XPB high expression vector for promoting angiogenesis and endothelial cell line

Abstract

The invention discloses an XPB high expression vector for promoting angiogenesis and an endothelial cell line. The invention provides an XPB DNA double-chain fragment carrying an HA tag, the sequence of the XPB DNA double-chain fragment is shown as SEQ ID NO: 4, the invention further provides an expression vector containing a target gene sequence shown as SEQ ID NO: 4 and an XPB high-expression microvascular endothelial cell line, the cell line can promote the proliferation capacity of HMEC-1 under an anoxic condition, the migration capacity of cells and the tubulation capacity of the cells, and in addition, the cell line has a good application prospect. According to the invention, the fusion tag technology is utilized to fuse the HA tag into the XPB overexpression recombinant vector, which is helpful for detecting the potential interaction mechanism of XPB with other key molecules in angiogenesis; the method has an important application value.

Description

Technical Field

[0001] The present invention relates to an XPB high-expression vector and an endothelial cell line for promoting angiogenesis, and belongs to the technical field of medical molecular biology and genetic engineering. Background Art

[0002] Ischemic diseases are diseases of hypoxia and ischemia of tissues and organs caused by restricted blood flow. They include ischemic heart disease, ischemic brain disease, and peripheral arterial disease, and they have a high incidence and mortality rate worldwide. Hypoxia or oxidative stress often causes damage to proteins, lipids, and DNA, but cells also have a damage repair system to respond to damaging stimuli. Homologous recombination repair (HRR) and nucleotide excision repair (NER) are two important pathways for DNA damage repair. The universal transcription factor TFIIH (transcription factor IIH) is a multiprotein transcription complex composed of 10 subunits. In addition to its function in initiating transcription, it also plays an important role in nucleotide excision repair. The XPB protein (xeroderma pigmentosum group B), the largest subunit of the complex (89 kDa), is located in the core region of TFIIH. It possesses ATP synthase activity and ATP-dependent single-stranded DNA (ssDNA) helicase activity in the 3'→5' direction. It is responsible for unwinding promoters and damaged DNA regions recognized by RNA polymerase II, thereby promoting transcription initiation and DNA repair. When mutations in the XPB gene cause functional defects in the protein it encodes, the cell's ability to repair damaged DNA is significantly impaired, often leading to diseases such as xeroderma pigmentosum (XP), Cockayne syndrome, and trichothiodystrophy. In recent years, the important role of XPB in the development and progression of cardiovascular diseases and the underlying mechanisms are gradually being revealed. For example, spironolactone-induced XPB degradation can improve endothelial function and control inflammation by inhibiting NF-κB and AP-1 signaling pathways.

[0003] Angiogenesis plays a crucial role in physiological and pathological processes, including tissue and organ growth and development, ischemic diseases, cancer, inflammation, and diabetes. During angiogenesis, endothelial cells are activated in response to external stimuli or signals, such as hypoxia, oxidative stress, and VEGF. These cells acquire enhanced motility, allowing them to migrate or proliferate to distant ischemic sites. They then sprout new capillaries, improving blood perfusion in the ischemic area and repairing damaged tissues and organs. The ischemic and hypoxic microenvironment often triggers the release of pro-angiogenic factors, promoting angiogenesis after injury. However, in most ischemic diseases, the extreme pathological microenvironment disrupts the balance between pro-angiogenic and anti-angiogenic factors, leading to endothelial dysfunction, impaired angiogenesis, and difficulty restoring or reestablishing tissue perfusion. Although therapeutic approaches targeting molecules to ischemic areas to promote angiogenesis and tissue revascularization have been gradually developed, the efficacy of angiogenesis-focused therapies remains to be improved and enhanced. It is crucial to screen the key molecules that regulate the angiogenesis process and reveal their underlying molecular biological mechanisms, which will help in the development and application of molecular targeted drugs that promote angiogenesis. Summary of the Invention

[0004] The present invention aims to construct microvascular endothelial cells with high XPB expression, thereby improving endothelial function and promoting angiogenesis.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides an XPB DNA double-stranded fragment carrying an HA tag, comprising a DNA strand as shown in SEQ ID NO: 4 and a complementary strand thereof.

[0007] In a second aspect, the present invention provides an expression vector comprising a target gene sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 4.

[0008] Preferably, the expression vector is selected from viral, fungal or bacterial expression vectors.

[0009] In a third aspect, the present invention provides a cell comprising the expression vector described in the second aspect, wherein the cell is a human microvascular endothelial cell HMEC-1.

[0010] In a fourth aspect, the present invention provides a method for preparing a recombinant lentiviral expression vector, comprising: digesting the lentiviral vector pHBLV-CMV-MCS-EF1-PURO with restriction endonucleases EcoRI and BamHI, and cloning the target gene shown in SEQ ID NO: 1 or SEQ ID NO: 4 into the digested lentiviral vector to obtain a recombinant lentiviral vector.

[0011] In a fifth aspect, the present invention provides a method for preparing a human microvascular endothelial cell line with high XPB expression, comprising the following steps:

[0012] Step 1: The recombinant lentiviral vector is combined with the viral packaging helper plasmids pSPAX2 and pMD2G to form a three-plasmid lentiviral system for viral packaging, and then transfected into 293T cells to obtain a virus solution; the recombinant lentiviral vector is a recombinant lentiviral vector prepared by the preparation method according to claim 5;

[0013] Step 2: The virus solution obtained in step 1 is used to infect the microvascular endothelial cell line, and the microvascular endothelial cell line with high XPB expression is screened, i.e., the microvascular endothelial cell line with high XPB expression is obtained.

[0014] In a sixth aspect, the present invention provides the expression vector of the second aspect, and the use of the recombinant lentiviral vector prepared by the preparation method of the fourth aspect in any one of the following A1) to A10):

[0015] A1): preparing a reagent for promoting XPB expression;

[0016] A2): Preparation of products for promoting the proliferation of microvascular endothelial cells under hypoxic conditions;

[0017] A3): Preparation of products for promoting the migration of microvascular endothelial cells under hypoxic conditions;

[0018] A4): Preparation of a product for promoting the tube-forming ability of microvascular endothelial cells under hypoxic conditions;

[0019] A5): Preparation of products for constructing cell models or animal models with high XPB expression;

[0020] A6): Promote XPB expression in vitro;

[0021] A7): Ability to promote proliferation of microvascular endothelial cells under hypoxic conditions in vitro;

[0022] A8): Promotes the migration of microvascular endothelial cells under hypoxic conditions in vitro;

[0023] A9): Promotes the tube-forming ability of microvascular endothelial cells under hypoxic conditions in vitro;

[0024] A10): Construct a cell model with high XPB expression.

[0025] In a seventh aspect, the present invention provides the cell of the third aspect and the use of a human microvascular endothelial cell line with high XPB expression prepared by the preparation method of the fourth aspect, wherein the use is selected from any one of the following B1) to B7):

[0026] B1): Preparation of a product for promoting the proliferation of microvascular endothelial cells under hypoxic conditions;

[0027] B2): Preparation of products for promoting the migration of microvascular endothelial cells under hypoxic conditions;

[0028] B3): Preparation of products for promoting the tube-forming ability of microvascular endothelial cells under hypoxic conditions;

[0029] B4): Preparation of products for establishing an animal model with high XPB expression;

[0030] B5): Ability to promote proliferation of microvascular endothelial cells under hypoxic conditions in vitro;

[0031] B6): Promotes the migration of microvascular endothelial cells under hypoxic conditions in vitro;

[0032] B7): Promotes the tube-forming ability of microvascular endothelial cells under hypoxic conditions in vitro;

[0033] B8): Study the molecular mechanisms and signaling pathways between XPB and angiogenesis.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] The present invention utilizes lentiviral vector overexpression technology to achieve overexpression of XPB in endothelial cells, and discovers for the first time that XPB is a key molecule that promotes angiogenesis after ischemia and hypoxia. High expression of XPB can promote the proliferation ability of HMEC-1 under hypoxic conditions, promote the migration ability of cells, and promote the tube-forming ability of cells. This will provide an effective tool for clarifying the relationship between XPB and angiogenesis in various pathological modeling environments in the future, as well as for exploring the potential molecular mechanisms and signaling networks between XPB and angiogenesis, greatly reducing the time and economic costs of early gene editing. In addition, the present invention utilizes tag fusion technology to fuse the HA tag into the XPB overexpression recombinant vector, which will help explore whether XPB can utilize its ATP synthase activity to directly interact with and modify key candidate molecules at the post-translational modification level, thereby mediating the regulation of various biological functions such as angiogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 To identify the effect of XPB overexpression by RT-qPCR;

[0037] Figure 2 Western blot was used to identify the effect of XPB overexpression;

[0038] Figure 3 The effect of XPB overexpression on the proliferation of human microvascular endothelial cells HMEC-1 under hypoxic conditions;

[0039] Figure 4 The effect of XPB overexpression on the migration of human microvascular endothelial cells HMEC-1 under hypoxic conditions;

[0040] Figure 5 This is the effect of XPB overexpression on tube formation in human microvascular endothelial cells HMEC-1 under hypoxic conditions. DETAILED DESCRIPTION

[0041] To make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings. Example 1: Design and Synthesis of Double-Stranded XPB DNA Fragments Carrying HA Tags

[0042] 1. Design of double-stranded XPB DNA fragments carrying HA tags

[0043]

[0044] This gene encodes a protein with the amino acid sequence shown in SEQ ID NO:2. The sequence of SEQ ID NO:2 is as follows:

[0045] MGKRDRADRDKKKSRKRHYEDEEDDEEDAPGNDPQEAVPSAAGKQVDESG

[0046] TKVDEYGAKDYRLQMPLKDDHTSRPLWVAPDGHIFLEAFSPVYKYAQDFLVA

[0047] IAEPVCRPTHVHEYKLTAYSLYAAVSVGLQTSDITEYLRKLSKTGVPDGIMQFI

[0048] KLCTVSYGKVKLVLKHNRYFVESCHPDVIQHLLQDPVIRECRLRNSEGEATELI

[0049] TETFTSKSAISKTAESSGGPSTSRVTDPQGKSDIPMDLFDFYEQMDKDEEEEEE

[0050] TQTVSFEVKQEMIEELQKRCIHLEYPLLAEYDFRNDSVNPDINIDLKPTAVLRP

[0051] YQEKSLRKMFGNGRARSGVIVLPCGAGKSLVGVTAACTVRKRCLVLGNSAVS

[0052] VEQWKAQFKMWSTIDDSQICRFTSDAKDKPIGCSVAISTYSMLGHTTKRSWE

[0053] AERVMEWLKTQEWGLMILDEVHTIPAKMFRRVLTIVQAHCKLGLTATLVRED

[0054] DKIVDLNFLIGPKLYEANWMELQNNGYIAKVQCAEVWCPMSPEFYREYVAIK

[0055] TKKRILLYTMNPNKFRACQFLIKFHERRNDKIIVFADNVFALKEYAIRLNKPYI

[0056] YGPTSQGERMQILQNFKHNPKINTIFISKVGDTFSFDLPEANVLIQISSHGGSRRQ

[0057] EAQRLGRVLRAKKGMVAEEYNAFFYSLVSQDTQEMAYSTKRQRFLVDQGYS

[0058] FKVITKLAGMEEEDLAFSTKEEQQQLLQKVLAATDLDAEEEVVAGEFGSRSSQ

[0059] ASRRFGTMSSMSGADDTVYMEYHSSRSKAPSKHVHPLFKRFRK

[0060] In order to achieve that the protein encoded by the exogenous XPB gene carries the HA tag, the HA sequence (SEQ ID NO: 3) was added before the stop codon "TGA" of the XPB protein coding region sequence: 5'-GGATCCTACCCATACGATGTTCCAGATTACGCT-3',

[0061]

[0062] 2. Obtaining cDNA

[0063] RNA from human microvascular endothelial cells (HMEC-1) was extracted using a Vazyme RNA extraction kit. The HMEC-1 cDNA library was obtained by reverse transcription using a reaction system consisting of 8 μl PrimeScript RT Buffer, 8 μl RT primer Mix, 2 μl PrimeScript RT Enzyme Mix I, and 2 μl nuclease-free water at 37°C for 15 min and 85°C for 5 s.

[0064] 3. PCR amplification of target fragment

[0065] Based on the designed XPB DNA double-stranded fragment sequence carrying the HA tag, a pair of specific primers were designed and synthesized. The forward primer sequence was designed as follows: 5'-CTAGAGGATCTATTTCCGGTGAATTCGCCACCATGGGCAAAAGAGACCG-3' (SEQ ID NO: 5),

[0066] The reverse primer sequence was designed as: 5'-AGCGATCGCAGATCCTTAGGATCCTCAAGCGTAATCTGGAACATCGTAT-3' (SEQ ID NO: 6).

[0067] Prepare a PCR amplification system: 25 μL 2X PCR Buffer, 1 μL dNTP mix (10 mM each), 2 μL forward primer (10 μM), 2 μL reverse primer (10 μM), 1 μL cDNA template, 18 μL ddH₂O, and 1 μL phanta Super-Fidelity DNA Polymerase. Amplify the PCR reaction using the following conditions: initial denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C–72°C for 30 s, extension at 72°C for 30–60 s / kb (27–35 cycles), and final extension at 72°C for 10 min to obtain a double-stranded XPB DNA fragment carrying the HA tag.

[0068] Example 2: Construction of XPB overexpression lentiviral vector and identification of its overexpression effect

[0069] 1. Vector digestion and fragment ligation

[0070] The reaction system of 1 μl vector DNA (1 μg / μl), 4 μl 10* buffer, 32 μl ddH2O, 1.5 μl EcoRI, and 1.5 μl BamHI was reacted in a 37°C water bath for 1-2 hours to digest pHBLV-CMV-MCS-EF1-NEO. After the digestion was completed, agarose gel electrophoresis was performed to recover the target fragment. TM The double-stranded XPB DNA fragment carrying the HA tag was ligated into the enzyme-digested pHBLV-CMV-MCS-EF1-PURO vector in one step to obtain the XPB overexpression recombinant lentiviral vector.

[0071] 2. Recombinant vector transformation

[0072] Thaw DH5α competent cells on ice, take 10 μl of the above-mentioned XPB overexpression recombinant lentiviral vector and add it to 100 μl competent cells, gently tap the wall of the EP tube to mix, and let it stand on ice for 30 minutes; heat shock in a 42°C water bath for 45 seconds, and immediately place it on ice to cool for 2 minutes; add 900 μl of LB liquid culture medium without antibiotics to the EP tube, and shake at 37°C 250 rpm for 1 hour; centrifuge the transformed bacterial solution at 5,000 rpm for 5 minutes, discard 900 μl of the supernatant and resuspend it with the remaining culture medium, and use a sterile spreading rod to gently spread the resuspended bacterial solution on an LB solid culture medium plate with ampicillin preheated in a 37°C incubator in advance; incubate the plate with the bacterial solution upside down in a 37°C incubator for 12-16 hours.

[0073] 3. Colony PCR identification and sequencing

[0074] Ten colonies from the recombination reaction transformation plate were transferred to 1.5 ml EP tubes containing 1 ml of LB liquid medium containing ampicillin. The cells were shaken at 37°C, 250 rpm, for 12 hours. A portion of the culture suspension was then assayed for colony PCR using the universal sequencing primers provided on the vector. The remaining culture suspension corresponding to colonies identified as positive by colony PCR was sequenced and aligned (Shanghai Sangon Biotechnology Co., Ltd.) to select the successfully constructed target plasmid.

[0075] 4. Preparation of Virus Concentrate

[0076] 10 μg of XPB overexpression recombinant lentiviral vector / pHBLV-CMV-MCS-EF1-PURO empty vector, 10 μg of viral packaging helper plasmid pSPAX2 and 5 μg of pMD2G (Shanghai Hanheng Biotechnology Co., Ltd.) were transfected into 293T cells according to the instructions of Lipofectamine 3000 transfection reagent (Shanghai Hanheng Biotechnology Co., Ltd.). After 48 h, the cell culture fluid was collected and centrifuged to obtain the viral supernatant, i.e., the XPB overexpression / overexpression control virus concentrate, which was stored in a -80°C refrigerator.

[0077] 5. Cell infection

[0078] Add 20 μl of XPB overexpression / overexpression control virus concentrate to 293T cells with a confluence of 70-80%; replace with fresh complete culture medium after 24 hours; use puromycin for positive selection and amplify culture after 48 hours to obtain the human microvascular endothelial cell line HMEC-1 after virus infection screening.

[0079] 6. Obtaining cDNA and identifying the effect of XPB overexpression by RT-qPCR

[0080] (1) RNA was extracted from human microvascular endothelial cells HMEC-1 infected with XPB overexpression virus concentrate and overexpression control virus concentrate using the Vazyme RNA extraction kit. The RNA was reverse transcribed to obtain cDNA using a reaction system of 8 μl PrimeScript RT Buffer, 8 μl RT primer Mix, 2 μl PrimeScript RT Enzyme Mix I (Takara Bio, Japan), and 2 μl nuclease-free water at 37°C for 15 min and 85°C for 5 s.

[0081] (2) RT-qPCR identification of XPB overexpression effect

[0082] A 10-μl RT-qPCR reaction system was prepared, consisting of 5 μl of TB Green Premix Ex TaqII (Tli RNaseH Plus) 2 (Thermo Fisher Scientific, USA), 0.3 μl of upstream primer, 0.3 μl of downstream primer, 4 μl of nuclease-free water, and 0.4 μl of the cDNA template obtained in Step 1. The prepared reaction system was placed in a fluorescent quantitative PCR instrument for RT-qPCR amplification. The relative expression of the XPB gene in human microvascular endothelial cells (HMEC-1) infected with the XPB-overexpressing virus was detected (using the β-actin gene as an internal reference gene). The mRNA expression of XPB was calculated using the 2-ΔΔCT method.

[0083] The upstream primer for amplifying the XPB gene was 5'-ATGGGCAAAAGAGACCGAGC-3' (SEQ ID NO: 7), and the downstream primer was 5'-GAGGTGTGGTCGTCCTTCAG-3' (SEQ ID NO: 8).

[0084] The upstream primer for amplifying the β-actin gene was 5'-GTTGTCGACGACGAGCG-3' (SEQ ID NO: 9), and the downstream primer was 5'-GCACAGAGCCTCGCCTT-3' (SEQ ID NO: 10).

[0085] (3) Experimental results

[0086] Test results such as Figure 1 The results showed that after the XPB overexpression virus concentrate was infected with human microvascular endothelial cells HMEC-1, the expression of XPB mRNA was significantly higher than that of the control, indicating that the XPB overexpression effect was significant.

[0087] 7. Western blot identification of XPB overexpression effect

[0088] (1) Western blot specific steps:

[0089] Total cellular protein in human microvascular endothelial cells HMEC-1 infected with XPB overexpression virus concentrate and overexpression control virus concentrate was extracted using neutral lysis buffer RIPA. After denaturation, SDS-PAGE electrophoresis was performed at 200V for 40 minutes; transfer to the membrane at 300mA for 100 minutes; the PVDF membrane was blocked with rapid blocking buffer for 30 minutes; incubated with 1000:1 diluted GAPDH rabbit primary antibody (Cell Signaling Technology, USA) and 1000:1 diluted XPB mouse primary antibody (Cell Signaling Technology, USA) overnight at 4°C; the primary antibody was recovered and the membrane was washed three times with 1×TBST solution on a shaker for 10 minutes each time; incubated with 5000:1 diluted mouse secondary antibody or rabbit secondary antibody (Cell Signaling Technology, USA) at room temperature for 1 hour, and developed.

[0090] (2) Experimental results

[0091] Test results such as Figure 2 The results showed that after the XPB overexpression virus concentrate was infected with human microvascular endothelial cells HMEC-1, the protein expression of XPB was significantly higher than that of the control.

[0092] Example 3: Effect of XPB overexpression on the proliferation of human microvascular endothelial cells HMEC-1 induced by hypoxia

[0093] 1. Human microvascular endothelial cell HMEC-1 hypoxia model

[0094] The XPB-overexpressing human microvascular endothelial stable cell line constructed in Example 2 and the control cell line were seeded into a 96-well cell culture plate at a ratio of 5,000 cells per well. After the cells adhered, they were treated with hypoxia under 5% CO2, 95% N2, and serum-free medium for 1 day.

[0095] 2. EdU cell proliferation ability detection

[0096] The Cell-Light EdU Apollo567 In Vitro Kit was used to detect the proliferation level of XPB-overexpressing human microvascular endothelial cells HMEC-1 in a hypoxic environment. The specific steps are as follows:

[0097] (1) EdU labeling of HMEC-1 cells

[0098] EdU reagent (reagent A) was diluted in MCDB131 culture medium at a ratio of 1000:1 to prepare 50 μM EdU culture medium.

[0099] (2) Add 100 μL of 50 μM EdU culture medium to each well and incubate for 2 hours. Discard the culture medium.

[0100] (3) Wash the cells twice with PBS, 5 min each time.

[0101] 3. Cell immobilization

[0102] (1) Add 50 μL of cell fixative (PBS containing 4% polymethanol) to each well and incubate at room temperature for 30 minutes. Discard the fixative.

[0103] (2) Add 50 μL of 2 mg / mL glycine to each well, incubate on a decolorizing shaker for 5 min, and then discard the glycine solution.

[0104] (3) Add 100 μL of permeabilization solution (0.5% Triton X-100 in PBS) to each well and incubate on a decolorizing shaker for 10 minutes; wash once with PBS for 5 minutes.

[0105] 4. Apollo staining

[0106] (1) Add 100 μL of 1× Apollo staining reaction solution to each well, incubate in a decolorizing shaker at room temperature for 30 minutes in the dark, and then discard the staining reaction solution.

[0107] (2) Add 100 μL of permeabilization agent (0.5% Triton X-100 in PBS) and wash on a decolorizing shaker 2-3 times, each time for 10 minutes, and discard the permeabilization agent.

[0108] (3) Each well was washed 1-2 times with 100 μL of methanol for 5 minutes each time; and washed once with PBS for 5 minutes each time.

[0109] 5. DNA staining

[0110] (1) Prepare an appropriate amount of 1x Hoechest 33342 reaction solution by diluting Reagent F with deionized water at a ratio of 100:1 and store in a dark place.

[0111] (2) Add 100 μL of 1× Hoechst 33342 reaction solution to each well, incubate in a decolorizing shaker at room temperature for 30 min in the dark, and then discard the staining reaction solution.

[0112] (3) Add 100 μL PBS to each well and wash 1-3 times.

[0113] 6. EdU Image Acquisition and Analysis

[0114] The stained cells were detected using a fluorescence microscope, and the proportion of EdU-positive cells in each well was calculated to reflect the cell proliferation level.

[0115] 7. Experimental Results

[0116] Cell proliferation level Figure 3 (XPB over is a stable human microvascular endothelial cell line overexpressing XPB, and XPB over NC is a control cell line.) The results showed that compared with XPB over NC, XPB overexpression significantly increased the proliferation of human microvascular endothelial cells under hypoxic conditions.

[0117] Example 4: Effect of XPB overexpression on migration of human microvascular endothelial cells HMEC-1 under hypoxic conditions

[0118] 1. Human microvascular endothelial cell HMEC-1 hypoxia model

[0119] The XPB-overexpressing human microvascular endothelial stable cell line constructed in Example 2 and the control cell line were treated with hypoxia for 1 day under the conditions of 5% CO 2 , 95% N 2 and serum-free culture medium.

[0120] 2. Transwell experiment

[0121] XPB-overexpressing human microvascular endothelial stable cell lines and control cell lines were digested with 0.25% trypsin and terminated with complete medium by centrifugation at 1000 rpm for 5 minutes. 20,000 cells were plated in the upper chamber of a Transwell chamber (Beijing Lanjieke Technology Co., Ltd.) using 200 μl of serum-free medium per well. 500 μl of MCDB131 medium containing 10% fetal bovine serum was added to the lower chamber of the Transwell chamber and cultured for 24 hours at 37°C in an oxygen-deficient environment with 5% CO2 and 95% N2. The cells were fixed with 4% PFA at room temperature for 30 minutes and stained with 0.1% crystal violet at room temperature for 10 minutes. Images were then acquired.

[0122] 3. Experimental results

[0123] Transwell migration status and statistical analysis can be found in Figure 4 (XPB over is a stable human microvascular endothelial cell line overexpressing XPB, and XPB over NC is a control cell line.) The results showed that compared with XPB over NC, XPB overexpression significantly promoted the migration of human microvascular endothelial cells (HMEC-1) under hypoxic conditions.

[0124] Example 5: Effect of XPB overexpression on tube formation in human microvascular endothelial cells HMEC-1 under hypoxic conditions

[0125] 1. Human microvascular endothelial cell HMEC-1 hypoxia model

[0126] The XPB-overexpressing human microvascular endothelial stable cell line constructed in Example 2 and the control cell line were treated with hypoxia for 1 day under the conditions of 5% CO 2 , 95% N 2 and serum-free culture medium.

[0127] 2. Tube forming experiment

[0128] Matrigel (Corning, USA) was spread onto a 96-well plate at a volume of 50 μl per well and placed in a 37°C, 5% CO2 incubator for 30 minutes to allow the Matrigel to solidify. The hypoxic cells were digested with 0.25% trypsin and centrifuged at 1000 rpm for 5 minutes after termination with complete culture medium. At a cell count of 30,000 cells per well, 50 μl of the suspension was inoculated into a 96-well plate covered with Matrigel and cultured in a 37°C, 5% CO2 incubator for 6 hours. Images were then acquired.

[0129] 3. Experimental results

[0130] Tubular formation is shown in Figure 5(XPB over is a stable human microvascular endothelial cell line overexpressing XPB, and XPB over NC is a control cell line.) The results showed that compared with XPB over NC, XPB overexpression significantly inhibited the tube formation of human microvascular endothelial cells under hypoxic conditions.

[0131] The XPB-overexpressing human microvascular endothelial stable cell line constructed in the present invention is an XPB-overexpressing cell strain, which lays a foundation for in-depth research on the molecular mechanism of angiogenesis after ischemia and hypoxia.

[0132] The above description is only a preferred embodiment of the present invention and does not constitute any formal or substantial limitation to the present invention. It should be noted that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A double-stranded XPB DNA fragment carrying an HA tag, characterized in that: It includes the DNA chain shown in SEQ ID NO: 4 and its complementary chain.

2. An expression vector, characterized in that: The expression vector comprises the target gene sequence as shown in SEQ ID NO: 1 or SEQ ID NO:

4.

3. The expression vector according to claim 2, characterized in that The expression vector is selected from a virus, fungus or bacteria expression vector.

4. A cell comprising the expression vector according to claim 2 or 3, characterized in that: The cells are human microvascular endothelial cells HMEC-1.

5. A method for preparing a recombinant lentiviral expression vector, characterized in that: include: The lentiviral vector pHBLV-CMV-MCS-EF1-PURO was digested with restriction endonucleases EcoRI and BamHI, and the target gene shown in SEQ ID NO: 1 or SEQ ID NO: 4 was cloned into the lentiviral vector after the digestion to obtain a recombinant lentiviral vector.

6. A method for preparing a human microvascular endothelial cell line with high XPB expression, characterized in that: The steps include: Step 1: The recombinant lentiviral vector is combined with the virus packaging auxiliary plasmids pSPAX2 and pMD2G to form a three-plasmid lentiviral system for virus packaging, and then transfected into 293T cells to obtain a virus liquid; the recombinant lentiviral vector is a recombinant lentiviral vector prepared by the preparation method according to claim 5; Step 2: Infect the microvascular endothelial cell line with the virus solution obtained in step 1, and screen and obtain the microvascular endothelial cell line with high expression of XPB, that is, the microvascular endothelial cell line with high expression of XPB.

7. Use of the expression vector according to claim 2 or 3, or the recombinant lentiviral vector prepared by the preparation method according to claim 5 in any one of the following A1) to A10): A1): preparing a reagent for promoting the expression of XPB; A2): Preparation of products for promoting the proliferation of microvascular endothelial cells under hypoxic conditions; A3): Preparation of products for promoting the migration ability of microvascular endothelial cells under hypoxic conditions; A4): Preparation of products for promoting the tube-forming ability of microvascular endothelial cells under hypoxic conditions; A5): Preparation of products for constructing cell models or animal models with high XPB expression; A6): Promote XPB expression in vitro; A7): Ability to promote proliferation of microvascular endothelial cells under hypoxic conditions in vitro; A8): Promotes the migration of microvascular endothelial cells under hypoxic conditions in vitro; A9): Promotes the ability of microvascular endothelial cells to form tubes under hypoxic conditions in vitro; A10): Construction of a cell model with high XPB expression.

8. The cell of claim 4 and the use of a human microvascular endothelial cell line with high XPB expression prepared by the preparation method of claim 6, characterized in that: The application is selected from any one of the following B1) to B7): B1): Preparation of products for promoting the proliferation of microvascular endothelial cells under hypoxic conditions; B2): Preparation of products for promoting the migration ability of microvascular endothelial cells under hypoxic conditions; B3): Preparation of products for promoting the tube-forming ability of microvascular endothelial cells under hypoxic conditions; B4): Preparation of products for constructing animal models with high XPB expression; B5): Ability to promote proliferation of microvascular endothelial cells under hypoxic conditions in vitro; B6): Promotes the migration of microvascular endothelial cells under hypoxic conditions in vitro; B7): Promotes the tube-forming ability of microvascular endothelial cells under hypoxic conditions in vitro; B8): Study the molecular mechanism and signaling pathway between XPB and angiogenesis.

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