PDHA1 (i.DELTA.EC/i.DELTA.EC) mice. After the identification is completed, the mice are observed whether the body types and the body constitutions of the mice are different.
PDHA1 protein expression levels in mouse lung tissue: western Blot results are shown in FIG. 2, PDHA1(iΔEC/iΔEC)Conditional knockout mice to PDHA1(iΔEC/-)The relative expression of PDHA1 protein in mice was compared, where t is 4.320 and P is 0.0025 (P)<0.05), the difference between the two is significant.
PDHA1 mRNA expression levels in mouse lung tissue: the PCR results are shown in fig. 3, and PDHA1(i Δ EC/i Δ EC) conditional knockout mice compare the two sets of ct values of PDHA1 mRNA of PDHA1(i Δ EC/-) mice with t being 2.272, P being 0.0297, and P <0.05 expression is significantly reduced, and the difference has significance.
Energy metabolism changes following gene knockout: hexokinase 2(hexokinase 2, HK2) is the key rate-limiting enzyme in glycolysis, and Isocitrate Dehydrogenase (IDH) is the key enzyme for aerobic phosphorylation. Following the PDHA1 gene knockout, the expression of HK2 in mouse lung tissue was elevated and IDH expression was reduced, indicating a decrease in aerobic phosphorylation metabolism and an increase in glycolytic metabolism in mitochondria, as shown in fig. 4. HK2 and IDH protein PDHA1(iΔEC/iΔEC)And PDHA1(iΔEC/-)Two comparisons, tHK2 ═ 2.488, P ═ 0.02, P<0.05；tIDH＝2.166，P＝0.0414，P<0.05; HK2 and IDH mRNA PDHA1(i ΔEC/iΔEC)And PDHA1(iΔEC/-)Two comparisons, tHK2 ═ 2.198, P ═ 0.04, P<0.05；tIDH＝2.778，P＝0.0141，P<0.05。
Immunofluorescence detection PDHA1 gene knockout: the vascular endothelial cell Cadherin VE-Cadherin is a protein specifically expressed on the cell membrane of vascular endothelial cells, and the PDHA1 protein is a protein expressed in cytoplasm. For PDHA1(i ΔEC/iΔEC)And PDHA1(iΔEC/-)The expression of PDHA1 protein in vascular endothelial cells after VE-Cadherin staining can be identified by immunofluorescence double-color staining of lung sections of mice, as shown in FIG. 5, PDHA1(iΔEC/-)The expression is obviously higher than PDHA1(i ΔEC/iΔEC)Mouse, indicating PDHA1(iΔEC/iΔEC)The mice successfully knocked out the PDHA1 gene on vascular endothelial cells.
The PDHA1 gene is important for aerobic respiratory chain of cells as a key subunit vector gene of aerobic respiratory pyruvate dehydrogenase complex PDHc. The PDHA1 gene is known to use conditional knockout of Cre/LoxP system in pancreatic islet beta cells to study the correlation between diabetes and pancreatic islet beta cell energy metabolism. The Cre/LoxP system has been able to successfully conditionally knock out DEPTOR (a DEPTOR-domain-containing mTOR interacting protein (DEPTOR) gene in vascular endothelial cells, which plays an important role in regulating vascular endothelial cell activation and in pro-inflammatory and angiogenic responses in vitro) genes, and lays the foundation for studies related to vascular diseases at the animal level in vivo. The knockdown of PFKFB3 (6-phosphofructose-2-kinase/fructose-2, 6-diphosphatase 3, which is a key glycolytic regulator) and HK2 in vascular endothelial cells can reduce the glycolytic capacity of the vascular endothelial cells and inhibit angiogenesis, and PFKFB3 can mediate the glycolysis of the endothelial cells to promote pulmonary hypertension.
The foreign substances are inhaled into the lung through the respiratory tract to stimulate the lung to generate immune inflammatory reaction, cells are subjected to inflammatory loss and repair, extracellular matrix deposition is caused, and pulmonary fibrosis is gradually formed through accumulation. In the process of pulmonary fibrosis, vascular endothelial cells play an important role, and the vascular endothelial cells directly participate in the deposition of extracellular matrix, which can synthesize connective tissue components such as collagen, fibronectin, laminin and the like, and a large amount of collagen fibers can be synthesized around the vascular endothelial cells during pulmonary fibrosis. Therefore, pulmonary microvascular formation and collagen deposition play an important role in the pathogenesis of pulmonary fibrosis, and the two are in a mutual relationship and mutual promotion relationship. Experimental results show that PDHA1(iΔEC/iΔEC)Specific knock-out mice show that the expression of the PDHA1 gene is reduced after tamoxifen injection, but the PDHA1 gene as an important gene of energy metabolism can not be completely eliminated, otherwise, the mice can not survive, so that the mice bred by the gene are all heterozygotes. By subjecting primary PDHA1(iΔEC/iΔEC)And PDHA1(iΔEC/-)Mating with each other to breed PDHA1(iΔEC/iΔEC)15 mice, PDHA1(iΔEC/-)2, both body weight and body surface at 8 weeks of age, in accordance with Mendelian's Law of inheritanceThe characteristic comparison shows that the striking of the PDHA1 gene by vascular endothelial cells has no influence on the body weight and the appearance of the mice. PDHA1(iΔEC/iΔEC)The gene identification result shows that the CreERT2 gene has a band of 334bp, while no band at 334 proves that the rat is PDHA1(iΔEC/-)A transgenic mouse. The expression of PDHA1 protein and mRNA is reduced in vascular endothelial cells of knockout mice, and PDHA1 is proved(iΔEC/iΔEC)The gene knockout mice are successfully bred.
The alteration of the energy metabolism of vascular endothelial cells was confirmed in the validation of the expression of the rate-limiting enzymes HK2 and IDH for glycolysis and oxidative phosphorylation, respectively. The previous 10X single cell sequencing results showed that the difference in PDHA1 gene expression by endothelial cells was significant in saline and silica treated mice. After the PDHA1 gene was partially knocked out in the vascular endothelial cell, the authors will perform silico-pulmonization at a later stage to determine the mechanism of action of the gene in the vascular endothelial cell participating in the pulmonary fibrosis.
From the above, vascular endothelial cells play an important role in diseases, and different energy metabolism pathways can be associated with different pathophysiology, wherein glycolysis is related to AS and myocardial ischemia, and the deletion of related metabolic gene PDHA1 researches more on the energy metabolism relationship between diseases and EC.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims (4)

1. The construction experiment method of the vascular endothelial cell PDHA1 gene specific knock-out mouse is characterized by comprising the following steps:
1) breeding sufficient amount of PDHA1-Tek-creERT2(f/f,+)The mice are combined with the 2 male mice and the 4 female mice, the pregnancy of the female mice is determined when the female mice have vaginal embolus, the birth and the birth number of the mice are observed after the birth of the newborn mice, and the mice use ear tags as ear tags after the mice are born for three weeksMarking and cutting toes to perform gene identification; carrying out intraperitoneal injection on tamoxifen 20mg/mL at the weight dose of 75mg/kg at the age of 6 weeks of newborn mice, injecting 1 time every 24 hours, continuously injecting for 5 days, and taking materials at the age of 8 weeks after injection; carrying out lung lobe perfusion 10% OCT on the right lower lobe of the lung tissue to carry out frozen tissue embedding, and directly putting the rest lung lobes at-80 ℃;
2) and (3) identifying the mouse gene:
2a) preparation of 10 × MGB solution: taking 1 clean centrifuge tube of 50mL, adding 0.05mol of Tris crystal into the centrifuge tube, dissolving the Tris crystal in 50mL of deionized water, measuring the pH value of the centrifuge tube, and adjusting the pH value of the centrifuge tube to 8.8; another 15mL clean centrifuge tube was added 0.02mL (NH)4)2SO4And 10mL of deionized water until completely dissolved; another 15mL clean centrifuge tube was added with 0.01mL MgCl2 and 10mL deionized water; after the solution was completely prepared, 6.7mL of Tris solution was placed in a clean centrifuge tube and 830. mu.L of (NH) was added4)2SO4Solution, 650. mu.L of MgCl2Solution and 1.82mL of ddH2Fully and uniformly mixing O;
2b) preparation of Lysismix: uniformly mixing prepared 1mL of 10 XMGB, 50 mu L of 100% TritonX, 100 mu L of beta-mercaptoethanol and 8.4mL of ddH 2O;
2c) mouse toe digestion to extract DNA: taking the prepared and calculated Lysismix, adding proteinase K into the Lysismix, adding 3 mu L of proteinase K into every 200 mu LLysimix, preparing the mixture on site, adding 120 mu L of the prepared solution into an EP tube containing toes of a mouse, carrying out water bath at 55 ℃ for overnight lysis, carrying out denaturation at 95 ℃ for 10min, then centrifuging at 11000r/min for 5min, and taking the supernatant;
2d) DNA replication and gene identification: respectively adding 10 mu L of Novovozam blue enzyme, 0.3 mu L of upstream primer and 0.3 mu L of downstream primer and 0.59 mu L of extracted DNA into an eight-connected tube of the PCR; pre-denaturing at 95 ℃ for 5min, 30s at 95 ℃, 30s at 60 ℃, 42s at 72 ℃, 40 cycles, preserving at 72 ℃ for 5min, and 16 ℃, then performing DNA amplification, performing 1% agarose gel electrophoresis on the amplified DNA, and exposing and observing DNA bands;
3) western blot detection of protein expression levels in mouse lung tissues: aseptically taking lung tissues of a mouse, shearing the lung tissues by using scissors, adding a phosphatase inhibitor, a protease inhibitor, PMSF and a lysine Buffer which are well proportioned, fully breaking histiocytes by using a high-shear dispersion emulsifying machine, and extracting the total protein of the lung tissues of the mouse; adding Loading buffer and ultrapure water into part of total protein to dilute the protein to 3 mu g/mL, and denaturing at 95 ℃ for 5 min; taking 10 mu L of denatured protein sample, converting the denatured protein sample into 120V after the constant voltage of 10% SDS-PAGE gel electrophoresis is 80V; transferring the protein of the gel onto a PVDF membrane, performing constant pressure 15V, 75min, sealing with 5% skimmed milk powder prepared by PBST buffer solution for 2h, and adding 1:1000 dilution PDHA1, 1:1000 dilution HK2, 1: shaking table incubating IDH diluted by 500 and GAPDH primary anti-dilution solution diluted by 1: 5000 overnight at 4 deg.C, and washing with PBST buffer solution for 3 times, each time for 10 min; incubating horseradish peroxidase-labeled secondary antibody for 2h at room temperature, and eluting with PBST buffer solution for 3 times, each time for 10 min; ECL chemical luminescence method development, exposure time is adjusted according to the intensity of a luminescence signal, and Image Lab measures a gray value;
4) real-time quantitative PCR detection of mRNA expression levels in mouse lung tissues: collecting lung tissues of a mouse into a 1.5mL EP tube, shearing the lung tissues by using scissors, adding about 1mL of lysis solution RZ into the EP tube, adding lysis solution RZ1mL into each 50-100mg of tissue samples, and homogenizing in a homogenizer; centrifuging at 4 ℃ and 12000r/min for 5min, and taking supernatant to a new RNA ase-free EP tube; adding 200 μ L chloroform, shaking, standing at room temperature for 3min, centrifuging at 4 deg.C 12000r/min for 5min, eluting the aqueous phase to obtain RNA, and storing; measuring A260/A280 value by using an ultraviolet spectrophotometer, selecting RNA with A260/A280 ratio between 1.8-2.1 for reverse transcription, synthesizing cDNA as a template for q-PCR reaction, wherein the reaction system is 20 mu L, TB Gerrn Premix Ex Taq II (2) is 10 mu L, the upstream primer and the downstream primer are 0.8 mu L respectively, the cDNA template is 2 mu L, and the rest is filled with sterilized water; the reaction conditions are 10s at 95 ℃, 30s at 60 ℃ and 42s at 72 ℃, the total is 40 cycles, and the preheating at 95 ℃ is carried out for 5min before the reaction starts; 3 replicate wells per sample;
5) mouse lung tissue immunofluorescence VE-cadherin and PDHA1 co-staining: taking out the frozen section of the mouse lung from-80 ℃, putting the section into a wet cassette to restore to room temperature and dry the tissue surface, putting the section into PBS (0.01 mol/L), rinsing the section for 5min by a shaking table, and reducing the self background; soaking and covering with 4% paraformaldehyde for 25min, and rinsing with PBS for 5min for 3 times; perforating 0.3% triton-100 cell membrane for 20min, and rinsing with PBS for 5min for 3 times; sealing with volume fraction of 5% fetal calf serum for 30min, adding 1: 200VE-cadherin and PDHA1 primary antibody, sealing at 4 deg.C overnight; the next day, the cassette was taken out and returned to room temperature, rinsed with PBS 3 times, incubated with secondary antibody in dark, incubated at 37 ℃ for 1h, and rinsed with PBS 3 times; adding DAPI, incubating for 3min in dark, and rinsing with PBS for 3 times; and (3) dropwise adding an anti-fluorescence quencher, covering the glass slide, sealing the glass slide by using nail polish, and observing the glass slide by using a Zeiss LSM800 laser confocal microscope.
2. The experimental method for constructing mice specifically knocking off the vascular endothelial cell PDHA1 gene according to claim 1, wherein the experimental method comprises the following steps: the primer sequence of PDHA1 is F: TGTGACCTTCATCGGCTAGAA and R: TGATCCGCCTTTAGCTCCATC.
3. The experimental method for constructing mice specifically knocking off the vascular endothelial cell PDHA1 gene according to claim 1, wherein the experimental method comprises the following steps: the HK2 primer sequence is F: ATGATCGCCTGCTTATTCACG; CGCCTAGAAATCTCCAGAAGGG is added.
4. The experimental method for constructing mice specifically knocking off the vascular endothelial cell PDHA1 gene according to claim 1, wherein the experimental method comprises the following steps: the sequence of the IDH primer is F: GTGGGCGTCAAGTGTGCTA; CCACCCAGAATGTTTCGGATG is added.
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