CN113116901A

CN113116901A - Application of LDN193189 and CHIR99021 in preparing medicine for inducing neuron regeneration

Info

Publication number: CN113116901A
Application number: CN202110276561.4A
Authority: CN
Inventors: 何成; 苏志达; 谭子健; 袁一旻; 秦尚尧
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 2021-03-15
Filing date: 2021-03-15
Publication date: 2021-07-16

Abstract

The invention relates to the technical field of medicines, and provides application of LDN193189 and CHIR99021 in preparation of a medicine for inducing neuron regeneration. Through in vitro experiments, LDN193189 and CHIR99021 can induce differentiation in vitro for 12 days to reprogram mouse cortical astrocytes into neurons; through in vivo experiments, the LDN193189 and the CHIR99021 can induce astrocytes to differentiate into new neurons in vivo and simultaneously induce the new neurons to differentiate, mature and survive for a long time in vivo. This result was also achieved in aged mice, although not as well as in young mice. Therefore, the invention provides a new basis for treating central nervous system injury by LDN193189 and CHIR 99021.

Description

Application of LDN193189 and CHIR99021 in preparing medicine for inducing neuron regeneration

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to application of LDN193189 and CHIR99021 in preparation of a regeneration repair medicine after central nervous system injury, in particular to application in preparation of a medicine for inducing reprogramming of astrocytes into neurons.

Background

Regenerative repair after injury to the Central Nervous System (CNS) has been a clinically problematic issue. Various acute and chronic central nervous system injuries or lesions are often accompanied with irreversible neuron death and loss, which are one of the main pathological reasons of poor prognosis and poor life quality of CNS injury patients. Therefore, the main goal of regeneration and repair after CNS injury is to reverse and compensate for neuronal loss, thereby promoting recovery and compensation of related nerve functions.

Classical cell replacement therapy is generally developed based on stem cell transplantation, but has not been the mainstream treatment for CNS injury due to its various disadvantages, such as potential tumorigenicity, unclear differentiation state of progeny cells, etc. Therefore, there is a need to find other effective treatment strategies.

Small Molecule Compounds (Small Molecule Compounds) refer to Compounds with molecular weights of less than 1000 Dalton units, and are widely used in the field of reprogramming. Especially, with the completion of small molecule compound libraries and the improvement of detection methods in recent years, it gradually becomes one of the standard methods for neuron reprogramming.

LDN193189, Chinese name 4- [6- (4- (piperazin-1-yl) phenyl]Pyrazolo [1,5-A]Pyrimidin-3-yl) quinolines of formula C₂₅H₂₂N₆CAS registry number 1062368-24-4, having the chemical structure shown in formula I below:

LDN193189 is a very strong small molecule inhibitor of Bone Morphogenetic Protein (BMP) type I receptors ALK2 and ALK3, and a signal path of the LDN193189 is a key regulation channel for determining cell fate in embryogenesis and tissue homeostasis, and can promote differentiation of neural progenitor cells and human pluripotent stem cells.

CHIR99021, Chinese name 6- [2- [4- (2, 4-dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) pyrimidin-2-ylamino]Ethylamino, formula C₂₂H₁₈ Cl₂N₂CAS registry number 252917-06-9, having the chemical structure shown in formula II below:

CHIR-99021(CT99021) is a potent selective GSK-3 α/β inhibitor, IC₅₀10nM and 6.7nM, which is more than 500 times more selective for GSK-3 than CDC2, ERK2 and other protein kinases. Furthermore, CHIR-99021 is a potent Wnt/beta-catenin signaling pathway activator, and can enhance the self-renewal of mouse and human embryonic stem cells and induce autophagy (autophagy).

There is no literature report on the use of the two small molecule compounds to achieve in vivo neuronal reprogramming.

Disclosure of Invention

The present invention has been made to solve the above problems, and an object of the present invention is to provide a novel medical use of the LDN193189 and CHIR99021 composition, a pharmaceutical composition containing the same, and a method for inducing neuronal regeneration in vitro.

In a first aspect of the invention, an application of LDN193189 and CHIR99021 in preparation of a regeneration repair drug for inducing neuron regeneration after central nervous system injury is provided, the regeneration repair drug is a drug for inducing neuron regeneration, and further a drug for inducing astrocytes to be reprogrammed into neurons, and the LDN193189 and CHIR99021 induce astrocytes to transdifferentiate into neurons by significantly up-regulating expression of Ngn2, NeuroD1 and Myt1l and slightly up-regulating expression of Brn2, Foxg1, NeuroD2 and Lmx1 a.

Experiments prove that the LDN193189 and the CHIR99021 can not only reprogram the mouse cortex astrocytes into neurons through inducing differentiation for 12 days in vitro, but also induce the astrocytes to differentiate into new neurons in vivo, and simultaneously induce the new neurons to differentiate, mature and survive for a long time in vivo. This result was also achieved in aged mice, although not as well as in young mice.

Therefore, in vitro and in vivo experiments show that LDN193189 and CHIR99021 are possible to become new drugs for regeneration and repair after clinical treatment of central nervous system injury.

In addition, the regenerative repair drug of the present invention is a drug containing LDN193189 and CHIR99021 as the only active ingredients or a drug composition containing LDN193189 and CHIR 99021.

In a second aspect of the invention, there is provided a method of inducing reprogramming of astrocytes into neurons in vitro, comprising the steps of:

A. astrocyte treatment

Washing the astrocytes collected at the early stage with DMEM culture solution for 1 time, centrifuging at 800rpm for 4min at room temperature, then resuspending with DMEM culture solution containing 10% FBS, and adjusting cell density to 4 × 10⁴cells/ml; after the cell suspension is pipetted and dropped on a rigid carrier (e.g., a slide glass) and incubated for 4 hours in a cell culture chamber, 10% FBS DMEM culture solution is added to each well of a multi-well culture plate (e.g., a 24-well culture plate) and placed in the cell culture chamber for use.

B. Induced differentiation

Dissolving LDN193189 and CHIR99021 by using DMSO, adding the dissolved solutions into a DMEM culture solution containing 10% FBS after the dissolved solutions are completely dissolved, enabling the final concentrations to be 0.1-1 mu M (preferably 0.5 mu M) and 1.5-20 mu M (preferably 10 mu M), and replacing the previously-cultured astrocyte culture solution which is completely adherent with the culture solution;

on the 3 rd day of induction, changing to a neuron induction culture solution, wherein the solution contains 90% of DMEM/F12 by volume fraction, 1% of double antibody by volume fraction, 0.1-1 mu M LDN193189 and 1.5-20 mu M CHIR99021 by volume fraction, and changing the solution once every two days;

on the 7 th day of induction, the culture medium is replaced by a neuron maturation induction culture medium, wherein the culture medium contains DMEM/F12 with the volume fraction of 78.6%, N2 with the volume fraction of 20%, B27 with the volume fraction of 0.4%, GDNF, BDNF, penicillin/streptomycin with the volume fraction of 1%, 0.1-1 mu M LDN193189 and 1.5-20 mu M CHIR99021, the culture medium is replaced by a half of the culture medium on the 10 th day of induction, and the induced differentiation is completed on the 12 th day of induction.

Further, the method also comprises the steps of cell culture and purification:

cutting the pia mater to less than 1mm using a sterile scalpel³Adding a proper amount of pancreatin, digesting for 15 minutes in a water bath kettle at 37 ℃, and then adding a DMEM culture solution containing 10% fetal calf serum to terminate the digestion process; transferring the tissue digestive juice into a centrifuge tube, centrifuging at the room temperature of 800rpm for 5 minutes, removing a supernatant, adding a fresh culture solution, repeatedly blowing and resuspending, standing for 10 minutes, sucking out the upper monolayer liquid, and adding the upper monolayer liquid into a culture bottle;

replacing fresh culture solution on day 2, replacing culture solution every 3 days, shaking the culture bottle manually before replacing the culture solution to remove the hybrid cells, wherein the visible cell fusion degree is 90% after about 7 days of primary culture, and then carrying out passage; culturing by the above method after passage, culturing until the cell reaches 3 rd generation, and performing subsequent experiment in DMEM culture solution containing 10% fetal calf serum at 37 deg.C and 5% CO₂Culturing under the condition, after the cells are paved on 90-95% of the bottom of the culture dish, digesting with 0.25% pancreatin, and collecting the cells for later use.

Further, before astrocyte treatment, the slide was treated: soaking the glass slide in PDL solution overnight, taking out, placing in a 24-well plate, blow-drying the surface of the glass slide in a sterile operating platform, adding 100 mu L matrigel, incubating in a cell culture box for 2 hours, absorbing liquid, blow-drying, and irradiating and digesting in the operating platform for more than 1 hour by using an ultraviolet lamp for later use.

The neuron prepared by the method can be used as an active component of a regenerative repair medicament to compensate for dead or missing neurons in the CNS injury process.

Thus, in a third aspect of the invention, there is provided the use of neurons obtained according to the above method for the manufacture of a medicament for the regenerative repair of damaged central nervous system.

The fourth aspect of the invention provides a pharmaceutical composition for regeneration and repair after central nervous system injury, which comprises active ingredients and pharmaceutically acceptable auxiliary materials, wherein the active ingredients comprise LDN193189 and CHIR99021 or neurons obtained according to the in vitro induction method.

The pharmaceutical composition for regeneration and repair after central nervous system injury is decoction, injection, tablet or capsule.

Action and Effect of the invention

Through in vitro experiments, LDN193189 and CHIR99021 can induce differentiation in vitro for 12 days to reprogram mouse cortical astrocytes into neurons; through in vivo experiments, the LDN193189 and the CHIR99021 can induce astrocytes to differentiate into new neurons in vivo and simultaneously induce the new neurons to differentiate, mature and survive for a long time in vivo. This result was also achieved in aged mice, although not as well as in young mice. Therefore, the invention provides a new basis for treating central nervous system injury by LDN193189 and CHIR 99021.

In addition, as natural small molecular compounds, LDN193189 and CHIR99021 are easy to permeate blood brain barrier to play the central drug effect, and the two substances can be obtained through a commercial way, so that the preparation cost of the disease treatment drug is reduced, and the medical expense of patients is reduced to a certain extent.

Drawings

FIG. 1 shows that LDN193189, CHIR99021 can reprogram mouse cortical astrocytes into neurons;

FIG. 2 shows the specific course of the change in the transdifferentiation of astrocytes into neurons;

FIG. 3 shows that LDN193189, CHIR99021 induce astrocyte transdifferentiation by up-regulating key neuron-specific transcription factors;

FIG. 4 shows that LDN193189, CHIR99021 can induce new neurons in mice;

FIG. 5 shows that LDN193189 and CHIR99021 can induce differentiation and maturation and long-term survival of neonatal neurons under in vivo conditions;

FIG. 6 shows that LDN193189, CHIR99021 induce long-term survival of neurons in vivo;

FIG. 7 shows confirmation of in vivo induction of neogenic neurons from astrocytes by LDN193189, CHIR99021 by cell-specific marker co-localization;

FIG. 8 shows that the in vivo induction of nascent neurons by LDN193189, CHIR99021 was confirmed to be derived from astrocytes by a lineage tracing method;

FIG. 9 shows that LDN193189 and CHIR99021 can induce new neurons in older mice.

Detailed Description

The present invention will be described in detail below with reference to examples and the accompanying drawings. The following examples should not be construed as limiting the scope of the invention.

The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Example 1: LDN193189, CHIR99021 can reprogram mouse cortical astrocytes to neuron one, experimental:

1. cells for experiments: neonatal mouse cortical astrocytes, derived from primary culture.

2. Experimental reagents, consumables and instruments: DMEM medium (invirigen), fetal bovine serum (Gibco), dpbs (thermo Scientific hyclone), EDTA-trypsin (Gibco); 10cm petri dishes (Corning), T75 flasks (Corning), centrifuge tubes (AXYGEN); a cell culture box (Thermo), a water bath (Shanghai Biotechnology), a low-speed centrifuge (Thermo) and a surgical instrument (astronomical instrument).

3. Experiment medication:

the medicine of the invention is: LDN193189, CHIR99021

Negative control drugs: DMSO (sigma)

4. Cell culture and purification:

killing a newborn mouse by a carbon dioxide asphyxiation method, placing the mouse in 75% alcohol for disinfection for more than 5 minutes, using an operation to cut off the head of the mouse, and placing the mouse on an ice board for later use; peeling skin and skull of a mouse by using iris scissors, taking out a brain, soaking the brain in precooled DPBS liquid, separating a cerebral cortex under a microscope and peeling a pia mater; cutting the cortex to less than 1mm using a sterile scalpel³After adding a proper amount of pancreatinThe digestion was carried out in a 37 ℃ water bath for 15 minutes, and then a DMEM medium containing 10% fetal bovine serum was added to terminate the digestion process.

Transferring the tissue digestive juice into a centrifuge tube, centrifuging at the room temperature of 800rpm for 5 minutes, removing a supernatant, adding a fresh culture solution, repeatedly blowing and resuspending, standing for 10 minutes, sucking out the upper monolayer liquid, and adding the upper monolayer liquid into a T75 culture bottle; after changing fresh culture solution on day 2, the culture solution was changed every 3 days, and the culture flask was shaken manually before the solution change to remove the foreign cells.

After primary culture, the visible cell fusion degree is about 90% after 7 days, passage can be carried out, the cells are cultured according to the method after passage, and the subsequent experiment can be carried out after the cells are cultured to 3 rd generation. In DMEM medium containing 10% fetal bovine serum, 5% CO at 37 ℃₂Culturing under the condition, after the cells are paved on 90-95% of the bottom of the culture dish, digesting with 0.25% pancreatin, and collecting the cells for later use.

5. Planting and inducing astrocytes:

soaking the glass slide in a PDL solution overnight, taking out, placing in a 24-well plate, blow-drying the surface of the glass slide in a sterile operating platform, adding 100uL matrigel, incubating in a cell culture box for 2 hours, absorbing liquid, blow-drying, and irradiating and digesting in the operating platform for more than 1 hour by using an ultraviolet lamp for later use.

Washing the astrocytes collected in the early stage with 10ml of DMEM medium for 1 time, centrifuging at 800rpm for 4min at room temperature, then resuspending the cells in 10% FBS-containing DMEM medium, and adjusting the cell density to 4X 10⁴cells/ml. After 100uL of the cell suspension was dropped into the center of the slide and incubated in the cell incubator for 4 hours, 500uL of 10% FBS in DMEM was added to each well, and the 24-well plate was placed in the cell incubator for use.

The LDN193189 and CHIR99021 were dissolved in DMSO, and after completely dissolved, they were added to a 10% FBS-containing DMEM medium to give final concentrations of 0.5uM and 10uM, respectively. The previously cultured astrocyte culture solution which has been completely adherent was replaced with this culture solution.

On the 3 rd day of induction, the medium was changed to neuronal induction medium containing DMEM/F12 (90% v/v), diabody (1% v/v), LDN193189(0.5uM), CHIR99021(10uM) and changed every two days.

On day 7 of induction, the medium was changed to a neuronal maturation induction medium consisting of DMEM/F12 (78.6% v/v), N2 (20% v/v), B27 (0.4% v/v), GDNF (20ng/ml), BDNF (10ng/ml), penicillin/streptomycin (1% v/v), LDN193189(0.5uM) and CHIR99021(10uM), and half the medium was changed on day 3.

The control group used DMSO as an inducing compound throughout, at a concentration consistent with the experimental group.

6. Identification of neonatal neurons

After discarding the original cell culture from the well plate, the plate was washed 3 times for 5 minutes each with 1 x phosphate buffer, and the slides were subsequently removed and placed on parafilm-coated slides. The slides were fixed by adding the appropriate amount of fixative at room temperature for 15 minutes, followed by repeated phosphate buffer washes. After the washing, triton-phosphate buffer (TritonX-100PBS) was added to the mixture and the mixture was subjected to washing with PBS after 15 minutes at room temperature.

Antigen blocking was performed for 1 hour at room temperature using antibody dilutions; after blocking, the antibody dilutions were aspirated and washed again with phosphate buffer, followed by addition of primary antibody as needed. And (3) placing the wet box with the glass slide in a refrigerator, performing low-temperature dressing overnight, removing the primary antibody solution in the next day, washing 3 times by using a phosphate buffer solution for 5 minutes each time, then adding the corresponding secondary antibody and Hochests, dyeing for 2 hours in a dark place at room temperature, and washing the mounting.

7. Specific change of new neuron process

To clarify the specific changes in the process of transdifferentiation of astrocytes into neurons, cells at different time points during induction were analyzed using TUBB3 and GFAP, and fixed and stained on

days

5, 7, 9, and 12, respectively, from the start of induction.

Second, experimental results

On the 7 th day of induction (fig. 1A), it can be seen that some dcx (deubelocin) -positive cells appeared, and the cell morphology was characterized by the immature neurons, i.e. cell body shrinkage, nucleus staining enhancement, multilobe generation, etc., while the control group cells did not; when the observation period was extended to day 12, some TUBB3 positive cells were visible in the LC group under the field of view (fig. 1-a).

At day 7 post-induction, cells co-expressing TUBB3, DCX appeared in the field, indicating that they are in the process of maturing (fig. 1C); on day 15 post-induction, they were immunohistochemically stained with markers specific for three mature neurons, TUBB3, MAP2, and NeuN, respectively, at which time point a large number of cells co-labeled with NeuN/TUBB3, MAP2/TUBB3 appeared, all characterized by soma, nuclear shrinkage, multiple cell processes, etc. (fig. 1-D and E); at day 28 post-induction, NeuN/TUBB3 double positive neurons induced by LDN193189 and CHIR99021 appeared morphologically more mature in cells and neuronal processes became longer and more complex (fig. 1F). None of the control groups showed this at the time points described above (FIG. 1B).

To clarify the specific changes in the process of transdifferentiation of astrocytes into neurons, we stained and analyzed cells at different time points during induction using TUBB3 and GFAP, see fig. 2A and B: at induction day 5, some TUBB3 positive cells were seen in the experimental cells; notably, most of these cells co-labeled with GFAP, indicating that these cells are still in the early stage of transdifferentiation and that their expression of astrocyte-associated genes has not been completely inhibited; moreover, the positive signal of TUBB3 was only expressed in the soma part, and the lack of processes also confirmed that these neurons were not mature (a 1).

Similar observations were made at

days

7, 9 of induction, but with increasing absolute numbers of TUBB3 positive cells, the proportion of TUBB3 and GFAP double positive cells in TUBB3 positive cells decreased significantly. In the GFAP and TUBB3 double positive cells, the cytoskeleton shown by GFAP is obviously reduced and does not show the typical hypertrophic form of astrocytes. In addition, TUBB3 positive cells gradually approached normal neurons in morphology, appearing with reduced nuclei and increased staining, and appearing as complex processes. These phenomena indicate that, as the induction time progresses, astrocytes gradually down-regulate GFAP expression, and turn on TUBB3 expression, thereby transdifferentiating toward neurons (A2-A5). At induction day 12, the vast majority of TUBB3 positive cells were GFAP negative, suggesting that the transdifferentiation process was substantially complete (a 6).

Example 2: LDN193189 and CHIR99021 induce astrocyte transdifferentiation by up-regulating key neuron-specific transcription factors

To further understand the molecular mechanism of the transdifferentiation of astrocytes during reprogramming, we first used real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) to detect changes in various neuronal-related transcription factors in cells, and then verified them using immunofluorescent staining methods. In addition, the gene expression levels of stem/precursor cell-associated markers and neuronal markers were also identified.

First, experiment method

1. Cells for experiments: the same as in example 1.

2. Experimental reagents, consumables and instruments: DMEM medium (Invitrigen), fetal bovine serum (Gibco), EDTA-trypsin (Gibco), Trizol (Invitrogen), Rapid plastication kit (Thermo), SYBR (TOYOBO). 10cm petri dishes (Corning), T75 flasks (Corning), 6-well plates (Thermo), and centrifuge tubes (AXYGEN). Cell culture incubator (Thermo), water bath (Shanghai Biotech), centrifuge (Thermo), fluorescent quantitative PCR instrument (Roche).

3. Experiment medication:

the medicine of the invention is: LDN193189(MCE), CHIR99021(MCE)

Negative control drugs: DMSO (sigma)

4. Cell culture: the specific cell culture method was the same as in example 1.

5. Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR): on

days

2,4, 6, 8, 10 and 12 of induction initiation, cellular RNA was collected using Trizol and reverse transcribed as described. The primer sequences are shown in table 1:

TABLE 1 summary of primer sequences

6. Immunofluorescence assay:

after discarding the original cell culture from the well plate, the plate was washed 3 times for 5 minutes each with 1 x phosphate buffer, and the slides were subsequently removed and placed on parafilm-coated slides. The slides were fixed by adding the appropriate amount of fixative at room temperature for 15 minutes, followed by repeated phosphate buffer washes. After the washing, triton-phosphate buffer (TritonX-100PBS) was added to the mixture and the mixture was subjected to washing with PBS after 15 minutes at room temperature. Antigen blocking was performed for 1 hour at room temperature using antibody dilutions. After blocking, the antibody dilutions were aspirated and washed again with phosphate buffer, followed by addition of primary antibody as needed. The wet box with the slide inside was placed in a refrigerator and incubated at low temperature overnight. The following day, the primary antibody was aspirated, washed 3 times for 5 minutes each with phosphate buffer, followed by addition of the corresponding secondary antibody, Hochest, and washing the mounting after 2 hours of light-shielding staining at room temperature.

II, experimental results:

on the 2 nd day of induction, the three transcription factors of Ngn2, neuroD1 and Myt1l are observed to be significantly highly expressed, the expression level is increased by about 1000 times compared with that of a control group, the expression level is still maintained to be higher along with the progress of the induction process, and only neuroD1 slightly falls in the late induction period, but is still obviously higher than that of the control group; other neuron-related transcription factors, such as Brn2, Foxg1, NeuroD2 and Lmx1a, were also slightly up-regulated in their expression levels (fig. 3A).

This result was verified by immunohistochemical staining: under normal conditions, astrocytes do not express Ascl1 and Ngn 2. At day 5 post-induction, induced cells were stained with Ascl1 and NGN2, respectively, with essentially no Ascl1 positive signal appearing in the field, while a large number of cells expressed NGN2 (fig. 3B).

Meanwhile, the expression condition change of the stem/precursor cell related marker is also observed. It is noted that there was a significant upregulation of Nestin expression during induction, at levels approximately 50-fold higher than the control, and maintained high throughout induction (FIG. 3C). While other stem/precursor cell markers, such as Sox2, Pax6, and BLBP, were expressed at levels that were not significantly different from the control.

In addition, we also used quantitative RT-PCR to detect the gene expression changes of neuron markers during the process of induced transdifferentiation. Consistent with the results of the previous immunohistochemistry, high expression of DCX was observed at day 2 after induction, which was about 1000 times as high as that of the control group and continued. Whereas the expression of mature neuronal markers such as TUBB3, NeuN, MAP2 and synapase (syn) did not change significantly before D6 compared to the control group, with the expression gradually increasing over the following time (fig. 3D).

Example 3: LDN193189 and CHIR99021 can induce new neurons in mice

To determine whether LDN193189, CHIR99021 could induce neuronal reprogramming when applied in vivo, this combination of compounds was studied in the spinal cord of mice.

First, experiment method

1. Experimental mice: C57B6/L wild-type mice, purchased from Shanghai Ling Biotech, Inc.

2. Experimental reagents, consumables and instruments: mouse surgical instruments (astronomical instruments) and mouse anaesthesia machines.

3. Experiment medication:

the medicine of the invention is: LDN193189, CHIR99021

Negative control drugs: dmso (sigma).

4. Constructing a mouse spinal cord injury model:

the preparation method of the mouse spinal cord injury model refers to the previous work of the room and is slightly improved. The method mainly comprises the following steps:

weighing female adult C57BL/6 mice, and performing subsequent experiments if the weight is more than 22g for anesthesia; fixing the mouse on a constant-temperature operating table in a prone position after anesthesia is successful, preparing skin, cutting back skin after disinfection, and positioning the T8-T10 segment according to normal protrusion of the back of the mouse; the spinal muscles on both sides were cut off after cutting back fascia to separate the spine, and the vertebral plates were cut off from the back to the front using iris scissors to expose the spinal cord.

Hemostasis and flushing the field of vision with pre-cooled sterile normal saline; after the front segment vertebra is lifted by using the toothless forceps, the T8 segment spinal cord is clamped for 15 seconds by using the special microscopic forceps (the distance between the two forceps tips is 0.45mm after the forceps are closed); stopping bleeding after clamping, suturing muscles and fascia on two sides layer by layer when no obvious bleeding point exists in visual field, and suturing skin. After the operation is finished, the analgesic and the antibiotic are injected into the fat tissue at the back of the neck of the mouse, and the mouse is placed into an animal incubator. And returning the animal to the animal house after the animal is recovered. Animals were observed daily after surgery, and the abdomen was squeezed twice daily, and the bladder was massaged to aid in urination until bladder function recovered.

5. Mouse spinal cord injury model:

administration mode of mice: LDN193189 and CHIR99021 are dissolved in DMSO, and after all the components are dissolved, the components are added into sterile physiological saline containing 0.5 percent Tween-80, and the components are respectively given to the abdominal cavity for injection according to the dosage of 10mg/kg and 20mg/kg once a day.

Control mice used DMSO as a control drug throughout.

6. Collecting, fixing, dehydrating and slicing spinal cord tissues of mice:

after reaching the observation period, anesthetizing the mouse, fixing the mouse on an operating table in a supine position, cutting the chest and exposing the heart; cutting off the right auricle, inserting the vein indwelling needle into the left ventricle, after blood is seen in the needle tube, performing perfusion flushing by using pre-cooled normal saline, and after the liquid flowing out of the right auricle is basically colorless, performing perfusion by using pre-cooled stationary liquid until the muscle of the mouse is rigid, and completing the pre-fixation. Then, cutting off tissues, removing muscles, bones and the like by using micro scissors and tweezers, and putting the spinal cord into a fixing solution in a 4-degree refrigerator for 8-10 hours for post-fixation; after the tissue fixation is finished, the tissue is put into a 20% sucrose solution for dehydration and stored in a 4-degree refrigerator. After dehydration was complete, serial sections were taken at 17um thickness on a Leica cryomicrotome. Immunofluorescent staining experiments were subsequently performed.

Second, experimental results

The experimental results showed that in the wild mouse, the continuous administration of LDN193189 and CHIR99021 did not induce DCX positive cells (FIG. 4A); on the other hand, in the spinal cord of the mouse with spinal cord injury, on the 7 th day after the injury, GFAP and DCX co-staining is carried out on the spinal cord tissue at the injury position, and DCX positive cells appear near the spinal cord injury position after administration (FIGS. 4B-C); the presence of DCX-positive cells was seen in the spinal cord of mice on both day 5 and day 14 post-injury (fig. 4D-E), and the number of DCX-positive cells peaked at day 7 post-injury and fell back slightly at day 14 post-injury (fig. 4F). However, in the control group, no DCX-positive cells appeared on

days

5, 7 and 14 after the injury.

Example 4: LDN193189 and CHIR99021 induce differentiation and maturation and long-term survival of newborn neurons in vivo

To determine whether the de novo neurons induced in vivo by LDN193189, CHIR99021 could differentiate into maturation, they were analyzed using the maturation neuron specific markers Tuj1, MAP2 and NeuN. Considering the complex conditions in spinal cord tissue compared to in vitro experiments, such as many cell types and many staggered cell processes. Therefore, we injected mice with BrdU for the labeling of nascent neurons, and only cells that were simultaneously labeled with TUBB3, NeuN, and BrdU were considered nascent mature neurons.

The first experiment method comprises the following steps:

to determine whether LDN193189, CHIR99021 could induce neuronal reprogramming when applied in vivo, we studied this combination of compounds in the spinal cord of mice.

1. Experimental mice: C57B6/L wild type mice.

2. Experimental reagents, consumables and instruments: mouse surgical instruments, mouse anesthesia machine.

3. Experiment medication:

the medicine of the invention is: LDN193189, CHIR99021

Negative control drugs: dmso (sigma).

4. Constructing a mouse spinal cord injury model:

female adult C57BL/6 mice were weighed and if the body weight was greater than 22g, the subsequent experiments were performed. Anaesthetizing, fixing the mouse on a constant-temperature operating table in a prone position after anaesthetizing successfully, preparing skin, cutting back skin after disinfection, and positioning the T8-T10 segment according to the normal protrusion of the back of the mouse. The dorsal fascia is cut open and the bilateral dorsal muscles are cut open to distract the spine. The vertebral plates were excised from posterior to anterior using iris scissors, exposing the spinal cord. Hemostasis and flushing of the field of view were performed using pre-cooled sterile saline. After the anterior segment vertebra was lifted with toothless forceps, the T8 segment spinal cord was clamped with special micro forceps (distance between the two forceps tips was 0.45mm after closing) for 15 seconds. Stopping bleeding after clamping, suturing muscles and fascia on two sides layer by layer when no obvious bleeding point exists in visual field, and suturing skin. After the operation is finished, the analgesic and the antibiotic are injected into the fat tissue at the back of the neck of the mouse, and the mouse is placed into an animal incubator. And returning the animal to the animal house after the animal is recovered. Animals were observed daily after surgery, and the abdomen was squeezed twice daily, and the bladder was massaged to aid in urination until bladder function recovered.

5. Administration mode of mice: LDN193189 and CHIR99021 are dissolved in DMSO, and after all the components are dissolved, the components are added into sterile physiological saline containing 0.5 percent Tween-80, and the components are respectively given to the abdominal cavity for injection according to the dosage of 10mg/kg and 20mg/kg once a day. BrdU was dissolved in sterile saline with heating and given to intraperitoneal injections twice daily at a dose of 10ug/kg to label novacells.

Control mice used DMSO as a control drug throughout.

6. Collecting, fixing, dehydrating and slicing spinal cord tissues of mice: when the observation period is reached, after anesthetizing the mice, they are fixed in their supine position on the operating table, the chest is cut open and the heart is exposed. Cutting off the right auricle, inserting the vein indwelling needle into the left ventricle, after blood is seen in the needle tube, performing perfusion flushing by using pre-cooled normal saline, and after the liquid flowing out of the right auricle is basically colorless, performing perfusion by using pre-cooled stationary liquid until the muscle of the mouse is rigid, and completing the pre-fixation. Subsequently, tissues were cut off, and muscles, bones, etc. were removed using microscissors, tweezers, and the spinal cords were put into a fixation solution in a refrigerator at 4 ℃ for 8-10 hours of post-fixation. After the tissue fixation was completed, it was dehydrated in a 20% sucrose solution and stored in a refrigerator at 4 ℃. After dehydration was complete, serial sections were taken at 17 μm thickness in a Leica cryomicrotome. Immunofluorescent staining experiments were subsequently performed.

II, experimental results:

after 4 weeks of injury/induction, positive cells co-labeled with TUBB3, NeuN, and BrdU were visible in the spinal cord of experimental mice, indicating that they are neonatal, mature neurons (fig. 5A); whereas no positive cells co-labeled with TUBB3, NeuN, and BrdU were found in the spinal cord of control mice (fig. 5C). After 8 weeks of injury/induction, a large number of nascent mature neurons were still visible in the spinal cord of the experimental mice (fig. 5B). By statistical analysis, we found that the number of neonatal mature neurons labeled TUBB3, NeuN, and BrdU peaked at 4 weeks post-induction and declined slightly at 8 weeks of induction (fig. 5D).

Furthermore, we extended the observation period and observed the survival of induced de novo neurons at 8 and 12 months post-induction, respectively. According to the previous method, the identification and analysis of the neonatal neurons in the spinal cord tissue of mice was performed using tissue immunofluorescence staining. The results show that at 8 months (fig. 6A) and 12 months (fig. 6B) from the initiation of induction, positive cells labeled with TUBB3, NeuN, and BrdU were still visible in the spinal cord, indicating that the pre-induced neonatal neurons were viable for long periods of time.

Example 5: the in vivo induced new neuron of LDN193189 and CHIR99021 is derived from astrocyte

To explore the origin of neonatal DCX positive cells, they were analyzed using astrocyte, microglia and oligodendrocyte lineage specific markers. In addition, cell sources were also analyzed using lineage-tracing experiments.

The first experiment method comprises the following steps:

1. experimental mice: C57B6/L wild type mice.

3. Medicine for experiment

The medicine of the invention is: LDN193189, CHIR99021

Negative control drugs: DMSO (sigma)

4. Mouse spinal cord injury model: the specific experimental procedure was the same as in example 3.

5. Mouse tail vein injection mode: the adeno-associated virus stock solution was diluted to the appropriate concentration using sterile physiological saline and placed on ice for use. Female adult C57BL/6 mice were fixed and tail exposed, tail skin was sterilized and blood vessels were exposed using a 70% alcohol cotton ball, and 100uL of virus solution was aspirated using an insulin syringe and injected intravenously at the tail. After the experiment, the mice are returned to the animal room for later use after observation of no abnormality.

6. Administration mode of mice: the specific experimental procedure was the same as in example 3.

7. Collecting, fixing, dehydrating and slicing spinal cord tissues of mice: the specific experimental procedure was the same as in example 3.

II, experimental results:

as shown in fig. 7-A, B, C, cells co-labeled with DCX and OLIG2 or IBA1 were not seen, and only a small fraction of cells exhibited co-labeling with DCX and ALDH1L 1. DCX-positive cell proliferation was analyzed using PCNA immunohistochemical staining (FIG. 7-D), and only a very small fraction of cells appeared positive for PCNA. At 3, 5 days post-induction (FIG. 7-E), only a small fraction of DCX-positive cells were BrdU-positive at the same time, which is consistent with the PCNA fluorescent staining results.

We further analyzed the source of cells inducing the new neurons using a lineage tracing experiment. AAV2/9-GFAP-mCherry virus was injected via tail vein 3 days prior to injury/induction to specifically label spinal astrocytes. Induction was then given with LDN193189 and CHIR99021 and subsequently analyzed as before. First, we validated the validity and specificity of this method. Spinal cord tissue was section stained 2 weeks after tail vein injection of AAV. The results showed that mCherry was expressed only in GFAP-positive astrocytes (fig. 8-a). Subsequently, we performed lineage-tracing experiments as described previously, as shown in fig. 8-B, C, positive cells of the TUBB3, NeuN and mCherry trimmers were visible in the spinal cord lesions of the experimental group at week 4 of injury, while no positive cells of the TUBB3, NeuN and mCherry trimmers were visible in the spinal cords of the control group at the margin of injury (C1) and at the tissue beside injury (C2).

Example 6: LDN193189 and CHIR99021 can induce new neuron in aged mouse

The first experiment method comprises the following steps:

1. experimental animals: aged C57B6/L mice, purchased from Shanghai Ling Biotech, Inc.

2. Experiment medication:

the medicine of the invention is: LDN193189, CHIR99021

Negative control drugs: dmso (sigma).

3. Mouse spinal cord injury model: the specific experimental procedure was the same as in example 3.

4. Administration mode of mice: the specific experimental procedure was the same as in example 3.

3. Collecting, fixing, dehydrating and slicing spinal cord tissues of mice: the specific experimental procedure was the same as in example 3.

II, experimental results:

after 4 weeks of injury/induction, positive cells co-labeled with TUBB3, NeuN, and BrdU were seen in the spinal cord of the experimental group of aged mice, indicating that they are neonatal, mature neurons, but in significantly lower numbers than normal mice (fig. 9A); whereas positive cells co-labeled with TUBB3, NeuN, and BrdU were not found in the spinal cord of control mice (fig. 9C). After 8 weeks of injury/induction, nascent mature neurons were still visible in the spinal cord of experimental mice (fig. 9B). By statistical analysis, we found that the number of neonatal mature neurons labeled TUBB3, NeuN, and BrdU peaked at 4 weeks post-induction and decreased significantly at 8 weeks of induction (fig. 9D).

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

<110> China people liberation army navy military medical university

Application of <120> LDN193189 and CHIR99021 in preparation of medicine for inducing neuron regeneration

<130> description

<160> 40

<170> SIPOSequenceListing 1.0

<210> 1

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

catgttccag tatgactcca ctc 23

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ggcctcaccc catttgatgt 20

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ccagctggga gaagagtttg 20

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tttctttgcc atcccacttc 20

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

acgcagaagg caaggtgtcc 20

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gttcctcgtc ctgagaactg 20

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

aagccagtgt ctcttcgtgg 20

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gccttggtca tcttgcgttt 20

<210> 9

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

acttgaactc tatggcgggt t 21

<210> 10

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ccagttggta aagtccagca g 21

<210> 11

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tggctggcat ctgctctatt 20

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

taggcattgt gacgaatctg 20

<210> 13

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gacaagatcg cagcgcaagg 20

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ggcttagggc atttgaggaa 20

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

caggagtcag ccacccaaca 20

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

tccagccgca taaggttcat 20

<210> 17

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tagcccagta taaacgggag tg 22

<210> 18

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

ccaggttgcg aagaactctg 20

<210> 19

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

acggcctgaa gatggagga 19

<210> 20

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

cagaaacctg tccgagatga c 21

<210> 21

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ccctacgcca acatgaactc g 21

<210> 22

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

gttctgccgg tagaaaggga 20

<210> 23

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

agcgacgacg tgttcatcg 19

<210> 24

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

cccgttgtaa ctcaaagtgc tg 22

<210> 25

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

gcggagtgga aacttttgtc c 21

<210> 26

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

cgggaagcgt gtacttatcc tt 22

<210> 27

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

cggcaagaat atctgctcca g 21

<210> 28

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

caaatcggct gaagtagtcc at 22

<210> 29

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

cattttgacg aacgagacaa agc 23

<210> 30

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

tggaagtcca ttcatccgtg a 21

<210> 31

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

tagaccccag cggcaactat 20

<210> 32

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

gttccaggtt ccaagtccac c 21

<210> 33

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

atgacaggca agtcggtgaa g 21

<210> 34

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

catctcggcc ctttggactg 20

<210> 35

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

atcgtagagg gacggaaaat tga 23

<210> 36

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

gttcccaggc ttcttattgg tc 22

<210> 37

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

ccctgaagtc gaggagctg 19

<210> 38

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

ctgctgcacc tctaagcga 19

<210> 39

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

agctcaacaa atcccagtct ct 22

<210> 40

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

cggatggtct cagctttcac 20

Claims

The application of LDN193189 and CHIR99021 in preparing the medicine for regenerating and repairing damaged central nervous system is disclosed.
2. Use according to claim 1, characterized in that:

wherein the regeneration repair drug is a drug for inducing the regeneration of neurons.
3. Use according to claim 2, characterized in that:

wherein the agent inducing neuronal regeneration is an agent inducing reprogramming of astrocytes into neurons.
4. Use according to claim 3, characterized in that:

wherein LDN193189 and CHIR99021 induce astrocytes to transdifferentiate into neurons by up-regulating expression of Ngn2, neuroD1, Myt1l, Brn2, Foxg1, neuroD2 and Lmx1 a.
5. Use according to any one of claims 1 to 4, characterized in that:

wherein the regenerative repair drug is LDN193189 and CHIR99021 as the only active ingredients or a pharmaceutical composition comprising LDN193189 and CHIR 99021.
6. A method for inducing reprogramming of astrocytes into neurons in vitro comprising the steps of:

A. astrocyte treatment

Washing the astrocytes collected at the early stage with DMEM culture solution for 1 time, centrifuging at 800rpm for 4min at room temperature, then resuspending with DMEM culture solution containing 10% FBS, and adjusting cell density to 4 × 10⁴cells/ml; sucking the cell suspension liquid and dripping the cell suspension liquid on a hard carrier, adding 10% FBS DMEM culture solution into each hole of a multi-hole culture plate after incubating for 4 hours in a cell culture box, and putting the cell suspension liquid into the cell culture box for later use;

B. induced differentiation

Dissolving LDN193189 and CHIR99021 by using DMSO, adding the dissolved solutions into a DMEM culture solution containing 10% FBS after the dissolved solutions are completely dissolved, enabling the final concentrations to be 0.1-1 mu M and 1.5-20 mu M respectively, and replacing the previously-cultured completely-adherent astrocyte culture solution with the culture solution;

on the 3 rd day of induction, changing to a neuron induction culture solution, wherein the solution contains 90% of DMEM/F12 by volume fraction, 1% of double antibody by volume fraction, 0.1-1 mu M LDN193189 and 1.5-20 mu M CHIR99021 by volume fraction, and changing the solution once every two days;

on the 7 th day of induction, the culture medium is replaced by a neuron maturation induction culture medium, wherein the culture medium contains DMEM/F12 with the volume fraction of 78.6%, N2 with the volume fraction of 20%, B27 with the volume fraction of 0.4%, GDNF, BDNF, penicillin/streptomycin with the volume fraction of 1%, 0.1-1 mu M LDN193189 and 1.5-20 mu M CHIR99021, the culture medium is replaced by a half of the culture medium on the 10 th day of induction, and the induced differentiation is completed on the 12 th day of induction.
7. The method of inducing reprogramming of astrocytes into neurons according to claim 6, further comprising the steps of cell culture and purification:

cutting the pia mater to less than 1mm using a sterile scalpel³Adding an appropriate amount ofDigesting the pancreatin in a water bath kettle at 37 ℃ for 15 minutes, and then adding a DMEM culture solution containing 10% fetal calf serum to terminate the digestion process; transferring the tissue digestive juice into a centrifuge tube, centrifuging at the room temperature of 800rpm for 5 minutes, removing a supernatant, adding a fresh culture solution, repeatedly blowing and resuspending, standing for 10 minutes, sucking out the upper monolayer liquid, and adding the upper monolayer liquid into a culture bottle;

replacing fresh culture solution on day 2, replacing culture solution every 3 days, shaking the culture bottle manually before replacing the culture solution to remove the hybrid cells, wherein the visible cell fusion degree is 90% after about 7 days of primary culture, and then carrying out passage; culturing by the above method after passage, culturing until the cell reaches 3 rd generation, and performing subsequent experiment in DMEM culture solution containing 10% fetal calf serum at 37 deg.C and 5% CO₂Culturing under the condition, after the cells are paved on 90-95% of the bottom of the culture dish, digesting with 0.25% pancreatin, and collecting the cells for later use.
8. Use of neurons obtained by the method according to claim 6 or 7 for the preparation of a medicament for the regenerative repair after a central nervous system injury.
9. A pharmaceutical composition for the regenerative repair after central nervous system injury, consisting of an active ingredient comprising LDN193189 and CHIR99021, or neurons obtainable by the method according to claim 6 or 7, and pharmaceutically acceptable adjuvants.
10. The pharmaceutical composition for post-central nervous system injury regenerative repair of claim 9, wherein:

wherein, the regeneration and repair medicine composition after the central nervous system injury is decoction, injection, tablet or capsule.