CN105331634A - Method for inducing fibroblasts into neuronal cells by transdifferentiation and application of fibroblasts - Google Patents
Method for inducing fibroblasts into neuronal cells by transdifferentiation and application of fibroblasts Download PDFInfo
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Abstract
The invention provides a composition used for transforming fibroblasts into PV (parvalbumin) neurons, also provides a method for transforming the fibroblasts into neuronal cells and further provides the neuronal cells prepared according to the method. The composition contains one or more of Ascl1 gene and FSK (forskolin). The invention further provides application of the composition and the neuronal cells to preparation of medicines for treating nervous system diseases. According to experiments, the PV neuronal cells can be efficiently obtained by means of transdifferentiation to treat epilepsy after transplantation, memory improvement can be realized by reducing frequency of epileptic seizures, and accordingly a new approach is created for treatment of epilepsy and related diseases.
Description
Technical field
The present invention relates to biotechnology and neurodevelopment field.The present invention is specifically related to method and the application thereof that a kind of induced fibroblast transdifferentiation is neuronal cell.
Background technology
Regenerative medicine based on stem cell transplantation technology is that the mankind defeat intractable degenerative disease to bring huge hope.Induced multi-potent stem cells, owing to breaching the ethical hindrances of embryonic stem cell, is once considered to the new hope of regenerative medicine.But its induced efficiency low, the unstable between batch and high tumorigenicity make it be difficult to be directly used in disease treatment.
Since 2010, some scientists achieve by expressing specific transcription factor the adult cell or the adult stem cell that the adult cell of a type are changed into another kind of type both at home and abroad.This method is called as " transdifferentiation ".This technology does not need this intermediateness of experience multipotent stem cells, therefore can reduce carcinogenic risk.If can directly change other cells in human body into required for disease treatment cell type, then can avoid the series of problems that Transplanted cells brings.Therefore transdifferentiation technology is likely for the diseases such as degenerative disease and organ physical abuse provide good methods for the treatment of, has wide clinical value.
Parvalbumin Parvalbumin (PV) is important neurone special in a kind of brain.Parvalbumin is a kind of aqueous acidic protein separated from the muscle of the frog and fish at first, and the about 12K of molecular weight, is called as " low molecule albumin ".1981, find that PV is present in nervous tissue, and find that the neurone distribution containing PV is distributed with very high consistence with GABA is neuronic, thus start in nervous tissue and contain the neuronic research of PV, also researched and proposed new content to GABA is neuronic.The distribution in the brain of PV neurone is relatively extensive, and PV neurone belongs to GABA neurone family, plays unique function in brain, maintain the balance of electrical activity of brain, and having been reported epileptic condition may cause due to the neuronic exception of PV.
Epilepsy is one of neural system common disease, and epilepsy has become the second largest common disease of Neurology Department, and morbidity is only second to cerebral apoplexy.The World Health Organization adds up, and by the end of 2013, about there were nearly 1,000 ten thousand epileptics in China, wherein 6,000,000 reactivity epileptics by name, and annual newly-increased epileptic 400,000, bring white elephant to society, family and individual.
Epilepsy is chronic recurrent of short duration brain function imbalance syndrome.Cause repeatedly epilepsy outbreak for feature with brain neuron paradoxical discharge.The sickness rate of epilepsy is relevant with the age.Within it is generally acknowledged 1 years old, morbidity is the highest, is secondly to reduce gradually for 1 ~ 10 years old later.
Epilepsy, by cerebral neuron paradoxical discharge, causes the chronic encephalopathy that of short duration central nervous system function is not normal, and basic reason is the unbalance of excitability and inhibitory neurotransmitter.Treat this type of disease by transplanting inhibitory neuron and achieve good effect.But the treatment of epileptics of the source of inhibitory neuron great speed limit.
Summary of the invention
Therefore, the object of the invention is, for the current deficiency that efficiently cannot obtain inhibitory neuron, to provide a kind of induced fibroblast transdifferentiation to be the method for neuronal cell.Meanwhile, this application provides the purposes described neuronal cell being used for the treatment of neural system epilepsy relative disease, thus open up new therapy approach for the treatment of epilepsy or other relevant nerve diseases.
Unless specifically stated otherwise, " PV " neurone in this specification sheets all refers to parvalbumin neurone.
Unless stated otherwise, " FSK " in this specification sheets all refers to Fu Sikelin (Forskolin), be a kind of natural diterpene product be separated from Indian plant (Coleusforskohlii), in stechiology experimental study, Forskolin is commonly used to improve cyclic amp (cAMP) level.
For above-mentioned purpose, technical scheme provided by the invention is as follows:
On the one hand, the invention provides a kind of composition, it is neuronal cell that described composition is used for inoblast transdifferentiation, and described composition contains Ascl1 gene and FSK.
Preferably, described neuronal cell is PV neuronal cell.
On the other hand, present invention also offers the purposes of above-mentioned composition, described composition for the preparation of being neuronal cell by inoblast transdifferentiation, the reagent of preferred PV neuronal cell.
Present invention also offers a kind of for being the substratum of neuronal cell by inoblast transdifferentiation, containing FSK in described substratum, preferably, the concentration of described FSK is 5-30 μM, preferably 10 μMs;
Preferably, FSK is water-soluble, be prepared as certain density liquid storage, then for the preparation of above-mentioned substratum, preferably, the concentration of described FSK liquid storage is 0.5M-1M;
Preferably, described neuronal culture is made up of DMEM substratum, B-27 additive, Brain Derived Neurotrophic Factor (BDNF) and FSK, wherein, the ratio of described each composition is the DMEM substratum containing 980 milliliters in every 1 liter of substratum, 20 milliliters of 50 × B-27 additives, the FSK liquid storage of 10 microlitre 10 μ g/mlBDNF and 5-30 microlitre 1M.
Again on the one hand, the invention provides a kind of is the method for neuronal cell by inoblast transdifferentiation, said method comprising the steps of:
1) by Ascl1 channel genes inoblast;
Preferably, described inoblast is l cell;
Preferably, described inoblast uses DMEM substratum to add 10% foetal calf serum culture medium culturing;
Preferably, described Ascl1 gene uses adenovirus pAD vector introduction inoblast;
2) will import the inoblast of Ascl1 gene, use the neuronal culture containing FSK to cultivate; Obtain neuronal cell.
Preferably, described method also comprises step 3) qualification neuronal cell, preferably, describedly identify that neuronic method is Immunohistochemical Method.
Preferably, described step 1) be that method by comprising the following steps realizes:
A. adenovirus vector construct: Ascl1 gene is building up to adenovirus pAD carrier by BamHI+EcoRI double digestion;
B. the preparation of l cell cell: the mice embryonic choosing 14.5 days, gets skin histology, uses 0.5% trysinization, uses DMEM to add 10% foetal calf serum and cultivates postdigestive skin flbroblast, go down to posterity once, obtain inoblast after 2 days;
C. virus infection: use the Ascl1 adenovirus particles that step a builds, infects inoblast, thus by Ascl1 channel genes inoblast;
Preferably, described step 1) be that method by comprising the following steps realizes: by Ascl1 channel genes inoblast after 6 hours.Use culture medium culturing of the present invention, within every two days afterwards, change a subculture, after induction 10, obtain neuronal cell.
Preferably, described qualification neuronal cell is that method by comprising the following steps realizes:
Neuronal cell is fixed by 4% paraformaldehyde, close with 5%BSA afterwards, use the Tuj1 primary antibodie overnight incubation of rabbit afterwards, within second day, wash 3 times with PBS, then anti-hatch 1 hour with two of the donkey anti-rabbit of cy3 link, under fluorescent microscope, observe cellular form and coloration result.
Again on the one hand, present invention also offers prepared according to the methods of the invention neuronal cell.
The invention provides the application of above-mentioned composition in the medicine for the preparation for the treatment of nervous system disorders.
Present invention also offers the application of prepared according to the methods of the invention neuronal cell in the medicine for the preparation for the treatment of nervous system disorders.
Preferably, described nervous system disorders is epileptics.
The present invention passes through test to be proved, described transdifferentiation technology can efficiently obtain PV neurone, well treats epileptic condition after transplanting, by the reduction of epileptic seizures number of times, improves memory, thus is that the treatment for the treatment of epilepsy and relative disease opens new approach.
Accompanying drawing explanation
Below, describe embodiment of the present invention in detail by reference to the accompanying drawings, wherein:
Fig. 1 is the neurocyte that the present invention obtains;
Wherein, A figure is the GFP cell infected;
B figure is neuron-specific antibody Tuj1 immunohistochemical staining;
C figure infects GFP cell and Tuj1 to dye common marking;
The inoblast that " GFP " representative is infected, " Tuj1 " represents the neuronic Tuj1 antibody staining of transdifferentiation, and the cells infected of " GFP/Tuj1 " green has how many transdifferentiations to be neurone;
Fig. 2 shows that transdifferentiation neurocyte is PV neurone;
Wherein, A figure is PV neuron-specific antibody PV immunohistochemical staining;
B figure is neuron-specific antibody Tuj1 immunity compound staining;
C figure is that PV and Tuj1 antibody staining is marked altogether;
Wherein: " PV " represents PV neuron-specific mark PV antibody staining, and " Tuj1 " represents the neuronic Tuj1 antibody staining of transdifferentiation;
Fig. 3 is the schematic diagram of Nerve Graft cell, and wherein 1 place is transplanted cells, and 2 places are that hippocampus DAPI dyes;
What Fig. 4 showed is the model making epilepsy mouse, and wherein the left side is normal mouse control group, and the right is epilepsy mouse model group;
After Fig. 5 shows that epilepsy mouse transplants inhibition PV neurone, the experiment of spacious field shows that mouse epilepsy syndromes is restored wherein: " Control " represents Normal group, and " Epilepsy " represents epilepsy mouse group, and " PV-HC " represents transplantation group;
Embodiment
Unless specifically stated otherwise, C57BL/6J mouse used in following examples is all purchased from Beijing Vital River Experimental Animals Technology Co., Ltd..
Unless specifically stated otherwise, composition used in following examples is the composition of analytical pure rank, and can purchase from Sigma and obtain.
embodiment 1 induced fibroblast transdifferentiation is PV neurone
Specific experiment flow process:
1) adenovirus vector construct: Ascl1 gene is building up to adenovirus pAD carrier by BamHI+EcoRI double digestion, carries out the packaging of virus and the mensuration of virus titer respectively;
2) preparation of l cell cell: the mice embryonic choosing 14.5 days, decaptitate, after tail, backbone, internal organ and sexual gland, get skin histology, with 0.5% trysinization after 5 minutes, postdigestive skin flbroblast is taped against in 10 cm dishes and adds 10% foetal calf serum with DMEM, cultivate, go down to posterity after 2 days once, finally obtain inoblast;
3) virus infection: the hole 60,000 inoblasts being taped against 24 orifice plates, by packaged Ascl1 adenovirus particles, infect inoblast, after 6 hours, be replaced by neuronal culture (DMEM, 1 × B-27,10 μ g/mlBDNF), in neuronal culture, add little compound F 17-hydroxy-corticosterone SK (10 μMs), within two days afterwards, change a subculture, after induction 10, be detected as fibrocyte whether transdifferentiation neurone;
4) after within 10 days, cultivating, transdifferentiated cells is fixed by 4% paraformaldehyde, close with 5%BSA afterwards, use the Tuj1 primary antibodie overnight incubation of rabbit afterwards, within second day, wash 3 times with PBS, then anti-hatch 1 hour with two of the donkey anti-rabbit of cy3 link, under fluorescent microscope, observe cellular form and coloration result afterwards.
Experimental result:
Determine the neuronic number that transdifferentiation is formed and form.As the detection of the neurone marks such as Tuji1, PV, the results are shown in Figure 1 and Fig. 2, wherein Fig. 1 is the neurocyte that the present invention obtains; A figure is the GFP cell infected; B figure is neuron-specific antibody Tuj1 immunohistochemical staining; C figure infects GFP cell and Tuj1 to dye common marking; The inoblast that " GFP " representative is infected, " Tuj1 " represents the neuronic Tuj1 antibody staining of transdifferentiation, the cells infected of " GFP/Tuj1 " green has how many transdifferentiations to be neurone, shows that the present invention acquires the mark Tuj1 that neuronal cell expresses neuron-specific.
Fig. 2 shows that transdifferentiation neurocyte is PV neurone; Wherein, A figure is PV neuron-specific antibody PV immunohistochemical staining; B figure is neuron-specific antibody Tuj1 immunity compound staining; C figure is that PV and Tuj1 antibody staining is marked altogether; Wherein: " PV " represents PV neuron-specific mark PV antibody staining, and " Tuj1 " represents the neuronic Tuj1 antibody staining of transdifferentiation; Show that the present invention acquires the mark PV (Fig. 2) that neuronal cell expresses PV neuron-specific.
embodiment 2 transplants PV neurocyte treatment epileptic condition
Specific experiment flow process:
1) foundation of epilepsy mouse model.By abdominal injection pilocarpine medicine (280mg/kg) in 6 weeks large adult C57 mouse, injectable drug is after 2 weeks, and epilepsy mouse model is set up in induction.The mouse one of this test is divided into 3 groups, comprises 10 normal mouse groups, 10 epilepsy mouse groups, and 10 Nerve Graft cells are to epilepsy mouse group.
2) Nerve Graft cell.By stereoscopic localized and micro-injection system, transplant 50,000 neurocyte to the hippocampus in mouse brain.
Concrete hippocampus of mice location: with pilot pin 2mm after bregma, sagittal suture is other opens location, 2.5mm place a bit, is the planimetric position of hippocampus, then on this aspect, bores a small sircle hole with needle for trephination on skull.
Transplanted cells: hippocampus of mice is then positioned at 2mm under this circular hole, and operating instrument makes syringe needle be expelled to mouse brain hippocampus by completing during mouse brain drill hole decline 2mm.As shown in Figure 3, wherein 1 place is transplanted cells to result, and 2 places are that hippocampus DAPI dyes;
3) study of behaviour normal form detects mouse disease recovery extent.The study of behaviour detecting mouse by spacious field experiment study of behaviour normal form changes.Catch mouse tail root 1/3rd place to mention, put into spacious field experimental box (50 cm x 50 cm x 50 centimetres) center lattice gently, record the movement locus of mouse in the box of spacious field in 5 minutes.
Experimental result:
By by neuron transplantation to hippocampus of epileptic mouse region, detect the behavioral indexes of mouse after 2 months.Tested by spacious field, we observe normal mouse group, epilepsy mouse group, transplantation group respectively in 5 minutes move distance be 11,000,15,000,14000 centimetres; Treat that in the ratio of central zone time be 3.7%, 9.1%, 4.2% respectively; Treat that in the ratio of central zone distance be 8.2% respectively, 20.3%, 10.4%, result as shown in Figure 5, after showing that epilepsy mouse transplants inhibition PV neurone, the experiment of spacious field shows that mouse epilepsy syndromes is restored wherein: " Control " represents Normal group, and " Epilepsy " represents epilepsy mouse group, and " PV-HC " represents transplantation group.
Spacious field experiment display epilepsy group run duration is many, and move distance is long, and detecting, the time for the treatment of in the middle of box is obviously many, and display epilepsy mouse has the activity of high frequency.But after neuron transplantation, transplantation group move distance has and to a certain degree reduces, and stay in central section time and distance, transplantation group is significantly improved.
Claims (10)
1. a composition, it is neuronal cell that described composition is used for inoblast transdifferentiation, and described composition comprises Ascl1 gene and FSK;
Preferably, described neuronal cell is PV neuronal cell.
2. the purposes of composition as claimed in claim 1, described composition is for the preparation of being the reagent of neuronal cell by inoblast transdifferentiation, and preferably, described neuronal cell is PV neuronal cell.
3. for being a substratum for neuronal cell by inoblast transdifferentiation, wherein, described substratum contains FSK, and the concentration of described FSK is 5-130 μM, preferably 10 μMs;
Preferably, FSK is water-soluble, be prepared as certain density liquid storage, then for the preparation of described substratum, preferably, the concentration of described FSK liquid storage is 0.5M-1M;
Preferably, described substratum is made up of DMEM substratum, B-27 additive, Brain Derived Neurotrophic Factor (BDNF) and FSK;
Wherein, the ratio of described DMEM substratum, B-27 additive, Brain Derived Neurotrophic Factor (BDNF) and FSK is the DMEM substratum containing 980 milliliters in every 1 liter of substratum, 20 milliliters of 50 × B-27 additives, the FSK liquid storage of 10 microlitre 10 μ g/mlBDNF and 5-30 microlitre 1M.
4. be a method for neuronal cell by inoblast transdifferentiation, said method comprising the steps of:
1) by Ascl1 channel genes inoblast;
Preferably, described inoblast is l cell;
Preferably, described inoblast uses DMEM substratum to add 10% foetal calf serum culture medium culturing;
Preferably, described Ascl1 gene uses adenovirus pAD vector introduction inoblast;
2) will import the inoblast of Ascl1 gene, use neuronal culture as claimed in claim 3 to cultivate, obtain neuronal cell;
Preferably, described method also comprises the step of qualification neuronal cell, and preferably, the step of described qualification neuronal cell is undertaken by Immunohistochemical Method.
5. method according to claim 4, wherein, described step 1) be that method by comprising the following steps realizes:
A. adenovirus vector construct: Ascl1 gene is building up to adenovirus pAD carrier by BamHI+EcoRI double digestion;
B. the preparation of l cell cell: the mice embryonic choosing 14.5 days, gets skin histology, uses 0.5% trysinization, uses DMEM to add 10% foetal calf serum and cultivates postdigestive skin flbroblast, go down to posterity once, obtain inoblast after 2 days;
C. virus infection: use the Ascl1 adenovirus particles that step a builds, infects inoblast, thus by Ascl1 channel genes inoblast.
6. method according to claim 4, wherein, described step 2) be that method by comprising the following steps realizes:
After 6 hours, using neuronal culture as claimed in claim 3 to cultivate Ascl1 channel genes inoblast, within every two days afterwards, changing a subculture, induce after 10 days, obtain neuronal cell.
7. method according to claim 4, wherein, the step of described qualification neuronal cell is that the method by comprising the steps realizes:
The neuronal cell obtained is fixed by 4% paraformaldehyde, close with 5%BSA afterwards, use the Tuj1 primary antibodie overnight incubation of rabbit afterwards, within second day, wash 3 times with PBS, then anti-hatch 1 hour with two of the donkey anti-rabbit of cy3 link, under fluorescent microscope, observe cellular form and coloration result.
8. neuronal cell prepared by the method according to any one of claim 4-7.
9. a composition, described composition comprises neuronal cell as claimed in claim 8.
10. neuronal cell as claimed in claim 8 or the application of composition as claimed in claim 9 in the medicine for the preparation for the treatment of nervous system disorders;
Preferably, described nervous system disorders is epileptics.
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CN110665059A (en) * | 2019-10-17 | 2020-01-10 | 苏州大学 | Tissue engineering nerve transplant and preparation method thereof |
CN110872576A (en) * | 2019-06-06 | 2020-03-10 | 中国科学院广州生物医药与健康研究院 | Method for transdifferentiation of mouse fibroblasts into dopaminergic neurons |
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CN110872576A (en) * | 2019-06-06 | 2020-03-10 | 中国科学院广州生物医药与健康研究院 | Method for transdifferentiation of mouse fibroblasts into dopaminergic neurons |
CN110665059A (en) * | 2019-10-17 | 2020-01-10 | 苏州大学 | Tissue engineering nerve transplant and preparation method thereof |
CN110665059B (en) * | 2019-10-17 | 2021-09-28 | 苏州大学 | Tissue engineering nerve transplant and preparation method thereof |
CN113278585A (en) * | 2021-03-26 | 2021-08-20 | 广西大学 | Method for efficiently inducing human body cells to reprogram into neuronal cells |
CN113278585B (en) * | 2021-03-26 | 2023-09-15 | 广西大学 | Method for in vitro induction of reprogramming of human cells into neuron cells |
CN114231494A (en) * | 2021-11-29 | 2022-03-25 | 中国科学院动物研究所 | Application and method of USP10 gene and/or Ascl1 gene in inducing fibroblast to transdifferentiate into neuronal cell |
CN114231494B (en) * | 2021-11-29 | 2023-09-05 | 中国科学院动物研究所 | Application and method of USP10 gene and/or Ascl1 gene in inducing fibroblast to transdifferentiate into neuron cell |
CN114958747A (en) * | 2022-06-08 | 2022-08-30 | 中国科学院动物研究所 | Method for inducing pluripotent stem cells to generate excitatory and inhibitory neurons |
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