CN115813951A

CN115813951A - Transgenic stem cells and their use for treating insomnia or sleep disorders

Info

Publication number: CN115813951A
Application number: CN202211406260.XA
Authority: CN
Inventors: 王杰; 李连宽; 李玉同
Original assignee: Guangzhou Lianda Technology Co ltd
Current assignee: Beijing Yulong Shengshi Biotechnology Co ltd
Priority date: 2022-11-10
Filing date: 2022-11-10
Publication date: 2023-03-21
Anticipated expiration: 2042-11-10
Also published as: CN115813951B

Abstract

The invention provides a transgenic stem cell and application thereof in treating insomnia or sleep disorder. The anti-insomnia medicine is constructed on the basis of mesenchymal stem cells, particularly bone marrow mesenchymal stem cells, so that the sleep quality can be continuously and effectively improved, and the sleep condition can be improved; the NGF amino acid sequence is subjected to site-directed mutagenesis, so that the nerve repair capability is improved, and the pain-causing function is reduced; the mesenchymal stem cells are subjected to gene modification, so that the mesenchymal stem cells carry the mutated NGF gene and can play a long-acting role in vivo. The medicine can effectively prolong sleep time, improve sleep quality, and regulate DA, 5-HT, GABA and other factors to express.

Description

Transgenic stem cells and their use for treating insomnia or sleep disorders

The technical field is as follows:

the invention belongs to the field of medicine research, and particularly provides a transgenic stem cell and application thereof in treating insomnia or sleep disorder.

Background art:

insomnia is the most common sleep disorder, and insomnia is characterized by difficulty in initiating and/or maintaining sleep associated with daytime impairment, rather than by environmental disturbances such as inadequate sleep opportunity, mental stress without drowsiness, and the like. Chronic insomnia is characterized by at least 3 symptoms per week for at least 3 months, with the main manifestations of decreased sleep quality or sleep time disturbances, and an estimated 30% of all people worldwide having one or more symptoms of insomnia. The insomnia can seriously affect the physical and mental health of a patient, and the chronic insomnia can cause the reduction of sleep time, thereby increasing the risks of diseases such as coronary artery disease, myocardial infarction, type II diabetes, obesity, systemic hypertension and the like. In addition, insomnia can also induce a variety of mental disorders, for example, persistent sleep disorders increase the risk of recurrence of depression, while insomnia is also a significant risk factor for suicide.

In order to effectively treat and relieve insomnia, researchers have developed many therapies or medicines, wherein Cognitive performance therapy (CBT-I) is considered as a first-line treatment for insomnia, and the curative effect thereof is well-known. CBT-I is a multi-modal combination of relaxation training, cognitive remodeling, stimulation control, sleep restriction, and sleep hygiene educational intervention, typically lasting 5 weeks, that has been shown to be at least as effective as drugs in ameliorating insomnia with fewer associated side effects. Despite the advantages of CBT-I, many medical personnel prefer to prescribe hypnotics for the initial treatment of patients with insomnia, which is done because CBT-I therapy is limited by multiple factors, including limited availability of trained patients, time and cost factors, e.g., CBT-I may be difficult to perform, especially for patients in rural areas, which can be a time consuming and expensive intervention, limiting the effectiveness of insomnia patients to adequately participate in treatment. Furthermore, some studies in recent years have shown that although CBT-I can improve sleep, which can be evidenced by an improvement in the subjective sleep rating, objective data on the positive impact of CBT-I on sleep is less reliable (Mitchell LJ, et al. The impact of cognitive sleep therapy for inorganic on objective sleep parameters: A meta-analysis and systematic review. Sleep Med Rev.2019; 47. Hypnotics are therefore considered essential in many cases and continue to be widely used by clinicians.

The medicines for treating insomnia have a relatively long research and development history, barbiturates and related compounds become the most commonly used medicines for treating insomnia in the early twentieth century, the use of barbiturates begins to decline in the middle of the 20 th century, and people can better know the possibility of adverse side effects and lethal overdose of barbiturates; in 1963, the first benzodiazepine drug, chlordiazepoxide, was introduced into the us market, in 1970 flurazepam was the first benzodiazepine drug approved by the us Food and Drug Administration (FDA) as a hypnotic, which was rapidly used in the treatment of insomnia more than barbiturates due to the perceived better safety; zolpidem was the first non-benzodiazepine receptor agonist (nBBRA) hypnotic marketed in the united states in 1992, and to date, zolpidem remains one of the most widely prescribed drugs for the treatment of insomnia.

However, the use of drugs to treat insomnia is not a trivial undertaking, and a number of prospective and retrospective studies have shown that the long-term use of benzodiazepines and nBBRA hypnotics increases the Risk of falls, psychiatric disorders, cardiovascular disorders, dementia and mortality (Kripke DF. Mortality Risk of Hypnotics: strengths and Limits of Evaporation. Drug Saf.2016;39 (2): 93-107). In addition, some studies have shown that benzodiazepines or other hypnotics are used in increasing amounts several weeks before the death of the patient, indicating that these drugs may cause serious adverse effects. Therefore, there is an urgent need to develop a novel therapeutic method or drug for treating insomnia, which can reduce the toxic and side effects as much as possible while maintaining the effectiveness.

In recent years, stem cell therapy becomes a novel tool for basic research and clinical treatment of diseases, wherein Mesenchymal Stem Cells (MSCs) are a type of pluripotent Stem Cells, have strong self-replication, renewal and multidirectional differentiation capabilities, and can be differentiated into nerve Cells under specific conditions, so that the Stem Cells are applied to treatment of various nerve-related diseases, and insomnia is one of application scenes of the Stem Cells. The clinical observation of the intravenous infusion of human umbilical cord mesenchymal stem cells for treating chronic insomnia, china journal of clinical psychology, 2017, 25 (02): 378-385) of 39 chronic insomnia patients are selected, randomly divided into 19 patients in a transplantation group and 20 patients in a control group, the transplantation group is subjected to human umbilical cord mesenchymal stem cell transplantation treatment for 1 time, the control group is orally taken by alprazolam for 1 month, two groups of patients adopt SF-36 health survey profile and Pittsburgh sleep quality index to evaluate the clinical treatment effect, and the two groups of patients follow-up observation is carried out for 12 months; finding that the quality of life score and the sleep score of the cell transplantation group patient 1 month after the cell transplantation treatment are obviously better than those before the treatment, and the quality of life score and the sleep score of the cell transplantation group patient are continuously better than those before the treatment in subsequent follow-up visits; the sleep quality score of the control group after 2 weeks of oral alprazolam treatment is obviously better than that before the treatment, but the life quality and the sleep quality score of the patient after 3 months of treatment are reduced and have no difference with those before the treatment and continue until the follow-up visit is finished; the two groups find that the sleep quality score of the control group is obviously higher than that of the transplant group 2 weeks after treatment, the sleep quality score and the life quality score of the two groups are not different after 1 month after treatment, and the sleep quality score of the cell transplant group 2 months after treatment is obviously better than that of the control group and continues until the follow-up visit is finished. The quality of life score of the transplanted group is obviously better than that of the control group after 3 months of treatment, and the treatment is continued until the follow-up visit is finished; after alprazolam is stopped taking the medicine, 80% of patients in a control group have rebound insomnia, while the improvement effect on the sleep quality and the life quality of the patients treated once by the cell transplantation group can last for 12 months without obvious adverse reaction. Boika et al found that transplantation of MSCs reduced the severity of sleep disturbance in studying the therapeutic effects of bone marrow MSCs on Parkinson's disease (Aliakscandr Boika et al, sensory stem cells in Parkinson's disease: motor and non-Motor systems in the early posttransplantation period, surg neural int.2020; 11. Zhoubangyin et al (CN 110791476) and Wangning et al (CN 111759863) also applied for Chinese patent respectively for the stem cell treatment of insomnia, and are used for protecting the technology of MSCs for treating insomnia. Despite the progress of MSCs in treating insomnia, the treatment method still needs to be further optimized because the MSCs still face the difficulties of unstable curative effect, difficult administration and the like.

Nerve Growth Factor (NGF) was the first member of the neurotrophic Factor family to be discovered, and R.Levi-Montalcini started pioneering studies on experimental animals and isolated cells in the early 1950 s with an emphasis on the biological role of NGF, which showed protection not only in the survival of degenerated peripheral Nerve cells, but also in the regulation of neurotransmitter and neuropeptide synthesis in sympathetic and sensory Nerve cells (Shooter E.M.early days in Neurosci.2001; 24. Exogenous NGF administration affects the plasticity of neurons, enabling the adult nervous system to change its structure and function upon stimulation, and furthermore demonstrates that constitutive synthesis of NGF in adult tissues is associated with Peripheral Nervous System (PNS) neuron phenotypic characteristics, such as innervation density, cell size, axonal terminal sprouting, dendritic growth, induction and/or inhibition of neuropeptides and neurotransmitters or enzymes producing transmitters. NGF also plays an important role in Sleep regulation, and studies have shown that nerve growth factor is some of the cytokines that have been shown to enhance Sleep (Kuem Sun Han, et al, stress and Sleep Disorder, exp neurobiol.2012Dec;21 (4): 141-150), but this role again seems to be controversial, as NGF also enhances the sensitivity of the body to pain, thereby interfering with the Sleep process. Therefore, researchers have attempted to modify NGF by means of gene mutation, and have made beneficial attempts to reduce the sensitivity of NGF to pain while retaining its trophism, promotion and repair effects on the central and peripheral nervous systems, such as CN108314723A, CN107286233A, etc.

In order to improve the treatment effect on insomnia, the invention provides a modified mesenchymal stem cell, which is derived from bone marrow tissue and carries a genetically modified nerve growth factor gene, can effectively secrete the nerve growth factor in vivo, promotes proliferation and modification after damage of nerve cells, and has the functions of neuroprotection and repair, immunoregulation, influence on sleep-wake related neurotransmitters and the like, so that the treatment method can effectively prolong sleep time, improve sleep quality, regulate secretion of cell factors and comprehensively inhibit adverse insomnia.

Disclosure of Invention

In order to solve the technical problem, the invention provides a pharmaceutical composition for treating insomnia, which comprises a stem cell which is subjected to genetic modification, wherein the stem cell contains a polynucleotide for encoding a nerve growth factor mutant with an amino acid sequence shown as SEQ ID NO. 2.

The nerve growth factor is a factor which has a wide regulating effect on a nervous system, has a nerve repairing effect, can promote neurotransmitter, and can improve pain sensitivity, so that the effect of treating insomnia by using natural NGF is not good enough; according to the present invention, based on the existing research results, the structure characteristics of the nerve growth factor are combined, and the nerve growth factor is creatively subjected to site-directed mutagenesis so as to reduce pain or pain sensitivity, and further develop the drug for treating insomnia.

Furthermore, the nucleotide sequence of the nerve growth factor mutant is shown in SEQ ID NO. 3.

Further, the stem cell is a mesenchymal stem cell.

Further, the mesenchymal stem cells are bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells and/or adipose mesenchymal stem cells.

Further, the mesenchymal stem cell is a bone marrow mesenchymal stem cell, and the preparation method comprises the following steps: putting human marrow blood into a clean sterile centrifuge tube, adding human lymphocyte separation liquid, and separating human marrow mesenchymal stem cells by using a density gradient centrifugation method; inoculating said stem cells in DMEM/F12 medium containing 10% FBS, culturing at 37 ℃ and 5% CO2 at saturation humidity, replacing the culture medium after culturing for 72 hours, discarding the suspended non-adherent cells, and replacing the medium every two or three days for 1 time; observing the growth state of the cells under an inverted phase contrast microscope, and carrying out subculture when the fusion degree of the cells reaches more than 80%; detecting the surface antigen expression of CD44, CD90 and CD34 cells by a flow cytometer, and screening to obtain high-purity human mesenchymal stem cells

Further, the method for introducing the polynucleotide encoding the nerve growth factor mutant into the stem cell comprises the following steps: constructing a recombinant lentiviral vector carrying the nerve growth factor mutant polynucleotide, adding the recombinant lentiviral vector at a ratio of cell/virus 1 of 10, 37 ℃,5% CO ₂ Culturing for 12h under saturated humidity, discarding cell culture solution, washing with sterile PBS for three times, adding 0.25% pancreatin, digesting at 37 deg.C for 5min, adding complete culture medium containing serum to stop digestion, and centrifuging at 1000rpm for 5min to collect cells.

According to the invention, the NGF gene is carried by the mesenchymal stem cell, and the mesenchymal stem cell has a sleep regulation effect, can break through a blood brain barrier, and plays a treatment role in a central nervous system, so that the sleep condition is obviously improved, and the sleep quality is improved.

Further, the pharmaceutical composition also comprises a medicinal carrier, an auxiliary material or a diluent.

Provides the application of the pharmaceutical composition for treating insomnia in the preparation of the drugs for treating insomnia.

Further, the insomnia includes acute insomnia, subacute insomnia and chronic insomnia.

Advantageous effects

The application provides a medicine or a medicine composition for treating insomnia or sleep disorder, which has the following advantages:

(1) Carrying out site-directed mutagenesis on the NGF amino acid sequence, improving the nerve repair capability and simultaneously reducing the pain-causing function;

(2) The anti-insomnia medicine is constructed on the basis of the mesenchymal stem cells, so that the sleep quality can be continuously and effectively improved, and the sleep condition can be improved;

(3) The mesenchymal stem cells are subjected to gene modification, so that the mesenchymal stem cells carry the mutated NGF gene and can play a long-acting role in vivo.

Drawings

FIG. 1: MWT experiment;

FIG. 2: the NGF mutant has the cell proliferation promoting capacity;

FIG. 3: detecting sleep time;

FIG. 4 is a schematic view of: (ii) a DA expression level;

FIG. 5:5-HT expression level;

FIG. 6: GABA expression level.

Detailed Description

The following non-limiting examples are presented to enable those of ordinary skill in the art to more fully understand the present invention and are not intended to limit the invention in any way. All the technologies implemented based on the above-mentioned contents of the present invention should fall within the scope of the claims of the present application.

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagent biomaterial and the detection kit are commercially available without specific instructions.

Example 1 nerve growth factor mutant design and screening

1.1 nerve growth factor mutant design and acquisition

The nerve growth factor comprises three subunits of alpha, beta and gamma, the beta subunit is an active region and is formed by combining two single chains through non-covalent bonds, and the amino acid sequence of natural NGF is shown in SEQ ID No:1 is shown. NGF causes more severe pain during use and is partially intolerant to patients, resulting in limited clinical use, particularly in the treatment of insomnia where severe pain responses or pain sensitivity effects mediated by NGF may cause adverse effects and thus require appropriate means to "inactivate". In the prior art, point mutation is proposed to reduce the pain-causing effect of NGF, for example, CN112409471A proposes point mutations of D30N, T27E, K34A, E35A and I31N to suppress pain, CN108314723A proposes R100W/E/P to alleviate pain, CN102911265A, CN102898514A proposes deletion of amino acids at the C-terminal or N-terminal part to improve the stability of NGF, and based on the above research, the present application creatively proposes new mutation sites or site combinations to alleviate pain and improve NGF activity, as shown in table 1. The mutant adopts a criprpr technology to carry out site-directed mutagenesis, related genes are introduced into an escherichia coli expression vector, NGF mutant protein is obtained through cation exchange chromatography purification, and the specific operation steps are carried out according to a known mature protein preparation and separation purification method.

TABLE 1 NGF mutant design

Mutant name	Mutation design
		NGF-M01	K32P，E35S，Q96T
NGF-M02	D30E，K34H，R100W
		NGF-M03	K32G，V87P，R100S，△C5
NGF-M04	K32W，A89T，,R100S
		NGF-M05	I31S，K34V，A89N
NGF-M06	I31P，E35P，R103P，△N3
		NGF-M07	K34E，V87H，R100F，△N3

1.2 experiments on pain sensitivity of nerve growth factor mutants

A clean male SD rat with the weight of 250-300g is selected, the rat is raised at the indoor temperature (23 +/-1) DEG C and the illumination is carried out for 12 h/is dark, and an animal experiment is carried out after the rat is fed for a week and is adapted to the environment. In this example, the sensitivity effect of NGF mutants on pain was examined by mechanical hyperalgesia.

Rats were injected with 1 μ g of NGF and its mutants on the soles, and the day of the experiment was recorded as day 0, and mechanical with paw reflex thresholds (MWT) were measured on day 0, day 3 and day 6, respectively. The rat is placed in a plastic cage with a wire mesh at the bottom, after adaptation is carried out for 30min, the VonFley fine needle wire is adopted to stimulate the right hind toe surface of the rat, each time lasts for 4-6s, and the minimum interval of each stimulation is 2min. The small needle thread is slightly bent to be seen with naked eyes as a complete stress standard, the rat is positive when the rat rapidly contracts feet, the stimulation strength is recorded at the moment, the MWT (g) of the rat is calculated, and the average value is taken by 5 times of testing of each rat. The results are shown in figure 1, which, after point mutation, resulted in varying degrees of pain sensitivity in rats, where the pain sensation was significantly reduced in the M03, M04 and M05 mutants, while the remaining mutants had no significant improvement in reducing pain sensation, even the M07 mutant was more sensitive than native NGF.

1.3 experiments on the proliferation of neural growth factor mutants

PC12 is a commonly used nerve cell line, has high similarity with nerve cells in vivo in metabolic mode and physiological characteristics, and is easier to obtain and culture than primary cultured nerve cells, so PC12 cells are selected in the present example to verify the cell proliferation promoting effect of NGF mutants.

PC12 cells (purchased from ATCC) were recovered and cultured, and when the cell fusion rate reached 90%, they were subcultured. Cells were harvested, resuspended in culture medium, and cell density adjusted to 5X10 ⁶ Per mL, which was then plated in 96-well plates at 100. Mu.L/well, 37 ℃ 5% CO ₂ After 12h incubation at saturation humidity, 50. Mu.g/mL of NGF mutant and wild-type NGF were added separately and 3 replicate wells per set, with zero and blank control wells (equal amounts of cell culture medium) being set. After 24h of treatment, 10. Mu.L of MTT solution and 90. Mu.L of basal medium were added to each well, and after further culturing for 4h, DMSO solution was added and shaken up for 10min. The absorbance (OD value) was measured at 490nm, andcell viability, cell viability (%) = [ A (medicated) -A (zero-set)]/[ A (blank control) -A (zero set)]×100％。

As shown in FIG. 2, after NGF is added, the proliferation activity of PC12 is enhanced, but the proliferation promoting capability of each mutant shows different trend, wherein the proliferation promoting capability of M03 and M05 is stronger, the proliferation promoting capability of M02, M04, M06 and M07 has no significant difference compared with that of natural NGF, and the proliferation promoting activity of M01 is obviously reduced.

In conclusion, the M03 mutant with low pain sensitivity and strong nerve cell proliferation promoting activity is selected for subsequent experiments.

EXAMPLE 2 preparation of genetically modified Stem cells

2.1 extraction of mesenchymal Stem cells

Taking human bone marrow blood (from 5 healthy adults, the average age is 35 years), putting the bone marrow blood into a clean sterile centrifuge tube, adding 10mL of human lymphocyte separation liquid (purchased from Beijing Solebao science and technology Co., ltd.), and separating the human mesenchymal stem cells by using a density gradient centrifugation method. Inoculating said stem cells into DMEM/F12 medium containing 10% FBS, at 37 ℃,5% CO ₂ And culturing at saturated humidity, replacing the culture solution after culturing for 72h, discarding the suspended non-adherent cells, and replacing the solution for 1 time every two or three days later. And (5) observing the growth state of the cells under an inverted phase contrast microscope, and carrying out subculture when the cell fusion degree reaches more than 80%. The expression of CD44, CD90 and CD34 surface antigens of the cells is detected by a flow cytometer, and the result shows that: the positive expression of CD44 is more than 95%, the positive expression of CD90 is also more than 94%, and CD34 is not expressed basically, which indicates that the bone marrow mesenchymal stem cells with higher purity are obtained by culturing.

2.2 preparation of Lentiviral vectors

Adding HindIII and BamHI enzymolysis sites to two ends of the NGF nucleotide sequence of the neural stem cell factor mutant by a PCR method, and carrying out HindIII/BamHI double enzyme digestion on the obtained mutant nucleotide and a pFLAG-CMV-2 carrier at 37 ℃; after the enzyme digestion product is purified by gel, T4 ligase is used for carrying out reaction at 4 ℃ overnight for connection, and the plasmid vector is obtained. Then, the plasmid vector DH 5. Alpha. Competent cells were transformed by electroporation under 1.8kV, 25. Mu.F and 200. Omega. Conditions, 100. Mu.L of the bacterial solution was applied to LB plates containing ampicillin resistance, cultured overnight at 37 ℃, single clones were picked up for sequencing and identified, and positive clones with correct sequencing results were stored.

Frozen 293T cells (purchased from ATCC) were removed from liquid nitrogen, rapidly thawed in a 37 ℃ water bath, centrifuged at 1200rpm for 5min to collect the cells, then placed in 10% FBS-containing DMEM for culture, and the cell contents were 5% CO at 37 ℃% ₂ Culturing under saturated humidity, and subculturing when the cell confluency reaches above 90%, and culturing for 3-5 generations. 293T cells in good cell status were collected and plated at 6X 10 in each dish ⁶ The number of individual cells was seeded in a petri dish at 37 ℃ and 5% CO ₂ Culturing in an incubator overnight; removing the original culture medium, washing the cells three times by using sterile PBS, and then adding 10mL of Opti-MEM culture solution; mu.g of lentiviral expression plasmid, 10. Mu.g of packaging plasmid Del8.9, 20. Mu.g of Vesicular Stomatis Virus Glycoprotein (VSVG) (available from Clontech, USA) and 40. Mu.L of lipofectamine2000 were added to 1mL of Opti-MEM culture solution and mixed to obtain a mixed solution; slowly adding the mixed solution dropwise into 293FT cell culture solution, mixing, and adjusting the content of CO at 37 deg.C ₂ And culturing in a saturated humidity cell culture box.

After culturing for 72h, preparing the lentiviral vector by using an ultracentrifugation method, which specifically comprises the following steps: centrifuging at 4000g for 10min at 4 deg.C to remove cell debris; filtering with 0.45 μm and 0.22 μm filters to obtain supernatant, and placing into 40ml ultracentrifuge tube; centrifugation is carried out at 20000g for 2h at 4 ℃, after centrifugation is completed, the supernatant is discarded, and a virus sample is collected, and the lentiviral vector is resuspended and dissolved by using a cell culture medium. Subpackaging the virus, storing in an ultra-low temperature refrigerator at-80 ℃, and determining the virus titer by adopting a fluorescence method, wherein the virus titer is determined to be 2.8 multiplied by 10 ⁹ TU/ml, meeting the requirements of subsequent experiments.

2.3 genetic modification of mesenchymal Stem cells

Inoculating the mesenchymal stem cells obtained in the 2.1 step into a culture dish, and culturing at 37 deg.C with 5% CO ₂ Culturing at saturated humidity until cells are fusedThe degree reached more than 80%, using a pipette to aspirate the cell culture fluid, using sterile PBS to wash three times, adding fresh culture fluid, adding recombinant lentiviral vector according to a cell/virus 1 ₂ Culturing for 12h under saturated humidity, discarding cell culture solution, washing with sterile PBS for three times, adding 0.25% pancreatin, digesting at 37 deg.C for 5min, adding complete culture medium containing serum to stop digestion, centrifuging at 1000rpm for 5min, and collecting cells as MSC-muNGF.

Using the same method, mesenchymal stem cells carrying the natural NGF nucleic acid sequence were prepared, denoted as MSC-NGF.

Example 3 genetically modified Stem cells in animal models for treating insomnia

3.1 animal model preparation

In this example, a rat insomnia model was prepared by modeling chlorphenylalanine (PCPA). Selecting healthy SD rats, normally feeding for one week to adapt to the environment, wherein the feeding conditions are as follows: the room temperature is 20-26 ℃, the relative humidity is 40-60%, and the food is fed regularly and freely by natural illumination in a daytime circulation mode. Preparing PCPA alkalescent solution, injecting SD rat (300 mg/kg) into abdominal cavity 1 time per day, and continuously injecting for 2 days. After about 30 hours of the injection at the 1 st time, the circadian rhythm of the rat disappears, while the circadian rhythm of the control group of rats is normal, which indicates that the establishment of the rat insomnia model is successful.

3.2 treatment with drug administration

The model animals were randomly divided into 4 groups of 10 animals each, each of which was: MSC group: tail vein injection of 1X 10 ⁶ A plurality of MSC cells; MSC-NGF group: tail vein injection of 1X 10 ⁶ MSC-NGF cells; MSC-muNGF group: tail vein injection of 1X 10 ⁶ MSC-muNGF cells; control group: equal volume of saline was injected. The subsequent detection is carried out after one week of injection of the therapeutic agent.

3.3 behavioural testing

And evaluating the behavioral performance of the experimental animal by adopting a Y-shaped maze experiment. Before the experiment is formally started, the rat is put into the maze to adapt for 5min, a safety area and an electric shock area are arranged in the maze, and the rat is determined to finish the test after escaping to a light area under the electric shock and keeping for more than 10 s. The number of false reactions was observed and 30 tests were performed per experimental day. The experimental results are shown in table 2, the control group animals have a large number of errors, the condition is improved by injecting the MSC cells, the effect of using the MSC cells carrying nerve growth factors is more obvious, which shows that on one hand, the MSC cells have better nervous system repair effect, on the other hand, the MSC cells can be used as a delivery carrier, break through the blood brain barrier and enter the central nervous system, and further release the NGF or a mutant thereof, so that the system repair effect is exerted, and the nerve injury symptom is further relieved, but the MSC-NGF and the MSC-muNGF have no significant difference in the experiment.

TABLE 2 maze Experimental observations

3.4 sleep time observations

After 1 week of administration, the time when the righting reflex disappears and the time when the righting reflex appears are recorded for each group of rats, the rats enter sleep when the righting reflex disappears for more than 30s, and the rats enter sleep until the righting reflex reappears, namely the sleep time. Finally, the sleep latency and the sleep duration of the rats in each group are calculated. The results are shown in fig. 3, after the model is successfully made, the sleep time of the rat is obviously shortened, and the sleep latency is prolonged, which shows that the deep sleep time is shortened; after the administration treatment, the sleep condition is improved, but the sleep improvement effect of MSC-NGF in three treatment groups is the worst, probably because NGF has double functions of promoting nerve repair and causing pain sensation sensitivity, the use of NGF with a natural structure is difficult to obviously improve the sleep quality, and the use of MSC-muNGF treatment not only furthest retains the nerve repair function of NGF, but also weakens the pain sensation sensitivity effect, so that the sleep latency and the sleep duration of model animals are greatly prolonged and are close to the normal state.

3.5 molecular level detection

After the administration, the rats were sacrificed by removing their necks, the whole brain tissue was dissected out, about 20mg of the cerebral cortex tissue was cut, a certain amount of PBS (pH 7.4) was added, and the mixture was placed on ice and homogenized using a homogenizer; centrifuging at 4 deg.C for 20min at 3000r/min, and collecting supernatant; ELISA kit (purchased from R & DSystems of America) is used for detecting the concentration of factors such as Dopamine (DA), 5-hydroxytryptamine (5-HT), gamma-aminobutyric acid (GABA) and the like, and the specific steps are carried out according to the kit instruction.

DA is a neurotransmitter from the catecholamine family, considered to be a key neurobiological substrate, and has traditionally been the target of pharmacological treatment of movement disorders, mental disorders and, to a lesser extent, depression. There is also evidence that DA is a component of promotion and maintenance of wakefulness, DA neurotransmission is thought to be the primary mechanism by which exogenous stimulants (such as amphetamine, cocaine, and methylphenidate) promote wakefulness, and researchers have found that both methamphetamine and modafinil increase extracellular DA by blocking DA transporter (DAT) receptors that regulate DA reuptake; DAergic tone and wakefulness increased in narcolepsy dogs following treatment with amphetamine and modafinil, whereas neither drug had a wakefulness-promoting effect in DAT knockout mice. As shown in fig. 4, the DA content in the model group was greatly increased and the DA level was reduced after treatment with MSC, compared to the normal group; but the reduction amplitude of the MSC-NGF group is smaller, and the reduction amplitude of the MSC-muNGF group is the largest, so that the MSC-muNGF group can block neurotransmitter transmission, reduce the clear-headed degree of the body and contribute to sleep.

5-HT is an inhibitory neurotransmitter involved in the regulation of circadian rhythm systems and cognitive functions, high levels of 5-HT are thought to mediate the prolongation of sleep time, and it has been reported that drugs with sleep-promoting functions such as zolpidem, diazepam, lotus seed, etc. all act by regulating the level of 5-HT. As shown in fig. 5, the DA content in the model group was significantly reduced relative to the normal group; there was only a small recovery after treatment with MSC, but after treatment with MSC-NGF and MSC-muNGF, the 5-HT level rose dramatically, approaching the normal range.

GABA is an important central nervous system inhibitory neurotransmitter, researches show that insomnia is closely related to 3 aspects of anabolism, conduction and transportation of GABA in a gamma-aminobutyric acid (GABA) energy system channel, GABA has 3 receptor subtypes in the period of promoting 'wake-sleep', wherein GABA A receptors belong to ion channel type receptors, play an important role in the process by activating chloride ion channels, and GABA AR alpha 1 and GABA AR gamma 2 receptors play an important role as action targets for screening nerve and mental drug activities and participate in regulating inhibition/excitation balance. As shown in fig. 6, GABA expression levels in brain tissues after molding were significantly decreased, and GABA expression levels were significantly upregulated after treatment with MSC, although the effect of MSC-muNGF appeared more significant, but did not show significant difference.

While this invention has been particularly shown and described with references to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

1. A pharmaceutical composition for treating insomnia, which comprises a genetically modified stem cell containing a polynucleotide encoding a nerve growth factor mutant having an amino acid sequence shown in SEQ ID NO. 2.

2. The pharmaceutical composition for treating insomnia according to claim 1, wherein the nucleotide sequence of said nerve growth factor mutant is represented by SEQ ID NO. 3.

3. The pharmaceutical composition for treating insomnia according to claim 2, wherein said stem cells are mesenchymal stem cells.

4. The pharmaceutical composition for treating insomnia according to claim 3, wherein said mesenchymal stem cell is a bone marrow mesenchymal stem cell, an umbilical cord mesenchymal stem cell and/or an adipose mesenchymal stem cell.

5. The pharmaceutical composition for treating insomnia according to claim 4, wherein said mesenchymal stem cells are bone marrow mesenchymal stem cells, and the preparation method comprises: cleaning human marrow bloodAdding a human lymphocyte separation solution into an aseptic centrifugal tube, and separating the human mesenchymal stem cells by using a density gradient centrifugation method; inoculating said stem cells into DMEM/F12 medium containing 10% FBS, at 37 ℃,5% CO ₂ Culturing at saturated humidity, changing the culture solution after culturing for 72h, discarding the suspended non-adherent cells, and changing the solution for 1 time every two or three days; observing the growth state of the cells under an inverted phase contrast microscope, and carrying out subculture when the cell fusion degree reaches more than 80%; detecting the surface antigen expression of CD44, CD90 and CD34 cells by a flow cytometer, and screening to obtain the high-purity human mesenchymal stem cells.

6. The pharmaceutical composition for treating insomnia according to claim 5, wherein the method for introducing a polynucleotide encoding a nerve growth factor mutant into a stem cell comprises: constructing a recombinant lentiviral vector carrying the nerve growth factor mutant polynucleotide, adding the recombinant lentiviral vector at a ratio of cell/virus 1 ₂ Culturing for 12h under saturated humidity, discarding cell culture solution, washing with sterile PBS for three times, adding 0.25% pancreatin, digesting at 37 deg.C for 5min, adding complete culture medium containing serum to stop digestion, and centrifuging at 1000rpm for 5min to collect cells.

7. The pharmaceutical composition for treating insomnia according to claim 6, wherein said pharmaceutical composition further comprises a pharmaceutically acceptable carrier, adjuvant or diluent.

8. Use of the pharmaceutical composition for the treatment of insomnia according to any one of claims 1 to 7 for the preparation of a medicament for the treatment of insomnia.

9. The use of claim 8 wherein the insomnia includes acute insomnia, subacute insomnia and chronic insomnia.