WO2022148500A2

WO2022148500A2 - Use of polypyrimidine-tract-binding protein in preparation of medication repairing spinal cord injuries

Info

Publication number: WO2022148500A2
Application number: PCT/CN2022/084398
Authority: WO
Inventors: 杨日云; 潘静莹; 包璟崟; 夏盼慧
Original assignee: 南通大学
Priority date: 2021-04-16
Filing date: 2022-03-31
Publication date: 2022-07-14
Also published as: CN113171369A; WO2022148500A3

Abstract

Disclosed is a use of polypyrimidine-tract-binding protein silencer combined with retinoic acid and purmorphamine in the preparation of a medication for repairing spinal cord injuries, being related to the technical field of biomedicine. In the present invention, a virus is used in vitro to silence polypyrimidine-tract-binding protein (PTB), and small molecules retinoic acid (RA) and purmorphamine (PMA) related to motor neuron differentiation are added in combination, thereby successfully reprogramming mouse spinal cord reactive astrocytes into motor neurons, and facilitating the research on the effect of PTB combined small molecule reprogramming strategies in the repair after spinal cord injuries, thus allowing for better spinal cord injury repair and functional reconstruction.

Description

Application of Polypyrimidine Sequence Binding Protein in Preparation of Repairing Drugs for Spinal Cord Injury

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to the application of a polypyrimidine sequence-binding protein silencing agent combined with retinoic acid and purmorphamine in the preparation of a spinal cord injury repair medicine.

Background technique

Spinal cord injury (SCI) is a central nervous system injury disease with a high disability rate and serious consequences, which seriously impairs the patient's motor function and often leads to paralysis below the injury site. Currently recognized SCI treatment has three major difficulties: First, the rapidly activated reactive glial cells, especially astrocytes, will proliferate in large numbers to form dense glial scar tissue, so that the relatively slow-growing axons extend through the injury. At the same time, it encounters an insurmountable mechanical barrier and a chemical barrier formed by the inhibitory factors it secretes; second, some damaged neurons will be lost due to necrosis or apoptosis, and only a small amount of residual neural stem cells cannot adequately replenish the lost. The regenerative capacity of the damaged neurons that have not been lost is also very limited; third, there will be a variety of inhibitory regeneration factors and inflammatory factors at the injury site, forming a chemical microenvironment that is unfavorable for axon regeneration. These unfavorable factors are coordinated with each other and ultimately lead to difficult tissue repair and functional reconstruction in SCI.

When nerve tissue is injured, astrocytes will proliferate reactively and exhibit progenitor cell characteristics, indicating that they have a high degree of plasticity in cell transdifferentiation. If we manage to use reactive astrocytes that proliferate and have negative effects after SCI as target cells to directly induce reprogramming into neurons, or even motor neurons with corresponding functions in situ, we can achieve the goal of properly eliminating glia. The purpose of scarring, which can supplement the neurons lost due to SCI in situ, and at the same time improve the microenvironment of axon regeneration, should be a good method to solve the difficulties affecting repair and regeneration after SCI, and to replace the treatment and personality of SCI cells. The idea of chemical medicine R&D has contributed to a new way of realization.

SUMMARY OF THE INVENTION

PTB is an RNA-binding protein and plays an important role in the induction and differentiation of neurons. Current studies have found that silencing PTB can reprogram astrocytes into functional neurons and promote the corresponding disease model mice. function is restored. Spinal cord injury is a common neurological trauma that is difficult to treat. After spinal cord injury, astrocytes reactively proliferate and form glial scars at the injury site, thereby inhibiting the regeneration of neurons and axons; at the same time, a large number of motor neurons are lost. , the limited regeneration ability of damaged neurons, all of which bring great difficulties to the repair of spinal cord injury.

The purpose of the present invention is to provide the application of a polypyrimidine sequence-binding protein silencing agent combined with retinoic acid and purmorphamine in the preparation of a spinal cord injury repair medicine.

In the present invention, the polypyrimidine sequence-binding protein silencing agent is selected from lentivirus-packaged shRNA-PTB (5'-GGGTGAAGATCCTGTTCAATA-3').

On the other hand, the present invention also provides a method for inducing the differentiation of spinal cord reactive astrocytes into motor neurons in vitro. Amines promote differentiation.

The present invention silences the polypyrimidine sequence binding protein (PTB) by virus in vitro, and at the same time, the small molecule retinoic acid (RA) and purmorphamine (PMA) related to the differentiation of motor neurons are added together to successfully make the spinal cord of mice reactive The reprogramming of astrocytes into motor neurons provides help for further in vivo research on the role of PTB combined with small molecule reprogramming strategies in the repair of spinal cord injury, thereby achieving better repair and functional reconstruction of spinal cord injury.

Description of drawings

Figure 1 shows the establishment of a primary mouse spinal cord reactive astrocyte model. A is the GFAP staining of primary mouse spinal cord astrocytes; the mRNA level (B) and protein level (C) of GFAP in the spinal cord astrocytes of mice treated with 10 μg/mL LPS for 24 h were significantly higher than those in the control group rise. scale bar: 200μm.

Figure 2 shows the efficiency results of silencing PTB in shRNA-PTB lentivirus-infected mouse spinal cord reactive astrocytes. The protein (A) and mRNA (B) levels of PTB were significantly decreased after shRNA-PTB lentivirus infection of cells for 2 days. Fluorescence microscope image of shRNA-PTB lentivirus (C) after 2d infection of cells. scale bar: 200μm.

Figure 3 is a light microscope image of shRNA-PTB lentiviral reprogramming of reactive astrocytes in mouse spinal cord. scalebar: 200μm.

Figure 4 is a fluorescent image of shRNA-PTB lentivirus-induced reprogrammed neurons in mouse spinal cord reactive astrocytes. scale bar: 200μm.

Figure 5 is a fluorescence image of shRNA-PTB lentivirus combined with RA and PMA to reprogram reactive astrocytes into motor neurons in mouse spinal cord. scale bar: 200μm.

Figure 6 shows the transdifferentiation rate of mouse spinal cord reactive astrocytes directly reprogramming motor neurons.

Detailed ways

Polypyrimidine Binding Protein (PTB) is an RNA-binding protein and plays an important role in the induction and differentiation of neurons. Studies have shown that silencing PTB can reprogram midbrain astrocytes into dopaminergic neurons. Cells, while PTB silencing can also directly reprogram mouse retinas to ganglion cells. At present, most of the "subtractive" reprogramming strategies of PTB are located in the brain and retina, and there is no relevant report on its role in SCI repair. At the same time, studies have shown that small molecule retinoic acid (RA) is involved in inducing neural differentiation and the growth of motor neuron axons; Purmorphamine (PMA) is involved in neurogenesis and differentiation by activating the sonic hedgehog signaling pathway, promoting Differentiation of mesenchymal stem cells into motor neurons. Therefore, whether a certain number of motor neurons can be easily reprogrammed by PTB silencing of astrocytes combined with adding RA and PMA in situ after spinal cord injury deserves further study.

The invention silences PTB through virus in vitro, and at the same time, the small molecule retinoic acid and purmorphamine related to the differentiation of motor neurons are added together, and the reactive astrocytes of the mouse spinal cord are successfully reprogrammed into motor neurons. The in vivo study of the reprogramming strategy of PTB combined with small molecules in the repair of spinal cord injury provides help, and then achieves better repair and functional reconstruction of spinal cord injury.

The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but should not be construed as a limitation of the present invention. Modifications or substitutions made to the methods, steps or conditions of the present invention without departing from the spirit and essence of the present invention all belong to the scope of the present invention. In the examples, the experimental methods without specifying the specific conditions and the reagents without specifying the formula are all in accordance with the conventional conditions in the art.

Example 1

1. Primary culture to obtain mouse spinal cord astrocytes with high purity

Spinal cord tissue was obtained from neonatal mice. The spinal cord was placed in a petri dish filled with ice D-Hanks solution and rinsed twice. The meninges and blood vessels on the surface were carefully stripped off with microsurgical forceps. D-Hanks solution was rinsed 2-3 times and transferred to another. In a petri dish, chop the spinal cord to a chyle shape, transfer the chopped spinal cord block in the petri dish into a 15 mL centrifuge tube, then add an equal amount of 0.25% trypsin, digest it in a 37 °C water bath for 15 min, and mix by pipetting every 5 min. Homogenize until there is no obvious tissue block, and then add twice the amount of 10% FBS complete medium to stop the digestion of the enzyme; centrifuge at 1000 rmp for 5 min, then discard the supernatant, collect the cell pellet, and resuspend the cells with 5 mL of basal medium. Repeat the centrifugation twice; resuspend the cells with 10% FBS complete medium, and pipet the cell fluid to distribute evenly, and filter with a 200-mesh sieve to prepare a primary cell suspension.

Inoculate the primary cell suspension into a 25cm2 culture flask, incubate it upside down for 20 min in a 37°C, 5% CO2 incubator, then gently flip the culture flask, then take out the cell suspension in the flask, after removing the fibroblast components ; After centrifuging the cell suspension at 1000 rmp for 5 min, add 5 mL of complete medium containing 10% FBS, gently pipetting evenly, and count with a cell counter; then dilute it with complete medium containing 10% FBS to 5 × 105/mL, take 15 mL Inoculated in 75cm2 culture flasks. Then the medium was changed in full after every 2 days to remove the dead cell debris that did not adhere to the wall, so that the glial cells could fully grow. Observe under the microscope during each medium change. After about 1 week, the cells covered the bottom of the bottle, and then further purified and cultured . When the glial cells are cultured for 7 to 9 days, after the cells are covered with the bottom of the culture flask, place them on a constant temperature shaking culture bed at 37°C, with a rotation speed of 280 rmp/min, for 16 to 18 hours (the constant temperature culture bed is UV sterilized in advance). After shaking, take out the culture flask and sterilize it with 75% alcohol; after removing the cell suspension (mainly oligodendrocytes and microglia) in the culture flask, rinse twice with fresh medium, The remaining underlying cells are mainly astrocytes. Add 1 mL of 0.25% trypsin for digestion, gently shake the culture flask back and forth, left and right, observe the cell body shrinking and rounding under the microscope, immediately stop the digestion with 10% FBS complete medium, and then gently scrape the cells with a cell scraper. Then, use a sterile pipette to collect the suspended cells in a 15 mL centrifuge tube, centrifuge at 1000 rmp/min for 5 min, repeat twice, and finally inoculate the cells in a 75 cm2 culture flask for culture.

In order to ensure the viability and purity of cells, 2-3 passages of spinal cord astrocytes are generally selected for subsequent experimental studies.

2. Establishment of a primary mouse spinal cord reactive astrocyte model

Spinal cord astrocytes of passage 2 to 3 were selected, and the cells were stimulated with 10 μg/mL lipopolysaccharide (LPS) for 24 hours. The astrocyte-specific gene GFAP after LPS stimulation was detected by real-time RT-PCR and Western blot. Changes in the expression of , activate cells into reactive astrocytes.

As shown in Figure 1, the mRNA level (B) and protein level (C) of GFAP in the spinal cord astrocytes of mice treated with 10 μg/mL LPS for 24 h were significantly higher than those in the control group (blank), indicating that the construct was successfully constructed. Primary mouse spinal cord reactive astrocyte model.

3. shRNA-PTB silences the PTB of reactive astrocytes in the spinal cord of mice

The lentivirus-packaged shRNA-PTB (sequence 5'-GGGTGAAGATCCTGTTCAATA-3'SEQ ID NO.1) produced by Shanghai Heyuan Biotechnology Co., Ltd. was infected with reactive astrocytes 2d, and real-time RT-PCR was used The mRNA and protein expression changes of PTB were detected by techniques such as Western blot to determine the silencing efficiency of shRNA-PTB.

As shown in Figure 2, the protein (A) and mRNA (B) levels of PTB were significantly decreased after shRNA-PTB lentivirus infected cells for 2 days, indicating that shRNA-PTB lentivirus could reduce the reactive astrocytes in mouse spinal cord in vitro PTB expression, and the silencing efficiency is about 50%

4. shRNA-PTB lentivirus reprograms mouse spinal cord reactive astrocytes

The packaged shRNA-PTB lentivirus was used to infect reactive astrocytes, and after 2 days, the infection medium was replaced with induction medium (N3/basal medium: Insulin, sodium selenite, Retinoic acid, putrescine, ChIR99021, SB431542, Db-cAMP, FGF-basic, GDNF), half-change medium for induction medium every 2 d, induction culture for 7 d, 14 d, 16 d, 21 d, 28 d; immunocytochemical technique was used to detect MAP2 pan-neuronal markers and ChAT movement Neuronal marker expression.

As shown in Figure 3, in the control group, the cell bodies of the shCtrl group were flat, and the shape was the same as that of astrocytes; while the cell bodies of the shPTB group gradually took on a spherical or pyramidal shape, with different numbers and lengths protruding from the cell bodies. protrusions, showing a neuron-like morphology.

As shown in Figure 4, cells reprogrammed with shRNA-PTB for 16d were GFP/MAP2 double positive, while the shCtrl group was only GFP positive.

It can be seen that shRNA-PTB lentivirus can gradually change the morphology of reactive astrocytes in mouse spinal cord to neuron-like, and directly reprogram them into mature neurons of MAP2+ and motor neurons of ChAT+.

5. shRNA-PTB lentivirus combined with RA and PMA to reprogram mouse spinal cord reactive astrocytes

The packaged shRNA-PTB lentivirus was used to infect reactive astrocytes, and after 2 days, the infection medium was replaced with induction medium (N3/basal medium + 1 μM RA + 0.5 μM PMA, that is, N3/basal per 1000 mL). 1 micromolar RA and 0.5 micromolar PMA were added to the medium, and the medium was half-changed every 2 d, and the culture medium was induced for 7d, 14d, 16d, 21d, and 28d; immunocytochemical techniques were used to detect MAP2 pan-neural The expression of meta-markers and ChAT motor neuron markers, and the transdifferentiation rate of motor neurons was calculated.

As shown in Figure 5, cells in the shRNA-PTB+RA+PMA group and shRNA-PTB group reprogrammed for 28d were ChAT positive, while the shCtrl group was only GFP positive.

As shown in Figure 6, after 28 days of reprogramming, the ratio of directly reprogramming spinal cord reactive astrocytes to ChAT-positive cells in the shRNA-PTB+RA+PMA group was significantly higher than that in the shRNA-PTB group.

Based on the current situation of spinal cord injury repair and the progress of inducing in situ reprogramming, the invention achieves silencing of PTB in vitro, and simultaneously adds small molecules RA and PMA, and finally successfully reprograms the reactive astrocytes of the spinal cord into motor neurons; It provides a theoretical basis for the in vivo application of the reprogramming strategy of PTB combined with small molecules, and strives to achieve a better effect of spinal cord injury repair and functional reconstruction.

The present invention combines the two issues of suppressing hyperproliferative reactive astrocytes and replenishing lost motor neurons, and designs a method that can not only replenish the number of motor neurons lost due to spinal cord injury in situ, but also Simultaneously reducing the number of reactive astrocytes activated and proliferating due to spinal cord injury to alleviate the rapid proliferation of glial scars and improve the chemical microenvironment for axon regeneration, ultimately promoting functional recovery after spinal cord injury. .

Claims

Application of polypyrimidine sequence-binding protein silencing agent combined with retinoic acid and purmorphamine in the preparation of spinal cord injury repair drugs.
The application according to claim 1, wherein the polypyrimidine sequence-binding protein silencing agent is a lentivirus-packaged shRNA.
The application according to claim 1, wherein the concentration of the retinoic acid is 1 μM, and the concentration of purmorphamine is 0.5 μM.