Tracer agent for synaptic formation in-vivo visualization and construction method thereof

Abstract

The invention discloses a tracer agent for synapse formation in-vivo visualization and a construction method of the tracer agent. The method comprises the following steps: constructing a tool virus vector for conditional expression of a tyrosinase (TYR) reporter gene, wherein the tool virus vector carries a conditional expression type promoter; conditionally expressing the TYR reporter gene in the neural stem cell by using the tool virus vector; therefore, the tracer agent which can be combined with a nuclide-labeled probe targeting melanin to realize mediation of an image signal only under the condition of synaptic formation is obtained. The tracer agent provided by the invention can be combined with a nuclide labeled probe of targeted melanin, and effectively penetrates through blood brain barrier targeted melanin to realize that neural stem cell synapses are transplanted in the brain to form dynamic functional tracing. The construction of the system provides an effective research tool for the research of regenerative medicine and neural stem cell transplantation therapy.

Description

Technical Field

[0001] The present invention belongs to the field of nuclear medicine molecular imaging, and in particular, relates to a tracer for in vivo visualization of synapse formation and a construction method thereof. Background Art

[0002] Neural stem cell transplantation has become a potential new strategy for the treatment of many central nervous system diseases (such as ischemic stroke and Parkinson's disease), and neural stem cell synapse formation and integration are important mechanisms of cell transplantation therapy. A series of studies have shown that after neural stem cells are transplanted into the brain, they can differentiate, mature, integrate with the host neural circuits, and play a role in specific neural functions. However, at present, our understanding of the dynamic changes in synapse formation of transplanted neural stem cells in the brain is still insufficient. The key to this problem is the lack of effective means of evaluating synapse formation in vivo. Therefore, the construction of dynamic tracing technology for synapse formation of transplanted neural stem cells is crucial to explore the in vivo therapeutic mechanism of neural stem cell transplantation and promote the further development of cell therapy for nervous system diseases.

[0003] Positron emission tomography (PET) is a representative nuclear medicine molecular imaging technique that can visualize and quantify pathological and physiological processes at the cellular and molecular levels. Nuclear medicine reporter gene imaging is an indirect imaging method that uses exogenous reporter genes encoding specific enzymes, transporters or receptors to infect target cells simultaneously with the target gene to be tested, so that the proteins encoded by the reporter gene and the target gene are expressed synchronously. The reporter protein can be detected by nuclear medicine molecular probes to evaluate the expression of the target gene and the state of the cell. After years of development, PET reporter gene imaging has become an important technology for in vivo tracing of neural stem cells.

[0004] However, compared with the tracing of transplanted neural stem cells in the periphery, the tracing of transplanted neural stem cells in the brain is more challenging due to the limitation of the blood-brain barrier. At present, there is a lack of reporter gene systems that can be used for the central nervous system. Most of the existing reporter genes are not suitable for nervous system research for various reasons. For example, the most widely used HSV1-tk reporter gene currently lacks a PET probe that can penetrate the blood-brain barrier. The dopamine type 2 receptor reporter gene is highly expressed in the basal ganglia, which limits the application of the reporter gene in the brain or related brain regions. The in vivo visualization of neural stem cell synapse formation is an important direction to promote the further development of neural stem cell research and clinical transformation. How to achieve in vivo visualization of neural stem cell synapse formation based on the existing reporter gene system is of great research significance. Summary of the invention

[0005] The object of the present invention is to provide a tracer for in vivo visualization of synapse formation and a method for constructing the same.

[0006] The present invention adopts the following technical solutions:

[0007] A method for constructing a tracer for in vivo visualization of synapse formation includes: constructing a tool virus vector driven by a conditional expression promoter hSyn to express a tyrosinase (TYR) reporter gene; and transfecting neural stem cells with the tool virus vector to make the neural stem cells express the TYR reporter gene under synapse formation conditions.

[0008] The constructed neural stem cells have a TYR reporter gene that catalyzes tyrosine into melanin through a multi-step reaction in the cells; thus, melanin can be detected by PET molecular imaging to achieve in vivo visualization of synapse formation of transplanted neural stem cells in the brain.

[0009] As an embodiment, the tool viral vector includes one of a lentivirus, an adenovirus, and an adeno-associated virus.

[0010] As an embodiment, the expression of the tyrosinase reporter gene in the cell is driven by a conditional expression promoter of synapse formation, and the conditional expression promoter is hSyn.

[0011] As an embodiment, the gene sequence of the tyrosinase is shown in Sequence 1.

[0012] As an embodiment, the amino acid sequence of the tyrosinase is shown in Sequence 2.

[0013] As an embodiment, the tool virus vector expresses a fluorescent protein gene, including but not limited to GFP, RFP, BFP, EGFP, mCherry, mStrawberry, mApple, mRuby or EosFP.

[0014] As an embodiment, the tool virus vector carries a puromycin resistance gene and is driven solely by a constitutive promoter, which includes but is not limited to CMV promoter, EFla promoter, EFS promoter, CAG promoter, Cbh promoter, SFFV promoter; SV40 promoter, Ubc promoter, Ubi promoter, hPGK promoter, β-actin promoter, so as to facilitate the successful transfection of cells by puromycin screening after cell transfection in in vitro experiments.

[0015] As an embodiment, the PET molecular imaging probe is 18 F-FPABZA.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. The tracer provided by the present invention can realize in vivo visualization of long-term synapse formation after brain transplantation. The TYR reporter gene can be integrated into the genome of neural stem cells through a tool virus vector. The reporter gene can be continuously expressed in daughter cells without signal dilution due to division.

[0018] 2. The present invention selects the hSyn promoter specific for mature neurons and utilizes its biological characteristics closely related to synapse formation and synaptic function to construct a conditionally expressed reporter gene imaging system, providing a new in vivo evaluation technology for synapse formation.

[0019] 3. The tracer provided by the present invention directly binds to the PET molecular imaging probe 18 F-FPABZA can realize in vivo visualization of synapse formation in the brain. The probe has good blood-brain barrier penetration ability and does not require additional physical or chemical operations (such as ultrasound microbubbles, small molecule drugs) to open the blood-brain barrier.

[0020] 4. The tracing method provided by the present invention is not limited by the transplantation depth of neural stem cells in the brain, and does not require the insertion of optical fibers or lenses into the brain for invasive imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings.

[0022] Figure 1 This is the principle of in vivo visualization of synapse formation by reporter gene-modified neural stem cells. After transplantation, neural stem cells survive in the brain, differentiate into neurons, and form synaptic connections with surrounding neurons. Driven by the hSyn promoter, neurons derived from neural stem cells expressing synapses synchronously express tyrosinase and catalyze the production of melanin. 18 F-FPABZAPET combined with melanin can achieve in vivo visualization of synapse formation in neural stem cells.

[0023] Figure 2 A rat model of cerebral infarction transplanted with hSyn-TYR modified neural stem cells for one month 18 F-FPABZA PET images (a) and in vitro immunofluorescence staining verification results (b). Compared with the control group one month after cerebral infarction, the hSyn-TYR neural stem cell transplantation group showed higher 18 F-FPABZA uptake (a). The in vitro results showed that some cells in the cell transplantation area (left side of the dotted line in b) expressed synapse-related proteins SYP and EGFP, and co-localized well, further verifying that the technology described in this patent can visualize the synapse formation of transplanted neural stem cells in vivo. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and are not to be construed as limiting the present invention. It will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to these embodiments without departing from the principles and purposes of the present invention, and that the scope of the present invention is defined by the claims and their equivalents. The experimental methods for which specific conditions are not specified in the following examples are usually tested under conventional conditions or under conditions recommended by the manufacturer.

[0025] The present invention utilizes a tool virus vector for transfection to make neural stem cells express tyrosinase (catalyze the synthesis of melanin) under synapse formation conditions. After being transplanted into a rat model of cerebral infarction, the neural stem cells survive, differentiate into neurons, and form synaptic connections with surrounding neurons. Driven by the hSyn promoter, neurons derived from neural stem cells expressing synapses synchronously express tyrosinase and catalyze the production of melanin. 18 F-FPABZA PET combined with melanin can realize in vivo visualization of synapse formation of neural stem cells. The specific operation example is as follows:

[0026] 1. Lentiviral particle production and packaging

[0027] In the present invention, the hSyn-TYR-P2A-EGFP reporter gene is first cloned into a tool virus vector plasmid, and then the recombinant plasmid is co-transfected with PxpaX2 (packaging plasmid, expressing the transactivator protein required for HIV-1 replication) and PMD2g (envelope protein plasmid, expressing the envelope protein of VSV-G) into HEK 293T cells, and the viral supernatant produced after transfection is collected, and finally a high-titer lentivirus is obtained through concentration and purification steps.

[0028] 2. Neural stem cell reporter gene transfection and intracerebral transplantation in rat models of cerebral infarction

[0029] The present invention uses lentivirus to perform reporter gene transfection of neural stem cells, and transplants the neural stem cells into the cerebral infarction peripheral area of ​​a cerebral infarction rat model, and evaluates the in vivo visualization effect of neural stem cell synapse formation one month after transplantation.

[0030] 3. 18 F-FPABZA PET imaging

[0031] The invention is carried out by injecting the rat tail vein 18 After the injection of F-FPABZA, the rats were returned to the cage and allowed to move freely. After 60 minutes, the rats were anesthetized and 18 F-FPABZA PET static scan for 10 minutes.

[0032] 4. In vitro immunofluorescence staining of rat brain slices transplanted with stem cells

[0033] The invention obtains the brain of rats transplanted with neural stem cells through transcardial perfusion, makes frozen sections, performs synaptic protein SYP staining, and performs co-localization evaluation with GFP fluorescent protein of neural stem cells.

[0034] See the following embodiments for details:

[0035] Example 1: Construction of a lentiviral vector plasmid for overexpression of a tyrosinase reporter gene

[0036] 1. Synthetic gene: Synthesize tyrosinase reporter gene (sequence 1) by DNA synthesizer

[0037] 2. Amplification of tyrosinase reporter gene: The tyrosinase reporter gene was amplified in large quantities by polymerase chain reaction (PCR). The primers at the 5' and 3' ends contained Kpn1 and BamH1 restriction endonuclease sequences, respectively. The product TYR gene was purified using a gel recovery kit.

[0038]

[0039] 3. The PCR reaction system is as follows

[0040]

[0041]

[0042] 4. The PCR reaction procedure is as follows:

[0043]

[0044] 5. Insert the gene fragments after BamHl and Kpnl digestion into the multiple cloning site (MCS) of the prepared vector and connect them using DNA ligase. The vector sequence is hSyn-TYR-P2A-EGFP.

[0045] 6. The correct recombinant plasmid was screened by PCR and enzyme digestion. After sequencing confirmed that the vector was correctly constructed, the recombinant plasmid (hSyn-TYR-P2A-EGFP) was amplified in large quantities.

[0046] 7. Preparation of HEK-293T cells: Inoculate 4 million HEK-293T cells in a 10 cm culture dish, culture overnight to restore the cell state, and replace with fresh complete medium before transfection.

[0047] 8. HEK-293T transfection: Add three plasmids (7.5μg of PxpaX2 plasmid, 2.5μg of PMD2g plasmid, and 10μg of TYR recombinant plasmid) in proportion to a total volume of 500μL of DMEM serum-free and anti-dual antibody-free culture medium, recorded as A solution, and mix by pipetting; take 50-60μg (1μg / mL) of transfection reagent PEI and add it to 500μL of DMEM serum-free and anti-dual antibody-free culture medium, recorded as B solution, and mix by pipetting; add B solution drop by drop into A solution, mix gently with a pipette, and let stand at room temperature for about 15 minutes; slowly add the transfection reagent (A+B) that has been allowed to stand to the HEK 293T cell culture dish; 18h after transfection, replace the HEK 293T supernatant containing the transfection reagent with 10mL DMEM complete culture medium.

[0048] 9. Obtaining virus particles: About 36 hours after transfection, aspirate the supernatant into a new centrifuge tube and store at 4°C. Also add 10 mL of fresh complete culture medium to the cells, being careful not to wash away the cells. About 36 hours later, collect the supernatant into the same centrifuge tube. Filter the virus supernatants collected twice with a 0.45 μm filter and transfer them to a new centrifuge tube (you can also centrifuge the virus supernatant at 3000 rpm, 4°C for 5 minutes and discard the precipitate). At this point, the virus particles in the supernatant can be directly tested for titer or used to infect target cells. If the titer and purity of the virus are required to be high, they can be further concentrated and purified.

[0049] 12. Concentration and purification of lentivirus: Mix the supernatant with 40% PEG solution (PEG8000, 4000 or 6000 can be used), the final PEG concentration is 10%, and place on ice for 3 to 6 hours. Centrifuge at 2000g for 30 minutes using an Avanti J-15R centrifuge. Discard the supernatant and resuspend the precipitate formed after centrifugation with 1 / 20 of the original sample volume of PBS buffer. If further concentration is required, the resuspended virus sample can be transferred to a pre-sterilized ultracentrifuge tube and centrifuged at 100,000g (24,500rpm) in an Optima XPN-100 ultracentrifuge for 90 minutes. Carefully remove the supernatant, resuspend the precipitate with PBS or other virus storage solution, and store at -80°C for later use.

[0050] Example 2: Neural stem cell reporter gene transfection and intracerebral transplantation in rat model of cerebral infarction

[0051] 1. Neural stem cell transfection: Neural stem cells were seeded in 24-well plates at a density of 1 million cells / well. Fresh culture medium was replaced after 4 days of culture. A certain volume of lentivirus was added according to the titer of lentivirus with a multiplicity of infection (MOI) of 3. Complete culture medium was replaced about 8 hours after infection. If cell morphology changes were observed during the process, complete culture medium could be replaced in advance.

[0052] 2. Construction of rat cerebral infarction model: Rats of about 2 months old were anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital, with an injection dose of 50 mg / kg. The rats were fixed in a stereotaxic instrument, the anterior and posterior bregmas were leveled, and holes were drilled at the four corners of a square area on the right side of the skull. The coordinates were (2mm-5mm to the right of the midline, 2.5mm in front of the anterior bregma-1.5mm behind the anterior bregma), and the skull was opened. 1 ml of 20 mg / ml rose bengal solution was injected into the tail vein, and the skull opening area was irradiated with a 25 mw laser for 15 minutes. The skull was put back, the rat was removed, the skin was sutured for disinfection, and 200,000 units of penicillin were injected intraperitoneally for 3 days to prevent infection.

[0053] 3. Intracerebral transplantation of neural stem cells in rats with cerebral infarction model: Two weeks after the rats were modeled with cerebral infarction, the rats were anesthetized and fixed in a stereotaxic instrument. Three holes were drilled on the left edge of the craniotomy area of ​​the cerebral infarction (evenly distributed front and back). 0.67 μL of transfected neural stem cells (about 67,000) were aspirated with a microinjection needle. The neural stem cells were transplanted into the peripheral area of ​​the cerebral infarction at a speed of 0.2 μL / min in the brain tissue at a depth of 2.3 mm. After the injection was completed, the injection needle was slowly pulled out after standing for 5 minutes. The rats were sutured, disinfected, and intraperitoneally injected with 200,000 units of penicillin for 3 days to prevent infection.

[0054] Example 3: Tracer Injection 18 Synthesis of F-FPABZA

[0055] 1. After the fluorine source of fluorine-containing heavy oxygen water is adsorbed on the anion adsorption column (QMA column), 0.8 ml of eluent (15 mg K222, 3 mg potassium carbonate, 0.64 ml acetonitrile, 0.16 ml sterile water) is used to elute the QMA column into the reaction bottle. The liquid in the reaction bottle is evaporated at 110 degrees, and 1 ml of anhydrous acetonitrile is added during the azeotropic evaporation and repeated three times.

[0056] 2. After the coal is completely dried, add 1 ml of DMF solution containing 7.5 mg of precursor BrPABZA and react at 140°C for 30 min. Dilute the reaction solution and enter HPLC for purification and separation. Use a mobile phase of 7:3 acetonitrile and 0.5 / 1000 trifluoroacetic acid in water to collect the product peak at about 16 min.

[0057] 3. Dilute the product peak solution with 30 ml of pure water and pass it through a C18 separation column to adsorb the product on the C18 column. Then wash the C18 column with 10 ml of pure water. After eluting the product from the C18 column with 1 ml of alcohol, dilute it with 10 ml of saline containing 10 mg / ml ascorbic acid, and finally filter it through a 0.22 um sterile filter membrane to obtain the final 18 F-FPABZA injection.

[0058] Embodiment 4: 18F-FPABZA PET scanning to track transplanted neural stem cells

[0059] 1. 18 F-FPABZA PET scan: One month after neural stem cell transplantation, rats were anesthetized with 4% isoflurane and injected with approximately 18.5 MBq (500 μCi) into the tail vein. 18 After F-FPABZA, the rats were put back into the cage and moved freely. After 60 minutes, anesthesia was induced again with 4% isoflurane, and the rats were fixed in prone position on the small animal PET scanning window for static scanning. The scanning time was 10 minutes, and 2% isoflurane was given during the scanning to maintain anesthesia.

[0060] 2. 18 F-FPABZA PET reconstruction: The initial image obtained by scanning was reconstructed using the ordered subset maximum expectation method (OSEM), and the reconstructed image was processed using Pmod software. Figure 2 .

[0061] Example 5: In vitro immunofluorescence staining of stem cell transplanted rat brain slices

[0062] 1. Rats were deeply anesthetized with 1.5% sodium pentobarbital injected intraperitoneally, the cardiac field was exposed, and the perfusion needle was inserted from the apex of the rat heart. The needle was placed along the left ventricular outflow tract to the beginning of the aortic arch. 100 ml of normal saline was quickly perfused, and then about 50 ml of 4% paraformaldehyde was slowly perfused until the rat's forelimbs and neck were stiff. The intact brain tissue was carefully removed. The removed rat brain was placed in 4% paraformaldehyde solution, externally fixed in a 4°C refrigerator overnight, and then replaced with 30% sucrose solution for dehydration. After the rat brain was dehydrated, the brain tissue was embedded and frozen.

[0063] 2. Select brain slices, put them into a 24-well plate with 0.5ml PBS, shake them quickly on a shaker for 5 minutes, and repeat three times. Add 0.2ml of quick blocking solution to the 24-well plate, shake slowly on a shaker at room temperature for 1 hour, and wash them three times with 0.5ml PBS. Prepare the primary antibody in the appropriate ratio according to the antibody instructions, and incubate them in a 4°C refrigerator with slow shaking overnight. Recover the primary antibody, add 0.5ml PBS to each well and wash them three times. Prepare the secondary antibody in the appropriate ratio according to the primary antibody species and the secondary antibody instructions, and incubate them slowly at room temperature for 1 hour. Recover the secondary antibody, add 0.5ml PBS to each well and wash them three times. Add DAPI stain, incubate at room temperature for 10 minutes, and add 0.5ml PBS to each well and wash them three times. Flatten and mount the brain slices in a culture dish filled with PBS. Add anti-fading sealing agent to the brain slices, seal them, and observe them.

[0064] Sequence 1

[0065]

[0066]

[0067] Sequence 2

[0068]

[0069]

Claims

1. A method for constructing a tracer for in vivo visualization of synapse formation, characterized in that: These include: Constructing a tool virus vector for conditional expression of a tyrosinase (TYR) reporter gene, wherein the tool virus vector carries a conditional expression promoter; The tool virus vector is used to conditionally express the TYR reporter gene in neural stem cells, thereby obtaining a tracer that can combine with a nuclide-labeled probe targeting melanin to mediate image signals only under the condition of synapse formation.

2. The method for constructing a tracer for in vivo visualization of synapse formation according to claim 1, characterized in that: The tool virus vector is one of lentivirus, adenovirus, adeno-associated virus, and retrovirus.

3. The method for constructing a tracer for in vivo visualization of synapse formation according to claim 2, characterized in that: The conditional expression promoter carried by the tool virus vector is hSyn.

4. The method for constructing a tracer for in vivo visualization of synapse formation according to claim 2, characterized in that: The tool virus vector also expresses a puromycin resistance gene for screening successfully transfected cells in vitro.

5. The method for constructing a tracer for in vivo visualization of synapse formation according to claim 1, characterized in that: The gene sequence of the tool virus vector expressing the TYR reporter protein includes the sequence shown in Sequence 1.

6. The method for constructing a tracer for in vivo visualization of synapse formation according to claim 1, characterized in that: The tool virus vector also expresses a fluorescent protein gene, and the fluorescent protein gene is selected from GFP, RFP, BFP, EGFP, mCherry, mStrawberry, mApple, mRuby or EosFP.

7. The method for constructing a tracer for in vivo visualization of synapse formation according to claim 1, characterized in that: The radionuclide labeled probe targeting melanin is a PET probe 18 Fluoro-pyridine amide-benzamide ( 18 F-FPABZA).

8. The tracer obtained by the method according to any one of claims 1 to 7.

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