CN110283788A - A kind of induced myeloid astroglia reprogramming is the method for motor neuron - Google Patents
A kind of induced myeloid astroglia reprogramming is the method for motor neuron Download PDFInfo
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Abstract
The present invention provides the method that a kind of reprogramming of induced myeloid astroglia is motor neuron, comprising the following steps: (1) Primary rat astroglia purifying culture；(2) substance needed for forming the stage of development according to Neuronal induction and motor neuron has chosen 7 kinds of small-molecule drugs SB431542, LDN-193189, RA, bFGF, Purmorphamine, Forskolin, VPA, these small-molecule drugs are by carrying out induction reprogramming to astroglia in vitro；(3) metamorphosis and the analysis of Immunofluorescence technology of cell are observed, real-time fluorescence quantitative PCR analysis and statistical analysis method carry out analysis astroglia to the reprogramming of motor neuron and differentiation situation.Small-molecule drug combination of the invention can in vitro successfully reprogram Rat Astroglia induction for motor neuron, and transdifferentiation rate is more than 75%；May in spinal cord injury reparation and the treatment of reconstruction more practicable thinking.
Description
Technical field
The invention belongs to medicine technology fields, and in particular to a kind of induced myeloid astroglia reprogramming is refreshing for movement
Method through member.
Background technique
The clinical symptoms of spinal cord injury (spinal cord injury, SCI) are from slight feeling, injury gained in sports to limbs
Complete paralysis, depend primarily on the degree of injury and range of injury.Astroglia is right under normal physiological conditions
Nutrition, support and adjusting of neuron etc. all play a decisive role, however when spinal cord is once damaged, astroglia is thin
Born of the same parents will prompt activation be reactive astrocytes and to be constantly proliferated, glial scars are formed around damage location.Although
The cells such as sprawling that glial scars can control inflammation and the neuron for protecting survival, but its formation constitutes one again simultaneously
The barrier of a physics and chemistry, seriously hinders the regeneration of aixs cylinder, and has research it has also been found that some reactive spongiocytes can
It can generate neurotoxic effect.Since dead or apoptosis can occur for the neuron being damaged after spinal cord injury, neuron is caused
The decline of quantity.Even if also remaining a small amount of neural stem cell in adult spinal cord, also enough the neuron lost can not be supplemented.In this way
Result finally make the neuronal quantity of damaged spinal cord that can not be restored to normal level, add adult neurons axon regeneration
Inferior capabilities, so that the reparation of entire damaged spinal cord nerve fiber and reconstruction be made to become medicine a great problem.
Cell transplantation, especially neural stem cells transplantation have always been considered as being substitution damaged cell, protection neuron and rush
Into a kind of possible ways of nerve regneration reparation.But after transplanted cells enter damage spinal cord local organization, it is more difficult to orientation point
Turn to thesocyte (randomness of differentiation is larger).And after stem cell lacks stable cell origin, Medical Ethics, transplanting
The problems such as immunosupress, limits its clinical application.2006, induce multi-potent stem cell (induced pluripotent
Stem cells, iPSCs) appearance of technology opens the new era of regenerative medicine.However when it is applied to spinal cord injury reparation
When, researcher is it has been found that there are many applications limitations.Firstly, being usually used in the transcription factor of reprogramming in various types of tumours
In high expression, iPSCs Tumor formation with higher can be made in this way；Secondly, after spinal cord injury about 2 weeks or so be neural restoration
Best period, and iPSCs entirely induces the overlong time of differentiation, can not complete to break up within best repair period and repair；Again
Person, iPSCs not can guarantee orientation at all in complicated spinal cord and be efficiently divided into neuron, especially kinesitherapy nerve in environment
Member.Finally, neural stem cells transplantation process certainly will cause secondary damage to nerve fiber again.Recent study personnel are successfully led to
It crosses directly reprogramming and Exocrine Pancreas In Rats is transformed into beta cell, or fibroblast is induced to differentiate into cardiac muscle cell and mind
It is reported through member etc..So why not gather materials on the spot, directly damage location is activated and largely the astroglia that is proliferated as
Inducing target cell, reprogramming become neuron (induced neurons, iNs), on the one hand can mitigate and hinder axon growth
On the other hand glial scars can also supplement the neuron lost by damage with direct in-situ, that kills two birds with one stone reaches solution spinal cord
Two hang-ups of reparative regeneration are influenced after damage.
Summary of the invention
It is kinesitherapy nerve the technical problem to be solved in the present invention is to provide a kind of reprogramming of induced myeloid astroglia
The method of member, to solve the problems, such as proposed in background technique.
In order to solve the above technical problems, the embodiment of the present invention provides a kind of reprogramming of induced myeloid astroglia is
The method of motor neuron, which comprises the following steps: (1) Primary rat astroglia purifying culture, with new
Target cell of the primary astroglial cells of raw rat spinal cord as induction reprogramming, by the in vitro culture of differential attachment method with
Microwave shake culture obtains the astroglia of purity is high；(2) development is formed according to Neuronal induction and motor neuron
Substance needed for stage have chosen 7 kinds of small-molecule drug SB431542, LDN-193189, RA, bFGF, Purmorphamine,
Forskolin, VPA, these small-molecule drugs are by carrying out induction reprogramming to astroglia in vitro；(3) cell is observed
Metamorphosis and Immunofluorescence technology analysis, real-time fluorescence quantitative PCR analysis and statistical analysis method carry out
Astroglia is analyzed to the reprogramming of motor neuron and differentiation situation.
Further, the step (1) specifically includes following procedure:
(1-1) superclean bench shifts to an earlier date ultraviolet light irradiation 30min, the ice platform for taking out -20 DEG C of pre-coolings, the surgical device sterilized
Tool, HBSS buffer and plastic culture dish etc. are placed in superclean bench, and appropriate HBSS will be added in culture dish and be placed in ice
It is spare on platform；
(1-2) takes newborn 1 age in days rat to be placed in the 50ml centrifuge tube for being placed with appropriate ether, after anesthesia with 75% alcohol
Disinfection, and dried with sterile cotton balls；
While (1-3) is breaked end using tissue shear, sterile cotton balls hemostasis by compression, fixing rat body with hand makes its holding
Stretch posture；
(1-4) cuts off skin along backbone direction with tissue shear, and musculi colli is cut off；
(1-5) cuts off back spinal and tissue along backbone direction from being broken with staight scissors from neck, exposing spinal cord；
Two sides dorsal root ganglion is truncated with microforceps and is chosen spinal cord spare in the HBSS for be placed in pre-cooling by (1-6)；
Spinal cord is moved in another culture dish equipped with pre-cooling HBSS and is placed under stereomicroscope by (1-7), is shelled with microforceps
The spinal cord being stripped clean taking-up is placed in the HBSS of pre-cooling by the blood stains point of spinal meninges and surface from surface, and whole process exists
It is carried out on ice platform；
Spinal cord is shredded using microscissors and is placed it in 15ml centrifuge tube by (1-8), and the pancreatin of 2ml 0.25% is added,
Centrifuge tube is placed in 37 DEG C of water-baths, every 5min concussion mixes until tissue block decomposes, and then into centrifuge tube, addition 5ml is complete
Full nutrient solution terminates digestion, after moving into centrifuge, is centrifuged 5min with 1000rpm revolving speed, discards supernatant addition 4ml base training and is resuspended again
It is centrifuged 5min with 1000rpm revolving speed, is repeated 2 times, cell finally is resuspended with 4ml complete culture solution and is seeded to 25cm2Culture bottle
In；
Culture bottle is inverted in CO by (1-9)2Cultivate 20min in incubator, take out cell suspension in 15ml centrifuge tube with
1000rpm revolving speed is centrifuged 5min, and after removing supernatant and complete culture solution resuspension being added, 25cm is added in cell suspension again2Culture
In bottle, it is placed in 5%CO2It is cultivated in incubator, this cell marking is P0 generation；
(1-10) changes within every two days liquid until cell confluent cultures bottle, culture bottle is placed in 37 DEG C of microwave concussion and cultivate cases
Oligodendroglia and microglia are removed with 160rpm shake culture 18h；
Culture solution is discarded after (1-11) shake culture, and PBS is added and rinses 2-3 times, then adds the 1.5ml of pre-temperature
0.25% trypsin digestion cell is blown afloat adherent until cell can gently be blown down after addition 5ml complete culture solution termination digestion
Cell, cell suspension are pipetted into 15ml centrifuge tube, 1000rpm centrifugation 5min after discard supernatant, be added 1ml complete culture solution into
Row passage；
The culture of (1-12) astroglia is alternative in subsequent experimental to P2-P3.
Further, the step (2) specifically includes following procedure:
(2-1) takes out the coverslip being immersed in 75% alcohol and is placed in 24 porocyte culture plates, and sterile ultrapure water is added
Rinsing 3 times；
(2-2) exhausts ultrapure water, and the poly-D-lysine coating that appropriate 100ng/ml is added overnight, is drawn extra more for second day
Polylysine is added sterile ultrapure water and rinses 3 times, exhausts surplus liquid, dry spare；Cleaning process is soft, it is ensured that poly relies
Propylhomoserin is not fallen off；
(2-3) when step (1) astrocytes culture to P2-P3 for when, be added PBS rinse 2-3 times, be then added
0.25% trypsin digestion cell of 2ml of pre-temperature is added 5ml complete culture solution and terminates until attached cell can gently blow down
Cell suspension is added in 15ml centrifuge tube after digestion, is discarded supernatant after 1000rpm centrifugation 5min, 1ml complete culture solution is added
Cell is resuspended, with 4 × 104Cell concentration is inoculated in 24 orifice plates containing coating poly-D-lysine, is put into 5%CO2Incubator culture
Overnight；
(2-4) second day culture solution discarded in 24 well culture plates is rinsed 2-3 times with the PBS of pre-temperature, by drug-induced training
Nutrient solution one is uniformly mixed, and is added in culture hole, culture plate is put into 5%CO2Incubator culture；
After (2-5) drug-induced culture solution one is cultivated 2 days, drug-induced culture solution two is changed in cell culture well and is placed in
5%CO2Incubator culture；
After (2-6) drug-induced culture solution two is cultivated 8-10 days, drug-induced culture solution three is changed in cell culture well simultaneously
It is placed in 5%CO2Incubator culture changes liquid in every 2-3 days.
Further, the analysis of Immunofluorescence technology specifically includes following procedure in the step (3):
(3-1-1) takes out 24 porocyte culture plates, and the culture solution in hole to be dyed is exhausted, and the PBS rinsing of pre-temperature is added
It 2-3 times, takes out coverslip and is placed in another 24 orifice plates equipped with 4% paraformaldehyde of 300ml pre-temperature, fixed 10min；
(3-1-2) discards 4% paraformaldehyde, and every hole is added 300 μ l PBS and rinses 3 times, each 5min；
(3-1-3) discards PBS, and 300 μ l rupture of membranes liquid are added in every hole, and room temperature is incubated for 30min；
(3-1-4) discards rupture of membranes liquid, and 300 μ l confining liquids are added in every hole, and room temperature is incubated for 30min；
(3-1-5) discards confining liquid, and 300 μ l primary antibodies and confining liquid mixed liquor, 4 DEG C of overnight incubations are added in every hole；
(3-1-6) discards primary antibody for second day, and every hole is added 300 μ l PBS and rinses 3 times, each 5min；
(3-1-7) discards PBS, and the mixed liquor of 300ml secondary antibody and PBS is added in every hole, and 4 DEG C of incubation >=2h are carried out in the dark place
Reason；
(3-1-8) discards secondary antibody mixed liquor, and every hole is added 300 μ l PBS and rinses 3 times, and each 5min is carried out in the dark processing；
The Hoechst room temperature that PBS thinner ratio 1:1000 is added in (3-1-9) is incubated for 10min, discards Hoechst dilution, adds
Enter 300ml PBS to rinse 3 times, each 5min is carried out in the dark processing；
(3-1-10) takes adhesiveness glass slide to drip upper mounting liquid, and coverslip is carried out mounting, is protected from light processing；
(3-1-11) is observed and is photographed to record under laser confocal microscope.
Further, real-time fluorescence quantitative PCR analysis specifically includes following procedure in the step (3):
(3-2-1) takes out celliferous culture dish, discards culture medium, and rinsed 3 times with 37 DEG C of PBS are preheated to, 1ml is added
Trizol gently blows and beats and mixes cell, and collects cell suspension in 1.5ml EP pipe, stands 5min on ice；
(3-2-2) concussion of 250 μ l chloroforms is added into EP pipe and mixes, and is placed in -20 DEG C of refrigerators and stands 1-2h；Take out EP
Pipe 12000rpm, is centrifuged 5 minutes by 4 DEG C；
(3-2-3) goes supernatant liquor in another EP pipe, and isometric isopropanol is added thereto, is mixed by inversion,
12000rpm, is centrifuged 10min by 4 DEG C；
(3-2-4) carefully discards the ethyl alcohol that 1ml 75% is added in liquid, and 7500rpm, is centrifuged 5min, discards liquid by 4 DEG C,
Repeat this step；
1ml dehydrated alcohol is added in (3-2-5), and 7500rpm 4 DEG C, is centrifuged 5min, discards liquid, and EP pipe is placed in ultra-clean
It is dried up in platform；
(3-2-6) 20 μ l DEPC water dissolution RNA is added into EP pipe, and RNA is carried out reverse transcription, it is remaining be stored in-
80 DEG C of refrigerators；
(3-2-7) takes Reverse Transcriptase kit, and the system of following table carries out reverse transcription to RNA with when PCR reaction condition:
The cDNA that (3-2-8) reverse transcription obtains is expanded for qPCR, remaining to be stored in -80 DEG C of refrigerators；
(3-2-9) takes reagent in FastStart Universal SYBR Green Master to be placed on ice chest, following table
System carry out PCR amplification with when reaction condition:
Solubility curve is analyzed after (3-2-10) PCR amplification, it is ensured that PCR product specificity.
Further, statistical analysis specifically includes following procedure in the step (3): by software Graphpad
Prism5 carries out data statistics, for statistical analysis to all quantized datas and indicated with mean+SD；Sided t is examined
The significant property of statistics for calculating p value, p value < 0.05 have statistical significance.
Wherein, the complete culture solution is using 1% dual anti-of addition in DMEM/F12 basal medium and 10% FBS
Method be made.
10. wherein, the drug-induced culture solution one is using addition 2 μM of SB431542,1 μ in basic induction broth
M RA, it 5 μM of Forskolin, 20ng/ml bFGF, 0.25 μM of LDN-193189, is made；The drug-induced culture solution two is adopted
5 μM of Purmorphamine, 10 μM of RA, 20 μM of Forskolin, 200ng/ml are added in basic induced medium
BFGF is made；The drug-induced culture solution three is using addition 1mM VPA, 5 μM in basic induced medium
Purmorphamine, 10 μM of RA, 20 μM of Forskolin, 200ng/ml bFGF are made.
Above-mentioned, the basis induction broth is using dual anti-, the 1M for adding 1% in DMEM/F12 basal medium
B27、1M N2, 2mM Pidolidone, 3.5mM glucose is made.
The advantageous effects of the above technical solutions of the present invention are as follows: small-molecule drug combination of the invention can succeed in vitro
Ground reprograms Rat Astroglia induction for motor neuron, and transdifferentiation rate is more than 75%；In addition, the small molecule medicine
Object combination can also successfully complete the neuronotropic transdifferentiation of source of people astroglia；To the original position directly weight of astroglia
Be programmed to neuron, may in spinal cord injury reparation and the treatment of reconstruction more practicable thinking.
Detailed description of the invention
Fig. 1 is P0 in the present invention for the GFAP Immunofluorescence colored graph of astroglia；
P2 is dyed for GFAP the and Nestin Immunofluorescence of astroglia in Fig. 2 respectively present invention
Figure；
Fig. 3 is respectively small-molecule drug group astroglia aspect graph at 1 day, 3 days, 9 days, 20 days from left to right；
Fig. 4 is respectively control group astroglia aspect graph at 1 day, 3 days, 9 days, 20 days from left to right；
Small-molecule drug group GFAP and Tuj1 colored graph when Fig. 5 is respectively external evoked astroglia 6 days；
Control group GFAP and Tuj1 colored graph when Fig. 6 is respectively external evoked astroglia 6 days；
Fig. 7 is the partial enlarged view of Fig. 5；
MAP2 and NeuN colored graph when Fig. 8 is respectively the external evoked astroglia of small-molecule drug 10 days；
MAP2 colored graph when Fig. 9 is the external evoked astroglia of small-molecule drug 16 days；
Figure 10 is transdifferentiation rate (mean ± SEM, the n that small-molecule drug inducing astrocytes transdifferentiation is neuron
=3independent experiments)；
Figure 11 is relative expression's situation of small molecule inducing astrocytes different times related gene；
MAP2, HB9 and CHAT colored graph when Figure 12 is respectively small-molecule drug inducing astrocytes 16 days；
MAP2, HB9 and CHAT colored graph when Figure 13 is respectively small-molecule drug inducing astrocytes 30 days；
MAP2 and GAD65 colored graph when Figure 14 is respectively small-molecule drug inducing astrocytes 18 days；
MAP2 and VGLUT1 colored graph when Figure 15 is respectively small-molecule drug inducing astrocytes 18 days；
Figure 16 be small-molecule drug inducing astrocytes 16 days when Induction of neuronal HB9, CHAT, VGLUT1 and
GAD65 positive rate.
Specific embodiment
To keep the technical problem to be solved in the present invention, technical solution and advantage clearer, below in conjunction with attached drawing and tool
Body embodiment is described in detail.
A kind of induced myeloid astroglia reprogramming is the method for motor neuron, comprising the following steps:
(1) Primary rat astroglia purifying culture, using the primary astroglial cells of neonate rat spinal cord as
The target cell for inducing reprogramming obtains the astroglia of purity is high by the in vitro culture and microwave shake culture of differential attachment method
Cell；Specifically include following procedure:
(1-1) superclean bench shifts to an earlier date ultraviolet light irradiation 30min, the ice platform for taking out -20 DEG C of pre-coolings, the surgical device sterilized
Tool, HBSS buffer and plastic culture dish etc. are placed in superclean bench, and appropriate HBSS will be added in culture dish and be placed in ice
It is spare on platform.
(1-2) takes newborn 1 age in days rat to be placed in the 50ml centrifuge tube for being placed with appropriate ether, after anesthesia with 75% alcohol
Disinfection, and dried with sterile cotton balls.
While (1-3) is breaked end using tissue shear, sterile cotton balls hemostasis by compression, fixing rat body with hand makes its holding
Stretch posture.
(1-4) cuts off skin along backbone direction with tissue shear, and musculi colli is cut off.
(1-5) cuts off back spinal and tissue along backbone direction from being broken with staight scissors from neck, exposing spinal cord.
Two sides dorsal root ganglion is truncated with microforceps and is chosen spinal cord spare in the HBSS for be placed in pre-cooling by (1-6).
Spinal cord is moved in another culture dish equipped with pre-cooling HBSS and is placed under stereomicroscope by (1-7), is shelled with microforceps
The spinal cord being stripped clean taking-up is placed in the HBSS of pre-cooling by the blood stains point of spinal meninges and surface from surface, and whole process exists
It is carried out on ice platform.
Spinal cord is shredded using microscissors and is placed it in 15ml centrifuge tube by (1-8), and the pancreatin of 2ml 0.25% is added,
Centrifuge tube is placed in 37 DEG C of water-baths, every 5min concussion mixes until tissue block decomposes, and then into centrifuge tube, addition 5ml is complete
Full nutrient solution terminates digestion, after moving into centrifuge, is centrifuged 5min with 1000rpm revolving speed, discards supernatant addition 4ml base training and is resuspended again
It is centrifuged 5min with 1000rpm revolving speed, is repeated 2 times, cell finally is resuspended with 4ml complete culture solution and is seeded to 25cm2Culture bottle
In.
Culture bottle is inverted in CO by (1-9)2Cultivate 20min in incubator, take out cell suspension in 15ml centrifuge tube with
1000rpm revolving speed is centrifuged 5min, and after removing supernatant and complete culture solution resuspension being added, 25cm is added in cell suspension again2Culture
In bottle, it is placed in 5%CO2It is cultivated in incubator, this cell marking is P0 generation.
(1-10) changes within every two days liquid until cell confluent cultures bottle, culture bottle is placed in 37 DEG C of microwave concussion and cultivate cases
Oligodendroglia and microglia are removed with 160rpm shake culture 18h.
Culture solution is discarded after (1-11) shake culture, and PBS is added and rinses 2-3 times, then adds the 1.5ml of pre-temperature
0.25% trypsin digestion cell is blown afloat adherent until cell can gently be blown down after addition 5ml complete culture solution termination digestion
Cell, cell suspension are pipetted into 15ml centrifuge tube, 1000rpm centrifugation 5min after discard supernatant, be added 1ml complete culture solution into
Row passage.
The culture of (1-12) astroglia is alternative in subsequent experimental to P2-P3.
(2) substance needed for forming the stage of development according to Neuronal induction and motor neuron has chosen 7 kinds of small molecules
Drug SB431542, LDN-193189, RA, bFGF, Purmorphamine, Forskolin, VPA, these small-molecule drugs are logical
It crosses and induction reprogramming is carried out to astroglia in vitro；Specifically include following procedure:
(2-1) takes out the coverslip being immersed in 75% alcohol and is placed in 24 porocyte culture plates, and sterile ultrapure water is added
Rinsing 3 times；
(2-2) exhausts ultrapure water, and the poly-D-lysine coating that appropriate 100ng/ml is added overnight, is drawn extra more for second day
Polylysine is added sterile ultrapure water and rinses 3 times, exhausts surplus liquid, dry spare；Cleaning process is soft, it is ensured that poly relies
Propylhomoserin is not fallen off；
(2-3) when step (1) astrocytes culture to P2-P3 for when, be added PBS rinse 2-3 times, be then added
0.25% trypsin digestion cell of 2ml of pre-temperature is added 5ml complete culture solution and terminates until attached cell can gently blow down
Cell suspension is added in 15ml centrifuge tube after digestion, is discarded supernatant after 1000rpm centrifugation 5min, 1ml complete culture solution is added
Cell is resuspended, with 4 × 104Cell concentration is inoculated in 24 orifice plates containing coating poly-D-lysine, is put into 5%CO2Incubator culture
Overnight；
(2-4) second day culture solution discarded in 24 well culture plates is rinsed 2-3 times with the PBS of pre-temperature, by drug-induced training
Nutrient solution one is uniformly mixed, and is added in culture hole, culture plate is put into 5%CO2Incubator culture；
After (2-5) drug-induced culture solution one is cultivated 2 days, drug-induced culture solution two is changed in cell culture well and is placed in
5%CO2Incubator culture；
After (2-6) drug-induced culture solution two is cultivated 8-10 days, drug-induced culture solution three is changed in cell culture well simultaneously
It is placed in 5%CO2Incubator culture changes liquid in every 2-3 days.
(3) observe cell metamorphosis and Immunofluorescence technology analysis, real-time fluorescence quantitative PCR analysis and
Statistical analysis method carries out analysis astroglia to the reprogramming of motor neuron and differentiation situation.
Wherein, Immunofluorescence technology analysis specifically includes following procedure:
(3-1-1) takes out 24 porocyte culture plates, and the culture solution in hole to be dyed is exhausted, and the PBS rinsing of pre-temperature is added
It 2-3 times, takes out coverslip and is placed in another 24 orifice plates equipped with 4% paraformaldehyde of 300ml pre-temperature, fixed 10min；
(3-1-2) discards 4% paraformaldehyde, and every hole is added 300 μ l PBS and rinses 3 times, each 5min；
(3-1-3) discards PBS, and 300 μ l rupture of membranes liquid are added in every hole, and room temperature is incubated for 30min；
(3-1-4) discards rupture of membranes liquid, and 300 μ l confining liquids are added in every hole, and room temperature is incubated for 30min；
(3-1-5) discards confining liquid, and 300 μ l primary antibodies and confining liquid mixed liquor, 4 DEG C of overnight incubations are added in every hole；
(3-1-6) discards primary antibody for second day, and every hole is added 300 μ l PBS and rinses 3 times, each 5min；
(3-1-7) discards PBS, and the mixed liquor of 300ml secondary antibody and PBS is added in every hole, and 4 DEG C of incubation >=2h are carried out in the dark place
Reason；
(3-1-8) discards secondary antibody mixed liquor, and every hole is added 300 μ l PBS and rinses 3 times, and each 5min is carried out in the dark processing；
The Hoechst room temperature that PBS thinner ratio 1:1000 is added in (3-1-9) is incubated for 10min, discards Hoechst dilution, adds
Enter 300ml PBS to rinse 3 times, each 5min is carried out in the dark processing；
(3-1-10) takes adhesiveness glass slide to drip upper mounting liquid, and coverslip is carried out mounting, is protected from light processing；
(3-1-11) is observed and is photographed to record under laser confocal microscope.
Wherein, quantitative fluorescent PCR analysis specifically includes following procedure:
(3-2-1) takes out celliferous culture dish, discards culture medium, and rinsed 3 times with 37 DEG C of PBS are preheated to, 1ml is added
Trizol gently blows and beats and mixes cell, and collects cell suspension in 1.5ml EP pipe, stands 5min on ice；
(3-2-2) concussion of 250 μ l chloroforms is added into EP pipe and mixes, and is placed in -20 DEG C of refrigerators and stands 1-2h；Take out EP
Pipe 12000rpm, is centrifuged 5 minutes by 4 DEG C；
(3-2-3) goes supernatant liquor in another EP pipe, and isometric isopropanol is added thereto, is mixed by inversion,
12000rpm, is centrifuged 10min by 4 DEG C；
(3-2-4) carefully discards the ethyl alcohol that 1ml 75% is added in liquid, and 7500rpm, is centrifuged 5min, discards liquid by 4 DEG C,
Repeat this step；
1ml dehydrated alcohol is added in (3-2-5), and 7500rpm 4 DEG C, is centrifuged 5min, discards liquid, and EP pipe is placed in ultra-clean
It is dried up in platform；
(3-2-6) 20 μ l DEPC water dissolution RNA is added into EP pipe, and part RNA is carried out reverse transcription (according to each
RNA amount needed for testing is different, and RNA concentration is not less than 200ng/ μ l)；It is remaining to be stored in -80 DEG C of refrigerators；
(3-2-7) takes Reverse Transcriptase kit, and the system of following table carries out reverse transcription to RNA with when PCR reaction condition:
The part cDNA that (3-2-8) reverse transcription obtains (is carried out true for qPCR amplification according to cDNA amount needed for each test
It is fixed), it is remaining to be stored in -80 DEG C of refrigerators；
(3-2-9) takes reagent in FastStart Universal SYBR Green Master to be placed on ice chest, following table
System carry out PCR amplification with when reaction condition:
Solubility curve is analyzed after (3-2-10) PCR amplification, it is ensured that PCR product specificity.
Wherein, statistical analysis specifically includes following procedure: data statistics is carried out by software Graphpad prism5,
It is for statistical analysis to all quantized datas and indicated with mean+SD；Sided t examines the statistics for calculating p value aobvious
Property, p value < 0.05 have statistical significance.
Above-mentioned, the complete culture solution is using in DMEM/F12 basal medium addition 1% dual anti-and 10%
The method of FBS is made.
Above-mentioned, the drug-induced culture solution one is using addition 2 μM of SB431542,1 μM in basic induction broth
RA, it 5 μM of Forskolin, 20ng/ml bFGF, 0.25 μM of LDN-193189, is made；The drug-induced culture solution two uses
5 μM of Purmorphamine, 10 μM of RA, 20 μM of Forskolin, 200ng/ml bFGF are added in basic induced medium
It is made；The drug-induced culture solution three using added in basic induced medium 1mM VPA, 5 μM of Purmorphamine,
10 μM of RA, 20 μM of Forskolin, 200ng/ml bFGF are made.
Above-mentioned, the basis induction broth is using dual anti-, the 1M for adding 1% in DMEM/F12 basal medium
B27、1M N2, 2mM Pidolidone, 3.5mM glucose is made.
Small-molecule drug combination of the invention can in vitro successfully transport Rat Astroglia induction reprogramming
Dynamic neuron, and transdifferentiation rate is more than 75%；In addition, small-molecule drug combination can also successfully complete source of people astroglia
Neuronotropic transdifferentiation；The original position of astroglia is directly reprogrammed into neuron, may in spinal cord injury reparation and
More practicable thinking in the treatment of reconstruction.
The present invention can obtain following experimental results:
As a result 1, the form of spinal cord astrocytes and purifying rate:
The astroglia in rat spinal cord source is removed in being seeded to Tissue Culture Flask by differential attachment method a small amount of
Fibroblast, then add complete medium culture promote spongiocyte division growth.Since P0 is also deposited in cell
In a small amount of neuron, oligodendroglia and microglia, it is still in diversity (figure that cellular morphology is observed under phase contrast microscope
1).Until P0 reaches 80% or so for cell fusion degree, heteroproteose cell is removed by microwave shake culture and secondary culture is to P2 generation
Afterwards, with collagenous fibres acidic protein (GFAP) distinctive in astroglia dye, identification GFAP positive cell up to 95% with
Upper (Fig. 1, Fig. 2).P2 is in flat polygon shape for cellular morphology majority under phase contrast microscope, and cell body is larger, and nucleus is big
And it is obvious, cell has a small amount of irregular raised (Fig. 2).
As a result 2, the astroglia of purifying expresses Nestin situation
A small amount of neural stem cell may be mixed with by extracting in the astroglia that neonate rat spinal cord obtains.Nerve
Stem cell is a kind of mother cell with higher division potential and self-renewal capacity, can be led to when body meets with special circumstances
It crosses Proliferation, Differentiation and forms different types of neural tissue cell, such as neuron, astroglia, oligodendroglia.Greatly
Quantifier elimination find reactive astrocytes after injury region activation, also can self-renewing be proliferated and obtains similar to nerve cord
The potential of cell；In addition, studies have found that the astroglia cultivated in vitro, can make cell when certain conditions are suitable for
Form changes, and then the state activated when similar damage in vivo occurs, and finally express partial nerve stem-cell marker
Object is even dedifferentiated into class neural stem cell.Therefore before this experiment is reprogrammed, exclusion cell purification culture first
Cheng Zhong, the dedifferen tiation for the astroglia that may cause are thin to the astroglia for being ready for small-molecule drug induction
Born of the same parents carry out double mark immunocytochemical stains identification of GFAP and neural stem cell marker nestin Nestin.The results show that
Astroglia by purifying secondary culture does not express Nestin, this indicates that in the cell of in vitro culture and is not mixed into
Neural stem cell, and astroglia itself does not also occur being dedifferentiated into class neural stem cell phenomenon (Fig. 2).
As a result 3, the metamorphosis of small-molecule drug combination inducing astrocytes
The reprogramming of astroglia is not solely dependent on transcription factor, and small-molecule drug also has certain induction to rearrange
Cheng Zuoyong.This test we using SB431542, LDN-193189, RA, bFGF, Purmorphamine, Forskolin,
VPA is reprogrammed.Astroglia acts on 2 days first in drug-induced culture solution one, then replaces with drug and lures
It leads culture solution two to act on 6 days, finally replaces with drug-induced culture solution three and remake use, entire Induction Process changes liquid in every 2-3 days.I
Observe in the Induction Process of small-molecule drug, astrocyte morphology changes, and cell space is gradually reduced, cell is dashed forward
It rises and extends and attenuate；And although cellular control unit form also has part change, there is no to neuron Morphological Transitions, this may
It is that cell is in serum-free culturing conditions, cellular morphology is caused to change；Also, there is part thin with two groups of the extension of incubation time
Cellular lysate death (Fig. 3, Fig. 4).
As a result 4, small-molecule drug combination inducing astrocytes reprogramming is mature neuron
For astrocyte morphology to after neuron Morphological Transitions, we have found small point by immunofluorescent staining
Sub drug-induced 6 days or so astroglias also express Tuj1 other than expressing GFAP, show that these astroglias are opened
Begin gradually to change (Fig. 5, Fig. 6, Fig. 7) to neuron.Then detection in 10 days or so find inducing cell great expression MAP2 with
And cellular neural core marker NeuN (Fig. 8)；It is for statistical analysis to induction reprogramming efficiency, find the neuron of the MAP2 positive
Transdifferentiation rate is more than 80%, wherein almost all of MAP2 positive neuron all expresses NeuN, Induction of neuronal is prompted to convert
For mature neuron (Fig. 9).These results suggest that small-molecule drug can be gradually developed with inducing astrocytes for mature mind
Through member.
We are using RT-PCR technology detection different times neuron and astroglia related gene, in small molecule medicine
Cell expression in object Induction Process, as the result is shown with the extension of small-molecule drug induction time, neuron is relevant
The gene expressions such as NGN2 and NEUROD1 gene and motor neuron relevant NKX6.1, OLIG2 and ISL1 are all raised,
And ISL1 reaches to express after peak value when inducing the tenth day and gradually decreases (Figure 11).
As a result 5, small-molecule drug combination inducing astrocytes reprogramming is motor neuron
Neural precursor is into motor neuron atomization in vitro, the RA and SHH of commonly used low concentration or
The combined induction of Purmorphamine is completed.For this purpose, we are using high concentration RA and Purmorphamine and other are small
Molecular drug induces astroglia, and the cell of induction can be made to express general neuronal marker MAP2 and kinesitherapy nerve
First specific marker object HB9 and CHAT (Figure 12, Figure 13), and statistical analysis shows HB9 and CHAT positive cell rate > 90%
(Figure 16), and the marker GAD65 and VGLUT1 of glutamatergic neurons and GABAergic neuron are negative (figure
14, Figure 15).It is neuron that result above, which prompts small-molecule drug combination to can induce astroglia reprogramming, and several
All as motor neuron, without to other kinds of neuron differentiation.
The above is a preferred embodiment of the present invention, it is noted that for those skilled in the art
For, without departing from the principles of the present invention, it can also make several improvements and retouch, these improvements and modifications
It should be regarded as protection scope of the present invention.
Claims (9)
1. a kind of method that induced myeloid astroglia reprogramming is motor neuron, which is characterized in that including following step
It is rapid: (1) Primary rat astroglia purifying culture, using the primary astroglial cells of neonate rat spinal cord as induction weight
The target cell of programming obtains the astroglia of purity is high by the in vitro culture and microwave shake culture of differential attachment method；
(2) substance needed for forming the stage of development according to Neuronal induction and motor neuron has chosen 7 kinds of small-molecule drugs
SB431542, LDN-193189, RA, bFGF, Purmorphamine, Forskolin, VPA, these small-molecule drugs pass through body
Induction reprogramming is carried out to astroglia outside；(3) metamorphosis and Immunofluorescence technology point of cell are observed
Analysis, real-time fluorescence quantitative PCR analysis and statistical analysis method carry out analysis astroglia rearranging to motor neuron
Journey and differentiation situation.
2. a kind of induced myeloid astroglia reprogramming according to claim 1 is the method for motor neuron,
It is characterized in that, the step (1) specifically includes following procedure:
(1-1) superclean bench shift to an earlier date ultraviolet light irradiation 30min, take out -20 DEG C pre-cooling ice platforms, sterilized surgical instrument,
HBSS buffer and plastic culture dish etc. are placed in superclean bench, and appropriate HBSS will be added in culture dish and be placed on ice platform
It is spare；
(1-2) takes newborn 1 age in days rat to be placed in the 50ml centrifuge tube for being placed with appropriate ether, is disappeared after anesthesia with 75% alcohol
Poison, and dried with sterile cotton balls；
While (1-3) is breaked end using tissue shear, sterile cotton balls hemostasis by compression, fixing rat body with hand makes it keep stretching
Posture；
(1-4) cuts off skin along backbone direction with tissue shear, and musculi colli is cut off；
(1-5) cuts off back spinal and tissue along backbone direction from being broken with staight scissors from neck, exposing spinal cord；
Two sides dorsal root ganglion is truncated with microforceps and is chosen spinal cord spare in the HBSS for be placed in pre-cooling by (1-6)；
Spinal cord is moved in another culture dish equipped with pre-cooling HBSS and is placed under stereomicroscope by (1-7), removes table with microforceps
The spinal cord being stripped clean taking-up is placed in the HBSS of pre-cooling by the spinal meninges in face and the blood stains point on surface, and whole process is in ice platform
Upper progress；
Spinal cord is shredded using microscissors and is placed it in 15ml centrifuge tube by (1-8), and the pancreatin of 2ml 0.25% is added, will be from
Heart pipe is placed in 37 DEG C of water-baths, and every 5min concussion mixes until tissue block decomposes, and then into centrifuge tube, addition 5ml is trained completely
Nutrient solution terminate digestion, move into centrifuge after, with 1000rpm revolving speed be centrifuged 5min, discard supernatant be added 4ml base training be resuspended again with
1000rpm revolving speed is centrifuged 5min, is repeated 2 times, and cell finally is resuspended with 4ml complete culture solution and is seeded to 25cm2In culture bottle；
Culture bottle is inverted in CO by (1-9)220min is cultivated in incubator, takes out cell suspension in 15ml centrifuge tube with 1000rpm
Revolving speed is centrifuged 5min, and after removing supernatant and complete culture solution resuspension being added, 25cm is added in cell suspension again2In culture bottle, set
In 5%CO2It is cultivated in incubator, this cell marking is P0 generation；
(1-10) changes liquid for every two days until cell confluent cultures bottle, by culture bottle be placed in 37 DEG C of microwave concussion and cultivate cases with
160rpm shake culture 18h removes oligodendroglia and microglia；
Culture solution is discarded after (1-11) shake culture, and PBS is added and rinses 2-3 times, then adds the 2ml 0.25% of pre-temperature
Trypsin digestion cell is added after 5ml complete culture solution terminates digestion until cell can gently be blown down and blows afloat attached cell, carefully
Born of the same parents' suspension is pipetted into 15ml centrifuge tube, discards supernatant after 1000rpm centrifugation 5min, 1ml complete culture solution is added and is passed on；
The culture of (1-12) astroglia is alternative in subsequent experimental to P2-P3.
3. a kind of induced myeloid astroglia reprogramming according to claim 1 is the method for motor neuron,
It is characterized in that, the step (2) specifically includes following procedure:
(2-1) takes out the coverslip being immersed in 75% alcohol and is placed in 24 porocyte culture plates, and sterile ultrapure water rinsing 3 is added
It is secondary；
(2-2) exhausts ultrapure water, and the poly-D-lysine coating that appropriate 100ng/ml is added overnight, is drawn extra poly for second day and relied
Propylhomoserin is added sterile ultrapure water and rinses 3 times, exhausts surplus liquid, dry spare；Cleaning process is soft, it is ensured that poly-D-lysine
It does not fall off；
(2-3) when step (1) astrocytes culture to P2-P3 for when, be added PBS rinse 2-3 times, pre-temperature is then added
2ml0.25% trypsin digestion cell until attached cell can gently blow down, be added 5ml complete culture solution terminate digestion after
Cell suspension is added in 15ml centrifuge tube, is discarded supernatant after 1000rpm centrifugation 5min, 1ml complete culture solution is added and is resuspended carefully
Born of the same parents, with 4 × 104Cell concentration is inoculated in 24 orifice plates containing coating poly-D-lysine, is put into 5%CO2Incubator overnight incubation；
(2-4) second day culture solution discarded in 24 well culture plates is rinsed 2-3 times with the PBS of pre-temperature, by drug-induced culture solution
One is uniformly mixed, and is added in culture hole, culture plate is put into 5%CO2Incubator culture；
After (2-5) drug-induced culture solution one is cultivated 2 days, drug-induced culture solution two is changed in cell culture well and is placed in 5%
CO2Incubator culture；
After (2-6) drug-induced culture solution two is cultivated 8-10 days, drug-induced culture solution three is changed in cell culture well and is placed in
5%CO2Incubator culture changes liquid in every 2-3 days.
4. a kind of induced myeloid astroglia reprogramming according to claim 1 is the method for motor neuron,
It is characterized in that, the analysis of Immunofluorescence technology specifically includes following procedure in the step (3):
(3-1-1) takes out 24 porocyte culture plates, and the culture solution in hole to be dyed is exhausted, and the PBS rinsing 2-3 of pre-temperature is added
It is secondary, it takes out coverslip and is placed in another 24 orifice plates equipped with 4% paraformaldehyde of 300ml pre-temperature, fixed 10min；
(3-1-2) discards 4% paraformaldehyde, and every hole is added 300 μ l PBS and rinses 3 times, each 5min；
(3-1-3) discards PBS, and 300 μ l rupture of membranes liquid are added in every hole, and room temperature is incubated for 30min；
(3-1-4) discards rupture of membranes liquid, and 300 μ l confining liquids are added in every hole, and room temperature is incubated for 30min；
(3-1-5) discards confining liquid, and 300 μ l primary antibodies and confining liquid mixed liquor, 4 DEG C of overnight incubations are added in every hole；
(3-1-6) discards primary antibody for second day, and every hole is added 300 μ l PBS and rinses 3 times, each 5min；
(3-1-7) discards PBS, and the mixed liquor of 300ml secondary antibody and PBS is added in every hole, and 4 DEG C of incubation >=2h are carried out in the dark processing；
(3-1-8) discards secondary antibody mixed liquor, and every hole is added 300 μ l PBS and rinses 3 times, and each 5min is carried out in the dark processing；
The Hoechst room temperature that PBS thinner ratio 1:1000 is added in (3-1-9) is incubated for 10min, discards Hoechst dilution, is added
300ml PBS is rinsed 3 times, and each 5min is carried out in the dark processing；
(3-1-10) takes adhesiveness glass slide to drip upper mounting liquid, and coverslip is carried out mounting, is protected from light processing；
(3-1-11) is observed and is photographed to record under laser confocal microscope.
5. a kind of induced myeloid astroglia reprogramming according to claim 1 is the method for motor neuron,
It is characterized in that, real-time fluorescence quantitative PCR analysis specifically includes following procedure in the step (3):
(3-2-1) takes out celliferous culture dish, discards culture medium, and rinsed 3 times with the PBS for being preheated to 37 DEG C, 1ml is added
Trizol gently blows and beats and mixes cell, and collects cell suspension in 1.5ml EP pipe, stands 5min on ice；
(3-2-2) concussion of 250 μ l chloroforms is added into EP pipe and mixes, and is placed in -20 DEG C of refrigerators and stands 1-2h；Take out EP pipe
12000rpm, is centrifuged 5 minutes by 4 DEG C；
(3-2-3) goes supernatant liquor in another EP pipe, and isometric isopropanol is added thereto, is mixed by inversion, 12000rpm,
4 DEG C, it is centrifuged 10min；
(3-2-4) carefully discards the ethyl alcohol that 1ml 75% is added in liquid, and 7500rpm 4 DEG C, is centrifuged 5min, discards liquid, repeats
This step；
1ml dehydrated alcohol is added in (3-2-5), and 7500rpm, is centrifuged 5min, discards liquid, and EP pipe is placed in super-clean bench by 4 DEG C
Drying；
(3-2-6) 20 μ l DEPC water dissolution RNA is added into EP pipe, and RNA is carried out reverse transcription, remaining to be stored in -80 DEG C
Refrigerator；
(3-2-7) takes Reverse Transcriptase kit, and the system of following table carries out reverse transcription to RNA with when PCR reaction condition:
The cDNA that (3-2-8) reverse transcription obtains is expanded for qPCR, remaining to be stored in -80 DEG C of refrigerators；
(3-2-9) takes reagent in FastStart Universal SYBR Green Master to be placed on ice chest, the body of following table
System carries out PCR amplification with when reaction condition:
Solubility curve is analyzed after (3-2-10) PCR amplification, it is ensured that PCR product specificity.
6. a kind of induced myeloid astroglia reprogramming according to claim 1 is the method for motor neuron,
It is characterized in that, statistical analysis specifically includes following procedure in the step (3): being carried out by software Graphpad prism5
Data statistics, it is for statistical analysis to all quantized datas and indicated with mean+SD；Sided t is examined for calculating p
The significant property of the statistics of value, p value < 0.05 have statistical significance.
7. a kind of induced myeloid astroglia reprogramming according to claim 2 is the method for motor neuron,
Be characterized in that, the complete culture solution using 1% dual anti-of addition in DMEM/F12 basal medium and 10% FBS side
Method is made.
8. a kind of induced myeloid astroglia reprogramming according to claim 3 is the method for motor neuron,
It is characterized in that, the drug-induced culture solution one is using addition 2 μM of SB431542,1 μM of RA, 5 μ in basic induction broth
M Forskolin, it 20ng/ml bFGF, 0.25 μM of LDN-193189, is made；The drug-induced culture solution two is using in base
5 μM of Purmorphamine of addition, 10 μM of RA, 20 μM of Forskolin, 200ng/ml bFGF are made in plinth induced medium；
The drug-induced culture solution three is using addition 1mM VPA, 5 μM of Purmorphamine, 10 μM in basic induced medium
RA, 20 μM of Forskolin, 200ng/ml bFGF are made.
9. a kind of induced myeloid astroglia reprogramming according to claim 8 is the method for motor neuron,
It is characterized in that, the basis induction broth is used adds 1% dual anti-, 1M B27,1M in DMEM/F12 basal medium
N2, 2mM Pidolidone, 3.5mM glucose is made.
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| WO2020258946A1 (en) * | 2019-06-26 | 2020-12-30 | 南通大学 | Method for inducing reprogramming of spinal cord astrocytes into motor neurons |
| CN113116901A (en) * | 2021-03-15 | 2021-07-16 | 中国人民解放军海军军医大学 | Application of LDN193189 and CHIR99021 in preparing medicine for inducing neuron regeneration |
| WO2022077549A1 (en) | 2020-10-14 | 2022-04-21 | 中国科学院动物研究所 | Composition and method for transdifferentiating non-neuronal cells into neurons |
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