CN112480267A

CN112480267A - Promoter molecule capable of specifically recognizing endothelial cells in proliferation state and engineering cells

Info

Publication number: CN112480267A
Application number: CN202011414340.0A
Authority: CN
Inventors: 朱剑虹; 王智富; 钟俊杰; 陈柯竹; 李天文
Original assignee: Huashan Hospital of Fudan University
Current assignee: Huashan Hospital of Fudan University
Priority date: 2020-12-04
Filing date: 2020-12-04
Publication date: 2021-03-12

Abstract

A promoter molecule capable of specifically recognizing endothelial cells in a proliferation state and an engineering cell are fusion proteins and are obtained by fusing human apelin, intra-membrane hydrolysable polypeptide and deoxyribonucleic acid sequences corresponding to effector factors, wherein the intra-membrane hydrolysable polypeptide is a minimum transmembrane core domain of natural Notch, and the intra-membrane hydrolysable polypeptide is partially or completely covered by adjacent apelin and effector factors in a resting state. The kit has the characteristic of high functional modularization, identifies endothelial cells in a proliferation state, wherein the human apelin is combined with a neovascular surface molecular marker Apj receptor, and a cleavage site is identified and hydrolyzed, so that the neovascular can be specifically identified, the expression of a set gene is regulated and controlled, the kit can be used for tracing the proliferated endothelial cells, indicating the condition of vascular remodeling in an adult, and meanwhile, the kit can be used as a treatment means to realize the repair of a blood brain barrier.

Description

Promoter molecule capable of specifically recognizing endothelial cells in proliferation state and engineering cells

Technical Field

The invention belongs to the field of bioengineering, and particularly relates to a promoter molecule capable of specifically recognizing endothelial cells in a proliferation state and an engineering cell.

Background

The damage to the brain caused by Traumatic Brain Injury (TBI) is divided into two phases: direct injury and secondary injury. Direct injury is the destruction of brain tissue by external mechanical forces, resulting in the destruction of tissue structure and the direct death of cells by destructive blows, which also include vascular injuries, hemorrhage, etc. The secondary injury is caused by the disruption of the homeostasis of brain tissue, especially cerebral hemorrhage, blood brain barrier destruction, peripheral immune cell aggregation, secondary inflammation, and finally injury spread and tissue necrosis area enlargement.

Dysfunction of the blood brain barrier will cause leakage of various proteins and electrolytes, which disrupts the microenvironment of the cell and may trigger downstream chain reactions such as activation and recruitment of microglia. Secondary injury lasts much longer than originally thought, and can lead to neuropathy even after 40 years of separation after the primary injury.

The rate of recovery of the blood-brain barrier is quite different in TBI survivors, with some patients requiring more than one year in the longer recovery period and even 11 years after the injury, there are cases where the blood-brain barrier is broken down. In patients with brain trauma, the harm caused by secondary injury even exceeds that of direct injury, and the faster the blood brain barrier is repaired, the more inflammatory cells are prevented from gathering, the harm caused by secondary injury can be greatly reduced. After direct injury, blood vessels can be disorderly regenerated, the endothelial function of the new blood vessels is imperfect, the blood brain barrier is not established yet, and the acceleration of the maturation of the new blood vessels and the formation of the blood brain barrier are a large target of the repair of the brain trauma.

The synthesis of fusion proteins by synthetic biology is one of the common methods for developing new biological tools, and fusion proteins can have various characteristics through reasonable design. The engineered cell modified by the fusion protein is influenced by the fusion protein, and then the change of cell behavior is generated, wherein the change comprises the recognition behavior and the response behavior of the cell.

Neovascular endothelium possesses certain molecular markers that are not found in mature vascular endothelium, such as Apelin (Apelin)/Apj. Although the endothelial cells in the development process highly express the apelin, the expression level of the apelin in the mature endothelial cells is extremely low, and the endothelial cells can re-express the apelin only under pathological conditions, such as tumors, brain trauma, cerebral apoplexy and the like.

Apj is an endogenous receptor of apelin, is a G protein coupled receptor crossing endothelial cell membranes, is abundantly expressed in various tissues in the embryonic period, but in adult tissues, Apj is only expressed in budding blood vessels and can be used as a marker for endothelial cell proliferation. Therefore, the pair of mutually combined membrane protein molecules of apelin/Apj can be used as targets of targeting new blood vessels and subsequent treatment, and the sensitivity of the two is high and the combination is tight.

The synthetic Notch system is a chimeric protein receptor tool that can be engineered to regulate specific cellular signaling pathways by modifying natural Notch proteins. The endogenous Notch system is an important signal regulation system of mammals, and an endogenous Notch1 receptor comprises three structural domains, namely an extracellular recognition structural domain, a transmembrane structural domain and a transcription regulatory factor in cells, wherein the extracellular recognition structural domain and the transcription regulatory factor structural domain can be replaced by other structures to form a new synthetic receptor protein, so that cell targeted regulation and downstream signal response are realized.

In the prior art, no study on the identification of new vessels by using a synthetic Notch system has been reported.

Disclosure of Invention

The invention aims to provide a promoter molecule and an engineering cell capable of specifically recognizing endothelial cells in a proliferation state, wherein the engineering cell modified by the promoter molecule can be efficiently and stably combined with an Apj receptor to form cell-cell contact, specifically recognize a new blood vessel, and regulate and control the expression of a set gene in a downstream signal path; can be used for tracing the proliferated endothelial cells, indicating the condition of blood vessel remodeling in an adult, and simultaneously can be used as a treatment means to realize the repair of blood brain barrier.

In order to achieve the purpose, the invention provides the following technical scheme:

a starting molecule capable of specifically recognizing endothelial cells in a proliferation state is a fusion protein and is characterized by comprising human apelin protein, intramembrane hydrolysable polypeptide and an effector, wherein the human apelin protein, the intramembrane hydrolysable polypeptide and the effector are connected by two proteolytic cleavage sites in the intramembrane hydrolysable polypeptide; wherein the intramembrane hydrolysable polypeptide is the smallest transmembrane core domain of native Notch, and is partially or completely covered by adjacent apelin and effector in a resting state.

Preferably, the amino acid sequence of the apelin protein is shown in SEQ ID NO. 1; the amino acid sequence of the intramembrane hydrolysable polypeptide is shown as SEQ ID NO. 2.

The effector is selected from the group consisting of tetracycline transcriptional activator and Cre recombinase domains.

Preferably, the effector of the promoter molecule is Mfsd2a transcription promoter, and the amino acid sequence of the promoter molecule is shown as SEQ ID NO. 3.

The invention provides an engineering cell containing the promoter molecule capable of specifically recognizing endothelial cells in a proliferation state.

Further, the engineered cell is obtained by introducing the promoter into a eukaryotic cell by means of DNA recombination, DNA injection, plasmid transfection or virus transfection.

Preferably, the eukaryotic cell is an endothelial progenitor cell, a T lymphocyte, a neural stem cell, or a glial cell.

A method for preparing synNotch engineered endothelial progenitor cells comprises the following steps:

1) preparing endothelial progenitor cells;

2) construction of lentiviruses containing fusion proteins

Designing upstream and downstream specific PCR amplification primers containing the promoter molecules, introducing enzyme cutting sites, amplifying by utilizing overlap extension PCR, and calling a promoter molecule gene CDS region from a cDNA plasmid or a library template to connect into a T vector; cutting the CDS region from the T vector, and filling into a lentivirus overexpression plasmid vector; synthesizing a DNA neck ring structure corresponding to the siRNA, and inoculating a lentiviral interference plasmid vector after annealing;

preparing a lentivirus shuttle plasmid and an auxiliary packaging vector plasmid thereof;

after extracting the lentivirus over-expression plasmid vector, the lentivirus interference plasmid vector and the lentivirus shuttle plasmid respectively, co-transfecting the lentivirus over-expression plasmid vector, the lentivirus interference plasmid vector and the lentivirus shuttle plasmid to 293T cells to obtain a lentivirus containing a starter molecule;

3) transfection into eukaryotic cells

Transfecting the lentivirus with an endothelial progenitor cell and simultaneously transfecting a fluorescent reporter gene to obtain the engineered endothelial progenitor cell modified by the fusion protein.

Further, in the step 3), expanding the endothelial progenitor cells after transfection of the lentiviruses by using an EGM-2 culture medium, observing the expression condition of the labeled fluorescent protein by using a fluorescence microscope when the cell amount accounts for 80-90% of that of the culture bottle, identifying the marker of the transfected cell population by using a flow cytometer, and detecting the activation condition of the engineering cells.

The engineered cell is used for constructing a targeted new blood vessel, a mouse tumor model or a brain trauma model, reprogramming the cell, repairing cell damage, repairing blood brain barrier or regenerating the cell.

Further, the targeting of the new blood vessels refers to the targeting of the new blood vessels in subacute brain trauma.

According to the invention, a nucleic acid sequence is modified, apelin is used for replacing an extracellular segment of a Notch receptor, human-derived apelin, intramembrane hydrolysable polypeptide and deoxyribonucleic acid (DNA) sequences corresponding to effect factors are fused, and fusion protein which is obtained by partially or completely covering the intramembrane hydrolysable polypeptide in a resting state by adjacent apelin and effect factors is called as a nascent blood vessel recognition starter molecule, called as a starter molecule for short, and the sensitivity of the starter molecule to Apj is reduced by the special molecular structure, so that the specificity of the starter molecule is improved.

The fusion protein obtained by the invention has the characteristic of highly modularized functions, wherein the human apelin is combined with a neovascular surface molecular marker Apj receptor, then the cleavage site is identified and hydrolyzed, the neovascular is specifically identified, the minimum transmembrane core domain of natural Notch mediates the hydrolysis of the membrane inner segment to play a signal conduction function, further a downstream signal path is regulated and controlled, the expression of a set gene is regulated and controlled, and according to the difference of downstream effector genes, the starting molecular engineering cells can generate different cell behaviors.

Because the promoter molecule has the characteristic of targeting a new blood vessel, the target sequence is introduced into eukaryotic cells by using modes including but not limited to DNA recombination, DNA injection, plasmid transfection or virus transfection, and the like, so that the engineered cells are obtained.

In the engineering cell, the promoter is distributed on the cell membrane and spans the whole cell membrane, wherein the outer section of the cell membrane is a recognition domain and can be combined with an endothelial cell surface molecular marker Apj protein in proliferation, thereby endowing the ability of the promoter engineering cell for specifically recognizing new vessels. The binding of the promoter molecule and Apj results in the adhesion of the promoter molecule engineering cell to the endothelial cell in proliferation state, and the mechanical drawing exposes the hydrolysable peptide segment of the promoter molecule, so that the connection between the effector and the membrane segment is destroyed after the hydrolysable peptide segment is hydrolyzed, the effector falls off from the cell membrane and enters the cell nucleus to activate downstream effector gene, so as to realize the specific response of the promoter molecule.

The starting molecule needs to be in virtue of intercellular tension, which determines that the engineering cell can exert the effect only by contacting with the diseased region, greatly improves the accuracy, effectively avoids nonspecific expression, and the engineering cell modified by the starting molecule has the capacity of combining with endothelial cells of new vessels and leads the engineering cell to start downstream effector genes.

Because the fusion protein has the characteristic of targeting a new blood vessel, the target sequence is introduced into eukaryotic cells by using modes including but not limited to DNA recombination, DNA injection, plasmid transfection or virus transfection, and the like, so that the engineered cells are obtained. The engineered cells modified by the promoter molecules of the invention have a variety of uses, including but not limited to inhibiting tumor growth, detecting tumorigenesis, repairing blood brain barrier, reprogramming cells, and realizing cell damage repair or cell regeneration.

S1P is a molecule for promoting the closure of a blood brain barrier, and can be secreted by an endothelial progenitor cell, when an extracellular segment structural domain of a fusion protein is humanized Apelin protein, and an effect factor of an intracellular segment is Mfsd2a transcription initiation factor, an Apelin-synNotch receptor initiation molecule capable of repairing the blood brain barrier is constructed, in a craniocerebral injury model, the endothelial progenitor cell is modified by the initiation molecule, the cell is transfected with a gene Mfsd2a related to S1P, through an initiation molecule system, the extracellular segment of the initiation molecule can be combined with Apj protein, then a cleavage site is identified and hydrolyzed, the Mfsd2a transcription initiation factor enters a cell nucleus, the expression of an Mfsd2a gene is regulated, the expression of Mfsd2a is restarted, the secretion of S1P to an extracellular matrix is promoted successfully, the concentration of S1P is increased in an injury area, the closure is promoted, and the repair of the blood brain barrier is realized.

Blood vessels in an adult are in a resting state, and once pathological states such as tumors, trauma, inflammation, stroke and the like appear in vivo, an anoxic state appears in a focal region, so that endothelial cells are stressed to generate VEGF (vascular endothelial growth factor), the endothelial cells are stimulated to enter a proliferation state, a blood vessel sprouting process is started, and blood vessels in the focal region are remodeled. Thus, endothelial proliferation can serve as a marker to distinguish normal tissue from diseased tissue.

Endothelial progenitor cells, which are free progenitor cells in blood and can be freely transported in blood vessels, affect migration of neural progenitor cells in the infarct area and the peripheral area after stroke, which has positive significance for nerve regeneration after injury, and can be involved in natural injury repair, and methods for mobilizing endothelial progenitor cells from bone marrow and peripheral blood are reported in models of ischemic stroke and TBI at the injury site. Endothelial progenitor cells can express Spns2 and secrete S1P themselves, and therefore the selection of endothelial progenitor cells as engineered cells to be engineered is of great advantage.

The invention has at least the following beneficial effects:

the invention adjusts the amino acid sequence of the synNotch membrane inner structural domain, constructs a plurality of protein structures according to the amino acid sequence, selects the protein structure with higher coverage rate of the hydrolysis peptide segment, exposes the core regulation region of the starter molecule by pulling mechanical force, and in the core regulation region close to the transmembrane region, the protease recognizes and cuts to release the effect domain in the cell, enters the cell nucleus and regulates the downstream passage, thereby reducing the sensitivity degree of the starter molecule to Apj and obviously improving the specificity of the starter molecule.

According to the invention, through synNotch engineering, Apelin is used for replacing an extracellular segment of a Notch receptor to obtain a fusion protein which is used as a chimeric protein tool, can specifically and accurately identify an Apelin receptor, can regulate and control a cell signal path, regulates and controls the expression of downstream genes, has the characteristic of targeting a new blood vessel, and can be introduced into a eukaryotic cell to obtain an engineered cell capable of specifically identifying the new blood vessel.

The engineered cell of the invention can specifically recognize the new blood vessel, and has multiple purposes: neovasculature can be identified in adults, e.g., by vascular injection methods, and tumorigenesis detected; or can target tumor neovascularization, and utilize downstream response genes of engineering cells to play a targeted anti-tumor effect and inhibit tumor growth; or can be positioned in a brain trauma focus area, the Mfsd2a gene expression is regulated, the closure of a blood brain barrier is controlled, and the blood brain barrier is repaired; and reprogramming the cells to realize cell damage repair or cell regeneration.

Drawings

FIG. 1 shows the genetic constitution of a promoter molecule according to the present invention.

FIG. 2 shows the structure of 6 different proteins selected in example 1 of the present invention, wherein 1: apelin, 2: minimal transmembrane core domain of native Notch, 3: an effector factor.

FIG. 3 is a schematic diagram of the working principle of the engineered cell containing the promoter molecule in example 1 of the present invention, wherein, 1: apelin, 1': endothelial cell surface molecular marker Apj protein in proliferative state, 2: minimal transmembrane core domain of native Notch, 3: effector, 4: response element, 5: a targeted gene.

FIG. 4 shows the expression levels of Sphk1 and Mfsd2a in the engineered cells after the endothelial cells are stimulated and the secretion of S1P in different cells after the engineered cells are activated in example 2 of the present invention.

FIG. 5 shows the variation of the secretion of S1P in different cells in example 2 of the present invention.

FIG. 6 is the arrangement pattern of the endothelial cells of the cerebral blood vessels 4 days after the injury of the cranium brain in example 3 of the present invention, and also shows the basic principle of starting the molecular engineering cells to repair the blood brain barrier.

FIG. 7 is a graph showing the change in the concentration of S1P in the extracellular matrix after injection of promoter-modified endothelial progenitor cells following traumatic brain injury in mice in example 4 of the present invention.

Detailed Description

The present invention is further illustrated by the following specific examples.

The term "neovascular recognition promoter" appearing in the present invention is abbreviated as a promoter, i.e., a fusion protein capable of specifically recognizing endothelial cells in a proliferative state; the terms "engineered cell", "engineered cell" and "engineered cell" are used herein to refer to a cell obtained by introducing the promoter molecule into a eukaryotic cell by means of DNA recombination, DNA injection, plasmid transfection or viral transfection.

S1P is considered to be a key factor providing barrier function to endothelial cells and plays an important role in the maintenance of the blood brain barrier. The integrity of the blood-brain barrier is concentration dependent with S1P, i.e. the higher the S1P concentration, the higher the integrity of the blood-brain barrier, in a certain range (10nM-1 μ M). Endothelial cells in the brain express the transporter Spns2 of S1P, when the transporter is dysfunctional, S1P in extracellular matrix is obviously reduced, permeability of vascular endothelium is increased, tight junctions are also damaged, and exogenous S1P is supplemented to relieve vascular barrier leakage. As the major transporter of S1P in endothelial cells, Spns2 transports S1P from inside to outside the cell. In the existing research, Spns2 is not the only transporter of S1P, and Mfsd2a, which is the same protein as Spns2 in the MFS family, can significantly improve the transport efficiency of S1P and is specifically expressed in brain endothelial cells. Mfsd2a cannot be used as a direct carrier of S1P, but can significantly improve the transport efficiency of S1P. In brain endothelial cells, the highest concentration of S1P (600nM to 1. mu.M) was found in extracellular matrix when Mfsd2a was present with Spns2, whereas in the absence of Mfsd2a, the concentration of S1P decreased to between 100nM and 300nM (this range promotes transcytosis, but does not affect tight junctions). The efficient transport supported by Spns2 and Mfsd2a maintains a high concentration of S1P in the extracellular matrix, which is critical for the formation and maintenance of the blood-brain barrier.

The endothelial cells of the focal zone of the TBI model can start the expression of Apj, the promoter molecule can form cell-cell contact by combining with Apj, and the ligand is firmly combined with a receptor, so that the space condition is provided for the engineered cell to change the concentration of S1P in the microenvironment of a target cell. And the downstream response sequence of the starter molecule can be completed within twenty-four hours, at least twenty-four hours is required for the engineered cells to detach from the target cells, thus providing time conditions for changing the concentration of S1P.

The invention constructs a fusion gene by overlapping extension PCR, expresses a promoter molecule protein by a slow virus transfected cell, simultaneously transfects a fluorescent reporter gene to obtain an engineering cell modified by the promoter molecule, and co-cultures endothelial cells and the engineering cell in vitro to detect whether the engineering cell is activated; in vivo, a disease model is constructed, such as brain trauma, and the activation state of the engineered cells in vivo is detected. The status of the engineered cells was analyzed by immunofluorescence staining and flow cytometry. The engineering cells are delivered to the body of a model mouse through tail vein injection, and the functions exerted by the engineering cells are detected.

In the present invention, the preparation of engineered endothelial progenitor cells and their application in blood brain barrier repair are described in detail as examples, and the preparation and application of T lymphocyte engineering cells, neural stem cell engineering cells and glial cell engineering cells are similar to these.

The invention provides an endothelial progenitor cell modified by Apelin-synNotch receptor promoter, wherein the Apelin-synNotch receptor promoter is formed by connecting Apelin extracellular segment (the amino acid sequence of which is shown as SEQ ID NO.1 and the nucleotide sequence of which is shown as SEQ ID NO. 4) for identifying a new blood vessel, a minimum transmembrane core domain (a Notch core for short, the amino acid sequence of which is shown as SEQ ID NO.2 and the nucleotide sequence of which is shown as SEQ ID NO. 5) of natural Notch in an inner membrane segment and transcription regulation factors in series, the amino acid sequence of mature protein of the Apelin-synNotch receptor is shown as SEQ ID NO.3, the structure of the mature protein is shown as a figure 1, and the protein is converted into mature Notch receptor protein after signal peptide is cut off in a rough endoplasmic reticulum in cells after being translated and is secreted and output and positioned on the cell membrane of the endothelial progenitor cell.

The reagents used in the examples of the invention are as follows:

phosphate Buffered Saline (PBS), pH7.4, available from Gibco, cat # 10010049; double distilled water, available from Sigma-Aldrich, cat # DENWAT3 MSDS; 4% paraformaldehyde solution, available from Biosharp, cat # BL 539A; sucrose, available from Sigma-Aldrich, cat # S9378 MSDS; 10-kDadextran-tetramethylrhodomine available from Thermo Fisher corporation under item # D3312; isonectin GS-IB4 From Griffonia simplicifolia, Alexa Fluor^TM488conjugate (lectin), available from Invitrogen, cat # I21411; S1P Standard, available from Echelon Biosciences, cat # S-1000; fingolimod (FTY 720), available from MCE under # HY-12005; S1P enzyme-linked immunosorbent assay kit (ELISA kit), Shanghai Tong Yiyu Biotech Co., Ltd., cat # YY-ELISA-000479; oct (optical cutting temperature compound) embedding agent, SAKURA, cat # 4583; IP cell lysate, pecan day, cat # P0013; phenylmethylsulfonyl fluoride(Phenylmethylsulfonyl fluoride, PMSF), piceid, cat # ST 505; pierce BCA Protein Assay Kit, Thermo, cat # 23225; PageRuler^TMPrestained Protein Ladder, Thermo, cat # 26617.

Example 1 a method for preparing synNotch engineered endothelial progenitor cells, comprising the steps of:

1) preparation of endothelial progenitor cells

Taking a C57/BL6J mouse with the pregnancy period of 14-18 days, putting a pregnant uterus into precooled PBS containing 500-1000U of penicillin and streptomycin double antibody, soaking for 3-5 min, taking out a fetal mouse, and washing for 2 times by the PBS; fetal lung tissue is taken out under a microscope and put into PBS and cut into small squares with the diameter of about 1 mm.

Adding 0.25% pancreatin to fetal lung tissue, culturing in 10% FBS DMEM medium for 16-24 h, centrifuging at 1500rpm for 5min, discarding supernatant, collecting precipitate, suspending cells in 5ml EBM-2 medium containing growth factor and 5% FBS serum, and culturing at 10%⁶The number of cells/ml was seeded at T25cm²The cells were cultured in a 5% carbon dioxide incubator at 37 ℃. When the growth and fusion of primary cells reach 80-90%, the ratio is 1 multiplied by 10⁵Adding the cells per mL into a culture medium for subculture; and harvesting the cells when the cells to be transfected grow to 80% of the density, and freezing and storing the cells.

Identification of endothelial progenitor cell surface markers: 1) immunofluorescence: preparing cell slide, fixing with 4% paraformaldehyde for 10min, and rinsing with PBS for 3 times; adding 0.5% Triton X-100 for permeating for 15 min; adding goat serum, and sealing for 30 min; CD31, CD34, CD133, VEGFR2 primary antibody was incubated overnight at 4 ℃ and rinsed 3 times with PBS; adding fluorescent secondary antibody, incubating for 30min in dark, and rinsing with PBS for 3 times; adding DAPI to stain the nucleus, and rinsing with PBS for 3 times; sealing by using a sealing agent; images were observed and taken under a confocal microscope. 2) Flow cytometry: about 5 × 105 cells per tube, add antibody, incubate for 30min in the dark, rinse 3 times with PBS, and detect with flow meter.

2) Construction of promoter lentivirus

Referring to fig. 2-3, designing upstream and downstream specific amplification primers, introducing restriction enzyme sites, calling a CDS (coding sequence) of a target gene from a template (cDNA plasmid or library) by PCR (high fidelity KOD enzyme, 3K internal mutation rate of 0%), connecting the CDS to a T vector, cutting the CDS from the T vector, and loading the CDS into a lentiviral over-expression plasmid vector; wherein, the target gene is a gene which encodes the promoter molecule capable of specifically recognizing endothelial cells in a proliferation state, and the minimal transmembrane core domain 2 of the membrane hydrolysis polypeptide natural Notch is partially or completely covered by adjacent apelin 1 and effector 3 in a resting state, wherein 6 protein structures are shown in figure 2.

Synthesizing a DNA neck ring structure corresponding to siRNA, annealing, then connecting a lentivirus interference plasmid vector, preparing a lentivirus shuttle plasmid and an auxiliary packaging original vector plasmid thereof, respectively carrying out high-purity endotoxin-free extraction on the lentivirus overexpression plasmid vector, the lentivirus interference plasmid vector and the lentivirus shuttle plasmid, then co-transfecting 293T cells, replacing the vectors with complete culture media 6h after transfection, respectively collecting cell supernatants rich in lentivirus particles after culturing for 24h and 48h, and concentrating the viruses through ultracentrifugation of the virus supernatants to obtain the starting molecule lentiviruses.

The method comprises the following specific steps:

the slow virus package adopts 293T cells, the 293T cells in logarithmic growth phase are selected, trypsinized, inoculated in a T25cm2 culture bottle, and cultured in a 5% carbon dioxide incubator at 37 ℃; transfecting when the cell density reaches 60-70%, transferring the mixed solution of DNA and calcium phosphate into a culture medium, uniformly mixing, culturing for 12h, removing the old culture solution, and rinsing with PBS for 3 times; adding 5mL of fresh DMEM medium into each bottle of cells, and continuously culturing for 48 h; collecting supernatant of 293T cells after transfection for 72 h; centrifuging the collected supernatant at 4 ℃ for 10min at 4000g, and collecting the supernatant; filtering the supernatant of each different RNA interference plasmid; centrifuging in a 40mL ultracentrifuge tube at 25000r/min for 20min at 4 ℃; the ice PBS solution was dissolved in 500ul DMEM and 500ul PBS heavy rotaviral pellet, and then dissolved overnight at 4 ℃.

3) Promoter molecule modification of endothelial progenitor cells

Take 1X 10⁷-5×10⁷Discarding the old culture solution, adding 2-4mL of fresh EGM-2 culture solution, adding 200-300uL of the virus concentrated solution obtained in step 2), and obtaining a final concentration of 5 μ g/mLPolybrene, 5% CO at 37 ℃₂After 12-16 hours of infection in the incubator, the waste liquid is discarded, the cells are transferred to an uncoated culture bottle, 20-40mL of fresh EGM-2 culture solution is added, and 5% CO is added at 37 DEG C₂And after the continuous amplification culture in the incubator for 3-5 days, infecting to obtain the endothelial progenitor cells modified by the promoter molecules.

The method comprises the following specific steps:

(1) digesting endothelial progenitor cells by 0.25% pancreatin 18-24 hours before lentivirus transfection, adding EGM-2 culture solution after centrifugation to re-suspend and prepare single cell suspension for parallel cell counting, and enabling the cell suspension to be 1 multiplied by 10⁵Density per well was seeded in 24-well plates.

(2) After 24h of cell inoculation, the old medium was discarded and replaced with 2ml of fresh serum-free medium containing 5. mu.g/ml polybrene, the amount of added virus suspension required for a MOL value of 10 was calculated, added to the medium and mixed well by gentle shaking, placed at 37 ℃ and 5% CO₂And (5) incubation in an incubator.

(3) After 4 hours 2ml fresh medium was added.

(4) The culture was continued for 24 hours and replaced with fresh virus-free complete medium.

(5) After 3-4 days of transfection, puromycin is added into a complete culture medium, and the final concentration of puromycin is 5ug/ml, so as to screen a stable transfected cell strain and obtain an engineering cell containing the promoter molecule.

The engineering cell containing the promoter can specifically recognize the new blood vessel, regulate a cell signal path and regulate the expression of downstream genes, the working principle is shown in figure 3, the promoter is distributed on a cell membrane in the constructed engineering cell, the promoter stretches across the whole cell membrane, the outer section of the cell membrane is a recognition domain and can be combined with an endothelial cell surface molecular marker Apj protein 1' in a proliferation state, and therefore the capacity of specifically recognizing the new blood vessel is endowed to the promoter engineering cell. The apelin 1 on the starter molecule is combined with Apj to cause adhesion of the starter molecule engineering cell and the endothelial cell in a proliferation state, the minimum transmembrane core structure domain 2 of the natural Notch of the hydrolyzable peptide segment of the starter molecule is exposed by the traction of mechanical force, the connection of the effector 3 and the membrane inner segment is damaged after the hydrolyzable peptide segment is hydrolyzed, the effector 3 falls off from the cell membrane and enters the cell nucleus to activate the downstream response element 4 and the target gene 5, and the specific response of the starter molecule is realized.

Example 2 Co-culture of engineered cells with Apj-positive endothelial cells

1. Isolation and culture of endothelial cells

Taking a fresh umbilical cord of a postpartum mouse, and shearing a section with the length of 0.5 cm by using surgical scissors; injecting PBS into umbilical vein, and cleaning residual blood; injecting collagenase with the concentration of 0.1% into umbilical vein, and digesting for 5 minutes at room temperature; transferring the digestive juice containing the endothelial cells into a 15ml centrifuge tube, centrifuging for 5 minutes at 1000rpm, discarding the supernatant, adding sufficient 1640 culture solution for resuspension, inoculating into a T25 culture flask for culturing for 2-3 days, and carrying out passage when the cells grow to 80-90% fusion degree to obtain the endothelial cells.

2. Transfection and Co-culture

When the CMV promoter, TRE and Sphk1-Mfsd2a genes are transfected into prepared endothelial progenitor cells, the tTA will detach from the cells and enter the nucleus of the cells after the promoter binds Apj, and then bind to the response element TRE, so that the expression of Sphk1 and Mfsd2a is initiated.

Regulating the digested endothelial cells and engineered cells to a cell density of 1 × 10 with EGM-2 complete medium⁶Left and right, according to 1: 1 ratio endothelial cells were mixed with the engineered cells and added to a 6cm diameter dish to detect activation of the engineered cells, and after 24 hours of co-culture, activation was detected as well as the concentration of S1P in the culture medium, see fig. 4-5.

As shown in fig. 4, mRNA levels of Sphk1 and Mfsd2a were significantly increased, which represents that the presenting cells can activate the downstream response program of engineered cells, while ELISA measures S1P concentration in the culture medium, and as a result, as shown in fig. 5, S1P concentration in the culture medium was also significantly increased. Therefore, the engineering cell can be used for blood brain barrier repair in a new blood vessel.

Example 3 identification of neovascularization after brain trauma by engineered cells

(1) Construction of a brain trauma model

Pentobarbital sodium (0.4mg/10g) was intraperitoneally injected to anesthetize the mice, the mice were mounted on a stereotactic injection apparatus, after which a hole of approximately 5 mm diameter was opened in the right apical cortex using a burr drill, adjacent to the central suture. A transducing metal rod of 2.5 mm diameter was then placed over the skull opening and a 5 gram gravity hammer was released from a height of 15 cm to allow free fall, striking the transducing metal rod vertically.

(2) Transfecting the promoter, a transcription response element TRE and a red fluorescent protein RFP into the endothelial progenitor cells simultaneously;

(3) on the 3 rd day after the TBI mouse model is successfully constructed, engineering cells modified by endothelial progenitor cells are injected into tail veins, brain tissues of the mice are taken for observation after 24 hours, and the condition of the activation of the engineering cells in the damaged area is analyzed through the expression of a reporter gene.

The arrangement mode of brain vascular endothelial cells after 4 days of craniocerebral injury is shown in figure 6, and the basic principle of starting molecular engineering cells to repair blood brain barriers is shown.

Example 4 evaluation of the repair of blood brain Barrier by engineered cells

(1) Injection of engineered cells

The Mfsd2a-CreER and Rosa26-RFP mice are used for constructing a TBI model, and the TBI model is injected by a tail vein at the 3 rd day after being successfully constructed to be 1 multiplied by 10⁶Engineering cells, and detecting the expression of Mfsd2a in endothelial cells after injecting the engineering cells.

(2) ELISA for detecting S1P level in extracellular matrix

Detection tests are carried out according to the ELISA kit instructions, and as shown in FIG. 7, the ELISA detection tests prove that the concentration of S1P in the extracellular matrix is increased by about 3.4 times and is obviously improved after the engineering cells are activated in the damaged area compared with the injection of the common endothelial progenitor cells.

(3) Detecting engineered cell response

The qPCR method is used for detecting the expression conditions of the S1P synthetase Sphk1 and the S1P transport protein Mfsd2a of the engineering cells, and the qPCR detection shows that after the endothelial cells are excited, the expression levels of Sphk1 and Mfsd2a genes of the engineering cells are obviously improved and are respectively increased by about 3.4 times and 10.2 times.

(4) Detection of blood brain barrier recovery

Blood brain barrier was detected using a 10kDa dextran tracer, 10kDa dextran tracer was injected: after isoflurane anesthesia, the mice were fixed, the precordial skin and dermis were incised to expose the heart, 50 μ l of 10kDa dextran concentrator (2mg/ml) was injected to the apical site, after 15 minutes the mice were sacrificed by excess isoflurane, the intact brain tissue was stripped, soaked in 4% PFA solution and fixed in a refrigerator at 4 ℃ for 3-4 hours. And then soaking the cell in 30% sucrose solution at 4 ℃ for overnight dehydration, embedding the cell in OCT embedding medium, and carrying out immunofluorescence staining after frozen sectioning, wherein the result shows that the blood brain barrier is closed in advance after the engineering cell treatment.

The starting molecule chimeric protein developed by the invention not only accurately targets engineering cells to a damaged area, but also closes the blood brain barrier in advance in a TBI model, the starting molecule is not limited to be embedded into the cell membrane of endothelial progenitor cells, and can also be expressed on the surfaces of T lymphocytes, neural stem cells, glial cells and even more cells, and the extracellular apelin recognition structural domain can be replaced by other various potential ligand molecules, which means that the starting molecule has a very wide application range; meanwhile, the intracellular regulatory factor structural domain can be replaced by different reaction elements according to requirements to activate different downstream response programs, so that the function diversification of the starting molecules is realized.

Sequence listing

<110> Huashan Hospital affiliated to Fudan university

<120> a promoter molecule and engineering cell capable of specifically recognizing endothelial cells in proliferation state

<130> 2011245

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 77

<212> PRT

<213> human (Homo sapiens)

<400> 1

Met Asn Leu Arg Leu Cys Val Gln Ala Leu Leu Leu Leu Trp Leu Ser

1 5 10 15

Leu Thr Ala Val Cys Gly Val Pro Leu Met Leu Pro Pro Asp Gly Thr

20 25 30

Gly Leu Glu Glu Gly Ser Met Arg Tyr Leu Val Lys Pro Arg Thr Ser

35 40 45

Arg Thr Gly Pro Gly Ala Trp Gln Gly Gly Arg Arg Lys Phe Arg Arg

50 55 60

Gln Arg Pro Arg Leu Ser His Lys Gly Pro Met Pro Phe

65 70 75

<210> 2

<211> 713

<212> PRT

<213> human (Homo sapiens)

<400> 2

Pro Cys Val Gly Ser Asn Pro Cys Tyr Asn Gln Gly Thr Cys Glu Pro

1 5 10 15

Thr Ser Glu Asn Pro Phe Tyr Arg Cys Leu Cys Pro Ala Lys Phe Asn

20 25 30

Gly Leu Leu Cys His Ile Leu Asp Tyr Ser Phe Thr Gly Gly Ala Gly

35 40 45

Arg Asp Ile Pro Pro Pro Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu

50 55 60

Cys Gln Val Asp Ala Gly Asn Lys Val Cys Asn Leu Gln Cys Asn Asn

65 70 75 80

His Ala Cys Gly Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp

85 90 95

Pro Trp Lys Asn Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser

100 105 110

Asp Gly His Cys Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp

115 120 125

Gly Phe Asp Cys Gln Leu Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp

130 135 140

Gln Tyr Cys Lys Asp His Phe Ser Asp Gly His Cys Asp Gln Gly Cys

145 150 155 160

Asn Ser Ala Glu Cys Glu Trp Asp Gly Leu Asp Cys Ala Glu His Val

165 170 175

Pro Glu Arg Leu Ala Ala Gly Thr Leu Val Leu Val Val Leu Leu Pro

180 185 190

Pro Asp Gln Leu Arg Asn Asn Ser Phe His Phe Leu Arg Glu Leu Ser

195 200 205

His Val Leu His Thr Asn Val Val Phe Lys Arg Asp Ala Gln Gly Gln

210 215 220

Gln Met Ile Phe Pro Tyr Tyr Gly His Glu Glu Glu Leu Arg Lys His

225 230 235 240

Pro Ile Lys Arg Ser Thr Val Gly Trp Ala Thr Ser Ser Leu Leu Pro

245 250 255

Gly Thr Ser Gly Gly Arg Gln Arg Arg Glu Leu Asp Pro Met Asp Ile

260 265 270

Arg Gly Ser Ile Val Tyr Leu Glu Ile Asp Asn Arg Gln Cys Val Gln

275 280 285

Ser Ser Ser Gln Cys Phe Gln Ser Ala Thr Asp Val Ala Ala Phe Leu

290 295 300

Gly Ala Leu Ala Ser Leu Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu

305 310 315 320

Ala Val Lys Ser Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His

325 330 335

Leu Met Tyr Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly

340 345 350

Cys Gly Val Leu Leu Ser Arg Lys Arg Arg Arg Gln Leu Cys Ile Gln

355 360 365

Lys Leu Met Ser Asn Leu Leu Thr Val His Gln Asn Leu Pro Ala Leu

370 375 380

Pro Val Asp Ala Thr Ser Asp Glu Val Arg Lys Asn Leu Met Asp Met

385 390 395 400

Phe Arg Asp Arg Gln Ala Phe Ser Glu His Thr Trp Lys Met Leu Leu

405 410 415

Ser Val Cys Arg Ser Trp Ala Ala Trp Cys Lys Leu Asn Asn Arg Lys

420 425 430

Trp Phe Pro Ala Glu Pro Glu Asp Val Arg Asp Tyr Leu Leu Tyr Leu

435 440 445

Gln Ala Arg Gly Leu Ala Val Lys Thr Ile Gln Gln His Leu Gly Gln

450 455 460

Leu Asn Met Leu His Arg Arg Ser Gly Leu Pro Arg Pro Ser Asp Ser

465 470 475 480

Asn Ala Val Ser Leu Val Met Arg Arg Ile Arg Lys Glu Asn Val Asp

485 490 495

Ala Gly Glu Arg Ala Lys Gln Ala Leu Ala Phe Glu Arg Thr Asp Phe

500 505 510

Asp Gln Val Arg Ser Leu Met Glu Asn Ser Asp Arg Cys Gln Asp Ile

515 520 525

Arg Asn Leu Ala Phe Leu Gly Ile Ala Tyr Asn Thr Leu Leu Arg Ile

530 535 540

Ala Glu Ile Ala Arg Ile Arg Val Lys Asp Ile Ser Arg Thr Asp Gly

545 550 555 560

Gly Arg Met Leu Ile His Ile Gly Arg Thr Lys Thr Leu Val Ser Thr

565 570 575

Ala Gly Val Glu Lys Ala Leu Ser Leu Gly Val Thr Lys Leu Val Glu

580 585 590

Arg Trp Ile Ser Val Ser Gly Val Ala Asp Asp Pro Asn Asn Tyr Leu

595 600 605

Phe Cys Arg Val Arg Lys Asn Gly Val Ala Ala Pro Ser Ala Thr Ser

610 615 620

Gln Leu Ser Thr Arg Ala Leu Glu Gly Ile Phe Glu Ala Thr His Arg

625 630 635 640

Leu Ile Tyr Gly Ala Lys Asp Asp Ser Gly Gln Arg Tyr Leu Ala Trp

645 650 655

Ser Gly His Ser Ala Arg Val Gly Ala Ala Arg Asp Met Ala Arg Ala

660 665 670

Gly Val Ser Ile Pro Glu Ile Met Gln Ala Gly Gly Trp Thr Asn Val

675 680 685

Asn Ile Val Met Asn Tyr Ile Arg Asn Leu Asp Ser Glu Thr Gly Ala

690 695 700

Met Val Arg Leu Leu Glu Asp Gly Asp

705 710

<210> 3

<211> 790

<212> PRT

<213> human (Homo sapiens)

<400> 3

Met Asn Leu Arg Leu Cys Val Gln Ala Leu Leu Leu Leu Trp Leu Ser

1 5 10 15

Leu Thr Ala Val Cys Gly Val Pro Leu Met Leu Pro Pro Asp Gly Thr

20 25 30

Gly Leu Glu Glu Gly Ser Met Arg Tyr Leu Val Lys Pro Arg Thr Ser

35 40 45

Arg Thr Gly Pro Gly Ala Trp Gln Gly Gly Arg Arg Lys Phe Arg Arg

50 55 60

Gln Arg Pro Arg Leu Ser His Lys Gly Pro Met Pro Phe Pro Cys Val

65 70 75 80

Gly Ser Asn Pro Cys Tyr Asn Gln Gly Thr Cys Glu Pro Thr Ser Glu

85 90 95

Asn Pro Phe Tyr Arg Cys Leu Cys Pro Ala Lys Phe Asn Gly Leu Leu

100 105 110

Cys His Ile Leu Asp Tyr Ser Phe Thr Gly Gly Ala Gly Arg Asp Ile

115 120 125

Pro Pro Pro Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Val

130 135 140

Asp Ala Gly Asn Lys Val Cys Asn Leu Gln Cys Asn Asn His Ala Cys

145 150 155 160

Gly Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys

165 170 175

Asn Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly His

180 185 190

Cys Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly Phe Asp

195 200 205

Cys Gln Leu Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln Tyr Cys

210 215 220

Lys Asp His Phe Ser Asp Gly His Cys Asp Gln Gly Cys Asn Ser Ala

225 230 235 240

Glu Cys Glu Trp Asp Gly Leu Asp Cys Ala Glu His Val Pro Glu Arg

245 250 255

Leu Ala Ala Gly Thr Leu Val Leu Val Val Leu Leu Pro Pro Asp Gln

260 265 270

Leu Arg Asn Asn Ser Phe His Phe Leu Arg Glu Leu Ser His Val Leu

275 280 285

His Thr Asn Val Val Phe Lys Arg Asp Ala Gln Gly Gln Gln Met Ile

290 295 300

Phe Pro Tyr Tyr Gly His Glu Glu Glu Leu Arg Lys His Pro Ile Lys

305 310 315 320

Arg Ser Thr Val Gly Trp Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser

325 330 335

Gly Gly Arg Gln Arg Arg Glu Leu Asp Pro Met Asp Ile Arg Gly Ser

340 345 350

Ile Val Tyr Leu Glu Ile Asp Asn Arg Gln Cys Val Gln Ser Ser Ser

355 360 365

Gln Cys Phe Gln Ser Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu

370 375 380

Ala Ser Leu Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Lys

385 390 395 400

Ser Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr

405 410 415

Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly Cys Gly Val

420 425 430

Leu Leu Ser Arg Lys Arg Arg Arg Gln Leu Cys Ile Gln Lys Leu Met

435 440 445

Ser Asn Leu Leu Thr Val His Gln Asn Leu Pro Ala Leu Pro Val Asp

450 455 460

Ala Thr Ser Asp Glu Val Arg Lys Asn Leu Met Asp Met Phe Arg Asp

465 470 475 480

Arg Gln Ala Phe Ser Glu His Thr Trp Lys Met Leu Leu Ser Val Cys

485 490 495

Arg Ser Trp Ala Ala Trp Cys Lys Leu Asn Asn Arg Lys Trp Phe Pro

500 505 510

Ala Glu Pro Glu Asp Val Arg Asp Tyr Leu Leu Tyr Leu Gln Ala Arg

515 520 525

Gly Leu Ala Val Lys Thr Ile Gln Gln His Leu Gly Gln Leu Asn Met

530 535 540

Leu His Arg Arg Ser Gly Leu Pro Arg Pro Ser Asp Ser Asn Ala Val

545 550 555 560

Ser Leu Val Met Arg Arg Ile Arg Lys Glu Asn Val Asp Ala Gly Glu

565 570 575

Arg Ala Lys Gln Ala Leu Ala Phe Glu Arg Thr Asp Phe Asp Gln Val

580 585 590

Arg Ser Leu Met Glu Asn Ser Asp Arg Cys Gln Asp Ile Arg Asn Leu

595 600 605

Ala Phe Leu Gly Ile Ala Tyr Asn Thr Leu Leu Arg Ile Ala Glu Ile

610 615 620

Ala Arg Ile Arg Val Lys Asp Ile Ser Arg Thr Asp Gly Gly Arg Met

625 630 635 640

Leu Ile His Ile Gly Arg Thr Lys Thr Leu Val Ser Thr Ala Gly Val

645 650 655

Glu Lys Ala Leu Ser Leu Gly Val Thr Lys Leu Val Glu Arg Trp Ile

660 665 670

Ser Val Ser Gly Val Ala Asp Asp Pro Asn Asn Tyr Leu Phe Cys Arg

675 680 685

Val Arg Lys Asn Gly Val Ala Ala Pro Ser Ala Thr Ser Gln Leu Ser

690 695 700

Thr Arg Ala Leu Glu Gly Ile Phe Glu Ala Thr His Arg Leu Ile Tyr

705 710 715 720

Gly Ala Lys Asp Asp Ser Gly Gln Arg Tyr Leu Ala Trp Ser Gly His

725 730 735

Ser Ala Arg Val Gly Ala Ala Arg Asp Met Ala Arg Ala Gly Val Ser

740 745 750

Ile Pro Glu Ile Met Gln Ala Gly Gly Trp Thr Asn Val Asn Ile Val

755 760 765

Met Asn Tyr Ile Arg Asn Leu Asp Ser Glu Thr Gly Ala Met Val Arg

770 775 780

Leu Leu Glu Asp Gly Asp

785 790

<210> 4

<211> 291

<212> DNA

<213> human (Homo sapiens)

<400> 4

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccgaatctga ggctctgcgt gcaggcgctg ctgctgctct ggctctcctt gactgcagtt 120

tgtggagtgc cactgatgtt gcctccagat ggaacaggac tagaagaagg aagcatgcgc 180

tacctggtga agcccagaac ttcgaggact ggaccaggag cctggcaggg aggcaggagg 240

aaatttcgca gacagcgccc ccggctctcc cataagggcc ccatgccttt c 291

<210> 5

<211> 1757

<212> DNA

<213> human (Homo sapiens)

<400> 5

ccctgtgtgg gtagcaaccc ctgctacaat cagggcacct gtgagcccac atccgagaac 60

cctttctacc gctgtctatg ccctgccaaa ttcaacgggc tactgtgcca catcctggac 120

tacagcttca caggtggcgc tgggcgcgac attcccccac cgcagattga ggaggcctgt 180

gagctgcctg agtgccaggt ggatgcaggc aataaggtct gcaacctgca gtgtaataat 240

cacgcatgtg gctgggatgg tggcgactgc tccctcaact tcaatgaccc ctggaagaac 300

tgcacgcagt ctctacagtg ctggaagtat tttagcgacg gccactgtga cagccagtgc 360

aactcggccg gctgcctctt tgatggcttc gactgccagc tcaccgaggg acagtgcaac 420

cccctgtatg accagtactg caaggaccac ttcagtgatg gccactgcga ccagggctgt 480

aacagtgccg aatgtgagtg ggatggccta gactgtgctg agcatgtacc cgagcggctg 540

gcagccggca ccctggtgct ggtggtgctg cttccacccg accagctacg gaacaactcc 600

ttccactttc tgcgggagct cagccacgtg ctgcacacca acgtggtctt caagcgtgat 660

gcgcaaggcc agcagatgat cttcccgtac tatggccacg aggaagagct gcgcaagcac 720

ccaatcaagc gctctacagt gggttgggcc acctcttcac tgcttcctgg taccagtggt 780

gggcgccagc gcagggagct ggaccccatg gacatccgtg gctccattgt ctacctggag 840

atcgacaacc ggcaatgtgt gcagtcatcc tcgcagtgct tccagagtgc caccgatgtg 900

gctgccttcc taggtgctct tgcgtcactt ggcagcctca atattcctta caagattgag 960

gccgtgaaga gtgagccggt ggagcctccg ctgccctcgc agctgcacct catgtacgtg 1020

gcagcggccg ccttcgtgct cctgttcttt gtgggctgtg gggtgctgct gtcccgcaag 1080

cgccggcggt ctagactgga caagagcaaa gtcataaact ctgctctgga attactcaat 1140

gaagtcggta tcgaaggcct gacgacaagg aaactcgctc aaaagctggg agttgagcag 1200

cctaccctgt actggcacgt gaagaacaag cgggccctgc tcgatgccct ggcaatcgag 1260

atgctggaca ggcatcatac ccacttctgc cccctggaag gcgagtcatg gcaagacttt 1320

ctgcggaaca acgccaagtc attccgctgt gctctcctct cacatcgcga cggggctaaa 1380

gtgcatctcg gcacccgccc aacagagaaa cagtacgaaa ccctggaaaa tcagctcgcg 1440

ttcctgtgtc agcaaggctt ctccctggag aacgcactgt acgctctgtc cgccgtgggc 1500

cactttacac tgggctgcgt attggaggat caggagcatc aagtagcaaa agaggaaaga 1560

gagacaccta ccaccgattc tatgccccca cttctgagac aagcaattga gctgttcgac 1620

catcagggag ccgaacctgc cttccttttc ggcctggaac taatcatatg tggcctggag 1680

aaacagctaa agtgcgaaag cggcgggccg gccgacgccc ttgacgattt tgacttagac 1740

atgctcccag ccgatgc 1757

Claims

1. A starting molecule capable of specifically recognizing endothelial cells in a proliferation state is a fusion protein and is characterized by comprising human apelin protein, intramembrane hydrolysable polypeptide and an effector, wherein the human apelin protein, the intramembrane hydrolysable polypeptide and the effector are connected by two proteolytic cleavage sites in the intramembrane hydrolysable polypeptide; wherein the intramembrane hydrolysable polypeptide is the smallest transmembrane core domain of native Notch, and is partially or completely covered by adjacent apelin and effector in a resting state.

2. The starter molecule according to claim 1, wherein the apelin protein has an amino acid sequence shown in SEQ ID No. 1; the amino acid sequence of the intramembrane hydrolysable polypeptide is shown as SEQ ID NO. 2.

3. The promoter molecule according to claim 1, wherein said effector is selected from the group consisting of tetracycline transcriptional activator or a domain of Cre recombinase.

4. The promoter molecule according to claim 1, wherein the effector is Mfsd2a transcription promoter, and the amino acid sequence of the promoter molecule is shown as SEQ ID No. 3.

5. An engineered cell comprising the promoter molecule of claim 1 that specifically recognizes endothelial cells in a proliferative state.

6. The engineered cell of claim 5, wherein the promoter is obtained by introducing the promoter into a eukaryotic cell by means of DNA recombination, DNA injection, plasmid transfection or viral transfection.

7. The engineered cell of claim 6, wherein the eukaryotic cell is an endothelial progenitor cell, a T lymphocyte, a neural stem cell, or a glial cell.

8. A method for preparing synNotch engineered endothelial progenitor cells comprises the following steps:

1) preparing endothelial progenitor cells;

2) construction of lentiviruses containing fusion proteins

Designing upstream and downstream specific PCR amplification primers containing the promoter molecule of any one of claims 1 to 5, introducing a restriction enzyme site, amplifying by overlap extension PCR, and calling a CDS region of the promoter molecule gene from a cDNA plasmid or a library template to connect into a T vector; cutting the CDS region from the T vector, and filling into a lentivirus overexpression plasmid vector; synthesizing a DNA neck ring structure corresponding to the siRNA, and inoculating a lentiviral interference plasmid vector after annealing;

3) transfection into eukaryotic cells

9. The method for preparing synNotch engineered endothelial cells according to claim 8, wherein in step 3), the endothelial progenitor cells after transfection of lentiviruses are amplified by EGM-2 medium, when the cell amount accounts for 80-90% of the culture flask, the expression of the labeled fluorescent protein is observed by a fluorescence microscope, and the transfected cell population is subjected to marker identification by a flow cytometer to detect the activation of the engineered cells.

10. The synNotch engineered cell of claim 5 for use in targeting neovasculature, in the construction of a mouse tumor model or a brain trauma model, in reprogramming cells, in cell injury repair, in repairing the blood brain barrier, or in cell regeneration.

11. The use of claim 11, wherein the targeted neovasculature is targeted in subacute-stage brain trauma.