CN116891831B - Active molecule for promoting neural differentiation of adipose-derived stem cells and application thereof - Google Patents

Active molecule for promoting neural differentiation of adipose-derived stem cells and application thereof Download PDF

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CN116891831B
CN116891831B CN202311164494.2A CN202311164494A CN116891831B CN 116891831 B CN116891831 B CN 116891831B CN 202311164494 A CN202311164494 A CN 202311164494A CN 116891831 B CN116891831 B CN 116891831B
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adipose
stem cells
derived stem
osteocalcin
gene
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CN116891831A (en
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王妍
江小霞
罗常溢
杜张珍
曹才卓
秦巧臻
徐振华
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Academy of Military Medical Sciences AMMS of PLA
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Abstract

The invention discloses an active molecule for promoting neural differentiation of adipose-derived stem cells and application thereof, and the invention discovers that osteocalcin can obviously promote neural differentiation of adipose-derived stem cells for the first time, and in an acute traumatic brain injury mouse model, the over-expressed osteocalcin gene can obviously relieve anxiety-like behaviors and can recover learning and memory ability faster.

Description

Active molecule for promoting neural differentiation of adipose-derived stem cells and application thereof
Technical Field
The invention belongs to the technical field of cell biology, and particularly relates to an active molecule for promoting neural differentiation of adipose-derived stem cells and application thereof.
Background
Clinically, the brain neuron loss caused by the reasons of craniocerebral trauma, stroke, tumor and the like is quite common, and the only weak regeneration capacity of the brain is difficult to play a role in compensation due to the change of intracranial immune microenvironment caused by the reasons. How to supplement the number of neurons after brain tissue injury and regulate the immune microenvironment to avoid apoptosis of neonatal neurons is one of the dilemmas of current clinical therapies. In recent years, the research of stem cells combined with tissue engineering provides more viable ideas for overcoming the above dilemma. Mesenchymal stem cells are a type of progenitor cells with multipotency, which can produce a variety of tissues and are widely available, such as bone marrow mesenchymal stem cells (mesenchymal stem cell, MSC), adipose stem cells (adiose-derived stem cell, ADSC), and the like. Among them, the fat stem cell obtaining route is the most extensive, easy to obtain, has better immunoregulation ability and differentiation ability, can be used as one of the best choices for supplementing brain neuron loss.
Osteocalcin (OCN), the most abundant protein active molecule in bone, can be used as a marker of bone metabolism, has three carboxyl sites, has strong calcium binding property when carboxylated at all three sites (cOCN), is deposited in bone matrix after being combined with calcium, and can undergo decarboxylation reaction under certain conditions, and the decarboxylated Osteocalcin protein (ucOCN) has reduced affinity with calcium ions, and is dissociated from the bone matrix and enters blood circulation to generate endocrine effect. Experiments prove that after the high expression treatment of the OCN gene is carried out on the adipose-derived stem cells, the apoptosis is reduced in the neurogenic differentiation process, and the number of the neuroid cells is obviously increased, namely, the OCN can promote the neurogenic differentiation of the adipose-derived stem cells, is further verified in an acute traumatic brain injury mouse model, and is expected to be applied to the treatment of neuronal loss caused by various craniocerebral injuries.
To date, no related study or report has been presented of the use of osteocalcin in promoting neural differentiation of adipose stem cells.
Disclosure of Invention
The invention aims to provide an active molecule for promoting the neurogenic differentiation of adipose-derived stem cells and application thereof, wherein the active molecule is osteocalcin, and the osteocalcin can be used for obviously promoting the neurogenic differentiation of adipose-derived stem cells and is expected to be applied to the treatment of neuronal loss caused by various craniocerebral injuries for the first time.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, the invention provides the use of an agent that overexpresses a osteocalcin gene in promoting neural differentiation of adipose-derived stem cells.
Further, the agent includes a substance that increases the level of osteocalcin gene expression, a substance that enhances osteocalcin activity, a substance that delays osteocalcin metabolism, and/or any combination thereof.
Further, the agents include natural purification substances, modified natural purification substances, semisynthetic substances, and/or chemically synthesized substances.
Further, the agent includes a vector expressing a osteocalcin gene, a nanoparticle carrying a osteocalcin gene, a viral vector carrying a osteocalcin gene, a PEG-modified protein encapsulating a osteocalcin gene or protein, a protein microsphere encapsulating a osteocalcin gene or protein, a liposome encapsulating a osteocalcin gene or protein, an extracellular vesicle encapsulating a osteocalcin gene or protein, and/or any combination thereof.
In some embodiments, the osteocalcin comprises a osteocalcin gene and a osteocalcin protein. The osteocalcin gene is transcribed and translated into osteocalcin protein product in the body of the study object. In a specific embodiment of the present invention, the osteocalcin gene (OCN) has GenBank accession No. nm_007541.
In some embodiments, the osteocalcin gene has a sequence known in the art or is a derivative thereof. In some embodiments, the osteocalcin gene is a molecule comprising the sequence: (a) A osteocalcin Gene molecule having a sequence shown as Gene ID 632 (human), gene ID 25295 (Norway mouse), gene ID 281646 (bovine), gene ID 12096 (domestic mouse), gene ID 792433 (zebra fish), gene ID 403762 (dog), gene ID 397530 (pig), gene ID 396348 (chicken), gene ID 718296 (rhesus monkey) and the like; (b) A molecule which hybridizes under stringent conditions to the sequence defined in (a); (c) Molecules having a sequence homology of more than 70% (e.g. 75%, 80%, 85%, 90%, 95%, 98%, 99%, 99.5% or more, or any value or range of values therebetween) to the sequence of the molecule shown in (a) or (b), e.g. a osteocalcin gene molecule obtained by codon optimisation.
In some embodiments, the agent that overexpresses a osteocalcin gene refers to a substance capable of increasing the level of osteocalcin gene expression, enhancing osteocalcin activity, and/or delaying osteocalcin protein metabolism, including, but not limited to: a small molecule compound, a vector expressing a osteocalcin gene, a nanoparticle carrying a osteocalcin gene, a viral vector carrying a osteocalcin gene, a PEG-modified protein encapsulating a osteocalcin gene or protein, a protein microsphere encapsulating a osteocalcin gene or protein, a liposome encapsulating a osteocalcin gene or protein, an extracellular vesicle encapsulating a osteocalcin gene or protein, and/or any combination thereof.
In some embodiments, the carrier includes, but is not limited to: lentiviral vectors, retroviral vectors, poxviral vectors, herpes simplex viral vectors, adenoviral vectors, adeno-associated viral vectors, DNA plasmid-binding liposomes, DNA plasmid-binding molecular conjugates and/or DNA plasmid-binding polymers, as long as the vectors that can be used to deliver the gene of interest (osteocalcin gene) of the invention into adipose stem cells are within the scope of the invention.
In a second aspect, the invention provides a method of promoting neural differentiation of adipose-derived stem cells.
Further, the method comprises the following steps: the osteocalcin gene is over-expressed in the adipose-derived stem cells, so that the adipose-derived stem cells over-expressing the osteocalcin gene are obtained, and are cultured in a pre-induction culture medium and then are cultured in a neurogenic induction culture medium.
Further, the pre-induction medium was DMEM-HG medium containing 10% FBS, 5 mM β -mercaptoethanol.
Further, the neurogenic induction medium is DMEM-HG medium containing 10% FBS, 2% DMSO, 200 μM BHA, 40 ng/mL bFGF.
Further, the time of culture in the pre-induction medium is 24 h;
the time of culture in the neurogenic induction medium was 7 days.
In a third aspect of the invention, a pharmaceutical composition is provided.
Further, the pharmaceutical composition comprises adipose stem cells over-expressing the osteocalcin gene.
Further, the adipose-derived stem cells over-expressing the osteocalcin gene are prepared by the method described in the second aspect of the present invention.
In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or adjuvant.
In some embodiments, specific illustrative examples of the pharmaceutically acceptable carrier and/or adjuvant include, but are not limited to: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifying agents, such as wetting agents, e.g., sodium lauryl sulfate; a colorant; a flavoring agent; tabletting and stabilizing agent; an antioxidant; a preservative; non-thermal raw water; isotonic saline solution; and phosphate buffer, etc.
In some embodiments, suitable pharmaceutically acceptable carriers and/or excipients are described in detail in Remington's Pharmaceutical Sciences (19 th ed., 1995) which are useful as needed to aid stability of the formulation or to aid in enhancing activity or its bioavailability or to impart an acceptable mouthfeel or odor in the case of oral administration, and formulations which may be used in such pharmaceutical compositions may be in the form of the original compound itself, or optionally in the form of a pharmaceutically acceptable salt thereof. The pharmaceutical compositions so formulated may be administered by any suitable means known to those skilled in the art, as desired, and in preferred embodiments, a safe and effective amount of the pharmaceutical composition of the present invention is administered to a human being when the pharmaceutical composition of the present invention is used.
In some embodiments, the pharmaceutical composition of the present invention is suitably administered in a variety of doses depending on the formulation method, the administration mode, the age, weight, sex, disease state, diet, administration time, administration route, excretion rate and response sensitivity of the patient, and the like, and the skilled practitioner can easily determine the prescription and the dose of the prescription effective for the desired prophylaxis and/or treatment.
In some embodiments, the pharmaceutical composition may further comprise a second therapeutic agent that can be used to treat and/or prevent central nerve damage. The second therapeutic agent includes any drug or agent that can be used in combination with the osteocalcin gene-overexpressing adipose stem cells of the present invention to treat and/or prevent central nerve damage, so long as the drug or agent that can be used in combination with the osteocalcin gene-overexpressing adipose stem cells of the present invention to treat and/or prevent central nerve damage is within the scope of the present invention.
In some embodiments, the drug or agent that is the second therapeutic agent includes, but is not limited to: aspirin, ibuprofen, tramadol, oxycodone, gabapentin, pregabalin, amitriptyline, doxepin, nortriptyline, duloxetine, venlafaxine, desmethylvenlafaxine, sirolimus, ceramides, mecobalamin, vitamin B1, vitamin B6, dibazol, resina Draconis capsules, aniracetam, oxiracetam, citicoline, idebenone, butylphthalide, beraprost, nicergoline.
In addition, the invention also provides a method for treating and/or preventing central nerve injury, the method comprising the steps of: administering to a subject in need thereof a therapeutically and/or prophylactically effective amount of an adipose stem cell overexpressing a osteocalcin gene according to the present invention or a pharmaceutical composition according to the present invention.
In some embodiments, the subject refers to any animal, and also refers to human and non-human animals. The term non-human animal includes all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any domestic animals or pets; and non-mammals, such as chickens, amphibians, reptiles, etc., in preferred embodiments, the subject is a human.
In addition, the invention also provides a method for screening candidate drugs for treating and/or preventing central nerve injury, which comprises the following steps:
(1) Treating a fat stem cell system expressing or containing a osteocalcin gene with a substance to be tested;
(2) Detecting the expression of a osteocalcin gene in said system;
(3) Selecting a test substance capable of increasing the expression level of osteocalcin gene in adipose-derived stem cells as a candidate drug;
more preferably, the test substance comprises a substance that increases the level of osteocalcin gene expression, a substance that enhances osteocalcin activity, a substance that delays osteocalcin metabolism, and/or any combination thereof.
In some embodiments, any substance that may have an enhancing effect on the directional differentiation of adipose stem cells into neural cells is a test substance.
A third aspect of the invention provides any one of the following applications, the applications comprising:
(1) Use of adipose-derived stem cells overexpressing osteocalcin gene in the manufacture of a medicament for the treatment and/or prevention of a neuronal loss related disorder;
(2) Use of adipose-derived stem cells overexpressing osteocalcin genes for the preparation of a medicament for improving anxiety, depression and/or learning ability.
In some embodiments, the neuronal loss related disorders include disorders associated with neuronal loss due to a variety of causes, including but not limited to central nerve injury, a common neurological disorder including but not limited to neurological disorders resulting from trauma (shock, cut, firearm, compression injury, etc.), congenital damage, alcoholism, lead poisoning, ischemia and other neurological sensory disorders, movement disorders, nutritional disorders, etc., and any common neuronal loss due to a variety of causes is within the scope of the invention.
In the specific embodiment of the invention, experimental verification shows that the fat stem cells over-expressing the osteocalcin gene can obviously improve anxiety and depression-like behaviors caused by TBI and can obviously restore the learning ability of a TBI mouse by adopting a traumatic brain injury (Traumatic Brain Injury, TBI) mouse model, and the functional reconstruction of the fat stem cells on the traumatic brain injury is proved by the osteocalcin.
In the present invention, traumatic brain injury (Traumatic Brain Injury, TBI) refers to injury of the brain due to external mechanical force, which can cause transient or permanent somatic dysfunction, cognitive dysfunction, psychological dysfunction, etc. in brain trauma survivors, and craniocerebral trauma is one of the important pathogenesis factors of neurodegenerative diseases.
Compared with the prior art, the invention has the advantages and beneficial effects that:
the invention discovers for the first time that osteocalcin can obviously promote the neurogenic differentiation of adipose-derived stem cells. The experiment proves that the over-expressed osteocalcin gene can obviously promote the expression of the neurogenic differentiation related markers (such as Nestin, MAP2 and NeuN) in the neurogenic differentiation process of the adipose-derived stem cells, has obvious anti-apoptosis effect, can obviously relieve anxiety-like behaviors and can recover learning and memory ability faster in an acute traumatic brain injury mouse model, and the invention provides a new thought for the treatment of neuronal loss caused by various craniocerebral injuries.
Drawings
FIG. 1 shows the morphology of mouse adipose-derived stem cells that gradually possessed neural cells as the induction time increased during the neurogenic differentiation process;
FIG. 2 shows that the number of cells and the number of neuronal-like cells were significantly increased in the over-expressed OCN group compared to normal adipose stem cells during the same induction process;
FIG. 3 shows the expression of markers associated with differentiation of adipose-derived stem cells and OCN genes at days 1, 4 and 7 of induced differentiation;
FIG. 4 shows the expression of marker molecules and OCN genes associated with the differentiation of the over-expressed OCN adipose-derived stem cells on days 1, 4 and 7 of induced differentiation, wherein each group of results is respectively blank, induced for 1 day, induced for 4 days and induced for 7 days from left to right;
FIG. 5 shows the conditions of cell proliferation and apoptosis marker gene expression of OCN adipose-derived stem cells on days 1, 4 and 7 of induced differentiation, wherein each group of results is respectively blank group, induced for 1 day, induced for 4 days and induced for 7 days from left to right;
FIG. 6 shows the expression of OCN proteins in adipose-derived stem cells on days 1, 4 and 7 of induced differentiation;
FIG. 7 shows the expression of apoptosis-related markers of cell proliferation and apoptosis in OCN-overexpressing adipose-derived stem cells on days 1, 4, and 7 of induced differentiation, wherein each group of results is blank, induced for 1 day, induced for 4 days, and induced for 7 days, respectively;
FIG. 8 is a open field experiment the next day after TBI mice transplanted with over-expressed OCN adipose stem cells;
FIG. 9 is a new object recognition experiment of the third day after TBI mice transplanted with over-expressed OCN adipose stem cells.
Detailed Description
The invention is further illustrated below in conjunction with specific examples, which are intended to illustrate the invention and are not to be construed as limiting the invention. One of ordinary skill in the art can appreciate that: many changes, modifications, substitutions and variations may be made to the embodiments without departing from the spirit and principles of the invention, the scope of which is defined by the claims and their equivalents. The experimental procedure, in which no specific conditions are noted in the examples below, is generally carried out according to conventional conditions or according to the conditions recommended by the manufacturer.
Example overexpression of osteocalcin Gene (OCN) can significantly promote neural differentiation of adipose-derived stem cells
1. Experimental materials
1.1 Model mouse feeding
Experimental mice were purchased from Si Bei Fu Biolabs, the mouse strain was C57BL/6. Animal feeding and experimentation met national standards and related requirements, mice were housed in ventilated isolation cages (4-5/cage), kept 7 a.m.: 00-7 pm: 00, the temperature is 21-23 ℃. Animal breeders provide standard food and water to animals daily and closely monitor their health and welfare. All animal experiments were performed in accordance with the "guidelines for care and use of laboratory animals" approved by the military medical institute.
Construction of a traumatic brain injury (traumatic Brain Injury, TBI) mouse model: mice were subjected to brain trauma using a controlled cortical impact (controlled cortical impact, CCI) injury model and a sham-operated control (atraumatic craniotomy), respectively. Prior to CCI injury, each animal was intraperitoneally injected with 2, 2-tribromoethanol (350 mg/kg, sigma-Aldrich, T48402). The animals were placed on a three-dimensional frame, linked to a temperature controlled heating pad (37 ℃), and the scalp dehaired. After exposing the skull, a diameter 4 mm craniectomy was performed at the left parietal bone from the herringbone 1 mm. The brain was then impacted with a pneumatic metal impactor (3 mm diameter) at a speed of 3.5 m/s with a depth of 1.0 mm below the dura mater for a time of 400 ms.
1.2 Primary adipose-derived stem cell-derived suckling mice acquisition
The 3-4 day old suckling mice with primary adipose stem cells were purchased from the company si Bei Fu, the mouse strain was C57BL/6. All animal experiments were performed in accordance with the "guidelines for care and use of laboratory animals" approved by the military medical institute.
Construction of adipose-derived stem cells overexpressing OCN gene: gene synthesis and construction were carried out by the overexpressed OCN plasmid entrusted Ji Kai Gene Limited company, and lentivirus containing the overexpressed gene was packaged with a viral titer of 2.5E+9 TU/mL lentivirus transfected fatThe stem cells had a multiplicity of viral transfection (MOI) of 20. P1 adipose-derived stem cells were counted after being resuspended by digestion centrifugation at 2X 10 5 Number of dishes the resuspended adipose-derived stem cells were re-inoculated into 6 diameter cm dishes and virus transfected after cell attachment. According to the formula: viral volume= (cell number×moi)/viral titer the viral volume required for transfection was calculated as: 2 mL DMEM-HG (serum free) +80. Mu.L polybrene+1.6. Mu.L virus per dish of cells. Supplementing the liquid after 4-6 hours of transfection, and supplementing DMEM-HG complete medium containing 20% FBS; after 24 hours of transfection, the liquid in the dishes was discarded, replaced with 4 mL DMEM-HG complete medium containing 10% FBS, and passaged after the dishes had grown up.
2. Experimental method
2.1 Mouse fat stem cell extraction and cultivation
New rats aged 3 days were purchased from Si Bei Fu Biotechnology, strain C57BL/6. Alcohol is sterilized for 5 min after the mice are killed by neck breaking, and adipose tissues between scapula and inguinal of the mice are respectively extracted and sheared. Type I collagenase digestion is performed for 40-60 min, during which 30 s are vigorously shaken every 10 min. The plates were then filtered, neutralized and centrifuged.
(1) Culture of Primary adipose-derived stem cells in mice
Adipose-derived stem cell culture medium: DMEM-High Glucose (HG) (Gibco) +10% (v/v) Fetal Bovine Serum (FBS), 100U/mL penicillin and 100 μg/mL streptomycin (Gibco).
Taking C57BL/6 milk mice within 3-5 days, soaking in 75% alcohol for sterilization, peeling the skin between scapula and inguinal region under aseptic condition, separating out adipose tissue between scapula and inguinal region, digesting with 0.2% type I collagenase (Biofrox) for 40-60 min, shaking vigorously for 30 s every 10 min during digestion, stopping digestion with DMEM-HG (Gibco) culture medium containing 10% Fetal Bovine Serum (FBS) after the massive tissue is chyle, and filtering with a filter screen. 1000 Centrifuging at rpm for 5 min, removing supernatant, adding DMEM-HG containing 10% FBS, re-suspending cells, inoculating into cell culture dish, and extracting 8×10 cells per mouse 5 The size of the required culture dish is estimated by each cell, the 3 rd cell is changed after inoculation, and the cell growth is observedIn long term, the cells fuse into a monolayer after 4-6 days of culture.
(2) Subculture of primary adipose-derived stem cells
Old medium in the dishes was discarded, washed 2 times with PBS, digested 1-2 min with 0.25% pancreatin, and the cells were observed under a microscope, immediately stopped with complete medium containing 10% Fetal Bovine Serum (FBS) when the cells were rounded, blown with a Pasteur pipette, resuspended with complete medium after centrifugation, and the cell suspension was split equally into two separate aliquots for inoculation into two new dishes.
(3) Neural differentiation induction culture of primary adipose-derived stem cells
Discarding old culture medium in the dish, washing with PBS for 2 times, digesting with 0.25% pancreatin for 1-2 min, observing cell condition under microscope, stopping digestion with complete medium containing 10% Fetal Bovine Serum (FBS) immediately when cells become round, blowing with Pasteur pipette, centrifuging, re-suspending with complete medium, concentrating with 2×10 5 The total culture medium is replaced by DMEM-HG pre-induction culture medium containing 10% of Fetal Bovine Serum (FBS) and 5 mM beta-mercaptoethanol after the cells are attached to the wall, the DMEM-HG pre-induction culture medium containing 10% of Fetal Bovine Serum (FBS), 2% of dimethyl sulfoxide (DMSO), 200 mu M of Butyl Hydroxy Anisole (BHA) and 40 ng/mL of basic fibroblast growth factor (bFGF) is replaced after 24 hours of action, and the culture is carried out in a cell culture box for 3-4 days, and the cell density is up to 80% for passaging.
2.2 qRT-PCR experiments
(1) Adding 200 mu L of chloroform per 1 mL of TRIzol, performing full vortex shaking, standing at room temperature for 10 min, centrifuging at 12000 rpm and 4 ℃ for 15 min, sucking up about 400 mu L of upper clear water phase to a new 1.5 mL RNase-free EP tube, adding 600 mL isopropanol, mixing uniformly upside down, standing at room temperature for 10 min,12000 rpm, centrifuging at 4 ℃ for 15 min, discarding the supernatant, precipitating to room temperature for airing, adding 75% alcohol (prepared by non-enzyme sterile water) 1 mL into each hole for washing, centrifuging at 7500 rpm and 4 ℃ for 7 min, discarding the supernatant, precipitating to room temperature for airing, adding 20 mu L of non-enzyme sterile water for redissolving, and measuring the RNA concentration and RNA quality.
(2) Reverse transcription was performed using RNA as a template, and 1. Mu.g of RNA was reverse transcribed into cDNA according to the reagent specifications recommended system. The reaction conditions were 50℃for 15 min and 85℃for 5 sec.
(3) The qRT-PCR reaction conditions were 95℃for 3 min, 95℃for 5 sec, 60℃for 30 sec,40 cycles, and a dissolution profile was obtained, and data was analyzed. The expression quantity of beta-actin is taken as an internal reference, the qRT-PCR is used for detecting the expression change of related genes of different groups of samples, and the primer sequences are shown in the table 1.
TABLE 1 qRT-PCR primer sequences
2.3 Western blot experiment
(1) Cell preparation (all manipulation on ice): the cell culture dish was taken out and placed on ice, the culture dish was washed 3 times with PBS after the medium was discarded, and PBS was removed from the dish as much as possible to avoid the PBS affecting the quantification of cleaved proteins. To each dish 50 μl of cell lysate was added, the cells at the bottom of the dish were scraped with a clean cell scraper, and the lysate containing the cells was aspirated to a 1.5 mL EP tube and allowed to lyse on ice for 30 min before being frozen at-80 ℃.
Placing the mixture of the cracked protein and the cracked solution into an ice-water mixture, and placing the mixture into an ultrasonic crusher for ultrasonic crushing, wherein 15 sec is one period, and the total period is five. After completion of the disruption, the mixture was centrifuged for 15 min (4 ℃ C., 12000 rpm), and the supernatant was aspirated into a new EP tube, and the pellet was discarded.
(2) Protein concentration detection: mixing the solution A and the solution B in the BCA kit according to the proportion of 1:50 to prepare a reaction solution, diluting protein standard substances with different concentrations by 25 times, mixing the diluted protein standard substances with the prepared reaction solution, and placing the mixture into a water bath kettle for water bath at 37 ℃ for 30 min. And then placing the sample into an enzyme-labeled instrument to measure the absorbance of the standard sample, and preparing a protein standard curve. And respectively adding 1 mu L of protein sample into the reaction solution, repeating the above operation, measuring different absorbance of the protein sample, combining the protein standard curve, and calculating the protein concentration of the sample.
(3) Protein electrophoresis sample preparation: a20. Mu.g protein sample was placed in an EP tube to a constant volume of 20. Mu.L, 5 Xloading buffer was added, the shortage was filled with protein lysate, and after transient centrifugation, the mixture was boiled at 100deg.C for 10 min to stabilize the protein denaturation during electrophoresis. 10 And taking out the sample after the min, rapidly placing the sample on ice for cooling, and placing the sample on ice for standby after the cooling by instantaneous centrifugation again.
(4) Protein electrophoresis: the kit (purchased from Beijing Yingshi Biotechnology Co., ltd.) is simply prepared by using SDS-PAGE gel with 15% concentration, equal amounts of separating gel A liquid and separating gel B liquid are mixed into 6 mL separating gel working liquid, 100X coagulant is added, separating gel is added into a gel-filling glass plate with a thickness of 1.5 mm, and the gel-filling glass plate is sealed with isopropanol liquid and then stands for 30 min. After the separating gel is solidified, the isopropanol is discarded, the equivalent concentrated gel A liquid and B liquid are mixed into 3 mL concentrated gel working liquid, 100 x coagulant is added, the concentrated gel working liquid is added into the solidified separating gel, and then 15-hole sample-adding combs corresponding to the glass plate with the thickness of 1.5 mm are inserted into the separating gel for standing for 30 min.
And (3) putting the solidified electrophoresis gel into an electrophoresis tank, and pulling out the sample adding comb, wherein the sample adding comb is pulled out from left and right at the same time so as not to damage the concentrated gel lanes. After pulling out the loading comb, the sample was placed in the corresponding lane, and 5 μl 180 kDa Prestained Protein Marker (Vazyme Biotech co., ltd.) was placed in the left and right lanes of the sample. After 800 mL electrophoresis liquid is added into the electrophoresis tank, the protein sample is run out of the concentrated gel with 80V voltage, and the voltage is raised to 120V after entering the separation gel to continue electrophoresis.
(5) Protein transfer: a 0.2 μm pore size polyvinylidene fluoride (Polyvinylidene fluoride, PVDF) membrane was cut to the same size as the separator gum and immersed in absolute methanol for at least 30 sec. Using wet transfer printing, soaking sponge and filter paper in an electrotransfer buffer solution, separating a glass plate by using a special glue shovel, separating electrophoresis gel from the glass plate in the electrotransfer buffer solution and attaching the electrophoresis gel to the filter paper, taking out a PVDF film from absolute methanol and tightly attaching the PVDF film to the electrophoresis gel, attaching another piece of filter paper and sponge, tightly attaching the sponge, the filter paper, the glue and the film, and transferring the sponge, the filter paper, the glue and the film into a transfer printing groove, wherein the transfer printing groove is provided with the following placing sequence from a negative electrode (black matrix) to a positive electrode: the method comprises the steps of pouring transfer printing buffer solution into a transfer printing groove, wherein the buffer solution needs to be used for overflowing the sponge gasket. The transfer current was set at a constant current of 0.2 mA for 1 h 40 min.
(6) Blocking and antibody incubation: after the protein transfer is finished, the PVDF film is placed into a pre-prepared TBST solution, the protein contact surface and the non-protein contact surface are distinguished, marks are made, and the PVDF film is rinsed for 1-2 min so as to wash out the buffer solution on the film. The TBST solution was discarded, and after adding 5% skimmed milk, the mixture was slowly shaken on a shaker and blocked at room temperature for 60 min.
According to the dilution factor recommended by the antibody specification, the primary antibody is diluted with 5% skim milk (1:2000), the diluted primary antibody is added immediately after the milk is removed from the block, and the mixture is placed on a 4 ℃ shaker and incubated overnight with slow shaking.
The next day the TBST solution was added and rinsed 3 times for 5 min each. According to the dilution factor recommended by the antibody specification, the secondary antibody (1:5000) is diluted with 5% nonfat milk powder, the diluted secondary antibody solution is added immediately after the membrane is rinsed, and the membrane is placed on a shaker to slowly shake at room temperature for 1 h.
(7) Chemiluminescent development: after recovering the secondary antibody solution, the solution of TBST was added for rinsing 3 times for 5 min each. Equal amounts of the solution A and the solution B of the hypersensitive luminescence solution (ECL) were mixed to prepare 400. Mu.L of working solution. And (3) sticking the PVDF film protein surface on a developing plate upwards and removing bubbles, then dripping ECL working liquid on the surface of the film, fully covering the film protein surface, and then placing the developing plate into a developing instrument for developing.
2.4 Immunofluorescent staining
(1) Cell sample preparation: primary adipose-derived stem cells were cultured to P2-P3, digested and counted, and the primary adipose-derived stem cells were grown to 2X 10 4 The cells of (2) are inoculated to a 24-well plate cell climbing sheet, and the neural differentiation culture of the adipose-derived stem cells is carried out according to a chemical induction method until days 1, 4 and 7.
(2) Cell fixation and blocking: cells were fixed with 4% paraformaldehyde for 20 min, and after removal of paraformaldehyde, a blocking solution containing 0.3% Triton X-100 was added for 20 min.
(3) Antibody incubation: after the blocking is completed, the blocking solution is discarded, the blocking solution is rinsed three times with PBS for 5 min each time, the primary antibody (1:200) is diluted with the blocking solution according to the recommended dilution ratio of the antibody specification, the diluted primary antibody is added, and the mixture is put into a shaking table at 4 ℃ to be slowly shaken and incubated overnight.
According to the recommended dilution ratio of the antibody specification, the secondary fluorescent antibody (1:200) is diluted with a blocking solution, and the secondary fluorescent antibody is rinsed three times with PBS for 5 min each time. After rinsing, the secondary antibody was added and the mixture was placed on a shaker and slowly shaken at room temperature for 2 h.
(4) Sealing piece: after the secondary antibody incubation is finished, rinsing with PBS for three times, each time for 5 min, dripping a drop of DAPI-containing sealing tablet on a glass slide after rinsing, attaching the cell surface of the 24-pore plate cell climbing tablet to the sealing tablet of the glass slide, and observing under a fluorescence microscope after the sealing tablet is dried.
2.5 Treatment of TBI mice
The common adipose-derived stem cells and the adipose-derived stem cells which over express the OCN gene are respectively used for carrying out therapeutic intervention on the damaged part in the acute phase, and the specific experimental grouping and treatment method is as follows: mice were divided into Sham surgery (Sham), surgical model (TBI), adipose stem cell intervention (TBI-ADSC), and overexpressed adipose stem cell intervention (TBI-OE-ADSC). In CCI injury model construction, 5. Mu.L Matrigel (Corning, 356, 234) was administered to each mouse of TBI-ADSC group and TBI-OE-ADSC group, respectively, and 5X 10 was mixed 5 Individual adipose stem cells or overexpressing OCN adipose stem cells are transplanted into brain tissue injury areas.
Open field experiments are carried out on the second day after operation, and new object identification experiments are carried out on the third day, wherein the specific experimental method is as follows:
open field experiment: mice were placed in a square box of 50 cm x 50 cm from one corner thereof, allowed to adapt to a 1 min open field environment in the square box, then were recorded with a camera for a 5 min walk trajectory, and analyzed for total distance, total time, distance and time in the center area for 5 min.
New object identification experiment: the same square box is used for open field experiment, two identical objects (blue cylinders) are placed in the square box in the afternoon of the next day, and then mice are placed in the square box respectively for free exploration for 10 min. The next day a new object (yellow cone) and an old object (blue cylinder) were placed in the square box, after which mice were placed in the square box for free exploration for 5 min, respectively, and recorded using a video camera. The exploration time of the mice for the new object within 5 min was analyzed and a cognitive index (Recognition Index, RI), ri=new object exploration time/(new object exploration time+old object exploration time)%.
3. Experimental results
3.1 Morphological observation of ADSC neurogenic differentiation
Under the microscope, it was observed that after 24 h of pre-induction in the pre-induction medium containing 5 mM beta-mercaptoethanol, the adipose stem cells lost the morphology of mesenchymal cells and the retraction became approximately circular. Some cells begin to appear as nerve cells one day after the change to the chemically induced medium, and dendrites begin to appear. Over time, an increasing number of neural cell characteristics appeared, and on day 7, the onset of synapses was observed (see FIG. 1), demonstrating that chemically inducing adipose stem cells into neural differentiation was morphologically effective.
The number of neuronal-like cells was significantly increased in the same induction process over-expressed OCN groups compared to normal adipose stem cells (see fig. 2), indicating that over-expressed OCN was able to significantly promote neural differentiation of adipose stem cells.
3.2 Increased OCN expression during ADSC neurogenic differentiation and a trend towards anti-apoptosis
qRT-PCR results showed that the expression of OCN was gradually increased as the neurogenic differentiation of adipose stem cells progressed (see FIG. 3). Under the same induced differentiation conditions, the expression level of the neural stem cell markers and the neuron markers of the over-expressed OCN genome (OE-ADSC) is obviously higher than that of the common adipose stem cell group (ADSC) (see figure 4), and the expression level of apoptosis-related markers such as Ki67, MCM2, PCNA, bcl-2, bax and the like is obviously higher than that of the common adipose stem cell group, which indicates that the over-expressed OCN gene can promote the neurogenic differentiation of adipose stem cells and has obvious anti-apoptosis effect (see figure 5).
3.3 OCN promotes ADSC neurogenic differentiation
The invention adopts Western blot experiment to verify that a large amount of OCN protein is generated in the process of neurogenic induced differentiation (see figure 6), and the over-expressed OCN group shows the tendency of anti-apoptosis in apoptosis proliferation related marker protein molecules in the process of neurogenic induced differentiation (see figure 7).
3.4 OCN promotes functional reconstitution of ADSC for acute traumatic brain injury
After the TBI of the mice, common adipose-derived stem cells and adipose-derived stem cells which over-express the OCN genes are used for carrying out therapeutic intervention on the damaged parts in the acute phase. Open field experiments were performed the next day after surgery, and the experiments found that the over-expressed cell intervention group was more pronounced against anxiety-depression-like behavior caused by TBI than the normal cell intervention group (see fig. 8); the new object recognition experiments performed on the third day found that the learning ability of the overexpressed cell-mediated group was recovered better than that of the normal cell-mediated group (see fig. 9). The above results indicate that OCN is capable of promoting functional remodeling of traumatic brain injury by ADSCs.
The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims (6)

1. The application of the agent for over-expressing the osteocalcin gene in promoting the neural differentiation of the adipose-derived stem cells cultured in vitro is characterized in that the agent for over-expressing the osteocalcin gene is a carrier for over-expressing the osteocalcin gene.
2. A method of promoting neural differentiation of adipose-derived stem cells cultured in vitro, comprising the steps of: the osteocalcin gene is over-expressed in the adipose-derived stem cells, so that the adipose-derived stem cells over-expressing the osteocalcin gene are obtained, and are cultured in a pre-induction culture medium and then are cultured in a neurogenic induction culture medium.
3. The method of claim 2, wherein the pre-induction medium is DMEM-HG medium comprising 10% FBS, 5 mM β -mercaptoethanol.
4. The method of claim 2, wherein the neurogenic induction medium is DMEM-HG medium comprising 10% FBS, 2% DMSO, 200 μΜ BHA, 40 ng/mL bFGF.
5. The method of claim 2, wherein the time of culturing in the pre-induction medium is 24 h;
the time of culture in the neurogenic induction medium was 7 days.
6. Use of adipose-derived stem cells overexpressing osteocalcin gene for the preparation of a medicament for the treatment of traumatic brain injury.
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