CN115813951B - Transgenic stem cells and their use for treating insomnia or sleep disorders - Google Patents
Transgenic stem cells and their use for treating insomnia or sleep disorders Download PDFInfo
- Publication number
- CN115813951B CN115813951B CN202211406260.XA CN202211406260A CN115813951B CN 115813951 B CN115813951 B CN 115813951B CN 202211406260 A CN202211406260 A CN 202211406260A CN 115813951 B CN115813951 B CN 115813951B
- Authority
- CN
- China
- Prior art keywords
- stem cells
- insomnia
- mesenchymal stem
- cells
- pharmaceutical composition
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The invention provides a transgenic stem cell and application thereof in treating insomnia or sleep disorder. Based on mesenchymal stem cells, in particular bone marrow mesenchymal stem cells, an anti-insomnia medicament is constructed, so that the sleep quality can be continuously and effectively improved, and the sleep condition can be improved; site-directed mutagenesis is performed on an NGF amino acid sequence, so that the nerve repair capability is improved, and simultaneously, the pain-causing function is reduced; the mesenchymal stem cells are genetically modified to carry the mutated NGF genes, so that the mesenchymal stem cells can play a long-acting role in vivo. The medicine can effectively prolong the sleeping time, improve the sleeping quality and regulate the expression of factors such as DA, 5-HT, GABA and the like.
Description
Technical field:
the invention belongs to the field of medicine research, and particularly provides a transgenic stem cell and an application thereof in treating insomnia or sleep disorder.
The background technology is as follows:
insomnia is the most common sleep-impaired disease, and is characterized by difficulty in initiating and/or maintaining sleep associated with daytime injuries, rather than sleep due to environmental obstructions, such as insufficient sleep opportunities, mental stress, and lack of drowsiness, etc. Chronic insomnia is the occurrence of symptoms at least 3 times per week for at least 3 months, mainly manifested by a decrease in sleep quality or a disturbed sleep time, and an estimated 30% of people worldwide have one or more symptoms of insomnia. Insomnia can seriously affect physical and mental health of a patient, and chronic insomnia can lead to reduced sleep time, so that risks of diseases such as coronary artery disease, myocardial infarction, type II diabetes, obesity, systemic hypertension and the like are increased. In addition, insomnia may also induce various mental diseases, for example, persistent sleep disorders increase the risk of depression recurrence, and insomnia is also an important risk factor for suicide.
To effectively treat and alleviate insomnia, researchers have developed many therapies or drugs, of which insomnia cognitive behavioral therapy (Cognitive behavioral therapy for insomnia, CBT-I) is considered to be a first-line treatment of insomnia, and its efficacy has been recognized. CBT-I is a multimodal combination of relaxation training, cognitive reconstruction, stimulus control, sleep limitation and sleep hygiene educational intervention, typically lasting 5 weeks, which has been shown to improve insomnia at least as effectively as drugs, with fewer associated side effects. While CBT-I has advantages, many medical personnel prefer to prescribe hypnotics for initial treatment of patients with insomnia, because CBT-I therapy is limited by multiple factors, including limited availability, time and cost factors of trained patients, e.g., CBT-I can be difficult to do, which can be a time consuming and expensive intervention especially for patients in rural areas, limiting the effectiveness of insomnia patients to adequately participate in the treatment. Furthermore, some recent studies have shown that while CBT-I may improve sleep, which may be evidenced by an improvement in subjective sleep ratings, objective data regarding the positive effects of CBT-I on sleep are less reliable (Mitchell LJ, et al impact ofcognitive behavioural therapy for insomnia on objective sleep parameters: A meta-analysis and systematic review. Sleep Med Rev.2019; 47:90-102). Hypnotics are therefore considered necessary in many cases and continue to be widely used by clinicians.
Drugs for treating insomnia have relatively long development history, barbiturates and related compounds become the most commonly used drugs for treating insomnia in the beginning of the twentieth century, the use of barbiturates begins to decline in the middle of the 20 th century, and people better understand the possibility of adverse side effects and deadly excess of barbiturates at that time; in 1963, the first benzodiazepine, chlordiazepoxide, was introduced into the U.S. market, 1970, fluazepam was the first benzodiazepine approved by the U.S. Food and Drug Administration (FDA) as a hypnotic agent, and the use of benzodiazepines for the treatment of insomnia has rapidly exceeded barbiturates due to perceived better safety; zolpidem was the first non-benzodiazepine receptor agonist (nbra) hypnotic marketed in the united states in 1992, and to date, zolpidem has remained one of the most widely used prescription drugs for the treatment of insomnia.
However, the use of drugs for the treatment of insomnia is also not a constant task, and several prospective and retrospective studies have shown that prolonged use of benzodiazepines and nbra hypnotics increases the Risk of falls, mental illness, cardiovascular disease, dementia and mortality (Kripke df. Molar Risk of hypnotics: strengths and Limits of evaluation. Drug saf.2016;39 (2): 93-107). Furthermore, some studies have shown that the use of benzodiazepines or other hypnotics has increased a few weeks before the death of the patient, indicating that these drugs may cause serious adverse effects. Therefore, there is a great clinical need to develop new therapies or drugs for treating insomnia, and to reduce the toxic and side effects as much as possible while maintaining the effectiveness.
In recent years, stem cell therapy is a novel tool for basic research and clinical treatment of diseases, wherein mesenchymal stem cells (Mesenchymal Stem Cells, MSCs) are a type of pluripotent stem cells, have strong self-replication, renewal capacity and multidirectional differentiation capacity, and can be differentiated into nerve cells under specific conditions, so that the novel tool is applied to treatment of various nerve-related diseases, and insomnia is one of application scenes. Wang Yali (Wang Yali, clinical observation of human umbilical cord mesenchymal stem cells for chronic insomnia, journal of clinical psychology in China 2017, 25 (02): 378-385) 39 cases of chronic insomnia patients are selected, randomly divided into 19 cases of a transplantation group, 20 cases of a control group, wherein the transplantation group is given 1 time of human umbilical cord mesenchymal stem cells for transplantation treatment, the control group is orally taken for 1 month by adopting alprazolam, and clinical curative effects are evaluated by adopting SF-36 health survey profile and Pittsburgh sleep quality index for both patients, and the two groups of patients are observed for 12 months in a follow-up manner; the quality of life score and sleep quality score of the cell transplantation group patients were found to be significantly better than before treatment 1 month after cell transplantation treatment and continued to be better than before treatment in subsequent follow-up; the sleep quality score of the control group after oral alprazolam treatment for 2 weeks is obviously better than that before treatment, but the quality of life and the sleep quality score of the patient after treatment for 3 months are reduced, and the control group has no difference with the control group before treatment and lasts until the follow-up is finished; the comparison of the two groups shows that the sleep quality score of the control group is obviously higher than that of the transplanted group 2 weeks after treatment, the sleep quality and life quality scores of the two groups are not different 1 month after treatment, and the sleep quality score of the cell transplanted group is obviously better than that of the control group 2 months after treatment and is continued until the follow-up is finished. The quality of life score of the transplanted group is obviously better than that of the control group 3 months after treatment, and the quality of life score is continued until the follow-up is finished; patients in a control group have rebound of 80% of insomnia symptoms after the alprazolam is stopped, while the sleep quality and life quality improvement effect of the patients in a cell transplantation group can last for 12 months after one treatment, and no obvious adverse reaction exists. Boika et al have found in studying the therapeutic effects of bone marrow MSCs on Parkinson's disease that transplantation of MSCs reduces the severity of sleep disorders (Aliaksandr Boika et al, mesenchymal stem cells in Parkinson's disease: motor and nonmotor symptoms in the early posttransplant period, surg Neurol int.2020; 11:380). Bangyin et al (CN 110791476), wang Ning et al (CN 111759863) also filed Chinese patent application for stem cells for treating insomnia, and techniques for protecting MSCs for treating insomnia. Despite the progress made by MSCs in the treatment of insomnia, there is still a dilemma in that the efficacy is unstable and administration is difficult, so that the therapy still needs to be further optimized.
Nerve growth factor (Nerve Growth Factor, NGF) is the first member of the neurotrophic factor family to be discovered, and in the early 1950 s, R.Levi-Montalcini began an initial study of experimental animals and isolated cells focusing on the biological effects of NGF, which showed that NGF had a protective effect not only in the survival of degenerated peripheral nerve cells, but also in regulating neurotransmitter and neuropeptide synthesis in sympathetic and sensory nerve cells (Shooter E.M.early days ofthe nerve growth factor proteins.Annu.Rev.Neurosci.2001; 24:601-629). Exogenous NGF administration affects neuronal plasticity, enabling the adult nervous system to alter its structure and function upon stimulation, and furthermore demonstrates that constitutive synthesis of NGF in adult tissues is associated with Peripheral Nervous System (PNS) neuronal phenotypic characteristics such as innervation density, cell body size, axon terminal sprouting, dendritic growth, induction and/or inhibition of neuropeptides and neurotransmitters or transmitter-producing enzymes. NGF also plays an important role in sleep regulation, and studies have shown that nerve growth factor is a cytokine that has been demonstrated to enhance sleep (Kuem Sun Han et al Stress and Sleep Disorder, exp neurobiol.2012Dec;21 (4): 141-150), but this effect appears to be controversial because NGF can also enhance the sensitivity of the human body to pain, thereby interfering with the sleep process. For this reason, researchers have attempted to modify NGF by way of gene mutation, and have made beneficial attempts in this respect to reduce the degree of sensitization to pain, such as CN108314723A, CN107286233a, while retaining the trophic, promoting, repairing effects of NGF on the central and peripheral nervous systems.
In order to improve the effect of treating insomnia, the invention provides a modified mesenchymal stem cell which is derived from bone marrow tissue and carries a nerve growth factor gene subjected to genetic modification, can effectively secrete the nerve growth factor in vivo, promotes the proliferation and post-injury modification of nerve cells, and has the functions of neuroprotection, repair, immunoregulation, influence on sleep-wake related neurotransmitters and the like, so that the therapy can effectively prolong the sleep time, improve the sleep quality, regulate the secretion of cytokines, comprehensively inhibit the adverse effect of insomnia, has no obvious addiction and withdrawal reaction, has lower toxic and side effects, and provides a new direction for treating insomnia.
Disclosure of Invention
In order to solve the technical problems, the invention provides a pharmaceutical composition for treating insomnia, which comprises genetically modified stem cells, wherein the stem cells contain polynucleotides for encoding nerve growth factor mutants with an amino acid sequence shown as SEQ ID NO. 2.
Nerve growth factor is a factor with wide regulation function on the nervous system, has nerve repair function, can promote neurotransmitter and can improve pain sensitivity, so that the effect of treating insomnia by using natural NGF is poor; according to the existing research results and by combining the structural characteristics of the nerve growth factor, the invention creatively carries out site-directed mutagenesis on the nerve growth factor so as to reduce pain or pain sensitivity of the nerve growth factor, and further develops the medicine for treating insomnia.
Furthermore, the nucleotide sequence of the nerve growth factor mutant is shown as SEQ ID NO. 3.
Further, the stem cells are mesenchymal stem cells.
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 cells are bone marrow mesenchymal stem cells, and the preparation method comprises the following steps: placing human bone marrow blood into a clean sterile centrifuge tube, adding human lymphocyte separating liquid, and separating human bone marrow mesenchymal stem cells by using a density gradient centrifugation method; inoculating the stem cells into DMEM/F12 culture medium containing 10% FBS, culturing at 37 ℃ and 5% CO2 at saturated humidity, replacing culture solution after culturing for 72 hours, discarding suspended non-adherent cells, and replacing the solution 1 time every two or three days later; observing the growth state of the cells under an inverted phase contrast microscope, and subculturing when the cell fusion degree reaches more than 80%; detecting cell CD44, CD90 and CD34 surface antigen expression by flow cytometry, and screening to obtain high purity human bone marrow mesenchymal stem cell
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 according to the ratio of cells/viruses of 1:10, and adding 5% CO at 37 DEG C 2 Culturing at saturated humidity for 12h, discarding cell culture solution, washing with sterile PBS three times, adding 0.25% pancreatin, digesting at 37deg.C for 5min, adding complete culture medium containing serum, stopping digestion, centrifuging at 1000rpm for 5min, and collecting cells.
The mesenchymal stem cells are selected to carry NGF genes, have sleep regulation function, can break through the blood brain barrier, and play a role in treatment in the central nervous system, so that the sleep condition is remarkably improved, and the sleep quality is improved.
Further, the pharmaceutical composition also comprises a medicinal carrier, auxiliary materials or diluents.
There is provided the use of the pharmaceutical composition for treating insomnia in the preparation of a medicament for treating insomnia.
Further, the insomnia includes acute insomnia, subacute insomnia and chronic insomnia.
Advantageous effects
The present application provides a medicament or pharmaceutical composition for the treatment of insomnia or sleep disorders, having the following advantages:
(1) Site-directed mutagenesis is performed on an NGF amino acid sequence, so that the nerve repair capability is improved, and simultaneously, the pain-causing function is reduced;
(2) The anti-insomnia medicine is constructed based on 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 genetically modified to carry the mutated NGF genes, so that the mesenchymal stem cells can play a long-acting role in vivo.
Drawings
Fig. 1: MWT experiment;
fig. 2: NGF mutant ability to promote cell proliferation;
fig. 3: detecting sleeping time;
fig. 4: DA expression level;
fig. 5:5-HT expression levels;
fig. 6: GABA expression levels.
Detailed Description
The following non-limiting examples will enable those of ordinary skill in the art to more fully understand the invention and are not intended to limit the invention in any way. All techniques implemented based on the above description of the invention should be within the scope of the protection claimed in this application.
The experimental methods described in the following examples, unless otherwise specified, are all conventional; the reagent biological material and the detection kit can be obtained from commercial sources unless otherwise specified.
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, wherein 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 the natural NGF is shown as SEQ ID No: 1. In the process of using NGF, serious pain is caused, partial patients cannot tolerate the NGF, so that the clinical application of the NGF is limited, and particularly in the process of treating insomnia, the serious pain response or pain sensitivity effect mediated by NGF can cause the adverse effect, so that proper measures are needed to be adopted for carrying out passivation treatment. In the prior art, a point mutation mode has been proposed to reduce the pain-causing effect of NGF, for example, CN112409471A proposes that D30N, T27E, K34A, E A, I31N and other point mutations can inhibit pain, CN108314723A proposes that R100W/E/P can relieve pain, CN102911265A, CN102898514A and other points can delete C-terminal or N-terminal part amino acid to improve the stability of NGF, and on the basis of the above research, the application creatively proposes a novel mutation site or site combination so as to relieve pain and improve the activity of NGF, as shown in the table 1. The mutant is subjected to site-directed mutagenesis by using a crispr technology, related genes are introduced into an escherichia coli expression vector, and then NGF mutant proteins are obtained through cation exchange chromatography purification, wherein the specific operation steps are carried out according to known mature protein preparation and separation and purification methods.
TABLE 1 NGF mutant design
| Mutant name | Mutant 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 pain sensitivity experiments with nerve growth factor mutants
Clean-grade male SD rats with weights of 250-300g are selected, the temperature (23+/-1) DEG C in a rat raising room is 12h illumination/12 h darkness, and animal experiments are carried out after feeding for one week to adapt to the environment. In this example, the mechanical sensitization method was used to detect the sensitization effect of NGF mutants to pain.
Rats were plantar injected with 1 μg NGF and its mutant, respectively, and mechanical stimulation footstrike reflex thresholds (mechanical withdrawal threshold, MWT) were examined on day 0, day 3, and day 6, respectively, when the day was recorded as day 0. Placing the rat in a plastic cage with a wire mesh at the bottom, after adapting for 30min, stimulating the right rear toe surface of the rat by using a VonFrey fine needle wire, continuously stimulating each time for 4-6s, and keeping the minimum interval between each stimulation for 2min. The MWT (g) of the rat was calculated by taking the slight macroscopic bending of the fine needle wire as a complete stress standard, taking a positive response when the rat became rapidly contracted, recording the stimulation force at this time, and taking an average value by testing 5 times each rat. As shown in FIG. 1, the pain sensitivity of rats was varied to different degrees after point mutation, wherein the pain feeling of M03, M04 and M05 mutants was significantly reduced, while the rest mutants were not significantly improved in terms of pain feeling reduction, even the sensitivity of M07 mutants was higher than that of natural NGF.
1.3 cell proliferation-promoting experiments with nerve growth factor mutants
PC12 is a common neural cell line, which has high similarity to in vivo neural cells in terms of metabolic mode and physiological characteristics, and is easier to obtain and culture than primary cultured neural cells, so PC12 cells were selected in this example to verify the pro-cell proliferation effect of NGF mutants.
PC12 cells (purchased from ATCC) were recovered and cultured, and subcultured until cell fusion reached 90%. Cells were collected, resuspended using medium, and cell density was adjusted to 5x10 6 Each mL was then inoculated into 96-well plates at 100. Mu.L per well, 37℃and 5% CO 2 After 12h of incubation at saturation humidity, 50. Mu.g/mL of NGF mutant and wild-type NGF were added, respectively, and zeroed wells and blank wells (equivalent amount of cell culture medium) were set, 3 multiplex wells per group. After 24h of treatment, 10. Mu.L of MTT solution and 90. Mu.L of basal medium are added to each well, and after further incubation for 4h, DMSO solution is added and shaking is carried out on a shaker for 10min. Absorbance (OD value) was measured at 490nm and cell viability was calculated, cell viability (%) = [ a (dosing) -a (zeroing)]/[ A (blank) -A (zero setting)]×100％。
As shown in fig. 2, after NGF was added, the proliferation activity of PC12 was enhanced, but the proliferation-promoting ability of each mutant exhibited different trend, wherein the proliferation-promoting ability of M03 and M05 was strong, while the proliferation-promoting ability of M02, M04, M06, M07 was not significantly different from that of natural NGF, but the proliferation-promoting activity of M01 was significantly 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
Human bone marrow blood (from 5 healthy adults, average age 35 years) was taken, placed in a clean sterile centrifuge tube, 10mL of human lymphocyte separation solution (purchased from beijing solebao sciences ltd) was added, and human bone marrow mesenchymal stem cells were isolated using density gradient centrifugation. The stem cells were inoculated into DMEM/F12 medium containing 10% FBS, 37℃and 5% CO 2 Culturing at saturated humidity for 72 hr, replacing culture solution, and discarding suspended non-adherent cells, and replacing solution 1 time every two or three days. Observing the growth state of the cells under an inverted phase contrast microscope, and subculturing when the cell fusion degree reaches more than 80%. Cell CD44, CD90, CD34 surface antigen expression was detected with a flow cytometer and the results showed that: the positive expression of CD44 is more than 95%, the positive expression of CD90 is also more than 94%, and CD34 is basically not expressed, which indicates that the bone marrow mesenchymal stem cells with higher purity are obtained by culture.
2.2 preparation of lentiviral vectors
Adding HindIII and BamHI enzymolysis sites at two ends of the NGF nucleotide sequence of the mutant of the neural stem cell factor by a PCR method, and carrying out HindIII/BamHI double digestion on the obtained mutant nucleotide and a pFAG-CMV-2 vector at 37 ℃; after gel purification of the digested product, the plasmid vector was obtained by overnight ligation at 4℃using T4 ligase. Then, the plasmid vector DH5 alpha competent cells were electrotransformed under the conditions of 1.8kV, 25 mu F and 200Ω,100 mu L of bacterial liquid was coated on LB plate containing ampicillin resistance, cultured overnight at 37 ℃, and monoclonal was picked up for sequencing identification, and positive clone strains with correct sequencing results were preserved.
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, and then placed in DMEM with 10% FBS at 37℃in 5% CO 2 Culturing under saturated humidity until cell confluence reaches 90%In the above steps, subculture is performed, and co-culture is performed for 3 to 5 generations. 293T cells in good cell status were harvested at 6X 10 per dish 6 The cell number was inoculated in a petri dish at 37℃with 5% CO 2 Is cultured overnight in an incubator; the original medium was removed, the cells were washed three times with sterile PBS, and then 10mL of Opti-MEM broth was added; 10. Mu.g of lentiviral expression plasmid, 10. Mu.g of packaging plasmid Del8.9, 20. Mu.g of Vesicular Stomatitis Virus Glycoprotein (VSVG) (available from Clontech Co., U.S.A.), and 40. Mu.L of lipofectamine2000 were added to 1mL of Opti-MEM culture solution, and mixed to obtain a mixed liquid; the mixed liquid is slowly dripped into a culture solution of 293FT cells, gently mixed, and stirred at 37 ℃ and 5% CO 2 Culturing in a saturated humidity cell incubator.
After 72h of culture, lentiviral vectors were prepared using ultracentrifugation, specifically comprising: centrifuging at 4deg.C for 10min at 4000g to remove cell debris; filtering with 0.45 μm and 0.22 μm filters to obtain supernatant, and placing into a 40ml ultracentrifuge tube; centrifugation was performed at 20000g for 2h at 4℃and after centrifugation, the supernatant was discarded to collect virus samples and the lentiviral vector was resuspended and lysed using cell culture medium. Packaging the virus, storing in-80deg.C refrigerator, measuring virus titer by fluorescence method, and measuring virus titer to 2.8X10 9 TU/ml meets the requirements of subsequent experiments.
2.3 Gene modification of bone marrow mesenchymal Stem cells
Inoculating the bone marrow mesenchymal stem cells obtained in step 2.1 into a culture dish at 37deg.C with 5% CO 2 Culturing under saturated humidity until cell fusion degree reaches above 80%, sucking cell culture solution with a pipette, washing with sterile PBS for three times, adding fresh culture solution, adding recombinant lentiviral vector at cell/virus ratio of 1:10, 37 deg.C, and 5% CO 2 Culturing at saturated humidity for 12h, discarding cell culture solution, washing with sterile PBS three times, adding 0.25% pancreatin, digesting at 37deg.C for 5min, adding complete culture medium containing serum, stopping digestion, centrifuging at 1000rpm for 5min, and collecting cells, which are marked as MSC-muNGF.
Using the same method, mesenchymal stem cells carrying the native NGF nucleic acid sequence, designated MSC-NGF, were prepared.
Example 3 genetically modified Stem cells animal model for treating insomnia
3.1 animal model preparation
In this example, a model of insomnia in rats was prepared by a phenylalanine (PCPA) molding method. Healthy SD rats are selected and normally fed for one week to adapt to the environment, and the feeding conditions are as follows: the room temperature is 20-26 ℃, the relative humidity is 40-60%, natural illumination is carried out in a daytime circulation mode, feeding is carried out regularly, and water is freely drunk. A weakly alkaline solution of PCPA was prepared and SD rats (300 mg/kg) were intraperitoneally injected 1 time a day for 2 days. After about 30 hours of the 1 st injection, the circadian rhythm of the rat disappears, and the circadian rhythm of the rat in the control group is normal, which indicates that the insomnia model of the rat is successfully established.
3.2 treatment for administration
Model animals were randomly divided into 4 groups of 10 animals each: MSC group: tail vein injection 1 x10 6 A plurality of MSC cells; MSC-NGF group: tail vein injection 1 x10 6 Individual MSC-NGF cells; MSC-muNGF group: tail vein injection 1 x10 6 Individual MSC-muNGF cells; control group: an equal volume of physiological saline is injected. One week after injection of the therapeutic agent, subsequent testing was performed.
3.3 behavioural detection
The behavioral performance of the experimental animals was evaluated using a Y-maze experiment. Before formally starting the experiment, the rats are put into a maze to be adapted for 5min, a safe area and an electric shock area are arranged in the maze, and after the rats flee to a light area under electric shock, the rats are kept for more than 10s, so that the rats are determined to finish one test. The number of false reactions was observed and 30 tests were performed per experimental day. The experimental results are shown in table 2, a large number of errors occur in animals in a control group, the situation is improved by injecting MSC cells, the effect of using MSC cells carrying nerve growth factors is more obvious, and the results show that on one hand, the MSC cells have better nervous system repairing 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 then release NGF or mutants thereof, thereby playing a role in system repairing and further relieving the symptom of nerve injury, but in the experiment, the MSC-NGF and MSC-muNGF have no obvious difference.
Table 2 maze test observations
3.4 sleep time observations
After 1 week of administration, the time when the specular reflection of each group of rats disappeared and the time when the specular reflection appeared were recorded, so that the mice were put into sleep after the specular reflection disappeared for more than 30 seconds, and the mice were put into sleep until the specular reflection was reproduced, so that the mice were put into sleep. Finally, sleep latency and sleep duration were calculated for each group of rats. As shown in the graph 3, after successful modeling, the sleep time of the rat is obviously shortened, and the sleep latency period is prolonged, which means that the deep sleep time is shortened; after administration of the treatment, sleep state is improved, but the sleep improvement effect of MSC-NGF is the worst in three treatment groups, probably because NGF has the dual functions of promoting nerve repair and inducing pain sensitivity, and the sleep quality is difficult to be remarkably improved by using NGF with a natural structure, and the treatment by MSC-muNGF not only maximally maintains the nerve repair function of NGF, but also weakens the inducing pain sensitivity, so that the sleep latency and the sleep duration of a model animal are greatly prolonged and the sleep state is approximate to a normal state.
3.5 molecular level detection
After the end of the administration, the rat is sacrificed by neck removal, the whole brain tissue is dissected and taken out, about 20mg of the brain cortex tissue is sheared, a certain amount of PBS (pH 7.4) is added, and the mixture is placed on ice for homogenization by a homogenizer; centrifuging at 3000r/min and 4deg.C for 20min, and collecting supernatant; the concentration of factors such as dopamine (dopamine, DA), 5-hydroxytryptamine (5-HT), gamma-aminobutyric acid (gamma-aminobutyric acid, GABA) was measured using ELISA kit (available from R & DSsystems Co., USA) according to the kit instructions.
DA is a neurotransmitter from the catecholamine family, which is considered a key neurobiological substrate, and has traditionally been the target of drug treatment for dyskinesias, psychotic disorders, and to a lesser extent depression. There is evidence that DA is a component of promotion and maintenance of the awake state, and that DA neurotransmission is considered to be the primary mechanism of promotion of wakefulness by exogenous agonists such as amphetamine, cocaine, and methylphenidate, and researchers have found that both methamphetamine and modafinil increase extracellular DA by blocking the DA transporter (DAT) receptor that regulates DA reuptake; following treatment with amphetamine and modafinil, the DAergic tone and wakefulness of the narcolepsy dogs increased, and neither drug had a wake-promoting effect in DAT knockout mice. As shown in fig. 4, DA levels in the model group were greatly increased relative to the normal group, and DA levels were reduced after treatment with MSC; however, the MSC-NGF group has smaller reduction amplitude and the MSC-muNGF group has the largest reduction amplitude, which indicates that the MSC-muNGF group can block neurotransmitter transmission, reduce the wakefulness of the organism and help sleep.
5-HT, an inhibitory neurotransmitter, is involved in regulating the circadian system and cognitive-functions, and high levels of 5-HT are thought to mediate the prolongation of sleep time, and drugs having sleep-promoting functions such as zolpidem, diazepam, lotus seed, etc. are reported to act by regulating 5-HT levels. 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, 5-HT levels increased significantly, 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 promoting a 'wake-sleep' period, GABA A receptors belong to ion channel type receptors, and play a role in activating chloride ion channels, GABA AR alpha 1 and GABA AR gamma 2 receptors play an important role in the process, and can be used as an action target point for screening nerve and spirit type drug activity and participate in regulating inhibition/excitation balance. As shown in fig. 6, GABA expression levels in brain tissues were significantly reduced after modeling, and GABA expression levels were significantly upregulated after treatment with MSC, although the effect of MSC-muNGF appeared to be more pronounced, but no significant difference was shown.
While the present invention has been particularly shown and described with reference 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 (9)
1. A pharmaceutical composition for treating insomnia comprises a stem cell 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.
2. The pharmaceutical composition for treating insomnia according to claim 1, wherein the nucleotide sequence of the nerve growth factor mutant is shown in SEQ ID NO. 3.
3. The pharmaceutical composition for treating insomnia according to claim 2, wherein the stem cells are mesenchymal stem cells.
4. The pharmaceutical composition for treating insomnia according to claim 3, wherein the mesenchymal stem cells are bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells and/or adipose mesenchymal stem cells.
5. The pharmaceutical composition for treating insomnia according to claim 4, wherein the mesenchymal stem cells are bone marrow mesenchymal stem cells, and the preparation method comprises: placing human bone marrow blood into a clean sterile centrifuge tube, adding human lymphocyte separating liquid, and separating human bone marrow mesenchymal stem cells by using a density gradient centrifugation method; the stem cells were inoculated into DMEM/F12 medium containing 10% FBS, 37℃and 5% CO 2 Culturing at saturated humidity for 72h, replacing culture solution, discarding suspended non-adherent cells, and replacing the culture solution 1 time every two or three days; observing the growth state of the cells under an inverted phase contrast microscope, and subculturing when the cell fusion degree reaches more than 80%; detection of cell CD44, CD90, CD34 surface antigens by flow cytometryExpressing and screening to obtain high-purity human bone marrow mesenchymal stem cells.
6. The pharmaceutical composition for treating insomnia according to claim 5, wherein the method for introducing the polynucleotide encoding the nerve growth factor mutant into stem cells comprises: constructing a recombinant lentiviral vector carrying the nerve growth factor mutant polynucleotide, adding the recombinant lentiviral vector according to the ratio of cells/viruses of 1:10, and adding 5% CO at 37 DEG C 2 Culturing at saturated humidity for 12h, discarding cell culture solution, washing with sterile PBS three times, adding 0.25% pancreatin, digesting at 37deg.C for 5min, adding complete culture medium containing serum, stopping digestion, centrifuging at 1000rpm for 5min, and collecting cells.
7. The pharmaceutical composition for treating insomnia as recited in claim 6, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, adjuvant or diluent.
8. Use of a pharmaceutical composition according to any one of claims 1-7 for the treatment of insomnia in the manufacture of a medicament for the treatment of insomnia.
9. The use of claim 8, wherein the insomnia comprises acute insomnia, subacute insomnia, and chronic insomnia.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211406260.XA CN115813951B (en) | 2022-11-10 | 2022-11-10 | Transgenic stem cells and their use for treating insomnia or sleep disorders |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211406260.XA CN115813951B (en) | 2022-11-10 | 2022-11-10 | Transgenic stem cells and their use for treating insomnia or sleep disorders |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115813951A CN115813951A (en) | 2023-03-21 |
| CN115813951B true CN115813951B (en) | 2023-07-21 |
Family
ID=85527607
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211406260.XA Active CN115813951B (en) | 2022-11-10 | 2022-11-10 | Transgenic stem cells and their use for treating insomnia or sleep disorders |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115813951B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101243178A (en) * | 2005-06-16 | 2008-08-13 | 特拉维夫大学拉莫特有限公司 | Isolated cells and populations comprising same for the treatment of CNS diseases |
| CN102898514A (en) * | 2011-07-28 | 2013-01-30 | 中国人民解放军军事医学科学院生物工程研究所 | Recombinant human nerve growth factor deletion mutant, its preparation method and application |
| CN108314723A (en) * | 2018-01-11 | 2018-07-24 | 温州医科大学 | A kind of people source saltant type nerve growth factor and its preparation method and application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114671941B (en) * | 2016-03-18 | 2023-09-26 | 舒泰神（北京）生物制药股份有限公司 | Nerve growth factor mutant |
-
2022
- 2022-11-10 CN CN202211406260.XA patent/CN115813951B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101243178A (en) * | 2005-06-16 | 2008-08-13 | 特拉维夫大学拉莫特有限公司 | Isolated cells and populations comprising same for the treatment of CNS diseases |
| CN102898514A (en) * | 2011-07-28 | 2013-01-30 | 中国人民解放军军事医学科学院生物工程研究所 | Recombinant human nerve growth factor deletion mutant, its preparation method and application |
| CN108314723A (en) * | 2018-01-11 | 2018-07-24 | 温州医科大学 | A kind of people source saltant type nerve growth factor and its preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| Taking Pain Out of NGF: A ‘‘Painless’’ NGF Mutant, Linked to Hereditary Sensory Autonomic Neuropathy Type V, with Full Neurotrophic Activity;Simona Capsoni等;《PLoS ONE》;第6卷(第2期);第1-12页 * |
| 静脉输注人脐带间充质干细胞治疗慢性失眠的临床观察;王亚莉等;《中国临床心理学杂志》;第25卷(第02期);第378-381,385页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115813951A (en) | 2023-03-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6948250B2 (en) | Compositions and Methods of Inhibiting the Activity of LAR Family Phosphatases | |
| US20060247195A1 (en) | Method of altering cell properties by administering rna | |
| DK2913059T3 (en) | Hitherto UNKNOWN PROCEDURE FOR TREATMENT OF SPINE INJURY USING HMGB1 FRAGMENT | |
| Chen et al. | Circuit-specific gene therapy reverses core symptoms in a primate Parkinson’s disease model | |
| CN115813951B (en) | Transgenic stem cells and their use for treating insomnia or sleep disorders | |
| KR20190090359A (en) | Mesenchymal stem cell expressing brain-derived neurotrophic factor gene and use thereof | |
| WO2014140930A2 (en) | Compostions and methods for enhancing the therapeutic potential of stem cells | |
| JP2002512627A (en) | CNS nerve regeneration composition and method of using the same | |
| Quan et al. | Hydralazine plays an immunomodulation role of pro-regeneration in a mouse model of spinal cord injury | |
| WO2016125330A1 (en) | Retinal regeneration promoting drug | |
| WO1992010200A1 (en) | Functional interactions between glial s-100b and central nervous system serotonergic neurons | |
| CN115475247A (en) | Pharmaceutical use of beta 2-microglobulin or inhibitor thereof | |
| JP6308699B2 (en) | Preventive or therapeutic agent for demyelinating disease | |
| CN111617254A (en) | Application of substance for reducing content or activity of MYSM1 in treating depression | |
| CN115006534A (en) | Application and pharmaceutical composition of potassium ion channel Kir4.1 inhibitor for treating depression | |
| Liu et al. | Effects of NT-4 gene modified fibroblasts transplanted into AD rats | |
| CN112451683B (en) | Application of TIMP-1 in preparation of medicine for preventing or treating traumatic brain injury | |
| US20230181699A1 (en) | Method and drug for treating spinal muscular atrophy | |
| CN110726844B (en) | siRNA targeting PSMC5 gene and application of PSMC5 | |
| CN101555284B (en) | Truncated-type human ciliary nerve nutrition factor active segment and fusion protein thereof | |
| EP4347645A1 (en) | Neuro-mesenchyme units control ilc2 and obesity via a brain-adipose circuit | |
| CN112516168A (en) | Mesenchymal stem cells for intervention in stress-induced cognitive disorders | |
| CN113679732A (en) | Application of ligustrin in preparation of medicine for preventing and treating calcified aortic valve diseases | |
| CN117500530A (en) | Composition for inhibiting alpha-synuclein and aggregation inhibition method | |
| AU2021422314A1 (en) | Pharmaceutical composition for prevention or treatment of inflammatory disease or pain, comprising mesenchymal stem cells expressing ptx-3, timp1 and bdnf as active ingredient |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20230629 Address after: 312, 3rd Floor, Building 9, Yanyu Qingyuan, No. 26 Qinghe Maofang Road, Haidian District, Beijing, 100083 Applicant after: BEIJING YULONG SHENGSHI BIOTECHNOLOGY Co.,Ltd. Address before: Shop 105, No. 3, Fengye Road, Tianhe District, Guangzhou, Guangdong, 510000 Applicant before: Guangzhou Lianda Technology Co.,Ltd. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |